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Total tobacco cessation is the only treatment that improves the symptoms and reduces the risk of amputation in Buerger's disease. Other forms of treatment are not well established or have unfavorable results. Surgical revascularization is often ineffective because the distal target vessels are often involved in this diffuse segmental disease. Calcium channel blockers, anticoagulants, thrombolytics, prostaglandin analogs, sympathectomy, adrenalectomy, spinal cord stimulators, omental transfers, and stem cell treatment have all been tried with limited success in decreasing rest pain and avoiding amputation. We report a case of an interventional endovascular procedure for revascularization. A 28-year-old male had been suffering from acute right foot pain at rest and coldness with dysesthesia, cyanosis on the right 1st toe, and claudication for 3 weeks. The ankle-brachial pressure index (ABI) was 0.6 on the right leg. As he was a heavy smoker, the patient's hands and feet were usually cold, and the symptoms became severe when he was exposed to a cold environment. He was free of other diseases, including autoimmune disease, hypercoagulable state, and diabetes mellitus. Hematological laboratory findings and renal function tests were normal. Computerized tomography (CT) angiography showed segmental obstructions of the below-knee popliteal artery, which showed intraluminal thrombosis, and in the posterior tibial artery. The anterior tibial artery was obstructed (). We decided to operate because of the severity of the symptoms and the short segmental obstruction with thrombus. The wall of the popliteal artery had an adhesion and had thickened. The intima of the popliteal artery had also thickened due to inflammatory changes. We performed endarterectomy and vein patch angioplasty of the obstructed popliteal artery with the greater saphenous vein. A Fogarty catheter was not passed under the obstruction of the posterior tibial artery. However, the arteriotomy site was closed with patch angioplasty because of the occurrence of a large amount of back bleeding. After the first operation, the patient's symptoms did not improve and the patient requested reoperation to alleviate the symptoms. The blood vessel had surrounding coalescence with an inflammatory reaction. We performed vein patch angioplasty in the segmental obstruction of the posterior tibial artery 2 weeks after the first operation. Because of the thick intima and fibrosis, endarterectomy was not possible. After the operation, the patient's symptoms improved and he was discharged on postoperative day 15. His ABI was 1.0. In the histopathological examination, segmentally resected and serially sectioned popliteal arteries showed subacute stages of Buerger's disease. The lumen was near-totally or totally occluded by organized thrombi that presented as recanalized vessels. Remarkable chronic inflammatory cells such as lymphocytes and plasma cells were observed in the recanalized vessel wall with less inflammation in the walls of the blood vessels. The symptoms recurred, and the patient was re-hospitalized after 3 months. CT angiography showed diffuse posterior tibial artery obstructions in the patch angioplasty of the posterior tibial artery in the upper direction (). A pre-procedural angiogram showed severe stenosis in the upper part of the vein patch angioplasty of the posterior tibial artery. The possibility of stenosis and obstruction always exists in the blood vessels of patients with Buerger's disease due to inflammatory reactions. Angioplasty of the posterior tibial artery was performed using a balloon catheter (4-40 mm, Ever Cross; ev3 Endovascular Inc., Plymouth, MN, USA). Angiography after this procedure demonstrated a good final angiographic result and the absence of critical stenosis. Improved blood circulation was observed in the CT angiography performed after 1 year (). The patient recovered completely with no further complications or symptoms. This patient quit smoking from before the first operation until the time of this report. For 2 months after the endovascular procedure, warfarin was used. From after the first operation until the present, aspirin and clopidogrel (Plavix) have been used. Buerger's disease is defined as a nonatherosclerotic segmental disease that is characterized by occlusive, inflammatory, and thrombotic changes. Several different criteria, such as those suggested by Shionoya [] and Olin [], have been proposed for the diagnosis of Buerger's disease. Our case met all of the diagnostic criteria. Typical angiographic findings were distal vessel disease with no presence of arterial wall calcification and the development of a rich typical subcutaneous network of collaterals, usually referred to as corkscrew collaterals []. Tobacco use, in any of its forms, plays a central role in the pathogenesis and progression of the condition []. Clinical presentation usually begins with ischemia of the distal small arteries and veins of the legs, arms, feet, and hands, which is manifested by claudication of the corresponding extremities []. Progression of the disease is typically characterized by calf claudication and, eventually, ischemic pain at rest. Ischemic ulcerations on the toes, feet, or fingers may develop. Superinfection often occurs, and the lesions progress toward necrosis and distal gangrene. The prognosis for patients with Buerger's disease with respect to limb loss is significantly worse than that for patients with either atherosclerosis or the various forms of necrotizing immune arteritis [,]. At present, the only proven strategy to prevent the progression of the disease and avoid amputation, in some cases, is the complete discontinuation of cigarette smoking or any other use of tobacco in any form []. Nevertheless, many patients continue to have a very poor quality of life owing to the persistence of symptoms such as intermittent claudication, Raynaud's phenomenon, or even amputations [,]. The risk of amputation is highly correlated with continuing to smoke; the amputation rate varies from 42% to 5% at the Cleveland Clinic Foundation according to tobacco use []. At the end of a 5-year follow-up, the median prevalence of amputation in patients with Buerger's disease, based on the most recently reported series, is reported to be as high as 24.4% for minor amputations and 8.6% for major amputations, for a total amputation rate of 33% for patients treated conservatively []. Surgical revascularization for patients with Buerger's disease is possible only in a few cases, as a result of diffuse distal involvement with no distal runoff vessels available for bypass surgery [,]. Consequently, surgical procedures are technically challenging, with very low feasibility and patency rates. Endovascular treatment for Buerger's disease is not generally reported in the medical literature as either feasible or effective. Graziani et al. [] reported that an endovascular procedure is safe, technically feasible, and effective, independent of the continued or non-use of tobacco, which leads us to argue that the procedure alone achieved these high rates of clinical success, the decreased rates of amputation, and the maintenance of clinical improvement during midterm follow-up periods. Although the physiopathology of the disease is characterized by the presence of inflammation arteritis and associated thrombosis, this did not preclude or affect the results of the primary angioplasty of the treated lesions. In our case, the patient was suspected of having a subacute form of Buerger's disease on the basis of the microscopic findings. It seems that inflammatory changes in blood vessel obstruction were in progress after the patient underwent patch angioplasty of the segmental obstruction. We resolved this by endovascular intervention, and we have been observing the patient's progress for about a year, but no strictures or other abnormalities have been found. Finally, although tobacco cessation is one of the most important steps for a significant reduction in disease progression, we believe that an endovascular procedure can be an effective treatment, even in addition to more conservative and surgical management, in patients with Buerger's disease and critical limb ischemia.

A 19-year-old female presented with recurrent hemoptysis and oral ulceration. Her first experience of hemoptysis had been 2 years earlier; she was admitted to a local hospital at that time. Bronchofiberscopy revealed diffuse alveolar hemorrhage; however, there were no specific findings for the cause of hemoptysis. The patient was diagnosed with Behcet's disease (BD) since she had oral ulcers and erythematous skin lesions. Medical treatment including prednisolone and azathioprine was started. Two years later, she presented with hemoptysis and went through an additional bronchofiberscopic exam. The exam revealed a hyperemic nodular lesion within the anterior basal segmental bronchus of the right lower lobe (RLL). Biopsy failed because of the little bleeding that occurred when the nodule was touched. The patient was discharged with the same medication after conservative treatment. However, the patient experienced another episode of hemoptysis and was referred to Seoul Asan Medical Center emergency room. A rheumatologist and an ophthalmologist were consulted for a differential diagnosis between BD and Hughes-Stovin syndrome (HSS). Further, since these two conditions have a strong predilection for the male gender, a gynecologist examined the patient for thoracic endometriosis. The patient's physical examination showed oral ulcers but no evidence of genital ulcers, uveitis, and skin lesions. According to her past history, she had had no skin lesions since 2 years earlier, and hemoptysis occurred without any association with her menstrual cycle. Chest computed tomography showed residual ground-glass opacity in RLL due to aspirated blood, but no definite endobronchial lesion or vascular abnormality were observed as the cause of hemoptysis. Chest X-ray showed no specific findings. The patient's blood test was normal except for a mild elevation of the erythrocyte sedimentation rate (29 mm/hr). For the differential diagnosis, anti-nuclear antibody, rheumatoid factor, serum complement levels (C3 and C4), and anti-neutrophil cytoplasmic antibodies (c-ANCA and p-ANCA) were tested; all of them showed negative results. Elective bronchofiberscopy was scheduled in the operation room. During the exam, a small polyp was observed at the anterior basal segmental bronchus of the RLL and excisional biopsy was proceeded (). After the excisional biopsy using laser, active bleeding occurred; therefore, emergent surgical resection was decided upon. Basal segmentectomy of the RLL was performed using video-assisted thoracoscopic surgery. After transection of the basal segmental bronchus, intraoperative bronchoscopy showed no bleeding focus in the endobronchial area. The patient's postoperative course was uneventful. Chest tube drainage was removed on postoperative day 3, and the patient was discharged the next day with 5 mg/day of oral prednisolone. Pathological examination revealed abnormally dilated vascular structures in the bronchial submucosa with rupture (). Considering pulmonary aneurysm, an oral ulcer with no evidence of systemic thrombosis, we diagnosed this patient as an atypical presentation of BD. Pulmonary involvement of BD and HSS is characterized by thrombophlebitis and pulmonary aneurysm. To distinguish HSS from BD, physical examination and close observation are important. Specific features to BD are known to be oral ulcers, recurrent genital ulceration, eye lesions, skin lesions, and a positive pathergy test. However, thus far, the diagnostic criteria for HSS have not been formally established as the etiology of HSS is still unclear and only a few cases of the disease have been reported []. Therefore, HSS is diagnosed if a patient presents with a combination of aneurysm and thrombosis without any specific features of BD []. However, other clinical features are not specific since thrombophlebitis and aneurysm can occur anywhere, including in the hepatic artery, iliac artery, vena cava, cardiac chambers, femoral vein, iliac vein, and dural sinuses []. The patients may present cough, dyspnea, pulmonary hypertension, intracranial hypertension, fever, chills, chest pain, and hemoptysis [,]. Laboratory findings are also not specific. The blood tests can show leukocytosis, elevated C-reactive protein, erythrocyte sedimentation rate, and anemia when hemoptysis or aneurysmal rupture occurs. Radiologic evaluations, including chest X-ray and chest computed tomography, could be useful only in few patients with large aneurysms. Conventional angiography is regarded as a gold standard of pulmonary artery aneurysms []. However, when the patient has thrombosis in the vena cava and has a high risk of aneurysmal rupture, angiography should not be performed. Bronchoscopy might be useful in patients who present hemoptysis with bronchial artery aneurysm, and a Doppler ultrasound of extremities is important for evaluating the presence of deep venous thrombosis. Some patients, however, do not meet the international classification criteria for BD, as in this case. They may be indistinguishable from patients with HSS since the radiological and histopathological findings are similar. Further, the final clinical stage in both conditions is aneurysmal rupture with massive hemoptysis, which leads to a high mortality rate []. Therefore, HSS is suggested as an atypical manifestation of BD. Considering the histopathological findings, the medical management is similar in both diseases: steroids alone or in combination with immunosuppressants []. However, a standard treatment of HSS has not been established because of its unknown etiology. Surgical treatments are performed when massive hemoptysis originates from one segment or one lung. Lobectomy or pneumonectomy could be performed to remove the aneurysm [,]. However, in cases of extensive bilateral multiple aneurysms, operative treatments might be associated with high mortality and morbidity rates []. For these patients, transcatheter arterial embolization or immunosuppressive therapy might be an option []. To conclude, BD and HSS are rare diseases that might be indistinguishable. Both diseases, however, are associated with high mortality because of the massive hemoptysis in the final stage. Therefore, early diagnosis and immediate surgical resection are crucial for prevention of disastrous complication and mortality. Further, as in this case, the surgical team should be well prepared since an invasive evaluation might cause unexpected complications when the patient is suspicious of pulmonary or bronchial artery aneurysm.

A five-month-old male infant (weight, 6 kg; height, 64 cm) underwent elective repair of a large perimembranous-type ventricular septal defect (VSD). He had no discernible anomaly other than VSD; his preoperative routine laboratory values were all within normal limits, and he had no family history of genetic disease including malignant hyperthermia (MH)-susceptible trait (which was inquired later). The echocardiography revealed a large VSD of the perimembranous type with pulmonary hypertension. Premedication was not given. Upon anesthetic induction, fentanyl sodium and ketamine were administered intravenously, followed by rocuronium bromide for intubation. Anesthesia was maintained with nitrous oxide and sevoflurane, along with incremental doses of fentanyl and ketamine. End-tidal PCO (ETCO) and oxygen saturation were monitored continuously with an intermittent analysis of the arterial blood gases. The operation was carried out under standard hypothermic cardiopulmonary bypass (CPB). The patient's body temperature was normal (36.8℃) before the induction of anesthesia, but it increased to 38.7℃ just before the bypass; no specific measures were taken as he was going to be on hypothermic CPB soon. Systemic cooling was applied shortly after the initiation of the bypass, and the temperature was maintained around 26℃ throughout the intracardiac procedure. The flow rate was appropriately controlled according to the body temperature, and there was no episode of a significant fall in the mean blood pressure during the entire course of CPB. The blood gas profiles were also in the normal range. Upon rewarming, although the standard rewarming protocol was applied, a very rapid and excessive elevation of the temperature was noted; it took only 17 minutes for the temperature to reach 36.6℃/36.2℃ (esophageal/rectal), and then, the temperature continued to rise and peaked at 38.2℃/39.1℃ 12 minutes later. Cooling measures were applied, and the temperature fell gradually to the normal level 35 minutes after peaking. The CPB weaning was smooth. Urine dropped well without any noticeable color change. The first sign of metabolic acidosis, along with hypoxia and hypercapnia, was noted in the blood gas sample drawn 44 minutes after the CPB weaning. Hypoxia and hypercapnia were corrected by the manipulation of the ventilator; however, a mild degree of metabolic acidosis persisted (). Nevertheless, the hemodynamic parameters were quite stable. The chest was closed, and the patient was transferred to the intensive care unit (ICU) in a stable condition. Initially, in the ICU, the only sign against normal recovery was metabolic acidosis, which continued until postoperative day 2. Intermittent pyrexia was managed conservatively. The initial profile of the cardiac enzyme (creatine kinase [CK]/CK-myocardial band [MB]/troponin-I: 1,100/190/98.7) was unusually elevated, showing results that did not match the clinical findings at that time. Mental recovery was delayed; the patient opened his eyes late in the evening. Nonetheless, he was sedated again overnight mainly because of the unexpected metabolic acidosis. The next day, sedation was stopped, but the patient's movement was rather sluggish. He opened his eyes, but his eye contact was uncertain. The ventilator weaning was not successful. The serum concentrations of the cardiac enzymes continued to increase. The patient's mental status deteriorated rapidly over time, and he became comatose. The computed tomogram of the brain taken on postoperative day 2 revealed a diffuse brain injury pattern. The CK level continued to rise, peaking at 21,880 IU/L on postoperative day 2; it gradually fell afterwards (). Nonetheless, the urine output was well maintained, without any noticeable color change and without a significant elevation of the serum blood urea nitrogen or creatinine. Neither was observed as a significant abnormality in the chest X-ray. Echocardiography showed good postoperative results; the VSD was closed well without a residual shunt, and cardiac contractility was preserved. Metabolic acidosis also resolved after postoperative day 2. However, the patient fell into full coma and never regained consciousness. The isoenzyme study of CK from a blood sample taken on postoperative day 5 revealed a significant elevation of the MM fraction (). The family did not approve a tracheostomy. Thereafter, the patient followed a gradual downhill course and eventually died on postoperative day 33. At first, upon reviewing this complicated and unhappy case, malperfusion came to our mind first as a culprit for the diffuse brain injury and massive rhabdomyolysis leading to death. However, according to the perfusionist's record, the pump speed and the flow rate were maintained within the acceptable range throughout the run of the bypass, and the blood gas profiles were absolutely within the normal limits. Moreover, the extent of rhabdomyolysis as represented by the CK elevation was too severe to be simply attributed to malperfusion []. It would be logical to conclude that the initial excessive elevation of serum CK-MB was only a reflection of the elevation of the total CK amount. As for MH, we were not aware of it until we were reminded by a text that MH was supposed to be the number-one disease to be ruled out in the differential diagnosis of rhabdomyolysis []. MH is a genetic disorder of the skeletal muscle that presents a high fever, usually after operation, as a result of the hypermetabolic state to the volatile anesthetic agents, such as halothane, sevoflurane, desflurane, and the depolarizing muscle relaxant succinylcholine, and rarely to stresses such as vigorous exercise and heat. This disorder is characterized by massive rhabdomyolysis and is likely to be fatal unless recognized early and treated appropriately. The classic signs of MH include hyperthermia to a marked degree, tachycardia, tachypnea, increased carbon dioxide production, increased oxygen consumption, acidosis, and muscle rigidity. The early diagnostic clue of the disorder is known to be elevated ETCO. However, the presentation of the symptoms is highly variable: The fever can be a mild one and even totally omitted, and the interval from the exposure to the symptom onset can be highly variable. Thus, MH is a disease of exclusion; a variety of conditions may resemble MH during anesthesia. Similarly, when severe rhabdomyolysis is encountered postoperatively, MH should be considered in the first line []. In this context, we scrutinized the case again and came up with the notion that the earlier abnormal pattern of rewarming, which was once thought to be insignificant, might be an early sign of MH. The ensuing events such as acidosis and rhabdomyolysis corresponded well to the clinical features of MH as described earlier. It might be argued that careless and very rapid rewarming was the principle cause of death. We insisted that the temperature had increased abruptly although we had followed the general guideline of rewarming. In addition, we believed that if MH had developed in the setting of CPB, one could hardly have detected the early signs largely because of the symptoms obscuring hypothermia. Thus, we systematically searched the literature focusing on MH and CPB and found some relevant articles. Recently, Metterlein et al. [], in their review article, have described 14 patients experiencing an episode of MH associated with cardiopulmonary bypass in 12 reports over nearly three decades. In general, when MH develops in patients undergoing cardiac surgery under cardiopulmonary bypass, the situation becomes more complicated. Classical clinical features are obscured by the effect of hypothermia, rendering the early recognition and timely management of MH extremely difficult [,]. They also pointed out that putting aside volatile anesthetics, rewarming per se can be a triggering factor or at least have a modulating effect on the development of a crisis []. The definite diagnosis of MH is currently made by an contracture test [], which unfortunately was not carried out in this patient because of the rapidly declining clinical course. Dantrolene, which is currently accepted as an effective treatment regimen [], was also not administered in time because clinical suspicion was lacking at that time. General measures for the management of MH are usually supportive and symptom-specific. All potent inhalation agents and muscle relaxants should be stopped immediately, and minute ventilation should be increased to lower ETCO. Cooling measures such as ice pack application and/or nasogastric lavage with an iced solution should be carried out. Measures to maintain the urine output should be encouraged, and the treatment of hyperkalemia, arrhythmia, and potential disseminated intravascular coagulation should be undertaken as needed []. In summary, although we could not explain the cause of the brain injury, the occurrence of MH was very probable in this case; the abnormal pattern of temperature rise upon rewarming, which was transient and did not draw much attention at that time, might have been an early sign of MH. The ensuing metabolic acidosis that was hard to correct and massive rhabdomyolysis also tied in very closely with the clinical situation in the case of MH. We might have missed the first sign of MH-the increase in ETCO by a simple manipulation of the ventilator, as pointed out by others []. One should be aware of the possibility of MH, although it rarely occurs, when unexplained metabolic acidosis and rhabdomyolysis develop and continue in association with CPB and rewarming.

An 86-year-old female was referred to Seoul St. Mary's Hospital because of abnormal chest X-ray findings. The chief complaints were intermittent cough and mild fever ongoing for one month. Computed tomography (CT) revealed a mass (3.3×2.2 cm) in the lingula of the left upper lobe (LUL) and a second lesion (2.2×1.6 cm) in the superior segment of the left lower lobe (LLL), which were most likely malignant (). Preoperative assessments included magnetic resonance imaging of the brain, bone scan, whole-body positron emission tomography-CT, echocardiography, and bronchoscopy. Clinically, an abscess of LUL and a cancer of LLL (stage cT1bN0M0) were suspected. Preoperative forced expiratory volume in one second (FEV) was 1.37 L. Echocardiography revealed normal ejection fraction (60%), but akinesia on the basal posterior wall and a ventricular premature complex were observed in the electrocardiography. For the complete resection, lingular segmentectomy and LLL lobectomy should be carried out. Postoperative FEV was estimated to be 0.9 L. Due to advanced age and poor respiratory function, the surgical plan was wedge resection for the LUL lesion first. If the lesion was not malignant on the frozen report, the next step was the superior segmentectomy of LLL. Under general anesthesia, a double-lumen endotracheal tube was placed, and the patient was transitioned to the right lateral decubitus position. Once selective lung ventilation was achieved, a 4-cm incision was made in the fifth intercostal space at the anterior axillary line with a wound protector (Applied Medical, Rancho Santa Margarita, CA, USA). The two distinct masses were digitally palpable, with a visible retraction of the lower lobe visceral pleura. The cavitary lesion in LUL was sizable. Simple wedge resection was difficult for complete resection. First, the anterior portion of the fissure was dissected and divided, exposing the interlobar artery (). The lingular segmental artery was then identified and divided, followed by the elevation and resection of the upper lobe mass. Then, complete resection was achieved, and the distance between the mass and the resection margin was 0.6 cm. After there was no evidence of malignancy on the frozen section report, the superior segmental artery (encircled by a drain catheter) was identified and divided using an endostapler (TriStapler; Covidien, Norwalk, CT, USA) (). The inferior pulmonary ligament was divided to reveal the inferior pulmonary vein. Eventually, the superior segmental vein was divided using a Hem-o-lok (Teleflex Medical Inc., Research Triangle Park, NC, USA). After identifying the superior segmental bronchus, the peribronchial tissue and lymph nodes were dissected. Then, the bronchus was clamped, and both lungs were ventilated (), enabling the visualization of the segmental plane and stapler division (). Mediastinal lymph node dissection (para-aortic, subcarinal, inferior pulmonary ligament, and paraesophageal lymph nodes) was performed (). A chest tube (24 Fr.) was placed in the pleural cavity through the utility incision, and the wound was closed (). The total operative time was 135 minutes. Histopathology confirmed an abscess of LUL and an adenocarcinoma (1.6×1.4 cm) of LLL with the invasion of the visceral pleura (stage T2aN0M0), and a distance of 3 cm was obtained between the tumor and the divided segmental plane. The patient was discharged on postoperative day 7 without complications. Since its introduction in the 1990s, video-assisted thoracoscopic surgery (VATS) has been an attractive treatment modality for thoracic surgery, resulting in shorter recovery periods and less postoperative pain than open procedures []. Although, generally, three or four incisions are used for VATS lobectomy or the segmentectomy of lung cancers, a report by Rocco et al. [] on uniportal VATS wedge resection, citing the potential to reduce postoperative pain and paresthesia, has caught the interest of thoracic surgeons. Since 2010, Gonzalez-Rivas et al. [] have adopted this technique for lung cancer surgery, confirming its feasibility in a recent publication. Recently, many centers have adopted the abovementioned technique []. Furthermore, segmentectomy has also been performed by single incision []. Although lobectomy is the standard surgical management for early lung cancer, lobectomy is not suitable for all patients. Most of these patients are elderly and have poor cardiopulmonary reserve. Segmentectomy is a reasonable alternative procedure. In a comparison of segmentectomy with lobectomy for non-small cell lung cancer <2 cm, Okada et al. [] found that the 5-year survival rates did not differ by procedure. In particular, in the older patients, segmentectomy leads to less complications and low comparable oncologic impact than lobectomy []. We extended our experience with VATS for lung cancer to include a single-incision approach using a 4-cm utility incision of the 5th intercostal space (at the anterior axillary line). A wound protector is employed to protect the camera lens from oozing by the intercostal muscle. The required instrumentation does not otherwise differ from that of conventional VATS (endograsper, 10-mm camera with 30° angle, right-angle clamp, and ultrasonic device). The operative procedure differs according to the affected lobe. During dissection, we use a harmonic scalpel (Ethicon Endo-Surgery Inc., Cincinnati, OH, USA). A harmonic scalpel is very useful and suitable for the dissection and coagulation of microvessels because of its blunt tip and scissors-like action. We preferred a long right-angle clamp with a blunt tip during the tunneling and encircling of the vessels and the bronchus. Single-incision VATS does have some limitations. Clearly, when the multiple views of conventional VATS are forfeited, obstruction of view and impingement of instruments commonly occur. However, the surgical technique is not different, including the identification and division of the vessels and the bronchus, and lymph node dissection. We generally center the camera on the incision, inserting instruments on either side of it; further, for better visualization, the stapler is placed in the thoracic cavity beneath the camera. Conducting extensive surgery through a single, small incision is often ergonomically problematic for surgeons and assistants. During an operation, the operator and the assistant are sometimes superimposed; therefore, it is important to prevent contamination. We made a utility incision in the 5th intercostal space. The procedure through the 6th intercostal space allows the division of the inferior pulmonary ligament and the inferior pulmonary vein more easily. However, our first step is the dissection of the interlobar space; this approach is easier through the 5th intercostal space. If a conversion to conventional VATS is required, a utility incision in the 5th intercostal space is more convenient because a utility incision in the 5th intercostal space is always used during conventional VATS. In our experience, this approach is safe, feasible, and reproducible. Although segmentectomy is generally expected to be more difficult than lobectomy, superior, basal, and lingula segmentectomies are more easily executed, owing to the readily defined segmental planes. However, without multiple views through a single incision, the division of segmental planes has to be performed in one direction. Division of segmental planes is slightly difficult during the division of segmental planes in single-incision segmentectomy. In conclusion, single-incision VATS superior segmentectomy of LLL for early-stage lung cancer was both feasible and safe in this instance. However, its merits in cancer treatment require further study.

Myelolipoma is a rare, benign tumor composed of hematopoietic tissue and mature adipose tissue. It is nonfunctioning and often detected in the adrenal glands during routine X-ray or computed tomography (CT) as incidentalomas. Extramedullary myelolipomas are extremely rare, and half of them are located in the presacral area. We report a patient with multiple bilateral mediastinal myelolipomas that were resected simultaneously using bilateral three-port video-assisted thoracoscopic surgery (VATS). This is the first case report of multiple bilateral myelolipomas that were simultaneously removed and diagnosed using VATS. We also present characteristic features of myelolipomas, which are helpful for diagnosis. The patient was a 79-year-old male with a history of hypertension, nephrosclerosis, and alcoholic liver hepatitis. Abdominal CT was performed routinely at a nearby hospital, and three bilateral paravertebral mediastinal tumors were detected incidentally. He had no symptoms due to the tumors. He was referred to Toranomon Hospital for further examination and treatment. Laboratory tests showed a slight elevation of serum liver enzymes and creatinine. Other laboratory tests including tumor markers were within normal limits. He had no history of hematologic disorder or chronic anemia. CT revealed three bilateral paravertebral thoracic tumors. Two of them were located on the right side of the vertebrae at the levels of Th-8 and Th-10. The tumors were 20 mm and 35 mm in diameter, respectively. The third tumor was located on the left side of the vertebrae, extending from Th-9 to Th-11, and was 75 mm in diameter. These tumors were heterogeneous. The CT density of the tumors ranged from -60.0 to +20.0 Hounsfield Units. There was no calcification in the tumors. Contrast medium was not used because of his poor renal function (). Magnetic resonance imaging revealed that the signal intensity of the tumors was mainly isointense with that of the bone marrow and partially isointense with that of the adipose tissue (). Differential diagnosis included neurogenic tumors, lymphomas, pleural mesotheliomas, and metastatic tumors of unknown origin, but none of them had any conclusive evidence. Bilateral VATS with the three-port technique was performed. The tumors were soft and fragile and bled easily. The tumor on the left side adhered to its surroundings firmly and hence, took several hours to resect. The operation time was 5 hours and 27 minutes, and the blood loss was 327 mL. The enucleated tumors were brown and partially yellow, and measured 19×15×11 mm, 40×34×15 mm, and 75×45×30 mm, respectively (). Microscopic examination revealed mature adipose tissue with hematopoietic elements consistent with myelolipoma (). The surgical margins of the tumors were all negative. The patient's postoperative course was uncomplicated, and he was discharged on the fourth postoperative day. It has been four years since the operation, and there has been no sign of recurrence. Myelolipomas are rare, benign tumors, and most of these tumors are detected in the adrenal glands as incidentalomas. Myelolipoma consists of mature adipose tissue and bone marrow cells. Symptoms caused by the tumor rarely occur; however, growth of the tumor may cause compression of the adjacent organs, resulting in several symptoms []. The proposed causes of myelolipoma include degenerative changes in hyperplastic tumor cells or adenomas of the adrenal glands, metaplasia in primary stem mesenchymal cells of the adrenal cortex, and displacement of differentiated bone marrow cells during embryogenesis []. Extra-adrenal myelolipoma is an extremely rare form of myelolipoma that can occur in the retroperitoneum, stomach, liver, or mediastinum. About half of all extra-adrenal myelolipomas are located in the presacral region []. In the thoracic area, they are frequently found in the mediastinum or the paravertebral thoracic region; however, sometimes they are located in the lung or the thoracic spine []. There are several distinctive imaging features of myelolipoma. It mostly occurs as a single, unilateral tumor and is often well-circumscribed and encapsulated. The ratios of bone marrow tissue and adipose tissue are different in each tumor. This reflects the heterogeneous nature of the tumors and explains the observation of varying CT densities. Calcification in the tumor is rare but has been reported []. The differential diagnosis includes neurogenic tumor, lymphoma, lymph node metastasis, malignant mesothelioma, and extramedullary hematopoietic tissue. Among them, differentiation between myelolipoma and extramedullary hematopoiesis is important. In cases of extramedullary hematopoiesis, there is often chronic anemia due to hematological diseases such as thalassemia or hereditary spherocytosis. Chronic anemia can be used to differentiate between myelolipoma and extramedullary hematopoiesis []. Most cases of myelolipoma are treated with surgical resection because myelolipoma is a rare disease and its preoperative diagnosis is difficult. However, there are some reports of successful diagnosis with CT-guided or endoscopic ultrasonography-guided biopsy [,]. There have been no reports of malignant myelolipoma. The decision for surgical resection is based on either increasing size or local symptoms such as chest pain, pleural effusion, or superior vena cava obstruction. Kenny et al. suggested that myelolipoma that is larger than 10 cm in diameter should be resected because the possibility of rupture and bleeding is higher with larger tumors []. Once there is a definitive diagnosis, and there are no signs or symptoms of increasing size, observation without any treatment can be a reasonable option. Surgical resection is the most common method of treatment, and a majority of resections are performed via thoracotomy. In this case, three bilateral myelolipomas in the posterior mediastinum were resected using bilateral VATS. To the best of our knowledge, this is the first case report of three bilateral mediastinal myelolipomas resected simultaneously using bilateral VATS. Mediastinal myelolipoma is a rare tumor, but it should be considered when there is a differential diagnosis of mediastinal tumors. Most reports are cases of single and unilateral tumors; however, bilateral and multiple tumors can occur, although they are extremely rare. When surgery is necessary for diagnosis or treatment, VATS can be an alternative choice to thoracotomy.

A 60-year-old male was referred to the Asan Medical Center for a nodule in the left lower lobe (LLL). The nodule was incidentally detected during the preoperative evaluation of gallstone, for which he underwent laparoscopic cholecystectomy at an outside hospital three months prior to his presentation to us. On his visit, he did not have any respiratory complaints such as productive cough or dyspnea. Medical history was unremarkable except hypertension. Further, the patient had been a non-smoker for 10 years although he used to smoke half a pack a day for 30 years before quitting. We found that the patient's physical examination was unremarkable except decreased breath sounds over the left lower lung field. The laboratory data were all within normal limits. Chest X-ray and computed tomography scan revealed a mass having the following dimensions: 20×10×5 mm. The mass obstructed the secondary bronchus entering into the LLL, which resulted in a total collapse of LLL (). A flexible bronchoscopy showed an endobronchial mass filling the basal segments of the LLL (). Further, a biopsy indicated a granular cell tumor. The patient underwent a left lower lobectomy via left posterolateral thoracotomy through the 5th intercostal space. The left thoracic cavity showed neither pleural adhesion nor seeding suggestive of malignancy. The LLL was heavily collapsed due to the obstruction by the endobronchial tumor. We divided the LLL bronchus at the level of the left upper lobe spur and performed a left lower lobectomy. The medial side of the left main bronchus was repaired using an interrupted anastomosis of 3-0 Vicryl. The resection margin of the bronchial stump was clear from the tumor on the frozen section. All five lymph nodes that were biopsied were tumor-free. The patient recovered well postoperatively and was discharged on postoperative day 5. Immunohistochemical staining demonstrated the positivity for S-100 protein, and the Ki-67 labeling index was low (1%), supporting the current diagnosis. The final pathology report confirmed the diagnosis of the granular cell tumor (). As of the writing of this paper, the patient has been free of tumor recurrence for six months. Granular cell tumor (GCT), a rare benign neoplasm that most commonly occurs in the tongue, skin, subcutaneous tissue, and breast, was first described by Abrikossoff in 1926. Pulmonary GCT, known to comprise 6% to 10% of all GTCs [,], was first reported by Kramer in 1938, and since then, less than 80 cases of GCT arising in the lung have been reported in the English-language literature []. In Korea, Seo et al. [] first reported a bronchial GCT arising in the left main bronchus in 2006. It was traditionally termed 'granular cell myoblastoma' until the late 1980s after Abrikossoff suggested that GCT had a myogenic origin []. This traditional theory was challenged by subsequent electron microscopic and immunohistochemical studies [,]. Now, it is believed that GCT has a neural cell origin, thus establishing the current nomenclature. Although it has been known that most pulmonary GCTs behave in a benign fashion, our review of the literature suggests that they have no unique clinical features. Most pulmonary GCTs are endobronchial, but sometimes, they can be located peripherally []. Pulmonary GCTs can be associated with synchronous extrapulmonary GCTs occurring in various organs, such as the tongue, kidney, or esophagus []. Pulmonary GCTs may occur metachronously in a single lung and thus, can be multicentric in 10% to 20% of the patients, although a majority of the GCTs tend to be solitary. Pulmonary GCTs can be associated with other malignancies or infectious diseases such as tuberculosis or human immunodeficiency virus []. Pulmonary GCTs can be diagnosed at any age, but most cases of pulmonary GCT present in the third or the fourth decade [,]. Previous clinical series showed that there is no gender predilection, and GCTs are equally distributed over both lungs with a predilection for the upper lobe []. However, another study found a slight predilection toward the left lung with a preference for the lower lobe, as presented in our case []. There are no commonly agreed risk factors, although the association of smoking with pulmonary GCTs was hypothesized in some studies. More than half of the GCT patients are asymptomatic at the time of diagnosis, and respiratory symptoms such as cough, dyspnea, hemoptysis, and wheezing present as tumor erosion and bronchial obstruction progress due to the growth of the endobronchial mass. With the onset of the symptoms, the bronchial obstruction causes the suppuration and destruction of the lung parenchyma distal to the obstruction; in this case, surgical resection is clearly indicated. However, the adequate treatment strategies for this disease remain controversial. Daniel et al. advocated bronchoscopic resection in asymptomatic patients with a tumor having a diameter of less than 8 mm [], and van der Maten et al. [] suggested that endobronchial therapy be the primary treatment. However, bronchoscopic removal cannot be a safe treatment option as it does not guarantee complete removal when GCTs infiltrate through the entire bronchial wall, which is evidenced by the fact that the incidence of recurrence was as high as 54% after bronchoscopic removal []. In addition, although extremely rare, it is possible that pulmonary GCTs can be malignant; the first case of malignant pulmonary GCT was reported in 2003 []. Being relatively young at the time of diagnosis can be a factor that favors complete surgical resection. In this regard, we believe that adequate surgical removal should be the preferred treatment option for all patients among whom GCTs are amenable to surgical resection. Based on the size, location, and number of masses, a surgical option, including either segmentectomy, lobectomy with or without sleeve resection, or rarely pneumonectomy, can be chosen with a lower incidence of recurrence and long disease-free survival.

Mediastinal paragangliomas are very rare neuroendocrine tumors. Most of the tumors are benign, but some of them can be diagnosed as malignant []. Complete resection is the standard treatment of paragangliomas []. However, the highly vascular nature of these tumors and their characteristic anatomic locations make complete resection difficult. Therefore, cardiopulmonary bypass through median sternotomy is often required to perform complete resection [,]. However, whether minimally invasive surgery is unsuitable for this highly vascular mass if the mass is sufficiently small for video-assisted thoracoscopic surgery (VATS) is still uncertain. Therefore, here, we report a case of the complete resection of a paraganglioma using VATS. A 55-year-old woman was admitted to the hospital because of a mass in the anterior mediastinum. Her past medical history was unremarkable, but she was suffering from ongoing cough. Computed tomography was performed in a local hospital, and an anterior mediastinal mass was found (). Physical examination revealed normal blood pressure, and the routine blood work results, including complete blood count, electrolytes, and chemistry, were within the normal limits. The mass was located under the sternum; therefore, percutaneous biopsy was not easy. Hence, to achieve the pathological and surgical goal, complete mass resection was planned. The mass was located near the great vessels (under the left innominate vein, immediately above the aortic arch), but there was no sign of local invasion. Therefore, we planned the surgical resection of the tumor using VATS. A double lumen ET tube was inserted under general anesthesia, and the position was changed to the right decubitus position. A 10-mm-scope port was created in the 4th intercostal space (ICS) close to the anterior axillary line, and a window measuring 30 mm was created in the 3th ICS close to the anterior scapular border. An additional 5-mm port for a left-hand instrument was created in the 5th ICS close to the mid-axillary line. The mass was totally surrounded by mediastinal fat tissue. After confirming that the mass did not invade the neighboring organ, we dissected the mass with the surrounding fat tissue. We tried not to manipulate the mass directly but to push or pull the surrounding fat tissue. The mass was completely resected using a Harmonic scalpel (Ethicon Endosurgery, Cincinnati, OH, USA). The mass was assumed to be thymoma type A by frozen biopsy. A small chest tube (12 Fr.) was inserted, and the operation was completed without any accident, including a hypertensive crisis or sudden bleeding. The chest tube was removed on post-operative day 2, and the patient was discharged without complications. The final pathological report revealed the typical characteristics of a paraganglioma (). The tumor cells stained positive for synaptophysin (). The resection margin was clear, but the tumor cells invaded into the pericapsular connective tissue and the mitosis was considerable (10/10 high-power field). Paragangliomas are rare neuroendocrine tumors that arise in sympathetic and parasympathetic paraganglia. Most of them (range, 80% to 85%) arise from the adrenal medulla, but the remaining 15% to 20% are located in the extra-adrenal chromaffin tissue. Malignancy is defined by the presence of metastasis and tumor invasion in sites where the chromaffin tissue is normally absent, such as lungs, liver, and bones. The mass considered in this study did not show metastasis but had an invasion into the pericapsular tissue and considerable mitosis. Therefore, after consulting a pathologist, we defined the mass as a malignancy. Complete resection of the paraganglioma using VATS was feasible. Complete resection is the treatment of choice in the case of paragangliomas. However, this treatment is sometimes difficult because of the tumor's vascular nature and anatomical location. Therefore, preoperative embolization or cardiopulmonary bypass is used for the safe resection of huge masses [,,]. However, minimally invasive surgery has also been attempted in the case of this risky tumor. Thus far, a laparoscopic attempt has been successful, but a thoracoscopic approach has not been successful [,]. An indirect manipulation of the tumor and the tumor's small size are thought to be the key to the safe and complete resection of this tumor. The prognosis in the case considered here is not certain. Prognosis after complete resection is encouraging. Lamy et al. [] reported the results of a follow-up of 79 patients with paragangliomas over a period of 180 months. In the case of patients undergoing complete resection, the survival rate was 84.6% and the mean survival time was 125 months; in contrast, in the case of patients undergoing incomplete resection, the survival rate was 50.0% and the mean survival time was 71 months []. However, the latest study on the subject reported that the large size and the anterior mediastinal location of this tumor were the poor prognostic factors []. The patient in this case had a relatively small tumor, but the mass was located in the anterior mediastinum. Moreover, pericapsular invasion and considerable mitosis were found. However, the role of adjuvant treatment was not established. Therefore, a short-term outpatient clinic visit without adjuvant treatment was decided upon as the follow-up plan. In summary, paragangliomas might be incorporated in the differential diagnosis of an anterior mediastinal mass. Surgical management using VATS can be successfully achieved for the complete resection of a small paraganglioma, and careful manipulation is required to prevent intraoperative complications.

Cancer morbidity worldwide is growing. However in the United States, the overall morbidity slightly decreased in 2013, (by >0.6%/yr in men, but it was stable in women), while the mortality rates decreased by 1.8%/yr in men and by 1.5%/yr in women []. The lung was the site of the highest cancer mortality in both the sexes (28% and 26% in men and women, respectively, while the second highest mortality rates, prostate cancer in men and breast cancer in women (10% and 14%, respectively), were nearly half those of the lung cancer rates []. While the disease, when recognized in its localized state, has good five-year survival (52%), the regional or far distant stages have survival rates of only 25% and 4%, respectively. However, at the first diagnosis, 56% of cases already show distant metastatic lesions, while only 22% are regional and 15% are local only []. This is why the overall survival data shows a sad picture: relative survival rates (male/female) are 29.4%/33%, 7.8%/9.3%, and 4.9%/5.9% for one-, five-, and ten-year-survival studied in 2005 to 2009 []. Due to the sad fact that lung cancer is one of the leading causes of mortality for humans, there have been numerous attempts to stop this trend []. Despite the well developing treatment modalities, the ratio of lung cancer mortality rate to the incidence (0.8) is more than double the average mortality/incidence ratio (0.3) among the population <65 years []. The incidence of lung cancer between the ≥65 years and <65 years old patients differs in 14%. The Korean statistics are similar to the worldwide trends [,]. Furthermore, special circumstances were observed in the case of Korean patients: The profiles of EML4-ALK fusion gene variants of non-small-cell lung carcinoma (NSCLC) may differ from those in other populations of other ethnicities []. The need to improve the rate of cure has remained of central importance in the field. The majority of cases (range, 75% to 85%) are of the non-small-cell histology, and half of these are adenocarcinomas []. The intense challenge is that most of the NSCLCs are first diagnosed when they have already in advanced stage or have already become rapidly metastatic []. The majority of patients present with either locally advanced disease (stage III) or metastatic disease (stage IV). The standard first-line strategy for the treatment of advanced stage NSCLCs is a limited number of chemotherapy cycles to achieve tumor regression or at least stabilization []. Importantly, patients who undergo curative surgical resection for apparent localized disease have survival rates ranging between 50% and 80%, implying the need for better systemic treatment to cure occult micrometastatic disease. Therefore, the majority of patients either presents with advanced disease requiring chemotherapy or require chemotherapy at the time of relapse after surgical resection. While efforts to cut down on smoking are crucial to the eventual control of the disease, newer treatments for patients who currently have the disease are critical. As a class, NSCLC is relatively insensitive to chemotherapy, compared to small cell carcinoma (SCLC). When possible, they are primarily treated by surgical resection with curative intent; however, chemotherapy is increasingly being used both preoperatively (neoadjuvant chemotherapy) and postoperatively (adjuvant chemotherapy). Modern lung-cancer treatment is based on platinum-containing doublets (carboplatin and cisplatin) and more recently on gemcitabine, paclitaxel (Taxol and Doxitaxel), vinorelbine (Navelbine). The analysis of 52 clinical studies shows the advantages of cisplatin-based therapies (10% 1-year survival increase), which reduce the risk of exitus by 27% [], compared to the applied supportive therapies. In general, the median survival with such chemotherapy is 7 months. The gemcitabine-based triplets and doublets (paclitaxel/carboplatin/gemcitabine; paclitaxel/carboplatin/vinorelbine; paclitaxel/gemcitabine; gemcitabine/vinorelbine); had 37%, 29%, 40%, and 49% one-year survival and 9.6, 9.9, 8.7, and 10.7 months median survival, respectively []. The gemcitabine-based doublets had a lower response rate (RR), but longer survival and less adverse effects. In general, the median survival ranges between 6 and 12 months, with 7 months on average. The one year survival is 24% to 51%, and 25% to 30% on average. An extended large study including in total 1,207 patients [] with advanced NSCLC was performed to compare the randomly assigned treatment to a reference regimen of cisplatin and paclitaxel or to one of three protocols: cisplatin and docetaxel, cisplatin and gemcitabine, or carboplatin and paclitaxel. The result was somewhat disappointing: none of the four chemotherapy protocols showed significant advantage over the others in the treatment of advanced NSCLC. The RR was 19%, with a median survival of 7.9 months, the first-year survival rate was 33%, while the second-year survival rate was 11%. Two other chemo-protocols were also compared, pemetrexed and docetaxel. The clinical efficacy was equivalent, but significantly fewer side effects were observed with the docetaxel regime in the second-line treatment of patients with advanced NSCLC []. Considering the side effects of the present treatment options, including surgery, radiation, and platinum-based doublet chemotherapy, another promising drug family was developed connected to growth factor receptors (GFR) to treat NSCLC. To increase the efficacy of such growth factor receptor tyrosine kinase inhibitors, the coinhibition of GFR signaling pathways and combination of inhibitors along with radiation or chemotherapy have been studied []. This is a clinically validated therapy targeting the signaling pathways of either the vascular endothelial GFR or epidermal GFR (e.g., gefitinib, cetuximab, erlotinib, bevacizumab), but the complex signaling system of solid tumors could adapt to the situation [], so the targeting of multiple regulatory pathways makes sense []. The development of multitargeted tyrosine kinase inhibitors (TKIs) is thus a concern. Although there is increasing research interest in these kinds of drugs, their therapy-related adverse effects and their safety remain controversial. As we discussed above, some clinical trials have been stopped early because of toxicity issues with multitargeted TKIs, and some other trials did not achieve primary improvement in overall survival; there is still a need for exploring more convenient, newer pathways as well as to develop insight into the coinhibition of existing pathways. It is important that greater attention be drawn to the signaling pathways that are modified by the use of kinase inhibitors. Genomic landscapes for patient-specific evaluation should be provided to appropriately select patients who are most likely to benefit from RTK (receptor tyrosine kinase)-inhibition therapy. SCLC comprises about 15% of all primary lung cancer cases, and it is usually the consequence of smoking. SCLC is a major clinical problem, with an aggressive clinical appearance and short survival time. Its treatment protocol is different from that of NSCLC []. Currently, the optimal treatment is platinum-etoposide chemotherapy (four cycles) applied with complementary thoracic irradiation in patients in early stages, but radiotherapy is not applied in the late stage. Many promising clinical approaches are available for lung cancer, but unfortunately, the breakthrough in this kind of malignancy has not been reached yet, and lung cancer treatments remain a high priority in clinical practice. Our present paper reviews the feasibility of oncothermia treatment for both NSCLC and SCLC. This study concentrates on the significance of the survival time as one of the most important factors for measuring the success of a treatment in oncology. Hyperthermia (HT), combined with radiotherapy and chemotherapy, seems to be a promising method for cancer treatment [], although many of the underlying molecular mechanisms of this combination treatment are still not clearly understood. Preliminary results with NSCLC recruiting a limited number of patients (range, 5 to 80 patients) also seem encouraging where HT was combined with chemotherapy, radiotherapy, or both. The co-administration of HT was proven to be safe [,,]. A great number of studies show that HT inhibits angiogenesis, enhances chemo- and radio-sensitivity, and induces a high concentration of drugs within a tumor [,,]. However, there are some restrictions on HT, in general, that hamper its use in lung cancer treatment. Namely, it could aggravate preexisting pleural liquids, the intensive cooling by actual breathing is contra-effective for the overall temperature increase. Furthermore, the increasingly constrained blood flow supports the tumor by glucose, which is an important contra-action, together with the increased risk of the dissemination. Focusing of the energy is limited by the intensive movement of the organ by breathing, and maintains the raised temperature on the place where the energy is focused. In fact, it has even more complications in a good heat-conducting environment than in thermal spreading in other organs. However, some successful clinical trials have shown the feasibility of the HT method for lung cancer. Most of these are combined with radiotherapy, having a 14÷70 Gy dose in the given session. The measured RR was surprisingly high: RR=75% (n=12) [] and RR=100% (n=13) []. In this last study, the mean survival time (MST) was 15 months, (mean-value, 17 months) on the tumors, which had a size on average of 22 cm. The average total dose was 60 Gy, average heating time was 52.3 minutes, and average total session in the cycle was 27. Others had a comparison to a control-arm (not randomized), studying the RR (). Other trials had a comparison to a control-arm (not randomized), growing the RR from RR=70% (n=30) and RR=53.8% (n=13), to RR=94.7% (n=19) [] and RR=76.9% (n=13) [], respectively. The second year survival also increased remarkably: from 15% and 15.4%, to 35% and 44.4%, respectively. The first year survival was measured as well, increasing from 30% to 55% []. A study on advanced NSCLC patients (n=13, capacitive coupling, f=8 MHz) to control local chest invasion [] showed similar good results, and the pain relief was also surprisingly good (). The chemo-thermotherapy combination was also investigated for NSCLC with success. In laboratory studies, cisplatin was shown to be effective [], so the clinical studies concentrated on this drug combination. A special case report has shown its feasibility [], and has demonstrated a median survival gain (from 15 [n=20] to 25 [n=32] months) []. The median survival was measured in another study [] as 19.2 months, with RR=73% and a 1 year-survival of 75%. The 13.5 months median survival of the historical control was increased to 17.5 months using postoperative (lobectomy or pneumonectomy) application of intrathoracic chemo-thermotherapy by capacitive coupled HT [,]. Local control by capacitive HT in combination with radiotherapy was also successful for locally advanced non-small lung cancer []. The complete remission rates were 26% and 0% with and without HT, respectively, while the full RRs were 95% and 70%, respectively. Others have also achieved good results from complementary radiotherapy with capacitive HT []. The survivals for the treatment group (resection+postoperative intrathoracic chemo-thermotherapy [PICT], n=32) and the historical control groups: resection only (n=20) or exploratory thoracotomy (n=11) had median survival rates of 25, 15, and 10 months, respectively [,]. The survival curve of the treatment group was significantly better than those of the control groups. Furthermore, the local relapse-free survival for the treatment group (resection+PICT, n=32) and for the historical control group (resection only n=20) was drastically and significantly increased, having more than double the relapsed-free survival in the HT treated group than in its historical counterpart after 48 months. The postoperative application was successful in other studies too [,]. The 5-year median survival was measured in another study [], showing rather high numbers (24.5%, for patients with N0+N1 status [n=14]). The median survival with PICT was measured, and definite gain was found (from 15 [n=20] to 25 [n=32] months) (capacitive coupling, 8 MHz and 13.56 MHz). The MST was 10, 15, and 25 months for treatment group (resection+PICT) and the historical control groups (resection only and exploratory thoracotomy only), respectively. The survival curve of the treatment group was significantly better than those of the control groups. Measuring the local relapse-free survival for the treatment group (resection+PICT) and the historical control group (resection only) also revealed a significant benefit with HT treatment. Another chemo-thermotherapy study [] showed fairly good results: an MST of 19.2 months, RR=73%, and a 1 year-survival of 75%. The chemo-thermotherapy combination was also investigated for NSCLC with success. In laboratory studies, the synergy between the gemcitabine and HT in NSCLC was shown , and in a nude mouse xenograft model []. The decrease in the tumor size and a significant inhibitory effect of growth were shown, and HT supported gemcitabine-induced apoptosis. Locally advanced NSCLC was studied (n=32) with fractional radiation (180.300 cGy/fraction, 5 fractions/wk, median dose 5.58 Gy) [] (). The results indicate differences, but they were not significant. To search for significant improvements in outcomes of thermotherapy, a large study involving two randomized, controlled, multicenter phase III studies was sponsored by a nonmedical international organization, the International Atomic Energy Organization to avoid any bias in medical sponsoring: for uterine cervix (n=110) [] and for NSCLC (n=80) []. Both studies were performed in combination with radiotherapy and with or without HT. The results of both studies were disappointing. The differences in the complete clinical response (complete remission) as well as overall survival rates in combined and single radiotherapy arms were statistically insignificant (p=0.49 and p=0.868, respectively), while the local progression-free survival was significantly better in the combined arm (p=0.036). The authors concluded that there was no substantial benefit in treating locally advanced NSCLC by adding HT to radiotherapy, although there was some improvement in local progression-free survival. Other kinds of HT (whole-body heating and very local ablative heating) have also been attempted. Whole-body HT was also applied to treating advanced lung cancer []. A benefit of HT was found (n=49), which was more substantial in elderly (>60 years) patients. The remission rate was 50%, and the MST was 7 months (mean is 12.7 months) in primary and 5.5 months for metastatic diseases. Percutaneous ablation by radiofrequency (RF) [,] and by laser-induced interstitial thermotherapy [] are also used for pulmonary tumors. In addition, intrapleural HT by perfusion is being used in clinical practice []. The breathable perfluorochemical liquid used in convective HT also appears to be a feasible alternative for lung cancer treatment []. There is a serious need for a stable HT method that is free from controversy and adverse effects, not only because of the high incidence of pulmonary malignancies, but also because of the variety of primary tumors that frequently metastasize to the lungs. The most common types include breast cancer, colon cancer, prostate cancer, and bladder cancer. A primary lung lesion can also quickly develop metastatic lung malignancies in other lobes. This causes the patient to progress quickly to advanced disease and requires a high stage of care (frequently stage IV). Furthermore, due to the lack of multiple effective therapies for high-line treatment (third-line or over), no real treatment options are available in such cases, which is another cause of the high mortality rate. Good supportive and sensitizing complementary therapies are missing from the application spectra. Another important factor demands new therapies: the quality of life. Patients with lung malignancies rapidly lose their Karnowski Performance Score (KPS), so any new therapy should have minimal adverse effects and improve the quality of life of the patient. One of the most advanced HT modalities devoted to oncology is oncothermia [,]. This method aims to preserve all positive effects of conventional HT while improving on the imperfections and answering the challenges posed by conventional approaches. The key is nano-range energy liberation instead of overall heating of the mass of the target []. This review will not explain the method of oncothermia in detail, which has already been done elsewhere [,], or describe its technical realization []. However, this paper will note some important factors of the method that are closely related to pulmonology applications. Oncothermia is a highly sophisticated method that addresses some of the limitations of HT [] and makes it possible to reintroduce HT by new standards []. It is based on a strong synergy between the temperature and the electric field []. The RF current is chosen with a proper amplitude-modulated RF (13.56 MHz) [], which is absorbed in the nanoscopic range by the malignant cells []. The physiological differences of the malignant cells from their healthy counterparts [] distinguish the malignancy, which is self-selected by additional time-fractal modulation [], which means that this is a highly customized treatment method []. The malignant cell is destroyed by apoptosis []. The apoptotic process uses an outer pathway [] (not the mitochondrial internal pathway that is used in normal HT []), forming a damage-associated molecular pattern [] and inducing immunogenic cell death [], which could be the basis of the directed abscopal effect too []. These effects start to emerge in lung cancer management [,], and could be the basis of a long-awaited feature of oncology: personalized care []. The treatment technique is simple and very user-friendly. It is safe and rarely has any side effects; patients are relaxed during the treatment thanks to the comfortable waterbed below containing the lower electrode. The upper electrode is a thermally and electrically balanced water bolus, which conforms to any body shape. The RF current flows through the patient self-selectively, irrespective of the movements of the tumor. The current flows along the best path for conduction, which is also the best pathway for treatment. v e n t h a t o n c o t h e r m i a i s u s u a l l y a p p l i e d i n v e r y a d v a n c e d c a s e s , m o s t l y t h i r d - l i n e t h e r a p y a n d b e y o n d , i t i s r a t h e r c o m p l i c a t e d t o a s s e m b l e a n a p p r o p r i a t e c o h o r t f o r a p r o s p e c t i v e s t u d y . T h e p r e s e n t c l i n i c a l r e s u l t s a r e d o m i n a t e d b y c a s e r e p o r t s a n d f e w m o r e e x t e n s i v e s t u d i e s . Several reports on cases of oncothermia application worldwide have been informative (, , , ). Some important messages from these cases are: 1) Do not evaluate too early by imaging, due to the slow apoptotic process []. 2) It is better to measure the actual change of the tumor marker from serum []. Their clinical effectiveness is relatively good [], and corresponds well with the genetic abnormalities of the cancer []. Tumor markers may be used to monitor any response to the treatment and determine whether the cancer has recurred after treatment, but in general, tumor markers alone cannot be used to diagnose cancer; they must be combined with other tests. The guidelines of the National Academy of Clinical Biochemistry USA are best followed []. 3) The optimization of the actual treatment has to be personalized. The trimodal therapies (oncothermia and two jointly applied 'gold standards' like radio- and chemotherapies) have good outcomes, but bimodal application is also suitable in most cases. In the cases when the gold standards are inapplicable, monotherapy with oncothermia also has good results, but in these cases, the number of sessions in a cycle is approximately doubled []. 4) In chemotherapy applications, old 'simple' drugs like mitomycin-C could be reapplied []. 5) The new chemo-categories (like the addition of bevacizumab to conventional platinum derivatives) are also applicable to oncothermia []. Independently of oncothermia, we expect various chemo-agents from the literature to be synergistic; for example, the addition of bevacizumab to cisplatin-based chemotherapy significantly prolonged progression free survival and overall survival in phase III [,] and phase IV trials [], proved to be safe in treating over 5,000 patients [,,], and in the maintenance setting, also demonstrated a beneficial effect on progression free survival [,], but the addition of oncothermia to this combination was recently approved for the first time []. 6) Even in the cases where the local control is not so effective (poor partial remission/response or no response), the quality of life improved, which is a great advantage of oncothermia. 7) The method is safe and apparently free of adverse effects in any combined or monotherapy applications. 8) In the mild trimodal application (low-dose radiotherapy, an immune activator, and oncothermia), the abscopal effect is clearly observable [], which was also seen in laboratory model experiments without the addition of ionizing radiation []. 9) Oncothermia can be applied as concurrent therapy even in a fifth-line chemo-companion [] in highly advanced disseminated cases. 10) The strategies of advanced cases have multiple vices [], but oncothermia could be successfully applied in far advanced cases too [,]. 11) Oncothermia can be applied not only in primary lung malignancies, but also for metastatic cases as well [,]. Advanced lung cancer was studied in a double-arm prospective study [] with real endpoints of survival time and quality of life. In the active arm (n=75), stage IIIb and IV lung cancer patients were included, while a same patient characteristics cohort (n=40) was on control arm (). These patients were inoperable. Oncothermia was applied in 20 sessions, every other day, 60 min/each. The treatment efficacy, survival rate, and life of quality were assessed before the treatment, after the treatment, and every sixth month. The local control (remission rate) (), as well as the overall survival (), was significantly superior in the oncothermia combined (active) arm of the study in comparison with the control. In addition, the treatment group showed statistically significant differences in quality of life functions, emotions, general conditions, pain, shortness of breath, loss of appetite, cough, hemoptysis, and chest pain. Another study examined SCLC []. The double-armed study included treatment with (n=23) and without (n=8) oncothermia complementary to chemotherapy for SCLC. Chemotherapy was the first-line treatment: irinotecan (60 mg/m), and cisplatin (60 mg/m) three times. The status was evaluated by chest computed tomography. When the progression of the tumor or metastases was detected, the chemotherapy was changed to etoposide (110 mg/m) and cisplatin (70 mg/m) in the second line. Oncothermia was performed from the first chemotherapy treatment period up to 150 W, 1,490.5 kJ energy for 60 minutes, every second day, with a rise in temperature to 38.5℃ to 42.5℃. In this study, we used an electrode with a 30-cm diameter applied to the thorax, with at least 12 sessions in 1 cycle. The results showed promising improvement of the survival compared to chemotherapy alone. The overall survival showed a significant benefit for the oncothermia treated group (). An extended clinical investigation is in progress []. Presently, 10 NSCLC patients and 3 metastatic lung malignancies are included to study the safety (phase I) and the efficacy (phase II) in a well-controlled study. The work is in progress. A retrospective clinical study of NSCLC patients was performed by two hospitals in Budapest [,,], clearly demonstrating the feasibility and efficacy of oncothermia for advanced cases []. The first study was obtained from an open-label, single-arm, monocentric study (n=61) carried out in Peterfy Hospital, Budapest, Hungary (referred to as 'PFY') [,]. The patients included were analyzed according to an intention-to-treat schedule. The primary endpoints of the study were the overall survival time and the survival time from the first oncothermia treatment. The dates of exitus were checked by the National Death Register, so that accurate data were collected. Inclusion criteria were: 1) Inoperable or sub-totally resected, or recurrent primary pancreas tumor, 2) progression after radio- and/or chemo-therapy, and 3) KPS ≥30%, and the inclusion was irrespective of the localization of the lesion in the lung. Patients started the oncothermia process in their late/advanced stages, where most of them had failed to respond to any of the applied conventional therapies. The exclusion criteria were only the well-known contraindications of the oncothermia method (metallic implants or replacements in the treated area, missing heat sensitivity in the treated area, or a pacemaker or other field-sensitive implant in the patient). The age distribution of n=61 patients was near normal (p=0.82); no outlier was present. The median age was 58 years (range, 38 to 77 years), the mean-age was 58.97 years (standard error [SE]=1.17). The gender distribution was 21/40 female/male (34.4%/65.6%). The proportion of the elderly (>68 years) patients was 21.3%. Oncothermia was performed in 2 to 3 sessions per week. The treatment time per session was 60 minutes. The power was gradually and linearly raised depending on the patient's tolerance from 40-80 W to 100-150 W. The applied average energy was 300 kJ/treatment (range, 250 to 450). The applicators used were 3.1 dm and 7.1 dm, depending on the tumor volume. Most of the patients (n=49, 80.3%) had distant metastases. They were heavily pre-treated; all of the patients had received at least one line of chemotherapy and had undergone other treatments (surgery, radio- or second-line chemo-therapy). The actual staging was made at the first diagnosis (44% was in advanced [World Health Organization IIIb or IV] stages) and at the first oncothermia treatment (75% was in an advanced stage). The median of the elapsed time from the first diagnosis to the first oncothermia treatment was 8 months (range, 0.4 to 172 months), while its mean was 16.3 months (SE=3.1). The elapsed time ratio to the overall survival was more than 50% (median, 59.9% [6.5-99.1]; mean, 59.4 [SE=3.5]); the patients received their first oncothermia in the second half of their survival time. The oncothermia treatment was provided twice a week; the treatment number averaged 8.1 (SE=0.55) sessions with a median of 8 (range, 2 to 23). The Kaplan-Meier plots of the overall survival (median, 16.4 months [1.7-181.9]; mean, 25.6 months [SE=3.8]) and the survival from the first oncothermia treatment (median, 5.7 months [0.1-44.9]; mean, 9.2 months, [SE=1.3]) are shown in . For the elderly patients, neither the overall survival nor the oncothermia survival differed between the groups (p=0.68). The elapsed time to the first oncothermia treatment represents an important parameter. Namely, this is of course smaller (p=0.0019) for the patients with advanced disease at their first diagnosis (n=34, median, 13.0 months [1.5-142]; mean, 24.0 months [SE=5.2]; and n=27, median, 6.5 months [0.4-19.9]; mean, 6.67 months, [SE=0.83] for non-advanced and advanced, respectively). However, the opposite was found (p=0.14) when the staging at the first oncothermia was studied (n=15, median, 4.10 months [1.5-29.3]; mean, 8.9 months [SE=2.3]; and n=46, median, 8.3 months [0.4-142]; mean, 18.78 months [SE=4.0]; for non-advanced and advanced, respectively). This tendency is more obvious when the overall survival and oncothermia survival are evaluated depending on the ratio of the elapsed time until the first oncothermia treatment to the overall survival, dividing the patients into 'early oncothermia' and 'late oncothermia' categories. The overall survival shows the expected result: the low survival cases start earlier (p=0.0065) with oncothermia (n=31, median, 16.4 months [4.7-79.7]; mean, 19.62 months [SE=2.61]) than those with long survival, (n=30, median, 17.4 months [1.7-182]; mean, 31.7 months [SE=7.07]). On the other hand, the oncothermia survival was opposite (p=0.073): those with an early start (n=31, median, 8.4 months [2.4-44.9]; mean 12.7 months [SE=1.9]) had longer survival, than those with a late start (n=30, median, 2.7 months [0.1-40.0]; mean, 5.6 months [SE=1.6]). The number of treatments does not influence the overall survival significantly (p=0.61), but the oncothermia survival (p=0.0023) and the follow-up time after the last oncothermia (p=0.01) greatly depend on the number of oncothermia treatments (). Operability was especially beneficial for survival (p=0.0005), but the other pre-treatments did not relate significantly to either the overall survival or the oncothermia survival rates. The effect of the length of treatment grouped by before and after the median time of the data was measured also. In the early experience group (n=33), the median overall survival was 22.3 months (1.7-181) and mean was 33.7 (SE=6.4); the oncothermia survival median was 8.0 months (0.1-45) and mean was 11.6 (SE=2.07); and the median elapsed time to the first oncothermia treatment was 10.3 months (1.5-142) and mean was 22.1 (SE=5.3). Later, according to our experience (n=28), the data were: median overall survival, 12.3 months (3.6-51.9) and mean, 15.9 (SE=2.2); median oncothermia survival, 5.0 months (0.1-25.1) and mean, 6.37 (SE=1.24); median elapsed time to first oncothermia treatment, 5.9 months (43.77) and mean, 61.1 (SE=1.8). The differences between the early and late experiences were significant in the case of overall survival (p=0.028) and the elapsed time to the first oncothermia treatment (p=0.012), but not significant for oncothermia survival (p=0.19). The significantly better survival values in the first half of the study time compared to the second one probably originated from the fact that the patient spectrum had shifted to more advanced cases. In the early experience group, the proportion of the advanced cases was 33%, while in the late experience group, 57% of the cases were advanced, but both of them increased (76% and 75%, respectively) when measured at the first oncothermia treatment. The nearly equal percentage of the advanced cases in both categories (rising from very different starting points) indicates that the patients can be assumed to start the oncothermia treatment at nearly the same stage irrespective of the elapsed time from the original diagnosis to the first oncothermia treatment. Another Hungarian study [,,,] was made by HTT-Med (HTT), a specialized HT clinic. The inclusion and exclusion criteria as well as the complete protocol were identical with the PFY study. The age distribution of patients (n=197) was acceptably normal (p=0.59); no outlier was present. The median age was 57 years (range, 16 to 84), the mean-age was 56.71 years (SE=0.77). The gender distribution was 62/135 female/male (31.5%/68.5%). The proportion of the elderly (>68 years) patients was 20.3%. Most of the patients (157, 79.7%) had distant metastases (one, two, and three metastases were observed for 101, 43, and 13 patients, respectively). They were heavily pre-treated; most of them (93.4%) had undergone surgery and subsequent radiation therapy. The actual staging was performed at the first diagnosis (46.2% were in advanced [World Health Organization IIIb or IV] stages) and at the first oncothermia treatment they were at a more advanced status. The median of the elapsed time from the first diagnosis to the first oncothermia treatment was 5.5 months (0.2-111.3), while its mean was 10.6 months (SE=1.0). The elapsed time ratio to the overall survival was near 50% (median, 45.4% [1.6-96.7]; mean, 45.7 [SE=3.9]). The oncothermia treatment was provided twice a week; the number of treatments was on average 7.9 (SE=0.4) and its median 6 (3.40). The median treatment time was 60 minutes and the mean was 69.6 minutes (SE=1.3), while the median equivalent temperature was 52℃ (43℃-59℃) and its mean was 51.4℃ (SE=0.3). Note that the equivalent temperature is not the real temperature. It is the calculated value from the actual energy absorption and the impedance, meaning the actual destruction rate, which is as high as it would have been in the purely temperature-oriented case. The Kaplan-Meier plots of the overall survival (median, 15.6 months [1.1-122.1]; mean, 22.4 months [SE=1.31]) and the survival from the first oncothermia treatment (median, 7.57 months [0.1-68.6]; mean, 11.8 months [SE=0.91]) are shown in . For elderly patients, neither the overall survival nor the oncothermia survival differed (p=0.37 and p=0.49, respectively). The differences between the patients without or with metastases in their overall survival and oncothermia survival were not significant (p=0.33 and p=0.07, respectively). The number of treatments significantly influences the overall survival (p=0.048) and the oncothermia survival (p=0.00046) and the follow-up time after the last oncothermia treatment (p=0.0017) greatly depends on the number of oncothermia treatments. The operability here also gave a significant benefit for survival. We studied the effect of the experience of treatment by the data before and after the median time of the study. In the early experience group (n=94), the median overall survival was 15.3 months (2.4-122.1) and mean, 24.0 (SE=2.17); the median oncothermia survival, 7.2 months (0.3-68.6) and mean, 11.8 (SE=1.5); and the median of the elapsed time to the first oncothermia treatment was 5.37 months (0.4-111.3) and mean, 12.2 (SE=1.8). In the late experience (n=103) the data were: median overall survival, 15.83 months (1.1-77.7) and mean, 21.0 (SE=1.5); median oncothermia survival, 8.13 months (0.1-43.9) and mean, 11.8 (SE=1.1); and the median elapsed time to the first oncothermia treatment, 5.6 months (0.2-64.8) and mean, 9.1 (SE=1.1). The differences between the early and late experiences were not significant in case of the overall survival (p=0.85), oncothermia survival (p=0.17), and the elapsed time to the first oncothermia treatment (p=0.21). Due to the identical protocols, a simple meta-analysis was performed on these data []. The age distribution of the 258 patients together was near to normal (p=0.71), and no outlier was present. The median age was 57 years (range, 16 to 84), and the mean-age was 57.2 years (SE=0.7). In the spectrum of the PTF, a slight shift to the elderly patients was observed. The overall gender distribution was 83/175 female/male (32%/68%), and no significant difference could be measured between the places. The ratio of the elderly (>68 years) patients was 20.5%, (20.3% and 21.3% in PFY and HTT, respectively). The PFY/HTT patient ratio was 61/197 (24%/76%). Eighty percent of the patients had distant metastases in both study locations and half of them were in an advanced stage at the first diagnosis of the disease. The patients were heavily pre-treated; in PFY, chemo-therapy, and in HTT, the surgery was the most frequent modality. The median elapsed time to the first oncothermia treatment from the first diagnosis (ETO) was significantly shorter in HTT than in PFY (p=0.028). The oncothermia treatment was provided twice a week, the number of treatments in average was more in the PFY than in the HTT procedures. The overall survival and the survival from the first oncothermia treatment (OSO) are shown in , . The overall survivals are significantly lower in HTT (p=0.044), but in the oncothermia survival there are no significant differences (p=0.53). Survival after the treatment was not different in the two locations (p=0.55). However, for elderly patients, neither the overall survival nor the oncothermia survival was different (p=0.38 and p=0.86, respectively). We added a Hungarian historical (retrospective) control (n=53), which was collected from the St. Borbala Hospital (Tatabanya, Hungary), for comparison. The data-set was collected from the patients of the leading author of a meta-analysis []. The overall survival Kaplan-Meier plot shows a significant benefit of the oncothermia (p=0.033; median, 15.8 months; mean, 23.1 months (SE=1.3) for oncothermia; median, 14.0 months; mean, 18.5 months (SE=2.3) for the historical control) (). The patients were divided to subgroups of advanced (III, IIIa, IIIb, IV) (n=140) and not-advanced (I, Ia, Ib, II, IIa, IIb) (n=77) stages (data were not available for a smaller group (n=41); their data were not used in this evaluation). It is not surprising that the two subgroups are significantly different (p=0.038) by their survival (). The non-advanced subgroup (stages I, Ia, Ib, II, IIa, IIb) had 87 patients altogether (77 active, 10 control) and the advanced subgroup (stages III, IIIa, IIIb, IV) had 183 patients (140 active, 43 control). When we compared both subgroups with the relevant subgroup of the historical control, the effect of oncothermia became more pronounced in advanced cases (III+IV) (active arm: median, 14.7 months [n=132]; control arm: median, 11.0 months [n=43]) The result is more significant, p=0.023 than in the non-advanced case subgroup, where the differences were not large, and were not significant . The above two studies were performed by the same guidelines but in entirely independent hospitals, with no overlap in medical personnel. The two retrospective data sets can be regarded as independent. The study of the expertise of the personnel in the two places was the same; their training was sufficient to make the optimal performance from the very start of the treatment. The patients' pretreatments were mostly dominated by surgery and chemotherapy in HTT and PFY, respectively. Like the ETO; surprisingly the overall survival was also significantly lower as significantly different having the earlier start of oncothermia in HTT. It seems the patients treated by HTT were more advanced at their first diagnosis (more metastases were detected) than the PFY counterparts, which explains the difference. Despite the difference in overall survival, the OSO does not differ significantly between the two places. The yearly survival rates could be regarded as identical (p>0.99) within the measurement error (). This could be indication of the oncothermia leveling action as well. r p r e s e n t p a p e r s h o w e d a s t r o n g i n d i c a t i o n o n t h e o n c o t h e r m i a b e n e f i t t o t r e a t p u l m o n a r y m a l i g n a n c e s . T h e r e s u l t s c l e a r l y i n d i c a t e t h e f e a s i b i l i t y a n d t h e b e n e f i t o f t h e o n c o t h e r m i a t r e a t m e n t f o r b o t h S C L C a n d N S C L C f o r a n u m b e r o f r e a s o n s : 1 ) o n c o t h e r m i a s h o w e d v a l i d t r e a t m e n t p o t e n t i a l a n d s a f e a p p l i c a t i o n ; 2 ) t h e s u r v i v a l t i m e w a s i n c r e a s e d b y o n c o t h e r m i a f o r t h e p a t i e n t s ; 3 ) o n c o t h e r m i a i n c r e a s e d t h e q u a l i t y o f l i f e o f t h e p a t i e n t s ; 4 ) d u e t o t h e l i m i t e d e f f e c t i v e n e s s o f t h e e s t a b l i s h e d t h e r a p i e s , o n c o t h e r m i a c o u l d b e o n e o f t h e i m p o r t a n t f u t u r e m e t h o d s t o i m p r o v e o u r t r e a t m e n t f a c i l i t i e s .

Partial anomalous pulmonary venous connection (PAPVC) is an anomaly in which some but not all pulmonary veins connect to the right atrium or its tributaries as opposed to the left atrium []. The most common type of PAPVC is related to a sinus venosus malformation; the right upper and middle pulmonary veins connect to the superior vena cava (SVC) or cavoatrial junction, and a sinus venosus-type atrial septal defect (ASD) is present in 90% of the patients []. The ideal surgical correction of SVC-connected PAPVC creates a normal physiological flow with separate pulmonary and systemic venous pathways, while avoiding complications such as sinus node dysfunction []. Various surgical techniques have been described for the correction of PAPVC that connects to the SVC. Conventional repair, which creates a pulmonary venous pathway with one or two patches, carries the risk of pulmonary venous obstruction, SVC stenosis, and sinus node dysfunction [,,]. In the Warden procedure, reported in 1984, the SVC is divided, and the cephalic SVC is anastomosed to the right atrial (RA) appendage, while the caudal SVC serves as a pulmonary drainage conduit []. This procedure has been widely used for surgically correcting PAPVC, which connects to the SVC. Although the Warden procedure has been effective in decreasing the risk of SVC or pulmonary venous obstruction, there have been a few complications after the Warden procedure, including pulmonary venous obstruction, SVC obstruction, and sinus node dysfunction [,,]. We have modified the Warden procedure by using a RA wall flap along with patch augmentation with autologous or bovine pericardium. In this report, we review the early and mid-term outcomes in patients with PAPVC connecting to the SVC treated using our modified Warden procedure. From February 2005 to July 2012, 10 patients at Samsung Medical Center were treated with a modified Warden procedure for PAPVC connecting to the SVC. Diagnoses were performed using echocardiography or computed tomography. Cardiac catheterization was performed in three patients for evaluating the associated anomalies and pulmonary hypertension (). After standard median sternotomy, a large pericardial patch was harvested and treated with a 0.6% glutaraldehyde solution. The SVC was dissected circumferentially from the SVC-RA junction to the level of the innominate vein. The azygos vein was divided. Cardiopulmonary bypass was established by cannulation of the ascending aorta, inferior vena cava, and innominate vein. If the patient had a persistent left SVC (four patients), the left SVC was directly cannulated (). Using moderate hypothermia, we achieved cardiac arrest with intermittent antegrade cold-blood cardioplegia. The SVC was divided just above the uppermost insertion of the anomalous pulmonary vein. The end of the proximal SVC was closed directly, and it served as a conduit for pulmonary venous drainage. Just below the oblique incision along the atrioventricular groove, another small longitudinal incision was made to create the atrial flap at the right atrial auricle (RAA) (). An autologous or bovine pericardial patch was sutured to the margin of the ASD on the SVC orifice to create a pulmonary venous pathway (). In the absence of an ASD, a large ASD was created in the fossa ovalis area and the sinus portion. If a small ASD was present, it was enlarged for creating a non-restrictive pulmonary blood flow pathway. A RA flap was prepared for the posterior wall of the neo-SVC. The pectinate muscle in the RAA was resected to create a smooth atrial flap. This atrial flap was anastomosed to the distal end of the SVC for reconstructing the posterior SVC wall. The anterior wall of the neo-SVC was reconstructed with a generous autologous patch or with prosthetic materials (). We used 5-0 or 6-0 polypropylene suture for the reconstruction. Details of the surgical techniques employed herein are presented in . There were 6 female and 4 male patients. The median age at the time of surgery was 5.7 years (range, 1.0 to 58.1 years). All patients had PAPVC to the SVC or near the SVC-right atrium junction. In eight patients, pulmonary veins from segments confined to the right upper lobe were connected to the SVC. In the remaining two patients, veins from segments in addition to the right upper lobe were involved. Seven patients had the sinus venosus-type ASD (70%), two patients (20%) had an intact atrial septum, and one patient (10%) had patent foramen ovale. The associated cardiovascular anomalies included persistent left SVC in four patients, transposition of great arteries with ventricular septal defect in one patient, and double outlet of the right ventricle in one patient. Preoperative EKG showed normal sinus rhythm in eight patients, 1st degree atrioventricular block in one patient, and chronic atrial fibrillation in one patient. We observed no early mortality, early reoperation, or surgical morbidities. At last follow-up, all patients were asymptomatic and had normal sinus rhythm on the EKG. All patients underwent postoperative echocardiogram. Nine patients had normal flow in the SVC, with unobstructed drainage of the pulmonary veins to the left atrium. In one patient, the SVC flow velocity was slightly increased (1.3 m/sec, estimated pressure gradient=6.76 mmHg), but there was no clinical evidence of SVC obstruction (). The eight patients with normal sinus rhythms did not develop any arrhythmias post-surgery. The PR interval of the patient with 1st degree AV block normalized after surgery. Chronic atrial fibrillation converted to a normal sinus rhythm after the Cox maze procedure. Thus, there were no new arrhythmias after surgery. No patient required re-operation or re-intervention for systemic or venous pathway stenosis at the last follow-up. Multiple surgical techniques have been previously reported for repairing PAPVC. The classic ones include intra-atrial baffling using one or two patches and repair with a RA flap. Warden et al. suggested a novel technique for connections of anomalous pulmonary veins []. The Warden procedure transfers the SVC to the RA appendage, while redirecting an anomalous pulmonary venous flow to the left atrium by using a single patch. This procedure essentially prevents the anatomical obstruction of the intracardiac blood flow. However, stenosis at the SVC-RA junction has been reported. Gustafson et al. [] reported only one patient with SVC obstruction and sick sinus syndrome postoperatively in a series of 40 patients who underwent the Warden procedure. However, SVC obstruction developed two years after the Warden procedure in one patient from among a series of five patients, as reported by Stewart et al. []. This was attributed to a technical problem resulting from failure to excise all the trabeculations, as reported by Warden et al. []. The potential causes of stenosis are trabeculations on the RA appendage or tension at the RA-SVC junction. Said et al. [] suggested the use of vascular grafts between the SVC and the RA to decrease tension at the anastomosis. However, this modification may not be appropriate in pediatric patients because it may become too small as the child grows. Therefore, we modified the Warden procedure using an RAA flap and an autologous pericardium. The technique called 'RA wall pedicled flap technique' has been described and clinically used by a few surgeons [,,]. Nakahira et al. [] used a pedicled autologus pericardial flap for anterior augmentation of cavoatrial anastomosis with the expectation of a sufficient channel and tension-free anastomosis growth potential. However, they did not use the 'RA wall pedicled flap technique.' Park et al. [] reported the use of a modified Warden procedure in 9 cases, with the techniques used in three of these cases being seemingly similar to our technique, for tension-free anastomosis between the cephalic end of the SVC and the RA appendage where the anomalous pulmonary vein enters the more cephalic portion of the SVC. Tao et al. [] reported that one patient from among their series underwent a similar modification as our patients did. In this study, we described the treatment of 10 patients with the same surgical technique, as well as their early and mid-term clinical outcomes. As expected, we have not been required to perform any re-operation or re-intervention for obstruction or stenosis in the cardiac venous circulation. The tension-free anastomosis may decrease the risk of postoperative bleeding and thromboembolism. Therefore, this technique has become the standard procedure for surgical correction of PAPVC at our institution. Sinus node dysfunction is a serious complication after the 1-patch or 2-patch technique, in which the incisions are placed close to the sinus node or its arterial supply [,,,]. No sinus node dysfunction was detected in any of the 11 patients from among a series undergoing the Warden procedure reported by Gaynor et al. []. Stewart et al. [] did not report sinus node dysfunction in any of the 5 patients in their series who underwent the Warden procedure. Our modification was similarly associated with a low incidence of sinus node dysfunction, and no patients in our series newly developed arrhythmia during follow-up. We believe that our technique may improve the classic Warden procedure in some ways. It always provides a wide tension-free anastomosis; there is no single circular suture line causing late stenosis; and patients do not need anticoagulation because of a widely patent pathway and low rate of atrial arrhythmia. Therefore, our modified technique can be a viable option for surgically correcting PAPVC in both pediatric patients and adults. Our study is limited by our experience and the retrospective nature of this chart review. Because PAPVC is a rare situation, multi-institutional studies may be necessary. Our modification requires more complex suture lines than those in the original Warden procedure or other previous modifications. This may lead to technical difficulties, a steeper learning curve, or higher chances of bleeding. However, we believe that most experienced congenital cardiac surgeons are familiar with even such a complex suture. In addition, we did not experience any technical errors or postoperative bleeding. Thus, we believe that this is a readily reproducible technique. In conclusion, the Warden procedure using the RA auricular flap and a biological patch for PAPVC treatment was correlated with satisfactory early and mid-term outcomes. Because this modification may decrease the risk of SVC obstruction and other possible complications associated with the Warden procedure, it can be considered a viable option in both pediatric and adult patients for surgically correcting PAPVC.

Oral diseases are one of the most prevalent conditions in the world and are largely preventable. Dental caries affects 60-90% of school children and most adults in industrialized countries; it is increasingly prevalent in developing countries and highly prevalent in some Asian and Latin American countries.[] Periodontal disease is prevalent globally, with severe periodontitis in 5-15% of most populations; clearly associated with diabetes and compromised immunity. According to the National Oral Health Survey, in India dental caries is prevalent among 63.1% of 15-year-old and as much as 80.2% among adults in the age group of 35-44 years. Periodontal diseases are prevalent in 67.7% of 15-year-olds and as much as 89.6% of 35-44 year olds.[] Edentulism is high in some countries among adults ages 65 and older. Oral cancer is the 8 most common cancer world-wide; 3 most common in South-central Asia and twice as prevalent in less developed countries than in more developed countries and has shown a sharp increase in incidence rates in some European and other industrialized countries. Dental trauma in industrialized countries ranges from 16% to 40% among 6-year-olds and from 4% to 33% among 12-14-year-olds; in some Latin American countries, about 15% of schoolchildren; in the Middle East, about 5-12% among 6-12-year-olds. Oral diseases restrict activities in school, at work and at home causing millions of school and work hours to be lost each year the world over. Moreover, the psychosocial impact of these diseases often significantly diminishes quality of life.[] Prevention of disease, disability and suffering should be a primary goal of any society that hopes to provide a decent quality of life for its people. Prevention on the community or population based level is the most cost effective approach and has the greatest impact on a community or population, whether it is a school, neighborhood, or nation. An effective community prevention program is a planned procedure that prevents the onset of a disease among a group of individuals. Many different approaches to preventing dental diseases exist and the most cost-effective method is health education. Health education is any combination of learning experiences designed to facilitate voluntary actions conducive to health. These actions or behaviors may be on the part of individuals, families, institutions or communities. Thus the scope of health education may include educational interventions for children, parents, policy makers, or health care providers. It has been well-documented in dentistry and other health areas that correct health information or knowledge alone does not necessarily lead to desirable health behaviors. However knowledge gained may serve as a tool to empower population groups with accurate information about health and health care technologies, enabling them to take action to protect their health. Treatments for all oral diseases are available generally in industrialized and more developed countries, but may be expensive and not always accessible, many individuals lack access to care, as well as insurance or finances to pay for care. In less developed and poor countries, appropriate treatments are generally not available at all. Diseases of the craniofacial complex greatly affect an individual's quality of life with nutritional, functional and psychosocial consequences. Further, oral diseases are a costly economic burden for individuals, families and nations-both industrialized and developing. The goal of oral health education is to improve knowledge, which may lead to adoption of favorable oral health behaviors that contribute to better oral health. A basic oral health care program introduced by World Health Organization for less industrialized countries includes oral health education and emphasizes on the integration of health education with other oral health activities such as provision of preventive, restorative and emergency dental care. In recent years, attention has been drawn toward assessing the effectiveness of oral health education programs. This is in line with demand for evidence based research and will help to inform policy makers on how to allocate resources. A number of systematic reviews have been conducted on the available evidence. These have shown that oral health education can be effective in increasing knowledge in the short term and to some extent, behavior such as tooth brushing and healthy eating. This review is an addition to the published literature on dental health education, because systematic reviews are only as good as the basic research underpinning them and previous reviews have unanimously pointed out the paucity of good quality studies in this field. e a i m o f t h i s p a p e r i s t o c o l l e c t a n d c o l l a t e i n f o r m a t i o n o n e f f e c t i v e n e s s o f o r a l h e a l t h e d u c a t i o n p r o g r a m s a n d t o p o o l d a t a f r o m t h e s t u d i e s , w h i c h w e r e d e e m e d e f f e c t i v e i n o r d e r t o l i s t v a r i a b l e s a s s o c i a t e d a n d w h i c h m a y h a v e c o n t r i b u t e d t o t h e s u c c e s s o f t h e s e p r o g r a m s . A search of all published articles in Medline was done using the keywords “oral health education, dental health education, oral health promotion.” The resulting titles and abstracts provided the basis for initial decisions and selection of articles. Out of the primary list of articles, a total number of 40 articles were selected as they fulfilled the following inclusion criteria: The full text of the articles was then obtained from either the internet or libraries of Dental Research Colleges and Hospitals in and around Bangalore. A set of important variables were identified and grouped under five headings to make them amenable for coding. The coding variables were then described under various subheadings to allow us to compare the chosen articles []. The studies were reviewed based on the mentioned variables and results were described and summarized under the same. Thirteen studies[] showed their effectiveness in terms of change in knowledge, the sample size ranged from 42 to 2678 participants. The oral health education group ranged from 14 to 1339. The target population was mainly schools children and care givers of children and the elderly. The follow-up period ranged from 6 weeks to 6 years. Six studies targeted a population in the age group 7-13 years old, two studies in the elderly, one study for care givers, one in children 3 years old, one in the infants, one targeted all age groups and one was done in children where the age group was not mentioned. One study was done in the low socio economic status population, one included all socio-economic status groups and the rest did not mention the socio-economic status of the population. All the studies were done involving both genders except one which was done in an orphanage exclusively for girls. The education level of the oral health education target group ranged from primary to professional education. One study was done in an uneducated population of 7-11-year-old orphan girls. Oral health education was delivered in all studies by professionals – dentists or dental hygienists. 10 studies were done in a city, one in a town, one in a rural area. In seven studies Oral health education was given in a school, two in nursing homes, one in a health center, one in an orphanage, one in a club, one was a campaign and the setting was not mentioned. Nine studies had received funding and the rest did not mention. Eight studies received additional support – in the form of voluntary organizations, Non-Governmental Organizations, local government etc. All studies delivered oral health education in the form of instructions, in addition to instructions four studies distributed written matter regarding oral health to participants and four studies demonstrated oral hygiene methods to the participants, three studies used videos to educate the participants, one study done by Vachirarojpisan .[] had group discussions and two studies had campaigns. Twelve studies provided education in groups whereas one to individuals and the training time ranged from 20 min to 2 h. Six studies did not mention the training time. Health promotion was done in four studies. An incentive was given only in one study by Freil .[] where a smile contest was held at the end of the study. No study had policy backing. Other than oral health education only one study Tai .[] provided preventive and curative intervention, one study by Freitas .[] provided oral prophylaxis to the participants []. All studies were effective in improving the knowledge. Eight studies did not give a quantitative estimate of the improvement, 85% improvement was seen in a case control study done by Buischi .,[] conducted in 126 children aged 13 years in a school setting for a period of 3 years, oral health education was given in the form of instructions to groups of children []. Four studies[] evaluated their effectiveness through change in attitude. The sample size ranged from 198 to 458. The number of subjects in case group ranged from 99 to 458 participants with an average of 239 and in the control group 99-215. Two studies targeted adolescents and two elderly. Follow-up period ranged from 6 months to 6 years. Three studies were case control and one was experimental []. Target population in two studies for oral health education was adolescents, one in care givers and one in older migrant adults. Socio-economic status was not mentioned. Education level of the oral health education target group was secondary in the adolescents and not mentioned in the other two studies. The oral health education in all studies was delivered by professionals. The setting was schools in two studies, one in a nursing home and one in clubs. Funding was provided in three studies. Additional support was given in two studies. Oral health education in three studies were in the form of instructions, written literature, one study even had demonstrations and one used a video to educate the participants. One study educated the participants by delivering lectures. All studies educated the population in groups. Training time varied from 25 min to 1 h. Health promotion was present in studies which involved adolescents. One study by Tai .[] provided preventive and curative intervention too. Two studies did not quantitatively give their results all showed significant improvement, one study showed 74% improvement, one study showed 17% improvement in the attitude of the subjects []. Fifteen studies[] evaluated their effectiveness through change in practices related to oral health. The sample ranged from 42 to 3967 participants, the case group ranged from 14 to 3291 participants. Four studies were done in adolescents, four studies were targeted at mothers and caregivers of infants, one study in the elderly, one among all age groups and five in children. The follow-up period ranged from 6 weeks to 6 months. The target population was adolescents in four studies, three studies in infants, one study in the elderly, one in migrant adults, one for all age groups and five in children. Low socio-economic status population was taken in studies done by Kowash .,[] Freitas-Fernandes .,[] and Azogui-Lévy .[] Oral health education in all the studies was provided by professionals. Eight studies used the school as a setting, one was done at homes of the participants and two studies were done at health centers, one at an orphanage, one at clubs and one at nursing homes. Funding was provided in nine studies. Additional support was provided in nine studies. The studies either educated the participants by giving instructions, showing videos, demonstrating oral hygiene technique or by distributing written literature. Some studies used a combination of these methods; a study by Mariño .[] used lectures as a medium of education. In studies by Friel .[] and Peng .[] campaigns were done. Vachirarojpisan .[] held group discussions for the participants. Education was imparted in groups in all studies except in Kowash .[] and Freitas-Fernandes .[] The training time ranged from 15 min to 11/2 h. Health promotion was provided in six studies. Incentives were provided in the study by Friel .[] were a smile contest was held at the end of the program and in the study by Azogui-Lévy .[] where reimbursement was provided for participants who visited the dentist. Vanobbergen .[] study was based on the Ottawa Charter. Tai .[] provided preventive and curative care, Freitas-Fernandes .[] provided oral prophylaxis for the participants and Azogui-Lévy .[] provided curative care []. Thirteen studies were found to be effective and two studies were not effective. Only five studies gave a quantitative estimate of the effectiveness. Of this Rong .[] showed 45% improvement in practice outcome and Petersen .[] showed 7% improvement. Other studies showed 30%, 35% and 20% improvement respectively []. Seven studies[] evaluated the change in gingival health. The sample size ranged from 68 to 283. The case group ranged from 39 to 228 participants with an average of 112. Four studies were conducted in children and adolescents, the age of the participants ranged from 5 to 15 years in one study to 11-14 years in another study. One study was done for caregivers of infants, two in adults and one in the elderly. The follow-up period ranged from 1 month in two studies to 3 years in a study done by Kowash .[] Two studies were done in low socio-economic status participants. One study targeted infants, one Chilean refugee, one adult, one elderly and the rest adolescents and children. One study was done exclusively in children. Two studies were conducted in schools, two in clubs, two at homes and one in nursing homes. Only the study conducted by Kowash .[] was funded. Oral health education in five studies was provided in the form of only instructions, the other studies had demonstrations, videos or printed matter or a combination of all methods. Training time ranged from 15 min to 1½ h. Zimmerman .[] and Sgan[] provided oral prophylaxis to the case group. And Kara .[] provided preventive and curative care along with oral health education []. All studies were effective in improving the gingival status. Five studies gave a quantitative estimate of the effectiveness. The most effective studies were by Zimmerman .[] and Ivanovic .[] which showed a 50% improvement and by Beisbork .[] which showed a 51% improvement. Zimmerman[] conducted a study which consisted of 87 Chilean refugees in the case group, for a period of 6 months, the intervention was in the form of oral health education video, instructions and group discussions for a period of 45 min. It was combined with an oral prophylaxis program. The study showed an improvement in knowledge. Ivanovic .[] conducted a study in adolescents of 160 participants in the case group for a period of 6 month in a school with funding; the intervention was in the form of instructions for a period of 15 min. The study showed an improvement in knowledge []. Ten studies showed effectiveness in the plaque outcome category. The sample size ranged from 42 to 2678 participants. The case group ranged from 14 to 1339 participants with the follow-up period ranging from 1 month to 3½ years []. The target population was adolescents and children in seven studies, the age group ranging from 5 to 15 years. Two studies were conducted on adults and one on diabetic patients. One study was done on females exclusively and one study on male diabetic patients. Five studies were conducted in schools, one in orphanages, one in clubs, one in a workplace and one in a hospital setting. Five studies received funding. Six studies received additional support. Six studies provided education in the form of instructions, whereas the other studies used a combination of demonstrations, video and printed matter. Three studies provided education to individuals whereas the others provided education to groups. The oral health education was around 20 min. Health promotion was provided in three studies. Oral prophylaxis was provided in studies done by Freitas-Fernandes .,[] Beisbork .[] and Sgan .[] whereas preventive and curative care was provided in the study done by Kara .[] Ten studies[] were effective in improving the reduction in plaque, one study did not show any statistically significant improvement. Studies by Almas .[] showed a 50% reduction in plaque scores. The study by Almas .[] was done in a sample of 40 diabetic male patients in the case group, for a period of 7 days in a hospital with additional support; education was given in the form of instructions only in groups. The study which was done by Frencken .[] did not show a significant improvement, oral health education was provided to school teachers of 450, 8-year-old children for a period of 3½ years, funding was provided along with additional support. The study did not show any improvement in caries increment when compared with the control group []. Seven studies[] evaluated the effectiveness of their studies through bleeding on probing of the gingiva. The sample size ranged from 42 to 803. The case group ranged from 14 to 404 participants. Two studies were conducted in children, one in adolescents, one in children and adolescents two in adults and one in Chilean refugees. The follow-up period ranged from 1 month to 3 years. Four studies targeted children and adolescents and three adults. Two studies were done in low socio economic groups. And a study by Freitas-Fernandes .[] was done in female orphans. Professionals provided oral health education in all the studies. The setting was a school in three studies, a workplace in one and an orphanage in one and a club in another. Funding and additional support was provided in studies done by Lim .,[] Freitas-Fernandes .[] and Petersen .[] Education in the form of instructions was given in all studies, along with a combination of printed matter, demonstrations and videos. The training time ranged from 15 to 45 min. Zimmerman .,[] Freitas-Fernandes .[] and Sgan .[] combined Oral prophylaxis with oral health education []. All the studies were effective. Study done by Zimmerman .[] and Freitas-Fernandes .[] showed 50% reduction in bleeding on probing. Zimmerman .[] had provided oral health education to a group of 87 Chilean refugees over a period of 6 months; the study was effective in improving the gingival status too. Freitas-Fernandes .[] had conducted an oral health education program in a case group of 14 orphan children for a period of 6 months. Funding and additional support was received. The study also showed a 35% improvement in plaque scores and a significant improvement in knowledge and practice outcome []. Nine studies[] showed effectiveness through caries increment. The sample in the studies ranged from as low as 81 to 12,500 participants. The case group ranged from 43 to 12,500 participants. The oral health education population ranged from school children, adolescents to teachers and mothers. The follow-up period ranged from 12 months to 6 years. Study done by Blair .[] was in low socio economic population. All the studies targeted either children or adolescents. In the study done by Guennadi .[] trained personnel gave oral health education. Seven studies were done in a school setting, one at home and one at a health center. Five studies had received funding and additional support []. All the studies had used instructions to educate the population; some gave printed material to participants while a study by Vachirarojpisan .[] held group discussions. Oral health promotion was provided in seven studies. study by Vanobbergen .[] was based on the Ottawa Charter. Axelsson .[] and Guennadi .[] used fluoride dentifrice as an additional intervention []. Five studies showed a significant decrease in the caries increment. The results of four other studies were not significant. A study by Blair .[] showed a 20% decrease in caries increment. Rong .[] had conducted a study in a sample of 731, with a case group of 361 participants and 370 control groups in a school for a period of 2 years in 3-year-old children. Education was done in groups using video and demonstrations. Funding and additional support was provided for the study. The salient features of this study were that it involved significant others like teachers and parents in the program. This showed a significant improvement in practice though. The study which was done by Frencken .[] did not show a significant improvement either in caries increment or in plaque scores. Oral health education was provided to school teachers of 450, 8-year-old children for a period of 3½ years, funding was provided along with additional support. The study did not show any improvement in caries increment when compared with the control group []. For most of this century, dental health education has been considered to be an important and integral part of dental health services and has been delivered to individuals and groups in settings such as dental practice schools, the workplace and day-care and residential settings for older adults etc., The population as a whole has also been targeted using mass media campaigns. The educational interventions used have varied considerably, from the simple provision of information to the use of complex programs involving psychological and behavior change strategies. The goals of the interventions have also been broad and hence knowledge, attitude, intentions, beliefs, behaviors, use of dental services and oral health status have all been targeted for change. These efforts are testimony to dentistry is long-standing and perhaps pioneering concern with the prevention of oral disease via changes in knowledge, attitudes and behaviors and the adoption of healthier life-styles. However, the increasing pressure on health care resources means that questions are being raised about the costs and effectiveness of all forms of health service provision. This is also the case with respect to preventive interventions since they have long been presumed to reduce disease and therefore lower the demand for health services and the resultant costs. Answers to questions concerning the effectiveness of health education will tell us whether or not it is worth doing and if so, what works best under what circumstances. Data from well-designed evaluation studies also have a role to play in the further development of these kinds of interventions. Over the past few years, a substantial literature has emerged describing studies purporting to evaluate the effectiveness of various types and combinations of educational and behavior modification techniques. A set of coding variables were drawn under which the articles were reviewed to make them amenable for coding, these coding variables were then described under various subheadings so as to allow us to compare articles based on these coding variables: These coding criteria were drawn so as to identify variables or factors which have contributed or influenced the effectiveness of the program. However, a number of problems were encountered in this systematic review: Similar to the present study Kay and Locker[] in their systematic review of oral health education programs faced the problem of summarizing their results due to the differences in which outcomes were measured and reported. A major limitation is this review is the search strategy which was limited to Medline so articles published in journals not included are either highly specialized and/or of low circulation or have not been peer reviewed. Many of the articles which passed the inclusion criteria during the initial search were available only on payment, mails were sent to the journals/authors requesting a waiver of the same but no response was received, as the study was not funded, these articles were not included. However, it is possible that relevant data may be included in these journals and inclusion of these articles could have thrown a better light on the effectiveness of the oral health programs. A manual search in libraries of the research colleges was just limited to Bangalore, instead extending to the whole of India could have been done but the non-availability of funds crippled the study. Furthermore, conference proceedings, dissertations and government reports are excluded from Medline and important information will undoubtedly be overlooked with a limited search strategy such as that used in the current study. Out of total of 40 articles 13 articles evaluated the effectiveness of the program through improvement in knowledge, 4 through change in attitude, 15 through improvement in oral health related practices, 8 through improvement in gingival health, 11 through reduction in plaque, 8 through reduction in bleeding on probing, 9 evaluated the caries increment and 9 used other outcome variables to evaluate the effectiveness of the program. All studies showed an improvement in knowledge, no matter what design, sample, organizational or interventional variables were used. Oral health education was effective in all sample sizes which ranged from as low as 14 to 1339, among all age groups and even over long evaluation periods like 3 years in a study done by Buischi .[] Oral health education in all settings was effective and funding and additional support did not seem to be a factor that influenced the improvement in knowledge in the oral health education. Health education was given in the form of instructions, demonstration of oral hygiene practices, group discussions and lectures. Other than oral health education only one study by Tai .[] provided preventive and curative intervention, a study by Freitas-Fernandes .[] provided oral prophylaxis to the participants. Since quantitative estimates of the effectiveness were not given for all the studies it is difficult to list out the factors that would contribute to a successful program. Brown who had reviewed 57 such studies published between 1982 and 1992 concluded that dental health education was less effective in changing the knowledge of the participants when compared to change in practice.[] Kay and Locker[] who reviewed 14 studies published between 1982 and 1994 concluded that knowledge could be improved through dental health education. The results of the present study are consistent with this study, which also concludes that oral health education is effective in improving the knowledge of the participants. Oral health education was shown to be effective in changing the attitude of adolescents and the elderly, even after a follow-up period of 6 years there was a significant change in attitude as shown in the study done by Tai .[] This review shows that immediate change in attitude is high, i.e. around 74% as shown in study by Laiho .,[] but the quantum of change in long follow-up periods like 6 years as shown in study by Tai .[] is less, i.e. around 17%. This review shows that change in attitude is possible in teenagers through a sustained oral health education program. Brown who had reviewed 57 such studies published between 1982 and 1992 concluded that dental health education was less effective in changing the attitude of the participants when compared to change in practice.[] Kay and Locker[] who reviewed 14 studies published between 1982 and 1994 concluded that attitude could be improved through dental health education. The results of the present study are consistent with this study, which also concludes that oral health education is effective in improving the attitude of the participants. Oral health education in a range of sample sizes were effective in improving oral health related practices. Studies were more effective when oral health education is targeted towards children and when significant others are involved. Studies by Alsada .,[] Kowash .,[] Vachirarojpisan .[] and Rong .[] showed a significant improvement in oral health related practices and all the above mentioned studies involved significant others like care givers and mothers of children in the education of the target groups which obviously influences the behavior of the target group. Studies which received funding and additional support were more effective. Brown who had reviewed 57 such studies published between 1982 and 1992 concluded that dental health education was less effective in improving behaviors of the participants which is not consistent with the results of the present study which showed that oral health education improves the behavior of the participants.[] Oral prophylaxis was done along with oral health education in a study done by Zimmerman .[] done in Chilean refugees who showed an improvement of 50% in gingival health, thus suggesting that an oral prophylaxis component in an oral health education program could contribute to the improvement in the gingival health of the subjects. Kay and Locker's[] systematic review of oral health education programs showed that out of 15 studies published between 1982 and 1994 only eight concluded that gingival bleeding scores could be improved through dental health education. The results of the present study are consistent with this study which also concludes that oral health education is effective in improving the gingival health of the participants after reviewing eight studies. Sample size of the oral health education group, their age and setting of oral health education did not seem to influence the effectiveness of the study. The range of effectiveness was 3% to a 50% reduction in plaque scores in studies that gave a quantitative estimate of the results. The effectiveness of the studies when the follow-up was of long duration for example a study done by Alabandar[] was lower. Frietas .[] showed a 35% reduction in plaque scores when evaluated at 6 months. Thus oral health education in long term studies was not effective in reduction of plaque. Studies which provided oral prophylaxis regularly along with oral health education were usually more effective. Kay and Locker's systematic review of oral health education programs showed that out of 15 studies published between 1982 and 1994 only eight concluded that oral health education programs were generally effective in short term but no long term benefits were seen. The results of the present study are consistent with this study which also concludes that oral health education is effective in reduction of plaque in short term studies but was not effective in studies with long follow-up periods.[] All studies were effective, the study done by Zimmerman .[] in 87 Chilean refugees evaluated after 6 months was the most effective, showing a 50% reduction in bleeding on probing of the gingival. The sample size, the target population, setting of the study, funding and additional support to the study seemed to have no effect on the effectiveness of the study. Studies in which oral prophylaxis was done along with oral health education showed a comparatively more reduction in bleeding on probing of the gingival as compared to studies in which only oral health education was done. Nine studies showed effectiveness through caries increment out of these there was significant reduction in caries increment in five studies and in four studies there was no significant change. Only one study gave a quantitative estimate of the effectiveness, i.e. the study done by Blair .[] in 7012 school children which showed a 20% decrease in caries increment at the end of the 6 year study. The review showed that studies done in schools were effective and health promotion was a salient feature in most of the effective studies. Seven studies used other outcome measures to evaluate their effectiveness; Laiho .[] showed an increase in utilization of dental services after an oral health education program in 458 adolescents where health education was done in their school. Guennadi .[] showed an improvement in oral health awareness after an oral health education program in 3-12 year old children after a 3 year study. Simons .[] showed a reduction in denture stomatitis in 39 elderly patients after a 12 month oral health education program for their caregivers. Nicol .[] showed a reduction in oral mucositis and a reduction in denture stomatitis but no significant improvement in denture hygiene in 78 elderly patients after an 18 month oral health education program for their care givers.[] Most of the studies reviewed in this study showed an improvement in the outcome measures no matter what design, sample, organizational or interventional variables were used. Although a few studies showed a better improvement in the outcome variables due to certain salient features: All studies were effective in improving knowledge outcome, change in attitude over a longer time period is possible only through a sustained oral health education program, the involvement of significant others in oral health education programs is more effective, bringing about a higher improvement in practice outcome. An oral prophylaxis component in an oral health education program has shown to bring about a higher quantum of change in the gingival status outcome, bleeping on probing of the gingiva and plaque outcome. Where health promotion was a salient feature a more significant reduction in caries increment was noticed. Certain studies evaluated their effectiveness through utilization of dental services, an improvement in oral health awareness, reduction in denture stomatitis, reduction and oral mucositis. These studies were reviewed in the study but were not discussed in this article as the outcome measures were beyond the purview of the outcome variables intended to be evaluated in this article. Oral health education is effective in improving the oral health; this review throws light on the effectiveness of oral health education programs and identifies important variables which contribute to the effectiveness of these programs. There is an urgent need for more systematic reviews on studies evaluating the effectiveness of oral health education and promotion in the India. Overcoming the limitations of this study, such as research funding and standardizing the outcome variables which, would enable us to have a common measurement tool and systematically reviewing the future programs would help formulate a public health program with the best design. Recommendations for action: This review emphasizes the need for further research in evaluating effectiveness of oral health education; it has shown the limitations in terms of the lack of standardization in evaluating the outcome measures and lack of funding in this field. The government has a key role to play in this process through its policy making. Such a step forward also demands collaboration between academics and professionals to ensure that strategies are developed upon a sound scientific basis and are subject to appropriate evaluation. This may include a range of methodologies which together will illuminate the full costs and benefits of individual health promotion interventions as well as the overall strategic framework. Oral health education is effective in improving the knowledge attitude and practice regarding oral health and in reducing the plaque, bleeding on probing of the gingival and caries increment and in improving the gingival health. The present review throws light on the effectiveness of oral health education programs and identifies important variables which contribute to the effectiveness of these programs. This review has shown that oral health education is effective in improving the knowledge and oral health related practices of the target population when significant others are involved, thus involvement of significant others like teachers and parents especially in oral health education of school children would bring about a higher quantum of change in improving the oral health in children. Including an oral prophylaxis component in oral health education programs would bring about a higher quantum of improvement in the gingival health. Since oral health promotion programs have shown to be more effective than just oral health education, this approach should be adopted for bringing about an improvement in the target population, in such programs health promotion commits us not only to improving lifestyles but also to improving the environment in which lifestyle choices can be made. There is indication in this review that the most successful oral health programs are labor intensive, have involved significant others and have received funding and additional support. A balance between inputs and outputs and health care resources available will determine if the program can be recommended for general use.

Injuries of the maxillofacial complex represent one of the most important health problems worldwide.[] Maxillofacial injuries, such as soft-tissue injuries, dental injuries, or maxillary, mandibular, and zygomatic fractures; are the most common injuries treated by oral and maxillofacial surgeons.[] The mandible is a unique bone having a complex role in esthetics of the face and functional occlusion. Because of the prominent position of the lower jaw, mandibular fractures are the most common fractures of the facial skeleton. It has been reported that fractures of the mandible account for 36% to 59% of all maxillofacial fractures.[] Despite the fact that it is the largest and strongest facial bone, it is the tenthth most often injured bone in the body[] and second to nasal bone fractures[] and it is fractured two or three times more often than other facial bones.[] The age distribution of persons sustaining craniomaxillofacial injuries differs from one country to another. Traditionally, there has been a high male-to-female ratio among craniomaxillofacial injury victims, ranging from 10:1 to 6.6:1. However, the recent literature shows a trend toward a more equal male-to-female ratio. This can be attributed to a changing workforce and the fact that more women work outdoors in more high-risk occupations, thus becoming more exposed to RTA and other causes of craniomaxillofacial fractures. Many causes of craniomaxillofacial fractures have been reported, including road traffic accidents (RTAs), assaults, sporting injuries, falls, and industrial accidents; and in some areas of the world, attacks by animals.[] Many epidemiological studies have been published from different countries about the pattern of maxillofacial injuries, but demographic data are difficult to evaluate because of the many variables.[] Studies around the world have shown that assaults are the predominant cause of maxillofacial fractures in developed countries, while motor vehicle accidents (MVAs) are the most common cause in developing countries.[] Also, in cases of multiple site fractures, association between specific anatomic sites is sought. The development of reliable predictors of injury patterns will be a useful guide to the prompt and accurate diagnosis and management of mandible fractures in the trauma patient population.[] For each patient, the combination of these factors determine the likelihood of a mandibular fracture. Mandibular fractures have been studied extensively, and some controversy remains regarding the ideal treatment approach. The advent of AO/ASIF (Arbeietsgemeinschaft fur Osteosynthesefragen/Association for the Study of Internal Fixation) and microplating systems has further increased debate as to whether open reduction is a better treatment option for mandibular fracture treatment compared with closed reduction.[] A clearer understanding of the demographic patterns of mandibular fractures will assist providers of healthcare as they plan the treatment of maxillofacial injuries. Such epidemiological information can also be used to guide the future funding of public health programs geared towards prevention of such injuries.[] The aim of this study is to determine the etiology, frequency of mandibular fractures among different age and sex, to determine the frequency of anatomic distribution, and to report the different modalities of treatment provided to the patients of mandibular fractures reported at Department of Oral and Maxillofacial Surgery at our Institution from February 2008 to September 2009. This study was designed to establish the current demographic and treatment patterns of mandibular fractures at the Department of our institution. This was an observational, descriptive, and epidemiological study that included all cases of mandibular fractures that were clinically and radiographically diagnosed and treated at our institution from february 2008 to september 2009. Patients from 1 to 70 year age group and with either sex were included. Patients who had refused to participate in the research or medically compromised were excluded from the study. Patient information was collected by means of a medical data form specifically designed for the present study. The data collected included age, sex, etiology of fracture, anatomic site of fracture, and the types of treatment provided. Thirty-five patients included in the present study were divided into groups according to age (1-10 years; 11-20 years; 21-30 years; 31-40 years; 41-50 years; and 51-60 years) and according to sex into male and female. Mechanism of injury was recorded and classified as RTA, fall, interpersonal violence, assault, and other causes. Anatomically, the mandibular fractures were classified into seven regions: Symphysis, parasymphysis, body, angle, ramus, coronoid, and condylar. After making final diagnosis, informed consent was obtained from each patient and appropriate management was done under suitable anesthesia. Out of 35 patients, 31 were males (88.57%) and were females (11.43%) with a male:female ratio of approximately 8:1 []. The difference between both groups was found to be statistically significant ( < 0.05). The age of patients ranged from 1 to 57 years, with a mean age of 27.09 years. The mean age for females was 32.5 ± 6.03 years and for males it was 26.39 ± 11.59 years. We found a peak occurrence in young adults, aged 21-30 years ( = 15, 42.86%). This was followed by 31-40 years ( = 8, 22.86%), 11-20 years ( = 7, 20%), 1-10 years, and 41-50 years age group ( = 2, 5.71%). Patients belonging to 51-60 years were the least involved group ( = 1, 2.86%). Amongst males, 21-30 years group were the most frequently involved followed by 11-20 years ( = 7, 20%); whereas, in females 31-40 years age group was most common followed by 21-30 years ( = 1, 2.85%). In case of etiology, RTAs were the most common ( = 25, 71.43%), whereas falls were the second most likely cause ( = 7, 20%). Interpersonal violence represented ( = 2) 5.71% and assault accounted for ( = 1) 2.86% of mandibular fractures almost 48% of RTAs were found in 21-30 age groups, 42.86% in 11-20 age groups, 50% of interpersonal violence were equally found in 11-20 and 21-30 age group; whereas, assault were found only in 31-40 age group. Statistically, no significant association existed between the age group involved and the etiology of fracture ( > 0.05). The data for causes of fractures distributed by gender showed that, RTA was the most frequent etiological factor irrespective of gender ( = 23, 65.71% for males and n = 2, 5.71% for females). Whereas, the second most frequent cause of fracture for males was fall ( = 6, 17.14%) and in females was fall ( = 1, 2.86%) and assault ( = 1, 2.86%). But the interpersonal violence ( = 2, 5.71%) was the third cause of fracture which was found only in males. Statistically, significant association existed between the genders and the etiology of fracture ( < 0.05) []. There were a total of 49 fracture sites in 35 patients. The condyle was most frequently involved site with ( = 19) 38.78% of the total mandibular fractures. This was followed by body ( = 11) in 22.45% and parasymphysis ( = 10) in 20.41%. Symphysis fractures accounted for ( = 5) 10.2%, angle for ( = 3) 6.12%, and the remaining 2.04% was involving the coronoid process ( = 1) of the mandible. Out of the patients with RTA, the parasymphysis/condyle (12%) was the predominant combination of fracture site involvement, followed by the body/condyle (8%). In case of fall patients, the most common pattern was body/condyle (28.57%); whereas, in case of patients with interpersonal violence a combination of angle/parasymphysis (50%) and condyle/parasymphysis (50%) were the involved sites. Statistically, significant association existed between the etiology of fracture and the fracture site involved ( < 0.05) []. Several different approaches were used for the reduction, fixation, and immobilization of mandibular fractures. In approximately half ( = 16, 45.71%) of the patients, an open reduction and rigid internal fixation using bone plate and screws were done. For the 11 patients (31.43%), treatment involved closed reduction of the fracture using arch bars or ivy loops and intermaxillary fixation, in three patients (8.57%) closed reduction using arch bars or ivy loops and short period of intermaxillary fixation followed by physiotherapy, two patients were treated using (5.71%) open reduction followed by physiotherapy. For the remaining three patients with high condylar fracture (8.57%), treatment involved only physiotherapy and soft diet . Regardless of geographical boundaries, injuries to the body are common.[] The human face constitutes the first contact point in several human interactions; thus, injuries and/or mutilation of the facial structures may have a disastrous influence on the affected person.[] As with other diseases and injuries, epidemiological data provide an important basis for the evaluation of access to treatment, resource allocation and planning within the health services. It may also be used to develop preventive strategies and may provide information about the quality of care provided.[] Maxillofacial injury occurs in approximately 5-33% of patients experiencing severe trauma.[] Moreover, maxillofacial fractures are often associated with severe morbidity, loss of function, disfigurement, and significant financial cost.[] Given that the mandible is the only facial bone that has mobility and the remaining portion is part of the fixed facial axis, the fracture is never left unnoticed because it is very painful, pain that worsens with mastication and phonation movements, and even respiratory movements; sometimes there are facial asymmetry complaints. Mandible fractures may lead to deformities caused by displacement or non-restored bone losses, with dental occlusion affection or temporomandibular joint disorder (TMJD). If not identified or inappropriately treated, these lesions may lead to severe sequelae, both cosmetic and functional.[] It has been reported that incidence of maxillofacial fractures varies widely between different countries.[] The large variability in reported prevalence is due to a variety of contributing factors, such as the environment, sex, age, and socioeconomic status of the patient, as well as the mechanism of injury. For each patient, the combination of these factors determines the likelihood of a mandibular fracture.[] Mandibular fractures occur in people of all ages and races, in a wide range of social settings.[] The results of the present study of patients with mandibular fractures, who were treated at our institution, are largely in agreement with those of previous reports, particularly with regard to age and gender of the patient.[] The finding that age group 21-30 years constituted the group with the highest frequency of jaw fractures is consistent with previously published studies.[] Trauma is now considered a problem of young people, which may be because of their aggressive nature and careless driving on roads.[] It has also been consistently shown that the frequency of mandibular fractures among male (88.57%) is far greater than that of female (11.43%). This was found to be statistically significant ( value = 6.45, < 0.05) in the present study. Reported overall ratios of male to female have ranged from 4:1 to 12:1 in other studies; similar to the ratios observed here (8:1).[] The predominance of male gender is due to the fact that this group make up the most active group in society,[] is more prone to traffic accidents since they drive motor vehicles carelessly and is most likely to be involved in interpersonal violence[] and is normally associated with use of alcoholic beverage.[] The higher frequency of mandibular fractures among males compared to females may also be attributed to the fact that the females, most often, are confined to housework and they drive vehicles less frequently and carefully, and are less exposed to accidents, fights, industrial works, and sports and more participate in trading or farming.[] The causes of fracture have extremely variable incidence depending on social, geographical, and economic characteristics.[] In the present study, RTAs was the most frequent cause of fracture which was found to be statistically significant in males and females ( = 0.039). These results were found to be in agreement with the studies conducted by different authors,[] which were most common in males than females in the age group of 21-30 years. This might be because a large proportion of the population uses a motorcycle on a daily basis.[] The increasing number of RTAs in developing countries like India may be attributed to many factors like sharing of roadways by pedestrians and animals with fast-moving vehicles, with almost no segregation of pedestrians from wheeled traffic; the large numbers of old and poorly maintained vehicles on road; large numbers of motorcycles, scooters, and mopeds; low driving standards; large numbers of overloaded buses; widespread disregard for traffic rules; defective roads; poor street lighting; and defective layout of cross roads and speed breakers. In addition, the increasing volume of traffic as a result of economic expansion and rapid increase in the density of urban population may also be the factors responsible for increasing RTAs in recent times.[] Perhaps, the lack of experience in traffic, imprudent driving, and the type of service for which the motorcycle is generally used (fast delivery) might explain the higher incidence in young drivers. High speed, imprudence, use of open helmets, or no use of helmets can explain the high number of fractures secondary to motorcycle accidents. Olson ., reported that wearing helmet decreases the mortality but does not reduce significantly the number of fractures and point to speed as a determinant factor for fracture occurrence. Other studies have reported an association between wearing a helmet and the decrease of maxillofacial lesions and the severity of skull injuries. A previous study showed that facial trauma incidence was lower when the victims wore helmets, especially if closed. Although the Traffic National Code imposes the compulsory use of helmets and seat belt and apply severe penalties for high speed and/or drunk driving, there are still people that do not follow the law.[] These were followed by falls in the age group of 1-30 years, interpersonal violence in the age group of 11-30 years, and assault in the age group of 31-40 years. In a retrospective study, Fridrick ., demonstrated that altercations accounted for 47% of fractures and automobile accidents for 27%.[] Thorn .,[] in Greenland (90%) and Lee in New Zealand (49%)[] reported that the major cause of mandibular fractures were due to interpersonal violence. In a study conducted by Czerwinski .,[] Alexander .,[] King .,[] and Dongas and Hall[] assault was the most common cause of fracture. There are countries whose main cause of mandibular fracture is related with sport activities, such as in Austria.[] With regards to the patient characteristics, there existed no significant association between certain age groups and etiology of fracture ( = 0.222) in the present study. Whenever the maxillofacial region is injured, the mandible is more vulnerable than the midface fractures. This could be because the mandible is mobile and has less bony support than midfacial bones. These fractures are, however, more common in certain sites of the mandible than others.[] In the present study, the condylar region of the mandible was the most commonly involved site which is similar to the results found in other studies in men aged 21-30 years.[] This might be because the force of a blow is transferred from the chin along the mandible to the condyle often causing fractures in the neck, which is one of the weak anatomical locations within the mandible.[] But certain studies found other sites of mandible to be most commonly involved.[] It is difficult to cite a reason for this difference; perhaps further study on the causes of the regional mandibular fractures would be useful. One can speculate that interpopulation difference in the sites of mandibular fractures partly related to the diverse etiologic factors involved.[] This allows the conclusion that the pattern of presentation is a multifactorial variable.[] These were followed by body, parasymphysis, symphysis, and angle. The least affected site was the coronoid process of the mandible. With regards to patient characteristics, there existed no statistically significant association ( = 0.79) of any patient age group with any specific mandibular fracture site. This suggests that fracture site is dependent primarily on the physics of the specific injury mechanism and not on any inherent characteristics of the mandible itself such as the absence of dentition or presence of unerupted third molars.[] No statistically significant association ( = 0.097) was found between males and females with a particular anatomic site involved, between the anatomic site and the side of involvement ( = 0.193), and between males and females involving either the right side ( = 0.086) or left side ( = 0.166). The involvement of mandible fracture site is variable, depending on the many different causes of the fracture. Therefore, the literature differs a lot concerning the affected sites.[] The mechanism of patient injury correlates significantly ( = 0.020) with the anatomic location of fracture, and knowledge of these associations should guide treating physicians in their diagnostic workup of all head and neck trauma patients.[] Automobile accident victims will more commonly have condylar and body fractures. Patients involved in accidents involving posterosuperiorly directed energy such as being struck by vehicles where the underside of the anterior mandible receives the primary force of impact should be suspected of having condylar and subcondylar injuries. Victims of falls are significantly more likely to suffer from symphysis, parasymphysis, and body fractures. Victims involved in interpersonal violence will more commonly receive a blow to lateral portions of the jaw, predisposing these patients to fractures at lateral locations such as the parasymphysis, angle, and condyle. Victims of violent crimes such as assault are statistically more likely to suffer from coronoid fractures. In the present study, the percentage of single mandible fracture site (62.85%) coincides with other mandible fracture indiceses reported in large centers.[] 34.28% patients presented with two fracture sites and 2.85% with three fracture sites in the mandible.[] These were found to be statistically insignificant ( = 0.402). The most common mandibular fracture combination in this study was condyle/parasymphysis followed by body/condyle. These often occurred as a result of RTAs, with the mandible presumably fracturing in areas deficient in strength. Regarding the severity of mandibular fractures, displaced and undisplaced fractures were most commonly seen in males than females which were not found to be statistically significant ( = 0.76). Treatment of mandibular fractures has changed over the last 20 years.[] In 1989, Arthur and Berardo introduced a simplified technique of maxillomandibular fixation (MMF) by the use of cortical bone screws and stainless steel wire. This technique offers several advantages over traditional closed reduction techniques; including ease of technique, reduced operative time, and diminished chance of glove penetration and transmission of human immunodeficiency virus (HIV) and hepatitis B virus.[] There has been a decrease in the use of wire osteosynthesis and intermaxillary fixation and an increase in preference for open reduction and internal fixation with bone plates and screws. This has helped to reduce malocclusion, nonunion, improved mouth opening, speech and oral hygiene, decreased weight loss, and increased the ability for patients to return to work earlier.[] Treatment of mandibular fractures continues to be a challenging problem for the facial trauma surgeon.[] There are many different therapeutic possibilities, but many authors disagree about the best treatment approach.[] The treatment of mandible fractures requires adequate fracture reduction and stabilization through a closed or open technique. Success relies on the restoration of normal dental occlusion and bony union.[] The treatment chosen may differ as there are many factors like cost of treatment, affordability by the patient, feasibility in the hospital, doctor's decision and skill, and patient's willingness to avail the treatment advised; all of which may vary from one country to another.[] In the present study, 11 patients (31.43%) were submitted to closed reduction and MMF (using arch bars or ivy loops), three patients (8.57%) were submitted to closed reduction and MMF (using arch bars or ivy loops) followed by physiotherapy, two patients (5.71%) were treated with open reduction and rigid internal fixation using bone plates and screws followed by physiotherapy, and 16 patients (45.71%) were treated with open reduction and rigid internal fixation (ORIF) using bone plates and screws; which is in agreement with other literature studies.[] And remaining three patients (8.57%) were treated with soft diet and physiotherapy alone. Closed reduction and MMF treatment was preferred in cases of single, simple, or bilateral fractures; with little deviation or when the number of teeth and dental support provide conditions for the stability of the occlusion. ORIF was advised in patients with partial dentition, multiple, displaced, or severely comminuted fractures. Subcondylar fractures were mostly treated by CR and MMF or ORIF followed by physiotherapy to avoid TMJ stiffness whereas high condylar fractures were treated by soft diet and physiotherapy. The current preference for the use of miniplates systems in the treatment of mandibular fracture is evident. Increasing cost of equipment and operating time have frequently been considered a disadvantage of miniplate fixation of mandibular fractures. The major advantage of osteosynthesis is the avoidance, or reduction of IMF duration.[] In the light of the present study, we speculate that socioeconomic reasons such as poor roads, inadequate enforcement of road safety regulations and speed limits, reluctance to use helmets, decreasing tolerance, and increasing personal competitiveness among young men, could be the possible explanations, in particular in this part of the country. Hence it is strongly recommended that improving the condition of the roads and driving skills, raising the traffic sense of the general public through campaigns, strict legislation about the use of helmets by motorcyclists and seat belts by front seat occupants, and restriction of use of mobile phones while driving may help to reduce the number of the injuries. In addition, the need to encourage massive investments in safer alternative transport system needs to be emphasized. Epidemiological reviews of these injuries are needed to identify the risk factors leading to such trauma and help to train medical and dental practitioners to diagnose facial trauma and to provide immediate and long-term treatment.[] These reviews are useful for reaffirming previously established trends and identifying new patterns of disease frequency. Additionally, the success of treatment and the implementation of preventive measures are more dependent on the epidemiological assessments.[] Mandibular fractures occur in people of all ages and races, in a wide range of social settings.[] It is hoped that such assessments as the one presented here will be valuable to government agencies and healthcare professionals involved in planning future programs of prevention and quantifying demands or services[] and treatment.[] Future scope for the present study includes data collection from all the trauma centers of a particular location with the study conducted for longer duration to confirm the present trend/pattern of variables associated with mandibular fractures of a particular region. In the present study, the incidence of mandible fractures was more prevalent in male patients, especially during the 3 decade of life. The most common cause was traffic accident and the more frequently affected region was condyle of the mandible. Open reduction and rigid internal fixation using miniplates and screws was the most commonly used treatment. The coordinated and sequential collection of information concerning demographic patterns of maxillofacial injuries may assist healthcare providers to record detailed and regular data of facial trauma. An understanding of the cause, severity, and temporal distribution of maxillofacial trauma will permit the clinical and research priorities to be established for effective treatment and prevention of those injuries. Since, the main cause of these fractures proved to be MVAs, any efforts made to enforce traffic and safety rules in the roads and improve traffic culture can be an effective measure to promote the present situation. In addition, the need to encourage massive investments in safer alternative transport system needs to be emphasized. Since, significant association was found between cause of fracture and the fracture site involved in the present study, more studies are needed to confirm these associations which will help the attending healthcare professional in making quicker and correct diagnosis in all head and neck trauma patients.

U n d e r s t a n d i n g c a r i e s e t i o l o g y a n d i t s p r o g r e s s i o n h a s b e e n m a d e e a s y w i t h c o n c o m i t a n t a d v a n c e s i n s c i e n c e a n d t e c h n o l o g y . H o w e v e r , u t i l i z a t i o n o f t h o s e a d v a n c e s i n c o m m u n i t y p r a c t i c e i s s t i l l d i f f i c u l t . A l t h o u g h m o s t o f t h e p o p u l a t i o n i n d e v e l o p i n g c o u n t r i e s l i v e i n i n d i g e n c e , i t i s m u s t f o r t h e c l i n i c i a n t o d e v e l o p r a p i d , i n e x p e n s i v e a n d y e t e f f e c t i v e m e t h o d s f o r c a r i e s c o n t r o l a n d p r o g r e s s . T h i s a r t i c l e p r o v i d e s a n i n s i g h t o f h o w a s i m p l e o r a l r i n s e t e s t c a n b e u s e d f o r d e t e c t i o n o f c a r i e s a c t i v i t y . In a period marked by brilliant achievements in the prevention and treatment of disease, dental caries still remains one of the most widely spread afflictions in human populations.[] Dental caries is one of the most prevalent infectious diseases and for practical purposes it can be considered ubiquitous in civilized population.[] The dental caries prevalence in the child population is increasing at an alarming rate, the prevalence varying from 33.7% to 90%.[] Furthermore, the inequalities in the distribution of dental care in rural and urban areas correlate well with the difference found in caries prevalence for the respective populations. Given the harsh realities of the maldistribution of resources and variations in individual susceptibility, dental caries is not about to become another extinct disease.[] Dental caries gets established in the mouth long before it becomes clinically manifested in the form of visible lesions. This means it should be possible to assess the seriousness of the caries in a patient or in a population. Such an assessment is rather like making a weather forecast.[] The concept of prediction of human dental caries risk has existed for many years. Particularly, during the past 10-15 years, there has been a surge of interest in this issue. This appears to reflect recent advances in our knowledge of the etiology of dental caries, the potential promise of certain risk factors or combinations thereof for caries prediction. There should be incessant desire to prevent rather than to treat dental caries, but at the same time the increasing need of cost containment should be understood.[] Even though, dental caries is known to be a multifactorial disease process, the vast number of caries susceptibility tests developed so far is based on the microbiological aspects of dental caries.[] Caries activity can generally be defined as the occurrence and the rate at which, teeth are destroyed by acid produced by the plaque bacteria.[] A good caries activity test should possess at least three characteristics, i.e. validity, reliability and feasibility. It should also have good sensitivity and specificity. It is very difficult to measure all the factors responsible for caries using one caries activity test. Hence until date, the ideal method to evaluate caries activity in terms of sensitivity, specificity and reliability has not been found.[] Currently, there are many caries activity test kits available in the market, e.g. Dentocult SM, Mucount, GC Saliva Buffer Check etc., Unfortunately, many of these caries activity tests require extensive work-up time and special equipments. In developing countries simple, inexpensive and quick techniques, which do not demand sophisticated skills or consume a substantial amount of chair-side time, are required for the caries activity tests. These deserve the status in the routine clinical practice and epidemiological screening programs. However, very few studies have been reported regarding their sensitivity and specificity to assess caries risk. In this study, an attempt has been made to explore the co-relation of simple oral rinse technique (Oratest) with the other caries activity tests in children. This study was carried out in the Department of Pedodontics and Preventive Dentistry in association with the College of Microbiology. A total of 120 healthy school going children of both sexes falling in the age group of 6-8 years were selected by randomized sample technique.[] Out of 120 children, 30 caries free children were randomly selected through a survey carried out in a Public School, Pimpri. An informed consent was obtained from the child's parents/guardians for the subjects who were fulfilling the inclusion criteria. Care was taken to ensure ethical protection of human subjects at all stages of the study. All the necessary ethical guidelines were followed as prescribed by the Institutional Ethical Committee in accordance with the Central Ethics Committee on Human Research, India.[] Results were analyzed by Software Package for Statistical Analysis [SPSS] version 17. The Oratest time in the control group had shown a range of 200-305 min and in test groups shown a range of 24-200 min. A statistically significant difference revealed with the mean Oratest scores revealed a statistically significant difference ( < 0.001) between the control and individual test groups. The Oratest time observed in the control group was significantly higher than the individual test groups []. A statistically significant difference obtained with the mean of salivary count between control and individual tests groups []. Snyder's test results are recorded for each group. The modal values of Snyder's caries activity test for the control group, sub-group A, sub-group B and sub-group C was found to be negative, slight, moderate and moderate respectively []. Chi-square test results showed a statistically high significant difference ( < 0.001) between the Snyder's test results of control and individual test groups. Pearson's correlation analysis revealed, the count in a statistically significant negative linear relationship with Oratest time []. All test groups computed together the Oratest time (min) was found to be in a statistically highly significant negative linear relationship with existing caries status. Thus, Oratest time was found to have a significant negative linear relationship with salivary colony count and deft/DMFT of the individual []. An overview of changes in dental caries confers a global decline in DMFT levels of individuals. Thanks to the 20 century which has given tremendous knowledge in the understanding of oral diseases and possibilities for preventing and treating these diseases. However, this knowledge cannot be put into action in a developing country like India due to financial constraints and lack of man power. In a country where 70% population inhabits the villages (Consensus 2001), the Dentist: population ratio is 1:35,000 and only one out of five Dentists return back to the rural area to serve, prevention of caries will be a more viable option than rendering restorative treatment. In the existing situation in India, it calls for real dedicated and strenuous effort to implement caries prevention programs. The cost involved in assessing the high risk group by test and other methods in Indian situation is so high that it cannot become a routine procedure in the near future. Western methods for caries prevention as such will not be successful here and they need to be modified to suit the local needs and habits. There is an acute need for simple, robust, inexpensive equipments for use in rural surroundings. Hence, this study was designed to evaluate the caries susceptibility of children using a non-invasive, simple and inexpensive Oratest and to compare it with two other caries activity tests. In comparison with the sole use of any one caries activity test, the combination of the information of different parameters has been shown to increase the power in caries prediction.[] In this study both the subjective and objective evaluation has been carried out for each subject and then compared with each other. The caries process is initiated by activity within the biofilm and manifested in the underlying enamel or dentin.[] In the present study, the mean salivary count for 0 deft/DMFT subjects was 3.12 × 10 . Although in test groups, it ranged from 1.4 × 10 CFU/ml to 1.76 × 10 CFU/ml of saliva. Similar results were also been observed by Loesche .[] and by Straetemans .[] Lactobacilli being acidogenic as well as aciduric possesses a strong potential to demineralize enamel surfaces. Significant relation between lactobacilli and caries experience was shown by Crossner, Holbrook,[] Kingman .[] and Zickert .[] A simple colorimetric test, i.e. Snyder's test was developed for practicing Dentist to serve as a valuable aid in detection of lactobacilli. The Snyder's test measures the rapidity of acid formation when a sample of stimulated saliva is inoculated into glucose agar adjusted to pH 4.7-5 and with bromocresol green as color indicator. Indirectly, the test is also a measure of acidogenic and aciduric bacteria. Overall distribution of Snyder's test results in the present study [] correlated well with the existing caries status of individual. Such relation had also been supported by Ali YA .[] In a study carried out by Ramesh . Sensitivity of Snyder's test was found to be 87.5% along with positive predictive value of 18.9%. Specificity of 18.9% was obtained with negative predictive value of 87.5% in caries free individuals. However, 100% sensitivity was shown in children with caries.[] There have been two main schools of thoughts on the role of plaque bacteria in the etiology of caries and periodontal disease. The “specific plaque hypothesis” proposed that, out of the diverse collection of organisms comprising the resident plaque microflora, only a few species are actively involved in disease. In contrast, the “non-specific plaque hypothesis” considered that disease is the outcome of the overall activity of the total plaque microflora. In this way, a heterogeneous mixture of micro-organisms could play a role in disease. More recently, an alternative hypothesis “ecological plaque hypothesis” has been proposed, which stated that plaque-mediated diseases are as a consequence of imbalances in the resident microflora.[] Oratest, a whole mouth rinse test in this regard provides a pooled sample of microbiota of oral cavity. Thus, the principle of Oratest is congruent with Philip Marsh's “ecological plaque hypothesis” proposed in early 1990s. In young plaques, streptococci usually predominate, but other organisms such as and species may also play a role in the initial development of plaque. is among the more prominent initial colonizers of tooth surface. As the plaque matures during 7-14 days, the rods and filaments increase proportionally. The changes in the morphological forms of the plaque flora have been attributed to changing metabolic and environmental conditions within the plaque over time. As plaque accumulates and matures it becomes more anaerobic. The degree of anaerobiosis of plaque is reflected in its redox potential (Eh). The Eh of early developing plaque is about + 200 mV, which is similar to aerobically metabolizing tissue. However, several days old plaque will have an Eh of about –100 mV. Growth of anaerobes is encouraged at low Eh levels. Oxygen depletion by aerobic plaque micro-organisms is a prerequisite for the development and progression of anaerobic growth conditions. Oratest is based on the rate of oxygen depletion by the microorganisms in expectorated milk samples. Once oxygen gets utilized by aerobic micro-organisms, methylene blue acts as electron acceptor and gets reduced to leucomethylene blue reflecting the metabolic activity of the aerobic organisms. In some of the previous studies strong correlations were observed between Oratest scores and counts of aerobic and aerotolerant micro-organisms.[] Also studies done by Patalay .[] and Bhasin .,[] Saxsena .[] have suggested that Oratest can be used as chair-side caries activity test. But the literature about its comparative evaluation with other caries activity tests seems to be lacking. Hence, in the present study for the 1 time Oratest has been compared with qualitative and quantitative characteristics of cariogenic bacteria. Milk is used in this study because it acts as a suitable liquid for dislodging micro-organisms and provides an excellent medium for subsequent metabolism. Furthermore, it is non-toxic and is readily acceptable by children. Moreover, milk being white in color, make it easier to detect the earliest color change from blue to white at the base of test tube, i.e. methylene blue to leucomethylene blue.[] Milk used for the present study was sterilized using Pasteurization method. Nearly 1% of methylene blue was used in the present study as suggested by Desai . and Rosenberg .[] Since, there is no oxygen in the methylene blue molecule; the reduction of this dye involves transference of hydrogen. Bacterial metabolites have been shown to act as hydrogen donators.[] The corresponding metabolism, which occurs will be a reaction which supplies energy for the growth of the organisms. Sensitivity is defined as the ability of a test to detect the presence of a disease in patients with the disease, while specificity is the ability of a test to detect the absence of a disease in patients without the disease. The Oratest by virtue of its simplicity and high degree of sensitivity–could be used to screen patients for dental caries. However, the lack of specificity of the Oratest can mean that a high number of false positive readings occurred. Some of the false positive results for the Oratest and count could be explained by the presence of interproximal caries that was not diagnosed clinically. In both cases, the number of false positive results would be reduced if radiographs were exposed to detect interproximal caries. Since, larger sample size and proposed use of Oratest at community level, no radiographs were made for assessing proximal caries in the individuals. However, dmft/DMFT score was used as useful predictor of caries.[] Higher levels of cariogenic organisms and higher plaque values in high risk group may have resulted into shortest Oratest time. Thus, in context to the observations of the present study and of earlier studies it is supported that Oratest can be used as one of the tool to estimate caries activity and oral microbial level. Oratest is proven to be a simple, inexpensive and a rapid technique for assessment of caries activity according to present study. We believe that this method will be useful, both at the individual level and community level, in an attempt to identify groups of persons with an increased risk to develop caries. In the earlier study carried out by Cardash .,[] patients were supplied with denture hygiene kits consisting of plastic test tubes containing 0.12 ml of methylene blue and plastic pipettes. If such oral hygiene kits made available to children, then it might be possible for parents to monitor oral hygiene. Performance of the test prior to the treatment session not only makes the waiting period interesting for the child, but also test results can provide the Dentist with something to comment upon either to reinforce motivation and plaque control or to reinforce a positive behavior.

Hepatitis B infection (HBI) is one of the major public health problems globally and is the 10 leading cause of death. Worldwide, more than two billion of the population have evidence of past or recent HBV infection and there are more than 350 million chronic carriers of this infection.[] The number of HBsAg carriers in India has been estimated to be over 40 million. Estimates indicate that annually over 100,000 Indians die due to illness related with HBV infection.[] HBV is a 42 nm DNA virus assigned to the family Hepadnaviridae. The inner core of the virus contains hepatitis B core antigen (HBcAg) ; hepatitis B e antigen (HBeAg); a partially double-stranded 3,200-nucleotide DNA molecule, and DNA polymerase with reverse transcriptase activity surrounded by an outer lipoprotein envelope containing the surface antigen (HBsAg).[] Humans are the only known natural host and the virus circulates in the blood in concentrations as high as 108 virions per ml. Transmission of HBV occurs through percutaneous or permucosal exposure to infective body fluids. In addition to sexual contact and drug injection, nosocomial transmission is also a possibility.[] Blood and blood products are the most common vehicle of transmission in healthcare settings.[] With the increasing number of invasive diagnostic and therapeutic procedures, there is an increasing risk of HBV infection to the auxiliary healthcare workers (AHCWs).[] The AHCWs constantly come in contact with blood and its products due to daily handling of biomedical wastes and while performing invasive procedures. Hence, it is necessary for them to be aware of HBI and its prevention. In the present study, we have aimed at investigating the HBV infection related awareness and occupational risk perception of the AHCWs. r o s s - s e c t i o n a l s u r v e y w a s c a r r i e d o u t i n M . S . R a m a i a h M e d i c a l a n d D e n t a l H o s p i t a l s a m o n g 3 0 0 A H C W s w h i c h c o m p r i s e d o f l a b o r a t o r y t e c h n i c i a n s , h y g i e n i s t s , l a u n d r y w o r k e r s a n d t h e h o u s e k e e p i n g s t a f f . A f t e r e t h i c a l c l e a r a n c e a n d w r i t t e n c o n s e n t , t h e y w e r e c o u n s e l e d a n d e x p l a i n e d a b o u t t h e o b j e c t i v e o f t h e s t u d y a n d w e r e r e q u e s t e d t o a n s w e r a s t a n d a r d q u e s t i o n n a i r e w i t h m u l t i p l e c h o i c e s . T h e d e v i s e d q u e s t i o n n a i r e c o m p r i s e d o f 2 0 q u e s t i o n s a n d w a s p r e p a r e d i n t w o l a n g u a g e s , o n e b e i n g E n g l i s h a n d t h e o t h e r i n t h e l o c a l l a n g u a g e , t h a t i s , K a n n a d a . T h e d a t a w a s c o m p i l e d a n d s u b j e c t e d t o s t a t i s t i c a l a n a l y s i s . T h e d a t a w a s a n a l y z e d t h r o u g h S t a t i s t i c a l P a c k a g e f o r S o c i a l S c i e n c e s ( S P S S ) p a c k a g e ( I B M S B S S S t a t i s t i c s - S t a t i s t i c a l P a c k a g e f o r t h e S o c i a l S c i e n c e s ) . The various groups of AHCWs have been depicted in and the overall responses have been depicted in . It was encouraging to find that 96.2% of the 300 AHCWs were vaccinated against HBV. Overall, an adequate HBV infection related awareness was found among the group. 96.9% of the respondents knew that the main mode of transmission of Hepatitis B was through blood and its products. More than three quarters (76.3%) of the AHCWs knew that HBI causes liver cancer. Majority of the respondents (93.9%) were aware that HBI could spread through contaminated needles and syringes. A moderate occupational risk perception was found among the group. Astonishingly, only 40.5% of the AHCWs knew the correct course of action after a needle stick injury. Just about 38.9% of the respondents knew that blood soaked cotton and dressings are discarded in yellow colored bags and 52.7% answered correctly that sharps and needles are disposed in white colored bags. 80.2% of the AHCWs used sharps container to dispose used needles and lancets. The questions on the awareness and occupational risk perception of HBV infection and the percentage of correct responses are enlisted in Tables and , respectively. AHCWs are at a greater risk at contracting blood borne diseases due to their constant contact with blood, body fluids, or sharps contaminated with blood. In the present study we found that there was adequate awareness among the group, but risk perception was found to be moderate. Information regarding biomedical waste management was particularly found to be inadequate and this is a matter of great concern. Suggestions for remedial steps to be taken to prevent nosocomial HBV infection in AHCWs are elaborated in . Hepatitis B vaccination protocol and needle stick injury prophylaxis are collated in Tables and , respectively[] . enlists the hospital waste management protocol.[] Waste not properly managed can lead to serious health implications; therefore, proper waste disposal is mandatory and is protective against blood borne diseases. In a similar study conducted by Singhal ., in 2011 they found that a significant number (41.7%) of HCWs were unvaccinated even at an apex healthcare center. Two HCWs were found to be HBsAg positive. The antibody levels were significantly lower in those who were vaccinated more than 5 years ago than those who were vaccinated in last 5 years.[] In the present study however, we found that 96.2% of AHCWs were vaccinated. Another survey conducted by Carvalho in 2012 in Brazil revealed a seropositivity of 0.5% in students and 8.8% in professionals. They concluded that seroprevalence for previous contact with HBV was 17.6 times higher in professionals than in students.[] Khakhkar Vipul ., in 2012 screened serum samples for presence of HBsAg, HBeAg, and anti-HBc with the help of enzyme-linked immunosorbent assay (ELISA). They inferred that the highest proportion of HBsAg positivity was found among laboratory technicians (4.1%) followed by nurses (1.7%). The distribution of the HBsAg was not associated with age and gender. However, the positive rates of HBsAg were the highest for the HCWs with more than 30 years in job, with overall positivity of 2.4% (1/41) suggesting a greater exposure to blood and other putative risk factors.[] In another survey done by Muhammad S Memon ., in 2007, a total of 596 (64.6%) participants were immunized for HBV infection and 392 (66.2%) were inoculated three or more than three doses of vaccine. The pre-vaccination HBsAg status was checked in 380 (41.2%) and it was positive in 18 (4.7%) participants. The frequency of immunization was highest in doctors (92.4%) and lowest in nursing assistants (18.9%).[] A cross-sectional questionnaire based study done by Habib ., among HCWs in 2011 revealed that overall knowledge were inadequate and behavior and attitude towards clinical practices were found compromised. Sixty-five percent believed that all HCWs are at risk and 89% believed that vaccination provides protection.[] In the present study overall awareness was found to be adequate with 90.03% of AHCWs well-informed about HBI. On the other hand, risk perception was found to be inadequate with only 67.2% of AHCWs being well-informed on biomedical waste management. In a survey conducted by Dannetun ., in Sweden in 2006, they found that 79% (293/369) of HCWs had received at least one dose of vaccine, but only 40% (147/369) reported that they were fully vaccinated and 21% (76/369) had not been vaccinated at all. The majority of unvaccinated HCWs (72/76, 95%) stated that they would accept vaccination if offered. They concluded that the main barrier to better compliance with the guidelines is not lack of acceptance among the employees, but the failure of the employer to ensure that policies are implemented.[] AHCWs' safety is often overlooked, but these AHCWs comprise an invaluable part of our medical fraternity. It is our duty to ensure their safety. Awareness, precaution, and protection should be advocated in order to prevent the nosocomial spread of HBI. Therefore, there is a need for well-planned and clear policies for HBV screening, vaccination, and serological response checkups for all HCWs. Also hospital waste management is an important aspect in preventing the HBI which should not be overlooked. The AHCWs should be trained and periodically monitored for effective hospital waste disposal. Health education campaigns and training programs should be regularly organized for health workers on hospital infection control. Staff safety is a challenge that must be met with a comprehensive effort. A reduction in health disparities can be brought about by a joint effort which should aim at the following: Staff safety is a challenge that must be met with a comprehensive effort and they should be made to understand the risks they are exposed to. We need to work towards building a healthcare workforce prepared to prevent and diagnose viral hepatitis and provide care and treatment to infected persons.

Hospital waste is often described as any residual matter, solid or liquid that is generated in the diagnosis, treatment or immunization of human beings or animals.[] According to World Health Organization estimates 85% of hospital waste is actually non-hazardous and around 10% is infectious while the remaining 5% is non-infectious, but consists of hazardous chemicals such as methylchloride and formaldehyde.[] The main concern of infectious hospital waste is the transmission of human immunodeficiency virus and hepatitis B or C viruses. In this context, syringes and needles have the highest disease transmission potential. Dental waste is included in hospital waste.[] It is of two types, liquid waste and solid waste.[] They are further classified into two main groups: Non-risk waste and risk waste. Risk waste is infectious waste and hazardous waste.[] Infectious waste contains a great variety of pathogenic microorganisms. Hazardous waste contains metals that are toxic and never degrade once they reach the environment. It consists of silver, lead, mercury, X-rays and cleaning solutions.[] Amalgam is an acute neurotoxin; it's the most toxic non-radioactive element and also the most volatile heavy metal. Mercury can pose a threat due to release of mercury into the environment from dental practices and industries due to poor disposal. Other materials may contain potential hazards such as polystyrenes, barium, strontium, which may cause harm if correct use and disposal is not instilled.[] Improper disposal of dental waste can cause harm to the Dentist, to the people in the immediate vicinity of the Dentist who handle the materials, to the waste handlers or the general public at large through production of toxins through incineration.[] Today, with the increase in demand for dental care, there has been a rapid growth of the dental clinic in recent years, which in turn led to an increase in the amount of biomedical waste generated in the clinics.[] Hence, the aim of the present study is to assess the awareness and practices regarding disposal of various types of wastes in dental clinics. A cross-sectional study was carried out among the private dental practitioners of Tricity (Chandigarh, Panchkula and Mohali). A total of about 396 dental practitioners were registered with the different Dental Councils in Tricity, out of which 100 private dental practitioners were selected for the study by simple random sampling. The study was carried out for a period of 3 months from November 2012 to January 2013. The questionnaire included different sets of questions regarding procedure used for disposal of waste in the clinics. A pilot study was carried out on a small group of Dentists after which the questionnaire was finalized. Each Dentist was given a copy of the questionnaire personally and was requested to answer it before being collected back from them on the same day or next day. The information was gathered through self-administered questionnaire, which included questions about the awareness and practice of waste disposal in dental clinics. The data was analyzed using SPSS version 13.0. (SPSS Inc., Chicago, USA) Out of the 100 dental practitioners, 52% were males and 48% were females. 68.6% had completed post-graduate education and the rest 31.4% were undergraduates. Distribution of practitioners based on the number of years of practice had been described in . Data regarding the awareness of dental care waste had been described in . Nearly 14% of dental practitioners were not aware of the different categories of the waste generated in their clinics. Only 24% of the Dentists were aware that expired or outdated medicines come under cytotoxic drugs. With regard to the question about the category of impression material and cotton only 16% said correctly that it falls under the category of soiled waste. 12% of practitioners were not aware of the color coding used to dispose the waste. Nearly, 72% of the Dentists were aware of yellow color coded container to be used for disposal of human anatomical waste. When asked about the color coding for disposing sharps, 58% were aware of it. Dentist's response toward practices of dental care waste management had been described in . 76% of the Dentists segregate different waste according to the laws of biomedical waste management. 66% Dentists used needle burner to destroy needles and 14% of them disposed the needles in common bin after use. 52% of the Dentists disposed developer and fixer by first diluting it. 14% of them reused it after adding some new mixture to it. 12% of them disposed it directly to the wash basin. 42% of the dental practitioners stored lead foils and X-ray films separately in red/blue color coding containers and then disposed it safely while 22% of them disposed it directly into the common bin. Out dated or expired medicines were disposed in common bin by 32% of the Dentists. 22% of them store it in black containers and then disposed it. 62% of the practitioners stored extracted teeth in yellow containers and then disposed them safely. About 44% of the Dentists disposed silver amalgam in common bin. 14% of them stored it in air tight container with water. 10% among them stored it in a fixer solution. 22% among them did not know the method of disposal of silver amalgam. 6% among the Dentist didn't use silver amalgam in their clinics. 40% of the Dentists disposed orthodontic wires and brackets by first deforming and then disposing it in blue color coded container. 28% of the dental practitioners did not know the method of disposal of orthodontic wires and brackets. This study was undertaken to assess the awareness and practices of dental care waste management among dental practitioners in Tricity (Chandigarh, Panchkula and Mohali). This study provided an important insight into the proper method of disposal of waste by private practitioners. In the present study, 14% of the dental practitioners were not aware of the different categories of the waste generated in the clinics which was quite similar to the study conducted by Sudhir[] in which 11.1% of the practitioners were not aware of the different categories. 12% of the practitioners were not aware of the color coding used to dispose the waste which was found to be low as compared with the study done by Sudhir.[] Moreover, 24% of the Dentist's did not segregate different waste according to the laws of biomedical waste management, which was found to be quite less as compared with the study done by Sudhakar and Chandrashekar.[] This study showed that a substantial percentage of practitioners dispose dental waste without segregation, which exposes garbage collectors to a high risk of getting infected from health-care waste. 14% of them disposed the needles in common bin after use. In this study done by Treasure and Treasure[] 24.4% disposed the needles in common bin. This showed that the knowledge regarding proper disposal of sharps was very less and knowledge regarding the same should be given. In the present study, 34% of the practitioners disposed lead foils and X-ray films by storing it separately and then disposing it in black coded containers, which was in contrast to study conducted by Sudhir.[] 22% of them disposed it directly into the common bin, which is not considered a safe method for disposal as it can affect neurological development and function as discussed by Hedge ..[] In our study, 32% of them disposed outdated or expired medicines in common bin, 22% of them stored it separately to be disposed safely. 24% of the practitioners were aware that outdated or expired medicines comes under the category of cytotoxic drugs, but surprisingly only 18% of the Dentists used the correct method of disposal that is to be disposed it in secured landfills. All the waste biomedical waste, which is to be disposed in secured landfills or being shredded was stored separately in specific coded containers and then disposed. The disposal process is being carried out by the biomedical waste experts accordingly and nor by the Dentist themselves. 26% of the practitioners disposed extracted teeth in common bin, which is considered to be in factious material by Occupational Safety and. Health Administration and should be disposed in proper color coded containers. However, 62% of the Dentists used proper color coded containers that is yellow containers to dispose extracted teeth. In our study, it was found that about 44% of the Dentists disposed excess silver amalgam in common bin which was found to be more in contrast with the study carried out by Sudhakar and Chandrashekar[] in which the 35.2% of the Dentists disposed excess silver amalgam in common bin. Orthodontic wires being sharp waste should be disposed according to the biomedical laws. In our study 12% of them disposed directly in the common bin and 40% of them first deformed it and then disposed it which is not considered to be a correct method. Orthodontic wires should always be disposed in blue color coded bags. The outcome of our study focused a definite need to enforce more strict laws and measures for disposal in India, so that it becomes mandatory for all private practitioners to register their clinics under bio medical waste management services. a r g e n u m b e r o f p r a c t i t i o n e r s w e r e a w a r e o f d i f f e r e n t c a t e g o r i e s a n d c o l o r c o d i n g o f d i f f e r e n t t y p e s o f w a s t e y e t h a v e f a i l e d t o p r a c t i c e t h e s a m e i n t h e i r c l i n i c s . T h u s , t h e r e i s a n u r g e n t n e e d f o r c o n t i n u i n g d e n t a l e d u c a t i o n o n d e n t a l w a s t e m a n a g e m e n t p r a c t i c e s t o t h e s e d e n t a l p r a c t i t i o n e r s .

xref sup italic #text xref sup #text presents the general profile of the study population. The age group of 35-44 years contributed for more than one-third of the sample size, whereas there were fewer subjects who belonged to the youngest age group (18-24 years). The overall prevalence of periodontal disease was 98.9%. A CPI score of 2 (bleeding and calculus) was widespread among the study population, whereas deep periodontal pockets were presented by 14.78% of the subjects. None of the subjects was obese, whereas almost two-thirds of the subjects (65.8%) belonged to the normal weight group. Mean subject characteristics with periodontal status are illustrated in . There was a significant difference for age between the groups, with the mean age of the periodontitis group being approximately 4 years more than that of the control group. An almost similar trend was observed for BMI, with subjects belonging to the periodontitis group presenting a higher BMI. Logistic regression analysis revealed that subjects had an increased risk of periodontitis by 56% for each 1 kg/m increase in BMI (adjusted odds ratio: 1.56; 95% confidence interval: 1.26-1.92). Moreover, though the risk of periodontitis increased with increase in age, its influence was not significant []. The literature shows that the prevalence of periodontal disease is greater among obese people, and studies have proposed that an increased BMI may be a potential risk factor for periodontitis. Previous studies[] have included either young or old subjects, and data from those studies on both the young and adult individuals had suggested that periodontal status deteriorates with BMI. The present study included a wide range of age groups, with the youngest individuals being 18 years of age, that is, minimum age of recruitment and the oldest being 58 years of age, that is, age of superannuation. The overall prevalence of periodontal disease was 98.2%, whereas this prevalence is 89.6% among the general population of India belonging to the 35-44 years age group.[] The higher prevalence of periodontal disease among the present study population can be attributed to a multitude of reasons like poor oral hygiene practices and not availing the present dental health-care facilities available. However, the widespread prevalence of score 2, namely, bleeding and calculus is in accordance with previous studies.[] None of the subjects was obese, whereas almost two-thirds of the subjects (65.8%) belonged to the 18.5-24.9 BMI group. It was observed that the subjects with higher BMI and age had an increased risk for periodontal disease. The influence of age on periodontal disease is consistent with previous literature. Miyazaki .,[] used the CPI to assess the periodontal profiles of adults and found that periodontal disease increased with increase in age. The present study population had an increased risk of periodontitis by 57% for each 1 kg/m increase in BMI, whereas the risk of periodontitis increased by 16% among young Japanese adults aged 18-24 years.[] This difference in risk might be due to difference in age composition, geographical status, oral hygiene habits, and dietary practices. Hypponen .,[] and Salekzamani .,[] in their studies, revealed that BMI increases with age. Al-Zahrani .,[] assessed the association of BMI and periodontal disease among adults aged 18-34 years and observed that the prevalence of periodontitis was 76% higher among obese individuals. Furthermore, Reeves .,[] studied the association of body weight and waist size with chronic periodontitis among individuals aged 17-21 years and reported that weight significantly influenced the periodontal status. However, the present study is not devoid of limitations, as the periodontal status was assessed using the CPI, which does not include all the teeth and does not measure attachment loss. i g h e r B M I c o u l d b e a p o t e n t i a l r i s k f a c t o r f o r p e r i o d o n t i t i s a m o n g a d u l t s a g e d 1 8 t o 2 4 y e a r s . T h u s , t h e e v a l u a t i o n o f B M I c o u l d b e u s e d i n p e r i o d o n t a l r i s k a s s e s s m e n t . L o n g i t u d i n a l s t u d i e s w i t h a l a r g e r s a m p l e s i z e a r e r e q u i r e d t o c o n f i r m t h e a s s o c i a t i o n o f B M I a n d p e r i o d o n t a l d i s e a s e .

The society has collective consciousness. The beliefs, practices and attitudes can be studied in a same way as can be an individual mind with appropriate methods. Absence of scientifically supported background knowledge of a society can severely hinder any attempt of change, whether good or bad in a society. This makes a complete understanding of social factors and fears an absolute necessity before any change can be brought about by active intervention. Our society in its attitude toward dental health has been giving it less importance as compared to general health. There has been a lack of public identification of oral health deterioration and wide acceptance of morbid mouths along with widespread prevalence of oral diseases and lack of reasonable oral health-care services in the past. Dental public health programmers have not been able to achieve the depth and penetration into society required to bring about the change in societal attitude. In this article, observations based on experience and interactions with the society have been enumerated. Need for a scientific study of such observations has been stressed as only with a good understanding of society mind, a change in its individual unit can be brought about. Peoples attitude is shaped by their beliefs and the common belief has been that dental treatment is unbearably painful, this has led to people ignoring their oral health to an extent that when they eventually come to the dental clinic some of their teeth are invariably advised for extraction, which again as painful event reinforced this belief and add to the general household reputation of dental treatment as painful which usually reason much for those who shy away from oral health both in rural and urban areas. This perception needs modification before any intervention. Inclusion of video-aids or demonstration on a subject would indeed help modify this attitude although a very successful accomplishment on a large scale can eliminate this perception over a time period. Another factor is nearly traditional addiction of tobacco in Indian masses.[] Tobacco consumption, although harmful for systemic health, has many other ramifications in oral health. Firstly, tobacco and tobacco products directly harm the oral tissue; secondly, oral cavity is reduced secondarily to the pleasure of consumption and largely ignored. This unhealthy attitude gradually builds up to extent when the consumer totally ignores his oral health. Thirdly, it becomes a habit, a habit both powerful and prevalent. This has been passing from generation to generation. This variable of tobacco consumption is seen at every level, form planning to execution of any intervention. Awareness and education are the only tools for masses at present. Although an in-depth understanding of tobacco use and related behavioral factors would help in modification of the health-care program in advance. Another factor is forgotten general awareness about the oral health and its contribution to overall health and longevity. This has largely been made possible by continual non-availability of oral health services in their proximity and lack of elementary education in such matters.[] Contribution of oral health to systemic health is undeniable. This has to be reinforced into common mind by physicians and dentists. A mandatory oral check-up facility for a person that appears for a systemic check-up and professional consultation among health-care team that include dentists for management of systemic and metabolic disorders would in the long run convince common man of this importance of oral health. This can thus be concluded that to deal with the oral problems in societal framework would involve a multi-pronged approach which would take into account not only the present knowledge/perception in masses, but to build upon such knowledge in a constructive way to develop a healthy attitude and acceptance of oral health in our culture and society. The scenario in which a patient reaches the clinic or nursing care in India is that of as a last option and not as a first reaction. This is mostly due to the expensive care as by the fee charges and expensive medicine which usually surpass the paying capacity of the patient. Patient shy away from reaching at once for fear of money loss in pursuit and most of the time they wait for themselves to heal on their own. When they clearly know that the disease is not going to heal on its own by other “Desi Nuskas” only then they find out a clinic in their closest proximity and head to it with a dull face. This delay in proper care results in added morbidity to the patient which in turn leads to added costs and which again continues the vicious cycle of the perception that the treatment is expensive. This attitude is more pronounced with dental health related behavior. “Desi Nuskas”: In an effort to avoid going to a dentist, the patient first try to solve his/her problem using local belief about the disease and its cure. He would naturally turn to herbs other local products in search of relief. A small fraction of patients might eventually find some relief in short-term like the use of alum mouth rinses in bleeding mouth has been known to reduce bleeding symptoms. Only after comprehensive dissatisfaction form these natural or more of these local remedies do a patient reach a clinic in his or her proximity. There is no denying that a useful professional opinion is deferred unless the application of local wisdom at least once. This has to be countered with the actual scientific proof of their non-effectiveness. The proof-of-effectiveness or non-effectiveness is lacking. To be able to fully convince their subjects health planners should first assess the local methods used be people and provide proof of their ineffectiveness wherever possible. It is not possible for people to discontinue something which is not even denied by a professional. Even after getting a professional opinion of the disease and formulation of standard treatment plan most patient ask for medicines and intend to get away with the problem after eating medicines, which is not usually the case with dental treatment. As dental treatment essentially involve some work either in the form of scaling or cavity preparation. The treatment even asks for patient time which is usually described as the period of stress in his worlds. This add to our knowledge that longer treatment time, increased patient clinic time and multiple appointment to the clinic apart from expense form the actual treatment are other factors, which make people shy away from dental care. Another factor is the lack of identification of disease in initial stages, which also contributes to neglect and add to disease burden and morbidity. Masses do not know when they should head towards a clinic for a check-up. Oral cancers which are the most prevalent in country eventually are the one which when detected in earlier stages are curable.[] However, this has not been possible due to lack of understanding and knowing of initial symptoms. This suggests area to work for health education providers. In short, this has be to put to Indian masses consciousness that when is time to seek dental-care and that too with the right reasons. The dental-care should generate immense stimulus in form of satisfaction so as to reinforce good habit to seek oral care. However, this stimulus is rarely provided as the quality of service offered is often poor and more so in rural areas. A good dental clinic is largely beyond the reach of the masses.[] The other reason being prevalence of unqualified practitioners prevailing in both rural and urban areas, which provide quick and inexpensive harmful substitutes and people usually move to them.[] Quacks usually enjoy a wide social acceptance and a brisk practice and usually offer dental extraction to most of their patients sometimes as primary and mostly as ultimate relief. In fact, the quacks those who have been practicing in rural and far-off areas should be educated of fundamentals and reference as a separate program for them to have a mass effect. Moreover, any attitude in society cannot be modified effectively if the quacks believe otherwise. The esthetic treatment demand is even much less and that reflects the ultimate appreciation of teeth as components of esthetics in society. Even if the society is keen and understands orthodontic treatment is usually even more expensive. Even people in urban areas tend to avoid orthodontic treatment. People keep mistaken beliefs about dental treatment which makes one wonder that in such a scenario where there are few actually interested and educated in oral health, people mostly have knowledge of many strange misconceptions about treatment. Fear of loss of eyesight is just one for instance. Another example is that oral cleaning (scaling and root planing) can make their teeth mobile and is harmful to teeth. This alerts a periodontist to tread his path in society with greater care as he can find a hostile reaction in society on the contrary of a welcoming one. It is difficult to get good role models of oral health in society as most locals and even popular local personalities are models of tobacco consumption and unkempt oral health. In the absence of a role model children have no one to learn from except morbid mouths. It is therefore necessary for the health-care provider especially in rural setting to select examples for rural setting and incorporate them for education purpose whenever possible. Indian system of medicines is thousands of years old and is still widely accepted and practiced.[] However, as of its nature it was never meant to be suitable for caries excavation. Western system of medicines took roots in the west and evolved slowly over few centuries, whereas no such evolution has ever been present in Indian society. Indian society mind does not have the bridge of knowledge that can link the past system of Indian medicine to the western advanced one which is only known to those which are imparted this western based dental education in dental schools. The result is that the society as a whole is ignorant of oral health and some might possess a PhD. This lack of bridge or elementary absence of knowledge is mainly responsible for this attitude. Observations listed above [] are not based on any survey with data obtained as a result of a questionnaire. These observations are made by direct experience based on practice and living in the Indian society. These few observations along with other yet unexplored societal differences, beliefs and other challenges along the length and breadth of the country call for an urgent need for scientific understanding of present scenario only then useful strategies to make dentistry more acceptable and larger active community participation can be expected. e r e a r e m a n y y e t u n e x p l o r e d c o n v i c t i o n s a n d b e l i e f s i n I n d i a n s u b c o n s c i o u s , w h i c h n e e d t o b e w o r k e d i n a m u l t i p r o n g e d s t r a t e g y . E v e n t h o u g h , I n d i a i s p r o g r e s s i n g a s n e v e r b e f o r e i n o r a l h e a l t h , a k e e n u n d e r s t a n d i n g o f s o c i e t a l a s p e c t s c a n c e r t a i n l y w o u l d b e h e l p f u l f o r b o t h o r a l h e a l t h p l a n n e r s a n d i m p l e m e n t e r s e s p e c i a l l y i n r u r a l a r e a s . A c h a n g e i n s o c i e t y o u t l o o k a n d a t t i t u d e i s s l o w , w h i c h r e q u i r e s p e r s i s t e n t e f f o r t s a n d c o n t i n u o u s e d u c a t i o n a n d a c t i v e p a r t i c i p a t i o n o f s o c i e t y i n i t s o w n o r a l h e a l t h i s o f p a r a m o u n t i m p o r t a n c e . I n b r i e f a c a r e f u l p l a n n i n g b a s e d o n e d u c a t i o n o f o r a l h e a l t h t h a t n o t o n l y i n v o l v e s o r a l h e a l t h e d u c a t o r b u t s o c i e t y i n i t s n o b l e c a u s e r e m o v i n g m i s c o n c e p t i o n s a n d p r o v i d i n g e f f e c t i v e a n d c o s t - e f f e c t i v e t r e a t m e n t i n c l o s e p r o x i m i t y i s a n e e d o f a n h o u r i n I n d i a n s o c i e t y

Dental caries has been described as a disease involving the localized destruction of the tooth tissue by microorganisms. It is a chronic disease process that usually progresses slowly and infrequently is self-limiting. Dental caries can affect enamel, dentine, and cementum. It manifests clinically along a continuum from initial loss of mineral to the complete tooth destruction.[] Over the last few decades, the pattern of dental caries has undergone a profound change, and consequently, there has been a large number of initial lesions (IL), a reduction in cavitated lesion and the predominance of activity on the occlusal surfaces. These modifications in the pattern of the dental caries are very relevant, not only for clinicians, but also for epidemiologists and oral health service planner.[] Not all non cavitated lesions progress to become dentinal lesions requiring restorative treatment; and, a good proportion of them remain static or even rematerialize, especially the smooth surface lesions. These lesions are thus reversible, as opposed to a dentinal lesion, which is generally considered irreversible. Because there are usually more non cavitated than cavitated lesions, at any one time in both high-caries and low -caries population,[] the decision as to whether to include or exclude them, can make a substantial difference in the oral health profile obtained. Scientific literature has discussed epidemiological studies which use diagnostic criteria that consider caries as a cavitated lesion, e.g. the World Health Organization (WHO) diagnostic criteria. The point is that these studies have underestimated the dental caries in populations/groups, since initial lesion have been more prevalent than cavitated lesions. Dental health programs that focus only on the treatment of cavitated lesions are not enough to re-establish health in an individual/ population because they do not consider the different stages of carious lesion progression.[] Surveys that include IL could be very relevant to show distinct preventive and operative needs. The present study aims to investigate the influence of different diagnostic thresholds and different settings (epidemiological and clinical) on caries detection in the primary and permanent teeth of 7-15 year old school children of Bangalore city, India. e s t u d y w a s a p p r o v e d b y t h e E t h i c a l C o m m i t t e e i n R e s e a r c h a t M . S . R a m a i a h D e n t a l C o l l e g e , B a n g a l o r e ( I n d i a ) . T h e s c h o o l s g r a n t e d p e r m i s s i o n f o r t h e s t u d y a n d i n f o r m e d c o n s e n t w a s o b t a i n e d f r o m t h e p a r e n t s . italic #text Surveys are used to monitor the trends in oral health and disease, to develop policy, to evaluate dental health programmes, and to assess the dental needs.[] However, when the epidemiological data are compared with those obtained in the standard clinical setting, epidemiological surveys underestimate the prevalence of the disease.[] Furthermore, authors such as Kassawara .[] Assaf .[] Lindwood .[] have justified that the epidemiological evaluation of dental caries is a poor indicator for determining the number of surfaces that will subsequently be treated[] and has no discriminatory power in the prediction of an individual's future restorative treatment.[] Difference in the examination methods of both the settings (epidemiological and clinical setting) may be a relevant factor in the accurate estimation of the disease magnitude in the surveys, for instance; artificial light, compressed air, radiographs and other diagnostic aids (Fibre optic transillumination - FOTI) are frequently used by dentists in clinical setting, while epidemiologists usually use only clinical examinations under conditions very different from those found in a clinical setting.[] In addition to these factors, the criteria employed in most of the cross- sectional surveys consider dental caries only at the point of cavitation, excluding the initial lesion, thus resulting in an underestimation of the disease magnitude which interferes with further planning. Many researchers have stressed on the need to introduce modifications in the diagnostic criteria for dental caries, mainly under epidemiological conditions. Because of the significant changes in the manifestation of the dental caries in the last few decades, such as a reduction in the prevalence of caries and decrease in the progressive speed of the lesion[] and remineralisation of initial lesions, recent epidemiological research has shown that initial lesions have become more prevalent than the cavitated dentin lesions[] Therefore, the real condition of the disease in the population has been underestimated, and this can consequently generate inadequacies in the data for implementing e the therapeutic non-invasive measures to control the progression of the disease.[] Thus, the objectives of the present study were; i) to compare the different caries diagnostic thresholds under epidemiological setting. ii) and, to compare these diagnostic thresholds under the epidemiological and clinical settings. The present study showed that both the settings (clinical and epidemiological) and diagnostic thresholds (WHO and WHO + IL) could influence on the detection of the carious lesions in 6 - 15 years old children. The inclusion of IL in the epidemiological examinations could be an important and relevant factor in the accurate estimation of disease magnitude. Mean Ds under WHO + IL criteria (3.92 ± 3.49) was nearly double of the WHO criteria (1.88 ± 2.73) [], and the influence observed was more in the age group of 14 year olds, where mean Ds under WHO + IL criteria (5.43 ± 2.5) was more than double of the WHO criteria alone (2.02 ± 1.9). Authors such as Nuttall .,[] Meneghim .,[] Andrea,[] Ismil[] have shown the need and justification for including IL in the epidemiological surveys. The reason is that there is a higher prevalence of IL compared to cavitated lesions, particularly in the low caries prevalence areas. Therefore, its inclusion would contribute to a decrease in the level of underestimation of the disease magnitude, and would provide a better classification of the dental caries levels in the population.[] Some other researchers demonstrated[] that the pattern of dental caries has been undergoing profound modifications in the industrialized countries over the last few decades, showing drastic decrease in prevalence and incidence of the disease, and consequently, increase in the number of children who are free of dental caries.[] Most of this epidemiological research still uses the criteria of dental caries as cavitated lesions. In the present study, examination was also performed in a clinical setting, with variation in the use of the light source and compressed air. In the epidemiological setting, natural light and chip blower were used, whereas artificial light and compressed air were used in the clinical setting. It was observed that when both the settings were compared using same criteria (WHO + IL), clinical examination showed almost 40% more carious lesion than epidemiological examination.[] This influence of the light source is well evaluated in the previous studies.[] White spot lesions, loss of discontinuity of the enamel surface, and determination of the depth of penetration can be well appreciated in the artificial light, and therefore helps in the diagnosis of the initial[] as well as cavitated lesions.[] In epidemiological examinations, this could be one more reason for the underestimation of IL.[] One of the previous studies has shown that the use of diagnostic adjuncts, such as prior tooth brushing and drying were more important than the employment of the Community Periodontal Index (CPI) dental probe, mirror or blade to diagnose non cavitated (NC) carious lesions in the enamel, mainly for the low caries prevalence group.[] However, even with the employment of the diagnostic adjuncts of dental drying and brushing, the results of these studies showed that none of the combinations for the epidemiological examinations approached the diagnosis obtained in the dental setting in relation to the IL diagnosis for any of the two prevalence groups.[] Such information, once again, confirms the difficulties in examining the dental caries and underestimation in epidemiological examinations. Epidemiological surveys are very important for obtaining the data of the disease magnitude in a population. Such data is frequently used in health care planning, monitor service delivery and track disease trends,[] to plan preventive programs for the school and community and to assess the effectiveness of heath programs at school and community levels. In spite of this, even with such solid justifications for including IL in epidemiological surveys, continued research is needed so that future epidemiological information may capture the carious conditions more accurately. Thus, such changes can aid in deciding how funds should be directed to adequately meet the needs of the individuals and groups in question.[] Furthermore, this would guide the public health service planning processes, and lead to improvements in the diagnosis and preventive-therapeutic treatments in the dental health programmes. Some of the studies have evaluated the conventional clinical examination by using a method of validation, such as the determination of the depth of the lesion by minimal operative intervention. Such validation methods are currently not used in clinical research, although, this is a ‘gold standard’ for detecting caries. In the present study, this was not possible due to the practical and ethical issues, such as the ethical problem of opening a lesion. Some epidemiologists consider that one of the major problems lies in the difficulty of diagnosing dental caries in epidemiological surveys because of the examination conditions, the resources employed, the inherent difficulties in diagnosing initial lesions, the time spent on the evaluation, as well as the high cost of diagnostic adjuncts.[] At the present time, it is necessary to make the changes in the caries diagnostic threshold, and in the examination conditions (good quality light such as FOTI, additional diagnostic adjuncts like brushing and drying during examinations), for the development of epidemiological survey techniques and to evaluate the accurate magnitude of the disease in different groups. In addition, further studies should be directed to analyze the feasibility of adopting this new measure. e r a l l , i t w a s f o u n d t h a t b o t h t h e s e t t i n g s ( c l i n i c a l a n d e p i d e m i o l o g i c a l ) a n d d i a g n o s t i c t h r e s h o l d s ( W H O a n d W H O + I L ) , c o u l d i n f l u e n c e t h e d e t e c t i o n o f c a r i o u s l e s i o n s i n t h e 7 - 1 5 y e a r o l d c h i l d r e n .

Agenesis of one or more teeth in primary or permanent dentition is known as hypodontia, while, hyperdontia is a condition that of having extra to the normal complement of teeth. Hypodontia and hyperdontia are two extremes in the development of the dentitions. The occurrence of both these identities in the same individual is a form of combined numeric variation. Although, the existing literature shows exclusively either hyperdontia or hypodontia, however, only a few studies have been accounted the incidence of both the anomalies.[] The term “concomitant hypo-hyperdontia” (CHH) has been used to describe the occurrence in the same individual.[] The aetiology of CHH remains unclear and it is not evidently documented in the literature. While, environmental and genetic factors have been anticipated to explain the occurrence of these anomalies. Furthermore, CHH has been reported in patients with Down syndrome, Dubowitz syndrome, Ellis-van Creveld syndrome, fucosidosis, G/BBB syndrome, Marfan syndrome and cleft lip and palate.[] The reported prevalence was between 0.002% and 3.1%, most of these were of CHH involved both the arches.[] Most of these cases were identified accidentally and/or regular examination. Most recently, it has been reported by several authors the combination of both hypodontia and hyperdontia in both arches. The occurrence of CHH in the same dental arch in a healthy patient is most likely to be a rare phenomenon and all existing reports are reported in the maxilla.[] The occurrence of CHH in the mandibular arch is extremely rare, however, most recently few cases has been reported on this identity involving congenitally missing both central incisors. The purpose of this article is to review the literature and to describe three rare identities of CHH in the anterior region of the mandibular arch. xref #text Gibson[] divided hypo-hyperdontia as pre-maxillary, maxillary, mandibular and bi-maxillary hypo-hyperdontia based on the site of occurrence. The three cases described in this article are good examples of mandibular CHH excluding the third molars in both the arches. Garn .[] have discussed the association of third molar agenesis with missing teeth from other classes of teeth. The authors concluded that the association among the reduction in the number of other teeth and third molars hysterics the hypothesis of a field of variable intensity, which, in its greatest degree of expression, eliminates all four third molar teeth. The distribution of missing third molars in the present report was teeth 18 and 28 (Case 1), 18, 28 and 48 (Case 2) and tooth 18 (Case 3) accordingly, if, third molars were taken in to account these cases were considered as bi-maxillary CHH. Several hypothesis, including atavism, dichotomy, hyperactivity of the dental lamina and the concept of multi-factorial inheritance, have been proposed to elucidate the formation of SNT that are common in males. Similarly, several theories have been propagated on the tooth agenesis; the conception of a polygenic multi-factorial model of etiology still provides a good explanation for hypodontia, which is common in females[]. However, the etiology of CHH remains unclear. Additionally, there is no family history in three cases was evident, it has been recommended that disturbances in differentiation, migration and proliferation of neural crest cells was associated with interactions between the epithelial and the mesenchymal cells during the initiation of odontogenesis may be responsible for CHH.[] A recent study from Poland reported that CHH is rare and sex-related, with a predominance of hypodontia.[] SNT may be single or multiple, unilateral or bilateral in distribution, can occur in both dental arches and either in the primary mixed or permanent dentitions. The mesiodens in maxillary arch are the most commonly occurring SNT type followed by mandibular supplemental premolars[]. Contrarily, the cases in the report exhibited SNT in the anterior region of the mandible. Occurrence of SNT in the mandibular anterior region is exceedingly rare condition, but when they do occur, they appear as one or more smaller cone-shaped teeth. Excluding third molars mandibular second premolars were the most commonly missing in Caucasian population, whereas, mandibular incisors in Asian populations[]. It has also been reported that the unilateral form of missing teeth were more common. Similarly, in three cases one mandibular incisor was missing congenitally. Most recently Zadurska .[] reported that the occurrence of CHH in both jaws or in the maxilla and the authors stated that CHH never occur only in the mandible. Nevertheless, this condition in mandibular anterior region has been reported[] [], where both mandibular central incisors were missing with the presence of SNT in the midline. In contrast, in present cases only one mandibular central incisor was missing with the presence of a SNT. Therefore, it is surprising to note that occurrence of agenesis of unilateral central incisor exhibited in all cases with the presence of SNT. This concurs with the reported findings that a SNT seldom approaches or equals normal tooth size and shape, microdontic and conical shaped mandibular incisors are extremely rare. Taurodontism has been frequently reported with the incidence rate of 0.25% and 18%.[] Several authors have studied the association among taurodontism and hypodontia. Seow and Lai[] reported 34.8% of patients affected by hypodontia had taurodontism while Gomes .[] found one-third of their patients. It has been reported that taurodontism has been associated with CHH[]; similarly, in the present report two of our patients with CHH exhibited taurodontism in maxillary first permanent molars. Furthermore, most recently densevaginatus[] and double tooth[] have been reported in association with CHH. The standard treatment protocols have not been discussed for the management of CHH in literature and a multidisciplinary approach is necessary; hence, the management of CHH is quite challenging. In the present scenario, no treatment, extraction of mesiodens and close the space with fixed orthodontics, extraction of mesiodens and Maryland bridge as short-term and implants in feature and composite build-up to mimic as incisor, were treatment options included. Among our patients two were declined the treatment and one patient received treatment. It was decided to have aesthetic restoration for the mandibular anterior region. Early diagnosis is a key for the successful management, since it permits the dentist to implement the most appropriative treatment options for the patient to minimize consequences. The treatments may differ from individual to individual. The diagnosis of CHH in all our cases was only an incidental finding, which is similar to most of the findings that have been reported. Moreover, it has been stated that a high proportion of patients with CHH may remain undiagnosed in the population.[] The cases in the present report created a dilemma for the first instance in a clinical scenario and it is very difficult to diagnosis the CHH in a growing child. Our three cases exhibited mesiodens in tooth 41 region, which was coincidental and the size and shape of the tooth in 41 regions was completely different than usual appearance of mandibular central incisor. The panoramic evaluations of these cases confirm the findings. Clinical examination along with radiographic evaluation drew the proper diagnosis of the dilemma in our cases. It has also been sugested that the use of radiograph along with the clinical examination may enhance the recognition of CHH which probably could modify in the treatment plan.[] C H H i s a n u n u s u a l c o n d i t i o n a n d o c c u r r e n c e i n t h e m a n d i b u l a r a r c h i s e x t r e m e l y r a r e . M o r e o v e r , t h i s c o n d i t i o n w h i c h m i g h t b e d i a g n o s e d b y r o u t i n e c l i n i c a l a n d r a d i o g r a p h i c e x a m i n a t i o n s . A g e n e s i s o f u n i l a t e r a l m a n d i b u l a r i n c i s o r , w i t h t h e p r e s e n c e o f S N T i n t h e a n t e r i o r r e g i o n o f t h e m a n d i b l e i s e x t r e m e l y r a r e . T h e p r e s e n t c a s e s a r e u n i q u e c a s e s o f m a n d i b u l a r C H H . F u r t h e r m o r e , t a u r o d o n t i s m a s s o c i a t i o n s w i t h C H H h a v e b e e n e v i d e n t , w h i c h n e e d e d f u r t h e r d i s c u s s i o n .

Significance of any disease in particular area can be gazed by its prevalence. This becomes even more important for developing country like India where oral health program and preventive measures are far from satisfying needs. Prevalence of malocclusion has been studied in adolescents of Cicero,[] Negro children of Columbia,[] Black American children in the Evanston-Oak Park of Illinois[] Minnesota,[] Indiana and Kikuyu tribe of Kenya,[] Korean cleft patients,[] Hvar Island Croatia,[] Italian students,[] Hungarian population,[] Naples,[] Iranian school children,[] and Tanzanian school children.[] Karnataka state, is an active agriculture place with people from a wide spectrum of cultural and religion background. To date there is no available data on status of prevalence of malocclusion in Karnataka. Thus aim of the study was to record prevalence of malocclusion and to define difference in malocclusion status in urban and rural population. #text 12.21% had class I normal occlusion. 15.43% of rural and 9% of urban population had class I normal occlusion with statistically significant difference between rural and urban population ( = 0.000) []. Class I sagittal occlusion was found in 89.45% of the subjects, Class II in 8.37%, and Class III in 2.14% with statistically significant difference ( = 0.00) between two groups [ and ]. Normal overjet was seen in 48.22%, excessive in 33.71%, and reduced in 18.07% []. The difference between urban and rural population was statistically significant ( = 0.000) with urban population having more of increased overjet. Normal overbite was seen in 49.87%, deep in 35.97%, and reduced in 14.15% of total sample with no statistically significant difference between urban and rural population ( = 0.083) [ and ]. The frequency of crowding was 58.12%. Urban population had 58.25% and rural had slightly less (57.37%), but difference was not statistically significant ( = 0.683) []. Midline diastema was present in 15.43% with no statistically significant difference ( = 0.551) between urban and rural population [ and ]. Anterior crossbite was present in 8.48% of subjects. Urban population had 8.25% and rural had 8.56% anterior crossbite with no statistically significant difference ( = 0.816) []. Posterior crossbite was present in 0.99%. Urban population had 1.25% and rural population of 0.69% had posterior cross bite with no statistically significant difference ( = 0.112) between two groups [ and ]. This survey provides the first estimate of prevalence of malocclusion in Karnataka state. Examination was confined to high school students because of ease of accessibility;[] with a complete permanent dentition as malocclusion occurring in the mixed dentition is sometimes transitional leading to erroneous conclusions. Qualitative and quantitative methods available for measuring malocclusion are not truly inclusive of all occlusal criteria,[] thus, an alternative approach was used to register malocclusion by using occlusal characteristics. Angle's classification that is reliable, repeatable,[] and idealistically oriented for a broad population study[] was used for checking sagittal occlusion. Malocclusion has often been referred to as a “disease of civilization”, signifying that it is found (or at least reported) primarily in urbanized populations. This calls for rural-urban dichotomy. Prevalence of malocclusion was high and comparative to studies[] conducted by Rajendra in Bangalore city, Kharbandha in Delhi, by Altemus in Negro children, and U.S Public Health Survey. Findings of this present study were in disagreement with Guaba[] in the district of Ambala (70.8 per cent had normal occlusion). Prevalence of malocclusion was comparatively less (7%) as compared to studies[] conducted on Chinese. Class I molar relation was almost the same as found in studies[] conducted in Minnesota. There was a significantly higher prevalence of Class III occlusion among the Chinese, Malays, and blacks as compared to the Indians.[] Prevalence of overjet and overbite found in present study was same as that in urban Iranian school children, Yoruba adolescents in Ibadan, Nigeria,[] and study conducted on three ethnic races; Chinese, Malay, and Indian in Malaysia.[] Crowding was present in 57.69% of subjects. This was similar to the finding of Usha Mohan Das and Ali Borzabadi that crowding anterior was most common finding in subjects with class I malocclusion.[] This was in accordance with study conducted by Woon[] that stated crowded dentition was also a norm for the three races: Chinese, Malay, and Indian. Prevalence of crowding was same as seen in the Hvar island, Croatia; among Lithuanian school children; in Naples; in Rio de Janeiro State, Brazil; and Jordanian subjects,[] Much less prevalence of crowding was seen among adolescents of Ibadan, Nigeria (20%),[] Present study found midline diastema in 15.65% of subjects. This prevalence was much more as compared to study[] conducted by Gnanasundaram and Hashim on prevalence of midline diastema (1.6%) in Chennai city. Prevalence of midline diastema was much less than adolescents of Ibadan, Nigeria (37%).[] Anterior cross bite was present in 8.46% and posterior cross-bite was present in 0.88%. Posterior crossbite was recorded much more (8.8%) among Lithuanian schoolchildren; Rio de Janeiro State, Brazil (19.2%); urban Iranian school children; and in Lahore city (24%), Pakistan[] Difference was much more as compared to Lahore city as in their study data was collected from patients who visited department of orthodontics. Urban population had twice the class II sagittal occlusion and increased overjet as compared to rural population. This difference was similar to the study[] done to assess occlusal variation in three southwest Pacific populations. Dietary consistency can be considered as viable reason as the posterior region of mandible is associated with muscles of mastication,[] As far as anterior region is concerned habits can be considered as reason for discrepancy in urban and rural population. Further research is needed to determine whether there is association between above written factors and malocclusion. This study is limited as it has only recorded malocclusion in age group of 13-17 years which cannot be generalized to entire population. Secondly, orthodontically treated cases were excluded which can underestimate the prevalence of occlusal traits. But the proportion of adolescents who underwent orthodontic therapy and excluded were relatively small and thus does not make statistically significant difference in results. Thirdly, there are considerable variations in figures obtained as different researchers have used different criteria for assessing the same occlusal trait. This lay emphasis on the need to standardize criteria for assessing malocclusion. l o c c l u s i o n i s w i d e s p r e a d i n p o p u l a t i o n e x a m i n e d a t K a r n a t a k a S t a t e , I n d i a . P r e v a l e n c e o f m a l o c c l u s i o n w a s m o r e i n u r b a n p o p u l a t i o n w h e n c o m p a r e d w i t h r u r a l p o p u l a t i o n . C r o w d e d i n c i s o r s w e r e t h e m o s t c o m m o n f e a t u r e a s s o c i a t e d w i t h c l a s s I m a l o c c l u s i o n . R e q u i r e f u r t h e r s t u d i e s i n a r e a t o f i n d e t i o l o g y f o r v a r i o u s o c c l u s a l t r a i t s .

The soft–shell turtle was purchased from a local farmer in Japan, and the green sea turtle was provided by the Genome 10K Project (originally collected in Ocean Park, Hong Kong). Genomic DNA was extracted from the whole blood of a female individual in each species, and we constructed a total of 18 (for the soft–shell turtle) and 17 (for the green sea turtle) libraries consisting of short–insert (170–bp, 500–bp and 800–bp) and long–insert (2–kb, 5–kb, 10–kb, 20–kb and 40–kb) libraries. Sequencing was performed using the Illumina HiSeq 2000 system, and read error correction was performed for the short–insert libraries (on the basis of the –mer frequency distribution curve; ). Data accession numbers are given in . Filtered and corrected data were assembled using SOAPdenovo. We first generated contigs by constructing a de Bruijn graph with the reads from the –mer–split short–insert library data. The graph was then simplified to generate the contigs by removing tips, merging bubbles and solving repeats. All sequenced reads were then realigned onto the contig sequences, and scaffolds were constructed by weighting the rates of consistent and conflicting paired–end relationships. Finally, we retrieved the read pairs with one end that uniquely mapped to the contig and the other end located in the gap region, and performed a local assembly for these collected reads to fill the gaps. Repeat detection was performed using the program RepeatMasker and the Genetic Information Research Institute (GIRI) repeat library. For homology–based prediction of repeats, we used the library of known repeats in the Repbase database (v2008–08–01, Repbase–16.02) with RepeatMasker (v3.2.6) and RepeatProteinMask to identify transposable elements at the DNA and protein levels, respectively. The prediction of repeats involved building a repeat library with RepeatModeler and subsequently employing RepeatMasker. Tandem repeats were searched with the Tandem Repeats Finder (TRF). Whole–genome pairwise alignments were generated by LASTZ. Gene prediction for the two turtle genomes employed both the approach (GENSCAN (v2.5.5) and AUGUSTUS (v1.0)) and a homolog–based approach against the repeat–masked genome, and gene sets predicted by these two approaches were further consolidated with the GLEAN program. For the soft–shell turtle, an additional 146.7 Gb of RNA–seq data was used. The proteins of other vertebrate species were mapped to the genome using TBLASTN (Legacy Blast v2.2.23). Aligned sequences were then filtered and passed to GeneWise (v2.2.0) along with the query sequences. The resulting data sets were integrated by GLEAN into a consensus gene set. The best BLASTP match to the SwissProt and TrEMBL databases was used to assign function. The motifs and domains of the gene products were annotated with InterProScan against the protein databases ProDom, PRINTS, Pfam, SMART, PANTHER and PROSITE. Gene Ontology IDs for each gene were obtained from the corresponding InterPro entries. The above prediction pipeline was applied to the saltwater crocodile and American alligator genomes (from the Crocodile Genome Consortium), except for the integration step in the latter case. Gene family identification was performed using TreeFam. For gene expression comparison analyses between soft–shell turtle and chicken embryos, we generated and used another soft–shell turtle gene set that was created by the same Ensembl pipeline as the chicken gene set (see URLs). Over–represented GO terms were investigated by testing (Fisher’s exact test) the bias in frequency toward other GO terms among certain gene sets, using the total set of defined GO terms as a control distribution. Developmental genes (5,659 in total) were defined as genes with developmental GO terms, and developmental GO terms were defined as those with GO:0032502 (developmental process) as an ancestor. Experimental procedures and animal care were conducted in strict accordance with guidelines approved by the RIKEN Animal Experiments Committee (Approval IDs H14–23 and H16–10). The coding sequences of single–copy gene families conserved among the soft–shell turtle, green sea turtle, anole lizard, saltwater crocodile, chicken, zebra finch, dog, human, platypus and were extracted and aligned with guidance from amino–acid alignments created by the MUSCLE program. Sequences were then concatenated to one supergene sequence for each species. PhyML was applied to construct the phylogenetic tree under an HKY85+gamma or GTR+gamma model for nucleotide sequences and the JTT+gamma model for protein sequences. aLRT values were taken to assess the branch reliability in PhyML. RAxML was also applied for the same set of sequences to build a phylogenetic tree under a GTR+gamma or JTT+gamma model for nucleotide and protein sequences, respectively, with 1,000 rapid bootstraps employed to assess the branch reliability in RAxML. The same set of codon sequences at positions 1 and 2 was used for phylogenetic tree construction and estimation of the divergence time. The PAML program (PAML version 4.5) was used to determine divergence times with the approximate likelihood calculation method and the ‘correlated molecular clock’ and ‘REV’ substitution model. Two independent runs were performed to confirm convergence. Protein sequences of the two turtles and related species (chicken, anole lizard, and zebra finch) were used in BLAST searches against human protein sequences (Ensembl Gene v.68), identifying homologs. Subsequently, human proteins that lacked homologs in both the turtle species but had homologs in the related species were identified as lost genes in turtle. For the statistical analysis of gene family expansion and contractions, we generated pairwise whole–genome alignments for anole lizard and soft–shell turtle and for anole lizard and green sea turtle using LASTZ and created three–way alignments using MULTIZ. When an anole lizard gene fell in an area of conserved sequence and there was no homologous gene in the corresponding aligned sequences of the two turtle species, we hypothesized that gene loss potentially occurred at that locus in turtle (). Frameshift mutations and those introducing premature stop codons in the coding sequences were also considered to represent gene loss. Olfactory receptor genes were identified by previously described methods, with the exception of a first–round TBLASTN search, in which 119 functional olfactory receptor genes from human, mouse and zebrafish were used as queries (). To construct phylogenetic trees, the amino–acid sequences encoded by olfactory receptor genes were first aligned using the program E–INS–i in MAFFT. We then constructed a phylogenetic tree using the neighbor–joining method with Poisson correction distances using the program LINTREE. The numbers of olfactory receptor genes in ancestral species and those of gene gains or losses in evolution were calculated by the reconciled tree method with 70% bootstrap value cutoff. Homologous genes in soft–shell turtle, green sea turtle and other vertebrate species (chicken, zebra finch, anole lizard, and platypus) were first identified with the all–against–all BLASTP program. Orthologs were defined by reciprocal best BLAST hits (RBBHs) in humans and the other species. The full orthologous gene sets were aligned using the program MUSCLE. We then compared a series of evolutionary models within the likelihood framework using the phylogenetic tree obtained by our analysis. A branch model was used to detect the average length () across the tree (), the value of the ancestor of all soft–shell turtles, the value for the green sea turtle branch () and the value for all of the other branches (). Fertilized soft–shell turtle and chicken eggs purchased from local farms in Japan were incubated and staged according to previous descriptions. Amniotic membranes were removed before mRNA extraction, and more than two individual embryos were pooled for each sample. The RNeasy Lipid Tissue kit (Qiagen) and the Ambion MicroPoly(A) Purist kit (Life Technologies) were used for mRNA extraction. Three different types of sequencing were performed for transcriptome identification in the soft–shell turtle: (i) Titanium sequencing (about 2 Gb of clean sequence data), (ii) HiSeq strand–specific paired–end RNA–seq (two libraries were prepared by methods that retain strand–specific information, including a dUTP–based method (19 Gb of clean data) that was modified to comply with the Illumina TruSeq RNA sample prep kit and an original method developed at BGI Sequencing that was performed with Illumina HiSeq 2000 (26 Gb of clean data) and (iii) HiSeq non–stranded RNA–seq (deep–sequencing data for gene expression analysis was also used for transcriptome identification). Further details are given in the . Biological replicates for each developmental stage were created from an independent sample pool. Extracted mRNA samples were then sequenced with an Illumina HiSeq 2000 instrument. We identified 11,602 one–to–one orthologous genes in the soft–shell turtle and chicken using RBBH information from BLAST+ (v2.2.25). Gene expression scores were obtained from RNA–seq data by mapping clean reads to the genome using Burrows–Wheeler Aligner (BWA) software (v0.5.9–r16). SAMtools, BEDtools and the DEGseq package for R (v2.14.2) were used to calculate the tag count data that were mapped to the coding regions. Normalization of the orthologous gene expression scores was performed with all samples at once by either RPKM or TMM normalization. Pearson’s correlation coefficients, Spearman correlation coefficients, total Euclidean distances (t–Euclidean) or total Manhattan distances (t–Manhattan) were used to estimate similarities in the gene expression profiles of the two samples being compared. Two independent random selections from all reads were performed to make the mapped–10M reads (sequencing depth–controlled data set based on randomly selected 10M tags mapped to the genome) data. The Welch two–sample test or the Wilcoxon signed–rank test was used to detect the most conserved stages. The Holm–corrected level was applied for these multiple comparisons. Only results reproduced by the data set from two different normalizations (RPKM and TMM) were considered to be significant. Turtle IAP genes were selected using the following criteria: (i) the mean expression level after the phylotypic period (TK15–TK23) was more than five times higher (Wilcoxon test) than during earlier stages (gastrula, neurula, TK7 and TK9) and (ii) the chicken orthologs of the turtle IAP genes (if any) did not show such increases in chicken (the average expression levels in HH28 and HH38 did not show more than five times higher expression than in the Prim–HH14 stages). In addition to constructing the predicted gene sets, we manually searched for Wnt genes using TBLASTN. Cloning of the probes and whole–mount ISH were performed using standard methods (). Small RNA was extracted from dissected tissues using the mirVana microRNA Isolation kit (Life Technologies). Small RNA libraries were prepared and sequenced using an Illumina HiSeq 2000 (>24 million reads per sample). These small RNA reads, together with the miRNA sequences from chicken, zebra finch and from miRBase (v.18), were used to predict miRNA sequences in the genome. The program miRDeep2 (v2.0.0.3) was used to predict miRNAs for this prediction. Only miRNA predictions that had value lower than a significant Randfold level ( < 0.05 mononucleotide shuffling and 999 permutations; see for details) were taken into account for subsequent comparisons. miRNA target prediction was performed with miRanda (v3.3a) using the annotated 3′ UTRs of soft–shell turtle genes. To avoid an inflated type I error rate, an level of 0.01 (further Bonferronni correction in case of multiple comparisons) was accepted for statistical significance throughout the analyses unless otherwise specified. Statistical methods were carefully chosen to properly reflect the population of interest. The Welch two–sample test was used for two–sample comparisons when the data passed the Kolmogorov–Smirnov test for normal distribution; otherwise, the Wilcoxon signed–rank test was used.

To investigate the effect of combined treatment of staurosporine and ATRA on NB4-R1 and NB4-R2 cells, we first tested the concentration of staurosporine studied in both cell lines. DMSO treatment was regarded as control since both ATRA and staurosporine were dissolved in it. 2 nM staurosporine was determined to be used since there were no obvious effects on cell proliferation () and survival at such concentration (). The combined treatment suppressed cell growth at the third day (). The proliferation inhibition rate calculated as Methods mentioned was 36.1 ± 3.1% for NB4-R1 cells and 34.5 ± 2.7% for NB4-R2 cells. However, cell viability maintained above 90% in these two cell lines with any treatment for 72 hours. Annexin-V assay also showed that more than 90% cells were PI negative and Annexin-V negative even with combined treatment (). Thus, the combined treatment had no effect on cell survival but only inhibited proliferation in these two cell lines. Both cell lines had a characteristic morphology of APL blast such as irregular nucleus and large nuclear/cytoplasm ratio (). After incubated with 2 nM staurosporine or 1 μM ATRA alone for 72 hours, no significantly morphological change was observed in NB4-R1 cells (). Meanwhile, most of the NB4-R2 cells retained the morphology of APL blast with any single treatment while some cells presented decreased nuclear/cytoplasm ratio with ATRA alone (). However, when simultaneously treated with staurosporine and ATRA for 72 hours, both cell lines displayed appearances of matured granulocytes, such as lobed nuclei accompanied by markedly decreased nuclear/cytoplasm ratio (). Nitroblue tetrazolium (NBT) reduction activity did not increase remarkably with any single treatment in both cell lines (). In addition, the expression of CD11b was somewhat enhanced with any single treatment in NB4-R1 and NB4-R2 cells (). However, with the combination of staurosporine and ATRA, a more than additive effect was observed. The combined treatment significantly increased the percentage of CD11b positive cells in both cell lines (ST + RA compared with DMSO, 73.9 ± 6.6% 6.9 ± 1.3%, = 9321.63, < 0.001 in NB4-R1 cells and 75.1 ± 2.3% 9.0 ± 1.6%, = 8965.07, < 0.001 in NB4-R2 cells, ). Accordingly, prominently enhanced NBT reduction activity was observed in both cell lines with combined treatment (ST + RA compared with DMSO, 0.129 ± 0.006 0.014 ± 0.002, = 0.0007 in NB4-R1 cells and 0.161 ± 0.014 0.018 ± 0.003, = 0.0006 in NB4-R2 cells, ). Thus, these results demonstrated that the combined treatment induced granulocytic differentiation and indicated that the combination of staurosporine and ATRA could overcome maturation block in ATRA-resistant APL cells. To explore the molecular mechanisms of combined treatment-induced differentiation, we first examined several proteins involved in granulocytic differentiation, especially which are critical for the promyelocyte to granulocyte transition. The induction of CCAAT/enhancer binding protein β (C/EBPβ) and C/EBPε expression is implicated in the later stage of granulocytic differentiation. Therefore, cells were treated with staurosporine, ATRA, or staurosporine plus ATRA for 24 hours and the protein levels of C/EBPβ and C/EBPε were detected by immunoblotting. As shown in , in NB4-R1 cells, ATRA but not staurosporine elevated C/EBPβ protein level while the cellular level of C/EBPε was enhanced by both single treatment. In NB4-R2 cells, only ATRA treatment increased the protein level of C/EBPβ and C/EBPε. However, the up-regulation of C/EBPβ and C/EBPε was much more remarkable with the combined treatment in both cell lines (). These results suggested that C/EBPβ and C/EBPε may be responsible for the differentiation effect of combined treatment in ATRA-resistant APL cells. The degradation of PML-RARα has been the crucial mechanism of ATRA-induced granulocytic differentiation of APL cells. Treated with ATRA for 72 hours, the band corresponding to PML-RARα was almost disappeared in NB4-R1 cells or decreased in NB4-R2 cells (). Meanwhile, the protein level of RARα was reduced in both cell lines with ATRA treatment (see online). Unexpectedly, the addition of staurosporine totally restored ATRA-mediated PML-RARα alteration in both cell lines (). Interestingly, in NB4-R2 cells, staurosporine alone could increase the protein level of RARα and inhibit ATRA-induced reduction of RARα (see online). These data indicated that the cooperation differentiation effect of ATRA and staurosporine was independent of the regulation of PML-RARα. Our previous study demonstrated that the non-genomic effect, MEK/ERK signaling pathway could positively regulate ATRA-induced differentiation in APL cells (unpublished data). To elucidate whether MEK/ERK signaling pathway was activated, the phosphorylated MEK and ERK1/2 were assessed by Western blot analysis in cells treated with ATRA or/and staurosporine for 24 hours. As illustrated in , the combined treatment increased the amount of phosphorylation of MEK and ERK1/2 more significantly than that with any single treatment in both cell lines. The total amount of MEK and ERK1/2 in both cell lines remained almost unaltered after any treatment (). Having shown that the combination phosphorylated MEK and ERK, we tested whether MEK/ERK activation was necessary for ATRA and staurosporine-mediated differentiation. Cells were incubated with 1 μM U0126, a specific inhibitor of MEK for 1 hour prior to other treatments. The effectiveness of U0126 was evaluated by ERK1/2 phosphorylation. U0126 did attenuate ERK1/2 activation in these 2 cell lines (). Moreover, it abrogated cell differentiation triggered by the combined treatment in both cell lines as assessed by morphology (), NBT reduction assay (U + ST + RA compared with ST + RA, 0.026 ± 0.003 vs 0.129 ± 0.006, = 0.0076 in NB4-R1 cells, 0.045 ± 0.008 vs 0.161 ± 0.014, = 0.0199 in NB4-R2 cells ) and CD11b expression (U + ST + RA compared with ST + RA, 23.0 ± 2.1% 73.9 ± 6.6%, = 5186.60, < 0.001 in NB4-R1 cells and 21.9 ± 2.6% 75.1 ± 2.3%, = 5665.58, < 0.001 in NB4-R2 cells, ). In addition, in the presence of U0126, the combined treatment-enhanced C/EBPβ and C/EBPε protein level were remarkably suppressed in both cell lines (). These results highlighted a major role of MEK/ERK signal pathway in the differentiation-inducing effect of ATRA and staurosporine in ATRA resistant-APL cells. In the present work, we demonstrated that the combination of staurosporine and ATRA could overcome granulocytic differentiation block in retinoid resistant APL cell lines. Staurosporine has been proven to be not only an effective apoptosis inducing agent but also a potent differentiation inducer in a variety of tumor cell lines. To our knowledge, it is the first time to show that staurosporine can restore ATRA sensitivity in retinoid resistant APL cell lines. However, the effect of the combination on primary leukemia cells from retinoid resistant APL patients needs further investigation. For the molecular mechanisms of the combination–induced differentiation, we focused on certain proteins or signaling pathways involving in granulocytic differentiation rather than PKC. C/EBPs are a family of transcription factors which regulate cell proliferation and differentiation. Among them, C/EBPα, C/EBPβ and C/EBPε are critical mediators of granulopoiesis. C/EBPα is required for the proceeding from myeloblast to promyelocyte while C/EBPβ and C/EBPε play central roles in terminal differentiation of granulocytes. Moreover, C/EBPβ and C/EBPε were demonstrated to be required for ATRA-mediated differentiation in APL cells. In addition, C/EBPε alone could suppress the leukemia phenotype of APL and imitate the differentiation-induction effect of ATRA in APL mouse model. Thus, C/EBPβ and C/EBPε are not only the crucial regulators of granulocyte development but also the potential therapy target for leukemia. In this work, compared with any single treatment, the combination of staurosporine and ATRA remarkably increased the protein levels of both C/EBPβ and C/EBPε in NB4-R1 and NB4-R2 cells. Though any single treatment to some extent did up-regulate C/EBPβ or C/EBPε, we speculated that such increase might not be enough to trigger maturation but only combined treatment resulting in significant up-regulation of C/EBPβ and C/EBPε could speed up terminal differentiation. It was well known that co-repressor release, co-activator recruitment, transcriptional activation of genes with the RAREs and PML-RARα degradation were principal mechanisms of ATRA-induced differentiation of APL cells. However, it was indicated that pre-genomic activation of kinase signaling cascades also contributed to ATRA-induced differentiation. Our previous studies showed that MEK/ERK was activated during ATRA-induced granulocytic differentiation in APL cell line, NB4. Furthermore, inhibition of MEK activation suppressed almost all differentiation induced by ATRA, suggesting the significant role of MEK/ERK signaling pathway in ATRA-dependent NB4 cells granulocytic differentiation (unpublished data). Interestingly, the activation of MEK/ERK signaling pathway was also observed in the combination treatment. Moreover, attenuation of the MEK activation inhibited not only the differentiation but also the increased protein level of C/EBPε and C/EBPβ. Therefore, the protein level of C/EBPε and C/EBPβ was regulated by MEK/ERK signaling pathway and the later was required for the combination-induced differentiation. Accumulating evidence showed that MEK/ERK signaling pathway could promote C/EBPβ expression and modify its activity. Moreover, in ATRA-treated APL cells, as the result of increasing protein level of C/EBPβ, enhanced C/EBPβ binding activity preceded that of C/EBPε and C/EBPβ itself could induce the expression of C/EBPε. Hence, there might be MEK-C/EBPβ-C/EBPε cascade in the combination-induced differentiation. PML-RARα fusion protein is believed to contribute to the pathogenesis of APL in a dominant negative fashion, disrupting the normal physiologic function of PML and repressing transcription by RARs. Either AsO or ATRA is regarded as one of the most successful examples of tumor targeted therapy since they both degraded APL pathogenic protein, PML-RARα. However, in this work, though staurosporine inhibited ATRA-mediated PML-RARα degradation, the combination of staurosporine and ATRA still synergistically induced differentiation. It meant that the combination could induce differentiation independent of PML-RARα degradation, suggesting that the degradation of PML-RARα might not be necessary for APL therapy. In consistent with our observation, other agents such as oridonin, G-CSF, STI571, pharicin B and TNF could cooperate with ATRA to induce differentiation in ATRA-resistant APL cell lines without any effect on PML-RARα. The increased protein level of C/EBPε and C/EBPβ by ATRA treatment was confirmed to depend on PML-RARα expression. Therefore, it remains to be further explored whether staurosporine prevented PML-RARα degradation contributes to the enhanced protein level of C/EBPε and C/EBPβ as well as the combination–induced differentiation. In conclusion, the combination of staurosporine and ATRA synergized to trigger differentiation in ATRA resistant APL cell lines by MEK/ERK signaling pathway. It remains to investigate whether such combination could induce differentiation in primary leukemia cells from retinoid resistant APL patients. The exact molecular mechanisms of how staurosporine overcomes ATRA resistance as well as more detailed mechanisms of the combination-induced differentiation need to be further elucidated. ATRA was purchased from Sigma-Aldrich (St Louis, MO) while both staurosporine and U0126 were obtained from EMD Chemicals (San Diego, CA). They were all dissolved by dimethylsilfoxide (DMSO) as a stock solution at 1 mM, 2 μM and 1 mM respectively. The ATRA resistant cell lines, NB4-R1 and NB4-R2 cells were obtained from Dr Michel Lanotte (Hopital Saint Louis, Paris, France) and were cultured in RPMI-1640, supplemented with 10% fetal calf serum (Thermo Fisher Scientific Inc, Waltham, MA) in a humidified atmosphere of 95% air/5% CO2 at 37°C. To avoid possible effects of cell density on cell growth and survival, cells were maintained at less than 5 × 10 cells/ml. Cell viability was assayed by trypan-blue exclusion. Actual viable cell numbers were calculated by multiplying diluted times with counted viable cell numbers. Proliferation inhibition rate (%) = (control group actual viable cell numbers-experimental group actual viable cell numbers)/control group actual viable cell numbers ×100%. Cell maturation was evaluated by cellular morphology, NBT reduction assay and the content of cell surface differentiation-related antigen CD11b. Morphology was determined with May-Grunwald-Giemsa's staining of cells centrifuged onto slides by cytospin (Shandon, Runcon, UK; 500 r.p.m., 5 min) and viewed at ×1,000 magnification. NBT reduction was performed as previously described. Briefly, 1 × 10 cells were collected and incubated with 1 mg/ml NBT (Sigma-Aldrich) solution containing 10 μM phorbol 12-myristate 13-acetate (Sigma-Aldrich) at 37°C for 1 hour. Cells were lysed by 10% sodium dodecyl sulfate (SDS) and 0.04 N hydrochloric acid (HCl). The absorbance at O.D 540 nm was detected by spectrophotometer (Beckman Coulter, Brea, CA). The expression of cell surface differentiation-related antigen CD11b was determined on flow cytometry (EPICS XL, Coulter, Hialeah, FL). Fluorochrome-labeled anti-human CD11b/FITC antibody was purchased from Coulter-Immunotech (Marseilles, France). Annexin-V assay was performed according to instructions provided in the Annexin V-PI Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA). Briefly, 5 × 10 cells were harvested and washed with binding buffer provided in the kit. Then, cells were incubated with 5 μl annexin-V and 10 μl propidium iodide (PI) at room temperature in the dark for 15 min. Fluorescent intensities was determined on flow cytometry (Coulter). Cells were washed with phosphate-buffered saline (PBS) and lysed with RIPA buffer (Sigma-Aldrich). Cell lysates were centrifuged at 13,000 rpm for 10 minutes at 4°C. Supernants were collected and quantified by Bio-Rad Dc protein assay (Bio-Rad Laboratories, Hercules, CA). 20 or 50 μg protein extracts were loaded on 8% SDS-polyacrylamide gel, subjected to electrophoresis, and transferred to polyvinylidene difluoride membranes (GE Healthcare UK Ltd, Buckinghamshire, UK). After blocking with 5% nonfat milk in PBS, the membranes were probed with antibodies against RARα (Santa Cruz Biotech, Santa Cruz, CA), C/EBPβ (Santa Cruz Biotech), C/EBPε (Santa Cruz Biotech), anti-β-actin (Santa Cruz Biotech), phosphor-p44/42 Erk1/2 (Thr202/Try 204) (Cell Signaling Technology, Beverly, MA) and phosphor-MEK1/2 (Ser217/Try 221) (Cell Signaling Technology). Subsequently, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare UK Ltd). Immunocomplexes were visualized by chemiluminescence kit (GE Healthcare UK Ltd) according to the manufacturer's instruction. To detect Erk1/2 and MEK1/2, the same membrane incubated with the phosphorylated Erk1/2 or MEK1/2 was stripped with stripping buffer (2% SDS, 100 mM beta-mercaptoethanol, 50 mM Tris, pH6.8) followed by blocking and probing with anti-Erk1/2 (Cell Signaling Technology) or anti-MEK1/2 (Cell Signaling Technology). For NBT reduction, two-tailed paired Student's test was used, n value was 3. The flow cytometric analysis of CD11b was analyzed by chi-square test, n value was 20,000. D . Z . G . a n d Y . S . c a r r i e d o u t t h e e x p e r i m e n t s . D . Z . G . p r e p a r e d a l l t h e f i g u r e s . X . e s i g n e d t h e s t u d y a n d w r o t e t h e m a n u s c r i p t . A l l a u t h o r s r e v i e w e d t h e m a n u s c r i p t .

As shown in , we proposed a composite structure including an open-end waveguide and a planar-spiral phase plate (planar-SPP) to generate OAM radio waves, which functions like a transmit array antenna and transforms an incoming phase front into a desired outputting phase front. The open-end waveguide is adopted to provide an incident spherical wave as a primary source, and the proposed planar-SPP is placed parallel to the -plane, which transforms the incident waves to OAM beams. The planar-SPP is a square plane with a length of , and the distance between the primary source and the planar-SPP is . Planar quasi-period air holes are drilled in the substrate to form the proposed planar-SPP, which is used to compensate the phase delay associated with incident radio waves from different paths and generates the desired outputting phase distribution. To simply the analysis, the aperture of the planar-SPP is divided into unit square cells with a side length Δ in both and direction, and fixed number of air holes are drilled in each cell as shown in . As the first step of the design, both the incident and the required outputting phases of all units need to be analyzed. Then compensated phases can be calculated for those units. Since the Laguerre-Gaussian (LG) modes can carry an OAM of ħ per photon and the phase of the electric vector fields in a plane perpendicular to the beam axis has a φ dependence, where is the OAM mode index and φ is the azimuthal angle, the outputting phase of the proposed planar-SPP is designed increasingly in proportion to the azimuthal angle φ around the center of the aperture, which can be expressed as: Ideally, the incident electromagnetic waves for traditional SPP design are required to be plane waves. However, practical primary sources such as horn antennas and open-end waveguides can only provide spherical wave radiation, especially when they are placed not enough far away from the plate antenna. Hence, there are some unequal phase-distributions need to be compensated on the incident surface of the proposed planar-SPP. The unequal phase distribution can be determined by using a geometrical optics approximation under the assumption that the spherical wave comes from the phase center of the feed: where (θ) is the inputting phase of each unit cell, λ is the free-space wavelength of incident spherical wave and is the distance from surface of the planar-SPP to the phase center of the primary source. To convert the inputting spherical wave into a spiral-type wave with different modes, each unit of the planar-SPP transforms the inputting phase into the required outputting phase. Then the required compensation phase of each unit can be obtained as a function of coordinate of the -plane: We used a quasi-periodical structure shown in to transform the incident spherical electromagnetic waves to OAM beams, in which the cell structure is made by four drilled air holes in the substrate. By changing the radius of the air holes, the local effective dielectric constant of the substrate can be adjusted. Then the local refractive index of the substrate, namely decided by the local effective dielectric constant, can be changed to control the outputting phase. The whole substrate with quasi-periodically drilled air hole cells can be designed to have desired effective dielectric constant distribution with the same thickness. When the incident electromagnetic wave goes through the substrate, desired transmitting phase shifts are achieved. Hence, OAM waves with arbitrary mixed modes can be generated by properly dividing the proposed structure into several small parts to implement individual OAM mode. Unlike the traditional SPPs which require substrate with varied thickness, the proposed planar-SPP can be used to generate arbitrary OAM waves without changing the substrate thickness and keeps in a planar form. We studied the transmission phase characteristics of the air hole drilled cell at the frequency of 94 GHz. The transmission phase characteristics of the cell are analyzed by considering a unit cell with periodic boundary conditions and under a plane wave incidence with the aid of a full-electromagnetic-wave simulation software CST Microwave studio. The Arlon AD600A substrate is used in the work, which has a dielectric constant of 6.15 and a thickness of 6.35 mm. By simply changing the radius of the four air holes, the corresponding transmitting phase shift is obtained as a function of the radius. As shown in , the desired transmitted phase shift can be achieved by using unique radius for the drilled air holes. In addition, a phase shifting range exceeding 360 degrees is obtained by adjusting the radius of drilled air holes from 0.2 mm to 0.32 mm, which is enough to realize the arbitrary required phase distribution on the planar-SPP surface. Also, we separate the whole planar-SPP into several sub-parts, and each part is designed respectively taking the method mentioned before to generate electromagnetic wave with a specific OAM mode. In this way, OAM waves with a superposition of multiple OAM modes can be obtained by using only one OAM antenna conveniently. As demonstrations, we designed three OAM antennas: generating radio beams with OAM mode of = 1; superimposed mode of = 2 and = 4; and superimposed mode of = 1, = 2 and = 4 respectively. All three antennas have the same aperture size of = 45 mm and are divided into 30 × 30 unit cells. Each element has a square lattice with a period of 1.5 mm (about 0.47 wavelengths in free space at 94 GHz). And all of the planar-SPPs are illuminated by the same standard W-band rectangular open-end waveguide placed 35 mm away from the planar-SPPs. The designed prototypes are simulated using CST software and fabricated using commercial printed circuit board (PCB) technology, and measured in an anechoic chamber. The measurement setup is made up of an Agilent PNX Series Network Analyzer 5245A which is equipped with an Agilent millimeter head controller N5161A and two OML WR10 frequency extension modules (75 GHz–110 GHz), a W-band standard horn antenna, and a 2-D electronically controllable scanning stander controlled by a computer, as shown in . One port of the frequency extension modules is connected to an open-end waveguide to illuminate the planar-SPP and the other port is connected to a standard horn antenna that fixed on the scanning stander to detect the OAM beams. To meet the far-field condition, i.e. , where is the aperture dimension of the planar-SPP, the horn antenna is placed 1.3 m away from the planar-SPP. A program is developed to control the horn antenna scanning on the -plane. Then the network analyzer measures the intensity and the phase of the radio beams as a function of the coordinates of the plane. The 94 GHz OAM beams were sampled on a square area of 200 × 200 grids with a step of 2.5 mm. The intensity and phase can be obtained from the measured transmission scattering parameter at each sampled point. Then the transverse density and phase patterns on -plane are plotted using a Matlab code. Using the , it is easy to obtain the compensated phase of each cell for = 1 mode. And by employing the design curve in , the required radius of the air holes in each cell can be obtained. The whole structure is simulated using CST Microwave studio software. The simulated 3-D pattern is shown in and transverse intensity pattern of the radio beams is presented in , which shows a typical doughnut-shaped intensity with singularity in the center. The proposed OAM antenna shown in is manufactured by using PCB technology. Deploying the experiment setup mentioned before, we can obtain both the transverse intensity and phase patterns, which are shown in , respectively. The measured intensity pattern is quite similar to the simulated one as well as the theoretical result. Also, the experimental phase data shows a total 2π phase change around the centre of the plate as expected. For superimposed modes of = 2 and = 4, the whole planar-SPP is divided into two parts: the inner and the outer parts. The inner part is a square area and made up of 20 × 20 cells in the center of the planar-SPP. This part is designed to generate OAM with a state of = 2. And the outer part is with an annular shape and made up of 500 cells. The outer part is designed to generate OAM with a state of = 4. Both parts are designed following the and then the compensated phase of each cell for the two modes can be obtained easily. Similarly, the radiuses of the air holes on each element are designed using the curve in , as shown in . The simulated 3-D intensity pattern is presented in , and both the simulated and the measured transverse intensity patterns of the two-mode-antenna are presented in , respectively, where opposite crescent-shaped intensity patterns can be found and they agree well with theoretical analysis. Also, the measured transverse phase pattern is presented in . The proposed antenna can be designed to generating mixed-mode OAM waves with arbitrary number of OAM states. For example, to design a OAM antenna having superimposed mode of = 1, = 2 and = 4, a planar-SPP is designed by using 900 unit cells which is divided into three parts: the inner, the middle and the outer parts. The inner part is a square and made up of 14 × 14 cells in the center of the planar-SPP, which is used to generate OAM with the state of = 1. The middle part is with an annular shape and consists 288 cells for generating OAM with the state of = 2. And the outer part is also with an annular shape and consists the last 416 cells for generating OAM with the state of = 4. All the three parts are designed following the and then the compensated phase of each cell for the three-mode-antenna can be obtained correspondingly. Again, by applying the curve in , the whole structure is finalized, as shown in . The simulated 3-D and the transverse intensity patterns of the three-mode-antenna are presented in . To our knowledge, it is the first report for generating three-mixed-mode OAM waves using single antenna. The proposed structure is also been manufactured using PCB technology and measured in the anechoic chamber. Measured results are illustrated in , respectively. As expectations, well agreements between measured and simulated results are found in . It can be seen from ,, that measured intensity and phase maps of single OAM mode antenna and mixed-mode antenna are much coincided with the theoretical results. That verifies the proposed planar-SPPs have a distinct behavior of OAM beams and can be used to generate beams carrying OAM with any number of desired mixed modes. It shows great prospect in future OAM applications such as high speed communication with a narrow radio frequency band. h a v e s u c c e s s f u l l y g e n e r a t e d e l e c t r o m a g n e t i c b e a m s w i t h O A M o f d i f f e r e n t s t a t e s b y p r o p o s i n g a n e w c o n c e p t o f p l a n a r - S P P . T h e p r o p o s e d p l a n a r - S P P s e m p l o y s t h e c o n c e p t o f t r a n s m i t a r r a y a n t e n n a s , w h i c h u s e p h a s e s h i f t i n g e l e m e n t s t o o b t a i n t h e r e q u i r e d o u t p u t t i n g p h a s e a n d c o n v e r t s p h e r i c a l w a v e i n t o s p i r a l t y p e w a v e d i r e c t l y . I t i s a c o n v e n i e n t a n d d i r e c t w a y t o g e n e r a t e b e a m s c a r r y i n g O A M . T o o u r k n o w l e d g e , i t i s t h e f i r s t w o r k t h a t s h o w s t h e e x p e r i m e n t a l i n t e n s i t y a n d p h a s e r e s u l t s o f b e a m s c a r r y i n g m i x e d O A M m o d e s . A n d t h i s w o r k w i l l g r e a t l y p r o m o t e t h e s t u d y a n d a p p l i c a t i o n o f O A M i n r a d i o f r e q u e n c i e s . The electromagnetic waves with arbitrary mixed OAM modes are realized by transmitting spherical wave through a planar-SPP. The planar-SPP is manufactured by drilling air holes on a 6.35-mm Arlon AD600A substrate using PCB technology. The measurement setup is based on an Agilent PNX Series Network Analyzer 5245A, and both phase and magnitude of the electromagnetic waves with arbitrary mixed OAM modes can be measured. W . H . p r o p o s e d t h e m e t h o d . Z . - C . H . a n d L . C . d e v e l o p e d t h e m e t h o d . L . C . s e t u p t h e e x p e r i m e n t s . A l l t h e a u t h o r s p e r f o r m e d t h e e x p e r i m e n t s a n d a n a l y s i s . Z . - C . H . a n d L . C . w r o t e t h e m a i n m a n u s c r i p t t e x t . A l l a u t h o r s d i s c u s s e d t h e r e s u l t s a n d c o m m e n t e d o n t h e m a n u s c r i p t .

Soil inorganic N content was higher in 2007 than 2008 ( = 28.9, < 0.001; ). Both N ( = 202.2, < 0.001) and water addition ( = 59.0, < 0.001) significantly increased soil inorganic N content (). Across all treatments, ANPP was higher in 2007 than 2008 ( = 9.9, < 0.01; ). Across the two years, water addition significantly enhanced ANPP ( = 80.6, < 0.001; ) and N addition had no impacts on ANPP ( = 1.9, = 0.19; ). Nitrogen and water additions interacted to affect ANPP ( = 4.4, = 0.05; ), to the effect that the increases in ANPP under water addition were significantly lower in the N fertilized than in unfertilized plots during the two years ( = 7.4, = 0.015; ). Nitrogen uptake by aboveground parts showed substantial inter-annual variation, with higher N uptake in 2007 than 2008 ( = 21.6, < 0.001; ). Both N and water additions significantly stimulated plant N uptake (N: = 24.5, < 0.001; Water: = 61.3, < 0.001; ). The interactive effect of N and water addition on plant N uptake was significant ( = 8.0, = 0.013), with higher enhancement of N addition without water addition (ambient precipitation) than with water addition (increased precipitation). There was inter-annual variation of plant N concentrations across the two years ( = 12.1, < 0.01; ). Nitrogen addition significantly increased plant N concentrations at the community level, whereas the effect of water addition was not significant ( = 159.7, < 0.001; ). There was a significant interaction between N and water addition on plant N concentrations at the community level ( = 33.9, < 0.001; ). For the eight dominant species that together contributed more than 85% of ANPP, N addition significantly increased plant N concentrations in most cases (all < 0.05; ). In contrast, the effects of water addition on N concentrations varied greatly among species, showing positive (A.c. and C.s.), negative (C.k.), and neutral effects (). Nitrogen limitation of primary production is widely distributed in various ecosystems. An increase in net primary production in response to the addition of a limiting nutrient is considered as the classic test of nutrient limitation in a particular ecosystem. Across the two years in this study, N addition showed no significant effect on ANPP, indicating that N would not be the primary limiting factor in this ecosystem. We suspect that the lack of response in ANPP after N addition resulted from an offset between enhanced plant N uptake and decreased plant NUE. Consistent with our hypothesis, we found that N addition enhanced both plant N concentrations and uptake. Positive effects of N addition on plant N concentrations at both community and species levels as found in this study suggest that N addition causes negative effects on plant NUE. Negative relationships between NUE and soil N availability were observed in a variety of ecosystems, including grasslands, forests, and peatlands. However, others found no relationship between NUE and soil N availability due to the inherent trade-offs between the two components of NUE, N productivity and mean residence time of N. Decreased NUE at the community level in response to N addition may result from lower photosynthesis under enriched N conditions. Nitrogen addition may stimulate the growth of annual forbs at the expense of grasses as found in a similar ecosystem, because grasses generally have higher NUE than forbs as reflected by the lower N status in grasses (). Further, NUE could decrease when plant growth becomes limited by other factors. For example, increased P limitation has been triggered by nitrate addition in an annual grassland in Central California. We attribute the increased plant N uptake to higher N availability in soil under fertilized conditions. Average soil inorganic N content was 2.5 times higher in the fertilized plots than that in the control in both 2007 and 2008. Nitrogen addition greatly enhanced the plant N pool, largely due to higher N concentrations in plant tissues in the N enriched plots (). In a previous study, we have shown that N addition significantly increased N concentrations in green leaves of four dominant species in the same ecosystem, which gives further evidence that N addition significantly enhanced plant N status in this ecosystem. As community biomass showed no response to N addition, the enhancement of plant N uptake was mainly driven by changes in plant N concentrations. Water addition significantly enhanced ANPP in this semiarid grassland, irrespective of the ambient precipitation in the two hydrologically different years. Similar observations were reported in other semiarid grasslands receiving various rates of water addition. Moreover, Niu et al. reported that precipitation plays an important role in regulating ecosystem C fluxes in the temperate steppe, such as net ecosystem exchange, ecosystem respiration, and gross ecosystem production. Water availability also has important implications for the responses of ecosystem C and water fluxes to climatic change in the temperate steppe. At the regional level, ANPP increased significantly with increasing mean annual precipitation in the Inner Mongolia grassland of northern China. Together, results from this study and others indicate that water availability is an important driver for primary production and ecosystem C sequestration in the temperate steppe of northern China. The potential changes in precipitation regimes as predicted by climatic models may exert significant impacts on ecosystem functioning of the temperate steppe. A variety of mechanisms have been proposed to explain the positive effects of increased precipitation on ANPP in the arid and semi-arid grassland. The most common explanation is that plants use limiting nutrients, such as N, more efficiently under increased precipitation. However, our results show that water addition did not alter plant N concentrations and consequently plant NUE at the community level in this semiarid grassland, which is also in contrast with our initial hypothesis. Water addition significantly enhanced N concentrations in both green and senesced leaves of four dominant species in this ecosystem. Higher foliar N concentrations could lead to higher photosynthetic rates under increased precipitation, which has been reported by Niu et al. in other water addition experiments carried out in the temperate steppe of northern China. In the present study, however, we found that water addition seldom altered shoot-level and community-level N status, implying a hierarchical response of plant N to water addition from organ to community level. The neutral response of shoot N concentrations and thus NUE to water addition would be a result of similar relative increases in photosynthesis and plant N uptake rates with increasing precipitation. Yuan and Li reported no effect of soil water status on NUE of six species growing in two habitat types with contrasting soil water supply due to the trade-off between N productivity and its mean residence time (MRT), in that species from habitats with high soil water status had higher N productivity but lower MRT than those from habitat with low soil water status. We attribute the higher ANPP in water addition plots to the increases in the plant N uptake. Several mechanisms may contribute to the greater uptake of N by plants under increased precipitation. First, the increase in soil water availability may accelerate soil N cycling. Water addition increased soil N availability, as observed in this study, and may also increase the rate of soil N mineralization in this temperate steppe. Consequently, more N would be supplied for plant uptake under increased precipitation. Secondly, water addition has been found to increase fine root production in nearby ecosystems, allowing plants to explore more of the soil volume for available N. Third, growth of mycorrhizal fungi may benefit from increased C allocation to roots and also from pulsed water availability, such as the water addition in this study. It is well-known that mycorrhizal fungi contribute to the uptake and transport of organic and inorganic N. Increases in plant N uptake and ANPP in response to increased precipitation would result in higher litter production both in quality and quantity, indicating that increased precipitation would lead to a positive plant-soil feedback with respect to ecosystem N cycling in this semiarid grassland. There is no consensus about the relative importance of N and water in regulating ecosystem function of grasslands. We found that water addition stimulated ANPP while N addition did not affect ANPP in this semiarid grassland, suggesting that water availability was more important than N availability in regulating primary production in this ecosystem. The effect of water addition on ANPP was much stronger than that of N addition. In the dry year 2007, ANPP in the +N and +W plots was 12.6% and 62.5% higher than that in the control plots, respectively. In the wet year, these numbers were 28.6% and 63.6%. These results are consistent with Niu et al. who reported that ecosystem carbon and water fluxes and their responses to climate change largely depend on water availability in the temperate steppe of northern China. It is notable that water and N availability interacted to affect plant growth and plant N uptake and use, as indicated by the significant interactive effects of N and water addition on those variables. For example, the enhanced ANPP with water addition was much lower in the fertilized plots than that in the ambient N plots. Those results suggest that the stimulation of plant growth with increased precipitation would be weakened with increased N availability resulting from both atmospheric deposition and global warming in this temperate steppe. The interactive effects between increasing water and N availability on plant N use and primary productivity deserve more attention in modeling responses of terrestrial ecosystems under scenarios of increasing N deposition and altered precipitation regime. It seems self-contradictory to our assertion that this ecosystem was more water-limited than N-limited when in fact ANPP was lower in the wet year than the dry year. We propose that the low ANPP in 2008 was related to the precipitation patterns but not to the precipitation amount in the growing season, especially in July and August. The January-July precipitation is considered the dominant driver for ANPP in this temperate steppe. The total precipitation from January to July was 151.5 mm in 2007 and 238.8 mm in 2008. There were seven precipitation events larger than 10 mm in 2008 (averaged 21 mm per event), accounting for more than 60% of the total amount of precipitation from January to July. In contrast, there were four precipitation events larger than 10 mm in 2007 (averaged 14 mm per event), contributing 38% of the total precipitation from January to July. It is easy to see that the amount of precipitation was largely dominated by large precipitation events in 2008. Pulsed water supply could lead to increased N loss in semiarid grasslands, which may explain our observation that soil inorganic N content was lower in the wet year than in the dry year. In fact, soil inorganic N content in the top 10 cm of the soil in all treatments sharply decreased after an extreme rainfall (63.3 mm) at the end of July in 2008. Decreased soil N availability is the main reason for lower plant N uptake in lower ANPP in 2008 than in 2007. While an increase in precipitation event size increased ANPP in a semi-arid grassland in Colorado due to the alteration in soil moisture, our results highlight the importance of N availability in mediating the response of ANPP to altered precipitation regime. In conclusion, we found that water addition rather than N addition enhanced ANPP in a temperate steppe of northern China. Given the predicted increases of precipitation in mid-latitude regions, the temperate steppe would play an important role in C sequestration due to the increased ANPP as illustrated in this study and the increased net ecosystem exchange found in nearby ecosystems. However, it should be noted that the positive effects of increasing precipitation would be lower under N enriched conditions. Nitrogen enrichment had negative, and increased precipitation had neutral, effects on plant NUE at the community level. However, both factors showed positive effects on plant N uptake, although these effects may occur through different mechanisms. The neutral effect of N addition on ANPP possibly resulted from the offset between enhanced plant N uptake and decreased plant NUE. On the other hand, increases in plant N uptake accounted for the enhanced ANPP after water addition. The variation of ANPP in the two hydrologically different years illustrates that precipitation patterns may exert a strong effect on primary production, largely through its effect on soil N availability. Our findings highlight the importance of plant N uptake in mediating the responses of ecosystem functioning to N enrichment and increased precipitation in the semiarid temperate steppe in northern China. Our experimental site was located in the Xilin River Basin, near the Inner Mongolia Grassland Ecosystem Research Station of the Chinese Academy of Sciences (116°42′E, 43°38′N and 1250 m a.s.l.). The mean annual temperature in this area is 0.3°C, with the lowest monthly temperature (−21.6°C) in January and the highest (19.0°C) in July. The mean annual precipitation is 346 mm, with about 80% occurring in the growing season from May to September. The field experiment was carried out on a natural steppe community, which has been fenced to prevent grazing by large animals since 1999. The soil is a dark chestnut according to the Chinese classification, or Calcic-orthic Aridisol according to US Soil Taxonomy, with 48.6% sand, 26.1% silt, and 25.3% clay in the top 10 cm soil. Mean soil bulk density of the top 10 cm soil-depth is 1.23 g cm, and the soil pH is 7.5. The plant community was dominated by P. Smirn., (Linn.) Keng, and (L.) Gaertn, which are widely distributed perennial C3 grasses in the Eurasia steppe. To examine the effects of increased N and water availability on community composition and ecosystem functioning of the temperate steppe we established five replicate blocks of four 4 m × 4 m plots, separated by 1 m aisles in 2007. Each treatment was replicated five times. We added N in May and July each year, as urea, in dry form in two applications totaling 17.5 g N m yr. This rate of N addition would alleviate N limitation in the grassland of this region, and therefore the amount of N added was much greater than the local atmospheric N deposition. For water addition, 10 mm of tap water was manually applied with a sprayer each week from May to September each year. Water was always applied after 16:00 h to prevent rapid loss by evaporation. In total, water was added 18 times in each year, amounting to an additional 180 mm precipitation, which is about 50% of the long-term mean annual precipitation in this region. The observations in the present study were from two hydrologically contrasting growing seasons, with a total precipitation of 240 mm in 2007 (dry year, with 192 mm in the growing season) and 362 mm in 2008 (wet year, with 295 mm in the growing season) (see online). Consequently, we took this opportunity to test the inter-annual variation of responses of ANPP and N use strategies to N and water addition. Data were tested for normality using the Kolmogorov-Smirnov test and for equality of error variance using Levene's test. For plant N concentrations in , we log-transformed our data to meet model assumptions, whereas for all other analyses we used untransformed data. Repeated measurement ANOVAs were used to examine the effects of N and water addition on soil inorganic N concentrations, ANPP, community N uptake and plant N concentrations at species and community levels, with block as a random factor. Between-subject effects were evaluated as N or water addition treatments and within-subject effects were year. Water effects on ANPP were calculated as [100 × (W - control)/control] in the unfertilized plots and [100 × (NW - N)/N] in the fertilized plots. Here, N, W, and NW refer to N addition, water addition and addition of both N and water, respectively, in the above calculations. Two-way ANOVA was used to examine the effects of N addition and year on the water-induced changes in ANPP. All analyses were conducted using SPSS 13.0 (SPSS Inc., Chicago, USA). The significance level was set at 0.05. X . T . L . a n d X . G . H . c o n c e i v e d a n d d e s i g n e d t h e e x p e r i m e n t . X . T . L . a n d D . L . K . c a r r i e d o u t t h e f i e l d e x p e r i m e n t . X . T . L . , D . L . K . , Z . W . W . a n d F . A . D . a n a l y z e d t h e d a t a . A l l t h e a u t h o r s c o n t r i b u t e d t o t h e w r i t i n g o f t h e m a n u s c r i p t .

Structure of the magnetic-assisted TENG is shown in , which includes the top steel mass with the weight of 18.0 g, top NdFeB permanent magnet with the radius of 9.5 mm and the height of 1.5 mm, silica layer, spiral-shaped electrode wrapped by polyimide, and the bottom NdFeB permanent magnet with the radius of 5.0 mm and the height of 0.5 mm. Each part is located inside a polytetrafluoroethene (PTFE) cylinder. The magnets are Nickel-plated to avoid being corroded. The top magnet also serves as the top electrode of the TENG and has an opposite polarity with the bottom magnet, thus providing a magnetic repulsive force. Silica was coated onto the top magnet as a friction material. To convert mechanical energy into electricity in both triboelectric and electromagnetic mechanisms, the bottom copper electrode was fabricated as spiral shape, forming a coil to generated electricity based on Faraday's law. Polyimide was then fabricated around the spiral-shaped copper electrode as another friction material. To enhance the triboelectric output, nanostructures were created on the polyimide surface using inductively coupled plasma (ICP). Photograph of the whole TENG is shown in . The fabricated TENG is cylindrical with the diameter of 2.4 cm and the height of 2.0 cm. Photograph and scanning electron microscopy (SEM) image of the spiral-shaped copper electrode are shown in . The width, height and spacing of the spiral are 150 μm, 18 μm, and 150 μm, respectively. SEM image of nanostructures on polyimide surface are shown in . Working principle of the magnetic-assisted TENG can be divided into two parts: the triboelectric part and the electromagnetic part. Current flow generated in the triboelectric part is caused by the coupling between the triboelectric effect and electrostatic induction. According to the triboelectric series, the silica surface will be positively charged while the polyimide surface is with negative charges after cycles of contact and separation of the two materials. In this case, if the top and bottom electrodes are connected, charges will redistribute on both electrodes due to the electrostatic induction. Then, as shown in , given an external force, the gap distance decreases, leading to increased electric field () between the top and bottom plates. To balance the electric field, electrons will flow from the top electrode to the bottom electrode. When the external force is removed, the magnetic repulsive force will push the top plate to the original position, as shown in . This brings about the decrease of electric field, in which case a reversed current flow is produced. To verify the change of electric field, finite element method (FEM) simulations were conducted in COMSOL software package. A 2D axisymmetric model was established and the electric potential at infinite was set as zero. Charge densities on the friction surfaces were set as 5 μC/m and −5 μC/m, respectively. By changing the gap distance, different electric field distribution is achieved as shown in , which is consistent with the analyzed working process. Current flow of the electromagnetic part is caused by the electromagnetic induction. As shown in , when applying an external force, the magnetic flux () increases with the decreasing of gap distance, thus inducing current flow in the spiral-shaped electrode. Then, by removing the external force, magnetic force makes the gap distance increase, as shown in . In this case, magnetic flux decreases, causing a reversed current flow in the spiral-shaped electrode. Correspondingly, FEM simulations about the magnetic flux density distribution were carried out. The geometry was also 2D axisymmetric with zero magnetic scalar potential at infinite. In this model, relative permeability of the magnetic material (, NdFeB) was set as 1.044. Magnetization of the top and bottom magnet was set as −838000 A/m and 838000 A/m in Z-direction, respectively. As shown in , when the gap distance decreases, large magnetic flux density is obtained. The output performance of the magnetic-assisted TENG was measured with a 5 Hz external force. As shown in , with a 100 MΩ probe, output voltage of the TENG reaches to 90 V. By connecting a 100 kΩ external resistance parallel to the TENG, output current is recorded with a peak value of 6 μA, as shown in . Based on the theoretical calculation, the output curve of TENGs is dependent on the external resistance. The external resistance will not only affect the peak shape, but also influence the output value. Therefore, to further characterize the output performance of the TENG, output voltages under different external loads were investigated. As shown in , peak output voltage raises with the increasing value of the external loads, and the growth rate slows down at high resistance. As a result, peak output power reaches maximum at 16.67 MΩ, with the value of 170 μW. Besides the triboelectric output, the movement of the magnet will induce voltage in the spiral-shaped copper electrode. The induced voltage was first measured with a 1 MΩ probe. As shown in , peak voltage of 71.86 mV is achieved in the electromagnetic part. Compared with the triboelectric output, voltage generated by the electromagnetic induction is relatively low. However, large current can be obtained thanks to the low resistance between the two ends of the spiral. The induced current was then measured with a 0.5 Ω external resistance and peak current of 12.66 mA is achieved as shown in . Since the inner resistance of the electromagnetic part is low, good output performance can be achieved at very low resistances even below 10 Ω. As verified in , the output voltage increases dramatically as the load resistance varies from 0 to 32 Ω, and remains almost unchanged at higher resistance. Maximum peak power of 204 μW is obtained at 16 Ω, indicating low optimized resistance of the electromagnetic part. In the separation process of two friction surfaces, gravity of the mass is used to balance the magnetic repulsive force. By reducing the radius and height of top magnet to 5.0 mm and 0.5 mm, the magnetic repulsive force can be reduced. Furthermore, we increase the weight of top mass to 36.0 g and the gravity can balance the magnetic force at a very short distance. When the TENG is inclined, influence of the mass gravity will be weakened, thus enhancing the separation speed and distance. The contribution of the magnetic force () and gravity along the motion path () can be expressed as: where is the vacuum permeability, is the magnetization of the magnet, and are the radius and height of the cylindrical magnet, is the distance between two magnet, is the value of top mass, is the acceleration of gravity, is the tilt angle of the TENG, and is the friction coefficient between the mass and PTFE cylinder. Based on the above equations, the displacement and speed of the moving mass versus time can be calculated through a Simulink model. When the tilt angle increases, gravity component reduces, which leads to the increment of maximum displacement. As shown in , the maximum displacement increases from 3.48 mm to 12.71 mm as the tilt angle changes from 0° to 90°. The time required to reach the maximum displacement also increases with the tilt angle. Due to the distance-dependent magnetic force, velocity of the moving mass reaches maximum at a certain position. shows the velocity-time curve at different tilt angles, indicating that larger tilt angle contributes to faster movement. Based on the theoretical study of the contact-mode TENG, relative movement between the two friction surface (, the movement of the top mass) will affect the output performance of TENGs. A differential equation of the transferred charges can be expressed as: where is the transferred charges, is the charges on the friction surface, is the movement equation, is the area of the friction surface, is the external load resistance, is the vacuum permittivity, and ( = 1, 2) are the relative permittivity and thickness of the two friction materials, respectively. Based on , time domain output current can also be obtained from the Simulink model. In addition, by varying the tilt angle, will be changed thus affecting the output. To demonstrate the magnetic-assisted TENG as a self-powered omnidirectional tilt sensor, output measurement under different tilt angles was first conducted. As shown in , normalized separation peak voltage versus tilt angle is investigated both experimentally and theoretically. When the tilt angle is small (, less than 30°), the peak voltage shows no significant change, due to the basically unchanged separation velocity. As the tilt angle increases (, from 30° to 90°), the peak voltage raises and shows a nearly linear relationship with the tilt angle. The measured time domain output voltage is shown in . For omnidirectional sensing, two TENGs perpendicular to each other were mounted to a cube. As illustrated in , in the original state, the tilt angles of the two TENGs are 0° and 90°, respectively. With an arbitrary incline (, tilted state in ), the tilt angles of the two TENGs will be changed, thus affecting their output voltages. An arbitrary tilt angle can be divided into two parts: rotating around X-axis, and rotating around Y-axis. Then the tilt angles of the two TENGs (, and ) can be expressed as: A plot demonstrating the relationship between , and , is shown in in the . By recording the output voltages of two TENGs, and can be estimated. On this basis, the value of and can be obtained to determine the specific tilt angle. Experimental measurement was conducted under 16 tilt angles in different directions (both and vary from 0° to 90°), as shown in and . In this way, both the magnitude and direction of the tilt angle can be obtained in this self-powered tilt sensor. In addition, a self-powered system based on this magnetic-assisted TENG was built for visual display (see Video S1). As shown in , the TENG was mounted to a slope. A height adjuster was used to change the tilt angle. The output voltage of the TENG is divided thorough a 5 MΩ resistor and then rectified through a diode, keeping only the voltage at the separation process. The half-wave rectified voltage was then utilized to power a LCD-based visual sensor, as shown in . At different tilt angles, the voltage applied to the LCD-based visual sensor changes, thus affecting the final display of the LCD. As shown in , when operating at the tilt angle of 60°, only the first square displays in the LCD screen. Then, as the tilt angle increases to 70° and 80°, the second and third squares in the LCD screen become visible. This self-powered system directly converts the tilt angle information to visual display, without the need to record and analyze the output signal. Therefore, complex measurement device can be removed, which makes the self-powered system totally electric-free. The demonstrated visualized system greatly promotes the development of self-powered system and shows potential applications in the field of self-powered intuitive sensing. This magnetic-assisted TENG has a wider working range against the load resistance compared with previous TENGs, as plotted in in the . When the external load is at MΩ level, the triboelectric part can provide considerable power output. As the load resistance drops to tens of ohms, power output of the triboelectric part decreases nearly to zero. Drastically reduced power output is a common problem for traditional TENGs. In this magnetic-assisted TENG, the moving magnet and spiral-shaped electrode contribute to electromagnetic output, which broadens the application field of the TENG at low resistances. In addition, in the working process, the restoring force is provided by two permanent magnets instead of traditional mechanical structures such as springs or cantilevers. The magnetic repulsive force is accurate and stable, which is not affected by the mechanical fatigue. Stability of the TENG was measured with a 5 Hz periodic external force. Detailed stability measurement is shown in in the . Compared with other tilt sensors based on electrolysis, induction and optics, this triboelectric-based self-powered tilt sensor has the following advantages. Firstly, structure of this tilt sensor is simple, without the complicated fabrication process and liquid environment. Secondly, high resolution of about 400 mV per 1° is achieved due to the high output voltage of the TENG. The high output voltage also makes this tilt sensor not easily affected by the noise signal. Thirdly, tilt angle as large as 90° can be measured in all directions thanks to the two mutually perpendicular TENGs. Finally, the tilt sensor is self-powered without any external power supply, which is energy-saving and can be applied in remote areas and emergency power outage situations. In summary, we present a magnetic-assisted TENG and demonstrate it as a self-powered visualized omnidirectional tilt sensing system. The specially designed spiral-shaped electrode enables this TENG to generate electricity through both triboelectric and electromagnetic mechanisms, with the peak power densities of 541.1 mW/m and 649.4 mW/m, respectively. Moreover, this TENG can work at a wide range against the load resistance thanks to the output of the electromagnetic part. By carefully controlling the mass and magnetic force, the TENG can be used as a self-powered tilt sensor, with a high resolution of 400 mV per 1°. Theoretical calculation and experimental measurement of the output voltage at different tilt angles are conducted and shows good consistency. In addition, omnidirectional tilt sensing is realized by two TENGs fixed perpendicular to each other. For visualized sensing, a self-powered system is established based on the LCD screen. This self-powered visualized system eliminates the complicated measure process, and brings great opportunity for the further development of self-powered systems. Fabrication of the poyimide wrpped copper electrode was conducted using a polyimide substrate with copper foils on both sides (). Copper was then electroplated on both sides with the thickness of 18 μm (). Next, photoresist was spin coated and patterned on both sides (). Later, the electroplated copper on both sides was wet etched by FeCl (). After removing the photoresist (), another layer of polyimide was coated on both sides as the insulate layer (). Nanostructures on the polyimide substrate was created using ICP. O with a flow rate of 10 sccm was introduced to the chamber. Power of 400 W and 100 W was set to generate and accelerate the plasma ions. After 600 s, nanostructures were fabricated on the polyimide substrate. To measure the triboelectric output voltage, a 100× probe (HP9258) was conneceted to the oscilloscope (Agilent DSO-X 2014A). The equivalent internal resistance is 100 MΩ and the corresponding settings in the oscilloscope was adjusted to 100×. To measure the triboelectric output current, a 100 kΩ external resistance was connected parallel to the TENG and the measured signal was calculated to the current value. In the electromagnetic part, 1 MΩ probe was used to measure the voltage and a 0.5 Ω external resistance was parallel connected to measure the current. The Simulink model includes two parts: mechanical part, and electrical part. The mechanical part is established to obtain the time domain movement equation of the TENG based on the distance-dependent magnetic force. Considering the magnetic repulsive force, gravity, and the friction force, the total acceleration applied to the top mass of TENG can be expressed as: where is the acceleration, and has been expressed in and. Then, after two integral, time domain displacement is obtained and can be used to recalculate the magnetic force. Detailed Simulink model is shown in the red box in . The parameters used in the calculation are listed in . Based on the movement equation , electric output of the TENG can also be calculated. Through the quasi-infinitely large plane model and Kirchhoff's law, the current can be expressed as: where is the transferred charges, is the charges on the friction surface, is the movement equation, is the area of the friction surface, is the external load resistance, is the vacuum permittivity, and ( = 1, 2) are the relative permittivity and thickness of the two friction materials, respectively. Combined with an ordinary differential equation of can be obtained as: where , , . The Simulink model to slove this differential equation is shown in the blue box in . Calculation results by the Simulink model is shown in . By changing the tilt angle , different movement equations are obtained, which will affect the final time domain output voltage shown in . Detailed time domain displacement and output voltage at the tilt angle of 90° are shown in , respectively. M . H . , X . S . Z . a n d H . Z . c o n c e i v e d t h e i d e a a n d d e s i g n e d t h e e x p e r i m e n t . M . H . a n d X . S . p e r f o r m e d t h e m e a s u r e m e n t s a n d a n a l y z e d t h e d a t a . B . M . d i d t h e f a b r i c a t i o n w o r k . W . L . s e t u p t h e t e s t p l a t f o r m . M . H . , X . S . Z . a n d H . Z . w r o t e t h e p a p e r a n d a l l a u t h o r s p r o v i d e d f e e d b a c k .

The selected maize inbred lines used in this study were recombinant inbred lines developed from F plants of the cross Mp715 × Va35. There were two identical copies for each gene in each maize inbred line. Only up to two different alleles for each gene were present among all the six maize inbred lines because they were offspring lines from a single cross. To determine the resistance or susceptibility of each maize inbred line, aflatoxin accumulation was evaluated using the mean values of aflatoxin concentrations per 50 g ground mature kernels. Aflatoxin concentrations of the six maize inbred lines (Mp718, Mp719, Mp04:104, Mp04:89, Mp04:85, and Va35) were evaluated along with other maize inbred lines planted in the field. Each maize inbred line had three replications and was subjected to two treatments (inoculated and non-inoculated with ).The six maize inbred lines exhibited four levels of aflatoxin accumulation (). Mp719 exhibited the highest level of resistance among the six maize inbred lines with a significantly low value of aflatoxin concentration (53 ng/g). The susceptible maize inbred line Va35 was highest in aflatoxin concentration with a mean value of 1821 ng/g. The significance levels in the differences of aflatoxin accumulation levels among the tested maize inbred lines have been consistent over field trials for multiple years. Total RNA samples were prepared from developing kernels of the six resistant and susceptible maize inbred lines resulting in a total of 72 samples. Quantitative RT-PCR analysis and standard curve assays were performed and PCR efficiencies were calculated (). Test of RT-qPCR primers was performed on a total of 66 maize genes initially, including 50 RNA transport pathway genes and 16 differentially expressed candidate genes identified from previous studies. Out of the 66 RT-qPCR primer evaluation assays, 56 gene primers yielded RT-qPCR data of good quality with a PCR efficiency (in r-squared value) >0.9 () and therefore were included for the subsequent whole plate data analysis. The primer sequences for the selected RNA transport pathway genes are listed in . The gene IDs and functions of all the 56 tested genes are listed in . There were three whole plate assays for the reference gene GAPDH. Missing values in one GAPDH assay were corrected by calculation of the corresponding values from the other GAPDH assays. The relative delta C values obtained from preprocessing the raw RT-qPCR data were used as the gene expression values for the subsequent descriptive statistical analysis, analysis of variance (ANOVA), correlation analysis, and network analysis. Summary of the distributions of the gene expression values were presented by boxplots in with the median, spread and outliers showing for each gene. Large amount of outliers in the expression values from RNA transport pathway genes were observed. Since there were only up to two different alleles for each gene being involved in all 72 samples, the abundant expression variations observed in these genes indicated that different gene regulating patterns existed in these maize recombinant inbred lines. Scatterplots were used to evaluate if there were any trends present in the regulating patterns for each gene related to resistant or susceptible maize inbred lines (). Four examples are shown in regarding the regulating trends in gene expression patterns discovered in this study. A translation initiation factor gene eIF5B appeared to express consistently among samples (). The nucleoporin Nup133 gene had significant variations in gene expression values with down-regulation patterns showing in resistant maize inbred lines (). AI664980 was another example showing significant variations among samples with down-regulation patterns in resistant maize inbred lines (). TC231674 was found highly expressed in the resistant maize inbred line Mp718. It showed significant variations among samples with up-regulation patterns in resistant maize inbred lines (). Analysis of variance (ANOVA) was used to determine the significant levels of the differentially expressed genes among different groups and to identify the sources of the variations associated with maize resistance to aflatoxin accumulation. Contrasts for ANOVA analysis were constructed among maize inbred lines (by pedigree) and between resistant and susceptible groups (by RES). shows the p values for each gene obtained from ANOVA analysis based on the general linear models by RES, pedigree, RES*INOC, or pedigree*INOC. Of the 56 genes analyzed, 23 were differentially expressed among the tested maize inbred lines at a significance level of p < 0.05 (, column Pedigree), and 17 were found significant in expression differences between the resistant group and the susceptible group at p < 0.05 (, column Res). These significant genes included the nucleoporins Nup133, Nup62, Nup160, Nup85, Nup88, Nup53, UBC9, SUMO, and Sec13; the Survival Motor Neuron complex genes Ran, TGS1, SPN1, IPOB, plcln, and PRMT5; the Exon-Junction Complex genes MAGOH, Sap18, Ref_Aly. Most of the significant genes identified among the RNA transport pathway genes were from the NPC and SMN protein complexes. Some of the previously identified candidate genes BG266083, CD443591, BE050050, TC231674, CA399536, AI065864, AI664980, TC238832, BM379345, and TC247683 were again found differentially expressed in this research that had a set of germplasm different from the previous studies. None of the translation initiation factors (EIFs) were found differentially expressed among the resistant maize lines and susceptible lines ( and ). To determine if there were co-expression patterns in gene expression between all pairs of the tested genes, correlation analysis was performed on gene expression values and the correlation matrices were visualized by using R package “Corrgram”. are correlograms displaying the correlation matrices of Pearson's correlation coefficients between selected pairs of genes. The genes displayed in were selected from the significant genes identified from the ANOVA analysis presented in different functional groups. Correlations displayed in a correlogram were organized in the order that genes have similar expression patterns were grouped together. The signs and values of the Pearson's coefficients were reflected schematically with the correlation coefficients and the 95% confidence intervals displayed at the lower triangle, whereas the color-coded pie graphs in the upper triangle. showed expression correlations among genes in the NPC and SMN protein complexes of maize RNA pathways along with other previously identified candidate genes. Resistance related genes BE050050, TC231674, and BM498943 were found positively correlated to each other with a coefficient being at least 0.66. Resistance related gene TC238832 was highly correlated with the SUMO gene, an ubiquitin related disease defense gene. Susceptibility related gene AI664980 was found positively correlated to a nucleoporin gene Nup62 (0.88) and negatively correlated with a resistance related gene BE050050 (−0.97). showed the expression correlations among selected RNA pathway genes in the EIFs, EJC, and TREX protein complexes. Susceptibility related gene BG266083 was highly correlated with Sap18 which was an Exon-Junction Complex gene. A TREX gene THOC7 was found negatively correlated with the resistance related genes TC231674, BE050050, and BM379345, which suggested that down-regulation of the THOC7 gene was likely involved in maize defense responses. is an eigenvector plot showing the results from a Principal Component analysis (PCA) analysis on the correlation coefficients of the selected significant genes. The distance between genes in the correlation coefficients was illustrated by the angle formed between the gene eigenvectors. The length of an eigenvector represents the largest variance for each gene in the correlation coefficients. Gene eigenvectors placed close to each other were more similar in the expression patterns and hence were more positively correlated. For instance, THOC7 and Nup62 were found to be highly correlated with the expression of the susceptibility related gene AI664980. On the other hand, UBC9 was found to be positively correlated with the resistance related genes TC231674, BE050050, and BM498943 genes. The inclusion of previously identified candidate genes in this research provided a way to make some of the observations found in this study experimentally verifiable. Based on the directions of the gene eigenvectors, the expression of resistance related gene TC231674 (on chromosome 5, highly expressed in resistant maize inbred line Mp718) was shown positively correlated with the expression of resistance related gene BE050050 (close to maize resistance SSR marker bnlg2291 on chromosome 4) and negatively correlated with the expression of the susceptibility related gene AI664980 (GRBP2, highly expressed in susceptible maize inbred line Va35) across all the tested maize inbred lines in this study. This observation was consistent with the previous findings on the expression patterns of these defense related genes in a different set of germplasm where TC231674 was found highly expressed in a different resistant maize inbred line Mp313E. The genes selected from the RNA transport pathways were considered as elements in a static gene network in terms of potential biological processes. In order to determine the dynamic roles and relations in expression of these genes responsive to infection, we wanted to explore methods to construct empirical gene relational networks that were based on the variations in the actual gene expression levels. To achieve this goal, we conducted PCA on the gene expression data. The scores of the first two principal components (pc1 and pc2) associated with each gene were used to calculate a Euclidean distance matrix between all pairs of genes for the network construction. are network graphs constructed based on the Euclidean distance matrices. The vertices in the network represented genes. The edges represented the Euclidean distance between each pair of genes on the pc1 × pc2 plane. To highlight genes by protein complexes or groups, the vertices were color-coded for the seven different groups where the genes were chosen from. Five of the groups (EIFs, EJC, NPC, SMN, and TREX) represented the genes from different protein complexes in the RNA transport pathways and two groups (RES and SUS) represented the candidate genes selected from previous studies which were included in this research. is a network constructed for all the 56 genes in this study. The connectivity threshold was set arbitrarily as the Euclidean distance value being 2 for an exploratory criterion. Twenty-four genes were connected in the network. Six resistance related genes, including TC231674 and BE050050, appeared as isolates in the network. Two susceptibility related genes BG266083 and AI065864 were not connected to any of the tested genes either. Further experiment with more genes will be required to reveal genes closely related in expression patterns with these genes in the empirical expression relational networks regarding maize defense. is a subgraph extracted from the same network dataset to show the genes that were connected in the network at a threshold of 1.6 in the Euclidean distance value. Seven genes (Nup88, eIF2, CD443591, CA399536, SPN1, AI664980, and MAGOH) had the highest vertex degrees and were clustered closely together, suggesting a co-expression pattern of these genes in response to infecton among the tested maize inbred lines. Two other centers (hubs) were also revealed in the subgraph in . The resistance related gene BM379345 was found at the center of co-expression with five tested genes (IPOB, Nup62, eEF1A, eIF2, and Nup88). Nucleoporin genes Nup160, Sec13, and Rae1 were hubs for co-expression with genes PYM, Ref_Aly, eIF5B, pinin, and TC247683. The hubs with multiple connections were considered as genes of important roles in the network of cellular functions. The susceptibility related gene AI664980 was found adjacent to multiple RNA transport pathway genes including the Nup88, MAGOH, PAPB, and SPN1 genes and appeared to play an important role in the defense related nucleocytoplasmic trafficking activities based on the statistical inferences from network analysis. Maize genes at the centers in the network will be considered as important candidate genes and will be used in priority for further maize DNA marker studies. Numerous studies have shown that the resistant maize inbred lines exhibited significantly low levels of aflatoxin accumulation. Determination of the mechanisms underlying such maize host resistance to aflatoxin accumulation has been proven difficult due to the complex nature of this quantitative trait. Many genes were found to be involved in the maize host plant resistance. The exploration for methods to describe functional roles and relations of genes statistically using effective experimental designs and empirical gene expression data will expedite the discovery of DNA markers and uncover the mechanism of maize host resistance. In this study, we conducted RT-qPCR gene expression analysis on 56 genes including genes from RNA transport pathways that comprise the potential components of maize host resistance. The functions and relations of the genes were examined from three aspects: 1) statistical analysis (ANOVA) on gene expression data for the identification of differentially expressed genes and the determination of the significance levels; 2) correlation analysis for delineation of genes positively correlated or negatively correlated in response to infection; and 3) network analysis for depiction of relations of genes in the empirical functional network. Significant genes related to maize defense to infection were identified. Through the application of multidisciplinary methods, a wealth of data was generated for data mining and experimental validation. Evidence and supportive data have already been found through complementary research projects. For example, two differentially expressed genes, AI664980 and BG266083, which were found significant in the susceptible maize inbred line Va35 from previous reports, showed high significance again from this study in susceptible maize inbred lines (Mp04:85, Mp04:89, Va35). These genes are known to be involved in plant responses toward various stress and pathogens. Statistical inferences drawn from our RT-qPCR gene expression analysis indicated that genes in RNA transport pathways, especially in NPC and SMN complexes, were highly significant and involved in maize resistance. One supporting example was that the resistance related gene TC231674 found previously in a different maize resistant inbred line Mp313E was highlighted again in the resistant maize inbred line Mp718. Interestingly, the highly expressed gene TC231674 found in two resistant inbred lines was homologous to the human nucleoporin Nup85 but it was not the same gene as the maize Nup85. The role of TC231674 gene in terms of interactions with maize nuclear pore complexes (NPCs) is yet to be determined. Numerous additional examples can be used to show the richness of data yielded through the combination of the multidisciplinary methods in this study. One resistance related gene TC238832 was found positively correlated to the SUMO gene in the nuclear pore complex of the RNA transport pathway (). showed the relationship between these two genes according to the respective eigenvectors. The small angle between the two vectors represented a comparison of the Pearson's coefficients and a positive correlation between the TC238832 and SUMO genes. A similar but stronger relationship was noticed between the resistance related gene BM379345 and the eIF5B gene (), a translation initiation factor in the RNA transport pathway. The nuclear cap-binding protein subunit 2 gene (CBC gene) was another gene found positively correlated to both BM379345 and eIF5B. The positive correlations among CBC, eIF5B, and the BM379345 genes showed relationships that hinted at the possibility of these genes being related to resistance. By comparing these genes to the differentially expressed genes associated with susceptibility or resistance, we can gain new insights on the functions of the genes involved in the RNA transport and the plant defense mechanisms. A network was constructed showing maize genes closely related in terms of the magnitudes and directions of their largest variances in the expression values among the resistant and susceptible maize inbred lines. Applying network-based methods to describe empirical gene expression data was an exploratory strategy we investigated to reveal genes potentially important in the regulation of host-fungus defense responses. It provided new strategies on prioritizing candidate genes. Information revealed by network analysis also provided more insights into the roles of highly expressed resistance related genes whose functions were yet to be characterized. While this study resulted in promising results, more research and analysis, such as testing of DNA markers associated with the resistance related genes, are required to verify the results and determine the mechanisms of maize host plant resistance to infection and alfatoxin reduction. Six maize inbred lines (Mp718, Mp719, Mp04:104, Mp04:85, Mp04:89, and Va35) were used in this experiment. Five of them (Mp718, Mp719, Mp04:104, Mp04:85, and Mp04:89) were recombinant maize inbred lines obtained by eight generation selfing from F plants of a cross of Mp715 × Va35 and were selected against aflatoxin accumulation under inoculation in field conditions. The maize inbred line seeds were maintained by the United States Department of Agriculture, Agricultural Research Service, Corn Host Plant Resistance Research Unit (USDA-ARS-CHPRRU) at Mississippi State University. Mp718, Mp719, and Mp04:104 were maize inbred lines showing resistance to infection and aflatoxin accumulation. Mp04:85, Mp04:89, and Va35 were susceptible to . All maize lines were planted at the R. R. Foil Plant Science Farm at Mississippi State University. The experimental design was a randomized complete block design including three replications and two treatments (inoculated and un-inoculated with ) for each maize inbred line and two sample collection time points (2 and 7 days after inoculation). All primary ears were self-pollinated. Fourteen days after pollination, the inoculation of was performed using the strain NRRL 3357 (ATCC # 200026; SRRC 167). The procedure of fungal culture preparation and the fungal inoculation with side-needle technique were the same as described previously. Two and seven days after inoculation, which was 16 and 21 days after self-pollination, developing kernels from inoculated and uninoculated primary ears were collected for RNA preparation. All remaining primary ears from each plot were harvested at maturity and processed for measurement of aflatoxin concentrations as previously described. Developing kernels were collected from the resistant maize inbred lines (Mp718, Mp719, Mp04:104) and the susceptible maize inbred lines (Va35, Mp04:85, and Mp04:89), flash frozen in liquid nitrogen in the field, and stored at –80°C for further analysis. Total RNAs were isolated from the kernels using the BioRad Aurum™ Total RNA Fatty and Fibrous Tissue kit. Frozen kernels were ground into powder under liquid nitrogen and combined with PureZOL for disruption. Chloroform was added to the sample for extraction of the aqueous phase containing the RNA. The sample was subjected to DNase I treatment and followed by a series of washes and centrifugation steps with solutions provided with the kit. Upon completion, total RNA concentrations were determined using a NanoDrop® ND-1000 Spectrophotometer. The Quality control assessments of total RNA was performed with an Agilent 2100 Bioanalyzer. RNA samples with a QC value RIN >8 were used for cDNA synthesis. ThermoScript RT-PCR system (Invitrogen,

To investigate phage resistance mechanism in , wild-type strain PA1 was infected with phage PaP1 (MOI = 1000) and screened for phage-resistant mutants, which could be consistently recovered in high frequency (3.02 ± 0.85 *10) (). Interestingly, a high percentage (31% ± 13.2%) of the mutants produced red pigment, and two coupled phenotypes can be stably maintained, indicating genetic mutations. One Red mutant, PA1r, was randomly selected for further analysis, and its PaP1 resistance, as well as red-pigment producing phenotypes, was confirmed by dot assay on an agar plate (). To identify the mutation(s) in PA1r that might confer PaP1 resistance, as well as red-pigment producing phenotypes, the genomes of both PA1 and PA1r were sequenced (GenBank: PA1: CP004054, PA1r: CP004055) and subjected to comparative genomic analysis. A 219,572 bp genomic fragment deletion (6,217,866 to 6,437,440) in PA1r compared with PA1 () was observed. Sequences flanking the end points are shown in . The deletion in PA1r was confirmed by pulsed-field gel electrophoresis (PFGE) () and polymerase chain reaction (PCR) (, Primer sets in Materials and Methods). The precise deletion site in PA1r was verified by PCR (, Primer sets 6U/6D). Results showed that the deleted fragment in PA1r was flanked by gene and compared with another type of strain LESB58. Bioinformatic analysis revealed that the deleted 219.6 kb genomic fragment contains 192 genes (), including , a gene encoding the enzyme homogentisate-1, 2-dioxygenase that converts homogentisic acid into 4-maleylacetoacetate. Inactivation of this gene results in the accumulation of homogentisic acid, which gives the cell a red pigmentation phenotype. An insertional deletion mutant (PA1) was generated. PA1 produced red pigment but still retained sensitivity to phage PaP1 (), and complementation of gene in PA1r background did not restore the phage-resistant phenotype (). These results suggest that gene deletion does not confer PaP1 phage resistance. A random transposon mutant library of PA1 was infected with phage PaP1 to identify genes in that are essential for phage PaP1 infection. Two phage-resistant mutants (B10 and T2) () were isolated and transposon insertion sites were determined by a two-step semi-degenerate PCR. The mutation in B10 and T2 was mapped to genes (wzz2) and , respectively, both of which have been shown to be involved in LPS biosynthesis. Given that LPS often serve as phage-receptors in a variety of bacterial species, our data suggested that PaP1 could potentially recognize PA1 LPS as receptors and initiate the infection process. Agglutination test was performed with wild-type PA1 and PA1r strains to determine whether the resistance of PA1r to PaP1 infection was due to its defect in LPS biosynthesis. Results showed that PA1 remained as single cells, whereas PA1r tended to form cell clumps (), suggesting that PA1r carries defective LPS. Adsorption assays also revealed a significant reduction in phage adsorption to PA1r compared with wild-type PA1 strain (). The result of the binding assay was further confirmed by transmission electron microscope data. After 3 min co-incubation, a significantly higher number of phage PaP1 particles can be seen attached to the surface of PA1 cell than those binding to PA1r (). Agglutination assay and phage adsorption data suggested that a possible defect in LPS biosynthesis might confer PaP1 phage resistance in PA1r. A detailed bioinformatic analysis revealed within the deleted fragment the presence of , a gene involved in LPS biosynthesis. This gene encodes UDP-D-glucose pyrophosphorylase which converts Glc-1-phosphate to UDP-D-Glc, which is the predominant sugar in the outer core of oligosaccharides. A mutant is devoid of O-antigen and synthesizes a defective LPS core. Thus, a insertional deletion mutant (PA1 ) was constructed. PA1 showed a defect in LPS and was resistant to phage PaP1 infection. Moreover, was complemented into PA1r resulting in PA1r:, which was sensitive again toward this phage (). Three more Red mutants were randomly chosen and subjected to PCR and PFGE for further analysis to determine if Red mutants carry the same deletion. All three Red mutants (PA1R-37, PA1R-57, and PA1R-61) revealed genomic deletions with sizes similar to PA1r (). The deleted fragments ranged from approximately 200 kb to 300 kb, encompassing the region containing both and genes. However, the exact deletion sites among these mutants were different. Our data suggested that PA1r acquired phage resistance by deleting a large genomic fragment that contains gene essential for LPS biosynthesis. Although the defect in LPS confers resistance to phage infection, this defect might reduce the mutant's ability to infect a host, because LPS is an important virulent factor in Gram-negative bacterial pathogens, including . To test this hypothesis, PA1r was used to infect mice intraperitoneally, and their survival was monitored. All eight mice died within 24 h after infection with either PA1 or PA1r:, whereas all eight mice survived the infection with PA1r or PA1 (). Fluctuation test was performed to demonstrate whether the genomic fragment deletion is induced after phage infection. As is shown in , the number of red colonies on each plate varied drastically, from 2 to 45 colonies, in Group A; while the variation from group B is very small. This implies that deletion mutation arises before phage infection. In this study, a series of phage-resistant mutants were isolated, and almost 30% of these mutants produced red pigment. However, by reviewing the phage resistance mechanisms, no gene has been related to pigment production. Comparative genomic analysis revealed a 219.6 kb genomic fragment deletion in resistant mutant PA1r, including , which has been demonstrated to be associated with red pigment production (). However, the loss of could not confer phage resistance to PA1 (). We further demonstrated that the loss of , a key gene involved in LPS biosynthesis, which is 19 kb in front of , confers phage resistance to PA1r by preventing phage adsorption because LPS is the likely receptor of PaP1 (). Adsorption to the receptor is the initial step for phage infection. Several phage receptors in Gram-negative bacteria, such as LPS, capsular polysaccharides, flagella, and outer membrane proteins, have been identified. Mutation of the receptor is a common strategy for bacteria to avoid phage predation. We used an agglutination assay to show that PA1r carried defective LPS (). Given that the core oligosaccharide is abundant with negatively charged groups of 2-keto-3-deoxyoctulosonic acid and phosphate, strains that lack O polysaccharide will expose core oligosaccharide to the cell surface and cover up the negative charges. Therefore, LPS negative strains could be agglutinated by high salt concentrations. One of the major concerns regarding phage therapy is that phage-resistant mutants might result in the failure of phage therapy. According to our experiment, phage-resistant bacteria are swift to emerge, and the frequency is much higher than those of drug-resistant mutants. However, phage-resistant mutant PA1r has defective LPS, and LPS is a major virulence factor in . Thus, LPS minus mutant PA1r is significantly attenuated in the mouse model (). The trade-off between phage resistance and fitness, such as growth rate or virulence, has also been reported in other phage-host experiments, which suggests an advantage of phage therapy. From a genetic perspective, the genomic fragment deletion as a response to phage predation and the underlying mechanism of phage resistance have not been reported. Phage resistance is often acquired by mutations, DNA fragment insertion (CRISPR), or genomic rearrangements. Our study demonstrated that bacteria could defend phage infection by deleting their own genomic fragment containing gene essential for phage infection. The two main causes of genomic fragment deletion are spontaneous and site-specific. Spontaneous deletion is caused by the dysfunction of DNA replication and repair system. In this case, the deletion site is random and the deletion rate is quite low. For example, Tanji isolated a phage-resistant strain D198, which revealed a 209.4 kbp spontaneous deletion. However, this deletion was spontaneous and not repeatable. In an evolution experiment with after 20,000 generations, only seven deletions were found, ranging from 1 bp to 23,293 bp. In , the DNA loss rate was estimated to be 0.05 bp per chromosome per generation. Sanna estimated the apparent deletion rate in with chromosomal variation ranging from 0.5 × 10 to 2.2 × 10/cell/generation. Another experiment with also revealed random fragment deletion, but the deletion sites were unspecific and the frequency was quite low. By contrast, site-specific deletion is mediated by prophage or other mobile genetic elements. These large DNA fragments are often genomic islands acquired by horizontal gene transfer and could be excised and integrated at a specific site mediated by integrase and other enzymes. This kind of deletion may usually occur at a high frequency. For example, an 89 kbp pathogenicity island in , which is flanked by a 15 bp direct repeat, could be excised from the genome with a certain frequency (~3.2 × 10). In our experiments, the deletion events showed three clear novel features: high frequency, large genomic fragment deletions (200 kb to 300 kb), and no site-specificity. Thus, this deletion is neither site-specific nor spontaneous. Interestingly, smaller deletions are certainly favored in bacteria because the cost would be smaller, although the deletions in our experiment ranged from 200 kb to 300 kb. However, by reviewing the genes within the deleted region, none of the genes, including toxin genes, are obviously deleterious (). Therefore, this might be a novel deletion mechanism, and it is currently investigated in our laboratory. Another interesting issue is whether the observed deletion was induced by phage infection or occurred before phage predation. The fluctuation test is a classical experiment to prove that phage resistant mutants arise before phage infection. If genomic fragment deletion was caused by an induced activation in bacteria, then each plate should contain roughly the same number of red colonies, because the deletion frequency should be consistent in the bacteria population. However, we found that the number of red colonies on each plate varied drastically, from 2 to 45 colonies in Group A; while the variation from group B is very small (). This implies that deletion mutation arises before phage infection. What's more, the replication time of bacterium PA1 is 40 to 60 min at log phase (), and the infection cycle of phage pap1 is about 40 min. Thus, within 40 min, the bacterium is unlikely to be able to delete its genomic fragment and losses LPS, because phage already killed bacteria before they replicate. Thus, it seems that the deletion mutants might have already existed in the bacterial population before phage infection. The more intriguing question is why bacteria delete such a big fragment of their genome in the absence of phage. It is likely that phage resistant mutants were selected before phage treatment due to as-yet-unknown fitness benefits. In an effort to determine whether the selection could be due to mutants' growth advantage in culture medium, we compared the growth rate of PA1 and the deletion mutant PA1r. Compared to PA1 wildtype, PA1r displayed growth defect. (). Furthermore, in a head-to-head competition experiment, PA1r was outcompeted by PA1(). The fact that deletion mutants did not rise to a high frequency in the absence of phage could be due to the growth cost incurred by this deletion. This pleiotropic effect has also been reported in other phage resistant bacteria. For example, phage resistant mutant of SBW25 was found to be less competitive when competing with wide type bacteria. Thus, the unknown fitness of the deletion mutants before phage infection still needs further investigation in this system. In summary, we reported a novel phage resistance mechanism in which evades phage infection by genomic fragment deletion, a finding that could provide valuable insight into phage therapy. Phages and hosts used in this study are listed in . and lytic phage PaP1 were stocked in our laboratory. strains were grown on Luria-Bertani (LB) agar plates or in LB broth with aeration at 37°C. When required, ampicillin, gentamicin, or tetracycline were used. Their concentrations in different media are described below. PA1 was grown at 37°C in LB broth with aeration. When the bacterial culture reached OD600 of 0.2, 10 μl of culture (approximately 1 × 10 cfu) was mixed with 100 μl and 10 pfu/ml phage PaP1. The phage-infected culture was incubated at 4°C for 5 min and spread to one LB agar plate. Plates were then grown at 37°C for 24 h. Phage sensitivity of the isolated colonies were tested as previously described. Briefly, 3 ml of 0.8% soft agar was mixed with 50 μl overnight culture of bacteria and was layered onto bottom agar. Finally, 10 μl of phages were deposited on the agar and incubated overnight at 37°C. Genomic DNA from wild-type strain PA1 and phage-resistant mutant PA1r were extracted using UNlQ-10 Column Bacterial Genomic DNA Isolation Kit (SK1202; Sangong Biotech, Shanghai) according to the manufacturer's instructions. The quality and quantity of extracted DNA were measured using spectrophotometry. Extracted DNA with a concentration of up to 500 ng/μl was used for the whole genome sequencing and PCR verification. The complete genomes of the wild-type strain PA1 and the PaP1-resistant mutant PA1r were sequenced using Solexa to a depth of 200× (median coverage 161×), according to the manufacturer's protocols. The reference genome of strain LESB58 (GenBank accessions: NC_011770) was downloaded from NCBI and used for mapping the short sequencing reads produced by the Illumina Genome Analyzer. The sequencing reads of each isolate were mapped separately to their corresponding reference genome. Primer sets (1U/1D, 2U/2D, 3U/3D, 4U/4D, and 5U/5D; ) were designed to amplify five different regions within the 219.6 kb fragment. Primer set 6U/6D () was used to detect the precise deletion site in PA1r. Primer sets (7U/7D, 8U/8D, 9U/9D, 10U/10D, and 11U/11D; ) were designed to detect different deletion sites in different Red mutants. Their locations are indicated in and . PFGE was performed as previously described with the following modifications. After digestion of the genomic DNA with SpeI, PFGE was performed at 14°C with CHEF MAPPER XA (Bio-Rad) at 200 V with switch times that ranged from 5 s to 52 s for 18 h. Transposon mutagenesis was performed as previously described with the following modifications. After mixing PA1 with strain SM10pir/pCM639 for 2 h, the cells were collected and resuspended in 1 ml of LB. Bacterial cells were then incubated with a 30-fold excess of the phage PaP1 for 20 min at 37°C and plated on LB medium containing tetracycline (65 μg/ml) and chloramphenicol (10 μg/ml) for the inhibition of the donor strain. The insertion of the transposon was identified by a two-stage semi-degenerate PCR and sequencing as described previously. Phage PaP1 and bacterial strain PA1 or PA1r were co-incubated for 3 min and placed on copper screen for 10 min before being negatively stained with 2% phosphotungstic acid (pH 4.8) for 1 min. The samples were then scanned under TECNAI 10 electron microscope (Philips, The Netherlands) at 80 kV. LPS minus bacteria were agglutinated by high salt concentrations. This procedure offers a simple way to distinguish LPS defective bacteria. Bacteria pellets from centrifuged cultures were resuspended in 4% NaCl for 5 min. Cells were then examined by microscopy. The cells of LPS defective strains were clumped together, whereas the LPS positive strains remained as single cells. Bacteriophage adsorption assays were performed to determine whether insensitivity to bacteriophage was caused by receptor mutation. Overnight culture of bacteria was collected by centrifugation at 13,000 × for 1 min, and the cell pellet was washed with LB and resuspended to a final concentration of 10 cfu/ml in LB. Bacteriophage was added at a concentration of 10 pfu/ml to the bacterial suspension and incubated at 37°C with 150 rpm shaking under aerobic condition for 3 min. The samples were then filtered through a 0.2 μm filter, and phage titer was immediately determined by plaque assay. Percentage adsorption of the phage was calculated using the following equation: Percentage adsorption = [(initial titer − residual titer in the supernatant)/initial titer] × 100. Error bars represent the standard deviation of the three independent experiments. insertional mutant was constructed by a described previously procedure. With the use of PA1 genomic DNA as a template, a 598 bp fragment of was PCR-amplified with primer set q-GalU-U2 (forward: 5′-AAGCTT AAGGAAATGCTGCCGGTGGTGA-3′) and q-GalU-D2 (reverse 5′-CTATTCTAGA TTGCCCGGTTCAGTCTGCTCGA-3′). PCR products were gel-purified and ligated into T vector (pMD®19-T Simple vector, TaKaRa Code:D104A), resulting in pT-Q-galU. Fragment containing gene on the plasmid pT-Q-galU was cut out with XbalI/Hind III, and ligated into the XbaI/Hind III digested pEX18Tc, resulting in pEX -. Then, pEX - was transformed into strain S17-1 and conjugated into PA1 to generate insertional mutant via single crossover. The gene was knocked out as described above with the primers (forward 5′-tcatAAGCTTCGAGCAGCCCACCGACTTCCT-3′) and (reverse 5′-ctatTCTAGA GACCGTGCCGATGGTGTTGAA-3′). The gene was PCR-amplified using forward (5′-GGATCCTATGATCAAGAAATGTCTTTTCCCG-3′) and reverse primer (5′-CTGCAGTCAGTGAGCCTTGCCGGTCTTGT-3′), and cloned into T vector (TaKaRa) resulting in pT-. The gene was then released from plasmid pT- via digestion with BamHI and PstI, and cloned into the same sites on pUCP24 to generate pucp-. Then, pucp- was electroporated into strain PA1r to create PA1r: strains. The was complemented into PA1r using the same method with the primers forward hmgA-u (5′-ggatcctATGAACCTCGACTCCACTGCCC-3′), and reverse hmgA-d (5′-CTGCAGTTATCTCCGTTGCGGGTTGAAG-3′). The Animal Research Ethics Committee of Third Military Medical University approved this mouse protocol. The animal experiment was performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Third Military Medical University. Bacteria were grown in 10 ml LB medium at 37°C until the early stationary phase. Cells were collected by centrifugation at 10,000 × for 1 min. The cell pellet was washed and resuspended in saline to a final concentration of 3 × 10 cfu/ml. Each strain (1 ml) was injected intraperitoneally into 6- to 8-week-old BALB/c female mice, and animals were observed for 24 h. Three independent experiments were performed. All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). Log phage bacteria PA1 was diluted to 10 cell/ml. Then inoculate 0.5 ml into each of the 20 tubes (Group A), and inoculate 5 ml into one tube (Group B). When the culture reach OD600 = 0.2, 20 μL of cell culture from each tube in Group A was plated onto agar containing the PaP1 phage. For group B, all the 20 replicates were performed used the bacteria from the same tube. Then the red colonies were calculated after 24 h incubation. S . L . , F . H . a n d W . S . c o n c e i v e d t h e s t u d y . S . L . , X . Y . , S . G . L . , J . W . , Y . Z . p e r f o r m e d t h e e x p e r i m e n t s . Y . T . , X . R . , M . L . , X . J . , N . W . a n a l y z e d t h e s e q u e n c e d a t a . R . L . p r o v i d e d i n t e l l e c t u a l s u p p o r t . S . L . a n d H . X . w r o t e t h e p a p e r .

Using time-lapsed imaging, we recorded the cell growth and examined the structure and cell division dynamics for both muscle stem cells from mouse and rat, and human mesenchymal stem cells. Time-lapsed images were acquired at 10-minute intervals over the course of 5 days. From these images, we obtained direct measurements of cell cycle times (division times) as time elapsed between cytokinetic events (). Based on these data, we were able to construct cell lineage trees of divisional history (). Heterogeneity was detected in cell lineage analysis of all stem cells (). Lineage history trees show the presence of both dividing and nondividing cells in the population. Variation in DTs is also detected. Proliferative heterogeneity was also identified by BrdU labeling as we observed both BrdU+ and BrdU– cells; this did not correlate well with molecular heterogeneity in expression of the cytoplasmic myogenic protein desmin (), which illustrates the complexity of understanding heterogeneous characteristics. For the stem cell DTs from all populations examined, we first estimate the empirical cumulative distribution function (see and ), the empirical probability of observing a cell DT larger than a given threshold (also called exceedance probability), then perform a maximum likelihood estimation (MLE) of well-known distributions and investigate their goodness-of-fit with respect to the actual data (see ). The main reason for analyzing the exceedance probabilities is to quantify whether the stem cell growth exhibits an exponential or a power law type of behavior. By analyzing the exceedance probability of cell DTs for all three species, we find that the cell DTs are well approximated by exponential type of distributions () as one would assume. This is also shown by the deviations of the empirical exceedance probabilities from the Gaussian (), log-normal () and gamma () distributions. Instead, the graphical analysis in , and the and the statistical results in show that the exceedance probabilities are better fitted by long-tail distributions such as α-stable, Student-t and generalized extreme value (Gev) distributions. However, to better assess the validity of modeling stem cell DTs via uni-modal distributions, we estimate the statistical goodness-of-fit for each MLE fit which implies analyzing the between the empirical and postulated probability density function. These results suggest that although the stem cell DTs exhibit a long-tail type of behavior, this cannot be well fitted by uni-modal distributions alone (see ). A similar conclusion can be reached by analyzing the graphical fittings in of several well known unimodal distributions (i.e., Gaussian, Gamma, Log-normal, Generalized extreme value, Student-t, Weibull, asymmetric α-stable distribution) for the mouse and human cell DTs. In addition to graphical evidence, the Kolmogorov-Smirnov test, the Akaike Information Criterion and Hartigan's Dip test (see ) suggest that stem cell DTs cannot be modeled by an uni-modal distribution. This suggests that there actually may exist two dividing subpopulations of stem cells, each characterized by distinct dynamics of stem cells division rate; this is reflected by different probability density functions (PDFs) of the DTs. To overcome the poor fitting of uni-modal distributions and explain the observed heterogeneity in the PDFs of stem cell DTs, we consider the bi-modal statistical investigation and two PDF candidates: ) a bi-modal Gaussian distribution () and ) a bi-modal asymmetric α-stable distributions (). By employing the MLE strategy for a mixture of two asymmetric α-stable distributions and selecting the best fit according to the best Kolmogorov-Smirnov (K-S) test result, we observe that the empirical PDFs of mouse, rat, and human stem cell DTs are well fitted by the bi-modal α-stable distribution. Indeed, both graphically and statistically by looking at the error bars, we can see that the fitting involving the α-stable distribution captures much better the overall trend than the Gaussian one (). In sum, the magnitude of p-values of the K-S tests imply not only that we cannot reject the bi-modal α-stable distribution as a viable model for stem cell DTs, but also that the stem cells population exhibits a structure consisting of at least two sub-populations, namely a sub-population of stem cells that divide faster (with an average DT of approximately 12 hours and corresponding to first peak in the PDF plot of ), and a second sub-population of stem cells with a slower dynamics (with an average DT of approximately 18 hours corresponding to the second peak in the PDF plot of ) which exhibits a long-tail type of behavior. Besides the heterogeneous structure of stem cells population, we also observe that the empirical PDF estimated from stem cell DTs exhibits a pronounced time dependent behavior (). This time dependent behavior is illustrated not only by the changes in the shape of the empirical PDF at different time windows, but also by the estimated values of the bi-modal PDF. Indeed, the PDF investigation shows that stem cell growth is a stationary process, but rather a highly dynamic one characterized by time-dependent PDF variation. To further investigate the existence of a non-stationary behavior, we measure the mean (), variance (), skewness (), and kurtosis () over a sliding window of 80 cell DT recordings (from the time-ordered mouse stem cell DT data). Additionally, show the error bars in the time variation of mean, variance, skewness, and kurtosis for mouse, rat, and human cells. We observe that these higher order moments vary with time which suggests that the cell division is a non-stationary process (see ). For instance, the mean of the rat stem cell DTs exhibit a wide range variation from 15 hours in the beginning to almost 19 hours at later times. Similarly, the variance, skewness, and kurtosis exhibit a spiky behavior which cannot be modeled if one assumes a stationary behavior. In addition, the non-zero values for skewness and kurtosis show that the rat stem cell DTs do display a Gaussian behavior; this supports our predictions concerning the existence of a non-Gaussian dynamics of stem cell growth shown in . Further, the non-zero skewness shows that the stem cell DTs are not symmetrically distributed around the mean and may exhibit different tails at both ends of the PDF curve. Similarly, the non-zero kurtosis implies that the stem cell DTs can exhibit large values and so a better modeling approach than the Gaussian-based framework needs to rely on long-tail distributions. Moreover, the observed variability and spiky dynamics of these higher moments imply that the stem cell DTs exhibit a heteroscedastic dynamics (i.e., some sub-populations exhibit different moment variability compared to others). An alternative proof of existence of heteroscedastic dynamics is shown in the where shuffled stem cell DT series lose their correlation structure when compared to the initial data sets. In general terms, the heteroscedastic dynamics means that the statistical moments of the stem cell DTs and, implicitly, those associated with stem cell growth exhibit a local variation which is very different from the global one or asymptotic limits. In addition, the very existence of this heteroscedastic behavior may not only confirm the heterogeneity of stem cell population, but also be indicative of an aging phenomenon that is characteristic to all multi-cellular organisms. As we later show in the paper, this aspect can play a crucial role not only in constructing an accurate mathematical model of stem cells growth, but also for predicting the structure and size of the entire population over time with high confidence while accounting through non-stationary methods and tools for the age of a patient. For a comprehensive investigation of the heteroscedastic dynamics of stem cell growth, we investigate the relationship between the higher order moments of stem cells dynamics and their order; we also estimate both the multi-fractal spectrum and generalized Hurst exponent function [A fractal is a geometrical object or stochastic process that displays self-similarity, on all scales and is characterized by a single fractal dimension. The fractal dimension is a mathematical concept measuring the unique features such as geometrical shape of an object or irregularity of a stochastic process. The object need not exhibit exactly the same structure at all scales, but the same “type” of structures must appear on all scales. Similarly, a stochastic process is regarded as fractal if its probability distribution function measured at two scales is self-similar and related to each other via a power law relationship. In contrast, a multi-fractal object or stochastic process is characterized by a series of fractal dimensions.]. The rationale for estimating the generalized Hurst function in is two-fold: It allows us to detect the existence of multi-fractality. For instance, if the generalized exponent is independent of the order of the moment (see ), then the stochastic process is mono-fractal. In contrast, if the generalized Hurst exponent is dependent of the order of the moment, then the process is called multi-fractal (see ). It allows us to compute the Hurst parameter and discriminate between short-range and long-range memory effects. For instance, if the Hurst exponent equals 0.5 then we can state that the dynamics of stem cell populations is governed by short-range memory or Markovian models. In contrast, if the Hurst exponent ranges between 0.5 and 1, then the dynamics of stem cell populations is said to possess long-range memory characteristics and non-Markovian dynamical models are needed. While mono-fractality implies that a single Lipschitz-Holder exponent (i.e., a function () is said to have Lipschitz-Holder exponent around point if ) characterizes the entire cell dynamics, the multi-fractal behavior shows that the existing temporal heterogeneity in stem cell population leads to multiple Lipschitz-Holder exponents. The multi-fractal spectrum encapsulates in its distribution of Lipschitz-Holder exponents information about multiple point correlations existing both in space and time. Implicitly, it is also a measure of the memory characterizing the stem cell dynamics. By measuring the dependency of the higher order statistical moments on their order value and using the Legendre transformation, we estimate the multi-fractal spectrum for the mouse, rat, and human stem cell DTs ( and ). One can clearly observe from not only that the stem cell DTs exhibit a wide multi-fractal spectrum, but also the Lipschitz-Holder exponents are mostly negative. The fact that the stem cell DTs display a significant range of negative Lipschitz-Holder exponents implies that the stem cell growth process exhibits an oscillating behavior consisting of active periods of growth (when the majority of the cells are actively dividing) followed by periods when fewer cells are recruited into the cell cycle. Alternatively, the results in (and ) display a complex nonlinear dependency between the generalized Hurst exponent and the -th order moment, which again supports the existence of multi-fractal behavior in stem cell growth. Based on these findings, our subsequent question is whether the current models relying on the assumption of either having constant or exponential growth in stem cell population size are suitable for predicting the population size; answering this question is critical for designing cell therapies or tissue engineered constructs for future personalized medicine. Therefore, we next considered the time lapsed traces of the number of stem cells in the population over time and investigate the goodness-of-fit for three mathematical modeling approaches: ) exponential growth associated with a non-fractal dynamics (); ) power law growth corresponding to a mono-fractal dynamics (); and ) a stretched exponential hinting towards a multi-fractal approach (). We observe that the stretched exponential and power law type of models offer better prediction capabilities than the state-of-the art exponential models (see ). By ignoring the multiscale biological dynamics and spatio-temporal correlations, the exponential models can be misleading. Indeed, the exponential growth assumption can lead to very optimistic predictions concerning the population size of stem cells and this may have detrimental effects on the therapeutic strategies. Our results suggest that the multi-fractal behavior of stem cell dynamics can be modeled as a superposition of several stretched exponential functions. Here, we show for the first time that distinct subpopulations of dividing cells exist within several heterogeneous stem cell populations. We report that the populations show time-dependent and fractal behavior. Both newborn derived mesenchymal stem cells and adult derived muscle stem cells demonstrate these population characteristics. By employing non-Gaussian statistics, statistical hypothesis testing and statistical inference, we identify the presence of two unique dividing subpopulations of stem cells, each with a distinct cell division rate and characterized by a different probability density function of DTs. Previous reports treated stem cells as homogeneous populations and assumed either a constant division time for the whole population or Gaussian distributed DTs. In terms of improving how stem cells are defined, our study strongly supports the notion of defining cells from a population level perspective in order to account for cell-cell interactions that affect the population structure (composition of different subpopulations) and dynamics (interactions of and changes in the subpopulations). Bacteria and yeast cell division and growth rates have been examined extensively for more than 50 years; the distributions appear to be rather homogeneous and, to the best of our knowledge, there are no reports of bi– or multi–modal growth rates. For instance, Tsuru et al. recognize the existence of fluctuations in growth rates and investigated how the changes in cell volume of individual bacteria are correlated with fluctuations in protein concentration. Complementing such a phenotypic diversity, a report by Niven et al. describes skewed cell division distributions in in the presence of environmental factors. The authors examined several distributions to fit their data (also obtained via time-lapsed cell imaging) and no definitive model distribution was identified, possibly due to the small sample size. More recent studies of differentiated cells, particularly those that use similar technology to permit the collection of a dataset of individual cell measurements, also did not report bi-modality. For instance, Tyson et al. emphasized the importance of investigating the statistics of intermitotic times and modeled these times via an exponentially modified Gaussian distribution. Based on their analysis, Tyson et al. concluded that their proposed exponentially modified Gaussian models are not definitive and require further improvement to accurately accommodate age structure and dynamics of stem cell populations. Along the same lines, Chang et al. demonstrate the existence of cell-to-cell variability in clonal populations of mouse hematopoietic progenitor cells and showed a link to heterogeneity of gene expression. Tan et al. used single-cell gene expression to show functional heterogeneity in epidermal cells, while Koschmieder et al. and Cantor et al. investigated it in the hematopoietic lineage. We see that such mechanism might be an underlying cause of the heterogeneity in our populations of stem cells. Furthermore, in our own previous reports, we used both standard Gaussian and non-parametric statistics to examine the distributions of cell cycle times in human and mouse stem cells. However, lack of sophistication in our previous statistical analyses and the small size, did not allow us to identify bi-modality (let alone multi-modality) in stem cells dynamics which we report here. Stem cell population heterogeneity may develop through the process of spontaneous progression of stem cell differentiation. It is worth mentioning that the interaction of the cells with the culture substrate and the rigidity of the substrate are important factors in modulating differentiation and cell phenotype heterogeneity as has been shown by Gilbert et al.. In most scenarios, non-dividing quiescent cells are presumed to become activated to slowly dividing multipotent stem cells which then progress to transiently amplifying (TA) committed progenitors and finally non-dividing differentiated cells. Here, we detected, in all populations the presence of a non-dividing quiescent subpopulation that could re-enter the cell cycle. The slowly dividing subpopulation for MSCs (16.8 hrs), rat stem cells (21.2 hrs), or mouse stem cells (21.9 hrs) was at least 4 hours slower than the fast diving subpopulation (human MSC 12.8 hrs, rat stem cells 11.7 hrs, mouse stem cells 13.2 hrs). It is possible that the slow dividing fraction represents multipotent cells, while the faster dividing cells represent committed precursors or transiently–amplifying cells. However, at this point, there are no clear methods to physically separate these subpopulations, and BrdU pulsing experiments () did not show a correlation between the myogenic precursor marker Desmin and BrdU rate of incorporation in stem cell populations. Nevertheless, the statistical analysis demonstrates the presence of two distinct populations which grow at different rates. This was detected in different species, and in different stem cell types, which suggests that it may be indeed a universal characteristic. The existence of these different phenotypes may allow for both short-term and long-term functions of the stem cell populations in response to tissue injury. Indeed, fast dividing TA cells can provide immediate restoration of damaged/lost differentiated cells, while the dynamic of TA cell depletion for acute repair may illicit a faster transition rate for cells in the multi-potent state to move to the TA state to re-establish some equilibrium of subpopulations. In this scenario, because dividing cells expand according to a stretched exponential, the quiescent stem cell activation could be conservative and occur only in the case of severe tissue loss or damage. In the case of normal tissue homeostasis, we could expect to observe oscillating dynamics ( or ) related to cell division behavior in the population, as normal cell death occurs, and lost cells are replaced. Further, our statistical analysis demonstrates that the population subsets display a non-stationary (i.e., time-dependent statistics). Non-stationary statistical analysis shows that not only do individual cells change over time, intrinsically, but the stem cell population also changes over time. In part, the non-stationary phenomenon finding shows that stem cells exhibit an aging process which is characteristic to all living organisms. Consequently, the rates of transition between subpopulation states will be important for understanding their evolution over time. The fractal behavior we observe in stem cell DTs identifies a stochastic process that displays a probability density function that is self-similar in nature, i.e., the distribution of cell DTs at one scale can be retrieved from that of another scale by using the fractal dimension and a scaling coefficient. Simply speaking, the existence of fractal statistics in stem cell DTs implies that the birth of a new cell is a random (i.e., uncorrelated) event from the development of previous ones. In other words, there is some form of correlation between cell divisions and this leads to a complex behavior which cannot be quantified by current mathematical models of stem cell growth. Nevertheless, due to numerous sources of variation (e.g., intrinsic differences, environmental interactions, nutrient availability, cell density), stem cell DTs cannot be characterized by a single fractal dimension, but by a set of scaling exponents which also demonstrates the existence of heterogeneity in stem cell population structure. Taking into account such characteristics allows us to construct an accurate dynamical system approach as described by Furusawa and Kaneko which may explain and, more importantly, predict the stem cell dynamics. Furthermore, the newly proposed model can be extended to include cell cycle and cell volume, along the lines of Halter et al. and Anderson et al.; these parameters may be used to show the multi-fractal behavior as the cells interact with each other. The value for teasing out the subsets of cells, their interactions, time-dependent and fractal behavior has implications for applied cell therapeutics. First, it is needed to identify/define the stem cell product. Second, it is necessary to accurately predict ex vivo growth rates of the stem cell populations. In regards to developing an FDA-approved cell therapeutic, purity and potency criteria need to be established. Recognizing that subpopulations, and hence multiple targets, exist in the stem cell population is essential to developing tools to control the fate of stem cells. Although, we and others have shown that the quiescent population exists and requires nonlinear growth models, our study is the first to show quantitatively and qualitatively that more sophisticated growth models are warranted due to the presence of multiple phenotypes. Models which account for population dynamics can better predict whether cytokines or growth factors that increase proliferation rates (e.g., for the purposes of obtaining certain cell doses for transplantations) cause perturbations in the dynamics of subpopulations, and how this affects the behavior of the entire cells population. This statistical approach may also be exploited for personalized medicine strategies. Using high throughput live cell imaging and our computational analyses, inter-patient variability and intrinsic intra-patient variation in stem cells dynamics can be studied. In the era of individualized medicine, the computational modeling may also include genomics and proteomics information and the dynamics of an individual biopsy-derived stem cell growth can be assessed before developing the cell therapy approach. Personalized medicine could also exploit our statistical investigation for estimating the richness of fractal behavior at the cellular level and make predictions at higher scales. By early detection of losing fractal richness at the level of stem cell evolution, one could predict the signs of genetic instability and onset of a disease well in advance of actual symptoms in the patient, when it may be hard, or impossible, to intervene. Further studies are needed to examine the molecular phenotype and transition rates of the two dividing subsets identified here. Epigenetic modifications may account for differences in phenotype or division rates, or the cell-cell communications may modulate division rates. The oscillating time-dependent behavior together with the finding that these subpopulations interact with each other (i.e., cells do not grow in isolation, but instead interact at various scales). In addition to that, the cells indeed interact with their microenvironment including the extracellular matrix, growth factors, chemokines, and the substrate they are adhering to. These interactions have been shown to affect the differentiation and fate decisions of stem cells. All of these factors call for developing new mathematical models describing their interaction and overall population growth. A challenge to these studies will be the isolation of these subsets and maintenance without re-establishing the heterogeneous parent population. We used time-lapsed imaging to directly examine the cell growth of mouse and rat stem cells isolated from skeletal muscle tissue and human mesenchymal stem cells. (Additional detail regarding imaging experimental set-up is included in and stem cell isolation is included in the ).Time-lapsed images were acquired at 10-minute intervals over the course of 5 days on collagen-coated surfaces. To determine the divisional status of the cells, and the cell cycle length of dividing cells, we used a custom developed program, i.e., ImageView (Karios Instruments, Harmar, PA). Cell cycle length was recorded as the time elapsed between cytokinetic events, as previously reported. Dividing cells were classified as cells with a visible DT. Non-dividing cells were classified as cells that were visible on screen for over twice the average DT. After determining DTs, the cells were tracked in order to perform lineage analysis and determine differences between dividing and non-dividing cells while also identifying the quiescent cells. A custom built image analysis program, CytoTracker, (Karios Instruments, Harmar,PA), was used to perform lineage analysis. A cell was selected and followed for the entire time it was visible on screen. When a cell divides, the division event is recorded, and each daughter cell was subsequently tracked. The entire cell lineage is tracked until either the end of the video or the cells are lost to follow up. Each cell is assigned a name in order to keep track of the lineage. The initial parent cell is 1.0. The two daughter cells are named 1.1 and 1.2. Their daughter cells are named 1.11, 1.12 and 1.21, 1.22 respectively. The tracks/locations ( and location) for the entire family of cells are recorded along with the cell name for each jpeg image.This allows a cells lineage to be determined. The cell histories can then be converted to a graphical display, or lineage tree which displays all the cells in the family, and their DTs, or their time on screen if the cell was lost to follow up (). In the graphical displays, the vertical lines represent a cell, and the length of the line corresponds to how long the cell was on screen. Lineage trees allow the divisional status of a cell to be determined. A tree with many cells represents a cell that is actively dividing and giving rise to progeny that continue to divide and contribute to renewal of the population. The lineage trees also allow DTs to be compared, or to identify cells that are non-dividing and visible on screen for over twice the division time. Also, asymmetrical divisions can easily be identified. #text

The ternary composites were prepared with a two-step protocol as shown in . After a first step of coating solution-exfoliated GO nanosheets with polyaniline, MnO nanorods were allowed to self-assembly onto the GO sheets and then wrapped by in-situ prepared polyaniline coatings in the second step. The MnO (in weight ratio) content in the ternary composites was determined with thermogravimetric (TGA) analysis (). Hence, the as-prepared MnO-PANI-GO composites (GOPM) are designated as GOPM-20, GOPM-46 and GOPM-70, meaning that mass percentage of MnO in the composites is about 20%, 46% and 70%, respectively. Typical X-ray diffraction (XRD) pattern of as-prepared hierarchical porous GOPM-46 and free MnO, PANI and GO are shown in . It can be seen that the XRD pattern of GOPM is similar to those of free MnO and PANI, indicating that the MnO particles have been well immobilized by PANI onto GO substrate. All of the major reflections [2θ = 21.2°, 28.0°, 37.5°, 42.5°, 59.0° and 69.7°] in the XRD pattern can be indexed to the orthorhombic phase of γ-MnO with lattice constants = 6.36 Å, = 10.15 Å and = 4.09 Å (JCPDS 14-644), confirming that γ-MnO crystalline phase forms in the synthesis, which is in good agreement with the reported patterns for γ-MnO. The broad peak centered at about 25.3°, attributing to the diffraction of plane of the as-formed PANI crystalline phase, suggests the successful synthesis of PANI. However, the sharp peak at 2θ = 11.0°, corresponding to its plane for pure GO, almost disappeared, which is attributed to the increase in inter-space distance of graphene layer due to intercalation of PANI/MnO2 between GO sheets. The Raman spectra of GOPM and pristine components are shown in . It can be clearly seen that both GOPM-46 and MnO samples feature a sharp peak at 640 cm, corresponding to the Mn-O vibration perpendicular to the direction of the MnO octahedral double chains of MnO. The Raman-active peaks at 1350 cm and 1600 cm, corresponding to the in-plane bond-stretching motion of C sp atoms (G band) and the breathing modes of or of benzenoid rings of GO (D-band), were significantly suppressed in intensity for GOPM-46 sample. The typical peaks for PANI also weakened in GOPM, mainly due to its low content in the hybrid composite. To confirm the XRD and Raman results, the compositions and the valence states of GOPM were further characterized with XPS and the results are shown in . The peak ~284.6 eV (for C 1s) originating from the graphitic sp carbon atoms and the peak at 531.9 eV corresponding to O1s in C–OH bond are observed for GO (). The existence of C, N, O, Mn in GOPM can be confirmed from . For GOMP, the XPS peaks of N 1s () are further decomposed into three Gaussian peaks with binding energies of 398.6 (-N = ), 399.7 (-NH-) and 400.1 (-N-). The peak at 400.1 eV is assigned to the quinoid amine and nitrogen cationic radical (N), while the one at 398.6 eV ( = N–) is due to benzenoid amine. The Mn 2p spectrum is analyzed in . Both Mn 2p peak at 643.0 eV and Mn 2p peak centering at 654.5 eV are clearly observed, which are in good agreement with the energy splitting of the standard spectrum of MnO. The peak-to-peak separation between Mn 2p and Mn 2p level is 11.5 eV, which is approximately the same value as that literature for MnO. The morphologies of dry MnO, GO-PANI and GOPM-46 are observed with FESEM and TEM (,). Pristine MnO nanorods with a length of 200 nm grow into nanospheres with diameters ranging from 300 to 600 nm (). The prepared PANI self-assembled on GO sheets like a broccoli composed numerous of small flower buds with a diameter of 50 nm (). When MnO nanorods were introduced to GO-PANI system, GOPM hybrid composites feature a porous yet densely packed structure (), with GO layers intercalated by porous interpenetrating networks of PANI-wrapped MnO nanorods. PANI functions as both protecting coating for MnO nanorods to avoid aggregation and adhesive to fasten GO sheets and MnO nanorods into densely packed structure. Exfoliated GO sheets feature transparent wrinkled tulle in TEM image (), while PANI formed a compact layer on top of GO sheets for GO-PANI composites. A close look at the TEM images of GOPM ( and ), one would clearly observed the existence of MnO nanorods, featuring a length less than 100 nm and a diameter ~20 nm. With the content of MnO nanorods increasing from 46% to 70% in the composites, the dimensions of MnO nanorods was observed to increase by 30% (). Nevertheless, the intimate contact between GO sheets and PANI wrapped MnO nanorods with different lengths can be clearly observed from both SEM and TEM images, which is important for improving electrical conductivity. A close look at the SEM image of ternary composites, GOPM-70 boasts increased porosity than GOPM-46, which can be confirmed by BET measurement. The GOPM-70 and GOPM-46 exhibit a surface area of 91.37 and 73.65 m/g, respectively, much higher than that for GO-PANI (~36.92 m/g). The increased surface area can be explained with the constitution of the ternary composites. Since PANI has the equivalent mass with GO, more MnO nanorods were uncovered by PANI with the increase of MnO content. More porous structure was therefore obtained to present higher surface area before heavy aggregation of MnO occurs. The capacitive performance of GO, MnO, GO-PANI, GO-MnO and GOPM were evaluated by cycle voltammetry (CV) and galvanostatic charge/discharge techniques in 1 M NaSO solutions. shows the CV curves of GOPM-46 at different scan rates. At low scan rates (≤50 mV/s), GOPM-46 exhibits slight redox peaks, suggesting the pseudocapacitance feature of GOPM. The asymmetrical CV curves can be attributed to the combined double-layer and pseudocapacitive contributions to the total capacitance. The high CV currents indicate the high conductivity and low internal resistance for GOPM as the electrode material. shows CV curves of GO, MnO, GO-PANI, GO-MnO and GOPM at 50 mV s, where GOPM-46 exhibits the highest capacitance than other electrodes. This can be attributed to the unique porous structure of hybrid GOPM, which effectively prevent the self-aggregation of GO sheets and MnO nanorods. More importantly, the hybrid GOPM with PANI-coated MnO nanorods intercalated GO nanosheets affords higher surface area to provide better conductive paths for fast electron transportation. The galvanostatic charge/discharge curves of GOPM in 1 M NaSO solution were carried out at a current density of 0.25, 0.5, 1, 2 and 4 A/g. As illustrated in , all the curves exhibit an equilateral triangle shape, indicating high reversibility of the hybrid materials during charge/discharge process. The charging/discharging process took longer time at lower current density, which is attributed to the sufficient insertion or release of Na during the charging/discharging process. The specific capacitance (C) of the electrode can be calculated according to equation: C = It/mV, where C is the specific capacitance (F/g), I is the charge-discharge current (A), t is the discharge time (s), V is the potential window (V), m is the mass of active material in the working electrode (g). The galvanostatic charge/discharge curves of MnO, GO-MnO, GO-PANI, GOPM at 0.25 A/g current density are compared in . The specific capacitance of GOPM (409 F/g) is much higher than that of MnO (150 F/g), GO-MnO (162 F/g) and GO-PANI (20 F/g) at the same current density. Same trend is observed for GOPM when further increasing current density in the range of 0.25~4 A/g (), indicating the robustness of the as-prepared hybrid GOPM as electrode materials. The specific capacitance of GOPM is found to increase with the content of MnO in the composite, as shown in . The specific capacitance of GOPM-70 (412 F/g), GOPM-46 (306 F/g) and GOPM-20 (252 F/g) is about 1~2 times higher than that of MnO (130 F/g) and GO-MnO (156 F/g) at the current density of 1 A/g. Further increase of MnO content over 90%, however, leads to decreased capacitive performance, e.g., the GOPM with 92% MnO delivered a specific capacitance of 382 F/g at 1 A/g current density. The specific capacitance of 412 F/g at 1 A/g for GOPM-70 is much better than graphene/MnO (78 wt.% MnO) binary composite under similar testing conditions. This improvement may be explained with the contribution of conductive PANI in facilitating charge transport and energy storage. Compared with the ternary composite sGMOPANI, our composite with MnO nanorods exhibited 11% improved capacitance in supercapacitors at same current density of 1 A/g. The specific capacitance of GOPM decreases with current density, with ~62% capacitance retained for GOPM-70 when the current density increased from 0.25 A/g to 4 A/g. The electrochemical stability of GOPM-70 nanocomposites was investigated at 4 A/g current density. As shown in , the capacitance of GOPM electrode retained about 97% of the highest capacitance even after continuous galvanostatic charge/discharge process for 5100 cycles, indicating a good cycling ability of the hybrid composites. The support carbon matrix GO allowed the strong deposition of PANI-protecting MnO nanorods on the surfaces of GO, which enhanced the mechanical strength of composite materials, resulting in the long charge/discharge ability. Interestingly, the specific capacitance showed slight increase in the first 150 cycles before decreased slowly in the later cycles, which may be explained with the insufficient contact of nanocomposites with NaSO aqueous solution at the beginning of electrochemical measurement. The electrochemical properties of GOPM were further evaluated with electrochemical impedance spectroscopy (EIS). The impedance spectra of composite electrodes before and after 3000 cycles were measured in the frequency range of 100 kHz–0.1 Hz at open circuit potential with an AC perturbation of 5 mV (). Theoretically, an ideal Nyquist impedance plot features a semicircle over the high frequency region and a linear part in the low frequency range. The larger semicircle observed for the electrode corresponds to higher interfacial charge-transfer resistance (R) for the layer on the electrode, attributed to the poor electrical conductivity of the materials. And the straight line of the Nyquist plot corresponds to the resistance () resulting from ion diffusion/transport, i.e., the more vertical line is indicative of an electrode more close to an ideal capacitor. The equivalent series resistance (ESR) of pristine MnO, GO-PANI, GOPM-46 and GOPM-70 obtained from the intersection of the Nyquist plot at the x-axis is 2.6, 0.7, 1.8, and 1.6 Ω, respectively. Considering the similar morphology of GO-PANI and GOPM, the difference in ESR of electrodes can be attributed to the different conductivities of electrode materials. The smaller ESR of GOPM than GO-PANI suggests the decreased charge transfer resistance in the presence of MnO nanorods as core. The high resistance of ion transfer in GO-PANI would be attributed to high charge density, resulting in low capacitance. In contrast, GOPM-46 exhibited a short diffusion path length of ions in the electrolyte, which could be seen from the low resistance of the capacitative part on the Nyquist plot. This may be explained by the structure of the composite: the formation of GOPM results in the surface charge of GO being compensated by the negative charge from both Cl doped PANI and MnO nanorods, leading to a lower resistance of ion transfer. Moreover, the presence of GO-PANI with high electrical conductivity resulted in a lower charge transfer. After 3000 cycles, the calculated ESR for GOPM-46 based electrode increased from 1.8 to 2.4 Ω, while the ESR increased from 1.6 to 2.1 Ω for GOPM-70. The increased resistance is probably attributed to the loss of adhesion of some active material with the current collector or the dissolution of some PANI/MnO during the charge/discharge cycling. sup #text Absorbent cotton (200 mg) was added to KMnO solution (40 mM, 200 mL) with simultaneous vigorous stirring and ultrasonic irritation for 20 min. The mixture suspension was then heated at 100°C for 24 h. A dark brown precipitate was obtained. The precipitates were collected by centrifugation and washed with deionized water and ethanol for 4 times. The product was dispersed in hydrochloric acid (1 M) for use. Freshly prepared GO (0.1 g) dispersion in water (100 mL) was ultrasonicated (250 W, 220 V) for 1 h to get an exfoliated yellow brown GO suspension. Aniline (0.12 g) was slowly added into the suspension and the stable GO/aniline suspension was obtained after stirring violently. A mixture of concentrated hydrochloric acid, ammonium persulfate (APS) in distilled water (10 mL) was then slowly added to the suspension under stirring. The molar ratio of aniline, hydrochloric acid and APS was 1:1:1. When the reaction was conducted in ice bath for 10 minutes, MnO nanorods (0.6 g) dispersion in hydrochloric acid (1 M, 50 mL) was added to the reaction system together with second addition of aniline (0.12 g). The reaction was stirred for 12 h. Finally, the composite was filtered and rinsed with distilled water and ethanol in sequence to afford the product (0.65 g) as a deep green solid. The electrochemical properties of the composite were investigated on a CHI660D electrochemical workstation (Shanghai, China) with conventional three-electrode system at room temperature. To prepare the working electrodes, the polytetrafluoroethylene with acetylene black (15 wt%) in ethylene solution were added to as-prepared composites to produce a homogeneous paste before pressed onto nickel foam current collectors. The electrodes were then dried under vacuum at 60°C for 24 h. The electrochemical performance of composites was investigated with standard CV, galvanostatic charge–discharge and EIS technique in 1 M NaSO solution. CV measurements were performed in voltage ranging from 0 V to 0.8 V at a scan rate of 10, 30, 50 and 100 mV/s, respectively. Charge-discharge processes were carried out galvanostatically at 0.25~4 A/g current density in 0~1 V voltage range. T . W . c o n c e i v e d t h e w o r k . H . G . , Z . L . , L . Y . a n d Z . S . p e r f o r m e d t h e e x p e r i m e n t s . H . G . , K . E . , T . J . a n d T . W . a n a l y z e d t h e d a t a a n d p r e p a r e d t h e m a n u s c r i p t .

Although most complex dynamic systems are nonlinear, the controllability of nonlinear systems is in many aspects structurally similar to that of linear systems. Actually, to ultimately develop the control strategies for complex nonlinear networks, a necessary and fundamental step is to investigate the controllability (especially structural controllability) of complex networks with linear dynamics. In this study, we thus consider the linear time-invariant nodal dynamics of a complex network with n nodes, where the activity of node (), can be described by the following equations where ≠ 0 if node j directly affects node i, that is, there is an arc from node j to node i in the network, and otherwise = 0. = 1 if input control signal () directly acts on node i and otherwise = 0. In this study, we are interested not in the complete controllability, but in the transittability of the system, which concerns the transition between two specific states by a suitable choice of input control signals (). Formally, the system is said to be transittable between two given specific states x and x if it can be transited between x and x in finite time by proper input control signals () (). Note that the system is transittable between any two states by simply acting one independent input control signal on each of n nodes. That is, all nodes are steering nodes, then we have = 1 for i = 1,2,…,n and thus . However in this study we are interested in finding the minimum set of steering nodes (called steering kernel) to achieve the state transition between two specific states, in other worlds, minimizing while the system is transittable between two given specific states x and x. The system can be rewritten in the vector-matrix format as follows where the n-dimensional vector () = ((), …, ()) represents the state of the network with n nodes at time t. The × matrix A = (a) describes the interaction relationship and strength between nodes. The × matrix B is called the input control matrix that corresponds to the steering nodes. The p-dimensional vector () = ((), …, ()) represents the input control signals. As in many situations, one controller cannot produce multiple independent input control signals. Here we assume that one controller can produce only one independent input control signal. Therefore, all elements in the j-th column of matrix B are all zeroes except for the s-th element if the j-th input control signal directly acts on node s. where () represents the subspace spanned by the column vectors of matrix C and is called the controllable subspace. Now finding the steering kernel to steer the system from state to can be formulated as a problem to find the matrix B with the minimum number of columns such that condition is true. However, the calculation of either is complicated and condition is computationally intractable (). Next, we state our first key result, i.e., we prove (see ) that the system is transittable between two specific states and with either belonging to () if and only if where . The transittability is typically considered between two stable states or one stable state and another state. Let us say that is a stable state, we can always assume that = 0 (the origin) without loss of generality, i.e., we can replace with – if ≠ , which does not affect the result. where = [, ]. Although the condition is much easier to be verified than the condition, the calculation of either () or () is still prohibitive because of the large size n of a complex network, the uncertainty, time dependence of the entries in matrices A and/or vector . Note that the transittability is not only to control a state to the origin, but also to control a state from the origin to other specific states. To overcome the computational impedance in verifying condition, we further introduce the structural transittability via the concepts of structural matrix and generic dimension of controllable subspace. M is said to be a structural matrix if its entries are either fixed zeros or independent free parameters. is called admissible (with respect to M) if it can be obtained by fixing the free parameters of M at some specific values. If A and B are structural matrices, system is called a structural system and is denoted by (A, B). Associated with a structural system (A, B), a directed graph G(A, B) = (V, E) can be defined with the set of nodes V = VUV, where V = {,…, }: = {,…, } is the set of state vertices, corresponding to the n state components while V = {,…, }: = {,…, } is the set of input vertices, corresponding to the p inputs, and the set of arcs E = EUE, where E = {(, ) = (, ) | a ≠ 0} is the set of directed edges between state vertices while E = {(, ) = (, ) | b ≠ 0} is the set of directed edges between input vertices and state vertices. We can also define a directed network G(A) = (V, E) with respect to a structural matrix A (). In a directed graph, an elementary path is a sequence of arcs where all vertices {, , …, } are different, and when = , it is called an elementary cycle. A stem is an elementary path originating from an input vertex in V. A structural system (A, B) is reducible if there exists a permutation matrix P such that with , , and , 1 ≤ ≤ and + = . Otherwise (A, B) is said to be irreducible. The dimension of the controllable subspace of structural system (A, B) varies as a function of free parameters in structural matrices A and B. That is, for different admissible systems (), the dimensions of their controllable subspaces might be different. As the maximum rank of matrix C is at most n, the dimension of controllable subspace of structural system (A, B) can reach the maximum value. We define this maximum value as the generic dimension of the controllable subspace of structure system (A, B) and denote it by GDCS(A,B). The GDCS(A,B) is a generic property in the sense that for almost all admissible systems (with respect to (A, B)) the dimension of their controllable subspaces takes a constant which is GDCS(A,B). Hosoe has proved that if (A, B) is irreducible. denotes the number of edges in . Applying to the structural system (A,B), identifying the steering kernel by which the network G(A) can be transited between a state x and the origin becomes finding a structural control matrix B with the minimum number of columns such that Our second key result is that we develop a graph-theoretic algorithm to identify the steering kernel by solving an optimal assignment problem of a weighted bipartite graph (). For details, see the Materials and Methods and the . We apply our algorithm to 27 complex networks to determine their steering kernels and the results are summarized in . These 27 networks are a portion of 38 complex networks in ref. , and the number of their nodes ranges from 32 to 27772 while the number of edges ranges from 96 to 352807. The phenotypes of complex networks are typically defined by a small portion of nodes. For examples, the number of molecules (such as genes or proteins) significantly involved in a specific human disease is only a small portion of all molecules in a network. Therefore, this study assumes that a transition between two specific states has 20% or 50% of nodes whose state values are changed, that is, in and has 20% or 50% of nonzero elements. The fraction of steering nodes is defined as the ratio of the size of steering kernel to the total number of nodes in the networks. Columns 5–8 in are the average results of 1000 randomly defined transitions of each network. Columns 7 and 8 respectively list the average fraction of steering nodes for transittability of 20% and 50% of nodes which are differently expressed at two states while Column 9 lists the fraction of driver nodes from Liu et al. Comparing Columns 7 and 8 to Column 9 concludes that the minimum numbers of steering nodes required for transittability is much less than those for complete controllability. For complete controllability, the generic dimension of controllable space is the number of nodes in the networks listed in Column 3. Columns 5 and 6 respectively list the average generic dimension of controllable space for transittability of 20% and 50% of nodes which are differently expressed at two states. Comparing Columns 5 and 6 to Column 3 concludes that the controllable spaces for transittability are much smaller than those for complete controllability. We employ four different biological systems with different phenotypes in order to demonstrate the applicability of our method, as well as validate our theoretical results. These four examples are p53-mediated DNA damage response network (three phenotypes), T helper differentiation cellular network (three phenotypes), yeast cell cycle network (three phenotypes), and epithelial to mesenchymal transition network (two phenotypes). shows the identified steering kernels for transition between two phenotypes. This network, consisting of 17 molecules and 40 interactions as shown in and , responds to cell stresses such as DNA damage and can stay at three phenotypes. If there is no DNA damage, the ATM is inactive (ATM2). The level of phosphorylated monomer (ATM*) is low, then the p53 remains inactive. The DNA damage can lead to two different cellular phenotypes: cell cycle arrest and apoptosis. At the cell cycle arrest phenotype, ATM is activated by DNA damage through auto-phosphorylation and transited from inactive dimer (ATM2) to ATM*. Subsequently, p53 is activated by ATM* and transited to p53*(p53 arrester). The expression levels of molecules represented by those green nodes in are oscillating. The p21 is the product of this state which induces cell arrest. In total, the expression values of 9 nodes are significantly changed when the normal phenotype is transited to the arrest phenotype. At the apoptosis phenotype, ATM* still activates p53 to p53*, but most p53* are in form of p53 killer. P53AIP1 is activated by p53 killer and finally activates Casp3 which induces cell apoptosis. At this state, PTEN contributes to full activation of p53. In total, the expression values of 12 nodes are significantly changed when the normal phenotype is transited to the apoptosis phenotype. In addition, comparing the arrest phenotype and the apoptosis phenotype, the expression values of all 17 nodes are significantly changed. Applying our methods to this network yields the steering kernel consisting of PTEN and p53DINP1 for the transition between normal and apoptosis phenotypes; the steering kernel consisting of Wip1 and p53DINP1 for the transition between normal and cell cycle arrest phenotypes; and the steering kernel consisting of Wip1, PTEN and p53DINP1 for the transition between apoptosis and cell cycle arrest phenotypes. These results are in great agreement with Zhang et al's results where PTEN and Wip1 are identified as key players for transitions of different states. On the other hand, if the complete controllability is applied to this network (), the minimum number of driver nodes is 3 while the driver nodes are not unique. For example, one minimum set of driver nodes consists of Wip1, PTEN and p53DINP1, which are the same as the steering nodes for transition between apoptosis and cell cycle arrest phenotypes. However, this set of nodes for other two transitions is redundant. Furthermore, the complete control strategy with these three driver nodes will affect the full state space during the phenotype transition as shown in , which is clearly undesirable in practice (see Discussion). T helper cells (Th cells) are a sub-group of lymphocytes, a type of white blood cells, which play an important role in the immune system, particularly in the adaptive immune system. They help the activity of other immune cells by releasing T cell cytokines. Matured Th cells express the surface protein CD4 and are referred to as CD4+T cells which can be classified as Th0 (precursor), Th1 and Th2 (effector) cells. Previously published experiments suggest that T-bet and GATA3 can induce both transitions from Th0 to TH1 and from Th0 to Th2. To deeply understand the mechanism of transitions among these phenotypes, Mendoza constructs a core network in charge of the differentiation of Th cells, which contains 17 nodes with 27 interactions as shown in and . Comparing among these three phenotypes, one can see that 5, 4, and 9 molecules are significantly differentially expressed, between Th0 and Th1 phenotypes, between Th0 and Th2 phenotypes, and between Th1 and Th2 phenotypes, respectively. Applying our methods to the T helper differential cellular network, we identify the steering nodes SOCS1 and T-bet for the transition between Th0 and Th1 and the steering nodes IL-4 and GATA3 for the transition between Th0 and Th2, which is in agreement with existing results. We also identify the steering nodes T-bet and GATA3 for the transition between Th1 and Th2, which is completely in agreement with the experimental data. However, if the complete controllability is applied to this network (see ), the minimum number of driver nodes is three and the three driver nodes are IL-12, IL-18 and IFN-β. Actually, without any one of these three nodes, this network cannot be completely controlled. Although it has been reported that IL-12 and IL-18 together can make the transition from Th0 to Th1, this complete control strategy will affect the full state space during the transition as shown in , which is undesirable in practice (see Discussion). The cell-cycle process is a vital biological process by which one cell grows to divide into two daughter cells. To study this process, Li, et al have established a molecular network consisting of 11essential molecules with 34 interactions as shown in and . Applying the logic-like operations and using the exhaustive search, they have found seven stationary states (attractors), each corresponding a stable phenotype. The attractor with the largest basin size corresponds to the G1 stationary state of the cell (denoted by phenotype 1). The next two largest attractors may represent some common disorder states of the cell (denoted by phenotypes 2 and 3). As other four attractors have small basin sizes, we do not consider them in this study. Comparing among these three phenotypes, we can see that 4, 1, and 5 nodes are significantly differentially expressed, between phenotypes 1 and 2, between phenotypes 1 and 3, and between phenotypes 2 and 3, respectively. On the one hand, the result by applying complete controllability to this network is that the minimum number of driver nodes is 1 which could be Cln3, MBF or SBF (). However, via either MBF or SBF this network cannot be completely controlled as either of them does not regulate node Cln3 from . Although via Cln3 the network can be completely controlled, the full state space will be affected as shown in , which is undesirable (see Discussion). On the other hand, we apply our methods to this network for studying the transitions among those three phenotypes. We found a single steering node MBF for the transition between phenotypes 1 and 3, and a single steering node SBF for both the transition between phenotypes 1 and 2 and the transition between phenotypes 2 and 3, which suggests that SFB and MBF play an important role for the transitions among these three phenotypes in the cell-cycle process. EMT is a phenomenon that cells change their genetic and transcriptional program leading to the alteration of phenotypes and functions. This change starts the metastatic dissemination which causes most human cancer deaths. To study EMT, Moes et al have constructed an EMT network consisting of 6 nodes and 15 interactions as shown in and . The expressions of MIR203, MIR200 and CDH1 are high and ZEB2, SNAI1 and ZEB1 are low at the epithelial phenotype while all are reversed at mesenchymal phenotype. Therefore, for this network, all 6 nodes are significantly differentially expressed between epithelial and mesenchymal phenotypes. Applying our algorithm, we can identify node SNAI1 as the steering node for the transition of these two phenotypes, which is completely in agreement with the experimental result verified by Moes et al that SNAI1 can activate the transition from epithelial to mesenchymal phenotype. Actually, by applying our algorithm, we can identify anyone of all nodes except for CDH1 as the steering node for the transition of these two phenotypes. From the other recent literature, MIR203 and MIR200 can also induce the transitions while leaving ZEB2 and ZEB1 deserving the further investigation about their function for the transition between these two phenotypes. In fact, controlling the transition between these two phenotypes is complete control of the network. When the minimum input theorem is applied to this network (), anyone of six nodes could be the driver node to steer the network from one phenotype to any other phenotype. However, acting input control signals on CDH1 cannot make the transition between these two phenotypes as CDH1 does not regulate any nodes in the network from . Transittability is at the heart of understanding state transitions of complex dynamic systems, especially cellular processes such as the cellular reprogramming and genetic disorder progressions. Besides the empirical studies, recently control theory for dynamical systems has been applied to complex systems. As indicated in discussions and , complete controllability of complex networks generally needs more steering nodes and its control affects the full state space (), and thus are not suitable to study the transittability and to identify the steering kernel for state transitions. Although the recently developed control strategy of nonlinear systems is applicable to study the transittability, it needs to know the exact expression of nonlinear functions and parameters in the model of complex systems, which is generally unavailable in practice. Instead of steering a directed network from any initial state to any desired state with the concept of complete controllability, transittability concerns the ability to steer a directed network from one specific state to another specific state. Obviously if a directed network is completely controllable, it can be steered from one specific state to another specific state, which indicates that complete controllability is sufficient for transittability. However, complete controllability is not necessary for transittability and should even be avoided in practice. For example, when considering the transition from a disease phenotype to a healthy phenotype, we expect to affect as a small state subspace as possible because side effects might be caused by unnecessarily changing some nodes in a large state subspace. The state subspace affected by a control law can be measured by the generic dimension of controllable (sub) space. The GDCS for complete controllability is always the full dimensional state space while the GDCS for two state transittability is generally a small subspace of the full state space (see and ). Therefore, in principle the control law based on the complete controllability affects states more than the one based on the transittability. In addition, as discussed in , a network cannot be guaranteed to transit from one specific state to any other state by acting the input control signals on only the minimum set of driver nodes identified by the minimum input theorem. Furthermore, although theoretically the steering kernel could be a subset of the minimum driver nodes, the minimum input theorem cannot be applied for efficiently finding the minimum number of steering nodes. Firstly, although the minimum number of driver nodes identified by minimum input theorem is unique, the maximum matching for a given network is not unique. Actually finding all maximum matchings for a given network is an NP-hard problem. This means that there is no efficient way to find all possible sets of driver nodes. Secondly, by the minimum input theorem, the minimum number of driver nodes is about 0.8 n for regulatory networks with n nodes. All possible combinations of 0.8 n driver nodes is at least 2, and thus it is computationally prohibitive to exhaustively check all of them. In this paper, we have systematically studied the transittability of directed networks and proposed an algorithm to identify the steering kernel for transitions between two specific states. To bypass the needs of knowing the exact expression of nonlinear functions and parameters in the complex systems, we have studied the transittability of directed networks with the concepts of structural linear systems and structural transittability. Our theoretical results provide the sufficient and necessary condition for determining the transittability, which is to check whether or not two GDCSs are equal. Although our theorems have been developed with continuous time-invariant linear systems, they can be directly applied to discrete time-invariant linear systems. Therefore, similar to the theorems, our results remain unchanged even if the free parameters in a linear system are allowed to vary with time. That is, our theoretical results are applicable to time-varying linear systems. To identify the steering nodes for the transition between two states, we have developed a graph-theoretic algorithm by solving an optimal assignment problem of a weighted bipartite graph. Applying our algorithms to 27 complex networks we have found that the minimum numbers of steering nodes for transiting two states are less than those for complete controllability and the controllable spaces for transittability are smaller than those for complete controllability. Furthermore we have applied our algorithm to 4 regulatory biomolecular networks and found that the numbers of steering nodes for transiting two cellular phenotypes are small, which is greatly in agreement with empirical studies on these networks. In addition, majority of steering nodes found by our method have been already reported in existing empirical studies while other new steering nodes are potentially important in corresponding cellular phenotype transitions. Therefore, we believe that our results also provide some fundamentals for understanding the mechanism of cellular phenotype transitions, and as such, are expected to have implications for network-based drug design. As can be seen, the theorems we developed in this study can directly be applied to any other complex networks, for example, social networks, power grids, food webs, the Internet, and electronic circuits, just named a few. In this paper, we mainly focused on studying transittability with suitable input signals, and the implementation or design of the control input signals as well as analysis of the dependence between the size of steering kernel and the degree distribution of networks could be one direction of future work. The T helper differentiation cellular network, the EMT network, and the yeast cell cycle network are directly from the published references without any change, respectively. The P53 mediated DNA damage response network are constructed from the differential equations in the of the paper. In such a construction, each variable in the differential equations corresponds to a node in the network. Node i regulates node j if the variable corresponding to node appears in the right-handed side of the differential equation corresponding to node j. All self-loops corresponding to the degradations are excluded in the constructed network as their weights may not be free parameters. For a network G(A) and structural state x, assume that the structure system (A, x) is irreducible. Let S be a subset of nodes corresponding to non-zero components in x. Let us define a weighted graph G′(A) as follows: 1) associate the weight = 1 with every edge e of G(A); 2) add the edge e = vv and associate the weight = if e = vv is not in G(A) for ; 3) add the loop e = vv and associate the weight = 0 if e = vv is not in G(A) for , where is a small positive number and less than 1/n for a network with n nodes. For simplicity, can take the value of 0.001, 0.0001, 0.00001 or the like. By solving an optimal assignment of a weighted bipartite graph representation of G′(A), we can find the maximum weight circle partition of G′(A). Assume that the weight of the maximum circle partition is + *, where r and s are integers and * < 1. Let t be the number of source strong connected components of G(A). Then, from .C, we have (, ) = (, ) = + and the size of steering kernel is . Note that the computational complexity of solving an optimal assignment of a weighted bipartite graph is O(n) according to reference for the worst cases in which a network is a complete graph. For the sparse networks which are true in most cases, our computational complexity is less than O(n). Actually and show that the our computational complexity is approximately O(n) for real complex networks with the number of nodes from 32 to 27772. F . X . W . , L . C . a n d J . L . c o n c e i v e d t h e s t u d y ; F . X . W . d e v e l o p e d t h e t h e o r e t i c a l r e s u l t s a n d w a s t h e l e a d w r i t e r o f t h e m a n u s c r i p t ; F . X . W . a n d L . W . a n d J . W . d e v e l o p e d t h e a l g o r i t h m s a n d d e s i g n e d t h e n u m e r i c a l s i m u l a t i o n a n d p e r f o r m e d t h e a c t u a l e x p e r i m e n t a l d a t a a n a l y s i s ; a l l t h e a u t h o r s m o d i f i e d t h e m a n u s c r i p t a n d a p p r o v e d t h e f i n a l v e r s i o n .

In recent works, integrated quantum circuits established on silicon waveguides are adequately investigated. Utilizing the femtosecond laser waveguide writing technology, on-chip devices to support and manuscript polarization-encoded qubits have been fabricated. A controlled-NOT gate has been experimental demonstrated with switchable entanglement. It is no doubt that silicon waveguides have excellent capabilities to confine and propagate light. However, limited by the inherent properties of silicon, they are not very convenient for fast manipulating and modulating entangled photons; such as the modulation scheme based on thermo-optical effect, the response time still has much room to improve in practical applications. In addition, to realize multifunctional integrated quantum circuits, entanglements of complicated formations are normally required such as the nonmaximally entanglement and mixed state entanglement. Under these circumstances, the LN-based system is considered as an alternative choice. Besides excellent nonlinear optical properties, EO effect in LN material provides an effective way to change the quantum state of photons through the photon interaction process. For a LN crystal, if we set its symmetry axis as the z-axis and apply a voltage along the y-axis, a variation is led into the dielectric constant. It could be written as with ( = 23, 32). In the equation, is the effective EO coefficient, and represents the applied electric field. The refractive indices of ordinary and extraordinary light are marked as and . This additional portion of permittivity corresponds to an equivalent polarization item, which is expressed as with . From , we obtain the interaction Hamiltonian for EO process as with In the equation, the quantized expression of electric field is utilized. is the overlap integral of waveguide modes expressed as . The function represents the structures of the inverted domains. The EO coefficient is periodically modulated, thus the o- and e-beams are coupled due to the periodic index ellipsoid deformation. The phase matching condition should be satisfied between the ordinary and extraordinary photons to ensure high conversion efficiency. The first item of the equation represents the annihilation of an e-polarized photon and the creation of an o-polarized photon, while the second corresponds to the inverse process. Accompanied by the two photon interaction process, the old photon state is substituted by new state which is desired to form target entangled state. Choosing proper EO processes to modulate entangled photon states supplies an innovative approach for entanglement architectures. As a simple illustration, the polarization qubit of photon is considered. Assuming is acted on a photon with polarization state , it would be transformed into polarization state with a certain possibility. Referring to the corresponding classical situation, we could obtain a superposition of and through modulating the applied voltage. The ratio of these two portions is determined by the value of applied voltage. The overall effect could be equivalent to = (), where is a standard rotation operator, and is the equivalent rotation angle. For a biphoton state, the modulation process could be written as with the matrix expression of the operator as where and are the corresponding equivalent rotation angles to determine the relative ratio of the two photon vector states, which are controlled by the applied voltage. It works similar to a half waveplate (HWP). However, there are still apparent differences. Assuming the input light is a linearly polarized light, a circular polarized beam is obtained if the wave plate thickness is only half. In contrast, the light passing through a PPLN with half-length is still linear-polarized, while the orientation angle is also half if the applied field is the same. For universal multiphoton entangled state, they could not be generated through SPDC directly, so the postprocessing modulations are quite critical. In multiphoton entanglement situation, only certain formations may lead to potential applications. We need to choose proper photons from the initial state , where represents the photon pairs from SPDC, and the function corresponds to their initial combination formation. After suitable modulations are applied based on the effective , the state thus could be transformed into a valuable formation such as GHZ state or cluster state. This process could be expressed as where ⊗ refers to the direct product of all . After the corresponding scheme is decided, the ferroelectric domains of the LN waveguide could be well-designed to satisfy the phase matching conditions of the related SPDC and EO processes. Through analyses of the set of {}, the matching vectors could be obtained as {}. Accordingly, the corresponding Fourier coefficients and the Fourier bases could be derivate. Based on these, the original function is recovered through where represents the actual domain arrangement of the entangled photon-pair source. It is worth mentioning that the correspondence between {} and {} does not have to be one-to-one. The choice of {} is flexible, which is an important part of domain engineering. As a detailed illustration, we consider the generation of an EO tunable polarization-entangled photon state with switchable characteristic. We consider the processes of photon interactions in a -cut, -propagating titanium in-diffused LN waveguide. There are well defined transverse electric (TE) and transverse magnetic (TM) propagation modes in the waveguide at near-infrared frequencies. In this work, only the fundamental modes of the TE and TM modes are discussed. For convenience, they are marked as o-polarized and e-polarized, respectively. Three photon interaction processes are included. They are two SPDC processes, which are expressed as , (, , and represent the pump, signal, and idler waves. and correspond to ordinary and extraordinary polarization); and an EO process takes place for the signal wave, namely (). If there is no voltage applied, only the SPDC processes happen in the waveguide. The power of the pump light is launched into the downconversion photons in corresponding propagation modes of the waveguide, the generated entangled state is type-II-like with the formation . In contrast, if there is an applied voltage, the EO effect is activated. The EO polarization rotation and SPDC processes happen simultaneously and are coupled everywhere in the crystal. In this case, the down-converted photons are equally generated everywhere and affected by the locally EO perturbed refractive index ellipsoid. The final obtained polarization state, including, e, o, e and o are the superposition result of all “sub-sources” inside the crystal. Therefore the rotation of polarization is realized through coherent superposition of all kinds of photons randomly collected in the sample. Corresponding to the coupling wave theory, that means the power of the fundamental TE mode () and the TM mode () at the signal frequency exchanges with each other. For a given sample length, the efficiency is dependent on the interaction strength, which is proportionate to the applied voltage. We could always find a proper voltage to make the efficiency reach ~100%, then the entangled state is switched to be type-I-like with the formation . Thus the entangled state is switchable through controlling the applied voltage on and off. To satisfy phase matching conditions of the three interaction processes, the domain structures need to be well designed. General speaking, we may design three sets of domain periods so that their reciprocal vectors are able to compensate these three wave vector mismatches. However, this straightforward scheme normally induces very thin domains and complicated structures. To solve this problem, several approaches have been proposed. For example, for the optimized wavelengths and domain structures, the needed domain periods could be reduced to two and they are of integer ratio, so that the domain structure could be easily fabricated. The schematic is shown in . Correspondingly, the sign of nonlinear coefficient and EO coefficient change periodically. The modulation functions are expressed as and , where . In the equations, is the substitute of nonlinear coefficient with the relationship /2. The effective component of and in our situation are and , respectively. Thus the reciprocal vectors are given as = , with their corresponding Fourier coefficients as . with where and represents the corresponding two modulation periods. Based on these analyses, the equations of QPM conditions could be expressed as In these equations, (j = p, s, i; σ = o, e) refers to the propagation constant of the corresponding waveguide mode, which is obtained based on the Hermite-Gauss formulations. and correspond to two SPDC processes, while corresponds to the EO interaction. (i = 1, 2, 3) are the required reciprocal vectors to compensate the phase mismatches. Through detailed calculations, we find an appropriate set of solutions for Eq.. The width and depth of the waveguide core are both set at 10 μm, and the maximum of index difference is set at 0.003. The pump, signal and idler wavelengths are 0.7335 μm, 1.6568 μm and 1.3162 μm, respectively. As room temperature operation is always more appreciated for future quantum circuits, the simulation temperature is chosen at 25°C. The photorefractive effect might bring some influences. However, the photorefractive damage of LN is not a simple process. It depends on many factors such as laser pulse width, operation wavelength and surface quality. Through recent technologies, the damage threshold has been increased to an acceptable degree for many applications. Moreover, the MgO doping is also an effective way to further avoid crystal damage. In this situation, the corresponding values for the () series are {() =, () = (3, −1), () =}. The dual-modulation periods are obtained at = 25.84 μm and = 154.96 μm, respectively. The ratio of these two periods is / = 6 and the duty cycle is 0.5, as is shown in . (More details about the selecting process could be referred to the section). To give a quantum mechanics description of our tunable entangled photon-pair source, we derive the state vector of entangled photons through the effective Hamiltonian. The total interaction Hamiltonian consists of two parts, which is expressed as . corresponds to the SPDC processes, while refers to the EO interaction. Both of them arise from polarization, namely, and . In detailed treatments, the pump field is usually treated as an undepleted classical wave. The signal and idler fields are quantized and represented by field operators. Thus the formulations of the pump, signal and idler fields could be written as where σ represents the polarization state or , and (j = s, i) is the corresponding refractive index. and are normalization parameters. , and correspond to the transverse mode profiles of pump, signal and idler field, respectively. For SPDC processes, if using the rotating wave approximation, is derived as with For the EO process, the interaction Hamiltonian is derivate as above. is expressed as with The entangled state vector could be obtained through . Detailed treatments thus could be divided into two cases: To ensure the quality of our tunable entangled photon-pair source, we investigate the corresponding properties. Firstly, the spectrum character is discussed, which is represented by the modulus squares of h-functions. For type-II-like polarization entangled photons, the corresponding expressions are and ; while those for the type-I-like polarization entangled photons are simultaneously affected by SPDC process and EO interaction, so the formulations are and , respectively. Calculation results are presented in . The natural spectral bandwidth of entangled source is mainly influenced by the group velocities of the signal and idler wavelengths. In our situation, these two wavelengths are close with each other, so the differences between the bandwidths for the two SPDC processes, , , + are relatively small. From and , when the waveguide length L is 3 cm, these two quantities are 0.35 nm and 0.27 nm, respectively. Compared with the previous report, these values are relatively smaller, which is beneficial to the improvement of entanglement degree. On the other hand, the natural bandwidth increases with the decrease of the waveguide length L. For instance, we could see that the value of bandwidth increase to ~0.9 nm for + process when the crystal length reduces to 1 cm. For pairs, as the EO process is integrated together, the natural bandwidths are further restricted through the corresponding h-functions. If the waveguide length L is 3 cm, the bandwidths for ordinary and extraordinary photon pairs are 0.20 nm and 0.23 nm, respectively. The difference between these two values is smaller, and the spectrum is almost symmetrical. The anticorrelation dip could be calculated based on the spectrum function, which is usually different for type-I and type-II SPDC. It is the Fourier transform of the SPDC spectrum. The corresponding formulation is . A narrower dip corresponds to a broader spectrum, so the width of anticorrelation dip is mainly determined by the natural bandwidth of the SPDC spectrum. For the orthogonally polarized entangled pair, corresponding to (Δ/2) and (Δ/2). We expand the phase mismatching to the first nonzero order of . The corresponding formulations are and . (j = p, s, i; σ = o, e) represents the dispersion parameter. For the parallel polarized entangled pair, as its spectrum is further confined by EO process, the spectrum property is naturally different from those of the traditional cases. The functions are written as (Δ/2)(Δ/2) and (Δ/2)(Δ/2). Similarly, we have and . As an illustration, the anticorrelation dips corresponding to (Δ/2) and (Δ/2)(Δ/2) are plotted in . Based on the discussions above, we calculate the von Neumann entropy of our source, in which the influences of (k, δ = o, e) are included. It is defined as = −(log). represents the reduced density operator for the subsystems. For a product state, the quantity vanishes. If the state is maximally entangled, the value of the entropy is 1. To describe tunable entangled source, the density operator of the entangled state is expressed as follows with The reduced density operator could be obtained through . Therefore the quantity is derived as where , , and . Besides, Θ = Θ + Θ and Θ = Θ + Θ. From the equation above, we could see that the value of S is mainly determined by Θ (i, j = o, e). In an ideal situation, , Θ = Θ and Θ = Θ, S would reach its maximal value 1, which corresponds to the maximal entanglement. If the natural bandwidth is narrow, and the ratio P/P, P/P is close to 1, Θ and Θ thus are almost equal to Θ and Θ, which makes S ≈ 1 and a very high degree of entanglement. On the contrary, wider bandwidth would affect the entanglement accordingly. As is discussed above, the natural bandwidth in our case is quite small. Following the treatments in Ref. , we simplify the calculation of into the perfect phase matching situation. Thus has the same formulation for two types of entangled states. This result verifies that EO process does not affect the entanglement degree. Besides, we could see that is mainly influenced by geometry and material parameters from Eq.. We find that the source has high tolerance for the geometrical variations. Until the waveguide modes of and are nearly cutoff, still stays above 0.99. The degree of entanglement maintains in a high level. What we have presented above is merely a simplest case for the two-photon entangled state, where the entangled states are generated and manipulated through EO modulation. Moreover, this type of system also could be used to generate multiphoton entanglement. A straightforward proposal is considered based on the switchable two-photon entanglement source proposed above. For our device, if the voltage is turned on, the generated entangled state would be . We may increase the pump power to improve the simultaneous generation possibility of double photon pairs in SPDC processes. In this situation, we may obtain four states with the same possibility, namely, , , and . To demonstrate the entanglement, we'd better erase influences of and . That could be realized through a post-selected detection scheme, which is widely used in energy-time entanglement. After the generated photon pairs pass through a single domain LN waveguide, the arrival time of o-polarized and e-polarized photons would be different due to the birefringence. If the frequencies of the down-converted photons are close and chosen around telecom band, the frequency dispersion would be small enough to be neglected. Therefore and could be detected the same time, while there would be time difference in detection of photons in and states. With a narrow time window that is usually used in energy-time entanglement, a four-photon GHZ state could be selected and detected. Moreover, with the help of on-chip optical elements, such as, mode analyzer, directional coupler, Mach–Zehnder interferometer and Y-coupler, entangled state of more than four photons and preselected entanglement scheme are designable. Moreover, with the help of artificially designed domain structures, the nonmaximally entangled state could also be easily realized, which can find applications in the loophole-free tests of Bell inequalities and quantum metrology with the presence of photon loss. The LN waveguide could be divided into several functional portions along the propagation direction. We may set the first portion to regulate the amplitude ratio of o-polarized and e-polarized pump light through EO interaction. Since both the o-polarized and e-polarized pump lights are involved, two corresponding SPDC processes, namely, o→ o + o and e → e + e may happen simultaneously in the second portion. They are dominated by the nonlinear coefficients d and d, respectively. In this case, the QPM conditions also could be satisfied for these two processes through suitable domain design. The non-maximally entangled state thus could be obtained that is expressed as . The relative ratio ε of amplitude may be controlled by the voltage applied on the first portion. As a consequence, an EO tunable non-maximally entanglement source is realized. The mixed state entanglement is not hard to be generated based on nonmaximal entanglement. A usual procedure is to create an entangled state with desired degree of entanglement, modulate the density matrix and then introduce decoherence. The first step could be realized through non-maximal entanglement with a suitable entanglement degree, and then we could utilize the EO polarization rotation effect to modulate the non-maximally entangled state to generate off-diagonal terms in the corresponding density matrix. To introduce decoherence into the system, experiments using bulk optical elements have been demonstrated based on the spacial properties of the SPDC emission cone. However, in waveguide systems, it's not convenient to use spatial decoherence directly. We could consider the temporal decoherence. Through introducing birefringent retardation, the vector and could be distinguished from and via the relative arrival time difference between two photons. Tracing over time, coherence between distinguishable terms in the density matrix could be erased, thus specific off-diagonal terms become smaller to form the target mixed entangled state. It's worth mentioning that besides the uniform domain periods we used all above, the domain structure of LN or similar materials could be elaborately prepared in more complicated patterns, such as quasi-periodic structures, which could provide complex reciprocal vectors to compensate the multi-process phase mismatching with high efficiency. We believe the domain engineered nonlinear waveguide is really a very promising platform for quantum integration circuits, which deserves more in-depth studies in both theories and experiments. As for practical applications of our device, the quantum cryptography protocol may be a potential area, which is the most realizable quantum information application nowadays. Based on the fast switching entangled state, our device provides a different strategy for quantum cryptography. Instead of switching the complementary analysis basis at the users' (Alice and Bob) places, we could switch between the states directly at the source while keeping the analysis basis always oriented along the same direction. Although this strategy may cause certain constrains, an advantage is that the operation conveniences for users may be improved. Another possible application example would be the classical and quantum communication without a shared reference frame. Alice may encode a logical qubit as , , and use it to communicate with Bob. If one applies a rotation to both of the photons, is unchanged, , it is invariant under arbitrary rotations of the reference frame around the propagation axis. Although will be changed, it is still orthogonal to . Therefore without a shared reference frame or the pre-alignment of Alice and Bob's polarization basis, a projective measurement on by Bob will distinguish the two logical bases. Furthermore, with small postprocessing modifications based on on-chip optical elements mentioned above, our source can switch between the generation of two entangled states with different symmetries and , both of which are invariant under the rotation of the reference frame. Thus a symmetry measurement by Bob will distinguish between the two states. The similar idea can also be applied to the error correction protocol in quantum computations. Moreover, in most of the integrated platforms to date, the quantum information is encoded in the spatial modes. Manipulating the polarization of photons on chip allows us to encode and decode classical and quantum information in the polarization degree of freedom. Fast switching means the shorter operation time of qubit, which is a desirable feature in practical applications. We believe over 1 GHz modulation bandwidth would be easily achieved in this case. In summary, we theoretically propose an effective approach to tailoring entangled photons through the combination of SPDC and EO effects. Based on the domain-engineered LN waveguide, an EO tunable polarization entangled photon-pair source is realized. It could selectively generate orthogonal-polarization or parallel-polarization photon by adjusting the applied voltage. The bandwidths and entanglement degrees of both types of entangled states are of excellent performances. In comparison with the bulk or in-fiber beam splitting, polarization handling, and wave plate elements, we proposed an integrated solution possibly on a single monolithic LN chip. This on-chip integration would be very desired in future quantum information related applications since it could be more compact, low loss and stable. In addition, the EO tunability is another attractive feature that supplies extremely fast manipulation on entangled photons, which is superior to the fixed wave plate solution. Moreover, the manipulation of some other entangled states are also discussed based on our proposals, including the multi-photon entangled state, the mixed entangled state and the nonmaximally entangled state, which show some attractive advantages. Introducing EO polarization modulation into domain engineered nonlinear waveguides offers opportunities for on-chip manipulation of quantum states and fast reconfigurability of the dedicated quantum chips, which is very desired for practical applications in future quantum circuits. To find a proper set of pump, signal and idler wavelengths, they are scanned carefully. Firstly, when the pump wavelength is fixed, calculation results reveal that and (for SPDC processes) are close but much greater than (for EO interaction). Thus they are assumed to correspond to the solutions (3, ±1) for (), respectively. For certain pairs of signal and idler frequencies, the values of and could be calculated through linear superposition of and , and then we compare the linear combination (1, ±1) of and with to see if they are matched. The corresponding expression is = − . For the wavelength satisfying = 0, three phase mismatches could be compensated simultaneously. In this study, we choose the pump wavelength at 775 nm. The values of against the signal wavelengths are plotted in (λ = 775 nm). It is seen from the figure that only the linear combination of and provides a solution for the equation . Thus are selected as the compensations of , and , respectively. Moreover, the phase matching conditions could be adjusted by the pump wavelength and the operation temperature, which are correspondingly shown in and . To ensure the ratio between and is an integer, we finally choose the pump, signal and idler wavelengths at 0.7335 μm, 1.6568 μm and 1.3162 μm, respectively. In this situation, , where the operating temperature is set at 25°C. For quantum communication applications, the operation wavelengths might be preferred to be chosen from the telecom-band. That could be easily realized by finding the solutions of Eq. accordingly, as is shown in . It is clearly from the figure that the whole telecom C band (1.53 μm–1.57 μm) and L band (1.57 μm–1.61 μm) could be selected for operation. Furthermore, the operating temperature could also be conveniently adjusted according the simulation results as shown in . Y . Q . L . a n d Y . M . c o n t r i b u t e d t o t h e o r i g i n a l i d e a . Y . M . , Z . J . W . a n d Z . X . C . d i d t h e o r e t i c a l a n a l y s i s , c a l c u l a t i o n s a n d i n t e r p r e t a t i o n s . A . H . T . p r o v i d e d t h e o r e t i c a l g u i d a n c e . Y . M . a n d Y . Q . L . w r o t e t h e m a n u s c r i p t t o g e t h e r . Y . Q . L . a n d F . X . s u p e r v i s e d t h e p r o j e c t . A l l a u t h o r s r e v i e w e d t h e m a n u s c r i p t .

To characterize the role of MreB in the GVE2 infection, the MreB gene was cloned from . E263. The results showed that the E263 strain contained MreB gene (), the sequence of which was highly conserved with that of mesophilic bacteria (data not shown), suggesting that MreB in thermophiles shared similar/same functions as that in mosephilic bacteria. In mosephilic bacteria, it is reported that the filament formation of MreB could be inhibited by A22 or MP265. E263 cells. E263 cells as helical structures, while the GFP alone showed no helix (). When A22 or MP265 was presented, the MreB-GFP could not form the helical conformation (). The results indicated that both of A22 and MP265 could inhibit the MreB polymerization in thermophiles as in mosephilic bacteria. As assayed, the optimal concentrations of A22 and MP265 were 40 μg/m and 70 μg/ml, respectively. The GVE2 virions were purified and observed to show the characteristic shape of bacteriophage (). Then the E263 cells were treated with A22 or MP265 and subsequently infected with the purified GVE2 at high temperature (60°C), followed by turbidity assays and plaque assays to evaluate the effects of MreB on the GVE2 infection. The results indicated that the addition of GVE2 led to a significant decrease of E263 growth rate due to the lysis ability of bacteriophage as evidenced by lower turbidity values and more plaques in samples incubated with the phage (). However, the A22-treated and GVE2-infected host cells (E263 + GVE2 + A22) grew normally, showing that the inhibition of MreB polymerization by A22 resulted in inefficient GVE2 infection. When the A22 in the A22-treated bacteria was removed and the bacteria were simultaneously infected with GVE2 [E263 + GVE2 + A22 (removed)], the turbidity values and plaques of the bacteria significantly decreased, suggesting that MreB was no longer inactive and that phage infection could proceed (). The MP265 yielded the same results as A22 (). The data presented that MreB played an essential role in the bacteriophage infection at high temperature and that this role could be withdrawn by addition of A22 or MP265. In an attempt to reveal the roles of MreB in GVE2 infection, the adsorption of GVE2 and replication of GVE2 genome were evaluated. In the GVE2 adsorption analysis, the MOI of GVE2 virions was 0.01. The results indicated that the copies of the free phages (phages not absorbed on E263 cell surfaces) were significantly increased in the A22-treated and GVE2-infected E263 cells (E263 + GVE2 + A22) at 15 min onwards post-infection of GVE2 (). The results showed that MreB played an important role in the absorption of GVE2 during virus infection. When A22 in the A22-treated bacteria was removed [E263 + GVE2 + A22 (removed)], the number of copies of free phages in this treatment was not significantly different from that in the positive control GVE2 only (). The data supported the conclusion that MreB was required for the adsorption of GVE2 in the host cells. It was found that when the MreB polymerization was inhibited by A22, the GVE2 genome copy numbers were significantly lower than those in samples with GVE2 only (), indicating the importance of MreB in the GVE2 genome replication. The results revealed that the recovery of MreB polymerization by removal of A22 led to significant increases of GVE2 genome copies (). One-step growth curve assays showed that the number of plaques was significantly decreased for the treatment E263 + GVE2 + A22 compared with those of the treatments E263 + GVE2 and E263 + GVE2 + A22 (removed) (), indicating that MreB might play essential roles in the GVE2 genome replication, particle assembly and host cell lysis. As assayed, MP265 yielded the similar results as A22 (). In order to characterize the effects of MreB on the distribution of GVE2 virions in host cells during phage infection, the A22-treated or MP265-treated E263 cells were infected with GVE2, followed by examination with confocal microscopy. The results showed that when the MreB polymerization was inhibited by A22 or MP265, the rod-shaped bacterial cells were changed into spherical cells (). However, the removal of the drug led to the recovery of bacterial cell shape (). The data indicated the essential role of the MreB polymerization in cell morphological change of bacteria. It was revealed that the morphological change of host cells led to the change of the GVE2 distribution in host cells during phage infection (). GVE2 virions preferred to localize in the cell poles of its host at the early stage of infection. However, the virions were distributed around the host cells when the MreB polymerization was inhibited by A22 or MP265 (). As controls, the samples were labeled with anti-GST antibody, and no signal was observed (data not shown). These results indicated that the cytoskeleton protein MreB, required for the morphology of bacteria, affected the distribution of phage in host cells during phage infection. Confocal microscopy data revealed that the percentage of GVE2-infected host cells was significantly decreased in the treatment E263 + GVE2 + A22 compared with that of the treatment E263 + GVE2 (). The findings suggested that the polymerized MreB could facilitate the GVE2 infection by affecting the distribution of virions during the phage infection. To evaluate the colocalization of MreB and GVE2, the thermostable MreB-GFP fusion protein was overexpressed in GVE2-infected E263 cells. The results revealed that the MreB-GFP was colocalized with GVE2 during the phage infection, while the controls presented no colocalization of GFP and GVE2 (), suggesting that the polymerization of MreB benefited the infection of GVE2. To determine the direct involvement of MreB in the polar localization of the GVE2 virions, the MreB-GFP -overexpressed E263 cells were treated with A22 or MP265, followed by infection with GVE2. The confocal microscopy results revealed that MreB-GFP was colocalized with the GVE2 virions and was distributed around the cells in the presence of A22 or MP265 (). In this case, the GVE2 virions were not localized in the cell poles of its host (), suggesting that MreB was directly involved in the polar localization of the virions. Collectively, these findings indicated that MreB played an essential role in the phage infection by direct interaction with the phage. Most studies on the interaction between virus and cell cytoskeleton protein come from eukaryotes. Viruses have evolved several ways to exploit the host actin cytoskeleton by using its essential functions in eukaryotic cells, in particular, the uptake and short-range intracellular transport. At present, homologues of at least three eukaryotic cytoskeletal protein families (actin, tubulin, and intermediate filaments) have been characterized in bacteria. Phages have co-evolved with their host bacteria, thus, it's not surprising that bacteriophages can take advantage of their host's MreB cytoskeletal systems, which are known scaffolds for some key processes of bacterial cells. It is believed that viruses exploit host actin to invade their hosts in eukaryotes. In bacteria, however, the roles of cytoskeletal protein MreB in phage-bacterium interactions have not been extensively investigated. Our study showed that during the GVE2 infection the host MreB was required for the adsorption of phage and the replication of phage genome in the host cells. Adsorption of bacteriophage, the first step of phage infection process, involves the recognition and contact of phage with the host cell surface. In general, adsorption of phage occurs in two steps, a reversible step and a subsequent irreversible binding step. The first contact of phage with its host cell surface is usually reversible binding. So the reversible step allows for the dissociation of phage from its host. Subsequently, the phage attaches irreversibly to specific receptors on the host cell surface, leading to the injection of the phage genome from its capsid into the host cell. Although DNA injection and irreversible adsorption has been characterized, it is still contentious. Phage adsorption to its host cell surface needs a specific interaction between the phage tail proteins and host receptors. Receptors are exposed in the outer membrane of Gram-negative bacteria and in the thick peptidoglycan cell wall of Gram-positive bacteria. Phage adsorption to Gram-positive bacteria is still poorly understood. It has been reported that the teichoic acid in the bacterial cell wall is essential for reversible binding of several phages in , such as phi 29, phi 25, and SPP1. Our study revealed that the host bacterial MreB was involved in the replication of GVE2 genome in host cells in high temperature environment. The cytoskeleton protein MreB might function as a primary organizer of the phage DNA replication because phage DNA and replication machinery components distributed in helical structures in a MreB-dependent way. As well known, the gene konck-out strategy is conventionally employed to investigate the function of a gene. However, the molecular biology of thermophiles is not intensively investigated, resulting in the lack of gene knock-out system, gene overexpression system and protein labeling platforms at high temperature. To reveal the role of MreB in the phage infection, the gene knock-out strategy was conducted, but it was not successfully carried out in this study, suggesting that the deficit of the MreB might be lethal for the thermophiles. These issues merit to be further investigated. In this context, the requirement of host MreB in the phage infection process resulted from the involvement of MreB in the adsorption and replication of GVE2 in its host cells. During the infection process, bacteriophages bind preferentially to host cell poles in some Gram-negative and Gram-positive bacteria. For example, phage lambda needs a polar localized inner membrane protein ManY to help the injection of phage genomic DNA. Therefore, it usually shows a polar adsorption at low multiplicities of infection. In the case of phage SPP1, both reversible adsorption step and irreversible binding to YueB occur preferentially near the cell poles, which are related to the initiation of DNA replication. It is evident that the MreB protein is involved in the pole targeting of phage in bacterial cells. In our study, the confocal microscopic images revealed that MreB played an important role in the polar distribution of phage in host cells. The MreB polymerization was required to keep the morphology of bacterial cells and the polymerized MreB facilitated the phage infection. This study indicated that MreB was an essential factor in both adsorption of GVE2 and replication of GVE2 genome. However, the mechanism of those processes and the detailed role of MreB in GVE2 infection are still unknown. The molecular events in bacteriophage infection process in high temperature environment need to be characterized in future. Thermophiles, the basis of the food chain in deep-sea hydrothermal vent, power the vent biological communities with the source of energy. In the vent ecosystem, the most significant players in nutrient and energy cycling are thermophilic viruses. Our findings provided insights into the interaction between bacteriophage and host thermophile in high temperature environment. This would be helpful to reveal roles of bacteriophages in controlling bacteria population and carbon turnover in deep-sea hydrothermal vent ecosystems. The deep-sea thermophile . E263 and its thermophilic bacteriophage GVE2 were isolated in our previous studies. The host E263 was cultured at 60°C in TTM medium (0.2% NaCl, 0.4% yeast extract, 0.8% tryptone; pH 7.0) supplemented with 25 mM MgSO. The host strain cultures in the mid log phase (OD = 0.4) were infected with GVE2 at a multiplicity of infection (MOI) of 0.01, 0.5 or 5. The GVE2 virions were purified as described previously. Virus samples were examined under a transmission electron microscope (JEOL 100 CXII) for purity. To determine the dosages of the MreB inhibitor, A22 [S-(3,4-dichlorobenzyl) isothiourea, Merck, Germany] or MP265 (4-chlorobenzyl chloride, Alfa Aesar, USA) with different concentrations (0.1–100 μg/ml) were mixed with cultures of E263 at the mid log phase (OD = 0.4). At different time after A22 or MP265 inoculation, the bacteria were collected and examined by microscopy to evaluate the optimal dosages. To assess the infection of E263 cells by GVE2, a turbidity assay was performed as previously described. E263 cells were cultured at 60°C. When the OD of the cultured bacteria was 0.3, the bacteria were mixed with A22 (final concentration 40 μg/ml) or MP265 (final concentration 70 μg/ml). Ten minutes later, the bacteria were infected with the purified GVE2 virions at a MOI of 5. To evaluate the effects of the removal of A22 or MP265 in the A22-treated bacteria or the MP265-treated bacteria on the GVE2 infection, the bacteria of the treatment E263 + GVE2 + A22 (removed) or E263 + GVE2 + MP265 (removed) were cultured for 1 h after the GVE2 infection and then centrifuged at 18,000 × g for 5 min to remove A22 or MP265. Subsequently the pelleted bacteria were resuspended and cultured in fresh TTM medium. At different times after the culture of E263, the optical densities (OD) of all treatments were measured by spectrophotometer (UV-1700 Shimadzu Corporation, Japan). The experiments were repeated three times. The E263 cultures (OD = 0.4) were mixed with A22 (40 μg/ml) or MP265 (70 μg/ml) and then incubated for 30 min. Then the bacteria were mixed with GVE2 virions at an MOI of 0.5 and subsequent with TTM soft agar medium (TTM medium with 0.5% agar). The soft media were spread on TTM solid medium plate, followed by incubation at 60°C overnight. For the treatment E263 + GVE2 + A22 or E263 + GVE2 + MP265, A22 or MP265 was dissolved in the TTM soft agar medium. For the treatment E263 + GVE2 + A22 (removed) or E263 + GVE2 + MP265 (removed), there was no A22 or MP265 in the TTM soft agar medium. Finally the plaques were examined. E263 cultures at OD of 0.4 were supplemented with 10 mM CaCl and 15 mM MnCl at 60°C. At the same time, 40 μg/ml of A22 or 70 μg/ml of MP265 was added into the cultures. After culture of E263 for 30 min, the bacterial cells were collected and infected with purified GVE2 virions at an MOI of 0.01. For the treatment E263 + GVE2 + A22 (removed), A22 was removed 30 min after addition of A22 (as described above). The treatment E263 + GVE2 + MP265 (removed) was carried out accordingly. Then the cells were infected with GVE2. At different time postinfection, the bacteria were collected. After 5 s of vortex, the bacteria were incubated for 5 min at room temperature, and then centrifuged at 15,000 × g for 5 min. The supernatant was subjected to quantitative real-time PCR to detect the free (non-adsorbed) phages. Cells of E263 at OD of 0.3 were infected with purified GVE2 virions at a MOI of 5. After culture at 60°C for 30 min, the cells were mixed with 40 μg/ml of A22 or 70 μg/ml of MP265. For the treatment E263 + GVE2 + A22 (removed) or E263 + GVE2 + MP265 (removed), A22 or MP265 was removed at 30 min after addition of A22 or MP265 as described above. The treated bacteria were collected at different times postinfection, vortexed for 5 s, lysed for 20 min at 99°C, and centrifuged for 10 s. The supernatant was subjected to quantitative real-time PCR for the detection of GVE2 genome copies. The experiments were conducted three times. The E263 cells were suspended in 800 μl of fresh TTM medium and then incubated with GVE2 at an MOI of 0.5 for 30 min at 4°C. Subsequently 10 μl of the antibody against VP371 (a capsid protein of GVE2) was added to terminate the adsorption. The mixture was centrifuged at 12,000 × g for 10 min and the pellets containing the GVE2-infected cells were resuspended in 20 ml of TTM medium. The bacteria were mixed with A22 (40 μg/ml) or MP265 (70 μg/ml) and incubated for 10 min at room temperature. For the treatment E263 + GVE2 + A22 (removed) or E263 + GVE2 + MP265 (removed), the bacteria were washed with TTM media by centrifugation at 1,000 × g to remove A22 or MP265. All the above bacteria were cultured at 60°C. At different time after culture, the bacteria were subjected to plaque assays. The experiments were biologically repeated three times. Real-time PCR was conducted to quantify the GVE2 genome copies in cell lysate using GVE2-specific primers (5′-ATCGGTTGTACTAACTTAAC-3′ and 5′-GCTTG TCGTATTCCTTATC-3′) and GVE2-specific TaqMan fluorogenic probe (5′-FAM CC GTCTTGTTCGTTGTCTCTGC-Eclipse-3′). The genomic DNA of GVE2 was collected from the GVE2-infected E263 samples by heating at 99°C for 20 min. The real-time PCR was conducted as described previously. Protein recombinant expression, antibody preparation and antibody labeling were carried out as before. Briefly the vp371 gene of GVE2 was cloned into pGEX-4T-2 vector (Novagen, Germany) and expressed in BL21 (DE3) as a glutathione S-transferase (GST)-tagged fusion protein. The recombinant VP371 protein was used as an antigen to immunize mice. The immunoglobulin G (IgG) fraction of the antiserum was purified with protein A-Sepharose (Bio-Rad, USA) and stored at −80°C until use. As determined by enzyme-linked immunosorbent assay, the antiserum titer was 1:10,000. The specificity of antibody was confirmed using Western blotting with the recombinant protein. Then the antibody labeling was conducted to label the antibody using fluorescent dyes. E263 cells at exponential growth phase were treated with 40 μg/ml of A22 or 70 μg/ml of MP265. At 30 min after the addition of the drug, E263 cells were harvested by centrifugation at 1000 × g for 1 min. Subsequently the A22-treated, MP265-treated and drug-free E263 cells were examined with a phase contrast microscopy (Nikon, Japan). Overnight cultures of E263 were diluted with TTM medium containing 0.01 M MgCl at 1:100 and then grown at 60°C. When the OD of bacteria reached 0.3, the bacteria were treated with 40 μg/ml of A22 or 70 μg/ml of MP265. After culture for 30 min, the cells were infected with GVE2 at an MOI of 5. At different times post-infection, the GVE2-infected bacteria were collected. For imaging, the E263 cells were immobilized with methanol for 30 min at 4°C. Then the labeled anti-VP371 or anti-GST antibody was added to the immobilized cells that were permeabilized by acetone (30 min, 4°C), and the cells were incubated overnight at 4°C. The cells were labeled with 10 μg/ml of 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen) for 10 min. Subsequently 10 μl of samples were dripped on slides (Sigma, USA) covered with a thin 1% agarose film and examined under a Leica TCS SP5 confocal microscope (Germany). The digital images were acquired by and analyzed using LAS AF version 2.0.0 software. In order to elucidate the effects of A22 and MP265 on the MreB polymerization, a thermosensitive promoter, a thermostable GFP gene (Los Alamos National Laboratory, USA) and the MreB gene of E263 were cloned into the pNW33N vector (Bacillus Genetic Stock Center, USA) to make the pNW33N-sgsE-MreB-GFP construct. Then the E263 cells were transformed with the plasmid and cultured at 65°C. E263 cells at exponential growth phase were treated with 40 μg/ml of A22 or 70 μg/ml of MP265 for 30 min. After immobilization with methaol for 30 min, the drug-treated and drug-free E263 cells were examined with a Leica TCS SP5 confocal microscope (Germany). The E263 cells were transformed with the pNW33N-sgsE-MreB-GFP plasmid and cultured at 65°C. Overnight culture of E263 harboring MreB-GFP was diluted with TTM medium at 100 folds, and continued to grow at 65°C. When the OD reached 0.6, the bacteria were infected with purified GVE2 at an MOI of 5. At different times, the bacteria were collected and the GVE2 virions were labeled with the anti-VP371 antibody as described above. Subsequently the bacteria were examined under a Leica TCS SP5 confocal microscope (Germany). The numerical data from three independent experiments were analyzed by one-way analysis of variance (ANOVA) to calculate the mean and standard deviation (SD) of the triplicate assays. M . J . , Y . C . a n d C . X . p e r f o r m e d t h e e x p e r i m e n t s u n d e r t h e s u p e r v i s i o n o f X . Z . , M . J . a n d Y . C . w r o t e t h e m a n u s c r i p t . A l l a u t h o r s r e v i e w e d t h e m a n u s c r i p t .

xref #text T h e r e a r e ( a t l e a s t ) t h r e e c o m m o n o b j e c t i o n s t o t h e v i e w t h a t n o n - m y o c y t e s m a y b e e l e c t r i c a l l y c o u p l e d t o m u s c l e c e l l s i n n a t i v e c a r d i a c t i s s u e . Gap junctions, in particular between cardiac myocytes, lend themselves to relatively easy identification. Fibroblast gap junctions are significantly smaller , and thus are more easily overlooked. One should further note that early immuno-histochemical characterisation of connexin distribution in cardiac tissue did not tend to include any form of cell labelling. In this setting, intercalated discs between myocytes are a prominent feature, while the small ‘speckles’ of fluorescence, indicative of fibroblast-based gap junctions, could easily be regarded as background noise . What's more, the absence of immuno-histochemically detectable gap junctions in confocal microscopy studies is not necessarily synonymous with a lack of connexin-based cell coupling, as shown for example in native arterial smooth muscle . Unless super-resolution optical techniques are used, fluorescently labelled Cx will be detected only if spatially-clustered, involving at least 80–120 channels (of note, smaller arrays, containing 15–20 Cx-channels, are regularly present in rat ventricular myocardium, as confirmed by EM ). Computer modelling studies suggest that as few as 10–30 connexin channels would provide sufficient coupling for electrophysiologically relevant effects of cardiac fibroblasts on myocytes . Possible sites for such coupling could be finger-like extensions of cardiac fibroblasts into the cardiomyocyte basement membrane — points of contact that may be more frequent in living (A), rather than the fixed and dehydrated tissue used in most histological analyses. , provide an alternative substrate for electrotonic coupling between different cell types. These structures may either form connexin-free direct cytosolic links , or involve Cx-coupling at the point of contact of two semi-tubes. The latter could occur ‘anywhere’ along the nanotubular connection, somewhere between apparently separate cells. Punctate immuno-labelling (in the absence of a cytosolic dye) would, in this setting, probably be regarded as background unrelated to intercellular connections . Evidence for the presence of nanotube coupling between cardiac fibroblasts and myocytes has been shown and (B; ), although their relevance for cardiac electrophysiological integration remains to be established. The notion of being able to explain cardiac electrophysiology, based solely on homocellular myocyte coupling, is not without limitations. For example, atrial ablation lines (scars, created to interrupt uncontrolled excitation waves) often become ‘electrically transparent’ with time, due to re-emergence of functional conduction pathways . While this may be due to incomplete lesioning, 10–20% of heart transplant recipients also show electrical coupling across the (unquestionably continuous) separation between donor and recipient tissue . Aberrant electrical coupling can arise also across suture lines after operations to fix cardiac birth defects . In all these cases, electrical conduction scar tissue. Possible explanations include direct ‘touch-and-go’ interaction of surviving myocytes either side of a cut, generation of cardiomyocytes that link the two tissue edges, or electrical conduction non-myocardial cells such as fibroblasts . Based on the histological appearance of post-transplantation scar tissue , the last option may offer the most straightforward explanation and, hence, be in keeping with Ockham. Fibroblasts in native heart tissue at rest have a membrane potential of - 10 to - 20 mV when electrically isolated from other cells . They are electrically non-excitable (though subsequent to long-term cell culture, expression of the voltage-gated sodium channel Nav1.5 has been reported in human atrial fibroblasts ). Fibroblasts are excellent ‘followers’ of an electrotonically provided waveform. Thus, if electrically coupled to a cardiomyocyte, the fibroblast's membrane potential will show a myocyte-like action potential shape, albeit with a slowed upstroke and reduced amplitude (as illustrated in double whole-cell patch clamp experiments ). The ability of fibroblasts to passively convey action potentials, together with their high membrane resistance that supports ‘low-loss’ long-range signal conduction, makes fibroblasts a tantalising potential conduit for electrotonic signal transmission. Unfortunately, this very behaviour also makes it nearly impossible to confidently explore cardiac fibroblast electrical integration , using classic electrophysiological techniques . While fibroblasts that are electrically isolated from cardiac muscle cells can be identified by their weakly polarised membrane potential and mechanically-induced polarizations in the rhythm of tissue contraction, those fibroblasts that are well-coupled (and, hence, at the centre of interest here) are indistinguishable from poorly impaled cardiomyocytes (; ). Nonetheless, indirect support for heterocellular connections in coupled fibroblasts has been found (, centre; note that upon electro-mechanical uncoupling of the preparation, the mechanically induced component ‘’ recedes, while ‘’ and ‘’ remain). Immuno-histochemical labelling studies for a range of cardiac connexins, combined with identification of cells on either side of connexin signals, suggest that Cx-localization at the point of contact between cardiac fibroblasts and myocytes is far from uncommon, both in healthy ( ) and post-infarct tissue ( ). Of course, presence of a protein ‘in the right place’ does not confirm its functionality. Direct evidence for heterocellular coupling in native tissue has thus far been obtained only in rabbit sino-atrial node, where dye transfer between myocytes and fibroblasts was observed . Clearly, further investigation is needed to explore developmental dynamics, anatomical distribution, physiological regulation, and response to disease progression or treatment attempts, but proof-of-concept for fibroblast–myocyte coupling has been established. A conceptual approach to myocardial function that excluded the possibility of heterocellular coupling would, therefore, be unnecessarily restricted. Given the principal difficulty of distinguishing between trans-membrane potential waveforms of (active) cardiac myocytes and (passive) fibroblast-followers in normal heart tissue, cardiac scars offer an interesting alternative study subject, because the ratio of electrically active and passive cells is shifted significantly towards predominance of the latter. A fine example of such work is the 2007 study of Walker ., investigating fully-transmural infarcts in adult rabbit left ventricle by optical mapping of voltage-sensitive dye signals (A). They observed propagation of cardiac excitation waves into scar tissue, even after chemical ablation of any surviving sub-endocardial muscle layers (B), performed to exclude possible contributions from these deeper myocyte-sheets to epicardially mapped potentials; ). The signals from within the scar resembled ventricular action potentials, albeit with slowed upstroke and reduced amplitude — very much in keeping with previous observations in neonatal rat fibroblast–myocyte cell pairs and adult rat atrial tissue . Subsequent work by other investigators confirmed that excitation waves can indeed invade and pass through cardiac scar tissue . These studies further showed that the myocyte-like action potentials inside four-week old ventricular scar tissue in rabbit ventricle are not accompanied by significant cyclic changes in intracellular free calcium concentration . As calcium transients are a signature activity of cardiomyocytes, the most straightforward explanation again is that non-myocytes passively display much of the electrical signals conducted inside scars , perhaps electrically integrating surviving myocyte islands that could act as active ‘repeater stations’ that maintain or boost electrical signal amplitude. #text There is a growing array of new experimental tools at our disposal to explore heterocellular coupling in native cardiac tissue. In coming years it is likely that we will see these tools provide unexpected advances in our understanding of the electrical interplay between the multiple cell types that make up the heart. In particular, it is anticipated that there will be definitive answers on where and when myocytes and non-myocytes are capable of electrophysiologically-relevant coupling , and what the context and the relevance are of such interactions. Answers here will provide new insight into normal cardiac function and into mechanisms of disease development. Perhaps more importantly, an understanding of the nature and dynamics of heterocellular electrical communication in the heart could give rise to novel or improved therapeutic approaches. This may range from amendments to established procedures, such as atrial ablation, to the vision of modifying cardiac scar properties in a targeted manner to reduce arrhythmogenesis following infarction. a s m o d e s t e q u i t y i n F i r s t S t r i n g R e s e a r c h I n c . c o m p a n y t e s t i n g C x 4 3 - b a s e d t h e r a p e u t i c s f o r w o u n d h e a l i n g ( c o - f o u n d e r , s c i e n t i f i c a d v i s o r y b o a r d m e m b e r ) .

It is established that kidney function is reduced with age, but since there is a co-variation with vascular disease, the precise ageing effect is uncertain. There are studies strongly supporting the hypothesis that a decline in kidney function is related to normal senescence [, ]. In the indigenous people of Kuna, who have no known vascular disease, there is an age-related decrease in glomerular filtration rate (GFR) as measured by inulin clearance []. In a subset of healthy participants aged 29–100 years from four cohort studies, there was a strong correlation between age and increasing cystatin C []. On the other hand, in the well-known Baltimore Longitudinal study of Ageing, where creatinine clearance was the measure of GFR and healthy participants were followed for up to 14 years, 36% showed no decline in creatinine clearance []. If there is an ageing effect on renal function in the absence of disease, there is little evidence as to what factors influence it. A sex effect has been proposed and rejected [, ]. Smoking seems a natural culprit, and it has indeed been shown that it induces microalbuminuria and that it deteriorates kidney function in those with CKD [], and there are indications that it also increases the risk of CKD in the general population []. Blood lipid levels, physical activity, BMI and blood pressure have all been investigated for their correlation with kidney dysfunction and evidence has been found [–]. Vascular risk factors causing atherosclerosis are thus likely to account for an important part of the decline in renal function with age, whereas the influence of sex is less certain. An age-associated decline in renal function has been interpreted to be linear as well as accelerating [, , ]. Another debate is whether or not there is an epidemic of chronic kidney disease (CKD). There is an escalating proportion of the population with CKD defined by estimated GFR (eGFR) <60 ml/min/1.73 m []. Nevertheless, it is uncertain whether an eGFR below this limit is always due to disease or if a normal ageing process can lead to such low filtration. In a large population study, it is usually not feasible to use true clearance methods since it is invasive. However, eGFR will never become more than an estimate and has weaknesses particularly in older people due to changes in body composition. Therefore, finding suitable formulas for all ages, body compositions, with or without chronic disease has not been possible. Cystatin C has arisen as a strong alternative, especially in older people since it is not dependent on muscle mass []. In the light of these uncertainties, we decided to study a representative group of non-diabetic older people without vascular disease to (i) explore the variations in plasma concentrations of cystatin C, (ii) study the effect of ageing on the level of plasma cystatin C, identify a possible impact of sex and evaluate whether smoking, physical inactivity and other atherosclerotic risk factors also affect the kidney function of those without known vascular manifestations, (iii) estimate the proportion that would be diagnosed as having CKD with the prevailing criterion. This is, to our knowledge, the first time cystatin C has been studied in a large population of healthy older people originating from one cohort. The original population of the Good Aging in Skåne (GÅS), part of the Swedish National Study on Aging and Care (SNAC), includes 2,931 participants aged 60–93 years old from 9 age cohorts, randomised from the municipality registers with a participation rate of 60%. The study has been described elsewhere in detail [] and creatinine and cystatin C has been studied for the whole cohort []. Plasma cystatin C was as one batch measured by a fully automated particle-enhanced immunoturbidimetric assay [] at Lund University Hospital, Laboratory Medicine using the Hitachi Modular P analysis system and reagents from DAKO (Dako A/S, Glostrup, Denmark). The total analytical imprecision was 2.1% (with concentration of 1.0 mg/l in control sample) and 1.7% (with concentration of 4.0 mg/l in control sample). Normal reference range: 0.63–1.44 mg/l (>50 years) []. Hereafter plasma cystatin C level will be referred to as cystatin C. Three formulas from the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) were used to calculate eGFR []. CKD-EPI creatinine = 141 × min(Scr/, 1) × max(Scr/, 1) × 0.993 × 1.018 (if female) × 1.159 (if black), is 0.7 for females and 0.9 for males, is −0.329 for females and −0.411 for males, min indicates the minimum of Scr/ or 1, and max indicates the maximum of Scr/ or 1. CKD-EPI cystatin C = 133 × min (Scys/0.8, 1) × max(Scys/0.8, 1) × 0.996 × 0932 (if female), min indicates the minimum of Scys/0.8 or 1, and max indicates the maximum of Scys/0.8 or 1. CKD-EPI creatinine–cystatin C = 135 × min(Scr/, 1) × max(Scr/, 1) × min(Scys/0.8, 1) × max(Scys/0.8, 1) × 0.995 × 0.969 (if female) × 1.08 (if black), is 0.7 for females and 0.9 for males, is −0.248 for females and −0.207 for males, min indicates the minimum of Scr/ or 1, and max indicates the maximum of Scr/ or 1. Scr is serum/plasma creatinine and Scys is serum7plasma cystatin C. eGFR was categorised in CKD stages according to Kidney Disease Outcomes Quality Initiative guidelines []. To assess the combined effect of age and the factors sex, smoking and physical activity on cystatin C, a multivariate regression analysis was performed with adjustment for main effects: age, sex, smoking, physical activity, in-office systolic and diastolic blood pressure, BMI, total cholesterol, HDL, lipid-lowering treatment as well as interaction effects between age and sex, smoking and physical activity (Table ). -values corresponding to age and interaction terms involving age were adjusted for multiple testing using Bonferroni correction. A -value of <0.05 was considered statistically significant. Analyses were conducted using the statistical software SAS 9.2, Cary, NC. The study was approved by the regional ethics committee at Lund University (LU 744-00). All subjects provided written consent to participate. sup ext-link table-wrap #text This study particularly focuses on the non-diabetic general older population without vascular manifestations and points to a decline in kidney function with age that is more pronounced in men. The ageing effect observed in population studies is explained by several structural changes seen in the ageing kidney: loss of renal mass, arterial sclerosis, arteriolar hyalinosis, loss of tubules, interstitial fibrosis and increasing glomerulosclerosis []. Even though male sex is associated with lower cystatin C levels, there is an additional ageing effect in men. The sex effect on GFR has been debated and results are diverse. Odden showed the opposite sex-dependent ageing effect on cystatin C levels in their population of healthy participants aged 28–100 years old, although the additional ageing effect in their study was clinically insignificant and as far as we understand not corrected for multiple testing as in the present study []. In addition, to our knowledge, this study is the first in which cystatin C is analysed in a large number of healthy persons older than 80 years originating from one general population study with one standardised study protocol and standardised laboratory methods. Although the effect of sex on GFR has been quite extensively researched for younger adults, there are much less data on the sex-specific effects of ageing. In a meta-analysis, male sex was associated with a more rapid rate of progression in CKD []. Berg confirmed this in healthy potential kidney donors and showed a significant decrease in men with age compared with females, but this was done in a group aged 20–50 years []. A slower decline in kidney function in women is explained by higher levels of oestrogen and lower levels of androgens []. The suggested mechanisms are that women have a more robust nitric oxide system [], lower renin–angiotensin–system activity [] and in the ageing women increased levels of metalloproteases preventing matrix expansion in the kidney []. Our data confirm previous findings that in the healthier older people, HDL seems to have a protective effect on renal function. [, ]. Smoking is related to a decline in kidney function in this population. However, when smoking interacts with the ageing effect, we cannot see a significant additional ageing effect of smoking. This might be due to our broad definition of smoking that includes all the people who have ever smoked. It could also be due to the fact that the smokers that have had vascular incidents are excluded by definition or survival bias. The increase in cystatin C nearly doubled comparing the young and intermediate age groups with the intermediate and oldest age groups, which may indicate an accelerating ageing effect. This corresponds to previous findings [, ]. However, in the study of the whole GÅS cohort including subjects with diabetes and vascular manifestations, this phenomenon was not obvious. One explanation might be that the ageing effect is diluted by vascular disease in the non-selected material []. Forthcoming longitudinal studies on subjects without vascular manifestations and risk factors may reveal a possible accelerating ageing effect on renal function. The ageing effect—whether accelerating or not—seems to be highly individual since the intra-age-group difference between the 5th and 95th percentile almost doubled from the youngest to the oldest age group in our study. In early longitudinal studies on ageing and kidney function, it was observed that although most people deteriorated a third did not []. At least half of the participants over 80 in this study demonstrated an eGFR < 60 and are thus regarded as having CKD in spite of being relatively healthy. It has been shown, however, that older individuals with deteriorated kidney function have a lower risk than younger of progression to end-stage renal disease []. In the whole GÅS cohort, 6.5% (CKD-EPI creatinine) and 18.9% (CKD-EPI cystatin C) of persons >80 years old were classified as CKD Stages 4–5 (severe kidney disease) [] which correspond with a Belgian cohort of 80+ years []. In this healthier subgroup, the prevalence was much lower than 3.9% (CKD-EPI cystatin C) and 3.4% (CKD-EPI creatinine), which could indicate that normal ageing normally does not result in severely reduced kidney function. Our study has some limitations. We do not use a confirmatory gold standard such as iohexol clearance. At the time for this study, there was no international calibrator for cystatin C which may be a concern when using the different formulas. The problem with the current use of estimation equations is highlighted by the fact that depending on which formula used half (CKD-EPI creatinine) or two thirds (CKD-EPI cystatin C) of the 80 years old in the study fall below the limit of eGFR 60 ml/min. Moreover, albuminuria was not measured, which would complement our measures of kidney dysfunction. The self-reported data could include underreporting of previous smoking. The cystatin C assay was run before the introduction of a calibrator for cystatin C. The participation rate in GÅS was 60% and visits at home were done to counteract selection bias of the more frail older people. In a study of a healthier subgroup, the participation rate is a smaller problem since there tends to be a selection bias with the participants being healthier. Inherent confounders to cross-sectional studies include specific birth cohort effects and survivors' bias. In this particular case, survivors' bias would work against the age effect since there is an increased mortality risk with reduced kidney function []. In conclusion, this cross-sectional study shows an effect of ageing on cystatin C levels in those without overt vascular disease or diabetes. This ageing effect is more pronounced in men and is also associated with lower HDL, higher diastolic blood pressure and smoking. This tendency has to be confirmed in longitudinal studies. The heterogeneity in plasma concentration of cystatin C seems to become greater with age. Finally, the incidence of severe CKD (eGFR < 30) in those over 80 years old is substantially lower in the healthier subset compared with the whole GÅS cohort. #text ext-link #text

The rising prevalence of chronic disease and disability with older age increases the need for healthcare services in the ageing population. Healthy living habits play a major role in the ageing process [, ], but other exposures that the persons face during the life course increase the risk of adverse health outcomes in old age. Both high mental and physical job strain contribute to increased sickness absence from work and early withdrawal from the workforce [, ]. Higher mental and physical job strain have been found to increase the prevalence of several chronic diseases and conditions such as coronary heart disease, diabetes and obesity as well as mortality [–]. Recent findings indicate that mental job strain and work-related stress [–] and physical work conditions [] have long-term effects on health and functioning also after retirement in old age. Although evidence on the long-term effects of job strain is accumulating, physical and mental job strain have less frequently been investigated simultaneously in a representative population using concrete public health indicators such as care use. These findings are of interest while simultaneously as the demands of the working life increase, the ageing workforce is expected to transition into retirement at an older age []. This might potentially exert negative effects to the health and functioning of the individuals who enter into third age. In the present study, we explore further the effects of physical and mental job strain assessed in midlife on the volume of hospital in-patient care use in old age among public sector employees working in blue- and white-collar professions. The Finnish Longitudinal Study on Municipal Employeesby the Finnish Institute of Occupational Health included 6,257 middle-aged municipal professions randomly chosen from all municipals in Finland [, ]. At baseline in 1981, 5,625 individuals aged 44–58 years had postal questionnaire data available on physical and mental job strain, for a response rate of 89.9%. Compared with the original sample, those with missing data on job strain in midlife were on average older (51.5 versus 50.3 years, -test < 0.001) and more often blue-collars (66.6% versus 44.5%, -test < 0.001), but there were no differences in gender or health behaviours such as smoking or alcohol consumption. The Ethical Committee of the Finnish Institute of Occupational Health approved the study. We used baseline data on job strain to avoid the ‘healthy worker effect’ [, ], which might have resulted in underestimation of the association between job strain and hospital care use. Job strain had most likely been relatively stable during the latest years of the work careers, because >70% of the male participants had held the current work position for the last 10 years. Mental job strain was examined with the job demand–control concept described by Karasek [, ]. The five items (Cronbach's alpha = 0.73) included questions on, e.g. pressures related to work, work-pace and time schedule. Participants were asked whether these items were present at work ‘not at all = 0’, ‘little = 1’, ‘somewhat = 2’ or ‘a lot = 3’. The summary score ranged between 0 and 15 with a higher score indicating higher demand. The 10-item scale termed job control (Cronbach's alpha = 0.85) included questions on, e.g. to what extent it was possible to influence the work environment or participate in planning of the work. Participants were asked whether the items were present at work ‘not at all = 0’, ‘little = 1’, ‘somewhat = 2’ or ‘enough = 3’. The summary score ranged between 0 and 30 with a higher score indicating higher level of job control. Summary scores on job demand and job control were calculated and divided into thirds indicating low, intermediate and high levels of demand or control. The mental job strain indicator was constructed as: low job strain (low demands with high or intermediate control), high job strain (high or intermediate demands and low control) and intermediate job strain (all other combinations of demands and control) []. Physical job strain at work was assessed with three items on cardiorespiratory strain [] and three on musculoskeletal strain, Cronbach's alpha = 0.87. Cardiorespiratory strain was assessed with the question: ‘How often does physical exertion in your work cause: (i) sweating, (ii) breathlessness and (iii) heart palpitation?’ The answering alternatives for these three items were: ‘never or very rarely = 0’, ‘rarely = 1’, ‘occasionally = 2’, ‘quite frequently = 3’ and ‘frequently = 4’. Musculoskeletal strain was assessed with the question ‘How much strain does work cause to your: (i) arms, (ii) legs and (iii) back?’ The answering alternatives for these three items were ‘not at all/ very little = 0’, ‘not much = 1’, ‘pretty much = 3’ and ‘very much = 4’. The summary score ranged between 0 and 24 with a higher score indicating higher physical strain. The summary score of these six items was calculated with a higher score indicating higher physical strain and it was further divided into distribution-based thirds indicating low, intermediate and high physical job strain. Hospital in-patient admission and discharge dates were extracted from the Finnish Hospital Discharge Register (FHDR) between January 1, 1981 and December 31, 2008 for all participants. A recent review showed that compared with external information on hospital care, >95% of discharges were identified in the FHDR []. One hospital care day was defined as an overnight stay or day surgery in a central, district or university hospital or health centre. The total volume of hospital care was calculated by adding together all hospital care days which had accumulated during the 28-year follow-up. Mortality dates were obtained from the Finnish National Population Register. Occupational class was formed from the 133 different occupational titles identified by clustering them into 13 occupations based on job analysis at the participants’ workplaces []. These were further collapsed into blue-collar (e.g. maintenance and cleaning), lower white-collar (transport work and nursing) and upper white-collar (e.g. administrator and physician) professions. Smoking (never smoked, ex-smoker or current smoker), alcohol consumption (never, twice a month at most or at least once a week) and physical activity during previous year (inactive, moderate activity once a week at most or vigorous activity at least once a week) were also inquired in the questionnaires. Main chronic illnesses diagnosed or treated by a physician such as cardiovascular diseases (e.g. hypertension), metabolic disorders (e.g. diabetes) and musculoskeletal diseases (e.g. arthritis) were inquired in the questionnaires. The association between physical and mental job strain in midlife and the volume of hospital care use were tested using negative binomial regression models, where the strength of an association is estimated as an incidence rate ratio (IRR) and 95% confidence interval (CI). IRRs are interpreted as relative risk estimates and represent the risk for persons in the predictor variable groups relative to those in the reference group. The analyses were conducted for subjects who died or survived over the follow-up period. An offset variable of the length of follow-up time was used to adjust estimation for a function of the number of days the subjects spent under observation. Volume of hospital care was a continuous variable with 1 day as a unit. Analyses were adjusted first for age and length of follow-up and then for occupational class, health behaviours and main chronic illnesses. We analysed men and women separately (Wald test = 0.051 for the interaction between gender and job strain on hospital care use). Modelling was performed in the R statistical programming environment (version 2.12.2) using the MASS-package (version 7.3–11) []. The mean age at baseline was 50.3 years (SD 3.6) and 45.0% were men. The characteristics in midlife according to gender are presented in Table . Over the course of 28 years, only a few individuals (0.5%) had no in-patient hospital care days. Duration of hospital in-patient care ranged between 0 and 2,875 days. Median duration of hospital care during the follow-up was 39 days (interquartile range [IQR] 13, 96 days) for men and 32 days (IQR 12, 78 days) for women. The median number of hospital care episodes during the follow-up was 6 (IQR 3, 12 episodes) and ranged between 0 and 123 episodes. Of the 5,625 participants, 1,738 (1,073 men and 665 women) died during the follow-up. For the deceased, median duration of follow-up was 19.5 years (IQR 13.5, 24.5 years) ranging from 20 days to 28 years. The ones who survived the follow-up were at baseline slightly younger (49.8 years SD 3.4 versus 51.6 years SD 3.6, -test < 0.001), more often women (78.5% versus 57.6%, -test < 0.001) and white-collars (61.2% versus 42.7%, -test < 0.001), reported more frequently low physical (34.3% versus 28.8%, -test < 0.001) and mental (21.1% versus 19.0%, -test = 0.017) job strain in midlife than the ones who did not survive. The rates of hospital in-patient care days per 1,000 person-years, presented in Table , for men were 7.78 (95% CI 7.71, 7.84) for low, 9.68 (95% CI 9.50, 9.74) for intermediate and 12.56 (95% CI 12.47, 12.66) for high physical job strain in midlife. For men, these rates were parallel but slightly lower for mental job strain. For women, the rates of hospital care were 6.63 (95% CI 6.57, 6.68) for low, 7.91 (95% CI 7.87, 7.95) for intermediate and 10.35 (95% CI 10.25, 10.42) for high physical job strain in midlife. Among women, there were no differences in the rates of hospital care according to mental job strain in midlife. The associations between job strain and volume of hospital in-patient care in the 28-year follow-up are presented in Table . Compared with those public sector employees with low job strain in midlife, men with high job strain had on average a 42% and women a 71% higher risk of spending an in-patient day in a hospital during the follow-up, IRRs adjusted for age and length of follow-up time were 1.42 (95% CI 1.24, 1.62) and 1.71 (95% CI 1.53, 1.92), respectively. For men the association between mental job strain in midlife and volume of hospital care was parallel but lower. For women, the association between mental job strain in midlife and volume of hospital in-patient care was less clear and not statistically significant. Further adjustment for occupational class, health behaviours and main chronic illnesses assessed in midlife attenuated the IRR's, but they remained statistically significant for physical job strain. Next, we included both physical and mental job strain simultaneously in the models to examine whether they modified each other's effect on the volume of in-patient hospital care, see Table . In the model adjusted for age and length of follow-up, the effects of physical and mental job strain in midlife on hospital care in old age were similar to the separate effects described above. Adjusting for occupational class, health behaviours and main chronic illnesses attenuated the associations particularly for men. Finally, we only included hospital care that took place after the employees turned 65 years ( = 5,097) to investigate the long-term effects of job strain on hospital care use. The results were similar, if not even more pronounced, compared with those reported for all hospital care during the follow-up. We found in this population-based cohort including male and female employees working in the public sector, that high physical job strain assessed in midlife predicted an increased need for in-patient all-cause hospital care in old age. Findings were parallel but less evident for mental job strain. To our knowledge, this is the first prospective study to investigate the long-term effects of both physical and mental job strain on the overall volume of hospital care use in a representative old population. Our study extends previous findings on the relation between job strain and morbidity by using hospital in-patient care days as a measure of the burden of morbidity over a long follow-up period. Earlier findings on the effect of higher physical job strain on increased cardiovascular morbidity and mortality have been reported [, ]. A number of epidemiologic studies have found that high mental job strain increase the prevalence of certain chronic diseases and conditions such as cardiovascular disease, diabetes and obesity [, ]. However, few studies have investigated the effect of both physical and mental job strain in the same cohort and little is still known about the effects of work strain on old age health. We found the rate of hospital in-patient care per 1,000 person-years to be almost two times higher for those with high physical job strain. Exposure to high job strain during the work career seemed to set individuals on a higher care use trajectory also years later, which was confirmed in the present analyses showing that employees with high job strain in midlife had an increased risk of hospital in-patient care also after turning 65 years. This is in line with earlier findings on the association between low work ability in midlife and higher prevalence of disability in old age [], which is a well-known risk factor for increased need of care in older age []. We found that the longer the person survived, the less hospital care days were needed. This can be explained by the increase in hospital care need in the last years of life due to decreasing health []. Contrary to the common view of death being a competing risk, the competing risk in our study was the end of the follow-up. This meant that most of the participants who survived until the end of the follow-up did not reach the last years of life where need for hospital care is highest. Further studies in unselected decedent populations are warranted to examine job strain as a predictor of hospital care use in the last years of life. The study strengths include a long prospective follow-up in a large population-based dataset consisting of a wide variety of professions. We had complete register-based data available until old age for all participants for the entire study period and did not lose anyone to follow-up. There are some limitations that need to be addressed. First, we imputed missing values in the job strain items at midlife which might have introduced some bias to the results. However, the results were similar when using non-imputed data. Second, the participants with data on job strain were younger and more often white-collars than those who did not have data available on job strain. Furthermore, our study included occupationally active public sector employees and should be considered when generalising the present results on a population level. In our study, high mental and particularly physical job strain in midlife increased the volume of hospital in-patient care use in old age. These findings are of clinical importance while the demands of the working life increase among the ageing workforce, which is expected to continue to work in older age in Western countries. A recent meta-analysis [] showed that a healthy lifestyle reduced the risk of coronary artery disease substantially among those with high job strain. This indicates that along with measures that improve work conditions and decrease job strain among the middle-aged employees, promoting a healthy lifestyle is likely to reduce the long-term negative effects of job strain. For example, work place physical activity programs have yielded positive results in terms of increased physical activity and decreased musculoskeletal disorders []. However, more research on the effectiveness of programs that help preserve the health and functioning of ageing employees is warranted in the effort to prevent increase in the volume of healthcare services needed in old age. #text N o n e d e c l a r e d .

Improving mental wellbeing throughout the life course is recognised as an important component of healthy ageing []. Healthy ageing is influenced by an individual's socioeconomic position throughout the life course [, ], but also by societal level factors such as the welfare state [, ]. Early old age, containing both the retired and those in the final stages of working life, is increasingly acknowledged as an important stage of the life course, as the labour force across Europe is becoming older []. Measuring wellbeing and its determinants among this population is therefore considered a high policy priority. The welfare state is thought to be a major determinant of the patterning of inequalities in health and wellbeing [], as it may modify the effect of socioeconomic position on these outcomes. It is unlikely that any single aspect of the welfare state is responsible for wellbeing, but rather its influence is likely to arise as a result of a combination of policies. Five welfare regimes have been characterised within Europe. These are based on the differing contributions of the family, market, and state to the welfare of individuals within a country []. Southern countries (including Spain and Greece) typically have fragmented income maintenance schemes and exhibit high dependency on the family and voluntary sector [, ]. Scandinavian countries are characterised by a more interventionist state that seeks to promote social equality via principles of redistribution, universalism, a commitment to full employment and income-protection [, ]. Germany and France exemplify Bismarckian regimes. Benefits are usually earnings-related and administered by the employer, a supportive role for the family is encouraged, and social divisions are maintained []. Post-communist countries (including Poland and the Czech Republic) are characterised by their transition from communism to market economies and have social security systems which provide limited coverage []. The UK and Ireland are considered to be part of a ‘Liberal’ regime type, characterised by market dominance and modest state benefits, which are often means-tested []. Our study aims to first examine the magnitude of socioeconomic inequalities in life satisfaction among Europeans in early old age, using measures of socioeconomic position from across the life course. Second, we investigate the influence of the type of welfare regime on socioeconomic inequalities in life satisfaction. Data were taken from Wave 2 (release 2.5.0) and SHARELIFE (release 1.0.0), the retrospective Wave of the Survey of Health, Ageing and Retirement in Europe (SHARE) collected during 2006–07 and 2008–09, respectively. SHARE is a longitudinal panel survey, which collected representative data from individuals aged 50 and over in 13 European countries. Further information about SHARE is found elsewhere [, ]. The population studied included individuals in early old age (50–75 years) participating in Wave 2 and SHARELIFE, who were born in their current country of residence ( = 18,324). During Wave 2 participants were asked: ‘On a scale from 0 to 10 where 0 means completely dissatisfied and 10 means completely satisfied, how satisfied are you with your life?’ Life satisfaction is a valid measure of wellbeing, which contains substantial information about how individuals evaluate their lives []. Life satisfaction was treated as a continuous variable to ease the interpretation and comparison of results [, ]. Childhood socioeconomic position was captured by the number of books in the household when the participant was aged 10 years, collected via retrospective recall. Education level was recorded using the International Standard Classification of Education (ISCED-97) []. Current household wealth (equivalised) was derived from a series of questions relating to financial and real assets, as well as liabilities []. Countries were grouped into four welfare regimes: Southern (Greece, Italy and Spain), Scandinavian (Denmark and Sweden), Post-communist (Czech Republic and Poland) and Bismarckian (Austria, Belgium, France, Germany, the Netherlands and Switzerland). To quantify socioeconomic inequalities in life satisfaction, slope indices of inequality (SIIs) were calculated []. Each measure of socioeconomic position was ranked from the least advantaged to the most advantaged (with the mid-point of their range in the cumulative distribution used for each category) []. These were standardised to produce a rank, where the theoretically most advantaged had a value of 1 and least advantaged a value of 0. The SII was calculated by running linear regression models using the socioeconomic rank to predict the outcome measure. It can be understood as the difference in mean life satisfaction between the hypothetically least and most socioeconomically advantaged. Since socioeconomic distributions varied by country, gender and cohort (pre-1946 and post-1945) separate ranks were calculated for each of these groups. Relative indices of inequality by gender and welfare regime were also calculated by dividing the SII by the mean life satisfaction. Single level linear regression models stratified by welfare regime and containing country fixed effects were run to calculate the SIIs. Multilevel (random-intercept) regression models were also calculated to test the statistical interaction between the welfare regime type and socioeconomic position. All analyses were stratified by gender and adjusted for age group in 5-year bands. Further methodological details available in the . The highest level of life satisfaction was found in Scandinavian countries and the lowest in Post-communist countries (Table ). Full-descriptive statistics are found in . Table displays the SIIs for each measure of socioeconomic position stratified by welfare regime (results from the multilevel models are found in ). Narrower socioeconomic inequalities in life satisfaction were found in Scandinavia, when examining the effect of the number of books in childhood. For men in Scandinavia, the SII was 0.08 (95% CI: −0.18 to 0.35) and for women 0.13 (95% CI: −0.11 to 0.37). Differences in life satisfaction between welfare regimes were most apparent at the least advantaged end of the socioeconomic ranking and narrowed as the rank increased (). SIIs were larger in each of the other regimes among both genders, largest for men in the Post-communist regime and for women in the Southern regime. Narrowest educational inequalities in life satisfaction were found among men and women in Scandinavia (Figure A and B). The SII for men in Scandinavia was 0.37 (95% CI: 0.09 to 0.65) and for women 0.13 (95% CI: −0.13 to 0.38). Among both genders, the Bismarckian regime also exhibited relatively narrow inequalities in life satisfaction. Largest education SIIs were found in the Post-communist regime for both men (SII = 1.11, 95% CI: 0.70 to 1.52) and women (SII = 1.39, 95% CI: 1.00 to 1.77). Wealth inequalities in life satisfaction were smallest in Scandinavia for both genders (Figure C and D). However, the SIIs for wealth were mostly larger across all regimes compared with the other measures. In Scandinavia, the SII for men was 0.73 (95% CI: 0.48 to 0.99) and for women 0.67 (95% CI: 0.44 to 0.91). These were not much larger in the Bismarckian regime and were largest in the Post-communist regime. Scandinavian countries also exhibited the narrowest relative inequalities in life satisfaction (). For most measures of socioeconomic position, the Bismarckian regime also displayed narrower relative inequalities compared with Southern and Post-communist countries. Socioeconomic inequalities in life satisfaction among Europeans in early old age were present in all welfare regimes. The Scandinavian welfare regime exhibited the narrowest inequalities in life satisfaction, using all three measures of socioeconomic position from across the life course. Post-communist countries generally exhibited the largest socioeconomic inequalities in life satisfaction. Inequalities in life satisfaction were largest using current wealth in most regimes, but inequalities by education level were large, particularly among women in the Post-communist and Southern regimes. Highest levels of life satisfaction were also found in the Scandinavian regime and the lowest in the Post-communist regime. This study has a number of strengths, including the utilisation of high quality and comparable survey data. Potential limitations include the risk of attrition and survival bias. However, the expected direction of bias is likely to underestimate the magnitude of inequalities [–]. Few studies have investigated the influence of the welfare regime on socioeconomic inequalities in wellbeing among older populations, with most studies using negative measures of health. Socioeconomic inequalities in poor self-rated health were found to be narrower in Nordic countries [, ], but others have had contradictory results [, ]. Our results have important implications for future research and policy, especially given the recent welfare policy changes across Europe []. As life satisfaction captures one aspect of wellbeing, further research using different indicators is needed to check the consistency of findings. The differing magnitude of inequalities by measure of socioeconomic position highlights the importance of using multiple measures when quantifying inequalities in health, especially among older populations. We recommend future research examines the impact of changes to welfare policy to try and unpack which policies may foster a more equitable distribution of wellbeing. Our findings suggest that mechanisms to buffer the effect of socioeconomic disadvantage in early old age specifically, perhaps through more redistributive fiscal policy and universal pensions, may help to reduce socioeconomic inequalities in life satisfaction for this age group. n e d e c l a r e d . #text uri #text ext-link

demonstrates the fabrication process of hybrid (ultra)thin films supported on porous anodic alumina (PAA) membranes. It should be noted that all of the thin films presented in this work are hybrid films using poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) as matrices. PEDOT:PSS is a conjugate polymer that is widely used in transparent conductive electrodes. Experimental details can be found in the . Other polymers such as polystyrene (PS), poly(methyl methacrylate) (PMMA), and polyvinyl alcohol (PVA) can be used as matrices depending on the applications. Polymer matrix not only promises flexible processability but also provides mechanical support to the hybrid thin film that is required in the solution-based transfer and washing processes. Unless stated otherwise, commercially available 20 nm (pore size) PAA membranes were used for vacuum filtration. Home-made PAA membranes (with pore size down to 5 nm) could be used under certain circumstances. Such PAA-based vacuum filtration is, in principle, suitable for any component with at least one dimension larger than 20 nm (pore size). This method is also benefited from the possible large-area and high-throughput production of thin films with controlled thickness. In-situ and post washing the thin films with volatile solvents such as ethanol would efficiently remove the remaining high-boiling-point dispersants, e.g., CHP or NMP, which facilitates the production of high-performance thin film-based structures/devices. A general method for in-situ transfer of the as-fabricated hybrid thin films was illustrated in . Sodium hydroxide (NaOH) aqueous solution was applied to etch away the PAA membranes. Prior to the etching step, a specific configuration of the samples was adopted. As shown in , the film on PAA was placed upside down onto the desired substrates, e.g., PET or glass, with one edge attached onto the support through double-sided scotch tape. Such simple but unique configuration enables the flexible fixation of the free-standing thin films on the substrates after removal of the PAA membranes. Another key function of such line-contact mode is to ensure that the free-standing thin films were directionally guided throughout the whole solution process, e.g., etching and washing step. It should be noted that 3M NaOH solution was used in order to quickly (in 10 seconds) detach the PAA membranes from the thin films, making the etching process very efficient. Furthermore, to keep the solution process safe and sufficient, a specific and repeatable trajectory, as shown in , was introduced. A tilting posture, rather than vertical placement, was adopted for both the putting-in and taking-out step during etching and washing. To ensure that no residual NaOH was left on/in the thin films, greatly excess amount of deionized water was used for the washing step. In specific, the hybrid thin films were washed sequentially in three filled large beakers (500 mL) and with three times for each beaker at a relatively slow movement along the trajectory (e.g., 3–5 seconds per cycle). The following drying process with argon is also delicate and cautions must be taken to avoid bubbles and wrinkles. Practice would be very helpful to make bubble-free and wrinkle-free transferred thin films. Such bubble and wrinkle issues could also occur during the above solution process. For this reason, a volume ratio (1:4) of ethanol to the aqueous solution was used to minimize the bubble and wrinkle issues, in case of PET substrates. Now the hybrid thin films have been transferred onto the desired substrates. When necessary, the scotch tape can be removed from PET or glass using cotton with ethanol or razor blade respectively. Based on the proposed method, hybrid thin films on arbitrary substrates/surfaces were obtained. shows the photographs of various transferred thin films. Thin film transferred onto a hole structure, as illustrated in , can function as free-standing thin films (in the unsupported hole area) which could be very useful in substrate-free applications. It should be noted that flow drying is not suitable in case of a hole structure. An alternative method, i.e., natural drying when taken out from pure ethanol, was used. Fully self-stretched free-standing thin film was formed after the ethanol was rapidly and completely evaporated under ambient conditions. The ultrathin film transferred onto PET (50 μm thick) () shows promising properties such as high transparency () and flexibility (radius of curvature down to mm scale, ), which will be detailed in the following sections. Thin film transferred onto curved surface is shown in . This suggests the possibility for production of thin film-coated complex optical components (e.g., hole/dot/rod arrays), which could be very useful in lasering, imaging, and sensing. Thin films can be transferred onto glass, as indicated in . Not only CNT-based () but also graphene-based () thin films were successfully fabricated and transferred. The proposed method applies to other 1D (e.g., molecular, polymeric, metallic, and metal oxide wires) and 2D (e.g., molybdenum disulphide (MoS), bismuth selenide (BiSe), and boron nitride (BN) monolayers) materials, as well as 0D (larger than 20 nm) materials, therefore could provide a powerful route for production of more complex thin films. For example, complex thin film incorporating graphene and dye molecule aggregates is presented in . Its complexity (or structural hierarchy) is clearly shown in . The dispersion systems play an important role on the quality-control of the resulting hybrid thin films. Transmission electron microscope (TEM) was used to characterize the CNT and graphene dispersions which were made as described elsewhere. clearly reveal that both CNTs and graphene are well-dispersed on the supporting copper grids. It is known that aggregation often occurs after the solvents are completely evaporated. In our case, no bundles of CNTs or bulk graphite were found by searching the entire copper grids. They are mostly individual CNTs or monolayer and few-layer graphene, indicating the high quality of the dispersion systems. The thin films formed from these dispersions were characterized by field emission scanning electron microscope (FE-SEM). It should be noted that all SEM measurements were done without metallic coatings owning to the conductivity of the hybrid thin films. To avoid the charging issue during SEM measurements, both ends of the thin films were connected to the conducting stage through double-sided conductive carbon tape. Therefore these SEM images present the real surfaces of the thin films. presents the effect of metallic (i.e., Au) coating on the SEM images of the complex thin film incorporating graphene and dye molecule aggregates (the same sample as in ). reveal that the surfaces of the CNT-based thin and ultrathin films are quite uniform over large areas, consistent with that observed from the photographs () of the thin films. To reveal the CNT networks on the surfaces of the thin films, InLens detector was used, as shown in the insets of . presents the direct comparison of the images taken from InLens and SE (secondary electron) detector respectively. The surface of graphene-based thin film, as presented in , is also very uniform over wide ranges. The inset indicates that graphene sheets are well-distributed on the surface of the thin film. Compared with graphene-based thin film (), more complex thin film () shows relatively rough surface with well-distributed dye molecule aggregates. Similar situation on graphene distribution (inset of ) was found in this complex thin film. As mentioned above, one of the promising applications for the hybrid thin films is transparent conductive electrodes. presents the absorption spectra of the thin films as shown in . The significantly low absorbance of the ultrathin film () indicates its high transparency. As shown in , the transmittance of the ultrathin film is above 90% in the visible range and above 80% in the NIR range. In particular, the transmittance at 550 nm is above 94%, suggesting its extraordinarily high transparency. presents the I–V curve from the 4-probe electrical measurement of the ultrathin film. The linear behaviour of the measured I–V response indicates that such ultrathin film is suitable as electrode materials. The sheet resistance was measured to be 1850 Ω/□, higher than the industry standard (i.e., 100–200 Ω/□). However, the conductivity was calculated to be as high as 1.54 × 10 S/m based on the ultrathin (35 nm, see ) feature of the film. Therefore, the ultrathin film could be used as transparent conductive electrodes for applications such as solar cell. One of the key parameters for practical applications is the durability of the thin films. demonstrates that the ultrathin film is highly solvent-resistant. A simple test (i.e., applying the scotch tape on the film, pressing hard, and slowly removing the tape from the substrate) reveals the relatively strong adhesion between the ultrathin film and the supporting PET film (50 μm thick). It should be noted that no observable damage on the ultrathin film was found after several cycles of rubbing the surface with gloved fingers (with nitrile gloves), indicating such ultrathin film is also durable. It should be noted that a dry transfer technique was recently reported, which offers an extremely simple (direct contact and press transfer) and rapid (<15 s) transfer method towards large-scale (28 × 28 cm) CNT networks. However such a dry transfer technique could suffer from similar drawbacks to the PDMS method, e.g., the difficulty to transfer onto arbitrary substrates such as hole structures or curved surfaces. In addition, the transferred film could show relatively weak durability, which might be improved by a post heat and pressure process. Furthermore, the success of such a direct contact transfer process was determined by not only the relative adhesive strength of the film on the target and source substrates but also the mechanical strength of the film, indicating the limited applications in such as 1D nanostructure/nanomaterial networks. Our solution-based in-situ transfer method overcomes the above drawbacks and provides a powerful technique towards diverse substrates, durable transferred films, and broad available film components. Both solution-based and dry transfer methods can be utilized for applications such as transparent, conductive, and flexible electrodes. For example, the ultrathin CNT hybrid film showed a relatively low sheet resistance of 1850 Ω/□ at a very high transmittance of 94%, similar to that (2700 Ω/□ at 90%) of the intrinsic CNT (3.3 μm) networks. The sheet resistance of the treated CNT networks was significantly decreased to as low as 110 Ω/□, approaching the industry standard. #text Vacuum filtration was applied to fabricate the hybrid thin films using poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) as the matrices. PEDOT:PSS was purchased from Heraeus under the trade name Clevios PH 500, with initial concentration of 10–13 mg/mL in water. Dye molecules (i.e., 5, 10, 15, 20-tetraphenyl-21H, 23H-porphine (PPH)) were purchased from Sigma Aldrich and used as received. To fabricate the complex thin film incorporating graphene and dye molecule aggregates, PPH was dissolved in NMP at a concentration of 0.05 mg/mL. The stock CNT and graphene dispersions were with concentration of 0.36 mg/mL and 0.4 mg/mL respectively. For CNT-based thin films, PEDOT:PSS/HO (50× (i.e., 50 times diluted), 1 mL) and SWCNT/CHP (45×, 1 mL) were mixed for vacuum filtration. For CNT-based ultrathin films, PEDOT:PSS/HO (500×, 1 mL) and SWCNT/CHP (450×, 1 mL) were mixed for vacuum filtration. For graphene-based thin films, PEDOT:PSS/HO (50×, 1 mL) and graphene/NMP (50×, 1 mL) were mixed for vacuum filtration. Note that the above mixtures were homogeneous systems within a period of up to 24 h, sufficient for vacuum filtration which usually took up to 30 min. For complex thin films, PEDOT:PSS/HO (50×, 1 mL), graphene/NMP (50×, 1 mL), and PPH/NMP (0.05 mg/mL, 1 mL) were mixed for vacuum filtration. It should be noted that a turbid system was obtained when mixing the three components, due to the formation of the dye molecule aggregates. Solution-based in-situ transfer method was applied for transfer of the hybrid thin films onto arbitrary substrates. Details can be found in the Results Section. The photographs of the hybrid thin films were taken by a digital camera. The hybrid thin films were characterized by field-emission scanning electron microscopy (FE-SEM, Zeiss Ultra Plus) at an acceleration voltage of 5 kV, transmission electron microscopy (TEM, JEOL 2100) at an acceleration voltage of 200 kV, and film-based absorption spectroscopy (Agilent Cary 6000i) through a 5 mm diameter hole (note that all samples were larger than the hole). Sheet resistance measurements were made using the 4-probe technique with a Keithley 2400 source meter. Y . Z . a n d W . B . c o n c e i v e d t h e i d e a a n d d e s i g n e d t h e e x p e r i m e n t s . Y . Z . p e r f o r m e d t h e e x p e r i m e n t s . J . J . M . c o n t r i b u t e d e x p e r i m e n t a l t o o l s i n c l u d i n g t h e v a c u u m f i l t r a t i o n a n d 4 - p r o b e s e t u p . Y . Z . a n d W . B . a n a l y z e d t h e d a t a a n d w r o t e t h e m a n u s c r i p t . A l l a u t h o r s d i s c u s s e d a n d a p p r o v e d t h e r e s u l t s i n t h e m a n u s c r i p t .

Epilepsy surgery is the most effective treatment option for pharmacoresistant epilepsy, but is often hindered by difficulty in accurately delineating the epileptogenic zone. Although there have been great advances in surgical localization from the use of magnetic resonance imaging (MRI) during the previous decades, MRI is sometimes insufficient to identify the epileptogenic zone or could be non-concordant/discordant to other localization techniques such as clinical semiology, electroencephalography (EEG), video EEG monitoring, and neuropsychology testing. At these times, testing using functional techniques can help provide additional information in identifying the epileptogenic zone. Some such techniques include positron emission tomography (PET), ictal single photon emission computed tomography (ictal SPECT) and magnetoencephalogram/magnetic source imaging (MEG/MSI) which have already found their place in routine clinical practice with varying degrees of use at different centers. In addition, newer techniques such as diffusion tensor imaging (DTI) and functional magnetic resonance imaging (fMRI-EEG) have been extensively used at a fewer number of surgical epilepsy centers to aid localization or for functional mapping to assess surgical safety. Functional connectivity magnetic resonance imaging (fcMRI), magnetic resonance spectroscopy (MRS), arterial spin labeling (ASL), near infra-red spectroscopy (NIRS) and magento nanoparticle (MNP) imaging are newer techniques that hold promise, but are yet to find their place in the pantheon of functional neuroimaging techniques available to today's epileptologist for identification of the epileptogenic zone or in helping the placement of intra-cranial electrodes. In addition, improvement of these methods may help avert the need for intracranial EEG, which can be an expensive and occasionally risky investigation on account of its invasive nature.[] In this manuscript, we review some of the functional neuro-imaging techniques including the history of development, principles and their current/potential role in the pre-surgical evaluation of epilepsy. Relevant articles were located on PubMed by searching with the keyword related to the functional neuroimaging technique and the keyword “epilepsy” and were selected for relevance to epilepsy, in particular pre-surgical work-up. PET conveys functional representations of various aspects of brain activity. Depending upon the radionuclide tracer utilized, PET has been applied to study local glucose utilization (fluorine-18 fluorodeoxy-glucose [FDG]) and quantification of specific neurotransmitter-receptor relationships e.g. [C] flumazenil with gamma-Aminobutyric acid (GABAA) receptors, [F] 2’-methoxyphenyl-(N-2’-pyridinyl)-p-fluoro-benzamidoethyipiperazine with serotonin receptions, [F] Fallypride with dopamine receptors, opioid-based ligands on opioid receptors. The focus of this review will be on glucose metabolism imaging with FDG-PET. Brain glucose metabolism is a representation of neural activity. As a glucose analog, FDG is transported into tissues and phosphorylated by hexokinase in the same manner as glucose. However, unlike glucose, FDG accumulates in the cell as it cannot undergo further metabolism. As proton-rich isotopes such as FDG decay, positrons are emitted. Subsequently, positrons annihilate on contact with nearby electrons. Each annihilation produces two 511 keV photons travelling in opposite directions and these photons are then captured by multiple pairs of oppositely situated detectors surrounding the subject.[] Utilizing MR images from the same patient, FDG-PET and MR images can be co-registered. Fused images are color-graded, such that each color change reflects a difference in FDG uptake.[] In cases for which visual asymmetry of FDG uptake across homologous structures cannot be confidently discerned, the use of standard uptake value ratios can be supplemented to discern areas of abnormality. A difference of more than 10% is considered to be significant.[] Hypometabolism on FDG-PET has been pathophysiologically attributed to factors such as neuronal loss or reduction in synaptic density.[] Correspondingly, brain regions containing the epileptogenic zone have been shown to have hypometabolism on inter-ictal FDG-PET, with an in-plane resolution of 2-3 mm. Temporal lobe hypometabolism ipsilateral to the seizure focus has been observed in 60-90% of patients with temporal lobe epilepsy (TLE).[] For patients with extratemporal lobe epilepsy, hypometabolism on FDG-PET correctly localizes the seizure focus in 67%.[] In presurgical evaluation for medically refractory epilepsy, FDG-PET is particularly advantageous for patients with apparently normal brain MRI findings. In these cases, FDG-PET has an overall diagnostic sensitivity of 44% in detecting “non-lesional” epileptogenic substrates.[] PET co-registered to MRI (PCOM) has been shown to offer the additional advantage in some clinical scenarios []. For focal cortical dysplasias, FDG-PET frequently demonstrates wider boundaries of abnormality than MRI. PCOM has the advantage of superimposing metabolic abnormalities upon anatomic landmarks (such as particular gyri or sulci) and thus may enhance surgical planning toward more complete lesional resection.[] Epileptogenic tumor's, particularly gangliogliomas and dysembryoplastic neuroepithelial tumor's, are frequently associated with cortical dysplasias that extend beyond the tumor's. PCOM can help identify and define the borders of cortical dysplasias surrounding these tumor's such that a more complete resection of epileptogenic substrate can be pursued.[] Moreover, in patients with tuberous sclerosis complex, multiple tubers are frequently present, but often only one of the tubers may be epileptogenic. PET co-registered to DTI sequences of MRI protocols has shown promise for distinguishing epileptogenic tubers.[] FDG-PET, when done during or shortly following an epileptic seizure, often reveals complex patterns of both hypometabolism and hypermetabolism. Therefore, caution should be exercised to rule out the occurrence of seizures near the time of FDG injection. Antiepileptic medications can decrease glucose metabolic rates and additional steps to analyze metabolic rates normalized to the whole brain metabolism may be necessary in some cases. SPECT is a functional neuroimaging modality that is able to measure or compare differences in blood flow through tissues and organs. More specifically, brain perfusion SPECT allows for a quantitative estimation of regional cerebral blood flow. When utilized in the pre-surgical evaluation of patients with refractory epilepsy, SPECT scanning is performed during both the ictal as well as the inter-ictal periods. Local neuronal hyperactivity during an epileptic seizure triggers an autoregulatory response that contributes to preferential hyperperfusion of the seizure onset zone. Taking advantage of this phenomenon, ictal SPECT is performed with injection of the radionuclide tracer during the electro-clinical manifestation of a seizure — preferably within 20 s of seizure onset to optimize seizure focus localization.[] Ictal SPECT requires video-EEG monitoring as well as appropriate logistical support to insure prompt injection of the radionuclide tracer. Ictal SPECT is the only routine clinical imaging modality capable of detecting ictal brain functional changes, allowing for visualization of the seizure onset zone during an actual seizure. Similar to PET imaging, ictal SPECT is particularly advantageous for patients with apparently normal brain MRI findings. The radionuclide tracers frequently used in SPECT, Tc-hexa-methyl-propyleneamine-oxime or Tc-ethyl cysteinate dimer, are characterized by the ability to cross the blood brain barrier rapidly due to their small molecular size and lipophilicity and are distributed proportional to the blood flow in cerebral tissues. Upon cerebral uptake and intracellular conversion to polar metabolites, they are retained with minimal regional redistribution or washout — allowing sufficient time for image acquisition up to 4 hours after injection. Once trapped in cerebral tissues, the gamma radiation emitted from the radionuclide tracer can be directly detected by a gamma camera. Symmetry in terms of perfusion across homologous structures is analyzed, with asymmetry of more than 10% considered as abnormal. Across several investigations of patients with unilateral TLE proven by either EEG, brain MRI, or post-resection seizure freedom, ictal SPECT has been claimed to correctly identify the seizure focus in over 90% of the cases.[] For patients with mesial temporal ictal onset zones, the area of hyperperfusion primarily involves the anterior temporal pole and mesial temporal structures, while the lateral temporal cortex is more variably involved. For patients with lateral temporal epileptogenic substrates, studies have shown bilateral temporal lobe hyperperfusion (greater on the side of the lesion) or posterolateral temporal hyperperfusion ipsilateral to the lesion.[] Because it may take 15-20 s for the tracer to reach the brain, the ictal SPECT image may display not only the seizure onset zone, but also the areas of seizure propagation. Similarly, longer injection delay from the time of seizure onset may also result in more likelihood of including seizure propagation areas. Another caveat associated with longer injection delays is the phenomenon of “postictal switch” in which the seizure onset zone switches to hypoperfusion, while seizure propagation regions become hyperperfused — resulting in false localization of the seizure focus. In patients with TLE, common areas of seizure propagation can include ipsilateral insula, basal ganglia, frontal lobe, as well as contralateral temporal lobe.[] Ictal SPECT of patients with extratemporal epilepsy has been shown to have a lower sensitivity (66%) for detecting relevant abnormality, when compared to patients with TLE.[] This lower sensitivity is possibly related to rapid seizure propagation and secondary generalization that are most frequently associated with extratemporal epilepsy. For example, in patients with occipital lobe epilepsy, very early ictal injection is recommended in order to avoid incorrect demonstration of hyperperfusion in the temporal lobe — representing propagated seizure activity.[] Moreover, for seizures arising from the supplementary motor area (SMA), seizure propagation can be very rapid, while the seizure duration is often very short. Such presentation lowers the usefulness of ictal SPECT, which correctly localizes the seizure focus in only 40% of such cases.[] The accuracy of ictal SPECT can be enhanced by subtraction ictal SPECT co-registered to MRI (SISCOM) analysis []. Using a computer-aided voxel-based co-registration technique, the inter-ictal SPECT is subtracted from the ictal SPECT and a mean image is generated using the mean and standard deviation of the differences in all brain voxels. This mean image is then co-registered to the patient's brain MRI. This technique has been reported to significantly improve seizure focus localization when compared with side-by-side visual inspection of ictal and inter-ictal SPECT images.[] For patients with extratemporal epilepsy where rapid seizure propagation occurs and where perfusion changes can be subtle, SISCOM analysis may be particularly useful.[] Inter-ictal SPECT has a sensitivity of only about 50% in identifying the seizure onset zone.[] Therefore, inter-ictal SPECT on its own has been considered insufficient for confident localization of the seizure focus. DTI is a relatively novel imaging method that provides improved summary of substrate cellular and molecular properties. This modality may allow for increased sensitivity for microstructural abnormalities, particularly in tissues with highly organized microstructure such as the white matter — allowing visualization of the location, orientation, and anisotropy of the axon tracts. DTI derives from novel algorithms built on a classic MRI protocol-diffusion weighted imaging (DWI). This DWI modality uses the motion of water molecules to probe cellular and molecular structures, and is presently used in clinical practice to identify ischemia. Whereas DWI measurements are made in three directions and the results are summarized by three DWI images, DTI derives from computational methods that integrate diffusion data acquired in more than three directions (usually 7 or more). These measurements are summarized using a Gaussian model of diffusion, specifically by a tensor (ellipsoid) that approximates how far the water molecules diffuse in all directions from a point. This computational integration of diffusion data, the diffusion tensor, yields a sensitive detector of tissue microstructural pathology that influences freedom of water molecular diffusion.[] Common ways to summarize the freedom of molecular movement in DTI include fractional anisotropy (FA-an index describing the degree of anisotropy or directional constraints) and mean diffusivity (MD-a measurement of the amplitude of diffusion of motion). Another approach to DTI analysis is the method of fiber tractography (FT), which allows for a measure of white matter tract integrity. FT is determined by a computational algorithm that determines the likely fiber patterns that pass through one selected region or between multiple selected regions []. Compared with the region of interest (ROI) analyses utilized to determine FA and MD measure, FT has been shown to be less operator dependent and can generally include a more homogeneous sampling of data to yield more robust results.[] Although the proximity of the sphenoid bone and air cells of the sphenoid sinus are known to produce susceptibility artifacts during temporal lobe imaging with DTI, successful investigation of the hippocampus has been well-documented. Pathophysiologically, it is hypothesized that the hippocampal cell loss and architectural disruption may correlate with expansion of extracellular space, allowing increased freedom of molecular diffusivity.[] The gliosis component of hippocampal sclerosis (if present) may add directional constraints and is reflected by a decreased FA. Among patients with known mesial temporal sclerosis (MTS), two investigators have independently shown significantly increased MD indices in the hippocampus ipsilateral to the known MTS, when compared with values in the contralateral hippocampus and with control subjects. Additional evidence suggests that the DTI may reveal temporal lobe abnormalities that are not detected on conventional MRI. Among two patients reported by Assaf . and one patient reported by Salmenpera . with mesial temporal epilepsy, but normal MRI findings, elevated MD indices were significantly evident.[] Furthermore, among another nine MRI negative patients with EEG documented seizures localizing to the left temporal region, ROI analysis within the left temporal white matter demonstrated both a significant increase in MD and a significant decrease in FA.[] Conventional MRI and fMRI methods provide limited information regarding anatomic relationships between the epileptogenic substrate and white matter fiber tracts associated with eloquent cortical areas. DTI tractography may be able to address this limitation through its mapping of white matter fiber anatomy in relation to planned resection zones. In a study of 49 patients undergoing surgical planning for extratemporal resective epilepsy surgery, DTI tractography assisted in the surgical decision making in two-thirds of the cases. More specifically, DTI tractography helped modify the surgical procedure in one-third of patients with frontal lobe resections, one-fourth of patients with occipital resections and two-thirds of patients with parietal or multilobar resections.[] MEG [] refers to the detection of minute magnetic flux induced by the electrical activity of cortical neurons. MSI is the co-registration of MEG data on the patient's structural MRI to help in the accurate localization of detected brain activity. As the cortical activity induced magnetic flux is only in the order of 10-100 fT (1 femtoTesla is 10 Tesla), extremely sensitive magnetometers are needed for its detection. The initial measurement of the MEG signal in 1968 by David Cohen at the University of Illinois required a magnetometer consisting of a ferrite core surrounded by 2 million turns of a copper wire.[] MEG became technically easier with the development of superconducting quantum interference devices (SQUIDs), which dramatically increased the sensitivity of magnetometers. By the 1980s commercial manufacturers had made arrays of multiple sensors that enabled the simultaneous measurement of MEG signals from different areas of the brain surface akin to the measurement of EEG signals from different surface regions using scalp electrodes. However, as of 2011, only 160 laboratories around the world have MEG capability.[] The principle of MEG is based on the induction of magnetic flux perpendicular to the electric current generated by post-synaptic (intracellular) currents in pyramidal neurons.[] Since cortical neurons on the gyral crests are oriented in a radial direction from the scalp surface, they would be expected to create the maximal magnetic field radial to the cortical surface. Similarly, neurons in the sulci are oriented parallel to the scalp surface and induce magnetic fields radial to the scalp surface, which are best detected by MEG.[] Since the cortical neurons create very minute magnetic fluctuations (in the order of fT), ambient magnetism in the atmosphere (in the order of microteslas) can significantly interfere with their recording. Hence a magnetically shielded room is necessary to perform testing, which represents a significant cost in installing and operating these machines. The method of analyzing MEG signals uses several techniques, the simplest and most powerful of which is the equivalent current dipole model (ECD). ECD analysis requires expertise, especially with multi-dipole analysis. Other approaches include minimum-norm estimates or minimum-current estimates and beamforming.[] Source analysis can be used to localize the MEG activity and to determine artifacts, (e.g., a source close to eyeballs or tongue is likely to be artifact).[] MEG has advantages in that the skull and scalp are “magnetically transparent” and do not attenuate the cortical signals as happens with EEG. The drawback of using MEG/MSI, as with EEG, for source localization is the so-called “inverse problem” where an infinite number of potential sources can explain a detected signal. MEG/MSI has been used since the early 1980s in identifying epileptic foci.[] MEG is used in detecting epileptic spikes. As tangential spikes are better detected, MEG can often detect spikes, which are completely missed by surface EEG. Inter-ictal spikes with MEG have been reported to correctly localize epilepsy in 52-89% of patients.[] Unique information was provided by MEG/MSI in 24-58% and altered or informed surgical decision making in 9-11%.[] It has been reported that the direction of the MEG dipole can help differentiate types of temporal lobe epilepsy into mesial, lateral and diffuse onsets.[] MEG/MSI also helps in accurate placement of intra-cranial electrodes.[] A high coverage of the MEG signal area by the surgical resection and a homogeneous distribution of MEG localizations correlate to a favorable outcome after surgery.[] Ictal MEG has been claimed to be a useful tool and potentially provides better localization than inter-ictal MEG.[] Other uses of MEG include real-time tracking of brain activity to study sensory, motor, language and cognitive functions and the study of brain rhythms.[] FMRI depends upon the property of differential magnetic susceptibilities of deoxygenated and oxygenated hemoglobin. In 1990, Ogawa used this concept to develop a contrast between oxygenated and deoxygenated hemoglobin called blood oxygenation level dependent (BOLD) contrast to demonstrate activity in the visual cortex.[] Deoxygenated hemoglobin is paramagnetic leading to distortion of magnetic fields and a shorter T2 relaxation time. At areas of increased brain activity, reactive vasodilatation occurs that overcompensates for the metabolic demand leading to an influx of oxygenated hemoglobin, thus prolonging the T2 relaxation time. Subtracting the BOLD image of rest from a specific task (such as a language paradigm) thus leads to a prolonged T2 signal in areas of the brain activated by the task. Language fMRI is the most frequently used fMRI paradigm in epilepsy and has been claimed to be a substitute for the Wada test for purposes of memory lateralization. Memory fMRI tests do not yield a robust activation although several groups are continuing research in making a useful paradigm. Newer modifications of fMRI such as fMRI-EEG and functional connectivity MRI are also reviewed below. EEG-fMRI is a special application of fMRI incorporating information from EEG and was developed in 1992 by John Ives . EEG-fMRI strives to merge the structural resolution of fMRI (in the order of 3 mm) and the temporal resolution of EEG (in the order of milliseconds).[] The technique generally uses epileptiform discharges in the EEG as triggers to evaluate changes in the BOLD signal in a fashion analogous to using a verb generation task as a trigger in the traditional task-based fMRI described earlier. The BOLD activations of several such discharges are grouped together by the image analysis software to show the location of the fMRI activation that corresponds to the EEG signal of interest []. There are several technical challenges that need to be overcome to record the EEG-fMRI signal.[] EEG-fMRI detected BOLD activity in patients with epilepsy has been reported to have concordance with the scalp EEG in 88% and was found to have contributed information in addition to the scalp findings about the epileptic focus in 64%.[] EEG-fMRI has been suggested as a very useful test to guide implantation of intra-cranial electrodes.[] More recent developments in EEG-fMRI include the advances in performing simultaneous intracranial EEG recordings as part of the technique.[] EEG-fMRI activation during incidentally recorded seizures has also been reported to be of value in the localization of epileptogenic cortex. FcMRI utilizes the principles of fMRI to demarcate brain networks and was first described in 1995.[] The basic underlying principle involves the correlation of signal changes over time (time series) in different voxels (the smallest individual unit of the acquired MRI volume) of the brain to detect correlated voxels. Such voxels are put together on a brain map to reveal the underlying network. Different techniques of fcMRI have been described including ROI/seed-based techniques, independent component analysis (ICA), graph theory based analysis, amplitude of low frequency fluctuations, regional homogeneity analysis and Granger causality analysis. Since epilepsy is thought to be a network disorder[] with structural changes demonstrated in different regions of the brain distant from the primary epileptic focus,[] techniques evaluating brain networks such as DTI and fcMRI assume significance in elucidating the pathophysiology of the disease and have the potential to guide surgical treatment. Using fcMRI, it has been shown that the language network is disrupted in left TLE compared with the controls.[] It has also been shown that the default mode network (DMN) shows disconnection between its anterior and posterior aspects as well as between the posterior DMN and the hippocampus.[] In right TLE, greater connectivity between the left MTL and the medial frontal cortex improves non-verbal memory, suggesting potential adaptive changes. However, in left TLE, greater connectivity between the left MTL and posterior cingulum worsens verbal memory suggesting maladaptive changes.[] Attempts have been made to lateralize TLE based on functional connectivity, although independent confirmation of results by other groups is lacking at this time.[] NIRS is a low resolution functional imaging technique which has advantages of being portable and in being easy to use with existing techniques. First described in 1977,[] the NIRS setup consists of a transmitter probe, which transmits near infra-red spectrum wavelength rays that pass through the cranium to a depth of approximately 2 cm during which phase it is absorbed by hemoglobin in the tissue. Reflected rays from here is detected by a sensor probe and the strength of reflected rays is inversely related to the concentration of hemoglobin in the brain tissue.[] The low resolution images obtained can then be co-registered to the structural brain images to lateralize and localize the signal changes. Such optical properties of the brain can be used to detect the several parameters including oxygenated hemoglobin concentration, deoxygenated hemoglobin concentration, total hemoglobin concentration, cytochrome-oxidase redox state, regional oxygen saturation, tissue oxygenation index, blood flow index and others.[] NIRS has been shown to correctly identify the affected hemisphere during seizures, as corroborated by simultaneous ictal SPECT and intracranial EEG.[] Studies have also shown the use of NIRS in detecting extra-temporal and temporal lobe seizures, including sub-clinical seizures.[] Although the literature in this field is still evolving, NIRS has the potential to be a useful addition to the pre-surgical work-up of epilepsy, particularly given its ease of use and ability to combine with other tests such as video EEG monitoring and SPECT scans. In addition to seizure detection and localization, NIRS has also been shown to be useful in language lateralization and could be an additional or alternative procedure to Wada and fMRI testing.[] One disadvantage of NIRS imaging is the inability to measure functional changes in buried cortex such as deep sulci, insula, parasagittal mesial cortex, mesial and basal temporal lobes, infra-tentorial cortex and the basal ganglia.[] xref #text xref #text xref #text

T e m p o r a l l o b e e p i l e p s y ( T L E ) , e s p e c i a l l y m e s i a l t e m p o r a l l o b e e p i l e p s y ( m T L E ) i s t h e m o s t c o m m o n f o r m o f f o c a l e p i l e p s y i n h u m a n s ( T a n d o n 1 9 8 9 ) . T h e p a t h o l o g i c a l s u b s t r a t e o f m T L E i s u s u a l l y h i p p o c a m p a l s c l e r o s i s , t h e m o s t f r e q u e n t e p i l e p t o g e n i c l e s i o n e n c o u n t e r e d i n p a t i e n t s w i t h m e d i c a l l y r e f r a c t o r y e p i l e p s y . T h e d i s a b l i n g s e i z u r e s a s s o c i a t e d w i t h m T L E t h a t a r e g e n e r a l l y r e s i s t a n t t o a n t i e p i l e p t i c d r u g s c a n b e a b o l i s h e d i n m o s t p a t i e n t s b y s u r g i c a l t r e a t m e n t . T h e b e n e f i t s o f s u r g e r y f o r T L E h a v e b e e n w e l l - d e m o n s t r a t e d i n t e r m s o f s e i z u r e c o n t r o l , c o g n i t i v e f u n c t i o n a n d q u a l i t y - o f - l i f e . One of the main goals of pre-surgical evaluation in refractory epilepsy is the localization of the epileptogenic zone that has to be resected surgically. The gold standard for surgical localization is the electroencephalographic pattern recorded during a habitual seizure. The electroclinical correlation during typical ictal behavior is the critical part of the pre-surgical evaluation. Most candidates for epilepsy surgery can be adequately investigated by a continuous scalp EEG recording over several hours to capture interictal and ictal EEG. The procedure involves continuous recording of synchronous scalp EEG and video over a period of 24hours to 7 days, with the aim of recording interictal EEG and clinical seizures with corresponding ictal EEG. The advent of digital technology has allowed easy recording, retrieval and reformatting of large amounts of EEG and clinical data. Medications are gradually withdrawn with the aim of recording habitual seizures. The clinical semiology and the electrophysiological data provide independent lateralizing and localizing information during a seizure. a n d a r d E E G w i t h 1 0 - 2 0 s y s t e m p r o v i d e s l i m i t e d c o v e r a g e o f t h e t e m p o r a l r e g i o n s d e t e c t i n g o n l y a b o u t 5 8 % o f t e m p o r a l s p i k e s o r i n t e r i c t a l e p i l e p t i f o r m d i s c h a r g e s ( I E D s ) . A d d i t i o n a l e l e c t r o d e s h e l p i n i n c r e a s i n g t h i s y i e l d . The interictal abnormalities on scalp EEG in TLE are: The seizures in TLE are typically complex partial, with or without simple partial onset. The semiologic features of TLE are highly varied. Various sensory phenomena or associated with mTLE. These include viscerosensory symptoms such as a rising epigastric sensation and experiential phenomena such as fear, and , oroalimentary automatisms are common, occurring in approximately 70% of cases of limbic (hippocampal) seizures compared with 10% of patients with extra-limbic seizures. They often involve the hands (fumbling, picking and fidgeting) or mouth (chewing, lip smacking, swallowing). Less common automatisms associated with temporal lobe seizures include vocalizations, ictal speech and affective behaviors (out of context fear). Rarely behavioral phenomena, such as, crying (dacrystic), laughing (gelastic) and so-called “leaving behaviors,” for example, running out of the house have been observed. Certain motor behaviors like late and sustained head and eye version, contralateral arm dystonia are features typical of but not exclusive to TLE. They also have a strong lateralizing value for Temporal lobe seizures []. Post ictal dysphasia in TLE has a strong lateralization to the dominant temporal lobe. Dysphasia is more common after dominant temporal rather than frontal lobe seizures. Postictal aphasia is a very reliable lateralizing sign (80-90%)[] but specific postictal language testing must be utilized by the EMU in order to detect this phenomenon. Anomia and paraphasic errors are easy to demonstrate during seizures.[] Ictal speech preservation reliably predicts seizures of the non-dominant hemisphere origin.[] Seizures of non-dominant hemispheric origin, however, may interfere with speech function on the basis of postictal confusion. Seizure semiology has some limitations in lateralizing and localizing the seizure origin. Many semiologic features have high positive predictive values; however, each feature has some potential to falsely localize the seizure onset.[] False localization should be suspected if the onset of clinical seizures occurs earlier than the onset of ictal EEG discharge. In majority (approximately 90%) with unilateral TLE (unilateral MRI abnormality and IEDs), the ictal lateralization corresponds to interictal IEDs and slowing. Ictal rhythms can be variable even within the same patient. Lateralization at the onset can be observed in only a third of unilateral TLE.[] Ictal EEG may not aid in differentiating the anterior from posterior lateral TLE.[] Ebersole and Pacia classified the ictal rhythms in TLE into three types.[] Typical ictal surface EEG with high interrater concordance consists of a rhythmic 5-9 Hz theta activity that slowly evolves and remains localized to the temporal or sub-temporal regions, which is termed type 1 ictal rhythm []. This pattern is most specific for hippocampal seizures. A lower frequency of 2-5 Hz irregular ictal rhythm with widespread temporal distribution is termed as “type 2 rhythm” () and is often associated with neocortical seizures. Diffuse ictal EEG changes or attenuation without clear lateralization (type 3 rhythm) can be seen both in hippocampal and temporal neocortical seizures. The overlap in EEG findings between mTLE and nTLE in limbic seizures does not allow confident distinction between the two by ictal EEG alone.[] Postictal EEG adds critical information particularly when seizure onset is unclear, or ictal changes are marred by the muscle artifacts. The accuracy of postictal findings for lateralization has a higher degree of interrater reliability particularly in TLE than extratemporal seizures.[] Postictal EEG findings include polymorphic lateralized delta activity, background suppression and postictal spikes 57%, 29% and 25%, respectively.[] Postictal spikes are most sensitive for lateralization but may be affected by seizures spreading to the contralateral temporal lobe. In about a third, there may be no distinctive postictal change. #text xref #text

Status epilepticus (SE) has traditionally been defined as more than 30 minutes of continuous seizure activity or two or more sequential seizures without full recovery of consciousness in between for a total of more than 30 minutes.[] In 1999, Lowenstein . proposed a new working definition that lowered the threshold to more than 5 minutes of persistent seizure activity.[] This definition is more relevant clinically given that majority of self-aborting seizures are brief, lasting less than two minutes and the response to therapy declines as the duration of the seizure increases. Refractory status epilepticus has lacked a consensus definition; most today regard it as status epilepticus that continues despite treatment with benzodiazepine and one antiepileptic medication (AED), e.g., Lorazepam + phenytoin. Others regard refractory status epilepticus as failure of benzodiazepine and 2 antiepileptic medications, e.g., Lorazepam + phenytoin + phenobarb. The first definition is often referred to as 2 AED failure and the second one is termed the 3 AED failure. o u g h t h e d e f i n i t i o n o f R S E v a r i e s , i t i s c o n s i d e r e d a m e d i c a l e m e r g e n c y r e q u i r i n g a g g r e s s i v e t r e a t m e n t t o a v o i d l o n g - t e r m c o m p l i c a t i o n s a n d r e d u c e m o r t a l i t y . B e n z o d i a z e p i n e s r e m a i n t h e s h o r t - a c t i n g m e d i c a t i o n s o f c h o i c e f o r t h e r a p y i n i t i a t i o n i n S E f o l l o w e d b y l o n g e r - a c t i n g m e d i c a t i o n s ( p h e n y t o i n o r f o s p h e n y t o i n , o r p h e n o b a r b i t a l i n t h e c a s e o f p h e n y t o i n f a i l u r e ) . T r e a t m e n t o p t i o n s f o r p a t i e n t s w h o p r o g r e s s t o R S E v a r y c o n s i d e r a b l y , t h o u g h m i d a z o l a m , p r o p o f o l , a n d p e n t o b a r b i t a l a r e t h e m o s t f r e q u e n t l y u t i l i z e d . U s e o f e a c h m e d i c a t i o n i n c l u d e s a u n i q u e s e t o f a d v a n t a g e s a n d d i s a d v a n t a g e s , a n d a s s u c h , t h e r a p y s h o u l d b e i n d i v i d u a l i z e d a c c o r d i n g t o t h e s e i z u r e e t i o l o g y ( i f k n o w n ) a n d t h e i n d i v i d u a l p a t i e n t ' s n e e d s . F a i l u r e o f o n e t h e r a p y d o e s n o t p r e d i c t r e s p o n s e t o o t h e r t h e r a p i e s a n d t h e y s h o u l d b e g i v e n a d e q u a t e t r i a l s s e q u e n t i a l l y u n l e s s c o n t r a i n d i c a t e d . T h e r e i s n o e v i d e n c e o n s u p e r i o r i t y o f o n e a p p r o a c h t o t h e o t h e r a s y e t .

There has been a progressive increase in life expectancy in the general population over the last several decades. This trend is likely to continue, and it has been suggested that the first person to live up to 150 years may have already been born! Epidemiological studies indicate that the incidence and prevalence of epilepsy is the highest in the elderly.[] Thus, among patients with epilepsy, elderly persons constitute the fastest growing segment. This group presents unique challenges in terms of diagnosis and treatment because of differences in the clinical presentation and etiology as compared to younger persons, presence of co-morbidities and cognitive difficulties in a significant proportion of patients, and physiological changes that affect pharmacological management. Despite the magnitude and complexity of the problem, surprisingly little clinical and basic research has been done in this population. This article reviews our current understanding of the special issues that need to be considered in the diagnosis and treatment of epilepsy in the elderly. The emphasis is on epilepsy beginning in the elderly, rather than epilepsy starting at a younger age and persisting into later life. #text Several studies in Europe and USA have consistently shown that the incidence of acute provoked and unprovoked seizures, epilepsy, and status epilepticus is higher in the elderly as compared to the younger population. In the USA, Hauser and Hesdorffer[] noted an increase in the incidence of seizures beginning after age 50, and rising to 127/100,000 person-years in those aged 60 or older. In another study of a racially diverse, community dwelling elderly cohort, Hussain .,[] found an incidence of 10.6/100,000 person-years in those between 45-59 years old, 25.8/100,000 person-years for the ages 60-74 and 101.1/100,000 person-years for the ages of 75-89. The prevalence rate of epilepsy in persons 65 years or older is approximately 1.5%. In a recent study,[] the annual mean incidence of epilepsy in the elderly was 2.4/1000 persons and prevalence rate 10.8/1000 among US Medicare beneficiaries. Similar numbers have been observed in the UK, with an annual incidence rate of 85.9/100,000 in those between 65 and 69 years, and more than 135/100,000 for those older than 80.[] In Finland, Sillampaa .,[] found that the incidence of epilepsy increased from 1986 to 2002 in the elderly, but decreased in children and younger adults. In a small study of 23 elderly patients from South India, Thomas .,[] noted that a first seizure was a frequent symptom among those attending Neurology clinics. The prevalence of epilepsy is higher among nursing home residents than in community-dwelling elderly people. Based on use of antiepileptic drugs in 10%-11% of nursing home dwellers, with phenytoin being prescribed for approximately 60% of these patients, it has been estimated that the prevalence of epilepsy may be at least 6% in this population.[] The true incidence and prevalence of epilepsy in the elderly may actually be two to three higher than the numbers quoted above because of difficulties in identifying seizures and diagnosing epilepsy.[] Status epilepticus has also been reported to be more frequent in the elderly. In patients who are 60 and older, the annual incidence is 86/100,000, which is almost twice that seen in the general population. It is even higher after the age of 70 years. Mortality also increases progressively with age, etiology, and duration of status. In general, the mortality in the elderly is 38%, but increases to almost 50% in those greater than 80 years old.[] Non-convulsive status epilepticus has a poorer prognosis in the elderly than in younger patients, because of underlying processes rather than the duration of status, and is associated with a high rate of hospital-acquired infections, which could be fatal.[] Epilepsy in the elderly is an important and growing problem. The clinical manifestations and causes are different from those in younger patients. The diagnosis of epilepsy can be challenging because of atypical presentation, different entities that need to be considered in the differential diagnosis, psychosocial factors, comorbidities including cognitive dysfunction, and non-specific abnormalities on routine investigations. Treatment can also be more difficult due to unique pharmacokinetic and pharmacodynamic changes, increased sensitivity to side effects, presence of comorbidities and drug interactions. Several questions remain unanswered because limited basic research and clinical trials have been performed in this group. Does the aging brain become more prone to epilepsy? If so, can anything be done to reduce the change in seizure threshold? What is the role of the aging hippocampus? Why does status epilepticus occur more commonly in the elderly? What are the basic mechanisms of epilepsy related to stroke and neurodegenerative diseases? What are the problems of performing clinical trials in the elderly? What are the chronic complications of AED on bone health and other systems? There are also likely to be several “unknown unknowns.”[] The natural history and prognosis of epilepsy in the elderly also need to be better understood. In the recently published study on epilepsy in US Medicare beneficiaries,[] an unexpected finding was that, even though the incidence rates of epilepsy were higher in the elderly and continued to increase with age, the prevalence rates were not much higher than in the general population, and did not vary greatly with increasing age. The reasons for incident cases falling off prevalence counts are not clear, but possibilities include increased mortality from epilepsy, resolution of epilepsy, over estimation of incident cases or subsequent correction of an initial misdiagnosis of epilepsy.[] More studies specifically addressing these issues need to be performed in the future. The pathophysiological mechanisms underlying epilepsy related to stroke and neurodegenerative diseases need to be better understood. Identification of genetic and other biomarkers would be helpful in developing novel preventive approaches. A greater effort should be made to include elderly patients in clinical trials and to better understand the chronic adverse effects of AEDs in this population. The role of epilepsy surgery needs to be more clearly defined. Psychosocial factors as well as adherence to medications should be addressed. A more comprehensive care model for inpatient and outpatient care including primary care physicians, neurologists, epileptologists and nursing staff would be helpful in providing optimal care to these patients.[]

xref #text T h e a n t e r i o r t h a l a m i c n u c l e u s i s t h e m o s t w e l l - s t u d i e d t a r g e t f o r s t i m u l a t i o n i n e p i l e p s y w i t h o t h e r r e p o r t e d t h a l a m i c t a r g e t s b e i n g c e n t r o m e d i a n ( C M ) n u c l e u s a n d r e t i c u l a r n u c l e i . #text

Magnetoencephalography (MEG) measures the magnetic fields generated by electric currents in the brain. The magnetic field measurements are in the range of femto-tesla to pico-tesla. MEG provides a very accurate resolution of the timing of neuronal activity.[] This is a non-invasive test. MEG is a direct measure of brain function and it has a very high temporal resolution. It also has a good spatial resolution. MEG is usually combined with a magnetic resonance imaging (MRI) Brain to get a good structural perspective. This combination of MEG and MRI is called magnetic source imaging (MSI). Currently, its approved clinical uses in the United States of America are in Epilepsy Surgery and for pre-operative brain mapping. Most clinical MEG's in the United States are associated with Epilepsy Centers.[] MEG records magnetic fields generated by electric currents in the brain. An electric current is always associated with a magnetic field perpendicular to its direction as per the right-hand rule. The magnetic permeability of biological tissues is almost the same as that of empty space and so the magnetic field is not distorted by scalp or skull. However, the magnetic fields diminish as 1/ with the distance of ‘’. When neurons are activated synchronously they generate electric currents and thus magnetic fields, which are then recorded by MEG outside the head. Like electroencephalography (EEG), the source of the magnetic fields is dendritic current of pyramidal neurons that fire synchronously and in parallel. Axonal and synaptic currents and their magnetic fields cancel out.[] The usual amplitude of magnetic fields created by the brain are extremely small, they do not exceed a few hundred femto tesla (10 T). Compared with this the Earth's magnetic field is between 10 and 10 T and an MRI is usually 1.5-3 T. To record these minute magnetic fields presents a unique engineering dilemma. There are two basic issues, one is to record these small magnetic fields and the other is to shield out the earth's relatively stronger magnetic fields. The technology that has helped record these minute magnetic fields is super-conducting quantum interference detector which is like a highly sensitive magnetic field meter. To maintain super conductors one needs to provide an extremely cold environment, which is achieved by using liquid helium which is only 3° about absolute zero (−452°F or −270°C). The gives a schematic representation of MEG. To attenuate the external magnetic noise the MEG is housed inside a magnetically shielded room. The actual sensors recording magnetic fields are magnetometers and/or gradiometers. There are 2 types of gradiometers — axial and planar. Magnetometers provide the best signal and are most sensitive to deep brain sources but are also more sensitive to competing magnetic noise. Gradiometers are better at noise reduction. MEG does not detect radial dipoles but tangential dipoles are seen on MEG, this is because the corresponding magnetic field of the radial dipole remains within the cranial cavity and thus cannot be detected by sensors on the outside.[] The MEG has a basic source identification problem. Determining the active site in the brain from magnetic fields recorded outside the head presents the “inverse problem”. This problem has no unique solution. Thus, source localization represents approximations based on certain assumptions that have to be made. A commonly used assumption is presuming simplified models of neuronal generators like equivalent current dipole (ECD) [Figures –].[] MEG fields pass through the head without any distortion. This is a significant advantage of MEG over EEG. MEG provides a high spatial and temporal resolution []. The decay of magnetic fields as a function of distance is more pronounced than for electric fields. MEG is therefore more sensitive to superficial cortical activity, which should be useful for the study of neocortical epilepsy. MEG data is always co-registered with MRI Brain to provide MSI. This provides a unique combination of good anatomical detail with neurophysiologic data. It is important to understand the differences between neurophysiological tools we know from a novel neurophysiological tool we are trying to learn about. The first obvious difference is that EEG records the electrical activity and MEG records magnetic activity of the brain. In EEG the electrodes are placed on the scalp. MEG is performed using a dewar that contains multiple sensor coils, which do not touch the patient's head. MEG primarily detects the magnetic fields induced by intracellular currents, whereas scalp EEG is sensitive to electrical fields generated by extracellular currents. Although MEG is more sensitive in detecting currents that are tangential to the surface of the scalp, EEG is sensitive to tangential and radial neuronal activities. MEG requires 3-4 cm of synchronized cortical epileptic activity to detect an epileptic spike, whereas at least 6-20 cm of synchronized cortical area is needed for scalp EEG spike detection. Magnetic fields are not distorted by the tissue conductivity of the scalp, skull, cerebrospinal fluid (CSF) and brain; in contrast, electrical fields may be distorted by the skull and CSF. MEG provides better spatial resolution of source localization (2-3 mm) than EEG (7-10 mm). A recent study comparing the signal-to-noise ratios (SNRs) of hypothetical sources in different areas of the brain indicated an advantage for MEG over EEG in the study of neocortical epilepsies, although the estimated SNRs were comparable in the temporal lobe. MEG data can be analyzed by source modeling techniques, which allow for localization in three dimensions. Although source modeling techniques are available for scalp EEG, MEG source modeling is generally simpler and more accurate for technical reasons. MEG is certainly more expensive than an EEG. Here in the United States MEG costs a few thousand dollars. u r r e n t l y h a s t w o a p p r o v e d i n d i c a t i o n s i n t h e U n i t e d S t a t e s , o n e i s f o r p r e - o p e r a t i v e B r a i n M a p p i n g a n d t h e o t h e r i s f o r u s e i n e p i l e p s y s u r g e r y . MEG provides an accurate means of functional mapping of eloquent cortex; this includes identification of motor, sensory, visual and auditory areas. Language cortex mapping can also be attempted. MEG studies have shown functional brain tissue inside brain tumors.[] The recording and identification information should be according to the ACMEGS guidelines published in 2011.[] The accuracy of MEG functional localization has been confirmed by intra-operative recordings. An interesting and critical finding has been the identification of functional brain tissue within tumors. About 8-18% of patients were found to have functional activity within a glioma. s a l s o s h o w i n g g r e a t p o t e n t i a l i n e a r l y i d e n t i f i c a t i o n o f A u t i s t i c c h i l d r e n b y s t u d y i n g a u d i t o r y p r o c e s s i n g b y M E G . I t i s a l s o s h o w i n g p o t e n t i a l u s e i n i d e n t i f y i n g m i l d c o g n i t i v e d e f i c i t p a t i e n t s i n A l z h e i m e r ' s . M E G i s s h o w i n g p r o m i s e i n t h e s t u d y o f P s y c h i a t r i c d i s o r d e r s a n d a l s o i n H e a d I n j u r y .

Surgical decision-making is a complex process. The parties involved are usually the surgeon, the patient, the families, society, insurance companies and the hospital administration. What are the possible outcomes of such situations when multiple parties are interacting in self-interest? What are the “optimal” outcomes? What are the economically “efficient” outcomes? What are the medically “efficacious” outcomes? These questions need a systematic review of such interactions. The current standard of medical decision-making is the use of evidence-based analysis.[] This is a good start but it is insufficient because it ignores the presence of multiple players and lack of full information. Decision analysis[] understands the presence of uncertainties and the temporal sequence of medical decisions. But it is insufficient because it ignores the presence of strategic actions by the parties involved. The Theory of games[] is designed to answer such questions. It combines evidence-based analysis, uncertainties, temporal sequencing and strategic interactions among multiple parties to arrive at outcomes where the aforementioned issues of “possible”, “optimal”, “efficient”, and “efficacious” can be addressed. Early surgery for epilepsy is medically beneficial to patients who are surgical candidates. Yet the average time from diagnosis to surgery for intractable epilepsy is 20 years in most countries.[] The decision of the physician for surgical or medical intervention is based on knowledge of basic science, together with evidence for the medical benefit of the intervention over its medical costs. Physicians usually practice evidence-based medicine. In budgeting for healthcare costs, the practice of evidence-based medicine alone should not be viewed as a touchstone for medical and surgical interventions. It is common for patients to reject evidence-proven treatments. For example, nearly half of the patients with stage III colon cancer in the U.S. do not undertake chemotherapy following initial treatment.[] It is also normal for evidence proven surgical intervention not being available to meet the demand. This is the case of epilepsy surgery. The market for medical services and drugs, functions under incomplete information. There is uncertainty about the medical value of a drug or procedure and drug/equipment testing or case reports of procedures provide the rationale for initial use and pricing under uncertainty. In such markets, risk-averse consumers are willing to pay a premium in exchange for less uncertainty. When level-1 evidence is presented for the medical efficacy of a drug or procedure, the medical uncertainty is grossly reduced. In a market with asymmetric information, this will inevitably raise the market price and hence the cost of caring for the patient.[] Left alone at this stage, the close nexus between healthcare costs and the practice of evidence-based medicine is obvious and is a natural consequence of markets with imperfect information. Should healthcare be practiced at all costs? This is not only an ethical, legal, and moral question, but is also a practical question that may ultimately help curtail healthcare costs. If medical evidence is viewed as a necessary condition of drug or procedure use when medical, economic and social impacts constitute sufficient conditions, then some procedures or drugs may be rejected even though they may be medically correct. This would bring down market cost of the drug and eventually the cost of healthcare. Nevertheless, the tool used by the literature and by physicians for surgical or medical intervention is evidence-based practice. Today there is ample evidence that, in appropriately selected patients, epilepsy surgery helps reduce seizure frequency and improve quality of life.[] Hence, as far as the physician is concerned, epilepsy surgery should be considered early in the course of patient management for epilepsy; however, in practice, it is not. #text The value of an individual's productivity to society may exceed the actual productive output of the individual due to social multiplier effects and Okun's law.[] Furthermore, this value may change with time due to social, scientific and technological evolution. One way to measure this, would be to quantify the impact of an individual on GDP. This is a challenge and the present value of future contributions need to be assessed and calculated accurately because of changes in the worker (for example learning curves, training and education), changes in society (for example technological and social evolution) and the changing impact of the individual on society. From 1963 to 2010, life expectancy in the U.S. has increased from 67 to 78 years.[] During the same time, the ratio of socially dependent workers to that of independent workers in the population has increased from 15 to 20.[] The GDP per capita has increased from about $3,000 to $45,000.[] Hence, during this interval, people have been living longer and have become more productive thereby increasing GDP and national prosperity. This means that the relative contribution to GDP by an individual in 1963 is much less than the contribution by the same person in 2010. The same individual producing the same output is worth more to society today than he was 47 years ago. One has to account for this increase in the future value of an individual. Among other variables, society is concerned about the impact of the individual on GDP over a period of time. #text Considerable work has been done in describing and computing the costs of epilepsy.[] Prevalence of epilepsy in India is about 10 million patients with an annual incidence of about one million. There are about 2.5 million surgical candidates. Costs of the disease include two broad components: Direct costs and indirect costs. Direct costs include medical consultations, laboratory services, hospitalization costs, and cost of travel to see a physician. Indirect costs include the costs of loss of productivity due to seizures, adverse effects of drugs andthe visits to a physician. In 2001, the estimated annual direct costs in India were about Rupees 4,000 per patient and the annual indirect costs were about Rupees 10,000.[] These costs add up and the annual economic burden of epilepsy in India was estimated in 2001 to be about 88% of GNP per capita.[] In the U.S.A., the direct and indirect costs were approximately the same amounts in U.S. dollars.[] In Europe, the estimated overall costs were about 84 billion euros per year, which results in the magic number of 14,000 euros per patient annually.[] Thus, the benefits of epilepsy treatment would be the savings of these direct and indirect costs. In addition, society would reap benefits of having a multiplier effect of individual productivity on social welfare. Even if the prevalence and incidence remain the same over time, and even if the real (inflation adjusted) direct costs of medical and surgical management remain the same over time, the total (direct + indirect) costs and benefits of treating epilepsy will keep rising because of improving health indices and life expectancies. Consider the decision-making that has taken place for glioblastomamultiforme (GBM). The current data of incidence of GBM is about 9,000 per year in the U.S.[] Death per year from GBM is about 8,000. The patients’ ages are typically in the range of 45-70. In Nova Scotia, the estimated direct costs are about $17,000 and the indirect costs are about $15,000.[] Thus, comparing epilepsy with GBM, incidence of GBM is lower, prevalence is lower, direct treatment costs are higher, indirect treatment costs are higher, benefits are lower, contribution to GDP is lower — yet why do we invest more readily in GBM treatment than in epilepsy surgery? There is medical evidence that surgery is better than medical management for properly chosen epilepsy patients. If the game theoretic decision analysis also points to the same conclusion, then surgery would indeed be the correct option for the early management of epilepsy in appropriately chosen candidates. Epilepsy has a devastating burden on individuals, families, society, nations and the world. In response, this paper proposes a multicenter, multi-country collection of data on epilepsy that would populate the game theoretic decision analysis required to answer the question: Should we invest more in early surgical treatment of epilepsy?

xref #text It is estimated that there are more than 10 million persons with epilepsy (PWE) in India. Its prevalence is about 1% in our population.[] The prevalence is higher in the rural (1.9%) compared to urban population (0.6%)[] In the Bangalore Urban Rural Neuro-epidemiological Survey (BURNS), a prevalence rate of 8.8/1000 population was observed, with the rate in rural communities (11.9) being twice that of urban areas (5.7).[] The estimated burden of epilepsy using the DALYs accounts for 1% of the total burden of disease in the world, excluding that due to social stigma and isolation, which further add to the disease burden.[] Here, we provide an overview of epilepsy in India, and the recent developments and their applications in the field of epilepsy. This review also deals with the epidemiological estimates, causes, and risk factors of epilepsy in India, and the role of epilepsy surgery in India and its need. In India, with less than 2000 neurologists and an estimated 5-6 million patients with active epilepsy, there is a huge need to strengthen epilepsy services, particularly in the rural and underserved areas, as the prevalence in rural areas is much higher [Tables and ]. It is estimated in various studies that the overall prevalence of epilepsy in India is 5.59-10 per 1000.[] The age-adjusted prevalence ratio of active epilepsy in Kerala is 4.7 per 1000 population.[] Methodological differences in prevalence estimation, such as variations in defining the epilepsy, failure to exclude pseudo-seizures, syncope, and other mimickers of epilepsy, and missed cases because of concealment may influence the exact prevalence. More number of males getting affected by epilepsy was reported in a study conducted in India by Bharucha .[] in which prevalence in males (5.1 per 1000) was significantly higher than that in females (2.2 per 100) [Tables , ]. There are very few incidence studies from India, and the most recent one suggests an age-standardized incidence rate of 27.3/100,000 per year.[] The exact magnitude of medically intractable epilepsy in India is unknown. Hot water epilepsy (HWE), a kind of reflex epilepsy in which seizures are induced by pouring hot water rapidly over the head, has been reported from South India, the prevalence of which varies from 1.14 to 2.99 cases/1000 population.[] HWE was reported to account for 3.6-3.9% of all epilepsy cases in rural South India.[] The 1993 ILAE recommendations for epidemiologic studies suggested to divide the symptomatic epilepsies into “remote” and “progressive” types, wherein remote symptomatic epilepsies encompass cases developing following insults resulting in static lesions [such as those attributable to conditions such as stroke, head injury, or central nervous system (CNS) infections] and progressive symptomatic epilepsy encompasses epilepsies associated with progressive illnesses (e.g. brain tumors or degenerative disease). Bharucha .[] reported that symptomatic epilepsy constituted 23% (progressive- 2%, remote symptomatic- 21%) and idiopathic epilepsy about 77%. They also reported that 54% had partial and 46% had generalized seizures. In another study by Koul .,[] the seizure types were found to be partial seizures in 12%, generalized seizures in 79%, and unclassified type in 9% in a population in rural Kashmir. All the three population-based case-control studies in India found febrile seizures to be a significant risk factor for developing epilepsy.[] The Chandigarh Study[] found in addition that head injury, developmental delay, and family history of epilepsy were significant risk factors. Among the risk factors of epilepsy, Kannoth .[] further reported along with the family history of epilepsy, antecedent history of febrile seizures, birth by complicated delivery, and neonatal seizures as strong independent predictors of epilepsy. There were more similarities than differences in the distribution of risk factors between generalized and localization-related epilepsy syndromes.[] Single computed tomography enhancing lesion (SCTEL) is defined as disc or ring CT measuring less than 20 mm in size and enhancing on contrast administration. Single enhancing CT lesions are the commonest radiological abnormality in Indian patients with new-onset partial seizures. Histological studies suggest that SCTEL represents dying cysticerci that will spontaneously resolve as a result of host response. Infections of CNS including SCTEL account for 77% of patients with acute symptomatic epilepsy. Small single cerebral calcific CT lesion (SSCCCTL) is defined as single calcific lesion measuring less than 20 mm. Seizures are the commonest presentation of neurocysticercosis (50-80%)[] Among 991 patients of symptomatic localization-related epilepsy studied, infections of CNS including single CT enhancing lesion (SCTEL) accounted for 77% of patients with acute symptomatic epilepsy. Cerebrovascular diseases were the risk factors in 48% of patients with remote symptomatic epilepsy. Neurocysticercosis, SCTEL, and SSCCCTL together accounted for 40% of etiological factors and neurotuberculosis for 10% of the factors. Infections of the CNS and SCTEL together were the putative risk factors in 52% of patients aged ≤40 years. Cerebrovascular diseases were the etiological factor in 64% of patients aged >40 years in South India, as reported by Murthy .[] Mani .[] reported a diagnosis of NCC in 2% of unselected series of epilepsy. At a tertiary referral center in New Delhi, NCC constituted 2.5% of all intracranial space-occupying lesions.[] In a community survey of 50,617 individuals from South India, the prevalence of active epilepsy was 3.83 per 1000 and NCC was detected in 28.4% of them by CT.[] Single cyst infection (range 47.7-53.4%) is the most common in the Indian subcontinent.[] Serial magnetic resonance imaging (MRI) was performed in a cohort of patients with solitary cerebral cysticerci manifesting with new-onset seizures from this center. It was reported that albendazole therapy may hasten resolution of inflammation around the lesion, but affects neither the morphology of the NCC nor the process of degeneration and subsequent healing.[] The CNS infections are a major cause for acute symptomatic status epilepticus (SE), with an prevalence varying from 17.5 to 39.8% in various studies.[] In an effort to understand the underlying pathogenesis of epilepsy, Sinha . studied the serum cytokine levels in 100 patients with epilepsy during the immediate post-ictal phase. They found that the detectable serum cytokines in the patient group ( = 100) were interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, IL-2, IL-4, IFN-gamma, and IL-1 compared to the controls.[] About 12-27% of HWE epilepsy patients have positive family history. In an ongoing effort to understand the molecular basis of HWE, seven HWE families with several of their members affected with the disorder were examined and two loci for HWE at chromosomes 10q21.3-q22.3[] and 4q24-q28[] were identified. Exome sequencing suggested or gene, a glutamate transporter gene, as the causative gene. Absence of GABRA1 Ala 322Asp mutation has been noted in families of juvenile myoclonic epilepsy (JME) from South India.[] Further, in another study, association of gene was found in JME patients from Kerala.[] There might be a protective role of CAG allele of hSKCa3 (calcium channel) gene in JME patients.[] There is a susceptibility effect of CAG and CAG alleles of hSKCa3 gene in JME patients from Kerala.[] A locus for JME has been mapped to 2q33-q36 and 5q12-q14 in South Indian population.[] In another study from northern India, it is reported that polymorphisms in BRD2 and LGI4 might be risk factors for JME.[] Lafora body disease (LBD) is caused by mutation in the PME2 gene () on chromosome 6q and gene.[] The official name of this gene is “epilepsy, progressive myoclonus type 2A, Lafora disease (laforin).” is the gene's official symbol. The gene located at 6q24 provides instructions for making a protein called laforin.[] Early brain developmental abnormalities involving neuronal migration and lamination are often implicated in childhood epilepsy. Reelin (), a neuronal-signaling molecule, plays a crucial role in these migratory processes and is located on human chromosome 7q22. It is considered as a potential candidate gene for childhood epilepsy. In a study conducted at West Bengal in India, 63 patients with childhood-onset epilepsy and 103 healthy controls were recruited. Case-control analysis revealed significant over-representation of G/C and (G/C + C/C) genotypes and C allele of exon 22 G/C marker (rs362691) in cases as compared to controls.[] xref italic #text xref #text Seizures precipitated by a specific sensory stimulus are described as reflex epilepsy. HWE is precipitated by the stimulus of bathing in hot water poured over the head, and is also variably known as water-immersion epilepsy or bathing epilepsy. It is from southern India that a large number of cases of HWE have been reported. The exact etiopathogenesis of this type of epilepsy is not clear, but several factors including genetic factors, environmental factors, consanguineous marriages, and habit of taking bath with high-temperature water has been postulated as probable reasons.[] Reports from our center suggest that the cohort of patients with HWE is mainly from two adjoining districts, viz. Mandya-Mysore belt in Karnataka state of South India.[] HWE was reported to account for 3.6-3.9% of all epilepsy cases in this part of India.[] Triggering factors were mainly temperature of the water and contact of water on the scalp. Studies have shown a strong family history of epilepsy in these subjects from India (7-18%).[] Even though the exact mechanism of HWE is not clear, many theories have been proposed to explain the peculiar nature of seizures. Earlier, Satishchandra .[] had postulated that patients with HWE probably have an aberrant thermoregulatory system and are probably sensitive to a rapid rise in temperature. The impaired sympathovagal balance is observed in HWE, characterized by increased sympathetic activity and reduced parasympathetic activity in patients with HWE. The hypothalamus is involved in both the pathogenesis of HWE and autonomic regulation. Intermittent therapy with clobazam, 1-1½ h prior to hot water head bath, is the method of treatment for this type of epilepsy. AEDs are required only if HWE is associated with non-reflex seizures. In 1984, 13 cases of eating epilepsy were reported from South India.[] It was very frequently reported from Sri Lanka. Recently, six patients with eating epilepsy were studied in depth using MRI, single-photon emission computed tomography (SPECT) scan, and video encephalogram (EEG) from NIMHANS, and presence of lesions around perisylvian area has been identified. There might be an involvement of complex neuronal circuits around the Sylvian fissure (near area of face) responsible for eating epilepsy.[] There are close to 1.5 million women with epilepsy (WWE) in reproductive age in India. About a sixth of WWE in the world are living in India. It is estimated that there are about 2.73 million WWE in India and 52% of them are in the reproductive (15-49 years) age group.[] Sahota . reported that primary generalized seizures were seen in 53.6% women in their series. Sodium valproate was the most commonly used primary AED, alone or in combination with lamotrigine, followed by phenytoin and carbamazepine. Menstrual abnormalities, hirsutism, or both were seen in 19.4% women on these AEDs as untoward effects; of these, 60.2% received valproate, 25.3% received carbamazepine, 13.3% received phenytoin alone or in combination, and 1.2% received phenobarbitone alone. Polycystic ovaries were detected in 23.5% of valproate-treated women and 25% of carbamazepine-treated women.[] Exposure to AEDs in the first trimester of pregnancy has been associated with an increased risk of major congenital anomalies (MCAs) in offspring. MCM is defined as an abnormality that can interfere with the quality of life and warrants definite management. Nearly all studies on the adverse fetal effects of AEDs have methodological shortcomings. Studies done up to late 1990s suggest that the risk of MCAs in infants exposed to older-generation AEDs in the first trimester of pregnancy is about two to three times higher than in the general population (4-10% vs. 2-5%, respectively).[] No single MCA has been associated most commonly with a specific AED, with the exception of spina bifida which is more common with exposure to valproic acid (1-5% of exposed offspring) and carbamazepine (0.5-1.0%) than with other AEDs. Valproic acid is also the only AED for which dose-dependency has been ascribed in several studies, with the increase in MCA risk, compared with other AEDs, being reportedly most evident at doses above 800-1000 mg/day.[] The data from Kerala Registry of Epilepsy and Pregnancy (KREP) indicate that anemia, ovarian cyst and fibroid uterus, and spontaneous abortions are more frequent in WWE.[] In the study carried out by KREP, congenital malformations were detected in 12.5%. The odds ratio for the risk of malformation was much higher with sodium valproate (OR-6) than with carbamazepine (OR-1.2) or phenobarbitone (OR-0.8).[] Among the predictors of seizures during pregnancy in WWE, the main observations were that women with partial seizures on AED polytherapy or with at least one seizure in the pre-pregnancy month had higher risk of seizures during pregnancy.[] In the study conducted by the same center regarding the recurrence of MCM, of 492 pregnancies in 246 women, 43 pregnancies had MCM (42 women). The MCM rates were 8.5% in index pregnancy and 8.9% in follow-up pregnancy. The data suggested that the malformation recurrence risk may be dose dependent, and at low dose, there may not be increased risk of recurrence.[] The AED usage may lead to folate deficiency which may predispose to neural tube defects.[] Other mechanisms under consideration include alteration in the homeobox genes, retinoic acid signaling pathways, histone deacetylators, and polymorphisms involving AED transporters.[] WWE who can potentially become pregnant need to be started on folic acid 5 mg daily, 1 or 2 months prior to anticipated pregnancy. All WWE who are using AEDs are advised to take vitamin K 10 mg intramuscular (IM) at 34 and 36 weeks of pregnancy. Epilepsy and seizures may alter the hypothalamo-pituitary-ovarian axis and can lead to derangement of reproductive hormone levels. In a study conducted in Kerala on WWE and infertility, it was observed that the WWE-infertility group had different reproductive hormone profile than others; the luteinizing hormone (LH)/follicular stimulating hormone (FSH) ratio was significantly abnormal among the infertility group. This ratio was significantly abnormal in the follicular phase and luteal phase. The WWE-infertility group had 8.5 times higher risk of abnormal LH/FSH ratio.[] The Commission on Classification and Terminology of the ILAE defines SE as a seizure that persists for a sufficient length of time or is repeated frequently enough that recovery between attacks does not occur (ILAE, 1981). Refractory status epilepticus (RSE) is a medical emergency with high morbidity and mortality, and the continuous or repetitive seizures are unresponsive to first- and/or second-line AED therapy. In a study involving 98 patients with RSE, Sinha . found that 34 had died and seizure control was achieved in two-third of patients.[] In the clinic-pathological study of 100 cases of SE conducted at NIMHANS, neuroinfection was found in 50% of cases of fatal SE followed by cerebrovascular accidents (21.2%). Patients with prior history of epilepsy had discontinued treatment in 71.4% of cases, resulting in SE. Majority of patients with SE had generalized convulsive seizures (75%).[] The details of aetiology of SE in India is provided in . Abnormalities close to the central sulcus may give rise to long-duration focal motor seizures. This condition is called epilepsia partialis continua (EPC). Analysis of 75 case records of patients with EPC with mean age at presentation of 30.2 ± 23.4 years and with duration of EPC of 47.02 ± 188.2 days was done. Majority of the patients had type I presentation (90.8%), while the remaining seven patients had Rasmusssen's encephalitis (RE) with type II EPC presentation. Patients of age >40 years had stroke, idiopathic, diabetic non-ketotic hyperosmolar coma, and metastasis as the common causes.[] In 68 patients with convulsive SE, seizures were aborted in 66% in the valproate group and 42% in the phenytoin group. As a second choice in refractory patients, sodium valproate may be preferred in convulsive SE because of its higher efficacy (79%) as compared to the phenytoin (PHT-25%)[] Mishra . conducted a randomized, open-labeled pilot study comparing the efficacy and safety of levetiracetam and lorazepam in SE in 79 patients. Consecutive patients with convulsive or subtle convulsive SE were randomized to levetiracetam (LEV) 20 mg/kg intravenous (IV) over 15 min or lorazepam (LOR) 0.1 mg/kg over 2-4 min. Both were equally effective. In the first instance, the SE was controlled by LEV in 76.3% (29/38) and by LOR in 75.6% (31/41) of patients. The authors concluded that for the treatment of SE, levetiracetam is an alternative to lorazepam and may be preferred in patients with respiratory compromise and hypotension.[] italic xref #text xref #text italic xref #text xref #text Nearly one-third of the patients with newly diagnosed epilepsy on long-term follow-up will have their seizures unsatisfactorily controlled by treatment with available AEDs.[] Comprehensive epilepsy surgery centers with advanced imaging tools such as MRI, SPECT, positron emission tomography (PET), and magnetoencephalography (MEG), as well as a multidisciplinary team of neurologists, neurosurgeons, neuroradiologists, electrophysiologists, psychologists, psychiatrists, and medical social workers are needed in a country like ours. The first epilepsy surgery in India was performed on 25 August 1952 at Christian Medical College (CMC) in Vellore. The first epilepsy surgery at Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTMIST), Thiruvananthapuram was conducted in 1995. Almost simultaneously, epilepsy surgery programs were started at the All India Institute of Medical Sciences (AIIMS), New Delhi and NIMHANS, Bangalore. During the last one and half decades, these three centers together have undertaken over 2000 epilepsy surgeries. India with over 1.2 billion people will have over 1 million people with medically refractory epilepsies, of which nearly one half are potential surgical candidates. The success of any epilepsy surgery program depends upon the early identification of potential surgical candidates. Eventually after temporal lobe resective epilepsy surgery, there is over 70% probability that the patient will be seizure-free and over 30% chance of being free of AEDs within 2 years after surgery.[] The decision making for epilepsy surgery needs a multidisciplinary approach in which different investigators involved with the program work in conjunction to create an integrated picture of epileptogenesis and its impact on the patient and care givers. Knowing when not to operate, because of the need for further investigations is as important as selecting which patient may benefit from surgery in a resource-limited setting. #text T h e b u r d e n o f e p i l e p s y c o u l d b e r e d u c e d i n I n d i a b y a l l e v i a t i n g p o v e r t y a n d b y r e d u c i n g t h e p r e v e n t a b l e c a u s e s , v i z . p e r i n a t a l i n s u l t s , p a r a s i t i c d i s e a s e s , a n d h e a d i n j u r i e s . E m p o w e r i n g p r i m a r y h e a l t h c a r e w o r k e r s t o d i a g n o s e a n d s t a r t t r e a t m e n t m i g h t s i g n i fi c a n t l y r e d u c e t h e t r e a t m e n t g a p a n d t h e d i s p a r i t i e s b e t w e e n r u r a l a n d u r b a n a r e a s . I n I n d i a w i t h o v e r 5 0 0 , 0 0 0 p o t e n t i a l e p i l e p s y s u r g e r y c a n d i d a t e s , n o t m o r e t h a n 2 0 0 e p i l e p s y s u r g e r i e s p e r y e a r a r e b e i n g u n d e r t a k e n t o d a y . T h u s , o n l y a m i n i s c u l e o f p o t e n t i a l s u r g i c a l c a n d i d a t e s i n o u r c o u n t r y e v e r g e t s a c h a n c e t o u n d e r g o p r e - s u r g i c a l e v a l u a t i o n a n d s u r g e r y . T h i s e n o r m o u s s u r g i c a l t r e a t m e n t g a p c a n o n l y b e m i n i m i z e d b y d e v e l o p i n g m a n y m o r e e p i l e p s y s u r g e r y c e n t e r s a l l o v e r I n d i a .

Drug-resistant epilepsy (DRE) is defined as failure of adequate trials of two (or more) tolerated, appropriately chosen and appropriately used antiepileptic drug regimens (whether administered as monotherapies or in combination) to achieve freedom from seizures.[] Establishing a diagnosis of DRE is an important milestone in the treatment of epilepsy as it marks the transition of a patient who is taking medications to control a condition and living a relatively normal life to someone who is at risk of worsening seizures, injuries or even death as well as social stigma and economic hardship associated with uncontrolled seizures. Defining the presence of DRE also serves as an important landmark for the treating physician to take stock of the overall situation and reevaluate the diagnosis of epilepsy and treatment plan. It should prompt the physician to evaluate for possible remediable causes of DRE such as inadequate trials of antiseizure medications, use of wrong medications for the type of epilepsy or even misdiagnosis of epilepsy. On the other hand, a firm diagnosis of DRE in an adult may provide a distinct opportunity to discuss other treatment options with the patient because seizure freedom is unlikely with the addition of other antiseizure medications. Other treatment options in cases of DRE include: ketogenic diet, neuromodulation therapy and neurosurgical interventions such as resective epilepsy surgery to provide potential cure from uncontrolled seizures and palliative surgeries to help reduce the severity and frequency of disabling seizures. Therefore, evaluation of a patient with suspected DRE fundamentally requires three initial steps. First, a detailed history to firmly establish the diagnosis of seizures, help classify epilepsy syndrome and severity of the condition. Information regarding semiology and frequency of seizures, precipitating factors for seizures, past medical history including birth and developmental history and details of data of the antiseizure medications all are important to help achieve this goal. Second, a prolonged video-electroencephalography (EEG) recording should be obtained to capture the electroclinical events to help determine the type and location of the seizure onset in case of focal epilepsy and decide whether surgical treatment would be an appropriate treatment route to pursue. Third, obtain additional investigations to help identify the underlying cause of focal seizures and also help with localization. High-resolution magnetic resonance imaging (MRI) is the ideal structural imaging modality as it provides the finest detail of the brain parenchyma. When MRI is negative in a patient with suspected or established focal epilepsy, further work-up is critical to help localize the epileptogenic zone, especially when surgical resection is being considered. From a surgical point of view, the primary aim of additional testing in patients with medically refractory epilepsy is to delineate the epileptogenic zone and excise it with minimal or no functional neurological deficit. An epileptogenic zone is defined as “cortex that is necessary and sufficient for initiating seizures and whose removal (or disconnection) is necessary for the complete abolition of seizures”.[] Thus defining the epileptogenic zone and surrounding eloquent cortex are the two pillars of evaluation in patients with drug resistant focal epilepsy. Overall, the success rate of surgical intervention is best in patients with an identifiable structural lesion that is responsible for their epilepsy. However, identifying the epileptogenic region is more difficult in patients with so-called cryptogenic partial epilepsy where no such structural lesion can be identified on pre-operative neuroimaging. Over the years, improved technology has diminished the pool of such patients and more and more epileptic lesions can now be successfully detected using newer imaging technologies. This field is constantly changing but the investigations to identify the epileptogenic lesion at present can be categorized into: Though MRI of the brain is much superior to computed tomography (CT) scans, it is generally more complicated to interpret, costlier and less widely available in rural areas. However, different MRI sequences can be obtained to evaluate for a specific question in mind. All MRI scans are not performed with the same diligence and sequences, as such; a “negative MRI” has to be viewed with some degree of suspicion. A routine MRI done with 10 mm thick slices and 15 mm interslice gaps with a T1-weighted and T2-weighted axial image acquisitions and diffusion-weighted images may be adequate to evaluate for a gross cerebral abnormality such a sizable tumor, major malformation of the brain or acute stroke, but may not be adequate to pick up subtle abnormalities that may be responsible for DRE. A routine MRI with standard sequences in axial planes read by a general radiologist who is not very familiar with epilepsy investigations may fail to detect up to half of the focal epileptogenic lesions.[] This is a common problem and therefore a properly designed MRI protocol looking for subtle structural lesions associated with DRE and review by an experienced physician is essential. Most institutions develop their own MRI protocol to choose appropriate sequences for their local machine and a reasonable time allotted to the study. In our opinion, high-resolution MRI with epilepsy protocol should include at least following sequences: Fluorodeoxyglucose (FDG)-PET is a test where [F]-labeled 2-deoxyglucose is administered intravenously. The FDG is transported by the same mechanism as glucose into the brain, but it cannot be utilized as a fuel and is trapped intracellularly. The amount of glucose uptake is proportional to the regional metabolic needs; hence PET imaging of the brain following FDG administration reveals a snapshot of metabolic activity in the brain. In cases of focal epilepsy, the area of seizure onset and at times surrounding areas are hypometabolic interictally and pick-up smaller amounts of FDG compared to surrounding normal brain. The precise mechanism for this reduced metabolic demand is unclear but thought to be due to excess inhibition. Hence, an interictal FDG-PET scan showing an area of hypometabolism (relatively cold area) roughly indicative of epileptogenic zone.[] If the scan is performed during a seizure (ictal PET scan) or in an individual with frequent interictal spiking, it shows increased metabolism (hot spot).[] Hence, EEG monitoring during FDG infusion is necessary to look for electrographic seizure or frequent spiking. Correlation of FDG-PET abnormality and final determination of epileptogenic zone is well established in temporal lobe epilepsy, especially in patients where MRIs and EEG monitoring are non-localizing.[] In patients with temporal lobe epilepsy and negative MRI but positive findings on FDG-PET scan surgical outcome is excellent and similar to patients with hippocampal sclerosis [].[] Over the years several investigators have tried to use various ligands to study neurotransmitter or receptor distribution. These are primarily used for research and commonly use a radiolabeled carbon molecule [C] in place of one of the native carbon molecules in a compound to be studied. One can use ligands to noninvasively image brain chemistry and distribution of various receptors and molecules by labeling a neurotransmitter or its precursor or a ligand with high receptor affinity. FMZ, a gamma-aminobutyric acid B (GABA-B) receptor agonist that can reveal benzodiazepine receptor distribution was one of the early compounds used in patients with epilepsy with good success.[] Role of GABA in neuronal inhibition and seizures is well known and extensively studied. Therefore, evaluating GABA receptor distribution in patients with epilepsy, especially focal epilepsy was quite natural. The FMZ-PET images show decreased FMZ binding in the epileptogenic area. FMZ scans often show more restricted abnormality when compared to FDG-PET scan and is better aligned to the epileptogenic zone [].[] AMT is an amino acid PET tracer which is not incorporated into proteins; instead, tryptophan (and also AMT) is transported in brain tissue via the large neutral amino acid transporter and can be metabolized via the kynurenine or serotonin pathways. In patients with focal epilepsy due to certain causes, such as tuberous sclerosis, cortical dysplasia and certain epileptogenic brain tumors, AMT-PET scan typically shows increased uptake in the epileptogenic area [].[] FCWAY is a selective 5-HT1A-receptor antagonist that has been studied in patients with temporal lobe epilepsy. 5-HT1A-receptor binding is reduced ipsilateral to the temporal lobe seizure focus, hence the PET scan using this ligand shows decreased binding on the side of seizure onset.[] PET scan using other tracers such as C-diprenorphine, F-cyclofoxy and C-carfentanil to investigate binding to opiate receptors; and C-deprenyl to measure monoamine oxidase B activity are performed at various institutes as part of ongoing research programs.[] Many of them show promising results but have not shown to surpass the clinical utility of FDG-PET scan. Therefore, they are not routinely used clinically, but remain a great tool to study neurochemistry in various disease states including epilepsy noninvasively. xref #text xref #text fMRI is a noninvasive technique to image task-related increased blood flow in a specific brain region. The technique commonly used is blood-oxygen-level-dependent (BOLD) contrast, which utilizes increased regional blood flow in response to metabolic demand associated with a task, e.g., finger tapping will increase blood flow in contralateral motor hand area. Individual movements lead to a very small change that is below detection sensitivity, however, if the scan is repeated multiple times when task is being performed, the task-related change can be detected. Normally, the BOLD scan obtained at the resting level is subtracted from the values obtained during the task to enhance visualization of the cortex involved in processing of the particular task. Thus, fMRI can be utilized for various brain regions by obtaining task related scans for different modality. Commonly used tasks include motor tasks to locate the motor homunculus, various language tasks to locate Broca's and Wernicke's areas. The findings are only as good as the task is, e.g., motor tasks are simple but language paradigm used to identify Broca's area is good only if the tasks provided require activation of only that area. This is difficult as paradigms used to check motor language processing may simultaneously activate primary auditory cortex and Wernicke's area when instructions are given and the subject is listening and interpreting it as well as it will activate the phonation mechanism used to produce the sound. However, with increasing experience, robust validated protocols have been established to test for commonly used tasks to locate the language areas as well as motor, visual and auditory cortex. The non-invasive nature of the technique and its co-registration with structural MRI gives a very precise understanding of relationship of a lesion is present or epileptogenic zone if defined prior to surgery. In addition, neuronavigation methods can identify the location of eloquent cortex in the operating room if fMRI information is available and co-registered with structural imaging. This leads to precise presurgical planning of resection margins and meaningful discussion regarding potential benefits versus surgical risks can be held with the patient and family members prior to epilepsy surgery. MEG is a technique that studies magnetic field associated with interictal spikes instead of the commonly studied electrical field by EEG. The main advantage of the technique is that the magnetic field does not attenuate due to intervening tissue (meninges, skull and scalp) as EEG signal does. Recording magnetic field with high density array over the scalp one can determine the origin of the magnetic source of the interictal spike in 3D space.[] If the MEG information is co-registered with MRI one can view the origin of the spike on brain structural imaging again in 3D space []. Again the problem of assumption that there is a single, identical source for interictal spike and seizure remains as MEG also studies interictal phenomenon.[] Recent advances in computer processing of the large amount of data from EEG signal has led to the creation of dense array EEG recording and from that information to plot electrical source of that activity. This is similar to MEG but here traditional EEG signal is used.[] W h e n a s t a n d a r d i z e d s e r i e s o f n e u r o p s y c h o l o g i c a l t e s t i n g i s a d m i n i s t e r e d b y a n i n d i v i d u a l w e l l v e r s e d i n t h e e v a l u a t i o n o f p a t i e n t s w i t h D R E m a y d e t e c t s u b t l e i m p a i r m e n t s o f n e u r o c o g n i t i v e f u n c t i o n i n g . A s d i f f e r e n t b r a i n r e g i o n s s p e c i a l i z e i n d i f f e r e n t c o g n i t i v e f u n c t i o n , t h e s e d i f f e r e n c e s c a n p o i n t t o t h e e p i l e p t o g e n i c f o c u s a s i t w i l l i n t e r f e r e w i t h n o r m a l f u n c t i o n o f t h a t r e g i o n . T h i s p r o v i d e s i n d i r e c t b u t a d d i t i o n a l e v i d e n c e f o r l o c a l i z a t i o n o f s e i z u r e o n s e t . W h e n n e u r o p s y c h o l o g i c a l f i n d i n g s a r e i n a g r e e m e n t w i t h t h e c l i n i c a l , e l e c t r o p h y s i o l o g i c a l a n d f u n c t i o n a l n e u r o i m a g i n g d a t a i n p a t i e n t s w i t h M R I - n e g a t i v e D R E , d e c i s i o n t o s u r g i c a l l y i n t e r v e n e i s s u p p o r t e d . C o n v e r s e l y , w h e n t h e r e i s i n c o n g r u e n c e b e t w e e n t h e n e u r o p s y c h o l o g i c a l f i n d i n g s a n d o t h e r d a t a , o r t h e r e i s s u s p i c i o n o f m u l t i f o c a l o r g e n e r a l i z e d d y s f u n c t i o n , f u r t h e r i n v e s t i g a t i o n s i s u s u a l l y r e q u i r e d t o d e t e r m i n e w h e t h e r t h e p a t i e n t h a s a s u r g i c a l l y - r e m e d i a b l e e p i l e p t i c f o c u s . It has been increasingly recognized that some patients with intractable focal epilepsy may have underlying immunological disorder with antibodies directed against various central nervous system targets. It may be prudent to looks for such derangements in appropriate clinical setting. Screening for antibodies such as anti-nuclear antibody, anti-deoxyribonucleic acid, extractable nuclear antigen screen, anti-thyroid antibody, anti-glutamic acid decarboxylase, anti-N-methyl-D-aspartate receptor antibody, anti-voltage-gated potassium channel, etc. can be helpful. If such disorder is established, treatment with immunological therapy such as pulse or long-term corticosteroids, intravenous immunoglobulins, plasma exchange or even immunosuppressive therapy with cyclophosphamide, rituximab, etc. have shown to provide benefit in some instances.[] Increasingly various genetic underpinning of focal epilepsy is being understood. Most of the single gene or chromosomal disorders where DRE is part of the phenotype present in childhood and seen by pediatric neurologists or geneticists. Some syndromes may present in adulthood or continue into adulthood and may present as MRI-negative DRE. In such a patient characteristic presentation of a known genetic syndrome or a strong family history, genetic testing might help to establish proper diagnosis and end search for a cause. More importantly, it may also prevent unnecessary and unsuccessful surgical intervention. Some of the genetic causes of MRI-negative DRE include autosomal dominant partial epilepsy with auditory features due to mutation in LGI1 gene, autosomal dominant nocturnal frontal lobe epilepsy with various mutations in nicotinic acetylcholine receptor α and β subunits CHRNA4, CHRNB2 and CHRNA2, focal epilepsy with variable foci with various mutations in DEPDC5 gene, reflex epilepsies such as hot water epilepsy, primary reading epilepsy, idiopathic photosensitive occipital lobe epilepsy, etc. Generalized epilepsy with febrile seizures plus syndrome with mutation in various sodium channel genes SCN1A, SCN1B, SCN2A or GABA receptor (GABRG2) genes is being increasingly recognized syndrome in children but semiology is variable and remains not completely understood story.[] A s s u g g e s t e d a b o v e , t h e r e a r e v a r i e d r e a s o n s f o r M R I - n e g a t i v e D R E . T h e p r i m a r y a i m o f t h e e v a l u a t i o n i n s u c h i n d i v i d u a l i s t o e s t a b l i s h e p i l e p t o g e n i c z o n e a n d d e t e r m i n e i f s u r g i c a l r e m e d y i s f e a s i b l e o r n o t a n d a t w h a t c o s t . G e n e r a l g u i d i n g p r i n c i p l e s a r e t o e s t a b l i s h t h a t s t e r e o t y p i c a l f o c a l s e i z u r e s a r e t h e c a u s e o f h a b i t u a l e v e n t s . T h e s e c o n d s t e p i s t o d e t e r m i n e t h e l o c a t i o n o f s e i z u r e f o c u s . F o c a l s e i z u r e s l e a d t o v a r i o u s f u n c t i o n a l i m p a i r m e n t s e v e n i n t h e a b s e n c e o f c l e a r l y v i s i b l e s t r u c t u r a l a b n o r m a l i t y o n M R I . S u c h c a n b e d e t e r m i n e d b y f u n c t i o n a l n e u r o i m a g i n g , e l e c t r o p h y s i o l o g i c a l a n d n e u r o p s y c h o l o g i c a l t e s t i n g . T h e m o r e c o n c o r d a n c e b e t w e e n t h e v a r i o u s m o d a l i t i e s o f t e s t s , t h e h i g h e r t h e c o n f i d e n c e i n l o c a t i o n o f s e i z u r e o n s e t a n d h i g h e r t h e s u c c e s s r a t e o f s u r g i c a l t r e a t m e n t . I f t h e r e a r e s o m e d i s c o r d a n t r e s u l t s , o n e m a y n e e d t o e v a l u a t e t h e s t r e n g t h o f s u c h d i s c o r d a n c e a n d p r e p o n d e r a n c e o f t h e e v i d e n c e b e f o r e m a k i n g a j u d g m e n t t o p r o c e e d w i t h s u r g i c a l r e s e c t i o n . O n e a l s o h a s t o d e c i d e w h e t h e r o n e - s t a g e s u r g e r y ( u s u a l l y m e a n s a t e m p o r a l l o b e r e s e c t i o n i n a p a t i e n t w i t h M R I - n e g a t i v e D R E ) o r t h e r e i s a n e e d f o r t w o - s t a g e d a p p r o a c h t o d e f i n e t h e e p i l e p t o g e n i c z o n e m o r e p r e c i s e l y . S o m e o f t h e o t h e r t e s t s h e l p t o e s t a b l i s h t h e l o c a t i o n o f t h e e l o q u e n t c e r e b r a l c o r t e x i n r e l a t i o n s h i p t o t h e e p i l e p t o g e n i c z o n e , w h i c h i n t u r n h e l p s t o d e t e r m i n e r e s e c t i o n m a r g i n s a n d r i s k o f p o s t - o p e r a t i v e d e f i c i t s a n d / o r s e i z u r e r e c u r r e n c e .

xref #text xref #text In cases of lesional epilepsy, it is generally believed that seizures arise from the vicinity of the lesion. However, the precise location is not certain and cannot be determined on the basis of imaging abnormalities. Over the years, many have tried to use EEG recording acutely during surgical intervention. A brief recording of cortical surface EEG using subdural grid/strip electrodes is attempted prior to removal of the lesion. Alternatively, a depth electrode may be inserted within a lesion to evaluate intrinsic epileptogenicity. This EEG recording is known as ECoG. If epileptiform activity is encountered on ECoG, a tailored resection can be performed to include the area of electrophysiological abnormality. This is shown to be helpful in lesional epilepsy cases[] or temporal lobe epilepsy.[] However, the success of this approach is limited. The main reason is that the interictal abnormality recorded during brief ECoG recording may not be indicative of the actual seizure onset zone.[] A presurgical evaluation using a variety of noninvasive tests (e.g., magnetic resonance imaging [MRI], positron emission tomography [PET], neuropsychology testing, magnetoencephalography, single photon emission computed tomography, etc.) may establish seizure onset from a temporal lobe, perhaps the medial temporal lobe in cases of hippocampal sclerosis identified on MRI. It can also identify other potential epileptogenic lesions such as cortical developmental malformation or brain tumors. In cases of medial or anterior temporal lobe seizures, one stage “standard” resection of the anteromedial temporal lobe or more selective amygdalohippocampectomy can provide lasting seizure freedom.[] However, at times the presurgical evaluation may fail to provide definitive information regarding margins of the epileptogenic zone. In such instances, iEEG is often employed to help determine the epileptogenic zone (areas of seizure onset and early spread). Though general principles are well established, what triggers implantation of electrodes and iEEG recording may differ among different epilepsy centers and depend upon individual experience and availability of advanced technologies locally. The choice of electrodes is also dependent upon the individual's experience and expertise. iEEGs are usually recorded with the help of subdural grid electrodes and/or depth electrodes. Subdural electrodes are small metal discs embedded in teflon or silastic sheath material and arranged in different configurations []. These electrodes are connected to a thin metal wire and they are individually insulated and ultimately bundled and covered with plastic material (tail). When the configuration of electrodes is in a single column, it is commonly referred as a subdural strip electrode. When it contains rows and columns, it is known as subdural grid electrode and is available in many different sizes and configurations. These grids or strips are implanted subdurally over the surface of the brain and their tails exit the meninges, skull and ultimately through the scalp to protrude on the scalp []. There they are connected to an amplifier through cables to record EEG signals from each individual electrode from the grid/strip. When the electrodes are placed on a hollow plastic tube that can be inserted in the brain tissue itself, the electrode is known as a depth electrode. The electrode usually comes with a stylet to provide necessary stiffness to insert in the brain. Depth electrodes can be placed in the deep structures (such as hippocampus, amygdala and insula) of the brain or within lesions and are commonly placed under neuronavigation guidance to optimize implantation into the desired location []. Epidural peg electrodes are another type of iEEG recording electrodes that are placed by creating a trephination of the skull with a small opening on the inner table and inserting the electrode in the trephine with the tip touching the dura. These electrodes stay extradural in the epidural space. Electrodes are usually made of stainless steel or platinum. The platinum electrodes are more desirable as they are non-ferromagnetic making them more compatible with MRI, but they are more expensive. Each of these electrode types provides certain advantages and is associated with its own challenges which are summarized in . This phase of iEEG recording is relatively short and consists of implantation of intracranial electrodes followed by extraoperative prolonged video-EEG recording using these electrodes to delineate the epileptogenic zone and also map the eloquent cortex. In a typical two-staged approach, a craniotomy is performed over the suspected epileptic hemisphere during the first stage of surgery to expose the cerebral cortex where subdural grids are to be implanted. Following implantation of the subdural grid, strip and/or depth electrodes, iEEG is recorded extraoperatively off antiseizure medications for an extended period of time (typically 3-14 days) to capture habitual seizures. Analysis of this data establishes the epileptogenic zone. During the extraoperative phase of video-EEG recording, intracranial electrodes can be stimulated with a square wave electrical pulse using an external electrical stimulator and clinical effects of such stimulation can be monitored and documented. Stimulation of various primary sensory cortices can lead to sensory experience of that modality, e.g., stimulation of visual cortex leads to visual experience of seeing phosphenes, colors, shapes, objects or even formed visual images depending upon the type of visual cortex being stimulated. If one stimulates the primary motor cortex of the precentral gyrus it can lead to time-locked motor movements of the corresponding body part on the contralateral side, e.g.; hand area stimulation resulting in twitching of hand/wrist. Similarly stimulation over the language area causes disruption of language tasks, such as dysnomia, interruption of spontaneous speech, etc. At the end of this phase of recording one expects to have a reasonably good idea of where the epileptogenic zone of habitual seizure is and where the eloquent cortex is located. At this point, the epilepsy team makes a decision regarding the resection margins and discusses that with the patient and his/her family members and caregivers. The risks of potential post-operative deficits can be discussed more precisely at this stage as one has a very good idea of where the eloquent cortex is located in relationship to the proposed resection margins for the epileptogenic zone. After this discussion one finalizes the resection margins on the basis of anatomical details and electrophysiological findings and resection ensues during the second stage of surgery. e r e a r e m a n y c i r c u m s t a n c e s w h e r e i E E G i s u s e f u l , b u t t h e m a i n i n d i c a t i o n o f i t s u s e i s w h e n e i t h e r s e i z u r e s c a n n o t b e l a t e r a l i z e d o r l o c a l i z e d w i t h c o n f i d e n c e w i t h n o n - i n v a s i v e m e t h o d s o r t h e e p i l e p t o g e n i c z o n e i s c o n s i d e r e d t o b e c l o s e t o e l o q u e n t c o r t e x a n d p r e c i s e m a p p i n g o f b o t h e p i l e p t o g e n i c z o n e a n d e l o q u e n t c o r t e x i s n e e d e d t o p r o v i d e t h e b e s t p o s s i b l e o u t c o m e . #text There are no set standards to describe when iEEG is required. Each center follows their own guidelines depending upon their own preferences and experience. However, there are some general approaches and principles underlying the decision to employ iEEG.[] Common scenarios where iEEG is used in our center and to a large extent at other comprehensive epilepsy centers include: T h e t e c h n i q u e s f o r c r a n i o t o m y a n d p l a c e m e n t o f i n t r a c r a n i a l e l e c t r o d e s a r e s t r a i g h t f o r w a r d . P l a n n i n g f o r t h e i E E G s e s s i o n i s t h e m o s t c r i t i c a l a s p e c t a n d r e q u i r e s c o l l a b o r a t i o n a n d a n e x c e l l e n t w o r k i n g r e l a t i o n s h i p b e t w e e n t h e e p i l e p s y n e u r o s u r g e o n a n d t h e e p i l e p t o l o g i s t . T h e f o l l o w i n g f a c t o r s a r e i m p o r t a n t t o c o n s i d e r : S u r g i c a l i n t e r v e n t i o n t o t r e a t i n t r a c t a b l e e p i l e p s y i s k n o w n t o b e e f f e c t i v e . C u r r e n t l y , t h e r e i s n o c l a s s I d a t a t o p r o v e t h a t t h e i E E G r e c o r d i n g i s e f f e c t i v e i n m a n a g e m e n t o f i n t r a c t a b l e e p i l e p s y a n d i f i t i s e f f e c t i v e w h a t i s t h e b e s t w a y t o a p p r o a c h i t . H o w e v e r , i E E G r e m a i n s a v e r y u s e f u l t o o l i n s u r g i c a l m a n a g e m e n t o f e p i l e p s y w i t h o u t w h i c h i t c a n n o t b e p e r f o r m e d a d e q u a t e l y . I t s r o l e i s w e l l e s t a b l i s h e d i n h e l p i n g t o d e l i n e a t e t h e e p i l e p t o g e n i c z o n e p r e c i s e l y . T h e p l a n n i n g f o r i E E G r e c o r d i n g r e q u i r e s e x p e r t i s e o f m a n y i n d i v i d u a l s a n d e p i t o m i z e s t h e t e r m “ t e a m a p p r o a c h ” . T h o u g h i t i s g u i d e d b y f e w b a s i c p r i n c i p l e s , i E E G i n d i c a t i o n a n d p l a n n i n g v a r i e s s i g n i f i c a n t l y b e t w e e n c e n t e r s d e p e n d i n g u p o n l o c a l p r e f e r e n c e s , e x p e r t i s e a n d b e l i e f s .

xref #text xref #text The underlying cause of SE determines its outcome to a great extent.[] When possible the etiology of SE should be treated promptly. The etiology of SE depends on the geographical region; in the Veterans Administration co-operative study, the etiology of SE was remote neurologic cause in 69.5% and acute neurologic cause in 27.5%.[] In a study from India, on 117 patients with SE, the most common etiology was CNS infection in 63 (53.8%), stroke in 11 (6.5%), metabolic factors in 12 (10.2%) and miscellaneous disorders in the remaining.[] High prevalence of infection in SE has also been reported from Africa.[] High frequency of CNS infection and stroke are common in the developing countries, but all of these do not have specific treatment. However in a small percentage of treatable disorders such as herpes simplex encephalitis, cerebral venous sinus thrombosis and pyogenic infection, treatment of primary disorder may improve the outcome of SE. In SE, a large number of investigations are recommended such as blood counts, blood sugar, blood culture, serum electrolyte, serum chemistry, AED level, cerebrospinal fluid (CSF) examination, coagulation parameters, imaging and EEG. All the investigations are not necessary in every patient. In a systematic review, the role of investigation in changing the outcome of SE was evaluated for the quality of evidence and the diagnostic yield of different investigations.[] The investigation should be done in the clinical context. If infection is suspected, blood culture and CSF examination may be appropriate. AED levels should be ordered if the patient was on drug prophylaxis and there is suspicion of non-compliance or toxicity as a cause of SE. Serum tests for toxins are recommended if no cause of SE is apparent. Screen for inborn error of metabolic or genetic abnormalities would be cost-effective if there is a family history. EEG is especially useful for the diagnosis of non-convulsive SE or in focal abnormality. Imaging is especially useful in the evaluation of first seizure/SE or if the clinical setting suggests CNS infections or a mass lesion.[] Ideally all patients with SE should be managed in intensive care unit (ICU) and especially those who have not responded to 2-3 AEDs and/or have persisted SE for 2 h (refractory SE). The management of refractory SE is by anesthetic drugs such as midazolam (MDL), propofol or PB. The drugs used for management of SE and especially anesthetic drug cause hypotension and cardiorespiratory depression which are sometimes severe and is the limiting factor. Artificial ventilation and vasopressor drugs therefore are essential. There have been questions to what extent the physiological information helps in improving the outcome, e.g., pulmonary artery pressure or intra-arterial BP monitoring, though provides continuous objective documentation of BP and help in titrating the dose of vasopressor drugs but has associated risks due to frequent withdrawing of blood for investigations resulting in anemia and need for blood transfusion, risk of vascular injury and thrombosis and high frequency of blood borne bacterial and fungal infections. In a systematic review, the odds ratio for bloodstream infection by intra-arterial catheter was 1.7 (95% confidence interval [CI] 1.2-2.3), venous catheter 0.5 (95% CI 0.2-0.7) and central venous catheter 2.7 (95% CI 2.6-2.9)[] and there has been appeal for a moratorium on pulmonary artery catheterization.[] xref #text Valproic acid is a short chain fatty acid with anticonvulsant properties. It acts through activated sodium channel, neuronal calcium channels or through GABA metabolism. The safety and tolerability of VPA have been reported and upto 6 mg/kg/min has been administered to a total loading dose of 45 mg/kg without adverse reaction.[] The chief advantage is safety and ease of administration and its efficacy range between 58% and 83% respectively.[] In a study, 68 patients with convulsive SE were randomized to VPA and PHT. Seizures were aborted in 66% in VPA and 42% in PHT group. As a second choice also VPA was more effective (79% vs. 25%) than PHT[] and was considered non-inferior to PHT in another study.[] In another study VPA was effective in 65% (15/23) patients as first line therapy.[] IV formulation of LEV was recommended earlier for those patients in whom oral therapy was not possible and up to 1500 mg IV was recommended to be safer if given in 15 min. Later studies reported safety of up to 2500 mg in 5 min and 4000 mg in 15 min in normal volunteer.[] Safety and efficacy of LEV was reported in 17/18 episodes in 16 patients who were refractory to benzodiazepine and intubation was avoided.[] In a randomized controlled trial on 79 patients with SE, LEV 20 mg/kg over 15 min was as effective as LOR 0.1 mg/kg in 2-4 min (76.3% vs. 75.6%) and 24 h seizure freedom was also comparable (79.3% vs. 67.7%). However, LOR was associated with significantly higher need for artificial ventilation and insignificantly higher hypotension. The authors feel that LEV was an alternative to LOR and may be preferred to LOR in patients with respiratory compromise and hypotension or in non ICU setting.[] Lacosamide is a N-acetyl-N-benzyl-D-homoserinamide, a functional aminoacid and its mechanism of action is selective enhancement of slow inactivation of voltage-gated sodium channels, resulting in stabilization of hyperexcitable neuronal membranes and inhibition of repetitive neuronal firing. The IV formulation of lacosamide is now available and its efficacy has been evaluated in refractory SE in some studies.[] More studies with the well-defined end points are needed. The dose and side-effects of various AEDs used in the management of SE are summarized in the . Comparative study, systematic review and meta-analysis revealed comparable efficacy of LEV, PHT and VPA, and VPA has been reported to be safer than PB.[] There are limited studies evaluating these drugs as first-line AED in SE. More studies are needed to evaluate the efficacy and safety of these drugs compared with benzodiazepines which may have wider implication especially in resource poor countries. Recently the adverse events of intravenous antiepileptic drugs (IV AED) in SE have been highlighted in a report from an academic medical center in Switzerland. In a retrospective review of ICU cases during 2005-2011, out of 171 patients 37% were treated with IVAEDs and death occurred in 18%. The patients with IVAED had more frequent intubation (90% vs 25%), severe hypotension requiring vasopressor (6% vs 1.9%), more infection (43% vs 11%) and 2.9 fold relative risk of death. The authors suggested the need of a randomized controlled trial (Sutter 2013)

Epilepsy that cannot be controlled with known pharmacological management, also referred as drug-resistant epilepsy (DRE), patients are recommended for epilepsy surgery.[] The genesis of DRE still eludes us, but abnormal synaptic transmission is one of the key features of this disorder. Epilepsy can be caused by multiple mechanisms and often they appear so diverse that one would suspect that no common hypothesis applies. However, one common principle that is applied to the process of epileptogenesis is disruption of mechanisms that normally create a balance between excitation and inhibition. Dysfunction of mechanisms that inhibit excitatory synaptic transmission or promoting the mechanisms that facilitate excitation can lead to epileptogenesis.[] Although the concept of a balance provides a useful model for mechanisms that can initiate epileptiform activity, it is daunting to consider the array of potential causes. There are various controls that keep the balance between excitatory transmission and inhibitory transmission in normal neuronal network. In addition to the two prevailing theories of DRE, target hypothesis and transporter hypothesis,[] another recently proposed hypothesis is neural network hypothesis.[] According to this hypothesis, seizure-induced alterations of brain plasticity including axonal sprouting, synaptic reorganization, neurogenesis, and gliosis could contribute to the formation of abnormal neural network. This in turn leads to loss of the inhibitory effect of endogenous antiepileptic system and also prevented the traditional antiepileptic drugs from entering their targets, eventually leading to DRE. It is important to understand the processes through which the neuronal activities are synchronized which in turn lead to generation of seizures.[] To this end, localization of epileptic networks will help in guiding epilepsy surgery, deploying antiepileptic measures, and elucidating mechanisms underlying the process of epileptogenesis. sup italic #text Development and application of integrated dynamic imaging approaches examining neuronal circuit function has significantly helped in our understanding of the mechanisms underlying epileptogenesis, epilepsy, and seizure generation [see ]. Progress in neuroimaging has led not only to successful identification of epileptic foci for surgical resection, but also to an improved understanding of the functional and microstructural changes in long-standing epilepsy. Positron emission tomography (PET), functional magnetic resonance imaging (fMRI), and diffusion tensor imaging are all promising tools that can assist in elucidating the underlying pathophysiology in chronic epilepsy.[] In addition to conventional MRI, functional neuroimaging using PET and single-photon emission computed tomography (SPECT) can provide complementary information to help localize the epileptic focus and often provides additional information that cannot be obtained from conventional MRI sequences. Magnetoencephalography (MEG) has recently emerged as a clinical tool for neurology and neurosurgery could also be helpful in dysfunctional neuronal circuit by localization of the irritative zone and through functional mapping of eloquent (sensory, motor, and language) cortex. Recent MEG studies have demonstrated dramatic plastic shifts in brain function due to lesions or developmental abnormalities but not necessarily predicted by the anatomical changes.[] The major challenge that the epilepsy surgeons across the world face are those 30%-40% patients who still continue to have seizures despite undergoing an adequate epilepsy surgery.[] The more straight forward pathologies have better outcomes, which are similar in most centers. The more complex pathologies, while requiring more extensive work up, does not translate into a better outcomes.[] Localizing the dysfunctional network can explain the reasons for surgical failure and provide solutions to reduce the same. It is critical for the neurosurgeon to determine, how far the epileptogenic area is form the lesion. Growing evidences suggests that there is focal area where the onset of spike wave discharge (SWD) is located and these local SWDs propagate very quickly throughout the cortex and to thalamus at the millisecond scale.[] It is important to determine with highest precision the extent of epileptogenic network while planning a surgical intervention. Quantitative methods of EEG signal analysis and epileptogenicity index are useful tools for functional analysis of neuronal networks associated with an epileptogenic lesion.[] Posttemporal lobectomy, the presence of diffused pathology, is one of the reasons for recurrence after surgery. As suggested by positive neuroimaging in this case, maximal networks are close to the lesion.[] Electrocorticography (ECoG) provides useful information for the prediction of surgical success in surgically remediable epilepsy. DRE and post resection ECoG correlation with its grade of severity and clinical outcome suggest network pattern of the lesions. Despite its widespread usage, there are still controversies regarding its utility especially keeping in mind its short period of application.[] It also should be kept in mind that any electrode used potentially records from millions of neurons, for example, a circular electrode of the EcoG grid sits over 10 neurons, each having about 10 connections. Thus, this is equivalent to hanging a microphone among a population of 1 million people, each talking to 10,000 persons! Thus, the need of hour is to develop better amplifiers and also technologies which can analyze meaningfully such a huge amount of data.[] The method of intraoperative coregistration of MRI, PET, and ECoG has shown to provide better objective localization of the epileptogenic foci.[] Localizing eloquent cortex lesions, which present a surgical challenge, could be successfully achieved by combination of neuronavigation aided fMRI and diffusion tensor tractography along with cortical stimulation.[] Source localization by the combination of MEG and EEG provided insight into the regional network associated with epileptogenic focus.[] Stereoelctroencephalography- mediated electrophysiological recording helps in the computation of epileptogenecity index which helps determine if the epilieptogenic network is confined to the lesion or it involves other distant structures.[] Recently, high frequency oscillations (HFOs) have been reportedly shown to arise from brain regions constituting epileptic networks and may be important to seizure generation.[] HFOs are brief 50-500 Hz pathologic events measured in intracranial field and unit recordings in patients with refractory epilepsy. Basic research studies on HFOs indicate these local oscillatory field potentials correspond with an increase in rate and synchrony of neuronal discharges. Because HFOs can facilitate synaptic transmission through local networks, these events are implicated with information processing and consolidation of memory. Alterations to neuronal networks associated with epilepsy can also generate abnormal or pathologic HFOs that are believed to reflect fundamental neuronal disturbances associated with brain areas capable of generating spontaneous epileptic seizures.[] There is compelling evidence supporting the view that normal hippocampal ripples and neocortical HFOs in the normal brain reflect inhibitory post-synaptic potentials (IPSPs) of interneurons that regulate the firing and timing of postsynaptic principal cells. Epileptiform synchronization could be generated due to loss of inhibition, reduction in after hyperpolarization, enhanced excitatory synaptic transmission, enhancement of inhibitory network activity, and depolarizing γ-amino butyric acid (GABA). The generation of interictal epileptiform discharges (IEDs) epilepsy is commonly ascribed to enhanced excitatory interactions within glutamatergic neuronal networks.[] In terms of both pattern and underlying mechanisms, IEDs are heterogenous in nature. Evidence suggests that core region in which focal seizures are generated is surrounded by an area that generates hypersynchronous activity (denominated the “irritative region”) interposed between the seizure-onset area and the surrounding normal tissue.[] IEDs are generated both in the epileptogenic zone and in the irritative region and can spread to (and thus be recorded from) adjacent “healthy” brain structures. Therefore, it is reasonable to conclude that interictal events are sustained by cellular and pharmacological mechanisms that vary according to the site of generation. It is possible that these differences may result in a different functional role with respect to seizure generation. Experiments on animal models with convulsants showed that the CA3 region of the hippocampus is involved in generating IEDs, which were soon related to abrupt “paroxysmal” depolarization shifts (PDSs) in pyramidal cells.[] Hyperexcitation contributed to the PDS produced by most acute convulsants.[] It has been found that the extensive axon collateral network of CA3 pyramidal cells, which connect with many other CA3 pyramidal cells in addition to their longer range projections to CA1, septum and contralateral CA3 is responsible to the aforesaid hyperexcitation. It has also been shown that integrity of CA3 region of the hippocampus is an important determinant of glutamatergic drive to the CA1 pyramidal neurons.[] Ablation of CA3 field in rat brain slices lead to the reduction of glutamatergic synaptic transmission onto CA1 pyramidal neurons.[] Altogether, these reports suggest that excessive excitatory drive generated from the CA3 region may contribute to the abnormal synchronous neuronal activity associated with epileptogenesis. These synaptic networks can play a role in the synchronization occurring during both seizures and IEDs. It is possibly strengthened by sprouting of new connections and other synaptic changes in chronic epileptic foci. The duration of epilepsy is the most important predictor for long-term surgical outcome and active networks may attain permanent bi-stable stage with time.[] It is also plausible that idling networks may return back to their active bi-stable stage after discontinuation of antiepileptic drugs (AEDs) in patients rendered seizure free by epilepsy surgery.[] Altogether, the network concept provides better understanding of the epileptogenic lesion. Nonregression analysis of intracerebral EEG signals helps in the identification of epileptogenic networks.[] In addition, fluctuation analysis has been an important aspect of research in neurophysiology.[] Advanced cellular electrophysiological experiments has enabled us to record currents through a single or a group of channels from single neurons under voltage clamped conditions.[] Recent development on quantification of noise at the ion channel level has thrown light on the phenomenon of transport of ions and metabolites across cell membrane and its mechanisms especially its relation to neuronal communications.[] Synaptic noise, a kind of channel noise, plays an important role in this process. Despite remarkable advances in epilepsy surgery, the principles of surgery have been only resective, disconnective, or neuromodulatory surgery. Since Wilkins .,'s[] description of the famous case in 1886 of a patient with focal motor seizures with depressed fracture, who was cured of his epilepsy, when the lesion and also the surrounding area was resected led to the birth of epilepsy surgery. From here on the concept of “epileptic zone” took birth, where the lesion was assumed to be placed in the center surrounded by structurally normal but functionally deranged parenchyma. Luders further refined this concept by creating the concept of lesional zone, surrounded by ictal onset zone (as detected by video EEG) and further surrounded by a large irritative zone (as defined by inter ictal EEG). The epileptogenic zone was said to be placed between the ictal onset zone and the irritative zone.[] Currently, there is no single investigation available to exactly delineate the epileptgenic zone. This is mostly because all the current investigations,[] have different levels of spatial resolutions (ability to delineate the focus from the level of neuron to the brain) and temporal resolutions (ability to distinguish the onset of epilepsy at different time periods). Thus, PET has a lower temporal and spatial resolution as compared with SPECT. Thus, different modalities of investigations provide different snapshots of the epileptogenesis at different time periods and at variable magnifications. With the biomarkers of epilepsy limited to a few options (ictal EEG, HFO, and MEG), options turned on to devise more complex methods of data analysis to allow a greater sampling rate and larger sampling area. Linear mechanics to explain epiletogeneis were obviously doubtful to explain real-time situations. Thus, (Wendling .[]) became more useful to develop computer models of epileptogenesis. This also led to use of more complex computational models like nonlinear dynamical systems and chaos theory which helped us understand epileptogenesis much better. These mostly included phase synchronization methods, generalized synchronized methods, and phase lag index.[] To understand epileptogenesis better, Aubert .,[] developed the concept of epileptogenic index, which is essentially the propensity of the brain to generate high frequency oscillations and the time for this area to get involved in the seizure. He found that only in 1/3 of cases with epilepsy there is a single epileptogenic zone, while in the rest cases; there was evidence of more than one area, which generated epilepsy. There is now cumulating evidence that the epileptogenic “zone” is essentially a simplified concept. In fact, the epileptic neurons are now recognized to have a bi-stable nature during epileptogenesis and during interictal period.[] Thus, a small area of neurons, which epileptogenic stimulate more distant neurons, and through reentrant pathways forms a reverberating circuit (also called a “node”). Such circuits progressively recruit more distant circuits forming larger and larger networks over a period of time []. In fact, clinical studies (though indirectly) do also suggest existence of such networks. McIntosh .,[] demonstrated best long-term outcome for temporal lobe epilepsy with presence of a demonstrable lesion, suggesting that the maximal networks are centered around the lesion. Another elegant study by Janszky .,[] demonstrated that the seizure freedom was inversely proportional to the duration of epilepsy (being 90% Class I Engel for patients with duration of epilepsy between 1 and 10 years reducing to about 30% for patients with duration of epilepsy being >30 years at the time of surgery). While the findings of the study cannot be explained with the “zone” hypothesis, the “network” hypothesis can explain this by the fact that more neurons become recruited into the “epileptogenic network” over a period of time if epilepsy is not controlled. Similarly another landmark study by Schmidt .,[] demonstrated that the seizure freedom falls down to 65% (Class I Engel) over 3 years after discontinuation of AEDs for temporal lobe epilepsy suggesting that “idling” networks may return back to the active bistable state. These studies suggest that the epileptogenic focus is not a “fixed” zone but a dynamic, constantly changing group of networks. The network hypothesis is further strengthened by Lin .,[] who demonstrated diffuse thinning of neocortical grey matter in mesial temporal sclerosis suggesting clearly the complexity involved even in focal pathologies producing epilepsy. The network concept is important for the surgeon to understand that the epileptogenic “focus” may be far removed from the lesion and separated by normal parenchymal tissue. This has been very elegantly demonstrated in case study by Stefan .,[] Here, the patient had a ventricular perinodular hetertopia (PNH). Placement of depths and a surface grid actually revealed ictal onset from the surface even though the lesion was deep within the ventricle. The patient underwent both resection of the PNH and surface neocortical temporal gyrus, following seizure freedom was achieved. We also had a similar case, where a distant focus was demonstrated []. #text A n a l y s i s o f h y p e r s y n c h r o n u s n e u r o n a l n e t w o r k s h o u l d b e w i d e l y u s e d f o r e p i l e p s y s u r g e r y . C u r r e n t l y , u t i l i t y a n d e f f i c a c y o f d e t e r m i n i n g e p i l e p t o g e n i c n e t w o r k p r i o r t o s u r g e r y i s c o n t r o v e r s i a l . B u t w i t h h i g h - r e s o l u t i o n r e c o r d i n g a n d i m a g i n g t e c h n i q u e s , s i g n a l a n a l y s i s m e t h o d o l o g i e s a n d t h e p o s s i b i l i t y o f s t u d y i n g d y n a m i c b r a i n s t a t e s n e u r o n a l n e t w o r k a n a l y s i s h a s g a i n e d s t r e n g t h . I n I n d i a , t h e u s a g e o f n o n i n v a s i v e t e c h n i q u e s l i k e M E G a n d f M R I i s n o w g a i n i n g p o p u l a r i t y t o i d e n t i f y n e t w o r k i n v o l v e m e n t s b o t h s t r u c t u r a l l y a n d d y n a m i c a l l y . M o r e o v e r , d e v e l o p m e n t o f n e w e r m e t h o d s t o a n a l y z e t h e d y n a m i c s o f n e u r o n a l n e t w o r k s h a s g a i n e d m o m e n t u m a n d h a s y i e l d e d a w i d e r a n g e o f c o m p u t e r t o o l s b e i n g t e s t e d i n c l i n i c a l a n d e x p e r i m e n t a l e n v i r o n m e n t s . T h i s h a s l e a d t o d e v e l o p m e n t o f n e w e r c o n c e p t s w h e r e t h e e p i l e p t o g e n i c f o c u s e a r l i e r t h o u g h t t o b e a s t a t i c z o n e c e n t e r e d a r o u n d t h e l e s i o n i s n o w c o n s i d e r e d a s a n e v o l v i n g a c t i v e a n d a d y n a m i c c i r c u i t o f n e t w o r k s .

Epilepsy is one of the most common serious neurological conditions, accounting for 1% of the global burden of disease, based on disability-adjusted life years, the number of person years lost due to disability and premature death.[] This is equivalent to breast cancer in women and lung cancer in men. Among primary disorders of the brain, it is equivalent to depression and other affective disorders, Alzheimer's disease and other dementias, and substance abuse. Ten percent of the world's population will have at least one seizure during their lifetime and one-third of these will develop epilepsy at any given time.[] One percent of the world's population has active epilepsy. In countries where adequate diagnosis and treatment are available, 30-40% of people with epilepsy have seizure that are uncontrolled by medication,[] which accounts for 80% of the cost of epilepsy in the United States.[] Medically refractory epilepsy, therefore, is a major health concern not only for patients and their families, but for society. Treatment objectives for epilepsy are no seizures, and no side effects, as soon as possible. Early effective interventions provide the best opportunity to prevent adverse psychological and social consequences of recurrent seizures, progressive deficits that lead to irreversible disability, and premature death. In particular, seizures that interfere with school, work, and interpersonal relationships, during adolescence and early adulthood, prevent the acquisition of vocational and social skills necessary to live independently. When available treatments with the potential to eliminate disabling seizures are delayed (for instance, in the United States the average time from onset of epilepsy to surgery is 22 years[]), patients who do become seizure free often cannot be rehabilitated and remain dependent on their families and society. Early identification of pharmacoresistant epilepsy and institution of alternative treatments can prevent a lifetime of disability. The term “pharmacoresistant epilepsy” can no longer be taken literally, as there are now so many antiseizure drugs that it would take a lifetime to try all of them alone and in combination in any given patient. The best predictor of pharmacoresistance is failure of the first antiseizure drug, due to efficacy and not intolerance. Given failure of one appropriate drug trial, only 11% of patients eventually become seizure free, while only 3% become seizure free after failure of two appropriate antiseizure drug trials.[] There are likely many reasons for pharmacoresistance and research to elucidate underlying mechanisms is important for the future development of more effective treatments.[] A simple-minded approach to the triage of epilepsies is that there are benign epilepsy syndromes, which are easily treated, and severe epilepsies, which are not. Of the latter, there are conditions that are remediable but require specialized diagnostic and therapeutic approaches, and conditions that are not remediable and require supportive care. Early distinction between the two is critical in maximizing quality of life. In most cases, the distinction can only be made by physicians and other healthcare workers trained in the diagnosis and treatment of epilepsy, at specialized epilepsy centers with facilities and support services not available in the community. Full-service epilepsy centers are staffed by a multidisciplinary team consisting of neurologists/epileptologists, clinical neurophysiologists, neuropsychologists, neuroradiologists, neurosurgeons, psychiatrists, social workers, and nurses skilled in the management of epileptic seizures and their consequences.[] Apparent pharmacotherapy failure does not necessarily mean that standard antiseizure drugs will not work. Alternative causes include noncompliance, seizures that are not epileptic, misdiagnosis of the seizure type or epilepsy syndrome, inappropriate use of medications such as inadequate doses or drug-drug interactions, and lifestyle issues such as alcohol binging, drug abuse, stress, and sleep deprivation. Differential diagnosis can usually be achieved with specialized approaches available at epilepsy centers, which often requires inpatient video-electroencephalogram (EEG) monitoring to capture the habitual events and characterize their electroclinical features. These approaches can permit recognition of nonepileptic seizures and their causes, diagnosis of specific seizure types, and epilepsy syndromes, and determinations of which patients who are truly pharmacoresistant might be candidates for surgical therapy. In addition, epilepsy centers have the ability to utilize specialized pharmacologic approaches, including enrollment in clinical trials of experimental antiseizure drugs, to provide alternative treatments other than surgery, such as vagus nerve stimulation (VNS) and the ketogenic diet, and to evaluate and address psychological and social problems resulting from uncontrolled seizures. Usually, patients seek help not because they are having seizures, but because their seizures are interfering with their lives. It is, therefore, necessary not only to direct therapy towards the epileptic seizures, but also each individual patient's predicament, in order to maximize quality of life. Concerning the diagnosis of drug-resistant epilepsy, the International League against Epilepsy (ILAE) has proposed, as a testable hypothesis: “That drug-resistant epilepsy is defined as failure of adequate trials of two tolerated, appropriately chosen, and used antiepileptic drug schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom”.[] Once this diagnosis is made, a number of options can be considered. It is most important to first determine whether the patient may be a candidate for surgical treatment, because this is the only therapeutic approach with the potential to cure epilepsy. Depending on the type of epilepsy and the surgical procedure, most patients experience elimination, or at least a reduction, in disabling seizures, and significant improvement in quality of life. For patients who are not surgical candidates, stimulation approaches have outcomes similar to those brought about by additional medication trials, but with different side effect profiles. Ketogenic diet, particularly in young children, can eliminate seizures, and various complementary and alternative treatments offer additional opportunities to reduce the seizure burden and improve quality of life. Surgically remediable epilepsy syndromes are conditions with a known pathophysiology and a predictable natural history that includes unresponsiveness to pharmacotherapy and progressive features, such as developmental delay in infants and young children, or interictal behavioral disorders, most commonly depression.[] Patients with surgically remediable epilepsy syndromes are the most cost-effective surgical candidates, because presurgical evaluation can be performed noninvasively in most cases; by definition there is a 70-90% chance of complete elimination of disabling seizures, and disabling psychological and social consequences can be avoided or reversed, but only if surgical intervention is early. Mesial temporal lobe epilepsy (MTLE) is the prototype of a surgically remediable epilepsy syndrome.[] Other surgically remediable epilepsy syndromes include focal epilepsies due to discrete resectable structural lesions, epilepsies due to diffuse hemispheric disturbances, such as hemimegencephaly, Rasmussen's encephalitis, Sturge-Weber syndrome, and large porencephalic cysts, and gelastic seizures with hypothalamic hamartomas, because seizures originate within this alien tissue.[] MTLE is the most common form of epilepsy in adolescents and adults, the most medically refractory, and the most easily treated surgically. Most patients with MTLE have hippocampal sclerosis,[] although other lesions within the hippocampus, or in neocortical areas that preferentially project to mesial temporal structures, can produce the same characteristic limbic seizures. Features of MTLE with hippocampal sclerosis that should prompt early referral for epilepsy surgery are shown in []. Surgical therapy can eliminate disabling seizures completely in appropriately chosen patients.[] Multiple types of surgical interventions are now offered [], depending on the type of epileptic seizures and their presumed underlying causes. Standardized resections include anterior temporal resections and amygdalohippocampectomy for MTLE,[] and hemispherectomy or hemispherotomy, for patients, usually infants and young children, with diffuse epileptogenic regions limited to one hemisphere.[] Presurgical evaluation requires demonstration that epileptic seizures are originating within the standard area of resection. Neocortical resections are always tailored, in which case the presurgical evaluation must not only localize, but also determine the extent of the epileptogenic region. This is defined as the area of brain necessary and sufficient to generate habitual seizures and is, therefore, the smallest amount of tissue that can be removed in order to achieve a seizure-free outcome.[] There are no diagnostic tests that reliably determine the extent of the epileptogenic region, and this is approximated using a number of different tests of function and structure, including inpatient video-EEG monitoring, neuroimaging, and neuropsychological evaluation []. Lesionectomies without removal of cortical margins are performed when adjacent tissue cannot be damaged, and in the case of hypothalamic hamartoma where seizures actually originate within the tumor.[] Multiple subpial transection (MST) is a technique that can be used in cortical areas with essential functions, such as motor or language cortex, to reduce epileptogenicity without producing serious deficits.[] Presurgical evaluation is the same as that for tailored resection. Corpus callosotomy is a disconnection procedure effective particularly against drop attacks.[] Presurgical evaluation need only demonstrate that the patient is not a candidate for another, more definitive localized surgical procedure. Corpus callosotomy is performed rarely today because VNS may be equally effective against drop attacks in most patients. Stereotactic ablative surgery can be performed with noninvasive stereotactic radiotherapy, also called gamma knife surgery (GKS).[] This produces edema, which can have serious side effects and the beneficial effects take months to a year or more. A more recent laser ablative approach requires an intracranial probe, but the results are more immediate.[] These treatments are particularly useful in patients who have medical contraindications to surgery. #text xref #text

T h e e p i l e p s y c o m p r i s e s a h e t e r o g e n e o u s g r o u p o f d i s o r d e r s c l i n i c a l l y c h a r a c t e r i z e d b y r e c u r r e n t u n p r o v o k e d s e i z u r e s a n d e l e c t r i c a l l y c h a r a c t e r i z e d b y a s u d d e n s y n c h r o n i z e d b u r s t o f e l e c t r i c a l d i s c h a r g e e m a n a t i n g f r o m a p o o l o f n e u r o n s . T h e r e c u r r e n t s e i z u r e s t h a t c o n s t i t u t e e p i l e p s y m a y b e l o c a l i z e d i n o r i g i n o r m a y b e g e n e r a l i z e d . A m a j o r i t y o f p a t i e n t s w i t h l o c a l i z a t i o n - r e l a t e d ( f o c a l ) e p i l e p s i e s h a v e a s t r u c t u r a l l y a n d / o r e l e c t r i c a l l y i d e n t i f i a b l e l e s i o n i n v o l v i n g t h e c e r e b r a l c o r t e x . A l t h o u g h a m a j o r i t y o f p a t i e n t s w i t h e p i l e p s i e s a r e c o n t r o l l e d b y a n t i e p i l e p t i c d r u g s ( A E D s ) , n e a r l y o n e - t h i r d o f t h e m c o n t i n u e t o h a v e r e c u r r e n t s e i z u r e s d e s p i t e o p t i m u m A E D t h e r a p y . A p r o p o r t i o n o f t h e s e p a t i e n t s w i t h d r u g - r e s i s t a n t e p i l e p s y ( D R E ) a r e c a n d i d a t e s f o r e p i l e p s y s u r g e r y . T h e o b j e c t i v e o f e p i l e p s y s u r g e r y i s n o t o n l y t o e l i m i n a t e e p i l e p t i c s e i z u r e s w i t h o u t c a u s i n g a n y n e u r o l o g i c a l o r c o g n i t i v e d e f i c i t s , b u t a l s o t o i m p r o v e t h e q u a l i t y - o f - l i f e a n d m a k e a n i n d i v i d u a l a p r o d u c t i v e m e m b e r o f t h e s o c i e t y . #text The success of an epilepsy surgery program depends upon the early identification of potential surgical candidates and selecting from them, ideal candidates for surgery, who are destined to have a post-operative seizure-free outcome without any unacceptable neurological deficits.[] Since epilepsy surgery centers in resource-poor countries will lack the full range of state-of-the-art technologies usually available in resource-rich countries to perform pre-surgical evaluation and surgery, the success of epilepsy surgery programs in a developing country like India will depend upon the ability of the team to select ideal surgical candidates using locally available technology and expertise without compromising on patient safety.[] Knowing when not to operate because of the need for further investigations is as important as selecting which patient may benefit from surgery with the available facilities. The selection of the pre-surgical evaluation modalities that are required to reliably localize the epileptogenic zone and to delineate its relationship to eloquent cortical areas in an individual patient with DRE can vary from simple and straight-forward to highly complex multi-stage approach []. Patients considered for focal resections such as those with mesial temporal lobe sclerosis and low-grade neoplasms and those with large hemispheric lesions considered for hemispherotomy could be selected for surgery by magnetic resonance imaging (MRI) and interictal and ictal EEG findings; many of them may not even require ictal video-EEG recordings. In contrast, patients with focal cortical dysplasias that are located close eloquent cortical regions and those with normal/indistinct MRI findings often require multiple non-invasive and invasive investigations. Epilepsy surgery centers in resource-poor regions should initially restrict their surgical candidates to patients with mesial temporal lobe epilepsy and those with circumscribed potentially epileptogenic lesions in whom the epileptogenic zone can be unquestionably localized by using locally available relatively inexpensive and non-invasive technologies and in whom excellent post-operative outcome can be guaranteed. In these regions, where more often the patients and their caregivers bear the cost of medical care, epilepsy surgery centers will have to evolve a cost-effective pre-surgical evaluation strategy by restricting the investigations to the minimum. The author and his colleagues have shown that judicious use of spenoidal electrode EEG recording in patients with suspected mesial temporal lobe epilepsy can obviate the need for invasive EEG monitoring in nearly one in five them.[] Similarly, patients who require ictal single photon emission computed tomography (SPECT) can be carefully selected to optimize the utilization and yield of this test.[] It is important for epilepsy surgery centers to regularly assess their capabilities and limitations and adopt a stepwise approach to increasing levels of complex pre-surgical evaluation and surgical treatment strategies [].[] In this era of rapid electronic communications and telemedicine, it is no longer necessary for an epilepsy surgery center to possess all the advanced technologies used in the pre-surgical evaluation by itself. The epilepsy surgery centers can pool their technological and human resources and partner with centers nationally or internationally to develop optimum usage of facilities to benefit their patients. For example, the R. Madhavan Nayar Center, Trivandrum, which has undertaken nearly 1500 epilepsy surgeries during the last 18 years, still does not possess 3T MRI, SPECT and positron emission tomography of its own, but routinely utilizes the services of other centers to get these investigations done. Similarly, an epilepsy center in Uganda, where no MRI facility is available in the whole country, utilized computed tomography and expertise from well-established epilepsy centers in the United States and Canada to select ideal candidates and to undertake epilepsy surgery in that severely resource-constrained country.[]

xref #text In general, the term HFOs refers to EEG activity >70 Hz, whereas activity in the 30-70 Hz band is considered as gamma.[] Our ability to record and analyze high frequency activity can be influenced by various factors. A thorough knowledge of such factors is important to navigate and interpret the expanding literature on HFOs, particularly since different groups of investigators use different methods to report their findings. A few basic guidelines helpful in understanding the literature pertaining to HFOs are outlined in . Although HFOs can be recorded from scalp, it is easier to record such low-amplitude, high-frequency waveforms in a relatively artifact-free manner intracranially because of proximity to the generators of those waveforms, which tend to be smaller and located within the brain or on the brain surface. The sampling rate is an important factor determining the recording of high frequencies. Theoretically, the maximum frequency (Nyquist frequency) of the oscillations that can be evaluated corresponds to one-half of the sampling rate, i.e., a sampling rate of 1000 Hz would allow activities up to 500 Hz to be assessed. However, due to the limitations of the amplifiers used in the EEG systems, the maximum frequency that can be clearly evaluated tends to be roughly one-third of the sampling rate such that a sampling rate of 1000 Hz allows evaluation of activity up to 333 Hz. The size of the recording electrode can greatly influence the frequencies encountered, with larger electrodes less likely to record higher frequencies []. Studies using microelectrodes have demonstrated that physiological HFOs, particularly in the gamma band (sometimes up to 80 Hz), are associated with somatosensory, cognitive and perceptual tasks.[] One such example is the ripple oscillations (~200 Hz activity) described by Buzsáki . in the microelectrode recordings from the CA1 pyramidal layer of the rat hippocampus, occurring in conjunction with sharp waves during states of immobility, consummatory behavior and slow wave sleep.[] Interestingly, Bragin . subsequently noted similar ripple oscillations in the microelectrode recordings from mesial temporal structures in patients undergoing pre-surgical evaluation.[] These studies reported by the University of California-Los Angeles group focused on interictal HFOs in rodents and humans recorded by depth microelectrodes (40-60 μm diameter, 0.001 mm surface area) implanted chronically in the mesial temporal structures (hippocampus and entorhinal cortex).[] Using 2000 Hz sampling rate, these investigators described HFOs up to 600 Hz and classified them into two frequency bands: Ripples (100-200 Hz); and fast ripples (FRs: 250-500 Hz). The ripples were considered as physiological (the equivalent of rat ripples), whereas the FRs were felt to be clearly pathological, being associated with epileptogenicity.[] Subsequently, using slightly larger, proprietary clinical macroelectrodes (0.8 mm, ~800× larger than microelectrodes), the studies from the Montreal Neurological Institute reported similar, predominantly interictal HFOs in the 80-500 Hz range in patients undergoing pre-surgical evaluation with chronically implanted depth and subdural electrodes in both mesial temporal and neocortical structures.[] Around the same time, using much larger, commercially-available clinical electrodes (4 mm, ~4000 - 9400× larger than microelectrodes), other investigators described HFOs in human depth and subdural recordings obtained from both mesial temporal and neocortical structures.[] These investigators described both ictal and interictal HFOs in the 70-500 Hz range in chronic extraoperative intracranial recordings as well as intraoperative electrocorticography (ECoG). The effect of electrode size on the recorded frequency band was further explored by Worrell . in recordings using simultaneous depth microelectrodes (40 μm, 0.001 mm) and macroelectrodes (9.4 mm, ~9400× larger than microelectrodes) implanted in the mesial temporal structures.[] They showed that FRs were most likely to be recorded by single microelectrodes, but only rarely recorded by the adjacent macroelectrodes and attributed their finding to the spatial averaging of local field potentials by the relatively large surface area of the macroelectrodes leading to spatial undersampling of the focal HFOs.[] The method of analyzing HFOs could potentially lead to some inevitable discrepancies in the reported findings. Visual analysis of HFOs [] consists of inspecting EEG traces with appropriate filter and time scale settings. Commonly, one uses a low frequency filter >50 Hz and a high frequency filter at 300 Hz or 600 Hz depending upon the sampling rate. A time scale of 1-2 s per page is required to adequately visualize the HFOs, being determined by the maximum horizontal resolution of the modern computer monitors. Despite following these recommendations, visual analysis can be subjective due to lack of an accepted definition of HFO, in turn leading to inter-observer variability. Visual analysis is also time-consuming (for e.g., it could take 10 h to analyze 10 min data containing 10 channels[]), limiting the overall analysis to only small samples. Fatigue from prolonged visual analysis can also lead to intra-observer variability. On the contrary, automated analysis of HFOs is more promising because it tends to be more consistent and objective and allows processing of large samples. However, multiple algorithms, implemented on various platforms, to accomplish automation can also lead to significant differences in the reported findings. The exact mechanism of generation of HFOs is unclear, but several theories have been postulated, as reviewed by Jiruska .[] It is believed that ripples result from the summation of inhibitory postsynaptic potentials generated by the interneurons on the pyramidal cells. Another theory postulates that HFOs are generated by synchronous firing of a group of principal neurons (pyramidal cells or granule cells), with each individual high frequency cycle representing a population spike probably generated by a small group of principal neurons. Three mechanisms have been proposed to underlie such fast synchronous firing of pyramidal cells: Excitatory coupling between pyramidal cells, facilitated by axonal sprouting, resulting in the formation of clusters of pathologically interconnected neurons; electrotonic coupling through gap junctions at the axonal level; and ephaptic transmission between adjacent neurons. s p i t e t h e u n c e r t a i n t y r e g a r d i n g t h e m e c h a n i s m s i n v o l v e d i n t h e g e n e r a t i o n o f H F O s , t h e r e i s c o n v i n c i n g e v i d e n c e t o s u g g e s t t h a t t h e y a r e e a s i l y r e c o r d a b l e a s l o n g a s a p p r o p r i a t e m e t h o d s a r e u s e d . R e p o r t s o f c l i n i c a l i m p l i c a t i o n s o f H F O s c o n t i n u e t o a p p e a r i n t h e l i t e r a t u r e o n a r e g u l a r b a s i s a n d c a n b e c l a s s i f i e d i n t o t h e f o l l o w i n g 6 c a t e g o r i e s . The term DC EEG has been widely used in the literature to imply a frequency response of the EEG with a minimum at 0 Hz, which is typically recorded using special DC-coupled amplifiers that preserve the slow baseline fluctuations from significant distortion by the built-in high-pass filters.[] During pentylenetetrazole-induced tonic-clonic seizures recorded with conventional and DC amplifiers, the depolarization of pyramidal neurons has been shown to cause a negative DC shift which gradually recedes and changes into a positive shift in the postictal period.[] In cats with penicillin-induced seizure foci, similar DC shifts were noted to be characterized by a horizontal dipole, having maximum amplitude at the focus with the field extending to about 10 mm.[] In the early studies, DC shifts were not seen on conventional EEG recordings obtained from the AC amplifiers, possibly because of the short time constant of 0.3 s (~0.53 Hz low frequency filter) used during that time,[] giving rise to the notion that dedicated DC amplifiers were needed to record such activity. However, Ikeda . refuted this by demonstrating that ictal DC shifts could be recorded in human scalp and intracranial recordings using conventional AC amplifiers with a 10 s (~0.016 Hz low frequency filter) time constant.[] More recent studies by other investigators further confirmed that DC shifts were indeed recordable using AC amplifiers if long time constants were used during acquisition, and appropriate filters and time scales were used for display.[] In line with these observations, more recent studies using dedicated DC-coupled amplifiers have also shown the occurrence of ictal DC shifts in both scalp and intracranial recordings in humans.[] To avoid confusion with the terminology when AC recordings are analyzed, the term IBSs was used to refer to the DC shifts.[] Besides using an AC amplifier with long time constant, successful recording of IBSs is facilitated by the use platinum electrodes (as opposed to gold or stainless steel), larger surface area of the electrodes (e.g., subdural as opposed to depth electrodes) and higher input impedance of the amplifier (usually >50 M Ω).[] shows IBSs at the onset of a focal seizure in the inferomesial temporal subdural contacts with a low frequency filter of 0.016 Hz and a time scale of 30 s per page. Compared with IBSs, there is much less information in the literature regarding background ISA. The same factors that determine the recording of IBSs also influence the recording of ISA. There is no accepted definition for ISA, but investigators have generally analyzed it visually using bandpass filters of 0.01-0.1 Hz or 0.02-0.2 Hz and longer time scales of 10 or 20 min per page.[] The origin of ictal DC shifts appears to be multifactorial. The proposed mechanisms of its generation include neuronal activity, glial activity and blood-brain barrier alteration.[] It has been shown that sustained neuronal depolarization during a seizure causes an increase in extracellular potassium, which in turn causes glial depolarization that is conducted electrotonically within the glial network.[] This neuron-glia interaction results in spatial buffering of potassium and manifests as a monophasic negative DC shift in the deep recordings and as a negative or positive shift in the superficial recordings during generalized tonic-clonic seizures in animal models.[] xref italic #text xref #text

India is the second largest populous country in the world with a current population of 1.2 billion. With a prevalence rate of five per 1,000 person-years and an incidence rate of 50 per 100,000 person-years, it is estimated that at any given time, India has at least 5 million people with active epilepsy, to which nearly 500,000 people are added annually. Considering that one-third of these patients have drug-resistant epilepsy, there are approximately one million people with epilepsy in India at any time, who are potential candidates for presurgical evaluation and epilepsy surgery. As against these large numbers of patients, currently less than 500 epilepsy surgeries are undertaken in India annually, resulting in an enormous surgical treatment gap. This also results in huge health care burden, as 80% of healthcare costs in epilepsy are accounted for by the patients with drug-resistant epilepsy. Evaluation of the patients with drug-resistant epilepsy requires a multidisciplinary approach within the framework of an organized comprehensive epilepsy surgery program. Currently there are less than five fully functional comprehensive epilepsy care programs in the country which can cater to a limited number of patients resulting in long waiting lists for the presurgical evaluation and epilepsy surgery in these centers. At the same time, only handful of patients have willingness and resources to travel to these centers which are located far off from their places of living, further discouraging many physicians and patients to approach these centers. The only solution to overcome this problem of large surgical treatment gap and to reduce the waiting lists is to establish comprehensive epilepsy care centers across the country that are capable of evaluating and selecting the patients for epilepsy surgery with the locally available technology and in a cost-effective manner. Such centers do not require elaborate and expensive technologies as majority of patients with well-defined surgically remediable epilepsy syndromes, which forms a large group of patients with drug resistant epilepsy, can be selected noninvasively without the need of additional investigations. However, there is a lack of trained professionals to initiate and maintain the epilepsy surgery programs in India. Even the trained epileptologists face problems in initiating an epilepsy surgery program due to the lack of trained professionals to form a multidisciplinary epilepsy surgery team. Proper guidance and support in the early stages of initiating an epilepsy surgery program can help in overcoming early hurdles in establishing successful epilepsy surgery programs which can subsequently become self-sustained. t i o n a l E p i l e p s y S u r g e r y S u p p o r t A c t i v i t y ( N E S S A ) i s a i m e d a t h e l p i n g t h e t r a i n e d n e u r o l o g i s t s a n d n e u r o s u r g e o n s i n i n i t i a t i n g a n d e s t a b l i s h i n g e p i l e p s y s u r g e r y p r o g r a m s w h i c h s u b s e q u e n t l y c a n b e s u s t a i n e d l o c a l l y . I t w i l l b e m o s t h e l p f u l f o r t h e p r o f e s s i o n a l s w h o h a v e b a s i c s h o r t - t e r m t r a i n i n g i n e p i l e p s y a n d t h e b a s i c i n f r a s t r u c t u r e f o r p r e s u r g i c a l e v a l u a t i o n w h i c h i n c l u d e s a f a c i l i t y f o r l o n g - t e r m v i d e o e l e c t r o e n c e p h a l o g r a p h y ( V E E G ) a n d a n a c c e s s t o g o o d q u a l i t y m a g n e t i c r e s o n a n c e i m a g i n g ( M R I ) . T h e h e l p w i l l b e t a i l o r m a d e a s p e r t h e l o c a l r e q u i r e m e n t s a n d m a y t a k e v a r i o u s f o r m s l i k e h e l p i n g i n e s t a b l i s h i n g e p i l e p s y m o n i t o r i n g u n i t s , s e l e c t i n g p a t i e n t s f o r e p i l e p s y s u r g e r y a n d p r o v i d i n g e x p e r t s u r g i c a l h e l p i n t h e i n i t i a l s t a g e s . T h e N E S S A t e a m a i m s t o e n c o u r a g e t h e c e n t e r s t o i n i t i a l l y o p e r a t e o n p a t i e n t s w h o c a n b e s e l e c t e d w i t h m i n i m u m i n v e s t i g a t i o n s a n d w h o a r e l i k e l y t o h a v e g o o d o u t c o m e . O n c e t h e p r o g r a m i s w e l l - e s t a b l i s h e d , t h e n N E S S A t e a m w i l l h e l p t h e c e n t e r s t o s e l e c t m o r e d i f f i c u l t c a s e s . N E S S A a l s o a i m s t o e s t a b l i s h a N a t i o n a l E p i l e p s y N e t w o r k o f e p i l e p s y p r o f e s s i o n a l s f o r t h e m a n a g e m e n t o f m o s t d i f f i c u l t c a s e s o f e p i l e p s y i n I n d i a w h e r e m o s t d i f f i c u l t c a s e s c a n b e d i s c u s s e d a n d m a n a g e m e n t p l a n s c a n b e f o r m u l a t e d i n c l o s e g r o u p m e e t i n g s . As per the local requirements, members of the NESSA team will visit the potential centers to help them in establishing epilepsy surgery programs. The different phases of such visits will be:

xref #text Several common questions are asked in deciding where and how to use intracranial electrodes []. The most common question, which is raised when there is a lesion (acquired, neoplastic or developmental) is, “Where do the seizures begin in relation to the lesion?” Answering the question will confirm that the lesion or perilesional cortex is the focus (not always the case) and determine the seizure onset zone in relation to that lesion, as the lesion itself can be electrically silent. Because the onset zone may be in or near essential cortex such as speech or motor function, the second question is, “Will the planned surgery have significant impact on the person's overall function and independence?” In this case, functional mapping may be needed to define safe boundaries around the seizure onset zone for a planned resection. The third question asked when the seizure focus is less clearly defined-because of either poorly localized EEG ictal changes on scalp recordings or there are several potential foci. In this case, the question is “Can I identify a candidate region where the seizures are arising?” This third question is often highly speculative and carries the risk that a seizure onset zone will not be identified. It is clear that direct recording and stimulation has opened up the possibility of surgery for many patients especially for the nontemporal lobe patients. Unfortunately, for these people, the chance for seizure freedom following surgery remains less than that for the temporal lobe epilepsies with identified structural abnormalities. One of the key goals for moving the field forward is to use these electrodes more effectively in defining the seizure onset zone, but unfortunately overall seizure freedom rates have not improved significantly and the reason remains unclear. Intracranial electrodes are expensive and a separate surgical procedure to implant the electrodes is needed. This method of localization, uses up significant medical resources, so that the potential benefits have to be weighed against the risks and costs. In many healthcare systems, the costs associated with intracranial monitoring, may be prohibitive or consume resources that could be used to make surgery available to more patients, if the evaluation were entirely noninvasive. s u m i n g t h a t t h e o d d s f o r s u c c e s s f u l s u r g e r y a n d i m p r o v e d q u a l i t y o f l i f e , f o l l o w i n g t h e u s e o f i n t r a c r a n i a l e l e c t r o d e s a r e g o o d , t h e m a j o r i s s u e s i n p r o c e e d i n g w i t h i n t r a c r a n i a l m o n i t o r i n g i s w h e r e t o p l a c e t h e e l e c t r o d e s a n d w h i c h t y p e t o u s e . I t i s n o t p o s s i b l e t o r e c o r d f r o m a l l o f t h e b r a i n , s o i t i s e s s e n t i a l t h a t c l e a r h y p o t h e s e s b e d e v e l o p e d r e g a r d i n g p o t e n t i a l s e i z u r e o n s e t r e g i o n s , t o s e l e c t t h e m o s t l i k e l y a r e a s . T h e s e h y p o t h e s e s c a n i n c l u d e d a t a f r o m i c t a l a n d i n t e r i c t a l E E G , m u l t i m o d a l i t y s t r u c t u r a l a n d f u n c t i o n a l i m a g i n g , a n d s e i z u r e s y m p t o m s a n d n e u r o p s y c h o l o g i c a l p r o f i l e . A s n o o n e t e s t i s s u f f i c i e n t t o d e f i n e t h e f o c u s , i t i s i m p o r t a n t t o w e i g h a l l f a c t o r s i n d e v e l o p i n g t h e h y p o t h e s e s t h a t w i l l g u i d e t h e p l a c e m e n t . I n c l u d e d i n t h e c o n s i d e r a t i o n i s w h e t h e r m a p p i n g o f t h e c o r t e x t o i d e n t i f y r e g i o n s t h a t s h o u l d n o t b e r e m o v e d b e c a u s e o f t h e i r f u n c t i o n . T h e c h o i c e o f e l e c t r o d e s w i l l t h e n b e d r i v e n b y l i k e l y o n s e t z o n e s , t h e n e e d f o r f u n c t i o n a l m a p p i n g , a n d t h e p r e f e r e n c e s o f t h e s u r g e o n . There are two basic types of electrodes for intracranial recording subdural and intracerebral []. Each type has multiple different configurations (number and spacing of electrodes), but there is a clear division of function and method of placement that separates the two. Subdural electrodes are intended to record from the cortical surface and provide a broad area of coverage as possible, whereas intracerebral (also known as depth) electrodes are designed to record from areas that are beneath the cortical surface (e.g. deep dysplasias) or from regions that are less accessible to strips (e.g. the mesial temporal/limbic structures). Depth electrodes provide much less cortical coverage than the subdural variety. There is an overlap in how these electrodes are used, which is related to the technical issues of recording from a specific patient and the surgeon's comfort in placing an electrode in a given area. Subdural strips are frequently placed through a burr hole, unless a grid is being placed, in which case, an appropriately sized craniotomy is required to place the electrode array precisely over the cortex under direct visualization. In some cases, it is not possible or is inadvisable to lift the dura for electrode placement. In these cases, epidural placement is possible with little degradation of recording quality. However, cortical stimulation and mapping is not possible with epidural electrodes because the electrical stimulation will activate the pain fibers in the dura. Intracerebral (or depth) electrodes are most commonly placed through small holes using stereotactic frame that is attached to the skull. On some occasions, they are placed freehand into deeper structures to be used simultaneously with subdural grids. In this situation, the depth electrodes are placed following a craniotomy over the target region and the insertion point, and angle and depth of insertion are under direct visual control. Originally, the electrode strands were rigid and had to be fixed in place to avoid damage to the brain that would result from an unintended movement laterally. Today the electrodes are predominantly limp strands that are aided in insertion by an internal removable wire or an external slotted needle. There is considerable effort and technical infrastructure involved in determining the coordinates for electrode insertion and care must be taken to ensure that major vessels such as the middle cerebral artery are not in the electrode trajectory. It is a common observation that the best success in seizure control is achieved when there is an identified lesion and seizures arise from within or adjacent to the lesion. For this reason, it is important to know precisely where the electrodes are located; so placement must always be confirmed with a postoperative scan, ideally an MRI, so that the relation of the electrodes to the desired targets and any known lesions can be confirmed. In addition, it is important to rule out small hemorrhages that can sometimes cause seizures that have nothing to do with the real seizure focus and could lead to a false localization. Although, the grids can be placed precisely under visual control, the strips, which are inserted through the burr holes are not placed under direct visualization, and may not always end up where desired. Because the strip electrodes are generally intended to include or exclude an area from early seizure involvement, exact placement is less of an issue. With depth electrodes, it is a common experience that slight shifts in the angles of the trajectory can result in the electrode's being off the target by a few millimeters that can significantly change the interpretation of the recordings. With grid electrodes, it is especially important to know the relationship of suspected lesions to the ictal onset, as the grid itself with the known location of the lesion becomes the guide to the resection. That is, the area of resection is defined by specific electrodes and once the grid is removed, the seizure onset zone can be lost. Without knowing the position of the electrodes in relation to key structures (e.g. hippocampus) or lesions suspected of playing a role in the initiation of the seizures, it is very difficult to determine the significance of the EEG, especially if the site of onset is not clear. In interpreting the seizure onset on intracranial monitoring for potential resections, there is one rule that must always be kept in mind: For an area to be considered as a candidate for the seizure focus, electrographic seizure onset must precede clinical onset. Although, resecting an area in which the seizure onset on EEG precedes clinical onset does not guarantee a successful surgery, resecting an area in which EEG onset follows clinical onset will almost certainly result in a poor outcome. Video EEG correlation is essential in interpreting the recordings. One of the great benefits of intracranial electrodes is their precise spatial resolution of interictal and ictal activity. It is a common observation that epileptiform activity can be quite prominent at one electrode and be completely absent at adjacent electrodes. For this reason, the absence of epileptiform activity at electrodes that are near but not in a likely site of seizure initiation, does not exclude that site as a candidate of onset. The issue is that the electrical fields that are recorded from the electrodes are limited to what is essentially in direct contact with the conductor. Activity that is more than a few millimeters away is invisible to that electrode. As illustrated in , from a patient with a malformation shows the seizure onset is limited to a pair of electrodes in the depth of the malformation. Recording sites on the surface showed no evidence of involvement. This case illustrates how focal seizure onset can be and how recordings at the cortical surface can miss seizure onset just below. Of further interest, the seizure first spread to the ipsilateral mesial temporal structures, and not the overlying cortex. This case also emphasizes the importance of knowing exactly where the electrodes are positioned. In recent years, there has been a growing interest in the potential of therapy that is delivered directly to the seizure focus, either with electrical stimulation or with infusion of a neuroactive compound which would suppress the development of seizures.[] There have been several clinical trials of focal electrical stimulation that have shown marginal benefit, even though some preclinical studies had shown promise.[] There have been a number of preclinical reports about the potential benefits of various focal drug infusions or recently,[] focal transfection with a light-activated protein,[] but no clinical trials have been reported with this approach. Animal stimulation studies have also had quite variable results.[] Although, there is great belief and hope for the potential benefits of direct and focal therapy, the results have been underwhelming. However, this lack of major efficacy should not be an indication that the approach is ill considered. Rather, the failures to date may result from the lack of understanding the circuits of epilepsy and its manipulation. This problem is compounded by the many types and locations of epilepsy, each of which likely has a unique pathophysiology and circuitry. It is very likely that part of the problem arises from a “one size fits all” approach, such that either the right treatment is given to the wrong region or the wrong treatment is given to the right region. Moving this type of treatment forward requires that we have a greater understanding of seizure circuitry and the ways of altering the physiology of any particular circuit.[] Thalamic involvement in seizures has been recognized for many years, first for spike and wave seizures and recently, for focal seizures. The role of the thalamus is not clear, but some studies suggest that it is part of a divergent-convergent excitation amplification circuit that prolongs the duration of excitatory drive on target structures.[] There is preclinical evidence that intervention in the thalamus, either by infusion or stimulation, can suppress seizures.[] However, it is also clear that the site specificity for either stimulation or infusion may be highly restricted so that any slight misplacement of electrodes or cannulas may result in a therapeutic failure.[] This issue is illustrated well in , in which, placement shifts of one millimeter can bring about very different results.[] This potential need for precise placement of electrode may be a reason for the modest benefit observed from thalamic stimulation in clinical trials. These observations suggest that a better understanding of the cortical-subcortical circuits is needed to guide therapy trials in the future, while we use intracranial electrodes to define seizure onset zones. It will be necessary to use that opportunity to define the circuits that may be targets for future therapies. The preclinical data are clear that it is necessary to alter the physiology of the circuits to have a therapeutic effect and it is possible to measure that effect.[] One of the problems with the clinical trials is that there is no evidence that the interventions do anything to the system. In designing future approaches to epilepsy treatment with intracerebral therapy, it may not only be necessary to have a more complete picture of the key seizure circuits and their modulations, but also demonstrate that the intervention also changes the physiology. Such a measure might be able to predict seizure suppression from the outset and if the physiology did not change, the measure would allow for an adjustment in the placement of the therapeutic device. Looking into the future, neurophysiology may allow us to identify the many components of the seizure circuits, to determine their roles in the seizure, and to test how our interventions affect circuit function and ideally lead to improved therapies. t e r a l m o s t 8 0 y e a r s o f m o d e r n e p i l e p s y s u r g e r y , e l e c t r o p h y s i o l o g y r e m a i n s a c e n t r a l c o m p o n e n t f o r t h e i d e n t i f i c a t i o n o f t h e s e i z u r e o n s e t z o n e . H o w e l e c t r o p h y s i o l o g y i s u s e d h a s c o n t i n u e d t o e v o l v e , b u t w h a t h a s r e m a i n e d u n c h a n g e d i s t h e n e e d t o h a v e h y p o t h e s e s a b o u t t h e f o c u s , s o t h a t t h e r e s u l t s o f t h e E E G c a n b e a p p r o p r i a t e l y i n t e r p r e t e d . I n t r a c r a n i a l E E G h a s c l e a r l i m i t a t i o n s w i t h r e g a r d t o t h e i d e n t i f i c a t i o n o f t h e t h r e e - d i m e n s i o n a l ( d e e p a n d s u r f a c e ) c o m p o n e n t s a n d t h e s e l i m i t a t i o n s m u s t a l w a y s b e k e p t i n m i n d . T h e t r e a t m e n t o f i n t r a c t a b l e e p i l e p s y i s a l s o e v o l v i n g , s o t h a t f o c a l d e l i v e r y o f d i f f e r e n t t y p e s o f i n t e r v e n t i o n ( e l e c t r i c a l o r p h a r m a c o l o g i c a l ) , a r e n o t a t a t o o d i s t a n t f u t u r e . P h y s i o l o g y w i l l b e e s s e n t i a l i n d e f i n i n g t h e t a r g e t s f o r t h e s e i n t e r v e n t i o n s , b u t a l s o f o r p r e d i c t i n g t h e s u c c e s s f r o m t r e a t m e n t a t o n e o f t h e n o d e s o f t h e c i r c u i t .

xref #text xref #text xref #text Lesionectomy for epilepsy is a surgical procedure that is directed at the structural lesion, believed to be the etiology of the seizure disorder.[] In the case of isolated structural lesions, such as dysembryoplastic neuro-epithelial tumors, low-grade astrocytomas, or focal vascular abnormalities, total macroscopic and radiologic evidence of lesion excision is associated with excellent seizure-free outcome. The surgical approach will often depend on the preoperative and intraoperative identification of the particular structural substrate. The extent of the resection may be determined by different criteria: xref #text Hemispherectomy or hemispherotomy is performed successfully to treat medically intractable hemispheric epilepsy in adolescents and older children, providing remarkable results in terms of seizure outcome and quality of life.[] Three different surgical techniques are performed according to the specific etiology and extension of anatomic abnormalities: N o n - l e s i o n a l f o c a l e p i l e p s y s u r g e r y u s u a l l y r e q u i r e s e x t e n s i v e i n v a s i v e p r e s u r g i c a l e v a l u a t i o n , w h i c h d i f f e r s f r o m p a t i e n t t o p a t i e n t a c c o r d i n g t o t h e n o n - i n v a s i v e f i n d i n g s . T h e o b j e c t i v e o f p r e o p e r a t i v e i n v e s t i g a t i o n s i n t h i s c o n t e x t i s t o i d e n t i f y a n d d e m a r c a t e t h e e p i l e p t o g e n i c z o n e a s a c c u r a t e l y a s p o s s i b l e . N o n i n v a s i v e m o d a l i t i e s l i k e V E E G , P E T , S P E C T a n d M E G w i l l b e a b l e t o p r o v i d e l o c a l i z a t i o n i n s o m e c a s e s . F u r t h e r , i n v a s i v e E E G w i t h g r i d - , s t r i p - , a n d d e p t h - e l e c t r o d e s , o f t e n i n c o m b i n a t i o n , w i l l b e n e c e s s a r y , t o d e l i n e a t e t h e e p i l e p t o g e n i c s e i z u r e o n s e t z o n e . #text Multiple subpial transections are intended particularly to be used in those cases where the epileptogenic lesion lies in “unresectable” cortex, i.e. those cerebral regions sub serving speech, memory, and primary motor and sensory function.[] The procedure is based on experimental evidence indicating The technique requires severing of tangential intracortical fibers while preserving the vertical fiber connections of both incoming and outgoing nerve pathways and of the penetrating blood vessels that also have a vertical orientation. MST may be considered when seizures originate from within eloquent cortex and more specifically in the Landau-Kleffner syndrome (epileptic aphasia) performed over the Wernicke area and deep into the Sylvain fissure. Specifically MST, alone with regard to seizure origin, appears to lead to an approximate 30% improvement in seizures, increasing to 60% when it is performed in conjunction with resection. Overall, MST should be considered where seizure onset is demonstrated to be within eloquent cortex, either alone or preferably in combination with resection. It remains the surgical procedure of choice in the Landau–Kleffner syndrome, although no doubt exists that specialist evaluation is required in a center used to evaluating such patients, in view of the detailed neurophysiology required. Its role in multifocal epilepsy with autistic regression is yet to be proven. a m e - b a s e d s t e r e o t a c t i c t e c h n i q u e s h a v e b e e n e x t e n s i v e l y u s e d i n I n d i a f o r b o t h t e m p o r a l l o b e e p i l e p s y a n d g e n e r a l i z e d s e i z u r e s . H o w e v e r , s i n c e t h e i n t r o d u c t i o n o f i m a g e g u i d a n c e a n d n e u r o n a v i g a t i o n , t h e s e t e c h n i q u e s a r e b e i n g u s e d c u r r e n t l y f o r t h e e l e c t r o d e i m p l a n t a t i o n o f b o t h d e p t h a n d s u b d u r a l e l e c t r o d e s , a s w e l l a s t o l o c a l i z e l e s i o n s a n d f a c i l i t a t e a n a t o m i c a l r e s e c t i o n s . #text

E x t r a t e m p o r a l l o b e e p i l e p s i e s ( E T L E ) a r e c h a r a c t e r i z e d b y t h e e p i l e p t o g e n i c f o c i o u t s i d e t h e t e m p o r a l l o b e . T h e y h a v e a w i d e s p e c t r u m o f s e m i o l o g i c a l p r e s e n t a t i o n d e p e n d i n g o n t h e s i t e o f o r i g i n . T h e y c a n a r i s e f r o m f r o n t a l , p a r i e t a l , o c c i p i t a l l o b e s . W e d i s c u s s i n t h i s r e v i e w t h e s e m i o l o g y o f d i f f e r e n t t y p e s o f E T L E e n c o u n t e r e d i n e p i l e p s y m o n i t o r i n g u n i t . #text Auras are relatively less often reported in patients with FLE. Palmini and Gloor examined experiential phenomenon in seizure patients and found that auras were found in 93% of patients with temporal lobe onset versus only 61% with frontal lobe onset.[] Non-specific auras have been reported in patients with seizure foci in the frontal lobe. Patients usually report non-specific, unexplainable feeling, often localized to the head (“cephalic aura”).[] Asymmetric tonic seizures originating in the supplementary motor area are often preceded by a somatosensory aura. Autonomic auras like nausea or palpitations as well as emotional auras such as fear are also reported from seizure arising from the frontal lobe. The duration of seizures are significantly shorter in the frontal lobe seizure compared those originating from temporal lobes and usually last less than 1 min. Secondary generalization occurs in 70% of patients with FLE. Nocturnal preponderance and a significant association with sleep-wake cycle have been reported[] Consciousness is usually preserved in seizures originating from the frontal lobe. Even if consciousness is impaired, it is for a very brief period. If seizures of frontal lobe onset spread to the temporal lobes, postictal confusion, as typical for temporal lobe seizures, can also be observed. Clustering of seizures is also frequent in FLE. Convulsive and non-convulsive status epilepticus are frequently observed in FLE. Clonic activity and asymmetric tonic posturing are typical of frontal seizures and of all the possible frontal seizure symptoms and signs, motor manifestations remain the most frequent and important, observed in 90% of patients.[] Complex motor automatisms, including bicycling and thrashing movements, sexual automatisms, laughter and vocalizations, may result in misdiagnosis as psychogenic seizures and when present early in the attack, seem to be particularly characteristic of certain types of FLE.[] Kotagal . compared the behavior in patients with mesial temporal lobe epilepsy to patients with FLE and found that repetitive, proximal upper extremity movements, complete loss of consciousness, complex motor and hypermotor (large repetitive movements of the proximal musculature, i.e., bicycling movements) activity were more common in the frontal lobe seizures.[] FLE can be further subdivided into: D o r s o l a t e r a l f r o n t a l l o b e c a n b e f u r t h e r d i v i d e d i n t o c e n t r a l l o b e , p r e f r o n t a l a n d p r e m o t o r c o r t e x . The mesial surface area can be further divided into primary motor cortex for the lower limb, the supplementary sensorimotor area (SSMA), the anterior cingulate cortex and the prefrontal cortex. Of these, the anterior cingulate cortex and the SSMA have been most extensively studied in terms of focal epilepsy. The seizures arising from the SSMA are generally frequent, occurs in clusters and are more common during the non-rapid eye movement Stage I and II of sleep. These seizures typically present with abrupt onset with bilateral, asymmetric, tonic posturing of the extremities lasting 10-30 s along with facial grimacing, with vocalization or speech arrest and absence of postictal confusion.[] The tonic posturing may be unilateral with involvement of a single limb but predominantly involves the proximal and axial musculature. Penfield and Jasper described “the arm being raised and the head and eyes turned as though to look at hand” which is called the “fencing posture.”[] Ajmone-Marsan and Ralston created the term “M2e” to describe tonic abduction and external rotation of the shoulder with flexion of the elbow.[] Before tonic posturing, a somatosensory aura consisting of “tingling” or a feeling of tension, pulling or heaviness in a limb or the impression of impending movement of the limb may precede the tonic seizure. The sensation may be relatively focal, involving a portion of a limb, lateralized with both upper and lower limbs involved simultaneously and a poorly defined bilateral sensation in the head. Postictal manifestations are absent or mild and short-lived. Their shorter duration, stereotypical nature, occurrence in sleep, and the presence of a tonic contraction of the abducted upper extremities distinguish SSMA seizures from non-epileptic attacks. These postures are often accompanied by vocalizations and preserved awareness. Other semiologies may also be associated with mesial frontal lobe onset, including: hypermotor seizures, dialeptic seizures, focal clonic seizures of the lower limb and negative myoclonus. Absence type seizures are characterized by altered consciousness and minimal motor manifestations. The patient loses contact with his environment. He may still respond to his surroundings to some degree but in a slow and inappropriate way. These seizures can be brief and distinct, but they may also last hours or even days. These episodes, when prolonged, can present like a type of non-convulsive status epilepticus. Although this condition has also been called “spike-wave stupor,” this term is somewhat misleading because of the great variability in the level of altered consciousness from a barely perceptible change to bland unresponsiveness. For example, during prolonged episodes of this type of non-convulsive status, patients may seem normal but complain of being mildly confused. They may also be partially or completely amnestic for events occurring during the episode despite appearing alert. The ictal EEG pattern consists of generalized disorganized spike-wave activity, a pattern similar to that described for absence status in adults. It is, therefore, important to differentiate this type of frontal lobes seizure from idiopathic generalized absence seizures, as they may be clinically and electrographically similar. Hyperkinetic seizures usually begin abruptly, almost explosively, with the onset of complex behavioral automatisms. Patients may jump around, thrash, rock to-and-fro, pound on objects or rock back and forth. Bicycling movements or stepping movements are frequently described. Along with the motor manifestations there is extensive vocalization with screaming, yelling, shouting, humming, grunting, laughing, or barking. The patient may swear and produce understandable speech. The prominent motor features distinguish them from the automatisms seen in temporal lobe seizures and have been called “hypermotor automatisms.” Hyperkinetic seizures further can be divided into two types.[] The first, manifests with marked agitation with body rocking, kicking, or boxing and associated with a facial expression of fear and is associated with ictal foci in the ventromesial frontal cortex, The second variety manifests with mild agitation with either horizontal movements or rotation of the trunk and pelvis and usually accompanied by tonic or dystonic limb posturing was seen primarily in seizures arising from the mesial premotor cortex, mesial intermediate frontal cortex and dorsal anterior cingulate cortex. Hyper kinetic seizures have been reported to be also associated with the origin in the orbitofrontal or anterior cingulate areas, frontopolar and dorsal frontal origin.[] Cingulate gyrus epilepsy frequently present with complex stereotypic movements, in particular thrashing, kicking, grasping and running with or without vocalization. Behavioral disturbances such as motor or verbal aggression, paranoid delusions and personality changes including autistic, obsessive-compulsive and self-mutilating behavior have been described. Fear with or without a matching facial expression and laughter without mirth may be an early manifestation in mesial frontal lobe seizures. Occipital lobe epilepsy (OLE) appears to account for 2-13% of extratemporal epilepsies.[] Ictal clinical symptoms of the occipital lobe seizures are subjective, objective, or both.[] The signs and symptoms of OLE can be divided into. #text xref #text Parietal lobe epilepsy (PLE) accounts for a very small percentage of extratemporal epilepsies. Clinical semiology is difficult to study as much of the parietal lobe is silent with respect to clinical seizure manifestations. There is also dearth of literature because less frequent involvement of this lobe. In addition, some aspects of seizure-induced parietal lobe dysfunction might not be apparent without specific testing like testing of various cortical somatosensory functions. These seizures emanating from the parietal lobe are mainly simple focal events that manifest with subjective symptoms, such as somatosensory, somatic illusions, vertiginous, visual illusions or complex visual hallucinations, receptive or conductive linguistic disturbances, in the order of prevalence. These clinical seizure manifestations are usually related to the epileptogenic location, anterior or posterior, of the dominant or the non-dominant parietal lobe. Onset with simple sensorimotor manifestation is usually associated with anterior parietal lobe foci, whereas more complex symptomatology emanates from the posterior regions. t i e n t s w i t h P L E m o s t c o m m o n l y e x p e r i e n c e s o m a t o s e n s o r y a u r a s . T h e s e i n c l u d e v a r i o u s t y p e s o f p a r e s t h e t i c , d y s e s t h e t i c a n d p a i n f u l s e n s a t i o n s . #text xref #text The prototypical seizure type associated with hypothalamic hamartoma is the gelastic seizures. They are usually the first seizure type and occur very early in life. The gelastic seizures are usually very brief (duration < 20 s) and frequent (usually with multiple gelastic seizures per day). They mimic true laughter but more often are peculiar and mirthless even to the casual observer, and may incorporate behavioral elements of grimacing or crying (dacrystic seizures). They may or may not be associated with altered consciousness, which is often difficult to determine in infants. Other additional types of seizures develop in 80% of patients. Complex partial seizure, and other generalized seizure types can include tonic-clonic, tonic, atonic, or even absence can be seen along with the classic gelastic seizures. summarizes the different localizing and lateralizing signs seen in ETLE.

xref #text The search strategy spanning from a period of 1970s to 2012 was conducted using MEDLINE via PUBMED and the Cochrane databases using following search terms: ‘vertical root fracture’, ‘diagnosis of vertical root fracture’, ‘management of vertical root fracture’. A hand search was also carried out of the last two issues of the following major endodontic journals: Journal of Endodontics; International Endodontic Journal; Dental Traumatology; Australian Endodontic Journal. The process of cross referencing continued to get relevant articles. A total of seventy seven articles were selected for the study, which included forty three , and thirty four studies. The studies included case reports[] clinical surveys[] and review articles[] and a histological study.[] The titles and abstracts of all identified articles were screened to determine the relevance of each study with regards to the diagnosis, contributing factors and management of vertical root fracture. Full texts of selected articles were collected and analyzed. These articles were selected based on their evidence based information in establishing definite criteria for the diagnosis, role of predisposing etiological factors, based on studies with large sample and adequate follow up. Relevant information from each study, such as evaluation techniques, tooth involved, recall time and findings were summarized in Tables and . #text Vertical root fracture associated with root canal treated teeth is one of the most difficult problems to diagnose and treat. Early detection has two fold advantages - it prevents unnecessary frustration and inappropriate endodontic treatment, and prevents extensive damage to the supporting tissues. Diagnosis is usually confirmed through the clinical signs and radiographic features. But not all the typical signs of a fractured root may be present in each case. So, the combination of clinical signs, symptoms and radiographic features may provide a clue for the diagnosis of vertical root fracture. Also, presently CBCT has been shown to be promising in the early detection of vertical root fractures. A periapical radiograph can detect a fracture line only in 35.7% cases. Cone Beam Computed Tomography has been used successfully for the early detection of vertical root fracture in both endodontically and non-endodontically treated teeth in recent studies. It is more sensitive and accurate in the detection of vertical root fractures as compared to conventional[] and digital radiography.[] This has been confirmed by clinical studies.[] It has been shown that the presence root canal filling does not significantly influence the accuracy but reduces specificity[] in detection of vertical root fracture by CBCT. In an vitro study, CBCT has been used successfully for diagnosing simulated vertical root fractures of thicknesses 0.5 mm,1 mm,1.5 mm and 2 mm in extracted teeth. CBCT has been shown to be more accurate in vertical root fracture with thickness of 0 to 2 mm.[] Coming to the technical aspects, it has been shown that the accuracy varies among different CBCT systems. Also, axial scans are shown to be more accurate than sagittal and coronal slices.[] Multi-detector CT has been compared with CBCT and digital radiography in a recent study on extracted teeth. In non root filled teeth group, MDCT has been shown to be most accurate in diagnosing vertical root fracture, where as its specificity is similar to CBCT and digital radiography. In this study CBCT is found to be most sensitive among the three in detecting vertical root fracture.[] Early detection of vertical root fractures avoids unnecessary bone loss, which can result in difficulty in reconstructing a bone area, where implants are the treatment of choice in future. In such cases CBCT can be recommended when conventional radiography is negative. p

Pulpstones are a group of calcified masses in the dental pulp of healthy, diseased and unerupted teeth.[] Stones may exist either freely within the pulp tissue or be attached to or embedded in dentin.[] A single tooth may have 1-12 pulp stones or even more. The size may vary from a small microscopic particle to large masses that almost obliterate the pulp chamber.[] Two types of pulp stones have been described:[] Denticles possessing a central cavity filled with epithelial remnants surrounded peripherally by odontoblasts and pulp stones being compact degenerative masses of calcified tissues. They are reported to occur more often in the coronal region, but are also found in the radicular pulp. Pulp stones have been reported in patients with systemic or genetic diseases such as van der Woude syndrome.[] Despite several microscopic and histochemical studies, the exact cause of such pulp calcifications remains largely unknown. However, a number of conditions have been claimed to predispose to pulp stone formation such as pulp degeneration, inductive interactions between the epithelium and pulp tissue,[] age,[] caries, operative procedures, periodontal diseases, epithelial rests in the pulp tissue, orthodontic tooth movement,[] circulatory disturbances in pulp tissue,[] idiopathic factors[] and genetic predisposition.[] Recent literature still suggests that pulp stones are a feature of an irritated pulp, attempting to repair itself.[] The prevalence of pulp stones varies from 8% to 90%.[] Many prevalence studies have identified pulp stones using radiography. The true prevalence is likely to be higher because pulp stones with a diameter smaller than 200 μm cannot be seen on radiographs.[] Some researchers have investigated the prevalence of pulp stones or association of this condition with other dental conditions. However, very few studies have assessed the prevalence of pulp stones in Indian population. The aim of this study was to determine the occurrence of pulp stones using radiographs and to correlate their association with age, gender, systemic diseases such as hypertension, diabetes and gastritis; tooth type, jaw type, caries and restored teeth. A total of 2000 patients were selected randomly from the outpatient Department of Radio diagnosis of three different institutions belonging to three different regions of Andhra Pradesh for a period of 6 months. Patients below the age of 10 and above the age of 60 were excluded from the study. Among these, 2000 patients a total of 4449 teeth were examined. All the patients who had undergone a diagnostic radiograph of the premolar and molar region were considered for the study. Patients were not subjected for radiographic examination for the purpose of the study. Patient's medical history was recorded for diseases such as diabetes, hypertension and gastritis. Patients with more than one systemic disease and patients with other medical problems apart from diabetes, hypertension and gastritis were excluded from the study. Patient's dental status was thoroughly examined for DMF, which were recorded in the proforma. Diagnostic radiographs were taken using an analog IOPA unit under ICRP standards protecting the patient and the operator with constant exposure parameters (70 KVp, 6 mA and 0.6 s pulse). All the radiographs were processed manually using visual method in a dark room under safe light. The radiographs were evaluated for the presence or absence of pulp stones and were documented in the proforma. Statistical analysis of the data was done using the statistical package for the social sciences (SPSS 15.0) using Chi-square analysis. Differences were considered as significant when ≤ 0.05. The distribution of patients with pulp stones according to the age groups is shown in . The occurrence of pulp stones was most significant in among the age groups of 31-50 years of age. Of the 2000 patients a total of 518 patients had pulp stones which accounts to 25.9% prevalence. A total of 4449 teeth were examined of which 18% of teeth showed pulp stones. The distribution of teeth with pulp stones in comparison to the medical history is shown in . Patients with diabetes, hypertension and gastritis showed higher prevalence of pulp stones with a < 0.05, which was statistically significant. The distribution of teeth with pulp stones according to the location and tooth type is shown in . When individual tooth type is taken into consideration, maxillary 1 and 2 molars showed a strongly positive significance with 33% and 31% prevalence respectively to the overall prevalence of 18%. Regarding the tooth pathology, of the 4449 teeth examined, 1870 teeth had dental caries out of which 224 teeth showed pulp stones which accounts to 11.9% prevalence and 105 teeth were restored out of which 18 teeth showed 17.1% prevalence. The prevalence of pulp stones were statistically significant [] in teeth with dental caries and teeth without pathology. Detection of pulp stones using dental radiographs is only possible when the diameter is more than 200 μm.[] Most of the studies in the literature used paralleling technique to take the radiographs. al-Hadi Hamasha and Darwazeh[] in their study used periapical and bitewing radiographs, Çolak [] used bitewing radiographs, whereas Satheeshkumar [] and Syryn΄ska [] used panoramic radiographs in their study for detection of pulp stones. One study had proved that there is no significant difference in the technique to identify the pulp stones. In our study as we considered all the intraoral periapical radiographs taken in the out-patient department of Radiology, we used bisecting angle technique to take the radiographs. Al-Hadi Hamasha and Darwazeh[] examined patient records of 814 Jordanian adults and found that pulp stones were present on radiographs in 51% of the patients and 22% of the teeth studied. Baghdady VS [] assessed 515 iraqi subjects and recorded that 19% of the teeth had pulp stones. Ranjitkar [] examined the prevalence of pulp stones in Australian population and found in 46% of the subjects and 10% of the teeth examined. Tamse [] evaluated full mouth radiograph of 300 patients and reported that 21% had pulp stones. Gulsahi [] in their study found pulp stones in 12% of the subjects and 5% of the teeth examined. Nayak [] in a study on Indian population, examined 1432 teeth and found 9.35% prevalence of pulp stones. Sisman [] examined bitewing radiographs of 469 Turkish patients and found 57.6% prevalence of pulp stones in their patients and 15% prevalence among the teeth examined. In the present study, the prevalence of pulp stones in 26% of the subjects and 18% of the total teeth examined. Our results are almost in par with many of the studies in the literature. Some investigators have reported that pulp stones were more common in females than in males.[] Some showed that there is no significance. In our study, we found a slight higher prevalence in females than in males. According to Ranjithkar S et al[] pulp stones are reported to increase in frequency with age. The present study also confirmed this finding. Generalized pulp stones are found in the dentition of individuals with various conditions. These include tumoralcalcinosis, dentin dysplasia Type II, Elfin facies syndrome, familial expansile osteolysis,[] Elhers-Danlos syndrome Type I, osteogenesis imperfecta Type I and otodental syndrome. In forensic dentistry, a radiographic matching of pulp stone configurations, along with other features recorded in dental records, may provide valuable information in the identification of a deceased person.[] The correlation of pulp stones with cardiovascular diseases and other systemic diseases has been investigated. Edds [] found a significant relation between pulp stones and presence of arteriosclerosis and other cardiovascular diseases. High incidence of calcification in the dental pulp of patients with coronary atherosclerosis upon radiographic examination were reported.[] Theyalso found calcification and lumen narrowing within extirpated dental pulp vessels, in both medium and small precapillary arteriole of cardiovascular patients. Nayak [] in their study on Indian population found the prevalence of pulp stone in hypertensive patients was 15.85% higher than normal subjects. The present study also showed a higher prevalence of pulp stones in patients with hypertension, diabetes and gastritis when compared to the overall prevalence of pulp stones. Type II diabetic patients, in the present study, were detected with pulp stones higher than the overall prevalence of pulp stones. Russell had investigated human pulp histologically on non-carious extracted teeth of seven patients suffering from diabetes for a long term duration and control group of 13 non-diabetics.[] He concluded that calcification in angiopathies and thickened basement membrane were noted in both large and small blood vessels and vascular changes seemed more pronounced in the central area of the pulp. Calcifications in diabetics were frequent and often sickle-shaped. In another histopathological study, conducted by Bissada and Sharawy's on 21 human dental pulps of diabetics and 20 matched controls, no vascular changes groups were found in the dental pulp of both. However, amorphous calcified bodies in the pulp of diabetics were found. Dental pulp of patients who suffer from diabetes mellitus tend to age more readily because it of obliterative endarteritis and because it has limited or no collateral blood circulation in fully developed teeth.[] T h e o c c u r r e n c e o f p u l p s t o n e s i n o u r s t u d y w a s s i g n i f i c a n t l y h i g h e r i n t h e y o u n g e r a g e g r o u p p a t i e n t s a n d a l s o i n t h o s e g r o u p s o f p e o p l e w h o h a d u n d e r l y i n g s y s t e m i c p r o b l e m s .

The restoration of the endodontically treated tooth is an important aspect of successful endodontic therapy. There are wide ranges of treatment options of varying complexity. The clinician must be able to predict the probability of restoring such teeth successfully. In general, endodontically treated teeth experience significant coronal destruction as well as loss of radicular dentin, secondary to endodontic treatment. There is evidence that these teeth have reduced levels of proprioception, which could impair normal protective reflexes.[] A post and core retained crown may be indicated to fulfill these requirements[] Clinical longevity of the post and core restoration can be influenced by many factors including magnitude and direction of the occlusal load, design of dowel, thickness of remaining dentin, quality of cement layer and creation of ferrule effect to enhance structural durability of the final restoration.[] A ferrule is a metal ring or cap intended for strengthening. The word probably originates from Latin, combining the words “” or iron and “” for bracelets.[] A dental ferrule is an encircling band of cast metal around coronal surface tooth. Sorensen and Engelmann define the ferrule effect as a “360° metal collar of the crown surrounding the parallel walls of the dentin extending coronal to the shoulder of the preparation. The result is an elevation in resistance form of the crown from the extension of dentinal tooth structure.” A protective or “ferrule effect” occurs owing to the ferrule resisting stresses such as functional lever forces, the wedging effect of tapered posts and the lateral forces exerted during the post-insertion[] The effectiveness of the ferrule has been evaluated by a variety of methods including fracture testing,[] impact testing,[] fatigue testing[] and photo elastic analysis.[] Majority of studies regarding the effectiveness of a ferrule support the need for at least 1.5 mm as ferrule height.[] This recommendation requires that the ferrule encompass the entire circumference of tooth. Methods of testing could include static loading to stimulate forceful clench.[] The purpose of this study is to investigate the effectiveness of uniform 2 mm and 1 mm ferrule height configurations of fracture resistance of endodontically treated teeth when subjected to static load. A total of 40 freshly extracted maxillary central incisors were collected from the Department of Oral and Maxillofacial Surgery. The selection of teeth was carried out on the basis coronal height was limited to 9-11 mm and root length was limited to 12-14 mm were allocated into four groups of each having 10 teeth. Inclusion criterion included teeth extracted for orthodontic needs or periodontally compromised, non-carious teeth, intact teeth, teeth with single canals. Exclusion criterion included carious teeth, tooth cracks/fractures, restored teeth, primary teeth and teeth with developmental anomalies. The selected teeth were stored at room temperature in distilled water until testing. Group 1: Teeth restored with crowns (CRN), Group 2: Endodontically treated teeth restored with crowns (RCT and CRN), Group 3: Endodontically treated teeth restored with cast dowel cores and crowns incorporating uniform 2 mm ferrule (2 FRL), Group 4: Endodontically treated teeth restored with cast dowel cores and crowns incorporating uniform 1 mm ferrule (1 FRL). The specimens were mounted in acrylic resin blocks, with the long axis of the block, midfacial extent of each tooth parallel to the long axis of the block and the mid facial extent of cemento-enamel junction located 2 mm coronal to acrylic resin. Specimens of Groups 2, 3 and 4 were instrumented to a working length using ProTaper file system (Dentsply Maillefer) following conventional principles of the crown down technique until ProTaper file F5. At each change of file size, canals were irrigated with 3% NaOCl (Vishal Dento Care Pvt. Limited) and root canal conditioning agents ethylenediaminetetraacetic acid (Glyde-Dentsply Maillefer). After chemomechanical preparation, the canals were dried with absorbent paper points (Dentsply Maillefer, Ballaigues) and obturated by lateral condensation technique with F5 ProTaper gutta-percha cones (Dentsply Maillefer, Ballaigues) and AH Plus Sealer (Dentsply-Maillefer, Switzerland). In Groups 3 and 4, Post-space preparation began with the removal of gutta-percha using heated plugger. Final gutta-percha removal was performed with drills (#3 Gates-Glidden; Mani, Japan) taking care to preserve 5 mm apical gutta-percha followed by Largo Peeso Reamer No. 3 (Dentsply Maillefer Ballaigues, Switzerland). The Largo Peeso Reamer was marked to a depth of 5 mm short of working length, with endoblock (Dentsply Maillefer) and stopper was fixed. Gutta-percha was removed from specimens leaving 5 mm of gutta-percha in the canal apex. Following the post-space preparation, the space was rinsed with 3% NaOCl (Vishal Dento Care Pvt. Limited, India). Final irrigation was accomplished with saline and post-space was dried using paper points (Dentsply Maillefer Ballaigues). In Group 3 and 4, 2 mm of incisal reduction and 1.5 mm of facial and lingual reduction and Ferrule height was measured and marked as 2 mm and 1 mm uniform circumferentially on the tooth surface with digital vernier calipers (Mitutoyo, Tokyo, Japan) for standardization of ferrule height. Further crown was reduced to leave 1 mm uniform ferrule with a high-speed diamond point under abundant air-water cooling. Post and core patterns were made from pattern wax (GC pattern wax) using the direct method to replace the coronal dentin that had been removed. Wax patterns of Groups 3 and 4 (to minimize batch variation) were invested in gypsum bonded investment material (Deguvest California USA) using a 1:1 liquid to powder ratio and allowed to set for 30 min. Then invested wax patterns were placed in pre-heated burn-out oven (Unident, India) at a temperature of 700°C and left for 45 min. The patterns were cast in non-precious gold alloy with the aid of a centrifugal induction casting machine (Unident, India). The castings were divested and cleaned. The post and cores were fitted to the respective teeth and subsequently cemented with glass ionomer cement (3M ESPE). A lingual ledge was added to create standard loading point. To summarize, a coping with the metal backing was prepared onto, which labial porcelain was veneered. Wax pattern from each group was invested and cast using the same protocol used with dowels and cores using Ni-Cr alloy. PFM crowns were fabricated and cemented with Resin modified glass ionomer cement (3M ESPE). Cemented crown samples were stored for 3 days in an environment with 100% humidity in a humidity chamber. Prepared specimens were mounted on a holder slot, which was fixed to the lower arm of the universal testing machine to present the lingual side at an angle of 45° to the metal indenter of 4 mm diameter. The indenter was fixed to the upper arm of the universal testing machine, which was set to deliver an increasing load until failure. Failure was defined as a 25% drop in the applied load. The cross head speed was 2.5 mm/min and the load was applied to lingual ledge at a 45° angle to the long axis of the tooth. The specimens were tested under random order (samples were pooled mixed and picked without visualization) and the operator was not informed of the group designation of the specimen being tested. Variable of interest was the load at failure measured in Newton. The statistical analysis applied was one way analysis of variance (ANOVA) to detect the presence of group differences and pair wise comparisons between groups with the (Tukey Kramer multiple comparison test) significant difference test for multiple comparisons. The mode of failure for each of the specimens was noted by visual inspection. Software used for statistical analysis is SPSS 16 version. #text The test specimens can be stored in various storage media such as, distilled water, ethanol and normal saline. Studies have shown that, normal saline and ethanol decrease dentinal permeability and affect fracture resistance with time.[] No significant difference was found with distilled water storage. Thus in the present study, maxillary central incisors were stored in distilled water. Maintenance of adequate obturation is critically resistance to bacterial microleakage. Although studies indicate that 4 mm of gutta-percha provides adequate seal, stopping precisely at 4 mm is difficult.[] Recent studies advocates leaving 5 mm of gutta-percha apically.[] In the present study, in Groups 3 and 4, 5 mm of gutta-percha was left in apical root end so that the sealed accessory canals and lateral canals remained untouched to prevent microleakage. Gates-Glidden drills were run at 800-1000 rpm with slow-speed handpiece to prepare post-space. Generating frictional heat that softens the gutta-percha and eases its removal without disturbing apical root filling.[] In the present study, Groups 3 and 4 gutta-percha was removed using Gates-Glidden drill followed by Largo Peeso Reamer No. 3 at 1000-1200 rpm and post-space was prepared according to manufacturer instructions. A 2 mm cervical ferrule is regarded as the key to restoration longevity because of its strengthening effect. Roots resist better multiple stresses, to which they are subjected when the tooth is in use, even to loads resulting from parafunctional habits. The role of the ferrule is similar to cast ring.[] In this study, 2 mm of incisal reduction, 1.5 mm of facial and lingual reduction was performed. Ferrule height was measured and marked uniformly as 2 mm and 1 mm circumferentially on the tooth surface with digital calipers for standardization of ferrule height further crown was reduced to leave 2 mm and 1 mm ferrule height for respective Groups 3 and 4 with a high-speed diamond point under abundant air-water cooling. Both zinc phosphate and glass ionomer have similar properties and commonly used because of their ease of use, coupled with their history of clinical success. In the present study, in Groups 3 and 4, glass ionomer cement was mixed to luting consistency and inserted in the canal using lentulo spirals. This may be an effective technique for reducing voids and bubbles within the luting agent. The posts were then seated into the canal by firm finger pressure and excess cement was removed along the peripheries. In the comparative analysis between cements, resin modified glass ionomer cement showed a pull force more than twice as that of zinc phosphate cement. In thicker layers, the better physical chemical properties of the resin modified glass ionomer cement become more evident.[] In this study, PFM crowns were fabricated and cemented with resin modified glass ionomer cement (3M ESPE). Tan stated after cementation of crowns with resin modified glass ionomer cement crowns should be stored in 100% humidity for 3 days,[] to simulate the humidity until returned for strength testing.[] Cemented crown samples were stored for 3 days in an environment with 100% humidity in a humidity chamber. In the present study, specimens were evaluated for their fracture resistance, using the universal testing machine. Static load was applied at the same angulation along the long axis of tooth in the study undertaken by Tan , Zhi-Yue and Yu-Xing.[] This angle is found to be optimal as this is the value of interincisal angle in most patients where the forces act more. The mode of failure for specimens was an oblique fracture extending from the lingual margin to the facial surface just below the tooth's insertion into the acrylic resin.[] In this present study, Groups 3 and 4 with ferrule and cast post and core, the mode of failure of specimens was an oblique fracture in middle third of roots inside the acrylic resin block. In root canal treated tooth with crown without post and core fracture was in cervical section of the root. Endodontically treated maxillary central incisors with a uniform 2 mm ferrule were more fracture resistant than those with a uniform 1 mm ferrule. Endodontically treated teeth restored with custom cast post core were as strong as endodontically treated teeth with crowns.

xref italic #text This study was conducted in the Department of Conservative Dentistry and Endodontics. Teeth sterilization (gamma irradiation at 25 kGy)[] was performed at Microtol, Bangalore. Data was obtained using an inverted confocal laser scanning microscope (CLSM) (ZEISS LSM 510 META. GmbH, Mannheim, Germany) at the Indian Institute of Science, Bangalore. A total of 60 human single-rooted teeth recently extracted for orthodontic reasons were collected for the study. After extraction, the teeth were stored in chlorhexidine solution. For adhesion, we used scoring criteria of 0 (for no adhesion) and 1 (for adhesion). For penetration measured in μm, we used the depth-measuring tool from the CLSM software. Accordingly, results were subjected to statistical analysis using Median test, ANOVA and Student's -test. shows live bacteria (green color) and dead bacteria (red color). shows depth of penetration of in root cementum. In group I (control group) no access cavity was prepared and at the same time apical one-third of the teeth were sealed with varnish. Results showed no adhesion [], [] and no penetration of into the root cementum in any of the samples [], []. In group II[] few samples showed adhesion, mean of adhesion is 0.55 [], [] but no penetration was seen in any of the samples [], []. This means that if an intervention like root canal treatment was done at early stage of infection when no apical changes like demineralization or resorption had taken place, there would be lesser chances of penetration and persistent infection or re-infection. In group III, apical one-third of the teeth were exposed to acid and apical cementum was roughed to mimic apical periodontitis. In this group, all the samples showed adhesion [], [] and highest values of penetration was up to 160 μm deep in root cementum [], []. This means that delay in treatment leads to changes in apical environment such as apical demineralization and apical resorption, which helps to penetrate deep into cementum and in favorable conditions chances of persistent infection or re-infection also increases. A comparison of values shows high significant difference ( < 0.01) between groups I and III and groups II and III and significant difference ( < 0.05) between groups I and II [Tables and ]. In this study, was chosen as the test organism because of its ability to penetrate the root dentin in vitro[] and it is found up to 90% in persistent infection.[] has unique properties such as production of ACE and SPR that are collagen binding proteins, which help to adhere strongly to mainly type I and IV collagen present in the root dentin.[] is a very virulent microorganism, which can survive in alkaline pH and during long starvation periods and then becomes viable in the presence of serum.[] can survive long periods without any nutrient availability because it can derive nutrients from hyaluronan, which is converted by enzyme hyaluronidase and also derive energy sources from dentinal fluid even in a well-sealed root canal system. Under stress, produces aggregation substance (AS), which helps in bacterial adhesion.[] Lipoteichoic acids protect against lethal conditions, cytolysin, AS-48 and bacteriocin, which inhibits other bacterial growth. Cytolysin destroys cells such as erythrocytes, PNM cells, macrophages and kills gram-positive microorganisms.[] Due to this effect there is a shift from gram-positive to gram-negative bacteria.[] Persistent can cause an inflammatory reaction and may start periradicular damage.[] The adhesion of to type I and IV collagen[] is the basis of the present study because apical one-third of the root is made of cellular cementum primarily composed of type IV collagen. Adhesion is the first step in colonization[] and our study confirms adhesion and invasion by up to 160 μm into the root cementum []. However, a previous study showed penetration only up to 150 μm deep into the root dentin.[] Deeper penetration of in our study is due to the change in apical environment such as demineralization and apical resorption. In group I, there was no invasion or adhesion of as it is a control group. In group II, only few samples showed adhesion, mean of adhesion is 0.55 [] and no samples showed penetration. On the other hand in group III, all samples showed adhesion [] and highest penetration into root cementum up to 160 μm []. This shows that if early treatment is done after primary infection as in group II, there are less chances of penetration of and re-infection or persistent infection. Whereas in group III, there was delay in treatment at early stages of infection, leading to change in apical environment like demineralization of root cementum and root cementum resorption. These apical changes helped to penetrate deep into the root cementum, thereby increasing the chances of persistent infection or re-infection. In our study, we cultured for 8 weeks twice because 18 weeks showed primary infection followed by biomechanical preparation and obturation giving the impression of normal root canal treatment done to subside primary infection. The next 8 weeks of culturing was done to show secondary infection. Group II showed only adhesion but no penetration [Figures and ], whereas group III showed deeper penetration. This means irrespective of the primary or secondary infection, what matters most is the apical changes in environment like demineralization or apical resorption, which help to penetrate deep into root cementum and is the cause for re-infection or persistent infection, as confirmed by the samples in group III. We used gamma irradiation to sterilize the teeth because it does not alter collagen characteristics of the teeth. produces collagen binding protein,[] with the help of which it adheres strongly to collagen. Other methods of sterilization of teeth samples are by autoclaving, using hot air oven, etc. The disadvantage of autoclave is that it collapses the collagen strands and use of hot air oven makes teeth dehydrated and more brittle.[] In our study, data was collected using a CLSM as it has advantages over other methods such as histological samples, which cannot distinguish between viable and dead bacteria. Scanning electron microscope has the disadvantage of multiple steps for sample preparation making it time consuming and also it cannot differentiate between dead and viable bacteria. Fluorescence probe has the disadvantage that it cannot distinguish between viable and dead bacteria and also it cannot show distribution of bacteria. The CLSM (ZEISS LSM 510 META GmbH, Mannheim, Germany) analysis has advantage over other methods to visualize bacteria.[] Our study confirms that CLSM can give a clear picture about viability and spatial distribution of bacteria. We used FDA (FDA, Sigma, St. Louis, MO) and PI (PI, Sigma) dyes. FDA is a non-fluorescent cell-permeable dye, which in viable cells crosses the cell membrane, gets metabolized by intracellular esterases and is converted to fluorescein (green). Viable cells appear green in color. PI is non-cell permeable fluorescent dye, which gets adhered to ruptured cell membranes and dead cells appear red in color.[] Our research confirms the ability of to infect the root cementum. We used an organic acid (lactic acid) for demineralization of root cementum because the end product of sucrose metabolism is lactic acid.[] italic #text

The origin of the fluorescence is not fully clear,[] though it seems unlikely that apatite is responsible for the basal values associated with healthy enamel.[] The explanation may be the result of the combination of the inorganic matrix with the absorption of organic molecules.[] Carious lesions fluoresce more strongly than healthy tissues at an excitation wavelength in the red and infrared part of the spectrum.[] This increase in observed fluorescence is related to two processes: Demineralization of the tooth and bacteria with their metabolic products (porphyrins).[] Most of the fluorescence is induced by the organic components,[] rather than by crystal disintegration and transmission through scantly homogeneous enamel.[] This hypothesis is based on the fact that laser fluorescence (LF) does not detect lesions caused in the laboratory with acids instead of bacterial activity.[] Bacteria responsible for caries would produce certain endogenous porphyrins (fluorophores) that fluoresce when excited by red laser light.[] The LF-based device for caries diagnosis is equipped with a laser diode providing the excitation light source, wavelength 655 nm, necessary for detection of these fluorophores. Most studies evaluating LF have been carried out ,[] though there are also a number of studies.[] The results of studies cannot be extrapolated to conditions and the limitations of these studies are well-known. In order to get the maximal sensitivities and specificities from the LF device and to obtain an appropriate guideline for the practitioner to interpret the results, several cut-off values were presented,[] thus making the diagnostic decision even more difficult. In many cases, the cut-off values determined proved to be lesser than the cut-off values obtained from clinical studies.[] This may be due to the way in which the teeth are preserved, or changes in their organic content after extraction, inducing alterations in dental tissue fluorescence. The mentioned organic matrix begins to degrade with time. These could promote variations in the optical properties of the teeth[] and consequently in their LF response. Teeth being the most essential part of the dental research methodologies invariably need to be stored under proper conditions in order for various studies to be carried out authentically. Tooth storage conditions in studies are yet to be standardized. There is a need to clarify the effect of different storage media on the fluorescence readings during a specified span of time. Hence, the prime objective of this investigation was to evaluate the change in LF for teeth stored in different storage solutions as a function of time and to give cut-off values for different storage media at different time intervals to get them at par with the clinical/ conditions and to see which medium gives best results with the least change in LF values and while enhancing the validity of DIAGNOdent in research. Extracted teeth were collected and subjected to an autoclave cycle for 40 min as per the guidelines of Centers for Disease Control, USA based on the study by Pantera and Schuster.[] Initially, all the teeth were frozen and stored at −20°C immediately after extraction and had no contact with any storage solution for 7 days. Following this, the teeth were then defrosted to room temperature over a period of 14 h placing them in plastic containers. Nearly 100% humidity was insured by placing a wet blotting paper at the bottom of each container making sure that no contact was made between the teeth and the blotting paper. All teeth were then cleaned with a toothbrush under tap water for 15 s. Only the teeth with an LF ≥30, indicative of requiring definitive restorative procedures,[] were used. 90 extracted teeth were selected from this pool and a specific site was selected and recorded for measurement. Each specimen had a mean fluorescence value of 92 varying from 85 to 96. All 90 samples were then divided randomly into 9 groups of 10 teeth each. Measurements were made for each tooth after which the samples were stored as follows: Group 1 in 1% chloramine, Group 2 in 10% formalin, Group 3 in 10% buffered formalin, Group 4 in 0.02% thymol, Group 5 in 0.12% chlorhexidine, Group 6 in 3% sodium hypochlorite, Group 7 in a commercially available saliva substitute-Wet Mouth (ICPA Pharmaceuticals) and Group 8 in normal saline. The storage temperature was 4°C for these 8 groups. Group 9 was stored without any storage solution at −20°C. Prior to each measurement, the LF device was calibrated according to the manufacturer's instructions. Measurements were repeated at 30, 45, 60, 160 and 365 days. In order to repeat the readings at the same site each time, an indentation was marked near the site on the teeth with a round bur [, ]. In order to allow comparisons between individual LF measurements with time, the baseline was set at 0.00 and curves were plotted []. The mean change in LF values for the eight storage media and frozen condition was calculated. Average change at different time intervals were used as cut-off values as compared to the condition. Statistical Package for Social Sciences for Windows®, version 20. Statistical significance was set at = 0.05. The mean change in LF values in different storage mediums at different time intervals were compared using two-way ANOVA. In all specimens the difference was statistically significant for all groups except for the frozen specimens. All curves dropped dramatically immediately after a storage in solutions in Groups 1-8. Maximum drop occurred between days 1 and 30 after which the drop was gradual. The change in LF values at 30 days as a percentage of total change was evaluated and presented in the form of a bar diagram []. It was discerned that specimens stored in Wet Mouth, sodium hypochlorite, chloramine and normal saline exhibited a slower initial decline in LF values (up to day 30) as compared to formalin, buffered formalin, thymol and chlorhexidine. Therefore, it can be clearly discerned from Figures and that for short-term storage purposes-Wet Mouth, sodium hypochlorite, chloramines or normal saline should be preferred while for studies requiring long-term storage of teeth as specimens-formalin, buffered formalin, thymol or chlorhexidine should be preferred. However, the best storage condition would undoubtedly be the frozen condition which showed a statistically insignificant change ( > 0.05) in LF values over the entire period of the study. Most of the research studies carried out in dentistry are first evaluated under laboratory or conditions and only after a hypothesis is tested under these conditions, is the methodology extrapolated onto the clinical or condition. Various factors such as room temperature, humidity and storage media need to be considered when working under laboratory conditions. In the condition, the variable hypothesized to affect differences in cut-off values in different studies[] has been the storage solutions. Most of the storage solutions, used in dental research for preservation of extracted teeth, are antiseptics or disinfectants which are basically a “protoplasmic poison” for the bacteria. This probably leads to decrease in the concentration of fluorophores in the specimen by dilution ultimately leading to a reduction in the LF signal. Moreover, the chemicals used might even cause a modification of the fluorophores or affect the optical properties of dental hard tissues like dispersion and scattering over time. On the other hand, freezing is known to exert a bacteriostatic effect, as this is commonly used for preservation of bacterial cultures.[] Furthermore, it is highly unlikely that bacteria or their endogenous products (fluorophores) will undergo a change if no solution is in contact with them, such as in the frozen condition. Very few studies have dealt with the issue of effect of storage on LF response. In a study[] spectroscopic changes in human dentine exposed to various storage solutions were measured in the ultraviolet, visible and near-infra-red spectral ranges with an integrating sphere spectrophotometer over a 28 day period. It was concluded that changes in surface chemistry and optical properties of dentine did occur as a function of storage solution and time, which must be considered when studying dentine. Another study[] had a conflicting finding of increase in fluorescence after formalin fixation. However, in that experiment, the specimens had been stored in thymol saturated saline followed by subsequent storage in neutral-buffered formalin unlike our study where no prior contact was made with any other solution. Hence, the two studies cannot be compared. Few other studies[] have been conducted in the recent past which concluded that a decrease in LF values takes place when teeth are stored in different storage solutions as functions of time which is in concurrence with our findings. Our study evaluated a change in LF values for eight different storage solutions in comparison with the frozen condition which has not been previously conducted. In our study, a temporal relationship has been established in the drop in LF values wherein the maximum drop was seen during the first 30 days of storage following which the drop in the readings was found to be gradual. The observed drop in the LF values was significant for each of the eight solutions while it remained insignificant for the frozen condition, which is in agreement with the previous studies of Lussi [] Since there was little or no change in the LF values for the frozen teeth, hence it may not be wrong to assume that the frozen condition may be comparable to the condition thus enabling the practitioner to interpret results and extrapolate them with more accuracy to the clinical condition. The study validates use of DIAGNOdent as a reliable evaluating measure in various researches being conducted globally in the field of dentistry. Recently, many researches have been conducted, utilizing DIAGNOdent as a primary tool for studying mineralization of dental specimens.[] It is our study that goes a step ahead in providing cut-off values for each case at different time intervals in an attempt to obtain appropriate guidelines for undertaking such studies in the future. Only a few studies can be found in the literature dealing with the issue of the influence of storage on LF response. The inevitable inherent change in LF due to various storage media commonly used, at different time intervals is put forth by the study. The values thus obtained can help remove the bias caused by the storage medium and the values of LF thus obtained can hence be conveniently extrapolated to the condition. The study enables better use of DIAGNOdent as an evaluating measure in various studies.

During the root canal preparation procedures, dentin chips, pulp tissue, microorganisms and/or irrigants may get extruded into the periradicular tissues. Though a thorough control of the working length (WL) may decrease the risk, but nevertheless extrusion of any debris may potentially cause post-operative complications such as flare-ups,[] which are characterized by pain, swelling causing unscheduled visits of the patients resulting in interappointment emergency.[] At present, all preparation techniques and instruments are associated with extrusion of debris, even when the preparation is maintained short of the apical terminus and manual instrumentation happens to produce greater extrusion when compared to engine driven rotary preparation.[] The studies so far have proven that none of the various techniques and instruments can clean and shape the root canal system without producing some apically extruded debris (AED).[] However, it has been proved that various instrumentation techniques have been associated with different amounts of AED.[] ProTaper™ (Dentsply Maillefer, Ballaigues, Switzerland) system exhibits progressively variable tapers of each instrument that develop a “progressive preparation” in both the vertical and horizontal directions. The ProTaper™ cross-sectional design mimics that of a reamer, with three machined cutting edges and convex core.[] Hyflex™ CM nickel-titanium (NiTi) Files (Coltene-Whaledent, Allstetten, Switzerland) is produced by an innovative methodology (patent pending) which uses a unique process that controls the material's memory (a complex heating and cooling treatment). The cross-sectional design of Hyflex™ files is very much similar to EndoSequence.[] WaveOne™ (Dentsply Maillefer, Ballaigues, Switzerland), the recently introduced single-file NiTi system is claimed to complete root canal preparation with only one instrument in reciprocating motion with adequate size and taper. These files are made of a special NiTi alloy called M-Wire that is created by an innovative thermal treatment process.[] It is available in sizes of 21.06, 25.08 and 40.08 and these are used in a reciprocal motion that requires a special automated devices. As AED generates an acute inflammatory reaction in the periapical tissues, it is considered as an important parameter to assess the efficacy of an instrumentation technique or instrument design during root canal preparation.[] The aim of this study was to compare the amount of AED during preparation of straight root canals in extracted human teeth using WaveOne™ compared with the rotary full-sequence HyFlex™ CM and ProTaper™. In this study, 60 freshly extracted human mandibular premolar teeth that were sacrificed for orthodontic and periodontal purpose were used. The inclusion criteria was the single rooted mandibular premolar teeth with single root canal and apical foramen with root curvature between 0° and 10°. Radiographs were taken both mesiodistally and buccolingually to assess internal resorption, root canal calcification and curvature of the root canals. The degree of root curvature was calculated from the buccolingual radiographs by using the method of Schneider.[] Teeth with signs of crack, internal and external resorption, root caries, canal calcifications and open apices were excluded from the study. The selected teeth were randomly assigned into 3 experimental groups (Groups 1-3) with 20 teeth in each group. The debris and soft-tissue remnants were cleaned from external root surface and then stored in phosphate-buffered saline solution. To maintain similar tooth lengths, all teeth were measured and the crowns were sectioned with a high-speed bur under copious water spray until equal lengths were obtained. Access cavity preparation was done in each tooth and all external tooth surfaces were covered with 2 layers of nail polish except for 1 mm around the apical foramen.[] A 15 K-file (Mani, Tochigi, Japan) was used to determine the WL until it was visible at apical foramen. The WL was re-established by subtracting 1 mm from this measurement. The debris collection apparatus was made according to the design described by Myers and Montgomery.[] Eppendrof tubes were taken and weighed by electronic microbalance (Single Pan K-Roy analytical balance, K Roy and Co, Kolkata, India). Each individual tooth was held in a preweighed eppendorf tube which was fixed inside a glass vial through rubber plug. It was seen that no possible contact was made between the tube and the glass vial. The tube was vented with a 25 gauge needle to equalize the pressure inside and outside. All instruments were set into permanent rotation with a 6:1 reduction using X-SMART Plus™ endo motor (Dentsply, Maillefer). For each file, the individual torque limit and rotational speed programmed in the file library of the motor were used, whereas Wave-One™ was used in a reciprocating working motion generated by the motor. All the preparations were made by a single operator. The preparation sequences is as follows: The extruded debris and irrigant during preparation were collected in eppendrof tube. A total volume of 7 mL of distilled water was used in each root canal for irrigation. The irrigation needle (NaviTip 31ga; Ultradent, South Jordan, UT) was placed short of WL or slightly coronal to the point where resistance was encountered.[] After canal preparation, the eppendorf tube was removed from the glass vial. Then the tooth was separated from the tube and the root apex was washed off with 1 ml of distilled water that was collected in the same tube. All the eppendorf tubes were then incubated at 37°C for 15 days to allow the evaporation of moisture before weighing the dry debris. For each eppendorf tube three consecutive measurements were taken on an electronic microbalance and the mean measurement for each tube was considered to be its weight. The weight of extruded debris in each tube was calculated by subtracting pre experiment weight of the tube from the weight of tube with dried debris. The mean weight of extruded debris was calculated for each group. The data were statistically analyzed using Statistical Package for Social Science 16 (SPSS, version 16; SPSS Inc., Chicago, IL, USA). Analysis was performed using Kruskal-Wallis one-way analysis of variance and post hoc Student-Newman-Keuls test at a significance level of < 0.05. The mean AED weight and standard deviation of the three experimental groups is shown in . The results showed that all instrumentation techniques produced a significant amount of extruded debris. The mean extruded debris weight of the three groups. The mean apically extruded weight of debris in WaveOne (0.0076 g) was more when compared with the Hyflex (0.0019 g). On post hoc student-Newman-keuls test it was found that AED produced by reciprocating single file WaveOne™ and ProTaper™ (0.0071 g) was significantly more when compared to Hyflex™ ( < 0.05). However, no statistical significant difference was obtained between WaveOne™ and ProTaper™ ( > 0.05). A major objective of root canal therapy is to obtain a clean root canal system. Debris such as dentine chips, necrotic pulp tissue, microorganisms and irrigants may be extruded into the periradicular tissue during canal instrumentation which leads to endodontic flare-up. Apical extrusion of infected debris to the periradicular tissues is possibly one of the principle cause of this post-operative pain.[] Many factors affect the amount of extruded debris such as the instrumentation technique, instrument type and size, preparation endpoint and irrigation solution.[] The main objective of the present investigation was to determine the apical extrusion of dentine debris as a result of canal shaping by different rotary systems. As per the results obtained, extrusion of debris apically occurred independent of the type of instrument used. The reciprocating single-file system showed significantly more debris extrusion compared with both the full-sequence rotary NiTi instruments ( < 0.05). The obtained differences may be caused by the preparation technique and/or the cross-sectional designs of the instruments.[] Hyflex CM™ files have a cross-sectional design very similar to EndoSequence.[] The cutting profile of each Hyflex CM™ file facilitates penetration in the canal and presents a root canal shape corresponding with the original anatomy. A study by Bürklein found that there was more debris in the apical part of the canals after canal preparation with WaveOne and ProTaper instruments as they are characterized by three cutting edges with radial lands to support the blades and a relatively small chip space.[] ProTaper™ and WaveOne™ are characterized by a triangular or modified triangular cross-section resulting in a lower cutting efficiency and smaller chip space.[] This design may enhance debris transportation toward the apex when used in combination with a reciprocal motion. Contrarily, in continuous rotation may improve coronal transportation of dentin chips and debris by acting like a screw conveyor.[] In this present study, the canal WL was 1 mm short of the apical foramen. Myers and Montgomery[] clearly showed that a WL 1 mm short of canal length contributed to significantly less debris extrusion. A certain degree of caution must be taken when these results are transferred to the clinical situation because of the zero back pressure used in this study design, gravity may have carried the irrigant out of the canal. This is a known drawback of designs with no periapical resistance. It has been suggested that using floral foam may simulate resistance of periapical tissues.[] However, foam may absorb some irrigant and debris when used as a barrier and therefore, no attempt has been made in this present study to simulate periapical resistance. Bidistilled water was used as an irrigant in this study to avoid any possible weight increase due to NaOCl crystal formation. The tubes were stored in an incubator in order to evaporate the moisture and weigh the dry debris. Myers and Montgomery suggested a reassessment of the apical dentinal plug because of the potential benefits of reducing the amount of AED and irrigants and the prevention of over instrumentation in combination with extrusion of filling materials.[] A study has shown that establishing apical patency in mesiobuccal roots of maxillary molars resulted in apical extrusion of sodium hypochlorite in 100% of the specimens.[] A similar study has shown that maintaining apical patency was associated with less AED when compared with teeth in which the constriction remained intact.[] Ruiz-Hubard [] found that extrusion of debris apically was less using a crown-down pressure less technique in curved and straight canals when compared with the step-back technique. Zarrabi [] compared ProFile, RaCe and Flex Master rotary instruments with the step-back technique using manual files and reported that the step-back technique extruded greater amounts of debris than the rotary instruments. Ghivari found that step-back technique extruded a greater quantity of debris and irrigant in comparison to the other hand and rotary Ni-Ti systems.-[] Garlapati showed that K3 rotary instruments using crown down technique extruded less number of bacteria.[] Earlier studies have shown that manual instrumentation produced significantly more debris than the rotary NiTi techniques and the balanced-force technique.[] It was observed that rotation during instrumentation, with both the rotary and balanced-force techniques, tend to pull dentinal debris into the flutes of the file and direct it toward the coronal aspect of the canal. In case of engine-driven instruments early flaring of the coronal part of the preparation may improve instrument control during preparation of the apical third of the canal. The rotary motion tends to direct debris toward the orifice, avoiding its compaction in the root canal.[] De-Deus in their study have reported no difference in debris extrusion between conventional ProTaper Universal technique and single-file ProTaper F2 used in reciprocating movement.[] However, the results of this present study are in agreement with previous studies by Bürklein and Schafer[] which showed reciprocating single-file systems extruded more debris compared with the full-sequence rotary NiTi instruments. In the present study, the Group 3 (WaveOne™) has shown more extrusion of debris compared with Group 1 (ProTaper™) followed by Group 2 (Hyflex CM™) suggesting that reciprocation motion might cause more apical extrusion compared with continuous motion during root canal preparation. d e r t h e c o n d i t i o n s o f t h i s s t u d y , W a v e o n e w a s a s s o c i a t e d w i t h m o r e d e b r i s e x t r u s i o n c o m p a r e d w i t h P r o T a p e r a n d H y f l e x s u g g e s t i n g t h a t r e c i p r o c a t i n g s i n g l e - f i l e s y s t e m e x t r u d e d m o r e d e b r i s t h a n c o n t i n u o u s f u l l s e q u e n c e r o t a r y N i T i i n s t r u m e n t s .

Root canal microflora is the foremost cause of periradicular pathosis. A complex root canal system anatomy makes elimination of micro-organisms difficult and irrigants are essential since canal instrumentation alone is unable to remove all tissue remnants and debris. The desirable properties of an irrigant thus include it's ability to flush loose debris, lubricate the dentinal walls, dissolve organic matter and possess antimicrobial properties. Sodium hypochlorite (SHC) is the most commonly used endodontic irrigant with good antimicrobial efficacy[] and tissue dissolution[] but with concerns for vital peri-radicular tissues.[] Chlorhexidine gluconate (CHX) is a broad spectrum anti-microbial bisbiguanide effective against bacteria and fungi.[] It has lower toxicity than SHC[] but lacks tissue dissolving property.[] The Neem ( A. Juss) tree has been described for its value in traditional Indian medicinal texts. In recent years, the chemistry and structural diversity of over 135 compounds isolated from different parts of the neem tree has been documented.[] The isoprenoid group of constituents of neem have anti-inflammatory,[] anti-bacterial,[] anti-fungal[] and immunomodulatory properties.[] Our research screened several neem-based products and found maximal anti-microbial properties with a leaf extract from the tree.[] We thus aimed to evaluate the antimicrobial properties of this neem extract as an irrigant during root canal treatment and compared it with SHC and chlorhexidine irrigants. Combinations of the leaf extract with standard irrigants were also evaluated. #text Our results indicate that the number of post-irrigant positive cultures (CFU/ml) were significantly lower than the pre-irrigant cultures in Groups 1-5 ( < 0.05). No significant reduction in colony counts was found between the pre-and post-irrigant cultures with saline. represents the percentage reduction in micro-organisms with the different irrigants and combinations. No significant differences were found between SHC, CHX and ELE. There were no statistically significant differences between SHC and SHC + ELE or SHC + CHX combinations. Even though CHX did not differ from the ELE + CHX combination ( = 0.473), we found it to differ significantly from a combination of ELE + SHC ( = 0.049). Similarly, ELE did not differ significantly with ELE + CHX combination ( = 0.265), but had a significant difference with the combination of ELE + SHC ( = 0.022). All irrigants and combinations differed significantly from saline. The results from microbial counts on Gram-stained smears [] indicate that the number of micro-organisms on the post-irrigant smears were significantly lower in all the groups. A statistically significant difference was found between saline and SHC ( = 0.009); a combination of SHC and ELE with saline ( = 0.017); and a combination of CHX and ELE with saline ( = 0.008). Comparisons between all other groups did not yield any difference. The results from Gram-stained smears [] indicate that tissue loads on the post-irrigant smears were significantly lower in all the groups. SHC + ELE differed significantly from saline ( = 0.026). Comparisons between other groups did not yield differences. A. Juss is a useful traditional medicinal plant in India. Each part of the tree has some medicinal property. It is now considered a valuable source of unique natural products for development of medicines against various diseases.[] Past research has shown ELE of the neem tree to be effective against several microbes.[] It's use within the root canal has been undocumented. The antimicrobial property of ELE against a mixed flora of anaerobic and facultative micro-organisms was tested in this study. ELE has low toxic potential and safety tolerance limits have been established at 176 mg/kg body weight.[]3 mL of the ELE irrigant was used in this study, which was within the safety criteria. The use of Gram staining to evaluate bacteria within root canals has been described.[] The technique has been used to assess the antimicrobial effect of irrigants within root canals and tissue clearance based upon staining characteristics of the smear.[] A similar approach has been used in the present study. The leaf extract has several bioactive compounds that possess antibacterial capabilities. This may be attributed to the tetranortriterpenes that include, nimbin, nimbinin, nimbidinin, nimbolide and nimbidic acid.[] The antibacterial action of the drug has been studied by Baswa who found inhibition of cell membrane synthesis.[] Besides the antimicrobial action, this group of compounds also demonstrates anti-inflammatory function (ability to prevent the production of prostaglandins, especially Prostaglandin E1 (PGEI) and 5-hydroxytryptamine) which is a desirable characteristic of the irrigant.[] An immunomodulatory function has also been suggested for the neem drug that enables antigen presentation to immunocompetent cells.-[] This study is the first to use a leaf extract within the root canal. The irrigant combinations (ELE + SHC and ELE + CHX) had better antimicrobial properties than individual irrigants. This is indicative of synergistic action between the herbal and chemical irrigants, which potentiates their individual anti-microbial efficacy. It could be hypothesized that using the SHC + ELE combination as irrigants would prove beneficial. Biofilms present within the root canal are hard to eliminate with current irrigants and techniques. They exhibit an increased resistance to all disinfectants.[] has demonstrable antifungal action against Candida albicans biofilm on tooth substrate.[] A neem-based irrigant has also shown prevention of adhesion to dentin.[] The effect of an aqueous neem leaf extract on extracellular glucan produced by Streptococcus sanguis, which is responsible for co-aggregation of microorganisms to form colonies, has been demonstrated.[] The gallotannin component of the extract had an inhibitory effect on insoluble glucan production and bacterial aggregation. Such results have also been reported by Wu-Yuan [] We can hypothesize similar action with ELE in our study. The extract may have disrupted the biofilm adhering to the root canal walls and aided in the permeation of SHC to produce it's antibacterial action. This may explain the synergism between the two irrigants. ELE has anti-microbial properties attributable to the neem drug component. Ethanol is the solvent for the neem drug in ELE. Our study demonstrated that ethanol itself lacked antimicrobial properties.[] It has been used to reduce the surface tension of SHC which helps increase spreading ability.[] However, such mixtures are unstable and lack shelf-life due to the loss of available chlorine from SHC. In our study, ethanol was an integral part of the leaf extract and could have increased the spreading ability of the neem drug within the canal. When mixed with SHC within the root canal (SHC + ELE sequential irrigation), ethanol helped reduce the surface tension of SHC. This could further explain synergism between SHC and ELE. The anti-microbial effect of the standard irrigants, SHC and CHX showed no difference even though the percentage reduction with SHC was higher (84.85%) than CHX (69.17%). Similar results have been previously reported,[] though other studies have found differing results.[] Gram-stained smears were used for the detection of root canal flora. Significant differences were found between SHC and saline as well as combinations (SHC + ELE, CHX + ELE) and saline. The smears did not find a significant reduction in microbial counts between either ELE and saline or CHX and saline. This data does not co-relate with the anaerobic culture phase of the study. A possible explanation for this difference in results between the two methodologies could be the incomplete transfer of micro-organisms from the paper points onto the slides for gram smears. For anaerobic culture, the barbed broach and paper points were fully immersed within the TGB without attrition in the number of organisms. The tissue clearing ability of the irrigant was additionally evaluated by the staining characteristics of the Gram smears. A statistically significant difference was found only between SHC + ELE and saline. This method did not suggest of any difference in reduction in tissue load between SHC and saline. SHC has established tissue dissolving properties.[] This suggests Gram smears are not reliable indicators of tissue clearance within the canal as it is dependent on the amount of tissue that adheres to the paper point. r s t u d y i n d i c a t e s t h a t t h e e t h a n o l i c n e e m l e a f e x t r a c t c a n b e u s e d s u c c e s s f u l l y w i t h i n t h e r o o t c a n a l a s a n i r r i g a n t w i t h d e m o n s t r a b l e a n t i - m i c r o b i a l e f f i c a c y . S H C i s i m p o r t a n t f o r g o o d a n t i m i c r o b i a l e f f i c a c y a n d t i s s u e d i s s o l u t i o n p r o p e r t i e s . A c o m b i n a t i o n o f S H C a n d E L E i s s y n e r g i s t i c a n t i m i c r o b i a l l y . F u r t h e r c l i n i c a l r e s e a r c h u s i n g t h i s c o m b i n a t i o n c a n a i m a t e v a l u a t i n g p e r i - r a d i c u l a r h e a l i n g .

Root canal shaping is one of the most important steps in canal treatment.[] It is essential in determining the efficacy of all subsequent procedures, including chemical disinfection and root canal obturation.[] This “ideal” preparation can be a difficult task to achieve in severely curved root canals or S-shaped canals, especially with traditional stainless steel hand instruments.[] Nickel-titanium (NiTi) instruments have been marked as a way to overcome these shortcomings. NiTi-alloy has the advantages of super elasticity and the shape memory effect,[] which can maintain the original canal curvature and create a tapered root canal shape. Root canal working length (WL) may be defined as the distance from a coronal reference point to the point of which canal preparation and obturation should terminate. Success has been found to be greatest in teeth in which the obturation material extended to within 2 mm of the radiographic apex but did not extend beyond the radiographic apex.[] Canal curvature is suspected to be the predominant risk factor for instrument failure because of flexural stresses and cyclic fatigue.[] The clinician can do very little to prevent or reduce such stresses. The reciprocating motion of the NiTi rotary instrument has been shown to decrease the impact of cyclic fatigue compared with rotational motion.[] Therefore, it has been recently proposed that the single-file shaping technique may simplify instrumentation protocols and avoid the risk of cross contamination. Moreover, the use of only one NiTi instrument is more cost-effective and the learning curve is considerably reduced.[] Now-a-days simplifying endodontic procedures with complete safety and effectiveness is our primary concern. Micro-Mega now offers One Shape, the one and only NiTi instrument in continuous rotation for quality root canal preparations. One Shape allows for curved canal negotiation with an instrumental and easy dynamic. Its non-working (safety) tip ensures an effective apical progression avoiding obstructions which are often preceded by instrument separation. Quality root canal shaping can be achieved with one single instrument with remarkable design. A root canal treatment is approximately 4 times faster than a conventional treatment with One Shape file. Overall duration of treatment is shortened. Simplification of the endodontic instrument sequence is possible. The new Wave One NiTi single-file system has been recently introduced by Dentsply Maillefer (Ballaigues, Switzerland).[] The system is designed to be used with a dedicated reciprocating motion motor. It consists of 3 single-use files: Small (International Organization for Standardization [ISO] 21 tip and 6% taper) for fine canals, primary (ISO 25 tip and 8% taper) for the majority of canals and large (ISO 40 and 8% taper) for large canals. The files are manufactured with M-Wire (Dentsply Tulsa Dental Specialties, Tulsa, OK) NiTi alloy.[] The purpose of this study was to compare the canal curvature modification of the Wave One Primary file with One Shape file system. Thirty ISO 15, 0.02 taper, J shaped Endo Training Blocks (Dentsply Maillefer) were used. The canal in the blocks were marked from 0 to 11 at an interval of 1 mm taking 0 as the termination/reference point for the WL. Each simulated canal was colored with Indian ink injected with a syringe. Each specimen was mounted on a stable support consisting of a rectangular slot the size of the specimen (30 mm × 10 mm) and a support for a digital camera (Nikon D70; Nikon, Tokyo, Japan) positioned centrally and at 900 to the specimen. Digital images of all specimens before instrumentation were obtained and saved as JPEG files. Specimens were then randomly assigned to 2 different groups ( = 15 each). WL was established in the canals until reference point 0 with a #10-K file (Dentsply Maillefer). In group 1, the glide path was created with PathFile 1, 2 and 3 (Dentsply Maillefer) at the full WL until reference point 0 using Glyde (Dentsply Maillefer) as the lubricating agent. Each canal was shaped using OneShape rotary file till the WL (reference point 0) with the X-Smart Plus motor (Micro mega) set to 350 rpm and a 5-Ncm torque with a 16:1 contraangle. Canal patency was checked with a #10 K-file (Dentsply Maillefer) before the glide path, after the glidepath, before using OneShape rotary file and after OneShape rotary file. In group 2, the glidepath was created with PathFile 1, 2 and 3 at the full WL till reference point 0 by using Glyde as the lubricating agent. Canals were shaped with Wave One Primary reciprocating files using a pecking motion till WL. The reciprocating motor X-Smart Plus (Dentsply Maillefer) of the Wave One file was used with the manufacturer configuration setup. Canal patency was checked with a #10 K file (Dentsply Maillefer) before the glidepath, after the glidepath and before using WaveOne Primary and after shaping with WaveOne Primary. New instruments were used in each specimen. After instrumentation, all specimens in each group were repositioned in the slot and photographed as described previously. By using digital imaging software Corel draw Graphic Suite X5 (Corel Corporation, Ottawa, Canada), Adobe Photoshop CS3 (Adobe Systems Inc. San Jose, CA) and Solid works student Edition software (Dassault Systems Solid Works Corp, S.A., Velizy, France) the pre and post-instrumentation images were processed and superimposed. The processing of the images was done in the following steps: Step 1 — Alignment and auto color: Alignment of the axis of blocks done with reference to the scales in the toolbox of Corel draw Graphic Suite X5 (Corel Corporation, Ottawa, Canada). Then, auto color is performed which is an image correction tool which provides better visibility and maintains the color gamut difference per pixel. Step 2 — Inversion of images: One image is set to inversion mode to differentiate the properties of both images from each other while overlapping using Adobe Photoshop CS3 (Adobe Systems Inc. San Jose, CA). Step 3 — Orbit shifting and superimposition with respect to the reference line: Superimposition is the placement of an image on top of an already-existing image (inversion mode) to add to the overall image effect. During the development of images, a reference measurement was undertaken into consideration for the experimental analysis based on the perpectual analysis with respect to the reference line. This is known as orbit shifting. Step 4 — Degree of angle of curvature-One shape: Two reference points were assumed to precisely calculate the angle of curvature. Two intersection points were defined for OneShape and the angles for the same were calculated via solid works student Edition software (Dassault Systems Solid Works Corp, S.A., Velizy, France). The Angular calculation was based on the principle of geometry. Furthermore, the angles were confirmed using the trigonometric formulas for each block. Step 5 — Degree of angle of curvature-Wave One: This is calculated in a similar manner as step 4. Pre and post-digital images were superimposed, processed with Corel draw Graphic Suite X5 (Corel Corporation, Ottawa, Canada), Adobe Photoshop CS3 (Adobe Systems Inc. San Jose, CA) and solid works student Edition software (Dassault Systems Solid Works Corp, S.A., Velizy, France). Statistical analysis was performed with mean, standard deviation, one way ANOVA, ( < 0.05) -test and Karl Pearson's correlation coefficient. #text The purpose of this study was to compare the ability of 2 NiTi instruments, the WaveOne primary and OneShape, in canal curvature modification after instrumentation. In the present study, simulated canals were chosen to standardize the conditions. Much care should be taken in the extrapolation of the results to the use of real roots due to the difference between resin and dentin.[] In this study, geometric variations of canal curvature in the middle plane of standardized resin blocks were analyzed through a 2-dimensional photographic method. Simulated root canals have been widely used to allow a direct analysis of post instrumentation changes in canal curvature and thus to evaluate the tendency of these techniques to maintain the original canal anatomy under standardized conditions.[] Ni-Ti alloys have been found to be 2-3 times more elastic than similarly manufactured stainless steel files. This property may allow Ni-Ti files to negotiate curved canals with less lateral stress but do not allow the precurving of Ni-Ti files. Whether the physical tendency of Ni-Ti files to remain straight, prevents ideal instrumentation or whether their high flexibility allows a better negotiation of curved canals despite the inability to precurve, still remains questionable.[] Previous studies have shown that preserving the original canal shape with a less invasive approach minimizes the risk of canal transportation with a subsequently lower incidence of canal curvature straightening, the formation of ledges and irregular apical enlargement.[] The prevention of apical transportation and irregular foramen widening may also lead to a well-sealed root filling with less extrusion of debris and reduced post-operative discomfort.[] Preservation of the original canal shape and the lack of canal aberrations are associated with increased antimicrobial and sealing efficiency[] and reduced weakening of the tooth structure. Besides canal anatomy, other factors contribute to optimal mechanical instrumentation outcomes, such as instrument design, instrumentation sequence, rotational speed, operator's experience and the use of irrigants.[] Recently, a new WaveOne NiTi single-file reciprocating system has been introduced to simplify root canal preparation. Only one single shaping file is required to provide the canal with an adequate size and taper. The main characteristics of this system are single use, a reciprocating action and M-Wire technology alloy manufacturing. Even more recently, One Shape file has been introduced in which complete canal shaping is possible with only one single file in continuous rotation. The main advantages of this file are unique, sterile, economic and innovative. As a result of clinical use and extensive publication, continuous rotation is a principle known and recognized for almost 20 years. The advantages of the reciprocating motion are based on the physics law of action and reaction applied to root canal instrumentation, which results in a balanced force, as theorized by Roane [] The reciprocating movement minimizes torsional and flexural stresses, increases the canal centering ability and reduces the taper lock within the number of instrument cycles within the root canal. Recent studies showed that an alternating rotary movement is a valid option to optimize endodontic instrumentation by reducing the risk of instrument fracture and root canal deformity.[] The use of the reciprocating motion instead of the continuous rotation method could be advantageous in terms of stresses and the time required for the preparation of curved root canals with a single use of a NiTi file.[] In our study, the single-file technique used with the reciprocating motion enhanced the canal centering ability, leading to less invasive root canal preparation. Deviation from the original curvature can lead to excessive or inappropriate dentine removal, straightening of the canal and creation of a ledge in the dentinal wall, a biomechanical defect known as elbow, which forms the coronal to the elliptical-shaped apical seal, canals with hourglass appearance in cross-section, which requires stripping and over-preparation that weakens the tooth, resulting in fracture of the root. The single use of endodontic instruments was recently recommended to decrease instrument fatigue and possible cross contamination, reducing the number of NiTi rotary instruments required for canal preparation. The single-file technique was also suggested as being cost-effective. The WaveOne technique is both a single-file and single-use concept. As stated, it is a single-file concept given that one single file is able to transition a secured canal to a well-shaped canal, in most instances. Further, appreciate that a single WaveOne file is frequently used to prepare multiple canals in a single furcated tooth, performing a significant amount of work. The WaveOne concept must be considered a single-use concept due to the obvious stress and wear on the active portion of the file. In conclusion, within the limits of this study, the new WaveOne NiTi Primary reciprocating single-file better maintained the original canal anatomy, with less modification of the canal curvature compared with OneShape continuous rotation file. Further investigations are needed to understand whether the better performance of the instrument may be attributed to the reciprocating motion, the variable section design, the Wire alloy or the reverse cutting blades, or a combination of these variables.[]

Pre-flaring is recommended for elimination of cervical interferences. One problem due to use of the instruments for pre-flaring is whether they could create an increased risk for perforations, especially in mesial canals of mandibular molars.[] One of the iatrogenic factors in different methods of root canal preparation is the residual root thickness, which is an indicator of the future fracture resistance of the root.[] The mesial canals of mandibular first molars are not located in the center of the root and the areas between the canals and also between the canals and the furcation area have thin walls and are therefore called danger zones.[] Danger zones have less dentin in the ramification areas in comparison with the peripheral safe root areas.[] Therefore, over-preparing the cervical and middle thirds of the root canal might result in thinning of the dentinal walls and sometimes in strip perforations in the furcation area.[] In addition, thin dentinal walls increase permeability and fracture rate of teeth.[] Correction the perforations is very difficult and the key is to prevent them from happening in the first place.[] The low price, high cutting potential and easy use of Gates-Glidden (GG) drills have made them widely-used instruments in coronal preparation of root canals.[] GG reamers to No. 2 in the coronal third of mesiobuccal canal did not significantly decrease the residual dentin thickness.[] Although the use of these drills is a relatively old method in root canal preparation,[] little has been done to evaluate their effect in the danger zones of mandibular molars. Despite their long-time existence, the sequence in which they are used is not yet clearly defined.[] Some studies indicate that the crown-down technique is a safer method due to the fact that bigger files have lower penetration depth and prevent over-removal of the dentin.[] Mesial canal of mandibular molars is very close to the distal surface of the root and the distal wall thickness beneath the furcation area is about 0.7 mm.[] It has been reported the thickness of distal wall of mesial roots in the mandibular molars was merely 1.2-1.3 mm in 1.5 mm apical to the furcation.[] Researches have shown that 3-4 mm apical to the canal orifice is the most sensitive area in the mesial roots of mandibular first molars.[] The serial sequence technique is the most-widely accepted technique[] and was used in some studies;[] however, some others recommend or used the crown-down technique.[] Computed tomography (CT) is a diagnostic method used in dentistry, especially in endodontics;[] due to its non-invasive nature, it has become very common in the evaluation of root canal preparations, internal and apical pathologies.[] The aim of this study was to evaluate the residual root thickness after pre-flaring using different sequences of GG drills. For this ex-vivo study a total of 107 mandibular first molars from 35 to 55-year-old patients were collected, disinfected by immersion in 5.25% NaOCl solution for 1 h. After removing the calculus by a periodontal scaler, the teeth were stored in normal saline. All the teeth with external or internal root resorption, open apices, visible cracks, fractures, caries and previous root canal treatments were excluded. Mesial roots with the similar root lengths (from furcation to the apex) were used in the study. Access cavities were prepared and presence of two separate mesial canals and patency was confirmed by simultaneous placement of two #10 K-files (Maillefer, Ballaigus, Switzerland). Furthermore resistance to #15 K-file has been checked. Using Schneider method, mesiolingual (ML) canal curvature was determined by parallel radiographs in bucco-lingual and mesio-distal directions and the teeth ranged 20°-35° curvature were included. Finally, 60 mandibular first molars were coded. The teeth were placed halfway into the acrylic resin molds with their buccal surface facing up. To facilitate orientation of the canal in the CT scan sections (Somatom Sensation 16 CT Scanner; Siemens, Berlin, Germany), a copper filament was inserted into the resin parallel to the long axis of the tooth close to the ML line angle. The samples were stabilized on some fiber boards in a manner in which all furcations were aligned. The sections were obtained from 1, 2, 3 and 4 mm of the furcation area and the minimum initial root thickness (MIRT) of the furcation area in ML canal (distance from the outer border of the canal to the outer border of the root) was evaluated by the (Siemens Medical Solutions, Erlangen, Germany). The samples were randomly divided into 2 groups and the mean of the MIRT was assessed and hence that there would be no significant differences between them. To compare the root thicknesses before and after preparation in each of the groups and in each section, paired -test was used. -test was used to compare the residual root thicknesses in the two groups in each of the sections; repeated measure ANOVA was used to compare the initial thickness, the residual thickness and the amount of dentin removed in each group. The data were analyzed with SPSS software version 14.0 (SPSS, Inc., Chicago, IL). The significance level was set at 5%. The relative percentage of dentin removal was calculated by dividing the amount of dentin removed to the initial root thickness multiplied by 100.[] The MIRT in the furcation area of the ML canals in groups G1 and G2 were determined for all the sections in each group (intra-group) and for the same sections in the two groups (inter-group) []. In intra-group comparison, there was a statistically significant difference ( < 0.001). In inter-group comparison of each section, no significant difference was detected ( > 0.05). No significant difference was observed in the intra-group comparison ( > 0.05). The lowest percentage of dentin removal relative to the initial thickness in the 1-mm sections in groups G1 and G2 were 37.5 ± 17.2% and 32.2 ± 20.1%, respectively. The greatest percentage of dentin removal relative to the initial thickness in the 4-mm sections in groups G1 and G2 were 48.8 ± 21% and 44.4 ± 22%, respectively. In the inter-group comparison there was no significant difference ( > 0.05). This parameter was assessed in the intra-group and inter-group comparisons []. None of them showed significant differences ( > 0.05). In comparison of before and after preparations, significant differences were detected ( < 0.05). In terms of preparation mishaps, deformity of instruments or breakage of instruments were not seen. Only one of the G2 samples had a strip perforation within 3-4 mm from the furcation with the 0.5 and 0.4 mm initial root thicknesses respectively. The aim of this study was to compare the effect of the sequence of using GG drills on the MRRT in the furcation area of ML canals of mandibular first molars by CT scan method. CT scan method is capable of providing a 3-dimentional description of changes in the root canal, especially in the coronal and middle-thirds, which are the major areas altered by endodontic instruments.[] The CT scan method provides a functional and non-invasive method in evaluating the intra-canal morphology before and after preparation.[] The samples of this study had root curvatures ranged 20°-35°; such a limited range can be seen in some studies.[] As reported in the previous studies,[] the area within 3-4 mm below the root canal orifice is the most sensitive area during preparation of the mesial roots of molars. Therefore in this study similar to some previous ones,[] evaluation was conducted on the danger zones in the furcation area. In order to avoid perforations and to achieve a straight line access, the GG drills were declined and pressed towards the ML wall as a safety zone. In the present study, the MIRT of the ML canals in the danger zone was more than 1 mm in all the levels except in the sections 3-4 mm from furcations in G1 and the amount gradually decreased from a distance of 1 mm to 4 mm from the furcation. Berutti and Fedon[] reported the root thickness of the mesial canals of mandibular first molars to be the least (1.2-1.3 mm) at a distance of 1.5 mm from the furcation. In a study conducted by Coutinho-Filho [] the average initial root thickness in mesial canals of mandibular first molars at a distance of 3 mm from furcation area was 0.8 ± 0.15 mm. In a study by Garala [] stated that the initial root thickness is the most important factor in determining the residual root thickness after root canal preparation. In the present study, in comparing the relative percentage of the amount of dentin removal to the initial thickness between the two groups, no significant differences were detected ( > 0.05). Furthermore, there were no significant differences for the MRRT in similar sections between the groups ( > 0.05). Coutinho-Filho [] in their study have reported the residual root thickness was significantly greater in the crown down technique compare with the step-back technique. The results of the current study revealed that using GG drills in anti-curvature directions can maintain an appropriate thickness in the root. Lim and Stock[] showed that the MRRT after preparation should be no <0.3 mm to resist forces during root canal obturation. According to their results, the ML canals of mandibular first molars do have sufficient resistance to root canal obturation after usage the GG drills. Wu [] reported that using GG drills in mandibular molars weakens the furcation area regardless of the size of the instrument used or the penetration depth. They concluded that using the anti-curvature method does not lower the risk. Carvalho-Sousa [] reported that the use of GG drills is as safe as ProTaper rotary files with respect to perforation on the distal wall of the mesial canals of mandibular molars. In the present study, the average of the MRRT in all the sections and groups was <1 mm. Raiden [] showed that a minimum 1 mm of tooth structure has to be preserved around the post for the tooth to resist vertical fractures. Akhlaghi [] indicated that the thickness of the mesial canals of mandibular first molars after preparation in the distal and distolingual surfaces was <1 mm. Therefore, it appears the mesial canals of mandibular first molars are not sufficiently resistant to vertical fractures when posts are used. s e d o n r e s u l t s o f t h i s s t u d y t h a t t h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e s t e p - b a c k a n d c r o w n d o w n t e c h n i q u e s i n r e l a t i o n t o t h e M R R T , w h e n o p t i m u m i n i t i a l r o o t t h i c k n e s s i s p r e s e n t , G G d r i l l s u s e d i n a n y s e q u e n c e m a y b e s u i t a b l e , s a f e a n d c o s t - e f f e c t i v e f o r p r e - f l a r i n g o f m e s i a l c a n a l s o f m a n d i b u l a r f i r s t m o l a r s w i t h p r e s e r v i n g t h e r o o t t h i c k n e s s o f f u r c a t i o n a r e a s .

White spot lesions are early signs of demineralization under intact enamel, which may or may not lead to the development of caries. White spot lesions occur when the pathogenic bacteria have breached the enamel layer and organic acids produced by the bacteria have leached out a certain amount of calcium and phosphate ions that may or may not be replaced naturally by the remineralization process. This loss of mineralized layer creates porosities that change the refractive index (RI) of enamel that is usually translucent.[] The causes of white spot lesions may include plaque accumulation particularly along the cervical margins of teeth, inadequate home oral care, consumption of diets rich in sugar and/or those that frequently lower the intraoral pH. White spot lesions may also be seen after removal of orthodontic bands and brackets. The progression of white spot lesions can be slowed or even arrested by non-operative measures that influence etiologic factors such as maintaining oral hygiene and use of remineralizing agents such as topical fluorides and casein phospho peptide-amorphous calcium phosphate. Although lesions can be arrested by these measures they still continue to pose esthetic problems referred to as “enamel scars”.[] At times they may not be effective and the carious lesions tend to progress. In such conditions, the infiltration of caries lesions with low viscosity light curing resins is considered as a treatment option for non cavitated lesions, which are not expected to arrest or remineralize.[] Caries infiltration is a novel treatment option for white spot lesions and might bridge the gap between non-operative and operative modalities. It is a micro-invasive technology that fills, reinforces and stabilizes demineralized enamel, without drilling or sacrificing healthy tooth structure. It has also been shown to inhibit caries progression in lesions that are too advanced for fluoride therapy. Caries infiltration involves the use of low-viscosity light curing resins composed of triethylene glycol dimethacrylate (TEGDMA) which completely fills pores within the tooth, replacing lost tooth structure and stopping caries progression.[] It penetrates into the lesion by capillary forces and creates a diffusion barrier inside the lesion and not only on the lesion surface.[] The use of 15% hydrochloric acid for etching the surface layer is effective and postulated to be beneficial for a deeper infiltration of the resin into the body of the lesion.[] The use of solvents such as ethanol, acetone and water in resin infiltrates show lower surface tension and viscosities compared with materials without solvents. These materials show higher penetration coefficient.[] Thus, the success of caries infiltration technique, depends on the efficacy of this low viscosity resin or “caries infiltrant” to penetrate up to the depth of the white spot lesion and not just mask the lesion. Studies which have assessed the caries infiltrate, have shown that resin infiltration of initial non-cavitated proximal lesions have a good clinical applicability and very high patient acceptance.[] A study by Wiegand showed that use of caries infiltrate before application of conventional adhesive does not impair bonding to sound and demineralized enamel and can be used as a pre-treatment in demineralized enamel.[] However, a study by Schmidlin showed that application of an adhesive, either alone or in combination with the caries infiltrant, is more effective to protect enamel dissolution than application of only the infiltrant.[] Although clinical studies have been done earlier,[] they focused mainly on the clinical success and outcome of the resin. Depth of resin penetration could be a key determining factor for the creation of a diffusion barrier and the success of infiltration. Hence, the aim of the present study was to determine the depth of penetration of a commercially available resin infiltrate. xref #text xref #text The treatment of white spot or non cavitated lesions should aim to arrest the lesion progression and improve the aesthetics by diminishing the opacity.[] Caries infiltration acts by arresting the lesion progression by occluding the microporosities that provide diffusion pathways for acids and dissolved minerals.[] It is a simple, painless, ultraconservative technique that allows immediate treatment of lesions, not advanced enough for restorative therapy. Infiltrants used in this technique are light curable resins that are optimized for rapid penetration into the capillary structures of the lesion body. These materials exhibit a very low viscosity, low contact angles to the enamel and high surface tension. These properties are important for complete depth of penetration of the resin infiltrant into the body of the enamel lesions.[] The partially mineralized intact surface layer could hamper the resin from penetrating into the lesion. Hence, this layer was removed by acid etching with 15% hydrochloric acid gel. Application of hydrochloric acid as an etchant has been demonstrated to be superior to 37% phosphoric acid gel in removing the surface layer of natural enamel lesions when applied for 120 s.[] The etching procedure removes superficial discolorations and the higher mineralized surface layer, which might hamper resin penetration. Hydrochloric acid in similar concentrations is widely accepted in aesthetic dentistry to remove superficial discolorations using enamel microabrasion. However, contact with soft-tissues may cause ulcerations if used for more than 30 s. Therefore, in clinical application, isolation with a rubber dam is mandatory.[] Sound enamel has a RI of 1.62, while the micro porosities of enamel lesions are filled with either a watery medium (RI: 1.33) or air (RI: 1.0). The difference in refractive indices between the enamel crystals and medium inside the porosities causes light scattering that results in a whitish opaque appearance of these lesions, especially when they are desiccated.[] The principle of masking enamel lesions by resin infiltration is based on changes in light scattering within the lesions. This novel technique involves infiltration of the carious lesions with resin (RI 1.46) that, in contrast to the watery medium, cannot evaporate. Therefore, the difference in refractive indices between porosities and enamel is negligible and lesions appear similar to that of the surrounding sound enamel.[] It has a chameleon effect and requires no shade matching. Lesions lose their whitish opaque color and blend reasonably well with the surrounding natural tooth structure. An immediate improvement in the aesthetic appearance is observed. Since many enamel lesions remain unchanged or progress very slowly over long periods, there is adequate time to assess caries risk and initiate preventive procedures. Furthermore, the percentage of radiographically visible proximal lesions in the outer half of dentin that are cavitated has declined over the past several decades to approximately 41%.[] The efficacy of caries infiltration has been shown to be limited when used in cavitated lesions.[] In our study, the infiltrant successfully penetrated into the artificially created white spot lesion and formed a homogenous resin layer. These findings were in accordance with earlier observations which reported that resin mixtures with high TEGDMA concentrations tend to show better inhibition of lesion progression than those with high concentration of bisphenol A glycidyl methacrylate. It was attributed to enhanced ability of the resin to penetrate after application of ethanol.[] The mean values observed in this study are similar to those obtained by Buonocore,[] Voss and Charbeneau[](5-10 μm) and Pahlavan [](7 μm) but lower than those reported by Wickwire and Rentz[](25 μm) and Arakawa [](50 μm). These studies were based on indirect decalcification procedures to assess the depth of penetration. Wetting and penetration of resins might have been impaired as contamination of enamel surface with traces of dust, water and organic substances could not be avoided. A proper decontamination of the lesion may be essential to determine complete resin penetration. In the oral cavity, the enamel lesions are exposed to several contaminants, which tend to reduce the surface energy of enamel.[] Other factors such as saliva, pellicle and intraoral pH may also influence the depth of penetration of the infiltrate. Lesser filler loading contributes to low viscosity and enables better penetrability.[] The low viscosity of the infiltrant, enables it to be applied even on tooth surfaces which are difficult to access, such as, interproximal surfaces. Recently, it was shown that caries infiltration reduced lesion progression of non-cavitated interproximal caries lesions extending radiographically into the inner half of enamel up to the outer third of dentin.[] Microinvasive technology can be an advantage in the pediatric population. The use of caries infiltration limits the use of dental drill, thus improving patient acceptance to treatment. It also avoids periodic recall of the patient compared with fluoride application as it is a single sitting procedure. This new technology allows for specific therapy of early carious lesions without the need to prepare cavities, thus protecting and fully preserving the hard tissue surrounding the lesion. Furthermore, it is virtually painless as it requires no anesthesia and the treatment duration is predictable, thus positively affect the compliance of young patients. Caries infiltration can also be used as an adjunctive therapeutic measure for white spot lesions in adolescents and adults following fixed orthodontic therapy and in the absence of good oral hygiene.[] T h e m a x i m u m d e p t h o f p e n e t r a t i o n o f t h e r e s i n m a t e r i a l w a s 6 . 0 6 ± 3 . 3 2 μ m . C a r i e s i n f i l t r a t i o n c a n b e u s e d a s a p a i n l e s s a n d e f f e c t i v e o p t i o n f o r t r e a t i n g w h i t e s p o t l e s i o n s .

Improvements in the mechanical properties of composite resins and esthetic needs have brought about an increase in the application of these materials. Weak bonding to dentin surface remains, however, is deemed as one of the major problems of composite resin restorations especially in gingival floor of cavities.[] Application of low elastic modulus materials such as flowable composites, glass ionomer (GI) or resin modified glass ionomer (RMGI) under the composite restorations, known as sandwich technique is one of suggested methods for improving the bond strength to dentin. In traditional sandwich technique introduced by McLean, conventional GI was used.[] The bond strength between GI and composite was weak and various methods have been suggested for improving the bond strength including application of acid or resin coating on unset or maturated GI.[] In spite of the fact that bond strength is fairly improved by these methods, low tensile strength of GI is still reported to cause cohesive fracture in GI before deboning occurred in the interface.[] Some studies suggest the application of RMGI instead of GI in sandwich technique due to better mechanical properties, more resistant to moisture and higher bond strength to composite,[] RMGI can bond chemically to composite through co-polymerization of un-reacted monomer (hydroxyethyl methacrylate [HEMA]) in air-inhibited layer of surperfacial surface of cured RMGI with adhesive systems or composite resins. Furthermore, it may provide covalent chemical bond between adhesive resin systems and residual monomer in polyacid chains within the cured RMGI.[] The total – etch adhesive systems that need etching before applying have widely been used in restorative procedures. The etching and rinsing processes may remove calcium and aluminum from GI and reduce the cohesive strength of it. However, there are contradictory results reported in the literature about the effects of acid etching on bond strength of RMGI to composite. Etching of RMGI may have no effect,[] adverse effect,[] or improving effect on bond strength.[] The self-etch approach either two-or one-step adhesives are more user-friendly due to the time-saving and simplified procedure. The application of self-etch adhesives on GI or RMGI improve the bond strength to composite in comparison with total etch adhesives.[] In co-curing technique, two different light-cured materials were coincidently polymerized.[] Knight el al.[] in their study suggested the application of co-curing technique for RMGI and composite can decrease the internal stress in composite restorations and also reduce the clinical steps. Furthermore, simultaneous curing of RMGI with composite increases the bond strength between GI and composite.[] A study by Tulunoglu [] applied a co-curing technique for coincident polymerization of different adhesive systems with RMGI and concluded that although this method may increase in micro-leakage in self-etch adhesive, but, it has no significant effect in three-or two-step total-etch adhesives. The authors’ assumption was that simultaneous curing of RMGI and adhesive systems may increase penetration of adhesive systems into RMGI before curing and so the bond strength will improve. Hence, the aim of the present study was to evaluate the shear bond strength (SBS) of RMGI to composite resin, using different adhesive systems with co-/pre-curing techniques. xref italic #text The SBS of various groups is presented in . Two-way ANOVA showed that both variables (curing technique and type of adhesive systems) had a significant effect on SBS ( = 0.04 for curing technique and = 0.00 for adhesive systems). Furthermore, there was an interaction between two variables ( = 0.00). Tukey's post-hoc test indicated that both self-etch adhesive systems provide higher bond strength than the total-etch adhesive system ( < 0.05) while there was no significant difference between two self-etch adhesive systems. Independent -test presented that the application of co-curing technique increased the SBS in the both of self-etch adhesive systems ( = 0.001 for AdheSE and = 0.04 for AdheSE One F) but, decreased the SBS in total-etch adhesive system ( = 0.015). The distributions of failure mode in the various experimental groups are displayed in . In the total-etch pre-cured group, severe crack and voids was observed in composite-adhesive interface while in the co-cured group of this adhesive, severe crack and voids appeared in RMGI-adhesive interface. In both self-etch adhesive systems either pre-cured or co-cured, no crack or void was observed in the two interfaces. In the co-cured two-step self-etch adhesive group, the obvious resin tags formation at resin-RMGI interface was observed. The results of current study reject both of the hypotheses: Among the three different adhesive systems applied, the total-etch adhesive has the lowest value of SBS. This finding is similar to the results of the other studies.[] Acid etching and rinsing are important factors that will possibly have significant effect on bond strength. A study by Bracket and Huget[] demonstrated that the application of acid etching can improve the bond strength between RMGI and composite. In contrast, Kerby and Knobloch[] demonstrated this procedure can decrease the bond strength through a partial elimination of HEMA and un-reacted methacrylate groups in air-inhibited layer. However, some studies showed the inhibition or decreasing of penetration of acid into RMGI due to the high resin content and formation of polymeric matrix.[] Therefore, they concluded that acid etching has no significant effect on bond strength of RMGI to composite.[] The application of adhesive systems may improve the bond strength between composite and RMGI or traditional GI.[] In the current study, both self-etch adhesive systems had higher SBS than the total-etch adhesive. This finding is consistent with the results obtained by Arora [] and Kasraie [] Similarly, Kandaswamy [] concluded that mild self-etch adhesive had higher bond strength than the strong self-etch or total etch adhesive when they were applied on the unset GI cement. Gopikrishna [] demonstrated that the application of self-etch primer on unset GI improved the bond strength in comparison with application of this adhesive or total-etch adhesive on set GI. Another important factor should be mentioned is viscosity of adhesive systems. The lower the viscosity adhesive used, the better the bond strength may be achieved,[] Uncured HEMA in the surface of RMGI may improve the wetting ability of adhesive resins.[] The observation of noticeable resin tags formation in SEM micrographs of co-cured two-step self-etch adhesive group may be due to lower viscosity or better wet ability of this adhesive. The penetration of resin into RMGIs may improve the strength of these cements and therefore, the failure mode may change.[] Evaluation of the failure mode in this group showed that the number of cohesive failure of RMGI decreased and mixed failure was the most frequently failure mode was observed. In the total-etch co-cured group, acid was applied before formation of the resinous matrix so, acid could penetrate more into RMGI. Effect of acid on this uncured RMGI is somewhat similar to the traditional GI. The formation of weak salt on the surface of RMGI,[] removing or decreasing calcium and aluminum ions that reduce the tensile strength of RMGI[] and reduction in HEMA content[] may be explanations for reduction of bond strength in this experimental group. Furthermore, the increase of the cohesive failure mode in this group, in comparison with the other groups, points out the weakening effect of acid application on the uncured RMGI structure. SEM analysis showed the cracks and voids in RMGI-resin adhesive interface that further confirmed this hypothesis. Polymerization stress of composite might create these cracks and voids. There are contradictions about the failure mode observed in the previous studies since they reported the cohesive failure of GI/RMGI,[] mixed[] or adhesive as the most failure mode observed.[] In the current study, the most of specimens in total-etch co-cured group fractured cohesively in RMGI that remarks the detrimental effect of acid etching on uncured RMGI. In other groups, adhesive or mixed failure modes were the most observed failure modes. In SEM micrographs of two self-etch groups, no cracks and voids in any interfaces was observed. These merged margins in SEM evaluation may indicate a chemical bond formation.[] Gopikrishna [] concluded that the carboxylic groups in self-etch primer can chemically bond with calcium in GI as they bond to calcium of hydroxyapatite in dentin. The other factor to keep in mind is that leaching the metallic ions during the etching and rinsing procedure can decrease the mechanical properties of GI.[] Hence, in the current study acid-etch was applied after 1 min - that's an acceptable time for clinical use-until acid-base reaction induced rather initial setting and reduced the leaching the metallic ions. However, this problem may be important only for the total-etch group. Thickness and tensile strength of GI cement, type, viscosity and stress polymerization of composite, handling procedure and total removing the residual acid (if acid etch was applied) are other certain factors may affect the bond strength between GI or RMGI and the composite.[] In the current study, all adhesives are chosen from the same manufacturer, so that they will be only slightly different in chemical compositions that may affect the bond strength. In this condition, different application methods of these adhesives may have more effect on SBS than the little chemical composition differences. The application of co-curing technique and self-etch adhesive systems may improve the SBS between composite and RMGI. The authors suggested to evaluate the effect of the co-curing technique and the self-etch adhesive in clinical situation in future research. Furthermore, the assessment of the application of co-curing technique on bond strength of GI based-adhesives is required.

The success of endodontic treatment is mainly dependent on thorough cleaning, shaping and disinfection of root canal system. The irrigation protocol thereby plays a key role in the disinfection of the root canal space.[] A clean root canal system along with a three dimensional seal is the clinician's road to success. Subsequent to biomechanical preparation, an amorphous irregular layer known as the “smear layer” is formed on the root canal walls. It contains inorganic and organic substances that include fragments of odontoblastic process, microorganisms and necrotic debris.[] Its presence increases microflora and the inorganic toxins, decreases the sealing ability and increases the potential for bacterial survival and reproduction.[] Until date, no single irrigant has been capable of demonstrating both tissue dissolving as well as demineralizing properties. Current methods to remove the smear layer might involve the use of a chelating agent during irrigation or as a final rinse in combination with other irrigants[] having tissue dissolving properties. Sodium hypochlorite (NaOCl) is the main endodontic irrigant[] used to dissolve the organic portion of the smear layer. To remove the inorganic portion of the smear layer, a decalcifying agent is used, which can be either a chelator or an acid.[] Currently, all the products in the dental market sold to dissolve smear layer are based on ethylenediaminetetraacetic acid (EDTA) or citric acid.[] Neither EDTA[] nor citric acid have strong antimicrobial properties.[] Therefore, it has been proposed to use peracetic acid (PAA) instead of these classical decalcifying agents to dissolve the smear layer and also to disinfect the root canal system.[] Apart from EDTA and PAA, a newly introduced QMix solution is also recommended chelator which is used in conjunction with NaOCl. Recent research indicates that Qmix (DENTSPLY Tulsa Dental, Tulsa, OK), an experimental irrigant containing a mixture of a bisbiguanide antimicrobial agent, a polyaminocarboxylic acid calcium-chelating agent and a surfactant, might be as effective as EDTA at removing smear layers when used after an initial rinse with NaOCl.[] It is well-known that some chemicals used for endodontic irrigation are capable of causing alterations in the chemical composition of dentin.[] Any change in the Ca:P ratio may in turn change the microhardness, permeability and solubility characteristics of dentin and may also adversely affect the sealing ability and adhesion of dental materials.[] No study until date has been done to compare the effect of PAA, QMix and EDTA on calcium loss and microhardness of root dentin. Therefore, the present study has been undertaken to evaluate the effect of different irrigation regimens on calcium loss and its effect on the micro-hardness of the root dentin. n i n t a c t , s i n g l e r o o t e d h u m a n p r e m o l a r s , w h i c h w e r e e x t r a c t e d f o r o r t h o d o n t i c r e a s o n s w e r e u s e d f o r t h i s s t u d y . T e e t h w e r e s t o r e d f o r 1 w e e k i n F o r m a l i n a n d t h e n i n n o r m a l s a l i n e u n t i l u s e . T h e s o f t - t i s s u e c o v e r i n g t h e r o o t s u r f a c e w a s r e m o v e d w i t h c u r e t t e s . T h e t e e t h w e r e d e c o r o n a t e d a t t h e c e m e n t o e n a m e l j u n c t i o n u s i n g a h i g h s p e e d c a r b i d e b u r u n d e r c o p i o u s w a t e r i r r i g a t i o n . T h i c k t r a n s v e r s e s e c t i o n s o f 2 m m w i t h a m a x i m u m a n d m i n i m u m w i d t h o f 3 m m a n d 2 m m r e s p e c t i v e l y w e r e o b t a i n e d f r o m t h e c o r o n a l t h i r d o f e a c h r o o t u s i n g a l o w - s p e e d s a f e s i d e d d i a m o n d d i s c . E a c h s e c t i o n w a s f u r t h e r d i v i d e d i n t o 4 q u a r t e r s , e a c h p a r t c o n s t i t u t i n g a s a m p l e s p e c i m e n f r o m t h e s a m e t o o t h f o r e a c h g r o u p . S p e c i m e n s w e r e h o r i z o n t a l l y e m b e d d e d i n a u t o p o l y m e r i z i n g r e s i n s o a s t o e x p o s e t h e c a n a l p a r t o f t h e d e n t i n . T h e s p e c i m e n s w e r e g r o u n d f l a t o n a c i r c u l a r w e t g r i n d i n g m a c h i n e w i t h a s c e n d i n g g r a d e s o f S i C a b r a s i v e p a p e r s ( 3 2 0 , 6 0 0 , 1 0 0 0 , 1 2 0 0 a n d 1 5 0 0 g r i t ) u n d e r c o n s t a n t w a t e r i r r i g a t i o n u s i n g L e c o g r i n d e r p o l i s h e r . A t o t a l o f 4 0 s a m p l e s w e r e p r e p a r e d , 1 0 f o r e a c h g r o u p . The mean calcium loss and dentin microhardness and its standard deviation along with intergroup comparison are given in . Mean calcium loss in NaOCl + distilled water group (2.79 ± 0.97 ppm) and NaOCl + QMix group (5.72 ± 0.91 ppm) are least followed by NaOCl + EDTA group (6.38 ± 1.76) and maximum in NaOCl + PAA group (9.00 ± 0.55 ppm). There was a statistically significant difference between all groups except between NaOCl + QMix group and NaOCl + EDTA group (F = 50.215, df-3, 36; < 0.001). The mean Dentin microhardness in NaOCl + distilled water group (77.39 ± 2.16 Vickers Hardness Number [VHN]) and NaOCl + QMix group (70.68 ± 4.97 VHN) were maximum followed by NaOCl + EDTA group (69.70 ± 4.14) and least in NaOCl + PAA group (62.98 ± 8.17 VKN). There was statistical significant difference between all the groups except between NaOCl + QMix group and NaOCl + EDTA group (F = 12.26, df-3, 36; < 0.001). A negative correlation existed between the calcium loss and reduction in the microhardness of root dentin. Dentin is composed of inorganic components of hard dental tissues, in which calcium and phosphorus are distributed in the form of hydroxyapatite crystals. The Ca/P ratio in hydroxyapatite is approximately 1.67 and it depends on many factors such as level of mineralization, type of crystals, age of tissue and anatomical site.[] During biomechanical preparation, removal of the smear layer requires the use of irrigants that can dissolve both the organic and inorganic components. Since chelating agents act only on the inorganic component of the smear layer, therefore they were used in combination with NaOCl which acts on its organic component altering the mechanical and chemical properties of root dentin. However, reports have indicated that the use of EDTA and NaOCl may lead to dentinal erosion of the root canal walls.[] Further, it has been reported that surface treatment of dentin with different agents may cause alterations in the chemical and structural composition of dentin, which in turn may change its permeability and solubility characteristics[] and subsequently a loss of Ca/P ratio of root dentin resulting in an impact on the microhardness. In the design of this study, the issue of biological variability among different teeth was addressed by comparing the effects of different solutions on the dentin sections from the same patient. This allowed the comparison of the demineralizing capacity of different irrigating solutions on identical sections having similar degree of calcification geometry and surface area. Also, coronal parts of the roots were used to prepare the dentin specimens because of the higher possibility of sclerotic dentin in apical root. The exposure time of all the irrigants was kept 5 min[] and was standardized for all the groups. Atomic absorption spectroscopy is the technique used in this study to evaluate the demineralization effect of the chelators used and to determine the concentration of calcium in each sample. It is a single element technique which is less cost-effective than newer multi-element, techniques such as inductively coupled plasma atomic emission spectrometry. In the present study, the Vickers microhardness test was done to evaluate the surface changes of dental hard tissue specimens, treated with the chemical agents. Further Microhardness determination can provide indirect evidence of mineral loss or gain in the dental hard tissues.[] A significant negative correlation between loss of calcium from root dentin and microhardness was observed in all groups. Panighi and G’Sell found a simple linear correlation between hardness of dentin and calcium ion concentration and between hardness of dentin[] and wettability. In this study, irrigation with 5% NaOCl followed by distilled water as a final rinse eluted minimum amount of calcium from the dentin (2.7 ppm mean) and when compared with other groups, the difference was statistically significant. This finding is in agreement with a study conducted by Lottanti where NaOCl and distilled water hardly eluted any Ca.[] In the control group, no chelating agent was used but still some calcium loss was seen as a result of its mechanical flushing action on smear layer formed on root dentin. Since chelating agents cause demineralization of dentin, resulting in its softening, we find that more Ca loss was seen in groups having chelating agents. This might be the reason why all experimental groups showed significantly lower microhardness when compared with the control group. When the mean Ca loss of Group 3 (5% NaOCl-PAA) was compared with Group 2 (5% NaOCl + EDTA) and Group 4 (5% NaOCl-QMix), the result showed that Group 3 extracted significantly more Ca ions. More Ca loss shown by PAA can be explained by the fact that because of its acidity, the calcium stays in solution and does not reprecipitate. Despite the weak chelating power of this agent, more amounts of Ca ions were eluted from the root canal as compared to EDTA, which is a much stronger chelator but can only be in solution at a slightly alkaline pH.[] However, in our study the pH used was neutral and thus EDTA could not remain precipitated in the solution. Lottanti also found that EDTA removed slightly more smear layer than PAA ( < 0.05).[] More Ca loss shown by PAA could have resulted in a significant reduction in microhardness when compared with EDTA and QMix group. An insignificant difference in Ca loss and microhardness was seen between 5% NaOCl-EDTA and 5% NaOCl-QMix groups. As QMix contains EDTA in its composition along with chlorhexidine and a detergent, the effect of QMix on root dentin could have been almost similar to EDTA. A study carried out by Dai showed that the smear layer removing ability of QMix was comparable to that of 17% EDTA.[] From the present study, it can be concluded that:

The success of endodontic treatment is directly influenced by proper debridement of root canal including smear layer.[] Smear layer acts as a barrier and prevents bacterial invasion of the dentinal tubules.[] However, bacteria might survive and multiply in the smear layer and can also penetrate into dentinal tubules. In addition, smear layer might decrease antimicrobial effectiveness of medicaments or sealing ability of root canal filling. Therefore, it becomes mandatory to remove smear layer from root canal for optimum success of treatment.[] Published literature showed that regardless of instrumentation and irrigation techniques, effectiveness of irrigating solution remains limited in prepared root canals.[] Therefore, improvement of irrigating protocols is essential during root canal treatment in order to achieve better cleaning efficiency in very complex area.[] Although Sodium Hypochlorite (NaOCl) remains gold standard as a result of its antimicrobial effect and tissue dissolution properties, it has no effect on inorganic portion of smear layer. Therefore, NaOCl has been used in association with ethylenediaminetetraacetic acid (EDTA) which acts on inorganic debris formed in instrumented root canals. These irrigants can be used with conventional syringe irrigation or with activation of different new and emerging irrigation activation devices. There are limited published scientific data to compare new and emerging devices and methods for disinfection with conventional syringe irrigation. Therefore, this study was planned to evaluate the efficacy of recently introduced irrigation activation devices EndoActivator (EA), F-File and CanalBrush (CB) on removal of smear layer in coronal, middle and apical third of instrumented root canal. A sample of 80 single rooted premolar teeth extracted for periodontal and orthodontic reasons were used in the study. Inclusion criteria were permanent teeth, with intact apices, no previous endodontic treatment and small restoration. Exclusion criteria were root length shorter than 17 mm, extensive restoration, root caries, cracks and fracture. Specimens were decoronated by diamond disc to get root of 17 mm and thus working length of 16 mm. Root canals were instrumented using crown-down technique with Protaper nickel-titanium (NiTi) rotary (Dentsply — Tulsa Dental, York, PA) F2 to working length. During instrumentation, irrigation was done with 1 ml of 5.0% NaOCl. Upon completion of canal preparation, apexes were sealed with wax to prevent extrusion during final irrigation. Now irrigation was done with 5 ml NaOCl and 5 ml EDTA, with each irrigant being activated according to their assigned group such as: Assessment of reliability of scoring by three observers was done by measuring inter-observer agreement. The level of agreement between any two observers did not exceed above fair agreement (κ < 0.4), hence the final scores were taken on basis of consensus of two observers or in case of total disagreement median value of three observations was taken as final score. Overall as well as for all three locations mean scores were maximum in control group []. Minimum mean score were observed in Group II at coronal and apical locations and overall assessment. Group III had minimum score at middle third. Groups difference in score were found to be significant statistically for all three locations as well as for overall assessment ( < 0.001) []. Inter group comparison revealed that control group had significantly higher scores as compared to all three study groups at all locations as well as for overall assessment ( < 0.001) []. Between different study groups, Group I had significantly higher scores when compared with Groups II and III at coronal location ( < 0.05). Intra group comparison showed that except for control group significant difference ( < 0.001) in scores was observed at different locations in all groups []. At all other locations, coronal location had minimum scores while apical location had maximum scores. Scores at coronal level were significantly lower when compared to middle and apical levels; ( < 0.001) whereas scores at middle were significantly lower as compared to those at apical levels in Group I, II and III []. The removal of any vital and necrotic pulp tissue, microorganisms and their toxins, along with smear layer is essential for endodontic success. Studies have shown that currently used method of instrumentation especially rotary instrumentation technique produce a smear layer that covers root canal walls and block opening of dentinal tubules.[] Therefore, this study evaluated the efficacy of irrigation activation devices F-File, CB and EA on removal of smear layer. SEM evaluation in control group revealed presence of smear layer in entire root canal wall and very few dentinal tubules were open [] because mechanical flushing action created by conventional handheld syringe needle irrigation is relatively weak []. After conventional syringe needle irrigation, irrigating solution was delivered only 1 mm deeper than tip of needle.[] Because needle tip is often located in coronal third of narrow canal or, at best, middle third of a wide canal,[] penetration depth of irrigating solution are therefore limited. Furthermore, irrigating solution may not have been able to penetrate deep into apical part of root canal because of high surface tension.[] Mean score was maximum in Group I at all three locations except control group [; ]. F-file is rotated at 600 rpm in order to agitate irrigant solution inside root canal to remove remaining dentine debris without further enlarging the canal.[] The tip size (20 mm) and taper (0.04) provide a better clinical relationship to rotary NiTi file presently available than do sonic or ultrasonic instruments. These might be the reasons for this improved irrigation than in control group. Minimum mean scores were observed in Group II at coronal, apical and overall assessment []. It showed that efficiency of CB is more than F-File and EA but the difference was not statistically significant ( > 0.05) []. Similar study[] had showed that Endobrush was significantly better than instrumentation alone in debriding root canal; however, Endobrush was shown to pack debris into apical section of canal after brushing. Our study showed that in all study group, smear layer was found in apical part (represented as high score). It is clear that cleanliness of the coronal part of tooth is more easily achieved than of middle and apical thirds. Group III had minimum scores at middle third. Hence this study revealed that EA also clear more smear layer especially in middle region than CB and F-File []. So removal of smear layer at different regions is as follows: Coronal > middle > apical. Other study showed that EA was reported to be able to effectively clean debris from lateral canals, remove smear layer and dislodge clumps of simulated biofilm within curved canals of molar teeth.[] During use, action of EA tip frequently produces a cloud of debris that can be observed within a fluid-filled pulp chamber. Vibrating the tip, in combination with moving tip up and down in short vertical strokes, synergistically produces a powerful hydrodynamic phenomenon.[] Inter group comparisons [] revealed that control group had significantly higher scores when compared to study groups at three regions and for overall comparison. It was evident that F-File remove more smear layer then control group, which might happened because F-File produce laminar flow[] and created larger amount of fluid shear stress in clinical motion than in static position. This happened due to fluid was displaced by file forcing rapid fluid flow through small gap between file and canal wall. Between control group and Group II, CB remove smear layer significantly []. Irrigating with CB tended to produce cleaner canal walls, but was not significantly better than irrigation alone in removing the smear layer on canal walls. There were limited studies in relation to CB. Further expanded studies are required to explore role in removing smear layer from root canal. No difference between score of F-file and EA as irrigant agitation was found. Paragliola et al[] in their study have assessed only at 1, 3, 5 mm from tip of apex and found similar observation. Another study concluded that EA did not enhance removal of smear layer as compared with conventional Max-I-Probe irrigation with NaOCl and EDTA. A final irrigation with 1 ml of 17% EDTA solution was necessary to remove the smear layer after rotary instrumentation of root canal with or without the use of the EA system.[] It has been reported to cause entrapment of gas in apical third, also referred to as vapor lock, thus hindering complete removal of smear layer from this region.[] This could explain higher debris score for all specimens in apical third. The greater taper of root canal preparation has been shown to result in a better removal of smear layer from apical third. This was attributed to greater penetration of the irrigation needle.[] In Vapor Lock effect liquid have to penetrate closed-end channels at apical portion of root and is dependent on contact angle of liquid and depth and size of channel.[] Under all circumstances, these closed-end microchannels will eventually be flooded after sufficient time[](hours to days). This phenomenon of air entrapment and time frame has practical clinical implications, because endodontic irrigation is performed within a time frame of minutes instead of hours or days, air entrapment in apical portion of canal might preclude this region from contact or disinfection by irrigant. In the classic study by Senia et al[], Gopikrishna et al[], Koçani [] NaOCl did not extend any closer than 3 mm from working length, even after root apex was enlarged to a size 30. This might be attributed to fact that NaOCl reacts with organic material in root canal and quickly forms micro gas bubbles at apical termination that coalesce into an apical vapor lock with subsequent instrumentation. Because apical vapor lock cannot be displaced within a clinically relevant time frame through simple mechanical actions, it prevents further irrigants from flowing into apical region. More importantly, acoustic microstreaming and cavitation can only occur in a liquid phase. Therefore, once a sonic or ultrasonically activated tip leaves the irrigant and enters the apical vapor lock, acoustic microstreaming and/or cavitation becomes physically impossible. A simple method to disrupt vapor lock might be achieved via use of a hand-activated well-fitting root filling material[] that is introduced to working length after instrumentation with corresponding NiTi rotary instrument. This method, although cumbersome, eliminates vapor lock because space previously occupied by air is replaced by root filling material, carrying with it a film of irrigant to working length. Thus, in this study, all teeth were found to have debris present, especially in apical third. All techniques tested showed different degree of effectiveness in smear layer removal of root canal system. The results demand need for better irrigant protocols to completely remove debris from apical third of canal. e m o v e s m e a r l a y e r m o r e e f f i c i e n t l y f r o m r o o t c a n a l t h a n F - F i l e a n d E A . C B r e m o v e s m e a r l a y e r b e t t e r i n c o r o n a l a n d a p i c a l r e g i o n a n d E A i n m i d d l e t h i r d r e g i o n .

A periapical lesion is a lesion involving the apical area of the tooth. The initial response of the dental pulp to injury is not significantly different from that seen in other tissues. However, the final result can be dramatically different because of rigid dentinal walls of the pulp chamber, which leads to the formation of periapical lesions, which comprises one of the most common infections seen in the oral cavity. Periapical lesions are not caused by microbial infection alone but also by other primary and independent cofactors, such as necrotic pulp, stagnant tissue fluid or root canal fillings.[] Imaging techniques play a very important role in the specialty of endodontics. Periapical lesions accompanying endodontic infections are usually diagnosed and treated based on the initial radiographic findings.[] There have been reports in the literature over the attempts to make a differential diagnosis between cyst and a granuloma based on the radiological features: A cystic image would exhibit well defined margins with hyperostotic borders whereas the granuloma shows indistinct borders. It is generally accepted that the etiology of periapical lesions is derived from the presence and colonization of bacteria in the root canal system. Microorganisms always present themselves in co-aggregation with each other and play a significant role in the ecological regulation and eventual development of an endodontic habitat adapted poly-microbial flora.[] Teeth with pulpal involvement and associated with large periapical radiolucency, that do not heal successfully with routine endodontic therapy are subjected to endodontic surgical procedures to completely eliminate the periapical lesion, thereby allowing complete resolution of the lesion and bone growth. Teeth with periapical lesion often diagnosed as cyst or granuloma or abscess on radiological findings may not be the same. Therefore it is necessary to send the curetted periapical specimen for histopathological examination, to determine the true nature of the lesion. Although the presence of cystic cavity with lining is considered to be diagnostic of periapical cyst, the presence of proliferating epithelium without cystic cavity is also considered to be potential to transform into periapical cyst. A subset of periapical granuloma with epithelium is designated to be early cystic changes or potential cystic changes. Attempts have been made in this regard to elucidate the nature of epithelium using various immunohistochemical markers like cytokeratins. This study was carried out to compare the differentiation of the periapical lesions by both conventional radiography and histopathologic examination in adjunct with the immunohistochemical analysis and also attempts to find a possible co-relation between the conventional radiographic techniques. Ethical clearance was obtained from the institutional ethical committee. Informed consent of each patient was obtained after explaining the clinical procedures and the risks involved. Thirty patients were selected for the study which included teeth with pulpal involvement and associated with large periapical radiolucency i.e. 6 mm to 25 mm, that do not heal successfully with routine endodontic therapy (symptomatic even after three months follow-up) in relation to either maxillary or mandibular anterior teeth. Intraoral periapical (IOPA) radiographs were taken using a bisecting angle technique. The clinical data was not disclosed to the two experienced and specialist observers, and were asked to make a detailed description of the periapical lesions including the size of the lesion mesio-distally and superior-inferiorly. Access cavity preparation, working length determination, thorough chemo-mechanical preparation was done using K files. The root canal space was obturated using standardized gutta percha points. The cavity was then sealed with cavit. (3M ESPE Dental St. Paul, MN USA) Local anesthesia was achieved and a full thickness trapezoidal flap was reflected by placing an incision from the marginal gingiva to the depth of the vestibule. A surgical curette was used to remove each specimen, which was immediately placed in 10% buffered formalin solution. The specimen was later sent for histopathological examination which was subjected to routine H & E staining. Depending on the diagnosis, if required the samples were subjected to immunohistochemical analysis by employing CK-14 stain. The statistical analysis was done using Z-test for proportions with SPSS13 version. A total of thirty patients were selected for the study. They were distributed according to various study groups. The sex distribution revealed that twenty cases were males and remaining were females. The lesions were divided into five different age groups. The lesions obtained were maximum in age range of 20-29 years. 90% of the lesions were found in the maxilla. The radiographs were viewed and four cases were diagnosed as periapical abscess [], eleven cases as periapical granuloma [] and fifteen cases as periapical cyst [] corresponding to 13.33%, 36.67% and 50% respectively. The lesions were measured mesio-distally and superio-inferiorly. The mean mesio-distal measurement was 9.47mm and its standard deviation 4.3mm. The mean superio-inferior measurement was 10.07mm and its standard deviation 4.2mm. The obtained Haematoxylin and Eosin stained sections of periapical lesions were evaluated histopathologically and assessed by Spearman's rank order co-relation test. Two cases were diagnosed as periapical abscess [], twenty cases as periapical granuloma [], five cases as periapical granuloma with cystic potential [] and three cases as periapical cyst [] corresponding to 6.67%, 66.67%, 16.67% and 10% respectively. Five cases of periapical granuloma with cystic potential on immunohistochemical analysis with CK-14 stain confirmed that all the samples were developing periapical cysts []. One case of periapical granuloma and one case of periapical cyst were used as negative control and positive control respectively [Figure ,]. The possible correlation between radiographic and histopathologic findings was calculated using Spearman's rank order co-relation study []. The calculations showed that the co-relation between radiography and histopathology is not statistically significant in most of the cases. There was co-relation in only nine cases (30%) of the total samples of which seven were periapical granuloma and two were periapical cyst. The comparison of results from radiograph and histopathology was done using Z test for proportions ( < 0.05) which indicated the difference in proportions was significant in the cases of periapical granuloma, periapical granuloma with cystic potential and periapical cyst []. Successful root canal therapy includes proper diagnosis, ideal access cavity preparation, thorough chemo-mechanical preparation and a three dimensional obturation with an impermeable seal. The development and persistence of periapical inflammation after completion of root canal treatment can be attributed to a number of factors such as toxic materials, remaining necrotic tissue, bacterial infection or a combination of these. These factors are capable of inducing non-specific inflammatory and/or specific immunologic reactions in the periradicular tissues.[] Various studies have indicated that 75% to 95% of the teeth requiring endodontic treatment can be successfully treated conservatively. The remaining non-resolving fraction of periapical lesions should be treated surgically.[] The principal modality available to manage failure of conventional orthograde endodontic treatment for a large nonhealing periapical lesion is apical surgery with the success rate being 86-92%.[] Endodontic diagnosis depends most commonly on patient's history, radiographs and vitality testing. Radiographs aid the clinician in diagnosing the lesion during intra or inter-operative procedures as well as post-operative evaluation following root canal treatment. The radiographic method can only depict the location and the size of the periapical lesion and not the exact nature of the pathology. Priebe et al in their study concluded that out of 55 lesions, only 13% were diagnosed as periapical cysts and 59% of the 46 lesions were diagnosed as periapical granulomas and abscesses.[] The problem of unreliability in the radiographic interpretation of periapical lesions has been addressed by numerous studies.[] The periapical tissue responses can vary and assessment of a persistent radiolucency can be difficult unless a biopsy is performed. A radiolucency that persists following root canal treatment may be due to the root canal system still being infected, an extra-radicular infection, a periapical true cyst or a periapical scar.[] Therefore it is prudent to submit the tissue postoperatively for the histopathological examination to derive at a final diagnosis. The significance of submitting the specimen for histopathological examination lies in the fact that the chance of accurately interpreting the cystic formation on the basis of appearance of radiograph is poor. It provides an excellent opportunity to obtain complete information regarding the pathology and the microbiology of the lesion. It sharpens the diagnostic acumen related to periapical cyst, periapical granuloma or periapical abscess.[] The incidence of periapical abscess reported in the present study is 6.67%, which is in contrary to the findings of Ramachandran Nair et al and Ricucci et al where it was found it to be 35% and 28% respectively.[] The incidence of periapical granuloma ranges from 46% to 84% according to various studies done by the authors. In the current study, there is 66.66% incidence of granulomas. Our study is in accordance with the previous studies of Sommer et al, winstock et al, Pattersen et al, Celia [] whereas findings are contrary to the study done by Bhaskar, Demenico Ricucci and Lalonde & Luebke et al where they found it to be 48%, 45% and 40% respectively.[] The large variation in the frequency of granuloma occurrence may be due to differences in methods of biopsy collection and histological criteria used for the diagnosis of periapical lesions or may be due to smaller sample size.[] Accumulated data in the review of cytokeratin patterns in the post formative reduced enamel epithelium and the cell rests of Serres and Malassez, indicated that the major keratins consistently found were 13, 14 and 19.[] Gao et al in their immunohistochemical based study on periapical lesions mention that expression of CK-14 in periapical granulomas would suggest an early change towards periapical cyst formation. Further they stated that this expression subsequently was replaced by CK-13 and CK-4, suggesting epithelial change to form a cystic lining is associated with more clearly differentiated phenotype of stratified non-cornifying epithelium but one in which some simple epithelial keratins were co-expressed.[] The findings of strong expression of CK-14 in five periapical granulomas with cystic potential supports the view of Gao Thus it appears that such periapical granulomas are more potential than the conventional granulomas to develop into cyst. In the current study three out of thirty cases (10%) were diagnosed as periapical cysts which is in the reported range of literature (6-84%).[] The inclusion of such potential granulomas along with the true periapical cysts in the present study would increase the incidence of periapical cysts to 27% which is relatively high. In views of Gao et al,[] Safi et al,[] Langeland et al[] factors like serial sectioning, characteristics of the population sample, microscopic interpretation of the specimen and adjunct employment of appropriate CK markers would influence the reporting of incidence rates of periapical cysts in periapical lesions. Regarding the location of the periapical lesions, a study by Nobuharo and Del Rio found majority in the anterior maxilla (47.3%) followed by posterior maxilla, posterior mandible and anterior mandible (8.7%).[] In this study the findings are similar to the above authors i.e 90% in anterior maxilla and remaining in anterior mandible. One of the reasons for the higher incidence of cysts in maxilla is due to the greater quantity of epithelial debris in the maxilla.[] Some earlier studies have attempted to relate histological and radiographic findings in periapical lesions; some authors have stated that a preliminary clinical diagnosis can be made when a lesion is greater than 20 mm in diameter or has a cross-sectional area ≥ 200mm[] White et al related the size and type of the lesion, establishing that cysts tend to be larger than granulomas.[] Mortenson et al stated that a lesion greater than 1.5 cm can be safely classified as cyst.[] In the present study an attempt was made to not only locate or depict the size of the lesion but also to diagnose and categorize them radiographically as periapical granuloma, cyst and abscess and then co-relate the findings with the histopathological diagnosis. The correlation was seen only in 30% of the cases. Although 75% of the teeth requiring endodontic treatment can be successfully treated conservatively the other 25% of the cases definitely require the histopathological examination for accurate and confirmative results thus suggesting that diagnosis cannot be made solely on radiographic examination and histopathological examination is mandatory for the confirmation of the diagnosis. The radiographic diagnosis of periapical lesions remains inconclusive. Although histopathologic examination of periapical lesions gives true nature, the precise nature of certain lesions may be achieved with adjunct use of immunohistochemical markers. Future studies involving cytokeratin like CK-14 and proliferation markers like Ki-67 in adjunct with radiography and extensive follow-up may give better insight in understanding such subsets of periapical granulomas.

The principal aim of dental operator is to carry out the dental procedure with as little pain and discomfort as possible. One of the most distressing aspects of dentistry for the average dental patient is the fear and anxiety caused by the dental environment, particularly the dental injection, i.e., syringe and needle which is referred as “NEEDLE PHOBIA” or BLENOPHOBIA.[] The feeling of needle being attached to a syringe and penetrating the oral mucosa is quite distressing and carries a negative impact on patient's psychology. Epidemiological studies have shown that most of the patients put off their dental visits primarily due to the fear of needles, pain and bodily injury from injection.[] John F. Roberts introduced the jet injection syringe in 1933.[] The introduction of needleless injection into the clinical use in 1947 was the first change in injection technique since Alexander wood's introduction of the needle in 1853.[] It has been used for variety of procedures in both medical and dental fields. In the year 1973, Santangelo, Mott and Stevenson reported 83% patients suffering from leukemia preferred pressure anesthesia over conventional needle anesthesia for bone marrow aspiration and lumbar puncture. In addition, it has been successfully used for insulin delivery, regional and digital blocks, incision of abscess, aspiration biopsy of lymph nodes, repair of laceration, mass immunization, vasectomy, cryosurgery, curettage and cyst excision.[] The first dental study using needleless jet injector was reported by Margetis in 1958.[] Conventional pain control techniques often deal with the pharmacological/sensory component.[] The dental restorative procedures requires a local anesthesia and the anesthetic procedures is most often perceived as invasive, painful due to needle phobia by the patients.[] In response to this perceived aversion, pressure anesthesia was introduced and it inspired many dentists to carry number of clinical and epidemiological studies on human and animal subjects.[] The most interesting thing with these needleless device is the use of pressure to force the anesthetic solution safely into oral tissues which is well-accepted by the patients.[] The anesthetic solution infiltrates the tissue in the tiny droplet form which is immediately taken up by the myelin sheath of the nerve with an onset of action of approximately 1 ms. This amount is most effective in localizing its effect without producing an effect on systemic blood level hence helpful in cardiac patients. In dentistry it can be successfully used as anesthetic device for curettage and scaling, mental and nasopalatine blocks, cementing crowns, jackets, bands and clamps; copper tube impressions, gingivectomies, direct pulp injections, biopsies and pointing abscesses for incision and drainage procedures.[] The objective of the needless jet injection is to deliver local anesthesia without subjecting the patient to the unpleasant experience of facing the “needle” and to achieve adequate anesthesia that should be acceptable to the patients.[] Further before it is likely to replace traditional needle injection, it should also be preferred by the patients. Thus, the present study was carried out with the following objectives: The permission for the study was taken from Institutional Ethics Committee. Informed written consent for carrying out the treatment and their willful participation in the study was taken from the patients prior to starting of treatment. #text Pain control during dental operative procedures is very challenging task for the clinician. Pain control using local anesthetics is widely accepted and use of “needle” for administration is considered to be a gold standard; even though, most of the patients are having fearful attitude toward it. To develop a more positive attitude of the patient toward dental procedure adequate anesthesia is essential. Painless and needless anesthesia administration will further enhance patient's co-operation and alleviate his fear. Hence this study was conducted to compare patients’ acceptance and preference to needleless pressure anesthesia with classical local needle infiltration and to evaluate the efficacy of the needleless anesthesia for dental restorative procedures. In our study, 20 non-fearful adult patients requiring bilaterally similar dental restorative procedures were selected. The study was carried out using split-mouth design in two dental visits using classical needle anesthesia and pressure anesthesia. This split-mouth design offers potential saving in the resources. This design removes all components related to differences between subjects from the treatment comparisons thus reducing error variance (noise) and subsequently obtaining a more powerful test.[] Various anesthetic solutions such as lidocaine, articaine, mepivacaine with different concentration ranging from 2% to 5% have been used in previous studies.[] The type amount and concentration of anesthetic solution along with the amount of vasoconstrictor affect the anesthetic result. Bennett in their radiographic and histologic study supported pressure anesthesia as it provides penetration and infiltration roughly comparable to that produced by needle injection to near 1 cm depth, with the use of quantities up to 0.2 ml/injection; as it is the concentration gradient of anesthetic solution diffusing into the surrounding tissue determines how much anesthetic reaches a nerve.[] Dabarakis in their study found that 3% mepivacaine used with pressure anesthesia did not produced pulpal anesthesia.[] Hence, in our study, 2% lignocaine with epinephrine 1:80,000 was used with both the anesthetic techniques for completion of dental restorative procedures. According to literature provided by manufacturer very little anesthetic solution is needed to form a wheal which effectively gives adequate anesthesia to carry out restorative procedures. Hence, 0.3 ml of anesthetic solution was deposited buccally and 0.1 ml lingually and no supplementary anesthesia was required. In the first visit, classical needle anesthesia was administered as it is a gold standard as inadequate anesthesia leads to the development of dental fear.[] Thus for ethical reasons to avoid the development of dental fear the administration of highly efficacious needle anesthesia was delivered during the first visit and pressure anesthesia in the second visit. Patient's acceptance was assessed using Universal pain assessment tool and the effectiveness of anesthesia was done using soft-tissue anesthesia and EPT. Universal pain assessment tool comprises of Verbal descriptor scale, Wong-Baker Facial grimace scale and activity tolerance test. It was found to be more sensitive as compared to Visual analog scale[] and hence it was used in this study. Our study found that 70% patients preferred pressure anesthesia; whereas 10% patients gave preference for both the anesthetic techniques. It might be possible that needle phobic patients might respond more positively to pressure anesthesia. In this study, patients reported significantly more discomfort ( < 0.001) during injection for pressure anesthesia; this may be due to the difficulty in the positioning of anesthetic equipment. The results of our study are in broad agreement with the previous study done by Saravia and Bush[] who reported 75% pediatric patient's preference for pressure anesthesia over the conventional method. Munshi in their study also reported 93% patients acceptance for pressure anesthesia.[] EPT results showed more profound pulpal anesthesia with needle infiltration. But simple restorative procedures such as Class I, Class II and vital pulp therapies can be comfortably completed with pressure anesthesia provided the procedure is completed in 20-25 min. However, for pulpectomy procedures it can be used for intrapulpal injections. These results are in accordance with the study of Dabarakis .[] The results of our study are contrary to Arapostathis who reported more negative experiences with pressure anesthesia using INJEX as 73% children preferred the traditional needle method.[] Similarly, Dabarakis in their study reported only 17.6% patients preference for pressure anesthesia; whereas 52.8% patients preferred classical injection technique.[] In these studies, the anesthetic device used was INJEX in which the anesthetic delivering segment forms a 90° angle with the main body contrary to MADA jet. Its delivery segment forms a 45° angulation with gingiva producing better and easy positioning with complete contact with gingiva, less pressure during administration resulting in less leakage and less bad taste compared to other pressure anesthesia systems which form 90° angle.[] This pressure anesthesia offers advantages such as no needle-stick injury, decreased fear of procedure, faster drug absorption at the injection site and is autoclavable. However the major disadvantage being the initial cost of the equipment, bleeding at the site of injection, popping sound while injecting and most of the clinicians are not familiar with its use.[] Due to rigorous standardization protocols it was difficult to get a large number of patients in stipulated time period; However, post-hoc analysis revealed that this study had sufficient power (>99%) to detect a significant difference in acceptability parameters (e.g., pain, fear, discomfort, bad taste) in the two anesthetic techniques. Two anesthetic techniques were performed at two different visits; hence acclimatization to the anesthetic procedure may have caused reduced fear/pain/discomfort during the second visit. Other methods of enhancing effects of local anesthesia have been earlier evaluated with various degrees of success. The important parameter in improving the quality of anesthesia is the role of premedication. Studies have proved that pre-operative medication improves the efficacy of inferior alveolar nerve block[] and maxillary infiltrations.[] In addition, other injection techniques like X-tip intraosseous injections have delivered high success rate in achieving profound pulpal anesthesia in patients with irreversible pulpitis.[] In this study, though neither the post-operative complications were reported by the patients immediately nor confirmed objectively by the operator. Thus to quantify this factor further studies requiring immediate post-operative examinations with sufficiently large number of patients are required. From the literature review of pressure anesthesia; it is noted that no instances of infections were reported even in immunosuppressive patients. Hence, in today's clinical scenario of rigid infection control, this pressure anesthesia device can play a vital role; however controlled microbiological studies may yield better information. This technique may be particularly beneficial in reducing fear from needle view and contribute to limited dose administration which is proved to be beneficial for patients suffering from systemic disorders. #text

To ensure a successful restorative procedure, the bonded interface area must be capable of withstanding the stresses caused by polymerization shrinkage, occlusal loading and thermal changes. Marginal discoloration, recurrent caries and post-operative sensitivity may be associated with the penetration of bacteria and oral fluid due to an incomplete marginal adaptation at the enamel-dentin resin composite interface.[] There has been increasing interest in the incorporation of fillers into dentin adhesives. These fillers originate from conventional glass fillers, silica fillers, to nanometer-sized aerosol silica.[] For self-priming adhesives and single-step systems that do not require an additional bonding resin layer for coupling resin composite to the primed dentin, inclusion of fillers increases their viscosity that tends to prevent over-thinning of unfilled adhesive layers, thereby preventing incomplete polymerization caused by oxygen inhibition.[] They may also provide stress relief capacities against shrinkage stresses generated during the polymerization of dental composites, in a way that is similar to the use of resin composite liners and flowable composites.[] Manufactures have added filler to adhesives in an attempt to increase the strength of the adhesive, to modify the viscosity and to provide fluoride-releasing and radio-opacity particles.[] The perceived advantages of filler inclusion in dentin adhesives as stress buffer remains unpredictable and has not been substantiated in a recent clinical trial.[] A recent study of Giannini concluded that filler addition to bonding agents can increase the flexural strength and modulus; however, results are product dependent.[] They also suggested that filler addition does not necessarily have to increase the flexural strength and flexural modulus.[] A previous study showed no significant deference in tensile bond strength of filled and unfilled adhesive using low-viscosity composites, while the failure mode of fractures were different.[] However, it is difficult to prove the effect of filler inclusion in dentin adhesives if they are used in combination with flowable composites or different filled adhesives are used or when they are compared with unfilled adhesives that have dissimilar resin compositions.[] Therefore, the objective of this study was to examine the microleakage pattern of resin-dentin/enamel interfaces created by a filled and an unfilled version of the same self-etch bonding (Clearfil SE Bond) after thermo-cycling. A total of 48 non-carious human premolars stored in 0.5% chloramine T at 4°C solution for maximum 1 month following extraction, were used for the experiments. After being cleaned and pumiced, the teeth, including the apical third of their roots, were embedded in cold cure polystyrene resin cylinders (Acropars, Tehran, Iran). The occlusal portion of the tooth was transversally sectioned under water irrigation, allowing the configuration of a flat standard “occlusal” surface 4 mm above the cemento-enamel junction (CEJ). Class V cavity preparations were performed on the buccal surface, with a high speed carbide bur (D and Z, Germany), under water spray. Each bur was used for the preparation of four cavities and was then replaced by a new bur. Preparations were centered on the CEJ and had the following dimensions: 1.5 mm axial depth, 3.0 mm occluso-gingival height (1.0 mm under CEJ and 2.0 mm above CEJ) and 2.0 mm mesio-distal width. Margins received 45° bevel width of 0.5-1.0 mm. Prepared specimens were randomly allocated into two groups ( = 24) in accordance with the filler existence of the adhesive system. For the first group Clearfil SE Bond (Kuraray Medical Inc., Kurashiki, Okayama, Japan), which is a filled two-step self-etch and for the second group experimental SE Bond provided by the same manufacture, as an unfilled bond with the same composition, were used following the manufacturer's instructions. Restorations were finalized with Filtek Z250 composite in 3 increments (first inciso-axially, second gingivo-axially and finally gingivo-incisally) and light cured for 40 s between each increment using a light-emitting diode curing unit (Bluephase, Ivoclar Vivadent, Schaan, Liechtenstein). Teeth were stored in deionized water at 37°C in a humidity chamber (maintained at 95% relative humidity) for 24 h prior to thermo cycling. Thermo cycling was achieved with a programmed robot (Vafaee Manufacture, Tehran, Iran) that alternated the specimens between two temperature controlled water baths at 5°C and 55°C, respectively with a dwell time of 30 s in each bath for 2 days (1,000 cycles). After thermal cycling, teeth in each group were divided into two subgroups ( = 12), specimens of the first subgroup were incubated for 24 h in distilled water at 37°C and for the second group 3 months in the same condition. After the corresponding period for each subgroup the entire surface of each tooth (with the exception of the restoration and 1 mm of tooth structure adjacent to the restoration) was covered with two layers of nail varnish (Max Factor, France) to prevent dye penetration into the tooth except at the resin-tooth interface. The specimens were placed in 50% silver nitrate for 24 h at 37°C. After washing the teeth, they were sectioned using an Isomet slow-speed saw (Buehler Ltd, Evanston, IL, USA) with a diamond blade in water, longitudinally in a bucco-lingual direction yielding a 1.0-1.5 mm section through the center of the restoration. Dye penetration was measured using a stereomicroscope at × 32 magnification according to the following 5 point interval scale: 0 = no leakage, 1 = leakage restricted to the enamel, 2 = leakage into dentin but not reaching the axial cavity wall, 3 = leakage reaching the axial cavity wall and 4 = leakage beyond the axial cavity wall reaching the pulp. All evaluations were carried out in a blind study with no information to the examiner about the identity of the adhesive system used. One examiner measured each section separately resulting in 24 data points for each subgroup (12 teeth × 2 sections). The microleakage index at resin-dentin/enamel interfaces at two different locations after 24 h and 3 months for an unfilled and filled bonding system are summarized in . The Mann-Whitney tests showed that there was no significant difference between the filled and unfilled adhesive systems ( = 2447.5, = 0.383). The non-parametric Kruskal-Wallis on ranks showed that specimens of 3 months at the cervical location had a significant higher microleakage index (3.1) compared to occlusal specimens at 24 h (1.7) and 3 months (0.6) and the cervical specimens at 24 h (1.2) ( = 38.9, ≤ 0.001). This implies that the microleakage index after 24 h for the cervical and occlusal location were not significantly different. Furthermore, there was no significant different between leakage at 24 h and 3 months at the occlusal surface. SEM analysis revealed that unfilled SE bond can better infiltrate the dentinal tubules than filled SE bond by having longer resin tags []. In the current study significantly greater leakage at the gingival wall was indicated compare to occlusal wall for all groups. This finding is in agreement with some authors, who used different combinations of bonding agents and resin composites in both Class II and Class V restorations.[] The higher leakage scores detected in gingival wall compare with occlusal wall can be related to the structure of these two walls. Enamel thickness is greater in occlusal wall compare to gingival wall and bonding to enamel is a relatively simple process without major technical requirements or difficulties. On the other hand, bonding to dentin presents a much greater challenge, as it contains a substantial proportion of water and organic materials, which presents a moist surface that impairs the bonding mechanism.[] Moreover, it has been indicated that marginal dentin reveals more sensitivity to microleakage after thermo-cycling. Microleakage is frequently used for assessing the quality of the tooth/restoration interface.[] Dye penetration measured on sections of restored tooth is the most common technique for evaluating microleakage at the tooth/restoration interface.[] Although this method is simple, economic and fast the subjectivity of reading the specimens has been noted as being a shortcoming relating to this methodology. To overcome this problem all evaluations were carried out in a blind study. Clearfill SE Bond was used in this study as it was used in different previous studies and the results could be compared in this way. Self-etching systems are generally considered less technique-sensitive, when compared with systems that utilize separate acid conditioning and rinsing steps. Clearfil SE Bond contains 10% silanated colloidal silica particles in micro size.[] To compare the influence of presence of fillers in adhesive system, an experimental unfilled SE bond was provided by the factory. Results indicated no significant difference between microleakage in SE Bond and experimental SE Bond. Cardoso in their study also found no statistical difference between microleakage in Prime and Bond NT and Exp. NT (NT contains nanofillers, Exp. NT without filler). They showed that these two adhesives were quite successful in reducing leakage at dentin margins and resulted in virtually no leakage at the enamel margins.[] Nunes evaluated the effects of adhesive composition on microtensile bond strength (μTBS) to human dentin and they reported no significant difference between μTBS of filled and unfilled NT.[] In contrast they found a statistically significant difference between μTBS of Single Bond and Exp. Single Bond (SB unfilled, Exp. SB 10% ZrO/SiO). This can be explained by the different in size of fillers as NT contains nanofillers, which theoretically can penetrate through the demineralized dentine substrate specially the spaces between collagen fibrils,[] while in Exp. SB Z100 fillers were added and they are in micro size (0.6 μm). The width of the interfibrillar spaces is about 20 μm,[] therefore, the etched dentin may act as a sieve, with filler accumulating on the top and obstructing the resin penetration into the dentin below. It needs to be mentioned that Tay found that also nanofillers from the adhesive layer were congested around the tubular orifices, but were not found within the interfibrillar spaces of the hybrid layer.[] SEM analysis in this study revealed, the unfilled adhesive performed slightly better than the filled SE bond, as the length of the resin tags were almost 3 times than the filled adhesive []. Both filled and unfilled SE bond revealed the presence of incompletely infiltrated collagen fibrils. More over the presence of resin tags in filled adhesive was more irregular, which can be a prove for congestion of filler particles within the interfibrillar space. There is an ongoing debate on the importance of the resin tags influence on the bond strength to dentine. Recently Lohbauer reported that resin tag formation did not influence the μTBS, which is also unrelated to the C-factor.[] Filled adhesives were expected to act as an intermediate shock-absorbing elastic layer between resin composite and dentin, thus increasing the bond strength to dentin.[] Many studies evaluated comparisons between filled and unfilled adhesives; however, the advantages of these adhesives as stress buffers remain unpredictable,[] and have not been substantiated with bond tests[] and a clinical trial.[] According to the results of the current study no statistically significant difference was observed between microleakage of SE Bond (filled) and Exp. SE Bond (unfilled) before and after 3 months water storage. These findings shows that presence of filler has no influence on microleakage of SE Bond immediately or after 3 months hydrolytic degradation. The simplified procedure for dental bonding systems are of great interest, but the presence of fillers appears questionable if the bonding systems are employed in only one layer. However, future studies are needed to confirm this. e a p p l i c a t i o n o f f i l l e r i n a s e l f - e t c h a d h e s i v e s y s t e m h a d n o i n f l u e n c e o n m a r g i n a l l e a k a g e a t b o t h t h e e n a m e l a n d d e n t i n m a r g i n s . W h i l e t h e u n f i l l e d a d h e s i v e i n f i l t r a t e b e t t e r t h a n t h e f i l l e d a d h e s i v e , i t s l o n g - t e r m p e r f o r m a n c e i s n o t p r o m i s i n g .

Successful endodontic therapy involves instrumentation of the root canal to produce a surface free from debris and organic matter and obturation to achieve an optimally-sealed canal.[] Gutta-percha has been universally accepted as the gold standard for root filling materials and the material against which others are compared. However, gutta-percha even with conventional sealers is not capable of preventing leakage. Hence materials with adhesive properties are advocated.[] The ability of root canal fillings materials to penetrate into dentinal tubules is regarded as a relevant aspect in prevention of reinfection of the dentinal tubules.[] A relatively new material, Resilon-Epiphany system promises such tubular adhesion. Scanning electron microscopy (SEM) has been a commonly used testing method to evaluate sealer-dentin interface. However, this technique needs a special processing which would lead to shrinkage of the lower half of the hybrid layer.[] This study evaluated the percentage and average depth of dentinal tubule sealer penetration in the coronal, middle and apical thirds of teeth obturated with the Resilon-Epiphany obturation system using confocal laser scanning microscopy (CLSM). Ten maxillary central incisors were collected from the Department of oral surgery and cleaned of soft tissue debris and calculus with a bur. The teeth were disinfected using 2.5% sodium hypochlorite (NaOCl) solution (Prevest denpro Ltd, India) for half an hour and then stored in physiological saline. Access cavities were prepared using Endo-Access bur (Dentsply-Maillefer, Ballaigues, Switzerland). A K-file (Mani Inc., Japan) was placed in the canal and a periapical radiograph was taken to determine working length. All the teeth were enlarged using ProTaper rotary files and X-smart, endomotor (Dentsply-Maillefer, Ballaigues, Switzerland) up to F3 file size. Along with this instrumentation, irrigation was done between uses of each file, delivering 10 ml of 3% NaOCl (Prevest Denpro Ltd., India). After the instrumentation was completed, all the specimens received a final flush with a 10 ml of neutralized 17% ethylenediaminetetraacetic acid (EDTA), (Dent Wash-Prime Dental, India) followed by 10 ml of NaOCl to remove the smear layer. After that, 10 ml of sterile water was used to remove any remaining NaOCl residue. The canals were dried with sterile paper points (Dentsply-Maillefer, Ballaigues, Switzerland). To apply the epiphany primer, (Pentron Clinical Technologies, LLC Wallingford, CT, USA) a paper point was placed to working length and primer was applied to the paper point, letting the point wick the primer to the apex. To facilitate fluorescence under Confocal Laser Microscopy, Epiphany sealer was mixed with Rhodamine B Isothiyocyanate fluorescent dye (maximum absorption = 570 nm, maximum emission = 720 nm) to an approximate concentration of 0.1%. A mixture of fluorescent dye and sealer (Pentron Clinical Technologies, LLC Wallingford, CT, USA) was placed along the entire length of the canal using a K-file in anticlockwise direction. Resilon size 30, 0.06 taper cones were selected as master cones and the fit was verified by placing it to the working length. Canals were obturated using lateral condensation technique along with the use of Resilon accessory cones (Pentron Clinical Technologies, LLC Wallingford, CT, USA). Excess of Resilon were removed using heated instrument and vertically compacted at the orifices using hand pluggers. The material at the orifice was light cured for 40 s. Radiographs were taken to ensure that no voids were present. Access cavities were sealed with Cavit (3M ESPE MN, U.S.A). o n s i s t e n t f l u o r e s c e n t s e a l e r r i n g w a s s e e n a r o u n d t h e c a n a l i n a l l 1 0 × s e c t i o n s . A l l s e c t i o n s w e r e c a l c u l a t e d f o r p e r c e n t a g e s a n d a v e r a g e d e p t h s o f s e a l e r p e n e t r a t i o n . It has been observed that, regardless of the chemical composition of endodontic sealers, no root canal sealer has all the ideal properties and most leak with time, either through poor initial adaptation to the canal walls or due to solubility and disintegration of the sealer. Although gutta-percha has many desirable properties, it does not bond to the internal tooth structure, resulting in the absence of a complete seal. This produces a poor barrier to bacterial microleakage and is considered to be one of the weakest points in root canal therapy. Many attempts have been made to resolve the problem through using different sealers and variations in obturation techniques. These methods have reduced microleakage to a certain degree but have still failed to eliminate the problem.[] The concept of adhesive dentin bonding procedures for endodontic treatment has been previously investigated. It was found that resin-based adhesive materials may have the potential to reduce the degree of microleakage from both apical and coronal directions of the root canal system. Resilon looks and handles like gutta-percha and is therefore called Resin-Percha.[] The penetration into deninal tubule of the self-etching primer and composite sealer may prevent shrinkage of the resin filling away from the dentine wall and aid in sealing the roots. These materials have been shown to be biocompatible, non-cytotoxic and non-mutagenic and have been approved for endodontic use by the Food and Drug Administration of the United States.[] Epiphany bonds both to primer and Resilon cones forming a Resilon monoblock system, which has good adaptation. As Resilon is applied using a methacrylate-based sealer to self-etching primer-treated root dentin, it contains two interfaces, one between the sealer and primed dentin and the other between the sealer and Resilon and hence may be classified as a type of secondary monoblock. Resilon-filled root canals have been found to be better than conventionally gutta-percha — filled canals in resisting bacterial leakage and improving the fracture resistance of endodontically treated teeth.[] A volume of 3 ml of 17% EDTA for 3 min, followed by a 3 ml rinse with 1% NaOCl, has proved to be sufficient to achieve complete smear layer removal which is necessary for adequate bonding. However, manufactures warn against using NaOCl as the final rinse after removal of the smear layer. They recommend 6% NaOCl followed by 17% EDTA with a final rinse with sterile water or Chlorhexidine.[] Several test methods have been used to evaluate the sealing ability of obturated root canals, like Linear measurement of tracer dye or isotope), Fluid filtration models, Bacterial leakage models, Electro-chemical models, Spectrophotometry and SEM. However a single conclusive, advocative method, technique, or material over any other has still not been reached. The variety of evaluative methodologies and their assessment parameters are major reasons for such disagreement.[] SEM has been a commonly used testing method to evaluate sealer-dentin interface. However, this technique needs a special processing of the specimens. The microscopic images would thus show reduced thickness of the hybrid layer as processing would lead to shrinkage of the lower half of the hybrid layer where as in confocal microscope no such processing is needed and possibility of evaluating the different bonding agents is that can be kept humid during the examination which results in decreased risk of shrinkage artifacts.[] Measurement of sealer penetration into the dentinal tubules was the main parameter in this study. Epiphany sealer was labeled with rhodamine B isothiyocyanate fluorescent dye to facilitate fluorescence under confocal laser microscopy. Since there is no specific methodology devised for incorporating dyes into dental materials in the literature,[] current study tried to maintain standardization for the amount of dye to be incorporated-for 10 parts of sealer, 1 part of dye powder was taken and mixed manually so that approximate concentration of 0.1% of dye is maintained. The amount of dye used is so less that it would not alter the properties of sealer.[] Sealer penetration was measured in two different ways to get accurate results. Firstly, sealer penetration of any distance was measured by using Adobe Photoshop CS3. Secondly, the average depth of sealer penetration was measured at four different standardized points using ruler tool in the LSM 5 image analyzer and mean value was taken. The results showed that higher percentage and average depth of sealer penetration was found in the coronal section followed by middle section and the apical section This might be explained by a significantly higher density of dentinal tubules with greater diameters in the root's coronal third (about 40,000) compared with middle and apical thirds (about 14,400). The results were consistent with the studies conducted by Chadha , Patel , Gharib and Chandra , wherein they found lower average depth of sealer penetration in the apical sections compared with middle and coronal sections and also lower percentage of sealer penetration in the apical sections.[] Pawinńska with SEM analysis found good adhesion of epiphany sealer to the canal walls with visible tags in dentine tubules. Good adherence was also found of epiphany to Resilon and Resilon to root dentin and according to Fathia Resilon/Epiphany sealer had better apical sealing ability than gutta-percha/AH-Plus sealer.[] Within the parameters of the study, it concludes that epiphany sealer penetrates higher in coronal thirds of root compared to middle and apical thirds of the root. These results can be attributed to better adaptation of the obturating material to the root canal.

Restoration of endodontically treated teeth may pose a restorative challenge because of damage by caries and endodontic procedures.[] To provide sufficient retention to the core, a radicular support is provided in the form of a dowel.[] Some authors believed that placement of dowel may reinforce the tooth,[] but unnecessary removal of radicular dentin during dowel space preparation may negate any reinforcement gains from dowel placement.[] Traditionally custom metallic dowels were used which involves a two stage fabrication and placement procedure. Subsequently prefabricated posts were used. These prefabricated posts were made up of metal, ceramics or glass fiber. The teeth restored with cast posts usually show higher fracture resistance than fiber post but fail in an unrepairable manner.[] This difference in failure mode is attributed to the difference in the modulus of elasticity of cast and fiber dowels.[] Because the fiber dowels have modulus of elasticity similar to dentin, there is an even distribution and transfer of stress to the radicular dentin.[] Various authors have documented the usefulness of placing a ‘360 degree of metal collar of crown surrounding the dentin’, also known as ferrule, to increase the resistance form and prevent fracture of the restored tooth.[] There are conflicting reports regarding the effect of ferrule on fracture resistance. One study concluded that 1 mm of ferrule can significantly improve the fracture resistance while some studies recommended 2 mm of ferrule height.[] Schmitter et al[] reported that teeth with 2 mm had fractures on remote areas which pose a problem during restoration. Some studies comparing uniform and non-uniform ferrule reported a higher fracture resistance in uniform ferrule group,[] yet one study[] evaluating fiber posts reported no change in fracture resistance in uniform and non-uniform ferrule. Some authors have reported that ferrule placement does not improve the fracture resistance of teeth restored with cast dowels and prefabricated metal dowels.[] However majority of the studies have utilized single static load to fracture the specimen, which is different from the actual clinical scenario. The present study evaluated the fracture resistance of teeth restored with cast and fiber dowels, with or without a 2 mm ferrule preparation, under cyclic loading. The fracture mode was classified as repairable or non-repairable. The null hypothesis was that the ferrule placement and/or type of post have no effect of fracture resistance. Fifty extracted human permanent mandibular premolars were used in the study. The selected teeth had approximately similar dimensions with a mesio-distal width of 5.0-5.5mm and bucco-lingual width of 7-8 mm. Teeth with caries or restoration on the cervical third, large root canals or cracks/fissures were excluded from the study. The external debris was cleaned with a hand scaler and the samples were used within one month of their extraction. Ten teeth were kept as control and received no treatment (Group I, control group). The anatomic crowns were sectioned perpendicular to the long axis of the tooth, up to 2 mm from facial cemento-enamel junction (CEJ) in the samples with ferrule ( = 20, FR groups), and up to Facial CEJ in samples without ferrule ( = 20, NFR groups) []. The roots were embedded in acrylic moulds with epoxy resin liner to simulate periodontal ligament, in a manner that 2 mm of tooth height apical to facial CEJ was exposed. The root canals were then manually prepared till apical size #60 using a balanced force technique. The canals were copiously irrigated between change of each file, with sodium hypochlorite (5.25%) and EDTA solution (17%) with a 27-gauge needle and a final rinse with 10ml of distilled water, followed by a thermo-plasticized gutta-percha filling. The canal entrances were sealed with a non eugenol temporary filling material (Cavit-G 3M ESPE). The specimens were stored at 100% humidity at 37°C for 7 days. The samples were divided into four groups ( = 10), according to the ferrule and dowel method used. Group II / CD-NFR (cast dowel no ferrule): Standardized dowel spaces were prepared to a depth of 9 mm using Gates-Glidden drills followed by a commercially available dowel drill (ParaPost Fiber Lux, Coltene Whaledent). The impression of the dowel space was made with a plastic burn-out casting dowel, adapted to the canal with Duralay acrylic resin (Reliance Dental Manufacturing Company, Chicago, IL, USA). The core was build up with Duralay acrylic resin with a height of 5 mm, corresponding to the core formers of fiber dowel system. A circumferential shoulder of 1 mm was kept. The resin patterns were cast in Ni-Cr alloy (Heraus Kulzer, Hanau, Germany), sandblasted and luted in the canals with zinc phosphate cement (SS White, Rio de Janeiro, RJ, Brazil). Group III / CD-FR (cast dowel 2 mm ferrule): A 2 mm high circular ferrule was prepared with a high speed flat end tapered diamond cutting instruments (Brassler, USA), with circumferential shoulder of 1 mm width. Dowel spaces were prepared to a depth of 11 mm. Cast dowel and cores were fabricated in a manner similar to group I. Group IV / FD-NFR (fiber dowel no ferrule): Standardized dowel spaces were prepared to a depth of 9 mm. The prepared dowel spaces were dried with #60 absorbent paper points. Commercially available dual cure resin cement (ParaCore automix dual cure, Coltene Whaledent) was used to cement the dowels. ParaBond Non-Rinse Conditioner was applied to the canals with the help of #60 absorbent paper points. Mixture of Adhesive A and Adhesive B was applied and excess was removed with brush. The pastes of ParaCore automix white were mixed with the help of mixing tip and were applied into the canal with Lentulo-spiral no. 40 (Dentsply Maillefer, Ballaigues, Switzerland). Small amount of the mixture of the cement was applied to the ParaPost and the dowel was gently seated into the dowel space with the help of finger pressure. The samples were cured for 40 seconds. Core build up was done with manufacturers’ supplied core formers and the core height was trimmed to 5 mm with circumferential shoulder of 1 mm width. The samples were stored at 100% humidity at 37°C for 7 days. Group V / FD-FR (fiber dowel 2 mm ferrule): As in group II, a circumferential ferrule of 2 mm height was prepared with 1 mm shoulder width. Fiber dowels were luted and core build up was done as in group III. Coronal impressions were made using a polyvinylsiloxane impression material and metal ceramic crowns were fabricated using nickel-chromium alloy and a veneering of ceramic. The crowns were cemented with glass-ionomer cement (GC Fuji I, Tokyo, Japan). The specimens were subjected to cyclic loading of 150,000 cycles at 60N (simulating 6 months of oral masticatory stresses) at a frequency of 50Hz. The fracture resistance was measured with Universal Instron testing machine (Zwick GmbH and Co., postf. 4350, D-7900u/m, Germany). A compressive load was applied on the lingual ridge of buccal cusp. The acrylic block was placed at an angulation to provide a 45° angle between the long axis of tooth and 5 mm wide spherical loading tip. The samples were stressed to failure at a crosshead speed of 0.5mm/minute until a first major load drop. The load was recorded in newtons. The specimens were analyzed to ascertain the mode of failure and were divided into two groups: Repairable (fractures above cemento-enamel junction (CEJ), horizontal cervical fracture, core-tooth fracture); and non-repairable (oblique fracture entirely below CEJ, fracture in the middle or apical third of the root, vertical root fracture) []. The data was recorded and was statistically analyzed using one way analysis of variance for the fracture resistance and Fisher's exact test for the mode of fracture. Pairwise multiple comparison procedures for the fracture resistance of different groups were carried out using Holm-Sidak method using the program BioEstat (version 4.0; Mamiraua Institute, Belem, Brazil). #text The endodontically treated are usually more prone to fracture because of loss of the structural integrity.[] Other factors that could affect the fracture resistance of root canal treated teeth are stress during obtuaration; post space preparation, inappropriate use of the tooth as abutment,[] age of the patient[] and reduced proprioception.[] The restoration of a root canal treated tooth should prevent the tooth from fracture under masticatory stresses. In cases of extensive coronal damage, dowel placement will provide support to the core.[] The null hypothesis that ferrule placement and/or type of post have no effect of fracture resistance was rejected on the basis of data obtained. However, the fracture resistance difference between the non ferrule groups was not statistically significant. The decision to place a dowel depends upon the extent of structural loss and amount of functional forces.[] It has been well reported in the literature that fractures resistance of endodontically treated teeth with loss of one or more walls, is improved after placement of dowel, core and a full coverage restoration.[] However, there several studies reporting that placement of dowel (irrespective of its type) does not positively affect the fracture resistance or fracture mode, and even considered dowel placement as a potential risk towards root fracture.[] The higher incidence of ‘catastrophic’ root fracture was reported with the use of metallic dowels.[] In the present study, the teeth restored with cast dowel and 2 mm ferrule demonstrated highest fracture resistance among all the restored groups. However, it gave maximum incidence of non-repairable root fracture. The metallic posts have higher modulus of elasticity (MOE) than radicular dentin and cause stress concentrations at the weaker sections of root.[] The fiber dowel with 2 mm ferrule had significantly less incidence of non-repairable fractures but had less fracture resistance than cast dowel group with ferrule. The fiber dowels have a low MOE and dissipate stress evenly along the root surface.[] Moreover, the low MOE acts as a protective mechanism fracturing the fiber dowel before tooth fracture. This was evident in the teeth restored with fiber dowels in the present study where majority of samples of FD-NFR group fractured at core tooth junction. Placement of ferrule enhanced the fracture resistance of restored teeth, both in cast and fiber dowel groups. Ferrule effect is provided by the encircling of parallel walls of dentin crown, thus decreasing the splitting stresses within the tooth.[] It has been shown that teeth with ferrule have high fracture resistance and could bear more load cycles than teeth restored with ferrule.[] Akkayan[] reported that 2 and 3 mm ferrule significantly improved the fracture resistance as compared with teeth with less/no ferrule. Some authors have even reported that if ferrule is provided, the placement of dowel does not enhance the fracture resistance and the ferrule is the only ‘key element’.[] Regarding the effect of ferrule on fracture mode, it has been reported that teeth with 2 mm ferrule failed in a ‘catastrophic’ manner.[] If the ferrule height is 1 mm or less, the fracture mode was generally decementation or fracture at core-tooth junction. In the present study, the non ferrule cast dowel group had a majority of non-repairable fracture while majority of the teeth restored with fiber dowels fractured at the core-tooth junction. During clinical function, the teeth are unavoidably subjected to dynamic masticatory stresses. These sub-critical stresses are not constant in nature and provide a fatigue loading. To simulate these stresses 150000 cycles of cyclic loading was applied. After cyclic loading, the teeth were subjected to a static load until fracture to determine the fracture resistance. The functional loads play an important role in stress generation in endodontically treated teeth restored with dowels. It has been reported that the effect of cyclic load was more significant than type of post design in generation of stress in the tooth.[] Xible et al[] reported that there was difference between the survival rate and fracture resistance of teeth restored with a 1.5 mm ferrule and different prefabricated posts after mechanical loading and an oblique, static load. A clinical trial with 2 years of follow up reported that there was no difference in the clinical outcome of teeth restored with titanium or glass FRC posts with 2 mm ferrule.[] There are several limitations to the present study. Only a single type of force (60N) was applied during cyclic loading, which does not exactly replicate the type of dynamic masticatory forces on the tooth during function. Also the thermal cycling was not applied which has been shown to affect the luting agent and affect the outcome. There are certain avenues for further research. These include thermo-mechanical loading of samples with variable mechanical loading, using the same research protocol. The effect of different ferrule heights, ferrule configuration and different post methods could be evaluated in a clinical research. #text

Bernard-Soulier syndrome (BSS) was first recognized by two French hematologists, Jean Bernard and Jean Pierre Soulier in the year 1948 in a young male patient who has prolonged bleeding time, mild to moderate thrombocytopenia and very large platelets.[] It is known by other names such as hemorrhagiparous thrombocytic dystrophy, congenital hemorrhagiparous thrombocytic dystrophy and giant platelet syndrome. This syndrome is extremely rare as only ˜100 cases have been reported in published articles, mostly in the populations of Japan, Europe and North America. Prevalence has been estimated at <1/1,000,000.[] In India the reported cases are 27 until date.[] The mode of inheritance is usually autosomal recessive with autosomal dominant pattern seen in isolated cases. The disorder is caused by a deficiency in glycoprotein (GP) Ib/IX/V,[] which is a protein found on the surface of platelets. The GPIb-IX-V complex is composed of 4 transmembrane polypeptide subunits, disulfide-linked alpha and beta subunits of the GPIb and the noncovalently associated subunits GPIX and GPV[] []. The gene of each of the subunits has been cloned and their chromosomal locations are identified as follows: GPIb-alpha gene (chromosome 17),[] GPIb-beta gene (22q11.2),[] GPIX gene (3q21),[] and GPV gene (3q29).[] This protein is essential to the aggregation of platelets around injured blood vessels via a receptor to the von Willebrand factor (vWF) residing on the GPIb-alpha subunits This complex is involved in hemostasis. BSS can occur due to a defect in any of the subunits of the GPIb/IX/V complex. The majority of cases are due to defect in the GPIb-alpha subunit. The defect in GPIb-alpha subunit can be grouped as either qualitative or quantitative. The definitive diagnosis of BSS is made by identifying isolated defective ristocetin-induced agglutination when placed in an aggregometer. The diagnosis may be confirmed biochemically or by genotyping. Neutrophil inclusions are absent in peripheral smear in BSS.[] Four different features of BSS may contribute to the hemorrhagic diathesis: Thrombocytopenia, abnormal platelet interaction with vWF, abnormal platelet interaction with thrombin and abnormal platelet coagulant activity. Oral health care providers must be aware of the impact of bleeding disorders. In BSS patients, even the routine dental treatment cause a life-threatening situation; hence, they must be given special care. There are no documented cases of root canal treatment performed in patient with BSS. Provided herein is a case report of a female patient who presented to our department with BSS. e p r e s e n t c a s e r e p o r t i s a b o u t a 3 0 - y e a r - o l d f e m a l e p a t i e n t w h o r e p o r t e d w i t h a c h i e f c o m p l a i n t o f s w e l l i n g i n r e l a t i o n t o r i g h t m a x i l l a r y c a n i n e f o r 5 d a y s . D e n t a l e x a m i n a t i o n r e v e a l e d a r i g h t m a x i l l a r y c a n i n e w i t h a d e e p c a r i o u s l e s i o n . T h e p a t i e n t a l s o g a v e h i s t o r y o f e p i s o d i c s w e l l i n g i n t h e i n v o l v e d t o o t h . C l i n i c a l d i a g n o s i s w a s p u l p a l n e c r o s i s w i t h p e r i a p i c a l a b s c e s s a n d s i n u s d i s c h a r g e . T h e p a t i e n t g a v e a h i s t o r y o f B S S w h i c h w a s d i a g n o s e d a t t h e a g e o f 5 y e a r s . H e r p a r e n t s , s i b l i n g s a n d h e r c h i l d r e n w e r e f r e e o f B S S s y m p t o m s . The management of patients with bleeding disorders depends on the severity of the condition and the invasiveness of the planned dental procedure. The patients require transfusion in most cases. The benefits of receiving the transfusions must be evaluated against the risks of exposure. Repeated exposure to blood products raises concern for alloimmunization and platelet refractoriness. Factor VIIa can also be used as alternative drug regimen, but at times needs to be supplemented with platelet transfusion. This can result in a reduction in the perioperative use of blood products. However, it should be noted that factor VIIa may result in an increased risk of thromboembolic events.-[] When platelet transfusion is planned, human leukocyte antigen matching should take place if possible. Desmopressin administration have been shown to shorten the bleeding time in some patients. As a rather simple genetic defect, BSS could be a candidate for future gene replacement therapy using virally transduced megakaryocyte progenitors. In patients with BSS, nerve-block anesthetic injections are contraindicated unless there is no better alternative and prophylaxis is provided, as the anesthetic solution is deposited in a highly vascularized area, which carries a risk of hematoma formation.[] Anesthetic infiltration and intraligamentary anesthesia are potential alternatives to nerve block in many cases. An anesthetic with a vasoconstrictor should compaction technique. The access cavity was then restored with posterior composite filling (P60; 3M Dental be used when possible. Alternative techniques, including sedation with diazepam or nitrous oxide-oxygen analgesia, can be employed to reduce or eliminate the need for anesthesia. The patients require infusion of 1 bottle platelet rich plasma prior to procedure and tranexamic acid post-operatively. In our case, we did not require local anesthetic infiltration since the tooth was non-vital. As no much bleeding was contemplated post-operatively, as the tooth was non-vital, we did not give tranexamic in our case. Care should be taken to avoid injuring the gingiva while placing rubber dam clamps, matrices and wedges, alternately widgets may be placed. A rubber dam should be used to prevent laceration of soft-tissues by the cutting instruments. Saliva ejectors and high-speed suction can injure the mucosa in the floor of the mouth and cause hematoma or ecchymosis; thus, they should be used carefully. A gauze piece may be placed below the saliva ejector to prevent trauma to the delicate tissues in the floor of mouth. Endodontic therapy is preferred over extraction whenever possible in these patients. Endodontic therapy does not usually pose any significant risk of bleeding and can be performed routinely.[] Preventive and restorative dentistry are of particular importance to BSS patient since early dental treatment minimizes the need for later oral surgery. Every restoration in a BSS risk of a potential extraction. Care should be taken when prescribing drugs in BSS patients. Non-steroidal anti-inflammatory drug are contraindicated and the patient may be given acetaminophen 650 mg thrice daily. Post-operative antibiotics were advised as this will reduce the late bleeding which is due to the infection. The finish line for crown preparations for BSS patients should be preferably supragingival, so as to reduce the chances of gingival bleeding. Observation time is required for patients with bleeding disorders, it could be hours for those patients with a mild bleeding tendency whilst those with more severe conditions may require supervision overnight in hospital.[] T h e e n d o d o n t i c t r e a t m e n t i n p a t i e n t s w i t h B S S r e q u i r e s a n i n t e r d i s c i p l i n a r y a p p r o a c h i n v o l v i n g a n e n d o d o n t i s t a n d a p h y s i c i a n . A d e q u a t e p r e c a u t i o n s n e e d t o b e t a k e n t o p r e v e n t p o t e n t i a l p r o b l e m s o f h e m o r r h a g e . D r u g s w i t h a n t i p l a t e l e t a c t i v i t y a r e u s u a l l y c o n t r a i n d i c a t e d . N e r v e b l o c k s m u s t b e a v o i d e d . T h e s e p a t i e n t s n e e d t o b e e d u c a t e d a b o u t t h e d i s e a s e a n d t h e n e e d t o a v o i d t r a u m a . O p t i m u m o r a l h y g i e n e p r a c t i c e s s h o u l d b e e m p h a s i z e d t o a v o i d d e n t a l d i s e a s e s .

xref #text A 38-year-old healthy Asian woman was referred to the endodontic clinic by a general dentist. The patient presented to her dentist with a severe pain in left mandibular region ten days ago. The dentist initiated the root canal treatment in left mandibular canine (tooth # 22). During the cleaning and shaping procedure, an ISO # 10 K-file was accidentally fractured in one of the canals. The dentist could not bypass the fractured instrument and hence the case was opted for the referral. On clinical examination, tooth # 22 did not appear to have any coronal morphological variations and was identical to its right counterpart except it was mesially rotated. It was tender to percussion without any evidence of mobility, swelling or sinus tract. The mucosa and the underlying alveolar bone were normal on palpation. Careful pre-operative radiographic examination revealed a two rooted three canaled (i.e. buccal, middle and lingual canals) mandibular canine with a fractured instrument in the middle canal []. Prior to the initiation of root canal treatment, patient was explained about the aberrant root canal morphology and presence of a separated instrument in the middle canal. Patient was administered local anesthetic solution with adrenalin (2% lidocaine with 1: 100000 epinephrine, LOX 2% Neon Lab, India). Under rubber dam isolation (Hygienic, Coltène Whaledent Inc., USA), access opening was re-defined with the help of ultrasonic tips (Pro Ultra Endo Tips No. 2 and 3, Dentsply Maillefer, New York, USA). Careful clinical explorations of access opening with a DG-16 endodontic explorer (Hu-Friedy, Chicago, IL, USA) revealed two root canal orifices in bucco-lingual direction and were present more towards the buccal side. Moreover, a bleeding point was noted lingually at the end of a developmental fusion line indicating the location of unexplored third root canal orifice. Troughing the developmental fusion line lingually with ultrasonic tips under a dental operating microscope (Carl Zeiss Surgical GmbH, Oberkochen, Germany) revealed a third root canal orifice []. All the three root canal orifices were coronally pre-flared using a nickel-titanium (NiTi) ProTaper SX rotary file (Dentsply Maillefer, Ballaigues, Switzerland) with a brushing outstroke action to improve the straight-line access. Dental operating microscope was used to visualize the fractured instrument present in the apical third of the middle canal. The maneuver to bypass the instrument was initiated in the presence of Glyde (Dentsply Maillefer, Tulsa, OK) by wedging an ISO # 8 K-file (Dentsply Maillefer, Tulsa, OK) between the fractured instrument and the canal walls with frequent radiographic checks. The file was then advanced and withdrawn repeatedly in an attempt to widen the canal space and loosen the retained fragment from the root canal. However fractured instrument retrieval was not successful. So it was decided to prepare the entire root canal system, and incorporate the fragment into the obturation. The space created between the fractured instrument and the canal walls was enlarged with the larger sized files sequentially till ISO # 25 K-file. Working lengths for all three canals were determined with an apex locator (Root ZX; Morita, Tokyo, Japan) and were confirmed radiographically []. All three canals were prepared using ProTaper NiTi rotary instruments (Dentsply Maillefer, Ballaigues, Switzerland) according to manufacturer's recommendations. ProTaper S1/S2 rotary files were used with brushing outstroke action. Finishing files F1 and F2 were used with pecking motions until working length was reached. Irrigation was performed using triple distilled water, 2.5% sodium hypochlorite solution (Cmident, India) and 15% EDTA (Largal Ultra, Septodont, Saint Maur des Fosses, France). 2% chlorhexidine digluconate (R4, Septodont, Saint Maur des Fosses, France). were used as the final irrigant. The canals were dried with sterile paper points (Dentsply Maillefer, Tulsa, OK). Calcium hydroxide (Prime Dental Products Pvt. Ltd., Mumbai, India) was used as an inter-appointment medicament. The access cavity was sealed temporarily with intermediate restorative material (IRM, Caulk Dentsply, Milford, DE). The patient was recalled after a week during which the tooth was asymptomatic. The root canals were again irrigated with triple distilled water to remove the intracanal dressing of calcium hydroxide. Canals were dried. Obturation was done by a single cone technique with the use of gutta-percha cones and epoxy resin-based root canal sealer (AH plus sealer, Dentsply Maillefer, Tulsa, OK). In the middle canal, the fractured instrument was incorporated in the obturation []. Tooth was restored using light cured composite resin (Z100; 3M Dental Products). Subsequent follow-up x-ray was taken at 12 months []. Essential pre-requisites for a successful endodontic treatment are careful interpretation of pre-operative radiographs, a thorough knowledge and detailed exploration of internal root canal morphology and its divergence under the magnification and illumination. Straight radiograph provides information about the mesio-distal root canal orientation where as angled radiographs (radiographs at two different horizontal angulations) provide information about the facio-lingual canal position. Any attempt to reduce the required number of radiographs runs the risk of missing information of the complex canal morphology. However, in the present case the mandibular canine was rotated mesially. So, two roots and three canals appeared separate on the radiographs. Hence, there was no need to take any additional angulated radiographs. Furthermore, a careful tracing of periodontal ligament space suggested the presence of two separate roots with three canals. The access cavity was extended buco-lingually with the help of ultrasonic tips in order to conserve the tooth structure while searching for an extra and elusive canal orifice. The internal and external morphological variations of the mandibular canine were clearly appreciable from the radiographic and clinical examination in the present case. Hence, Cone Beam Computed Tomography (CBCT) scan was not done to reduce any extra radiation dosages. Even though the mandibular canine shows a single root and a single root canal with single apical foramen in about 92.2% of cases, clinicians should always search for any possible extra root or canal.[] Root canal morphological variations reported in the mandibular canine include two canals and one apical foramen in 4.9% of cases, two canals and two apical foramens in 1.2% of cases and two different roots each one with one canal in 1.7% of the cases.[] Also two distinct case reports of mandibular canine with three canals in one root or two roots were reported in the past.[] Hence, it is always prudent to consider all anatomical variations in the mandibular canine while performing a root canal treatment. Any pre-determined assumption about the root canal morphology leads to failure to locate any morphological variation, if present. It hinders the effective cleaning and shaping and creates a nidus of infection that directly compromises the long term prognosis of the tooth. In the documented case, the fractured K-file in the middle canal prevented the negotiation and thorough biomechanical preparation of the canal. However, the chances of uneventful retrieval of the fractured instrument were not predictable considering the impossibility of visibility of instrument under dental operating microscope, strategic importance of tooth, the location of the instrument and the limited thickness of the root. Success in management of fractured instrument is defined as the complete removal or complete bypass of the fragment without creating a perforation.[] Hence, decision was made to bypass the instrument under dental operating microscope to enhance the visualization of operating field and to conserve the maximum root structure. During the bypassing maneuver, higher magnification was used with frequent radiographic checks to avoid intra-operative complications like root perforation, ledge formation, pushing the instrument more apically etc. The EDTA was used to soften the root canal wall dentin around separated instruments. It facilitated the negotiation of files for the negotiation of the fragment.[] Internal root canal anatomical variation is a rule rather than exception. So, a clear understanding of pulp anatomy and its variations is essential if effective cleaning, shaping and obturation of the pulp space are to be achieved to assure the successful and predictable outcome of endodontic treatment.[] Mandibular canine with variations in number of roots and root canals should be treated without any pre-operative assumptions regarding its typical single rooted and single canaled anatomy. Thus the present case report highlights the endodontic management of an unusual case of mandibular canine with two roots and three canals. It also highlights the need for use of dental operating microscope and ultrasonics in locating the elusive canal orifices. e s d o n ’ t s e e w h a t m i n d d o e s n ’ t k n o w . S o i t i s i m p o r t a n t t o n o t e t h e i n t e r n a l a n d e x t e r n a l r o o t c a n a l m o r p h o l o g i c a l v a r i a t i o n s s o t h a t s i m i l a r a n a t o m y m a y b e p r e d i c t e d a n d m a n a g e d s u c c e s s f u l l y . S o m e t i m e s r e t r i e v a l o f a f r a c t u r e d i n s t r u m e n t i s i m p o s s i b l e o r u n d e s i r a b l e . I n t h e s e c a s e s , b y p a s s i n g t h e i n s t r u m e n t u n d e r m a g n i f i c a t i o n i s a v a l i d a l t e r n a t i v e , w h i c h c a n l e a d t o a f a v o r a b l e o u t c o m e a s p r e s e n t e d i n t h e g i v e n c a s e .

We previously demonstrated that an inflammatory environment inhibits BMP-2-induced bone mass and osteoblastic differentiation of BMSCs through inflammatory cytokines, including TNF- and IL-1. However, the mechanism responsible for this phenomenon is complicated and unclear. Therefore, we examined the effect of TNF-/IL-1 on BMP-2-induced osteoblastic differentiation. We cultured the multipotent C2C12 cells or preosteoblastic MC3T3-E1 cells in BMP-2- and/or TNF-/IL-1-supplemented medium for several days to study the effects of these inflammatory cytokines on BMP-2-induced osteoblastic differentiation. After 7 days of co-culture, alkaline phosphatase (ALP) staining () demonstrated that presence of TNF- or IL-1 inhibited BMP-2-induced ALP expression in C2C12 cells. Furthermore, quantitation of ALP activity revealed a dose-dependent inhibitory effect of TNF-/IL-1 on BMP-2-induced ALP expression (). Consistent with the effects observed for the early marker ALP, Alizarin red staining and quantification were also decreased in the TNF-/IL-1-treated MC3T3-E1 cells (). We also quantified ALP activity in MC3T3-E1 cells after BMP-2 and/or TNF-/IL-1 treatment and found that the level of ALP activity in MC3T3-E1 cells was reduced and was similar to that observed in C2C12 cells (). These results demonstrate that TNF-/IL-1 alone inhibits BMP-2-induced osteoblastic differentiation and mineralization. To further verify these findings, we quantified the transcription levels of the osteogenic genes such as (), (), and () in C2C12 cells by real-time PCR. BMP-2 increased the transcriptional levels of these genes, but TNF-/IL-1 alone attenuated this effect. Taken together, these data indicate that TNF-/IL-1 alone has a crucial role in the inflammation-mediated suppression of BMP-2-induced osteogenesis. BMP-2 is a potent osteoblastic differentiation factor that signals though Smads. To determine whether TNF-/IL-1 exerts an inhibitory effect on BMP-2-induced osteoblastic differentiation through crosstalk with the canonical BMP/Smad signaling, C2C12 cells were treated with BMP-2 in the presence or absence of TNF-/IL-1. We then analyzed the time- and dose-dependent changes in Smad1/5/8 and Runx2. First, we treated C2C12 cells with BMP-2 in the presence or absence of TNF-/IL-1 for 0, 4, 8, 12, 24, and 48 h. When cells were stimulated with BMP-2 alone, Smad1/5/8 phosphorylation and Runx2 expression were significantly increased. The maximum Smad1/5/8 signal occurred at 2 h posttreatment, and the maximum Runx2 expression occurred at 48 h posttreatment (, left panel). Consequently, the presence of TNF-/IL-1 effectively blocked BMP-2-induced Runx2 expression until 48 h later but did not change the phosphorylation of Smad1/5/8 (, middle and right panels). Based on the results of the time-course study, we next treated the cells with BMP-2 in the presence or absence of TNF-/IL-1 at different concentrations for 2 h to measure the phosphorylation level of Smad1/5/8 and for 48 h to measure the expression level of Runx2 by immunoblotting. Treatment with TNF-/IL-1 alone for 2 h did not affect the BMP-2-induced phosphorylation of smad1/5/8, but the expression of Runx2 was decreased in a dose-dependent manner after treatment with TNF-/IL-1 alone for 48 h (). We next examined whether the BMP-2-induced activation of the BMP/Smad signaling and the transcriptional activity of Runx2 are affected by TNF-/IL-1 treatment. We performed luciferase reporter assays with the pSBE-Luc vector to monitor the activation of BMP/Smad signaling and with the p6XOSE2-Luc vector to monitor the transcriptional activity of Runx2. BMP-2 treatment alone enhanced Smad1/5/8- and Runx2-induced luciferase activity simultaneously. Notably, the presence of TNF- or IL-1 significantly suppressed the transcriptional activity of Runx2 but did not affect the phosphorylation of Smad1/5/8 (). These data indicate that TNF-/IL-1 alone inhibits BMP-2-induced Runx2 expression and activation but does not affect the BMP/Smad signaling. In the canonical BMP/Smad signaling, the phosphorylated Smad proteins are translocated to the nucleus, where they bind specific motifs in promoter regions, recruit , and regulate the transcription of target genes. To further confirm whether the inhibitory effect of TNF-/IL-1 on BMP-2-induced osteoblastic differentiation was mediated by BMP/Smad signaling, we examined the nuclear translocation of Smad1 by immunofluorescence staining. As shown in , BMP-2 induced strong nuclear accumulation of Smad1. However, pretreatment with TNF-/IL-1 did not affect the nuclear translocation of Smad1. Collectively, these data indicate that the inhibitory effect of TNF-/IL-1 on BMP-2-induced Runx2 expression and activation is independent of the BMP/Smad signaling. MAPK signaling exerts a wide range of functions and controls of proliferation, migration, terminal differentiation, and cell death. There is evidence suggesting that inflammatory cytokines, including TNF- and IL-1, activate the MAPK signaling pathway in various cell types. In this study, we observed that the addition of TNF-/IL-1 to the culture medium induced a rapid and strong activation of MAPK signaling, including p38, ERK1/2, and JNK1/2, in C2C12 cells (). The addition of BMP-2 similarly led to the phosphorylation of p38, ERK1/2, and JNK1/2 in C2C12 cells (). To determine whether BMP-2-activated MAPK signaling induces Runx2 expression, C2C12 cells were treated with specific inhibitors of the BMP/Smad, p38, ERK1/2, and JNK1/2 signaling pathways. Because of the positive role of BMP/Smad signaling in regulating Runx2 expression, we pretreated C2C12 cells with a specific Smad1 inhibitor DMH1 to neutralize BMP-2-induced Runx2 expression and activation. We then examined the changes in Runx2 levels or p6XOSE2-luciferase activity, both of which could be affected by MAPKs. Before stimulation, C2C12 cells expressed a basal level of Runx2 protein, pSBE-Luciferase activity, and p6XOSE2-Luciferase activity. After a 1-h pretreatment with DMH1, the BMP-2-induced phosphorylation of Smad1/5/8 ( lane 4) and the upregulation of pSBE-Luciferase activity () were significantly suppressed to basal levels. However, the protein and mRNA levels of Runx2 (, lane 4 and 4b) as well as p6XOSE2-Luciferase activity () were significantly lower in DMH1-pretreated cells than in BMP-2-treated cells. Nevertheless, the levels of Runx2 expression and p6XOSE2-Luciferase activity remained above basal level, suggesting the involvement of other signals in regulating Runx2 expression and activation. We next blocked the different MAPK signaling pathways with the p38-specific inhibitor SB203580, the ERK1/2-specific inhibitor PD98059, and the JNK1/2-specific inhibitor SP600125 in the culture medium. SB203580 or PD98059 displayed synergistic effects with DMH1 to inhibit the BMP-2-induced Runx2 protein and mRNA levels ( lane 5, lane 6, and 4b) to the basal levels. By contrast, SP600125 had no significant effect on Runx2 expression ( lane 7 and 4b). To investigate the potential role of MAPKs in the regulation of the transcriptional activity of Runx2, p6XOSE2-Luciferase activity was measured in cells after inhibitor and BMP-2 treatment. The p6XOSE2-Luciferase activity was significantly decreased by the pretreatment of cells with SB203580 or PD98059 compared with treatment with BMP-2 only, whereas the p6XOSE2-Luciferase activity was not affected by SP600125 inhibition (). We also used Smad1 siRNA to silence expression of Smad1 in C2C12 cells and then block BMP-2-activated BMP/Smad signaling in this study. The expression of Runx2 in mRNA and protein level were similar to that after DMH1 pretreatment (). These results suggest that p38 and ERK1/2 signaling are required in BMP-2-induced Runx2 expression and activation. To examine the effect of the TNF-/IL-1-activated p38 and ERK1/2 pathways on Runx2 expression and activation, C2C12 cells were treated with BMP-2 and TNF-/IL-1 in the presence or absence of p38 and ERK1/2 inhibitors. First, we pretreated C2C12 cells with TNF- for 1 h and then stimulated with BMP-2 for 2 h. We observed that p38 and ERK1/2 signaling were activated at 2 h, whereas the expression of Runx2 protein and p6XOSE2-Luciferase activity were significantly decreased 48 h later (). These results indicate that the activation of p38 and ERK1/2 signaling in these cells suppresses Runx2 expression and activation. Thus, TNF--activated p38 and ERK1/2 signaling opposes BMP-2-activated p38 and ERK1/2 signaling in controlling Runx2 expression and activation. To further investigate these opposing roles, we blocked p38 and ERK1/2 signaling with SB203580 and PD98059, respectively. Blocking of p38 and ERK1/2 signaling restored Runx2 protein levels and p6XOSE2-Luciferase activity but did not affect Smad1/5/8 phosphorylation (). Similar results were also observed in C2C12 cells stimulated with BMP-2 and IL-1 (). These results indicated that the BMP-2- and TNF-/IL-1-activated p38 and ERK1/2 signaling pathways may have an opposing roles in the regulation of Runx2 expression and activation. To further elucidate the role of p38 and ERK1/2 signaling in BMP-2-induced Runx2 activity in an inflammatory environment, we ectopically induced p38 and ERK1/2 signaling activation before exposure to BMP-2 by transfection with CA-MKK3 or CA-MEK1, respectively. Transfection with CA-MKK3 or CA-MEK1 induced strong expression and phosphorylation of p38 and ERK1/2 signaling, respectively, in C2C12 cells without BMP-2 or TNF-/IL-1 treatment (). However, overexpression of p38 and ERK1/2 significantly decreased BMP-2-induced Runx2 expression () and p6XOSE2-Luciferase activity (), indicating that strong activation of p38 and ERK1/2 signaling has a negative role in Runx2 expression and activation. To further investigate whether the activation of p38 and ERK1/2 signaling have any effect in BMP-2-induced osteoblastic differentiation, we measured changes in the early osteogenic marker ALP in C2C12 cells and the later osteogenic marker OCN in MC3T3-E1 cells. Consistent with Runx2, transfection with CA-MKK3 or CA-MEK1 also significantly decreased the expression of ALP () and OCN (). Therefore, these data suggest that strong activation of p38 or ERK1/2 signaling has an inhibitory effect on BMP-2-induced Runx2 expression and osteoblastic differentiation. Increasing exaggerated inflammatory responses and related complications continue to occur after BMP-2/ACS implantation in the clinic and are strongly suspected for the low osteoinductive efficacy of BMP-2. The differentiation factor BMP-2 and the inflammatory cytokines such as TNF- and IL-1 are proved to have opposing roles in osteoblastic differentiation. However, the exact role of TNF-/IL-1 in BMP-2-induced osteoblastic differentiation and the underlying mechanism have remained unclear. Herein, we first demonstrated that TNF-/IL-1 inhibits BMP-2-induced Runx2 expression and activation and subsequent osteoblastic differentiation independent of BMP/Smad signaling. Then, we found that TNF-, IL-1, and BMP-2 alone all could activate p38, ERK1/2, and JNK1/2 signaling, respectively. However, TNF-/IL-1 and BMP-2 has opposing roles on Runx2 expression and activation. Moreover, the presence of TNF-/IL-1 was found to diminish BMP-2-induced Runx2 activity through the activation of p38 and ERK1/2 signaling. Finally, we confirmed that strong activation of p38 and ERK1/2 signaling by transfection with CA-MKK3 or CA-MEK1 significantly attenuated BMP-2-induced Runx2 expression and osteoblastic differentiation, even without stimulation of TNF-/IL-1. Taken together, these data suggest that the TNF-/IL-1- and BMP-2-activated p38 and ERK1/2 signaling have opposing roles and converge on Runx2 to regulate BMP-2-induced osteoblastic differentiation. The study of the interplay between the inflammatory environment and BMP-2 in osteoblastic differentiation is challenging and complicated by at least two issues: the expression profile of inflammatory cytokines is complicated, and these cytokines have multiple roles in osteoblastic differentiation; and BMP-2-induced pathways engage in crosstalk with inflammatory cytokine-induced pathways. Clinical and experimental observations have demonstrated that inflammatory cytokines such as TNF-, IL-1, IL-8, and IL-6 are significantly elevated in sera after BMP-2/ACS implantation or in supernatant after LPS stimulation. Among these elevated inflammatory cytokines, TNF- and IL-1 can be produced by osteoblasts, surrounding stromal or inflammatory cells. The roles of TNF- and IL-1 in the osteogenic differentiation of progenitor cells and bone metabolism are complicated and biphasic. In this study, the presence of TNF-/IL-1 alone could decrease ALP activity and the expression of osteogenic genes in BMP-2-treated C2C12 cells and MC3T3-E1 cells. These results suggest that TNF-/IL-1 have negative roles in BMP-2-induced osteoblastic differentiation. Although TNF- has been shown to inhibit BMP-2-induced osteoblastic differentiation in different cell types, the effects of IL-1 on BMP-2-induced osteoblastic differentiation were not investigated in these previous studies, and the underlying mechanisms responsible for this inhibition are unknown. In the well-documented BMP/Smad signaling pathway, BMP-2 activates Runx2 via the BMP/Smad signaling and subsequently modulates the processes of osteoblastic differentiation. Our data suggested that the presence of TNF-/IL-1 decreased BMP-2-induced Runx2 expression and activation independent of BMP/Smad signaling. Our finding is further supported by one report demonstrating that TNF- decrease the expression of Runx2 and the transcription of osteogenic genes by interfering with the DNA binding of Smad proteins instead of inhibiting phosphorylation of Smad1/5/8 or nuclear translocation of the Smad1/Smad4 complex. However, several reports have demonstrated that BMP/Smad signaling is required for TNF--inhibited osteoblastic differentiation through suppressing Smad1/5/8 phosphorylation and translocation. We suspected the discrepancy between these studies, and our approaches are contributed to the cell types and the differentiation stages of the starting cells used. Therefore, these data suggest the existence of another pathway besides BMP/Smad signaling to regulate BMP-2-induced Runx2 expression and activation. MAPK pathways can be activated by several extracellular stimuli. In our system, we confirmed that TNF-, IL-1, and BMP-2 alone all can activate the p38, ERK1/2, and JNK1/2 signaling in C2C12 cells. In previous studies, MAPK pathways can be activated by inflammatory cytokines, particularly TNF- or IL-1, to inhibit osteoblastic differentiation. Conversely, MAPKs are also required for the induction of an osteoblastic phenotype by BMP-2, leading to mineral deposition . These paradoxical phenomena seem to support the idea that BMP-2- and TNF-/IL-1-activated MAPKs have opposing roles in modulating osteoblastic differentiation and have raised a new question: what is the exact role of MAPKs in osteoblast biology in an environment in which inflammatory cytokines and exogenous BMP-2 treatment co-exist simultaneously? To address this issue, we should first clarify the role of each MAPK cascade on osteoblastic differentiation following BMP-2 treatment. Although the roles of p38, ERK1/2, and JNK1/2 on BMP-2-induced osteoblastic differentiation in various cell types have been well studied, details remain controversial. For example, BMP-2 activates p38 and ERK1/2 but not JNK1/2 to induce osteoblastic differentiation in C2C12 cells. However, JNK is required for the BMP-2-induced osteoblastic differentiation of MC3T3-E1-clone 24 cells. In this study, BMP-2-induced Runx2 expression and activation were significantly suppressed by the inhibition of BMP/Smad signaling synergistically with p38 or ERK1/2 signaling but not with JNK1/2 signaling. Taken together, our data suggest that p38 and ERK1/2 signaling are required for BMP-2-induced Runx2 expression and activation. In the clinic, elevated inflammatory cytokines and high concentrations of exogenous BMP-2 unavoidably co-exist in the surrounding of the BMP-2/ACS implant. Therefore, we treated C2C12 cells with BMP-2 and TNF-/IL-1 simultaneously to mimic the clinical model and investigate the exact role of p38 and ERK1/2 signaling . Our observations suggested that TNF-/IL-1 inhibited BMP-2-induced Runx2 expression and activation, and p38 and ERK1/2 signaling, not JNK1/2 signaling, were required for this inhibitory effect. These observations are contrary to at least one previous report that TNF- inhibits BMP-2-induced osteoblastic differentiation by activating SAPK/JNK signaling in C2C12 cells. This discrepancy suggests that when C2C12 cells are treated with BMP-2 alone, BMP-2-acvativated p38 and ERK1/2 signaling act as non-canonical pathways and have a weak inductive role in the upregulation of Runx2 expression and activation. However, the addition of TNF-/IL-1 causes strong p38 and ERK1/2 signaling activation, which has a role in downregulation of Runx2 expression and activation. To further prove this hypothesis, we transfected C2C12 and MCT3C3-E1 cells with CA-MKK3 and CA-MEK1 to strongly induce the ectopic activation of p38 and ERK1/2 signaling before BMP-2 treatment. Under these conditions, independently of the presence of TNF-/IL-1, constitutively activated p38 and ERK1/2 signaling significantly attenuated BMP-2-induced Runx2 activation and osteoblastic differentiation. These data suggest that strong activation of p38 and ERK1/2 signaling by inflammatory cytokines, not only TNF- or IL-1, decreases BMP-2-induced Runx2 activation and inhibits the osteoblastic differentiation of osteoprogenitor cells in an inflammatory environment. This study, concerning the opposing roles of TNF-/IL-1 and BMP-2 in regulation of osteoblastic differentiation in clinical condition, has got several significant conclusions. First, inflammatory cytokines TNF- and IL-1 inhibited BMP-2-induced osteoblastic differentiation. This result provides an alternate interpretation for the low osteoinductive efficacy of BMP-2 under clinical conditions. Second, BMP-2, TNF-, and IL-1 alone can activate the p38 and ERK1/2 signaling but have an opposing roles in regulating Runx2 expression and activation (). Finally, when exogenous BMP-2 and inflammatory cytokines such as TNF- and IL-1 are all present, p38 and ERK1/2 signaling are strongly activated, resulting in a significant decrease in BMP-2-induced Runx2 activation and osteoblastic differentiation (). Our study highlights the negative effects of an inflammatory environment on BMP-2-induced osteogenic differentiation and may facilitate the development of new strategies to improve the osteoinductive efficacy of BMP-2 and to enhance bone formation in a clinical setting. The murine multipotent mesenchymal progenitor cell line C2C12 and the preosteoblastic cell line MC3T3-E1 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). C2C12 cells were grown in Dulbecco's modified Eagle's medium (DMEM), and MC3T3-E1 cells were maintained in ascorbic acid-free -MEM. Both media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B (cell culture reagents were obtained from Invitrogen, Carlsbad, CA, USA). For osteogenic differentiation, the medium was replaced with serum-free DMEM or -MEM containing 200 ng/ml rhBMP2 in the presence or absence of TNF- (50 ng/ml)/IL-1 (20 ng/ml) (cytokines were obtained from PeproTech, Rocky Hill, NJ, USA). For inhibitor treatment, cells were pretreated with 25 mM Smad1 inhibitor DMH1 (Sigma, St. Louis, MO, USA), 25 mM p38 inhibitor SB203580, 50 mM ERK1/2 inhibitor PD98059, or 25 mM JNK1/2 inhibitor SP600125 (MAPK inhibitors were obtained from Selleck, Shanghai, China) for the indicated times before BMP-2 stimulation. Before staining, cells were treated for the indicated times and washed with PBS and fixed with 4% paraformaldehyde for 30 min. For ALP staining, cells were stained with naphthol AS-BI alkaline solution for 45 min to visualize ALP activity. For Alizarin red, cells were stained with 40 mM Alizarin red S (Sigma) solution (pH 4.1) for 10 min to visualize matrix calcium deposition. Cells were exposed to BMP-2 and/or TNF-/IL-1 at the indicated concentrations for 3 days. The cells were then lysed, and cellular ALPase activity was measured with an ALP detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The amount of ALP in the cells was normalized against the total protein content. Total RNAs was isolated from the BMP2-treated cells cultured in the presence and absence of TNF-/IL-1. Briefly, cells were washed with PBS and lysed with TRIzol reagent (Invitrogen), according to the manufacturer's protocol. Then 2 g of total RNA was used for reverse transcription, and the product was then used for real-time PCR. The quantification levels of osteogenic genes were quantified with an ABI 7500 Real-Time PCR System (Life Technologies, Foster, CA, USA). PCR primer pairs were designed based on the sequences of different exons of the corresponding genes (). All PCR amplifications were performed with an initial denaturation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 34 s, and melting curve analysis at 95 °C for 15 s and 60 °C for 60 s continuous. Proteins were extracted with RIPA lysis buffer containing 1 mM PMSF (Beyotime, Lianyungang, China). The protein samples were subjected to SDS-PAGE/immunoblotting analysis using anti-phospho-Smad1/5/8 antibody, anti-Runx2 antibody, anti-phospho- and anti-total ERK1/2 antibodies, anti-phospho- and anti-total p38 antibodies, anti-phospho- and anti-total JNK1/2 antibodies, and anti-GAPDH antibody (all from Cell Signaling, Danvers, MA, USA). The relative integrated density of each protein band was determined using an Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). After various treatments, the cells were fixed in 4% paraformaldehyde and then blocked with 5% goat serum. The cells were immunostained with anti-Smad1 antibody (Epitomics, Burlingame, CA, USA) or anti-OCN antibody (Cell Signaling) followed by a goat anti-rabbit Alexa Fluor-555- or Alexa Fluor-488-conjugated secondary antibody (Invitrogen). The cells were covered with an Anti-Fade Reagent (Cell Signaling). Transient transfections were performed using FuGENE HD (Promega, Madison, WI, USA). The total amounts of transfected plasmids in each group were equalized by the addition of an empty vector. For each transfection, C2C12 or MC3T3-E1 cells were separately co-transfected with Smad1 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CA-MKK3 (Cell Biolabs, San Diego, CA, USA), or CA-MEK1 (Cell Biolabs) and a luciferase reporter plasmid containing pSBE-Luc (Promega) or p6XOSE2-Luc (Sangon, Shanghai, China). A dual-luciferase assay was performed by using the Dual Luciferase Reporter Assay kit (Promega). The pSBE-Luciferase reporter plasmid was used to monitor BMP signaling, and the p6XOSE2-Luciferase reporter plasmid, which contained six tandem repeats of a Runx2 binding element in the promoter of the mouse OC gene, was used to monitor the transcriptional activity of Runx2. All data are presented as the mean±S.D. of three independent experiments. Statistical evaluations were performed with the Student's -test. -values<0.05 were considered statistically significant.

As a mitochondrial chaperone in the mitochondrial matrix, mortalin is critically required for the proper import and folding of nuclear-encoded matrix proteins. We hypothesized that PD-associated loss of mortalin function initiates impaired mitochondrial protein homeostasis. We first sought to measure the ratio of nuclear-encoded ATP5A to the mitochondrially encoded MTCO1 to assess potential mitonuclear imbalance. Mitonuclear imbalance was recently reported to precede activation of UPR(mt), together comprising a stress-signaling pathway conserved across many species. We found reduced mitochondrially encoded MTCO1 protein levels in knockdown cells compared with controls, whereas the level of nuclear-encoded ATP5A remained the same (). To further investigate the relevance of loss of mortalin to mitochondrial quality control, we measured the levels of the mitochondrial chaperone Hsp60 as a marker for the UPR(mt) as this protein is upregulated in conditions of intramitochondrial proteolytic stress (reviewed in Broadley and Hartl). To assess the intramitochondrial stress response upon loss of mortalin function, we analyzed the Hsp60 protein levels after knockdown in human neuroblastoma (SH-SY5Y) cells and in human fibroblasts from a heterozygous carrier of the PD-associated A476T mortalin variant (hereafter named A476T-fibroblasts). To validate the efficacy of knockdown in human neuroblastoma cells, mortalin levels were quantified. Western blot analysis revealed ∼70% reduction in mortalin protein (Supplementary Figure S1a). Because the abundance of Hsp60 may depend on the mitochondrial mass, we normalized our results to a mitochondrial marker (Tom20). We observed increased levels of Hsp60 in SH-SY5Y cells with reduced mortalin levels (). Similar alterations in Hsp60 levels were found in A476T-fibroblasts compared with control fibroblasts that may reflect a chronic activation of UPR(mt) in mutant cells (). Our results on mitonuclear imbalance and upregulation of the stress protein Hsp60 suggest that loss of mortalin function induces a mitochondrial stress response arising within the mitochondria. We hypothesize that the mitochondrial stress, mitonuclear imbalance and UPR(mt) activation in cells lacking functional mortalin would arise as a result of proteolytic stress. Therefore, we investigated whether knockdown causes an accumulation of aggregated or insoluble proteins within mitochondria by silver staining. As a result, no differences in the levels of SDS-soluble proteins within the mitochondria of knockdown or control cells were observed, but a significant increase in the amount of insoluble aggregates from the same mitochondrial sample was observed (). This indicates an increase in aggregated proteins within mitochondria upon loss of mortalin function that might arise from the significant amount of unfolded or misfolded proteins that could not be degraded efficiently. Activation of the UPR(mt) might sensitize or protectively precondition cells toward a second insult that causes further intramitochondrial stress. To differentiate these two possibilities, we induced intramitochondrial stress genetically by expressing the mitochondrial matrix protein ornithine transcarbamylase (OTC) or its truncated variant (dOTC) harboring a FLAG tag in SH-SY5Y cells. The accumulation of the truncated variant in an unfolded state within the mitochondrial matrix has been previously shown to elicit an intramitochondrial proteolytic stress response paralleled by Hsp60 upregulation. Both native OTC and dOTC were correctly targeted to mitochondria upon overexpression (). As expected, overexpression of the truncated dOTC variant led to a more punctate pattern in both conditions, control as well as knockdown. Interestingly, we also observed a more punctate pattern after overexpression of native OTC in knockdown cells that was not obvious in controls cells. Therefore, we analyzed the area of mitochondria (Tom20 signal) that colocalized with the area covered by OTC signal as a readout for proper intraorganellar distribution of the protein. The mitochondrial area covered by the OTC signal was significantly reduced in knockdown cells (). This may indicate an increased tendency of native OTC to aggregate in a knockdown background and argues in favor of an imbalanced protein folding capacity within the matrix. Impaired intramitochondrial protein quality control affects mitochondrial morphology and dynamics. We next extended our previous findings on mitochondrial fragmentation in A476T-fibroblasts to a human neuronal cell line and confirmed the characteristic fragmentation of the mitochondrial network in response to knockdown as assessed by aspect ratio, form factor and mitochondrial area per single mitochondrion (mitochondrial size) (). In contrast to wt mortalin, the PD-associated mortalin variants were not able to rescue the disturbed mitochondrial network caused by reduced mortalin function. Next, we used dOTC as a tool to genetically induce intramitochondrial proteolytic stress and then investigated mitochondrial integrity in SH-SY5Y cells with or without knockdown. Expression of dOTC in SH-SY5Y cells treated with control siRNA resulted in the fragmentation of the mitochondrial network as described previously for nonneuronal human cells (). The expression of native OTC in control cells did not affect mitochondrial integrity. Interestingly, overexpression of dOTC in control siRNA-treated cells recapitulated the mitochondrial fragmentation seen in SH-SY5Y cells with reduced levels. Moreover, expression of dOTC in the knockdown background further exacerbated the mitochondrial fragmentation phenotype compared with native OTC. The effect of dOTC on mitochondrial integrity is similar in both control and knockdown cells with no significant synergistic effect in the latter condition. This may indicate that the altered mitochondrial morphology readout reaches saturation, although we cannot rule out the possibility that less dOTC is imported into the mitochondria in the knockdown situation or that the level of proteolytic stress is saturated under two equally toxic conditions. As fragmentation precedes the degradation of dysfunctional mitochondria, we investigated an additional downstream readout and measured whether the observed increased mitochondrial fragmentation translates into changes of mitochondrial mass. Knockdown of in SH-SY5Y cells reduced the abundance of the outer mitochondrial membrane protein Tom20 () and the total area of mitochondria per cell (), suggesting a reduction in mitochondrial mass. This was supported by live cell imaging analyses in A476T-fibroblasts stained with Mitotracker green, a mitochondrial dye not dependent on MMP (). In line with intramitochondrial proteolytic stress modulating mitochondrial dynamics, the expression of dOTC within control siRNA-treated cells led to a significant reduction of mitochondrial mass (). Again, combining knockdown and expression of dOTC resulted in a more severe reduction of mitochondrial mass. This indicates synergistic intramitochondrial proteolytic stress conditions in terms of overall mitochondrial mass. Because of compensatory induction of organellar quality control mechanisms, intramitochondrial proteolytic stress should promote the specific degradation of dysfunctional mitochondria through autophagic clearance. To address this possibility, we employed several measures to assess autophagy by quantitative fluorescence microscopy techniques: assessment of overexpressed GFP-LC3 puncta by confocal microscopy; endogenous WIPI-2 puncta formation by confocal microscopy; automated high-throughput imaging and analysis of endogenous p62; and colocalization of lysosomes and mitochondria. Indeed, we found that knockdown results in a significantly higher level of autophagic activity and mitochondrial-lysosomal colocalisation, a measure of mitophagy. In detail, we used transiently overexpressed GFP-LC3 in SH-SY5Y to assess the number of autophagosomes by quantitative fluorescence microscopy. We found that the number of GFP-LC3 puncta per cell significantly increased in response to knockdown (), and this was abolished by overexpressing wt mortalin, but only partially by any of the PD-associated mortalin variants (). An increased number of autophagosomes (GFP-LC3 puncta) per cell can either be the result of an increased level in autophagy initiation or of a block in later stages of autophagy when autophagosomes contact the lysosomal compartment for final cargo degradation (autophagic flux). Hence, we monitored the autophagic flux by standard cotreatments with the lysosomal inhibitor bafilomycin A1 (BafA1) that prevents the fusion between autophagosomes and lysosomes. As expected, the addition of BafA1 elevated the number of GFP-LC3 puncta per cell under control conditions (). The number of GFP-LC3 puncta per cell clearly increased upon BafA1-treated knockdown when compared with both untreated knockdown cells and control siRNA cells treated with BafA1 (). This result strongly indicates that the autophagic activity increases upon knockdown. For further autophagy assessments we employed SH-SY5Y cells with inducible expression of miRNA targeting ( and ). By analyzing endogenous LC3 lipidation, the conjugation of LC3 (LC3-I) to phosphatidylethanolamine (LC3-II), we confirmed proper autophagic activity in this cellular model (). Next, we monitored the specific localization of endogenous WIPI-2, a member of the WIPI protein family that functions as an essential phosphotidylinositol-3-phosphate (PtdInsP) effector during autophagy initiation. Upon autophagy initiation, WIPI-2 accumulates at early and late autophagosomal membranes, and hence autophagy initiation can also be assessed by quantitative fluorescent puncta formation. In line with our results using GFP-LC3, we found a significant increase of endogenous WIPI-2 puncta when is knocked down ( and ). Furthermore, using an automated high-throughput imaging and analysis platform, we analyzed the specific targeting of endogenous p62 by autophagic clearance and found an increase in p62 degradation in cells lacking endogenous mortalin (). In summary, our autophagy assessments strongly indicate that mortalin negatively controls the process of autophagy and that knockdown results in elevated autophagic activity. As autophagy controls both the function and numbers of cellular organelles including mitochondria, we addressed the question of whether or not autophagy activation upon knockdown would lead to an elevated level of specific mitochondrial clearance called mitophagy (). For this aim, we quantified the colocalization of mitochondria, marked with Tom20 antibodies, with lysosomes, marked with LAMP1 antibodies, as a measure of mitophagy. Indeed, we found a higher degree of colocalization in cells treated with siRNA compared with controls, indicating increased autophagic clearance of dysfunctional mitochondria (). This effect was abolished by wt mortalin overexpression into the knockdown background but not by mutant R126W or P509S mortalin variants (). Although mutant A476T mortalin was able to revert the phenotype of acute knockdown in human SH-SY5Y cells, the chronic model of human A476T fibroblasts revealed an increased mitochondrial–lysosomal colocalization compared with controls, and therefore supports a concept of compensatory upregulation of organellar quality control. Finally, we investigated the effect of knockdown in mouse embryonic fibroblasts (MEFs) from knockout (KO) mice that contain an inducible ATG5 cDNA under the control of doxycycline. ATG5 is an essential component of autophagy-specific conjugation systems that promote the conjugation of LC3 to phosphatidylethanolamine at the forming autophagosome, and hence ATG5 deficiency blocks autophagy at early stages. In the presence of ATG5, a significant increase in the number of GFP-LC3 puncta per cell upon knockdown of was found (), corresponding to our findings using the neuronal cell line SH-SY5Y ( and ). In contrast, in MEFs devoid of ATG5 the increase of GFP-LC3 puncta upon knockdown was completely abolished. This indicates that mortalin controls the canonical autophagic machinery. To investigate the potential relevance of changes of mitochondrial morphology and induction of autophagy after loss of mortalin function to cell death, we analyzed cellular viability by measuring the activation of caspase 3 and the appearance of apoptotic nuclei. Acute mortalin depletion was sufficient to induce apoptotic cell death (). To further challenge molecular quality control in the mitochondria, we chemically induced intramitochondrial stress via 17-allylamino-17-demethoxygeldanamycin (17-AAG) treatment of cells. Previously, 17-AAG was described as an inhibitor of the chaperone Hsp90 in cytoplasm and mitochondria, thereby eliciting a UPR(mt) that promotes apoptosis. Knockdown of sensitized SH-SY5Y cells to 17-AAG treatment as quantified by the percentage of apoptotic nuclei and the activation of caspase 3 (). It is noteworthy that overexpression of wt mortalin in control SH-SY5Y cells treated with 17-AAG apparently compensated for the reduced Hsp90 activity and protected against subsequent apoptosis. In addition, in human A476T-fibroblasts we observed an increased susceptibility to 17-AAG treatment with higher levels of apoptosis compared with control cells (). To investigate the specificity of the loss of mortalin function-mediated susceptibility toward increased mitochondrial proteolytic stress, we treated cells with thapsigargin (TG), an inducer of endoplasmic reticulum (ER) stress. ER-stress caused apoptosis to a similar extent in both control and mutant fibroblasts (). The application of more specific genetically induced intramitochondrial proteolytic stress by overexpression of the truncated dOTC protein increased apoptosis in control SH-SY5Y cells to a similar extent as the chemical treatment (). Increased apoptotic nuclei but not activation of caspase-3 by dOTC was further exacerbated in the mortalin knockdown situation, suggesting that the apoptotic mechanism promoted by mortalin-induced proteolytic stress does not require caspase-3 and can act via alternative execution pathways. Notably, the overexpression of native OTC in a knockdown background induced apoptosis to a similar extent as control cells transfected with dOTC. This indicates that proteolytic stress due to dOTC overexpression recapitulates the effect of mortalin knockdown in terms of cellular viability. Knockdown of in combination with the expression of the dOTC protein showed the most severe effect on cell viability. The enhanced mitophagy observed in response to knockdown might exacerbate defects because of a depletion of mitochondria. Alternatively, it might be essential to prevent further cellular damage. To address this question, we sought to modulate autophagy in cells affected by the loss of mortalin function. Chemical induction of autophagy by Rapamycin reduced apoptosis in knockdown models as revealed by decreased caspase 3 activation and a reduced number of apoptotic nuclei (), as well as a reduced Annexin V staining revealed by FACS analysis (). In line with these observations, an inhibition of autophagy by use of the lysosomal inhibitor BafA1 in knockdown cells exacerbated apoptosis (). This clearly supports a critical role of functional autophagic machinery to prevent further damage. Intriguingly, the mitochondrial quality control machinery is critically related to PD genes encoding PINK1 and Parkin as mediators of the autophagic degradation of dysfunctional mitochondria. Therefore, to reduce the proportion of nonfunctional mitochondria among the mitochondrial pool, we genetically upregulated mitochondrial quality control proteins by overexpressing either PINK1 or Parkin in cells treated with siRNA and monitored the resulting phenotypes. Both PINK1 and Parkin overexpression in a knockdown background resulted in significantly enhanced numbers of GFP-LC3 puncta compared with the control condition (). These results argue in favor of an activation of autophagy to degrade impaired mitochondria. Consistently, the mitochondrial fragmentation phenotype in knockdown cells was rescued by overexpression of either PINK1 or Parkin (). To define whether our observations on positive effects of PINK1 and Parkin on mitochondrial network homeostasis were related to increased clearance of fragmented mitochondria from the cell, we used the KO model in MEFs. In control MEFs expressing ATG5, PINK1 partially and Parkin fully rescued the mitochondrial phenotype upon knockdown, in line with our observations in human neuronal SH-SY5Y cells (). In contrast, neither PINK1 nor Parkin overexpression rescued the mitochondrial fragmentation in KO MEFs (). The wt mortalin overexpression resulted in a healthy mitochondrial network in both WT and KO MEFs. This may indicate that the mechanism of rescue by overexpression of wt mortalin is not dependent on autophagosome formation (organellar quality control), but may reduce the intramitochondrial proteolytic disturbances resulting from knockdown. Whereas the rescue by wt mortalin overexpression acts within the mitochondria, an overexpression of either PINK1 or Parkin acts at the autophagy stage and cannot be successful in cells devoid of autophagosomes. To define whether an increased mitochondrial clearance in knockdown cells may be beneficial or rather toxic for the cells, we measured apoptotic cell death by Annexin V analysis. We found reduced levels of apoptosis under conditions of induced autophagy after overexpression of Parkin in knockdown cells (). This argues for a role of Parkin in rescuing the loss of mortalin by induction of autophagy in siRNA-treated cells (). PINK1 overexpression showed an effect on cell survival, but was not statistically significant. Finally, we investigated the link between mortalin loss of function and Parkin-mediated mitophagy by assessing the effect of expressing myc-Parkin in our knockdown paradigm. Therefore, we assessed an early stage of Parkin-mediated mitophagy that is defined by clustering of mitochondria and that precedes mitochondrial removal. We knocked down using an inducible miRNA system in HeLa cells, which are devoid of endogenous Parkin, and introduced recombinant myc-Parkin in knockdown as well as control cells. In accordance with our previous finding in SH-SY5Y cells and patient fibroblasts, the mitochondria in HeLa cells were fragmented when is knocked down. However, a significant increase in mitochondrial clustering as a readout for the induction of mitophagy was only observed after the introduction of Parkin in knockdown cells (). These sequestered mitochondria were previously described in paradigms of carbonyl cyanide -chlorophenyl hydrazone (CCCP)-induced mitophagy. In sum, the functional quality control system, enhanced by the overexpression of PINK1 or Parkin, lead to the activation of autophagy and in consequence to a rescue of the mitochondrial network in knockdown cells, thereby explaining the ability of Parkin to rescue knockdown cells from apoptosis. The genetic basis for disrupted mitochondrial integrity as a cause of neurodegeneration in PD was uncovered by the functional characterization of mutations that cause loss of Parkin or PINK1 function in autosomal recessive forms of the disease. A linear signaling pathway, with PINK1 acting upstream of Parkin, was described. This pathway controls the proper clearance of dysfunctional mitochondria from the cell. In this context, PINK1 is stabilized on the outer membrane of defective mitochondria and activates the process that leads to the recruitment of the E3 ubiquitin ligase Parkin to the dysfunctional organelle, finally leading to lysosomal degradation. Recently, the PINK1-mediated phosphorylation of mitofusin 2 (Mfn2) was found to be the critical step for the initiation of mitophagy via ubiquitination of Mfn2, with phosphorylated Mfn2 acting as a receptor for Parkin. Our results now provide evidence for an involvement of impaired intramitochondrial molecular quality control in PD mediated by loss of mortalin function. Importantly, mitochondrial dysfunction and altered integrity were compensated by PINK1/Parkin-mediated autophagy, representing downstream organellar quality control. We found that loss of mortalin causes intramitochondrial proteolytic stress and as defined by increased expression level of Hsp60, a marker for the activation of the UPR(mt). Activation of the UPR(mt) was further supported by the observed mitonuclear protein imbalance and an accumulation of insoluble proteins within the mitochondria. Consistently, human neuronal cell lines with reduced levels of mortalin were more vulnerable to chemically or genetically induced intramitochondrial proteolytic stress, an effect that was also observed in fibroblasts from a carrier of the A476T variant and that was completely rescued by reintroduction of wt mortalin. In contrast, no rescue capacity for mortalin was observed for pharmacologically induced ER-stress in this model, indicating the specificity of mortalin for UPR(mt) and the importance of mortalin for the maintenance of proteostasis within the mitochondrial matrix. Thus, we propose a linear signaling cascade in which loss of mortalin triggers impaired mitochondrial proteostasis that is upstream of PD-related neurodegeneration (). In our experiments in different and models of loss of mortalin function, we found evidence for increased autophagy due to accumulating mitochondrial damage based on intramitochondrial proteolytic stress. We demonstrate that the accumulation of autophagosomes upon loss of mortalin is because of activated autophagy. Although the correlation between the effect on autophagic activation and mitochondrial clearance is difficult to assess, the consistent reduction of mitochondrial mass in different loss of mortalin function models suggests that the increase in mitochondrial clearance is effective and represents an initial compensatory mechanism to maintain organellar quality. Previously identified genetic defects in Parkin and PINK1 contribute to PD pathogenesis based on their role in mitochondrial clearance via autophagy and implicated impaired organellar quality control. This, however, was typically seen in response to toxic insults (i.e., via CCCP) that interfere with mitochondrial homeostasis , so that the pathophysiological relevance of these observations in neurons still remains to be determined. We found that PD-associated loss of mortalin function caused a primary defect in intramitochondrial molecular quality control that causes characteristic changes in mitochondrial function and dynamics, leading to an increased autophagic activity with increased Parkin-mediated clustering of mitochondria. These mitochondria were subsequently cleared from the healthy mitochondrial pool, leading to reduced mitochondrial mass in different cellular and models. No alterations in cell viability under basal conditions were observed in fibroblasts of the human carrier of the A476T mutation that serves as a model of chronic mortalin haploinsufficiency. However, A476T fibroblasts showed an increased susceptibility toward proteolytic perturbation, indicating compensatory mechanisms active over lifetime. Thus, we speculate that the chronic impairment of the organellar quality control machinery sensitizes cells toward apoptotic cell death (). Indeed, overexpression of Parkin and PINK1 in knockdown models was able to revert mitochondrial phenotypes and reduce the susceptibility toward apoptotic cell death. This protective effect of PINK1 and Parkin was dependent on an intact autophagic machinery, as it was absent in fibroblasts from KO mice that lack ATG5 as an essential factor for autophagosome formation. Similar protective effects on cells with reduced levels of mortalin were observed upon treatment with rapamycin, a compound known to induce autophagy by targeting mTORC1. Therefore, our results suggest that effective removal of dysfunctional mitochondria via genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes due to loss of mortalin. The loss-of-function mechanism of PD-associated mortalin variants was suggested by an incomplete rescue of loss of mortalin-associated mitochondrial phenotypes by mutant R126W, A476T and P509S mortalin compared with wt mortalin . Furthermore, characteristic mitochondrial alterations in fibroblasts from the carrier of the A476T variant were observed . Interestingly, a recent study in yeast also supported a loss of chaperone function for the PD-associated mortalin variants R126W and P509S using biochemical assays with purified recombinant proteins and a functional complementation assays in yeast. We also performed experiments to rescue the inviable yeast mtHsp70 (Ssc1)-null strain with either native Ssc1 or Ssc1 carrying the analogous PD-associated variants (R103W Ssc1 ≙ human R126W mortalin; P486S Ssc1 ≙ human P509S mortalin). We confirmed a severe growth defect for cells harboring the ATPase domain mutant R103W Ssc1 and consistently found no significant difference for cells complemented with P486S compared with native Ssc1 (see and Goswami ). Using A453T Ssc1 that corresponds to the A476T variant found in German PD patients, we found similar effects on growth as for the A486S Ssc1 variant, also located in the substrate-binding domain (see and Goswami ). Our findings support the notion of differential effects of mtHsp70 variants located in the ATPase domain or in the substrate-binding domain in yeast. Moreover, our results in human cellular and models clearly show that the substrate-binding domain variants A476T and P509S exhibit a loss of mortalin function that translates into a phenotype of impaired mitochondrial proteostasis and changes in mitochondrial dynamics in human cells. These results underscore a concept of rare heterozygous variants in the essential gene that mediate neurodegeneration in PD via haploinsufficiency and are in line with the recent concepts on ‘rare variants common disease' that were deduced from genomic approaches on neurological diseases and support the presence of rare variants with reduced penetrance contributing as risk factors to common disease (reviewed in Sharma ). Recently, two novel missense variants (p.T333K and p.L358P) in the gene have been described that challenged the concept of mortalin variants in PD. For these variants, no alterations in different functional assays based on overexpression in SH-SY5Y cells were found. These results underscore that mortalin variants are not a frequent cause of PD and that functional consequences have to be carefully explored for all variants. Our results indicate that knockdown models or patient-based material are useful to determine whether novel variants may be related to loss of mortalin function. Based on the PD-associated symptoms of knockdown in , together with the concept of haploinsufficiency for disease-associated human variants , we suggest that an age-related decline of chaperoning capacities and subsequent collapse of protein homeostasis (reviewed in Baker ) may be responsible for neurodegeneration and would be in line with aging as the most important risk factor for PD. Any mutation or insult lowering mitochondrial quality control capacity is predicted to preferentially affect neurons because of their increased requirement for energy and increased risk for accumulating mitochondrial damage. It is known that mitochondria in neurons exhibit a higher organellar half-life compared with other postmitotical tissues. One reason for the particular susceptibility of dopaminergic neurons toward loss of mortalin function may be because of a reduced mitochondrial mass of DA neurons in the substantia nigra compared with other neuronal populations as has been shown in mice. Translated into the human situation, the limited equipment of DA neurons in terms of mitochondria could argue in favor of an increased vulnerability of the cellular energy supply toward impaired mitochondrial homeostasis, a hypothesis that is supported by increasing evidence for an involvement of the PINK1/Parkin-mediated organellar quality control in PD. Moreover, neurons of the substantia nigra apparently exhibit a reduced mitophagic capacity that, in addition, may contribute to the selective vulnerability of DA neurons in PD. We have shown in this work that a loss of mortalin function is sufficient to induce intramitochondrial proteolytic stress, the autophagic machinery is activated in conditions of loss of mortalin function and Parkin directly enhances the clustering of mitochondria in cells with reduced mortalin levels (). Parkin is part of an important step of the autophagic clearance machinery of damaged mitochondria from the cell. Therefore, overexpression of Parkin in human and murine models of loss of mortalin function may help to more efficiently remove dysfunctional mitochondria. This would further revert the altered mitochondrial morphology and protect from cell death ( and ). Indeed, rescue experiments including both PINK1 and Parkin point to a protective role of the PINK1/Parkin pathway being essential for removal of dysfunctional mitochondria as a result of mortalin loss of function. In summary, our study emphasizes the importance of mortalin for mitochondrial proteostasis and underscores the relevance of impaired protein quality control within the mitochondria in initiating a cascade of events leading to cell death. Our findings further stage mortalin-mediated disruption of mitochondrial integrity into the context of Parkin- and PINK1-associated PD phenotypes. Cloning of mortalin cDNA and its mutagenesis for insertion of the R126W, A476T and P509S variants has been described previously. Skin biopsies were taken from one offspring of a PD patient, both carrying the heterozygous A476T variant in the gene, and an unaffected sibling control. The carrier of the A476T variant did not show signs of PD at that time. All biopsies were obtained with patient's consent and approval of the local ethics committee. Cell culture of SH-SY5Y and HEK293 cells and human fibroblasts has been described previously. The m5-7 MEFs were prepared to achieve a knockout of as previously described. Passage number of fibroblasts was <10 for all experiments. Only fibroblasts with the same passage number were taken. Cell lysis was carried out identically for SH-SY5Y cells, HEK293 cells and human fibroblasts as well as MEFs. Cells were washed with phosphate-buffered saline (PBS) and detached with Trypsin at 37°C for 3 min. After 5 min of centrifugation at 14 000 r.p.m., cells were lysed in buffer containing 0.1% Triton X-100 in PBS and 1 × protease inhibitor cocktail (Roche, Penzberg, Germany). Proteins were detected by using antibodies against mortalin (anti-GRP75, Santa Cruz Biotechnology, Dallas, TX, USA), -Actin (Sigma-Aldrich, Munich, Germany), GAPDH (Life Technologies, Carlsbad, CA, USA), Tom20 (Santa Cruz Biotechnology), Hsp60 (from A Azem, Tel-Aviv University, Ramat Aviv, Israel), activated caspase 3 (Cell Signaling Technology, Danvers, MA, USA), activated caspase 9 (Cell Signaling Technology), Flag (Sigma-Aldrich), -tubulin (Sigma-Aldrich), -Tubulin (anti--Tubulin, (E7), Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), GAPDH (anti-GAPDH (G9545), Sigma-Aldrich) and rodent OXPHOS (no. MS604 Mitoscience (Abcam), Cambridgeshire, UK). Secondary antibodies were purchased from GE Healthcare (Buckinghamshire, UK). Mitochondria were purified from tetracycline-induced (nontargeting or miRNA) knockdown SH-SY5Y cells in homogenization buffer containing 10 mM HEPES (pH 7.4), 50 mM sucrose, 0.4 M mannitol, 10 mM KCL and 1 mM EGTA, using a two-step differential centrifugation at 600 × and 3000 × , respectively, for 15 min at 4°C. The mitochondria-enriched fraction was resuspended in a buffer containing 20 mM HEPES (pH 7.4), 0.4 M mannitol, 10 mM NaHPO and 0.5 mM EGTA to equal protein concentration of 1 g/l. Equal amounts of this mitochondrial fraction were added to equal volumes of a lysis buffer containing 4% (w/v) SDS or 2% (v/v) NP40. NuPAGE gradient gels were first placed in 10% (v/v) ethanol for 10 min on a shaker and then in 1% (v/v) HNO (Roth, Karlsruhe, Germany) for 15 min before incubation with 0.2% (w/v) AgNO (Sigma-Aldrich) for 30 min. After a short wash with ddHO, bands were made visible with 3% (w/v) NaCO (Roth) with 50 l formaldehyde solution (Sigma-Aldrich) per 100 ml. The reaction was stopped with 10% (v/v) acetic acid (Merck, Darmstadt, Germany) for 2 min when the staining was sufficient and gels were kept in ddHO until digitalization. The mitochondrial SDS fraction of equalized lysates (described above) were resolved on NuPAGE 4–12% gradient gels (Life Technologies) and western blots probed for total rodent OXPHOS (AbCam 110413, MS604) that contains antibodies against ATP5A (nuclear encoded) and MTCO1 (mitochondrially encoded) according to Houtkooper Tom20 was used as an additional loading control (Santa Cruz Biotechnology). A representative western blot is shown. knockdown was achieved by transfection of Flexitube siRNA (Hs_HSPA9_1, 3′-AlexaFluor555) or chemically unmodified nontargeting control siRNA (AllStars Negative siRNA AF 555) from Qiagen (Hilden, Germany) into SH-SY5Y cells. A total of 70 000 cells were transfected with 160 nM of siRNA or negative control siRNA using HiPerFect transfection reagent (Qiagen), leading to a reduction in mortalin protein of >70% in cells transfected with siRNA compared with control cells (). For knockdown using a tetracycline-inducible miRNA system (BLOCK IT, Life Technologies), miRNA sequences were designed targeting two regions on the gene (nucleotide start sites 773 and 1939) that were chained together and under polycistronic control of the same promoter in the same pcDNA 6.2-GW/EmGFP-miR miRNA vector backbone. Induced expression was monitored by EmGFP fluorescence (EmGFP-miR vector, Life Technologies) and by western blot. Additional transfection of any plasmid was achieved using HiPerFect transfection reagent. For immunocytochemistry, cells were seeded on collagen-coated slides. For successful knockdown of , siRNA was transfected 5 h after seeding the cells. GFP-LC3 and respective plasmids were transfected in the ratio of 1 : 4 using HiPerFect transfection reagent 12 h after siRNA treatment to control for positive transfection. Cells positive for GFP-LC3 were considered as also harboring the other plasmid. Analysis of the number of GFP-LC3 puncta per cell was done via an unbiased automatical counting approach using ImageJ software. To chemically elicit a mitochondrial unfolded stress response, cells were treated with 0.25 M of the Hsp90 inhibitor 17-AAG for 30 h before fixation. To induce ER stress leading to Ca depletion, cells were treated with 30 M for 5 h. Lysosomal inhibition was evoked by treatment with 100 nM BafA1 for 1 h followed by incubation in normal medium for 4 h. BafA1 prevents the fusion between autophagosomes and lysosomes and is an established method to assess the autophagic flux. For activation of autophagy, cells were incubated with 100 nM Rapamycin for 24 h. Fixation procedure and microscopic analysis were performed as described previously. If needed, cells were incubated for 15 min in 100 nM Lyostracker Red DND-99 (Life Technologies) before fixation. Proteins were detected by using antibodies against mortalin (anti-GRP75, Santa Cruz Biotechnology), Tom20 (Santa Cruz Biotechnology), Lamp-1 (Hybridoma Bank of Iowa University), cleaved Caspase-3 (Cell Signaling Technology) and Flag (Sigma-Aldrich). Secondary antibodies were purchased from Life Technologies or Zymed (San Francisco, CA, USA). Hoechst 33342 (Life Technologies) was used to stain nuclei. To assess endogenous WIPI-2 puncta formation, SH-SY5Y cells with inducible expression of miRNA targeting mortalin were seeded on coverslips in 24-well plates (50 000 cells per well). Mortalin miRNA expression was induced for 24, 48 and 72 h using 1 g/ml tetracycline, followed by a 3-h incubation in starvation medium (Earle's balanced salt solution (EBSS)) including BafA1 (200 nM). Subsequently, cells were fixed in 4% paraformaldehyde and indirect immunofluorescence conducted as previously described using anti-WIPI2 antibodies (1 : 50, Abgent, San Diego, CA, USA) and anti-IgG-Alexa546 (1 : 200, Life Technologies). Using confocal microscopy (Zeiss LSM 510) and ImageJ analysis, the number of WIPI-2 puncta per cell as well as fluorescent puncta area was determined. To assess endogenous p62 localization, SH-SY5Y cells with inducible expression of miRNA targeting were seeded in 96-well plates (10 000 cells per well) and grown in tetracycline-containing medium for 72 h followed by a 3-h treatment with control medium (CM) or EBSS in the presence or absence of BafA1 include concentration of BafA (200nM). Cells were fixed in 4% paraformaldehyde and indirect immunofluorescence conducted as previously described using anti-p62 antibodies (1 : 50, Santa Cruz, Dallas, TX, USA) and anti-IgG-Alexa546 (1 : 200, Life Technologies) and DAPI to stain cell nuclei. Using an automated image acquisition and analysis platform, p62 inclusions (puncta) per cell were imaged and analyzed from up to 5000 cells as previously described. CCCP and Valinomycin are the only known inducers of Parkin-mediated mitophagy, yet there is a lack of accepted methods to assess Parkin translocation following induction with other toxins such as rotenone. Therefore, we assessed the intermediate clustering of mitochondria preceding mitochondrial removal. We knocked down using an inducible miRNA system in HeLa cells that are devoid of endogenous Parkin. Exogenous myc-Parkin was introduced and the mitochondria in cells positive for miRNA only or miRNA+myc-Parkin were analyzed for number per cell using ImageJ (threshold 18-infinity and number of mitochondrial particles). For mitochondrial morphology and mass as well as colocalization studies, mitochondria were visualized by 100 nM MitoTracker Green FM (Life Technologies), lysosomes by 100 nM Lyostracker Red DND-99 (Life Technologies) and nuclei by Hoechst 33342 after an incubation time of 15 min. In studies using Lyostracker Red DND-99, the dye was also present within the medium during imaging the cells for improved visualization of lysosomes. Live cell imaging analysis was performed as described previously. The series of images were saved uncompressed and analyzed with AxioVision software (Zeiss) and ImageJ 1.41o software, respectively. Analysis of mitochondrial morphology was done as described previously. Morphological characteristics such as form factor, aspect ratio, area circularity and mitochondrial mass were evaluated using ImageJ software. To analyze the mitochondrial mass, the total area of mitochondria per single cell was calculated. Briefly, SH-SY5Y cells were washed with PBS at room temperature and then incubated in either Annexin V binding buffer (Biolegend, San Diego, CA, USA) or the same buffer containing Annexin V-Pacific blue (BioLegend, according to the manufacturer's titration) for 15 min on ice. Cells were sorted on a CyAn ADP (Beckman Coulter, Brea, CA, USA) FACS machine according to their Annexin V-Pacific Blue signal above the precontrolled threshold set by the unstained control cells. The percent of cells in the population above this threshold was also gated for those cells containing control siRNA-647 or Mortalin siRNA-647 fluorescent signal. To assess endogenous enogenous LC3 lipidation, SH-SY5Y cells with inducible expression of miRNA targeting mortalin were seeded in 24-well plates (50 000 cells per well) miRNA expression was induced for 72 h using 1 g/ml tetracycline, followed by a 3-h incubation in control medium or starvation medium (EBSS) including BafA1 (200 nM) or not. Subsequently, cells were lysed in hot Laemmli buffer and resolved by SDS-PAGE. Using LC3 antibodies (Nanotools, Munich, Germany), unconjugated LC3 (LC3-I) and PE-conjugated LC3 (LC3-II) were detected by standard ECL procedures and PeqLab Fusion SL (Erlangen, Germany). The imaging data were analyzed using Student's test; all the statistical tests were nonpaired two sided and those with a value of <0.05 were considered to be statistically significant. Data are expressed as mean±S.E. values (*<0.05; **<0.01; ***<0.001).

Previous studies have shown that ER stress causes cell death through accumulation of unfolded or abnormal proteins in the ER and subsequent activation of ER stress-induced caspases. ER stress transducers modulate ER-specific stress; therefore, we investigated whether the main ER stress transducer IRE1 regulates ER stress-mediated cell death. After SH-SY5Y cells were transfected with IRE1-specific siRNA for 48 h, total IRE1 levels were reduced by 40–60% control siRNA-transfected cells, without changes in -actin expression ( and ). We used western blots to determine whether downregulation of IRE1 expression induces ER stress and observed marked induction of CHOP, an ER stress-related marker protein, as well as GRP78, an ER chaperone (). Next, we knocked down other ER stress transducers, PERK and ATF6, to test their ability to regulate ER stress. PERK- and ATF6-specific siRNA reduced their respective protein levels by 60–80%, without any change in -actin expression (). We found, however, unlike IRE1 KD, reduction of PERK or ATF6 did not induce ER stress (), suggesting that only IRE1 regulates ER stress under basal conditions. As IRE1 is localized in the ER membrane and the ER structure undergoes dramatic changes upon cellular damage, we examined ER morphology under IRE1 KD. Western blotting revealed no difference in the expression of ER membrane proteins, such as calreticulin or calnexin (). Immunofluorescence experiments using anti-calreticulin antibody as an ER indicator showed that ER morphology was slightly altered in IRE1-KD cells (data not shown). We used transmission electron microscopy to analyze IRE1-KD-induced changes in ER morphology. The electron micrographs of IRE1-KD cells showed ER enlargement and distension (ER expansion) (). Thus, IRE1 KD induced ER stress and caused ER expansion. ER stress induces cell death; therefore, we tested the effect of IRE1 KD on cell viability. The results of MTT and calcein-AM assays showed that reduction of IRE1 induced cell death (). To confirm the increase of apoptotic cell death under IRE1-KD conditions, we performed the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, a method for detecting DNA fragmentation. Consistently, TUNEL staining indicated increased apoptosis in IRE1-KD cells (). To determine whether this cell death was mediated by ER stress, the IRE1-KD cells were treated with the chemical chaperone tauroursodeoxycholate (TUDCA) to protect the cells from ER stress. Indeed, TUDCA alleviated ER stress induction by IRE1-KD () but did not rescue IRE1-KD-induced cell death (), suggesting that IRE1-KD-induced cell death could not be rescued by inhibiting ER stress alone. Tunicamycin, an inhibitor of protein glycosylation, causes ER stress-induced apoptosis by accumulating unfolded or misfolded proteins in the ER. In this study, tunicamycin not only induced cell death in the SH-SY5Y cells but also enhanced cell death in IRE1-KD control siRNA-transfected cells (). These data suggest that IRE1-KD-induced cell death is mediated by mechanisms other than ER stress caused by accumulation of abnormal proteins in the ER. Previous studies have shown that dysregulation of intracellular Ca levels ([Ca]) induces cell death. As IRE1 is a type I transmembrane protein localized in the Ca-storing ER, we examined the effect of reduced IRE1 levels on intracellular Ca levels. Using the Fluo-4 calcium assay, we observed that IRE1 reduction triggered [Ca] upregulation (). To determine whether dysregulated [Ca] induced by IRE1 KD causes cell death, the cells were treated with 1,2-bis(o-aminophenoxy)ethane-,,′,′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), a Ca chelator. BAPTA-AM treatment prevented IRE1-KD-induced cell death (), suggesting that dysregulation of [Ca] has a role in IRE1-KD-induced cell death. Neither PERK nor ATF6 induced ER stress (); we explored their role in cell death by MTT and calcein-AM assays and found that knockdown of these regulators did not induce apoptosis (). Consistently, reduction of PERK or ATF6 had no effect on [Ca] (). These results suggest that only IRE1 regulates cell survival by maintaining Ca homeostasis under basal conditions. To avoid off-target effects during siRNA treatment, we reintroduced IRE1 into IRE1-KD cells. After transfection with control or IRE1 siRNA for 24 h, an exogenous IRE1 construct was transfected into the cells for 24 h (). IRE1 expression was increased in these IRE1-overexpressing (IRE1 o/e) cells (). IRE1 re-introduction rescued calcein-AM signal loss in IRE1-KD cells (), indicating that altered IRE1 levels regulated cell death. show that IRE1 regulated cell survival by maintaining Ca homeostasis under basal conditions. IRE1 re-introduction also restored [Ca] in IRE1-KD cells, as demonstrated by Fluo-4 calcium assay (). These data indicate that IRE1 regulates cell survival by maintaining Ca homeostasis under basal conditions. As IRE1 is an ER membrane protein and the ER is a major Ca-storing organelle, we investigated whether IRE1-KD-induced [Ca] increases are caused by ER Ca release. Thapsigargin, an inhibitor of ER Ca-ATPase (SERCA), was used to determine the concentration of free Ca within the ER lumen ([Ca]) by selectively depleting ER Ca stores, whereas BAPTA-AM was used to deplete cytoplasmic Ca. The Fluo-4 assay showed that the IRE1-KD cells showed few Ca level released from ER, compared with control siRNA-transfected cells (), suggesting that increased [Ca] in the IRE1-KD cells was caused by ER Ca release. To confirm this result, we directly measured the effects of IRE1 downregulation by Ca imaging with the fluorescent dye Fura-2-AM. Although the IRE1-KD cells showed increases in the basal [Ca], the levels of [Ca] increases in the IRE1-KD cells after challenge with thapsigargin (a measure of [Ca] stores) was lower than that in the control siRNA-transfected cells (). The experiment was repeated three times; the average increase after thapsigargin challenge was 48% of the control level (average baseline level). These data indicate that reduced IRE1 levels caused [Ca] upregulation by accelerating Carelease from the ER. The ER is the most important intracellular Ca store. Regulation of intracellular Ca by the ER is mainly mediated by Ca uptake into the ER through SERCA Ca pumps and Ca release through Ca channels, such as InsP3Rs or RyRs. To determine whether [Ca] increases in IRE1-KD cells are caused by ER Ca-related channels, we treated the cells with Ca channel blockers dantrolene and 2-aminoethoxydiphenyl borate (2-APB) to inhibit RyRs and InsP3R, respectively. As shown in , 2-APB treatment blocked the increase of [Ca] in the IRE1-KD cells, whereas dantrolene did not. Notably, western blotting indicates no difference in RyRs and InsP3R expression between IRE1-KD and control siRNA-transfected cells (), suggesting that upregulation of [Ca] in the IRE1-KD cells is associated with InsP3R but does not alter the expression of ER Ca-related channels. Next, to determine whether InsP3R-mediated Ca release in IRE1-KD cells influences cell death, the viability of IRE1-KD cells treated with vehicle, dantrolene, or 2-APB was determined by calcein-AM assay. Inhibition of InsP3R rescued cell death in the IRE1-KD cells, whereas the blocker of RyRs (dantrolene) did not (). To confirm this result, caspase-3 and -9 activities were measured. Essential for apoptosis, caspases exist as inactive zymogens and require proteolytic processing for activation. Under apoptotic conditions, the activation of upstream caspases (caspase-8 and/or -9) proteolytically activates downstream caspases, such as caspase-3. In comparison to control siRNA-transfected cells, the IRE1-KD cells showed increased levels of cleaved caspase-3 and -9 (); these effects were rescued by 2-APB, but not dantrolene. To confirm these results, the cells were treated with xestospongin C (XeC), an InsP3R-specific antagonist. XeC reversed [Ca] increases and IRE1-KD-induced cell death in the IRE1-KD cells (). These data suggest that, upon IRE1 downregulation, InsP3R-mediated Ca release induces apoptotic cell death. To explore the mechanisms through which IRE1 KD promoted InsP3R-mediated Ca release, we tested whether IRE1 interacts directly with InsP3R. By co-immunoprecipitation (co-IP), no interaction was detected between IRE1 and InsP3R (). We next investigated whether IRE1 downstream signaling is associated with InsP3R-mediated Ca release in the IRE1-KD cells. When the IRE1 kinase activity was inhibited by addition of the ATP-competitive inhibitor 1NM-PP1 to the SH-SY5Y cells, [Ca] was not affected (), suggesting that the IRE1 kinase activity and its downstream signaling pathway are not associated with the opening of InsP3R. Previous studies have demonstrated that CIB1 binding to InsP3R led to an inhibition of Ca release from InsP3R. CIB1 was recently suggested to function as a Ca-sensitive modulator by interacting directly with ASK1. Based on these findings, we tested the association between the regulation of the opening of InsP3R and changes in binding partners. The co-IP analysis shows that the extent of the CIB1-ASK1 interaction was increased in the IRE1-KD cells, compared with the control siRNA-transfected cells. In addition, the IRE1-KD cells also showed increased CIB1-ASK1 interaction and decreased InsP3R-CIB1 interaction, indicating that IRE1 downregulation reduced recruitment of TRAF2-ASK1 to IRE1, thereby resulting in increases in free ASK1-CIB1 binding and decreases in the CIB1-InsP3R interaction (). Decreased CIB1-InsP3R interaction induces Ca release from InsP3R; thus IRE1-KD-induced Ca release from InsP3R may result from changes in CIB1-InsP3R binding. To visualize the ASK1-CIB1 and CIB1-InsP3R interactions under IRE1-KD conditions, we performed the proximity ligation assay. In this novel assay, close proximity of the target proteins generates punctate signals. The IRE1-KD cells produced more signals in the presence of ASK1 and CIB1 antibodies and fewer signals in the presence of CIB1 and InsP3R antibodies (), suggesting that reduced IRE1 levels enhanced ASK1-CIB1 interaction and inhibited CIB1-InsP3R interaction. To determine whether decreased CIB1-InsP3R binding upregulates [Ca] to trigger cell death, SH-SY5Y cells were transfected with CIB1-specific siRNA (CIB1-KD cells) for 48 h. Reduced CIB1 levels led to [Ca] increases and cell death; treatment of CIB1-KD cells with 2-APB, but not dantrolene, reversed these effects (), suggesting that reduction of CIB1 may upregulate Ca release from InsP3R, in turn enhancing cell death. To investigate the mechanism through which IRE1-KD-induced [Ca] increases mediate cell death, we focused on mitochondrial functions because of their role as modulators of the apoptotic process. Previous studies have shown that enhanced [Ca] mediated by InsP3R and RyRs increased sequestration of vast amounts of Ca in mitochondria ([Ca]), which subsequently triggered mitochondrial membrane permeabilization and led to apoptotic cell death. This pathway also depends on Ca-induced opening of the permeability transition pore (PTP). As the IRE1-KD cells showed increased mitochondrial fission (), stable Mito-DsRed-expressing cells were used to investigate the effect of IRE1 KD on mitochondrial morphology. Electron microscopy (EM) showed increased mitochondrial fragmentation in the IRE1-KD cells (). We also determined the effect of IRE1 KD on mitochondrial membrane potential. The tetramethyl rhodamine methyl ester (TMRM) assay showed depolarization of mitochondrial membrane potential in IRE1-KD cells (). Depolarized mitochondrial membrane potential induces ROS generation. Dichlorofluorescein diacetate (DCFDA) staining showed increased DCF fluorescence, representing increased ROS levels in IRE1-KD cells (). To detect mitochondrial superoxide accumulation, we performed MitoSOX Red staining and found that the IRE1-KD cells showed significantly higher levels of MitoSOX Red fluorescence in mitochondria (). To determine whether alterations in mitochondrial homeostasis induce cell death under IRE1-KD conditions, we performed the cell death assay with several blockers. Treatment with NAC, a well-known ROS scavenger, or Ru360, a blocker of the mitochondrial uniporter (MCU), rescued cell death caused by IRE1 KD (), indicating that IRE1-KD-induced [Ca] increases enhanced Ca load in the mitochondria, thereby leading to mitochondrial dysfunction and cell death. Notably, treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), the mitochondrial uncoupler, did not induce additional cell death in comparison with IRE1 KD alone (), suggesting that IRE1 KD induced cell death through mitochondrial dysfunction. Cyclosporin A (CsA), which inhibits mitochondrial permeability transition, partially rescued the IRE1-KD cells from cell death (). These results suggest that IRE1 KD induces mitochondrial dysfunction and cell death through increased ROS generation. Accumulating studies have shown that ER stress is closely associated with cell death. As the ER mediates protein synthesis, folding, and Ca maintenance, the ER disruption causes cell death through several mechanisms. In response to ER stress, the cells activate the ER stress-specific defense system. It is well known that IRE1 acts as the main ER stress transducer; however, its role in cell death is not yet fully understood. In this study, we chose SH-SY5Y cells based on previous reports on the roles of IRE1 in ER stress and mitochondria-ER crosstalk. Our results demonstrate that cell death was induced in IRE1 knocked down SH-SY5Y cells (IRE1-KD cells) compared with control siRNA-transfected cells (Con). As the ER is a Ca-storing organelle, [Ca] increased when IRE1 was knocked down, and treatment with a Ca-chelating agent rescued cell death induced by IRE1 KD. We explored the underlying mechanism of IRE1-induced [Ca] increases and found that IRE1-KD-induced [Ca] upregulation resulted from ER Ca release. Our results also indicate that the ER Ca-related channel InsP3R mediated IRE1-KD-induced [Ca] increases, thereby leading to cell death. Previously, abnormal Ca release from InsP3R has been suggested to act as an important apoptotic signal. Here, we found that treatment with InsP3R blockers inhibited [Ca] increases and cell death in the IRE1-KD cells. In addition, treatment of SH-SY5Y cells with the InsP3R agonist adenophostin A caused significant cell death, whereas co-treatment with BAPTA-AM inhibited cell death (). These results suggest that enhanced InsP3R-mediated Ca release may induce cell death in a Ca-dependent manner. We explored the underlying mechanism of IRE1-KD-induced InsP3R activation by co-IP and found that IRE1 did not interact with InsP3R directly. In previous studies of InsP3R's binding partners, CIB1 binding to InsP3R inhibited Ca release from InsP3R, and CIB1 is thought to function as a Ca-sensitive modulator by interacting directly with ASK1. We tested the association between opening of InsP3R and the CIB1-ASK1 interaction under IRE1-KD conditions and found that IRE1 KD enhanced the CIB1-ASK1 interaction but reduced the CIB1-InsP3R interaction, which in turn resulted in increased Ca release from InsP3R. There are previous studies that IRE1-ASK1 pathway mediates cell death under pathological conditions. In contrast, we focused on the role of IRE1 itself under normal condition. We compared with control siRNA-transfected cells and IRE1 siRNA-transfected cells without any stimuli. We found first that IRE1 regulates Ca homeostasis in the ER by trapping ASK1. The downregulation of IRE1 induced the increased ASK1-CIB1 interaction through the decreased IRE1-ASK1 interaction, resulting in the reduction of inhibitory roles of CIB1 in Ca release through IP3R. Consistently, CIB1 KD increased [Ca], likely through reduced interaction between CIB1 and InsP3R and thus induced cell death (). Mitochondria are the intracellular organelles associated with Ca handling. Mitochondrial Ca uptake regulates intracellular Ca signaling and cell survival by buffering cytosolic Ca levels. Previous studies have shown that Ca accumulation in mitochondria induced apoptotic cell death through Ca-induced MPTP opening. As IRE1 KD induced [Ca] increases and cell death, we focused on mitochondrial alterations, including abnormal mitochondrial fission and reduced mitochondrial functions in the IRE1-KD cells. In addition, IRE1 KD increased the levels of ROS, a well-known cell death-inducing factor. Ca accumulation in mitochondria occurs via the MCU across a steep electrochemical gradient. Treatment of IRE1-KD cells with MCU blockers inhibited cell death, indicating that Ca accumulation in mitochondria may act as a main apoptotic factor in the IRE1-KD cells. We also found that ROS scavengers reduced cell death in the IRE1-KD cells. Based on the finding that treatment of the IRE1-KD cells with 2-APB reduced ROS generation (), we suggest that IRE1-KD-induced [Ca] accumulation caused increased ROS generation and eventually induced apoptotic cell death. Notably, IRE1-KD-induced cell death was also mediated, at least in part, by calpain activation ( and ). Treatment of the IRE1-KD cells with a calpain inhibitor blocked cell death induced by [Ca] upregulation. These results are consistent with previous studies showing that excessive Ca binds and activates Ca-dependent enzymes, such as calpain, thereby activating caspase-12 and triggering the apoptotic pathway. Surprisingly, unlike IRE1, knockdown of the other two ER stress transducers, PERK and ATF6, did not lead to [Ca] increases or cell death. In conclusion, reduced expression of the ER stress transducer IRE1 induced ER stress and caused cell death by accelerating ER Ca release via InsP3R. InsP3R-induced ER Ca release in the IRE1-KD cells caused cell death via prolonged mitochondrial Ca accumulation and alterations in ER morphology and function (). Human neuroblastoma SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone, Irvine, CA, USA) supplemented with 10% fetal bovine serum (HyClone) and an antibiotic mixture of penicillin (100 U/ml) and streptomycin (100 g/ml). The control siRNA (sc-37007) and siRNA against IRE1 (sc-40705 and 1171247), PERK, ATF-6, and CIB1 (sc-43271) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and/or Bioneer Inc. (Daejeon, Korea). Cy3-tagged IRE1 siRNA was made by Bioneer, Inc. and IRE1 cDNA was purchased from Addgene (ID:20744; Cambridge, MA, USA). Cells were cultured for 48 h after transfection with Lipofectamine for cDNA and RNAimax for siRNA according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA) and treated with vehicle or appropriate concentrations of TUDCA (T0266), tunicamycin (T7765), thapsigargin (T9033), BAPTA-AM (A1076), CCCP (C2759), dantrolene (D9175), 2-APB (D9754), -acetyl--cysteine (NAC) (A7250), L-NG-nitroarginine methyl ester (N5751), CsA, and Calpain Inhibitor I (A6185) from Sigma-Aldrich (St. Louis, MO, USA); Xestospongin C (sc-201505) and Ru360 (sc-222265) from Santa Cruz Biotechnology; DEVD-fmk from BD Biosciences (Franklin Lakes, NJ, USA); 1-tert-butyl-3-naphthalen-1-ylmethyl-1 H-pyrazolo[3,4–d]pyrimidin-4-ylamine (1NM-PP1) from Cayman Chemical (Ann Arbor, MI, USA); and adenophostin A from Calbiochem (San Diego, CA, USA). Sequences were as follows: siRNA against IRE1 5′-CUGCUUAAUGUCAGUCUAC-3′ (sense), 5′-GUAGACUGACAUUAAGCAG-3′ (antisense); siRNA against PERK 5′-GAGAACACAGAAGAGUCUA-3′ (sense), 5′-UAGACUCUUCUGUGUUCUC-3′ (antisense); and siRNA against ATF-6 5′-CAGAGAACUGUCUCGUACU-3′ (sense), 5′-AGUACGAGACAGUUCUCUG-3′ (antisense). Cell pellets were prepared as described and western blotted with the following antibodies: anti-IRE1 (ab37073; 1 : 1500) from Abcam (Cambridge, MA, USA); anti--actin (A1978; 1 : 5000) from Sigma-Aldrich; anti-PERK (sc-13073; 1 : 1000), anti-ATF6 (sc-22799; 1 : 1000), anti-GADD153 (sc-575; 1 : 1000), anti-ASK1 (sc-7931 and sc-5294; 1 : 1000 for WB, 1 : 100 for IP and PLA), anti-caspase-12 (sc-70227; 1 : 1000), anti-GRP78 (sc-1050; 1 : 1000), anti-TRAF2 (sc-7346; 1 : 1000), and anti-calpain (sc-7530; 1 : 1000) (Santa Cruz Biotechnology); anti-ryanodine receptor (MA3-916; 1 : 1000) (Thermo Scientific, Hudson, NH, USA); anti-InsP3R (07-1210; 1 : 2000 for WB, 1 : 300 for IP, 1 : 100 for PLA) and anti-CIB1 (MAB2601; 1 : 1500 for WB, 1 : 500 for IP, 1 : 100 for PLA) (Millipore, Schwalbach, Germany); and anti-calreticulin (2891; 1 : 2000), anti-calnexin (2433; 1 : 2000), anti-cleaved caspase-9 (9501; 1 : 2000), anti-caspase-9 (9502; 1 : 2000), and anti-caspase-3 (9662; 1 : 2000) (Cell Signaling Technology, Beverly, MA, USA). Immunoreactive bands were photographed and quantified on LAS-3000 with MultiGauge (Fuji Film Inc., Tokyo, Japan). To measure cell viability, calcein-AM, MTT, and TUNEL assays were performed. The calcein-AM assay was performed according to the manufacturer's instructions (C3099, LIVE/DEAD Viability and Cytotoxicity Kit; Molecular Probes, Invitrogen, Carlsbad, CA, USA). Briefly, 5 × 10 cells were incubated for 24 h after seeding in 96-well plate, and then transfected with 20–50 pM siRNA for 24–48 h. Treatments were administered after transfection at optimal dose (see figure legends). Calcein-AM reagent in phenol red-free media (1 M) was added, incubated for 1 h at 37 °C, and washed three times with PBS. Fluorescence was measured at excitation and emission wavelengths (ex/em) of 485 nm/530 nm on a fluorescence plate reader (Infinite M200 Pro; TECAN, Männendorf, Switzerland). The MTT assay was performed as described, Briefly, after transfection and drug treatment, 2.5 mg/ml MTT (M2003; Sigma-Aldrich) in phenol red-free medium was added and incubated for 2 h at 37 °C, followed by aspiration of the MTT solution, addition of isopropanol to dissolve the formazan crystals, and incubation at 37 °C for 1 h. Absorbance was measured at 540 nm. Experiments were independently repeated at least three times, and data were expressed as a percentage of the control (control siRNA-transfected or vehicle-treated cells). The TUNEL assay (G7361; Promega, Madison, WI, USA) was performed according to the manufacturer's protocol. Cells (1 × 10) were incubated for 24 h after seeding in 96-well plates and transfected with control or IRE1 siRNA for 48 h. TUNEL-positive cells were counted under a fluorescence microscope (Olympus, Tokyo, Japan) and expressed as the percentage of apoptotic cells relative to counted cells (=500) in 96 wells. Hydrogen peroxide levels were determined using DCFDA (C6827; Invitrogen). In brief, treated cells were incubated with 1 M DCFDA for 30 min and washed with PBS. Fluorescent signals were captured using a fluorescence microscope. Changes in mitochondrial oxidant production were measured using MitoSOX Red staining (5 mM for 15 min at 37 °C; M36008; Invitrogen), according to the manufacturer's instructions. In depolarized cells, mitochondrial labeling with potential-indicating probes like TMRM disappears; therefore, red fluorescence serves as an indicator of mitochondrial membrane potential. The medium was replaced with phenol red-free medium containing 500 nM TMRM (100 l/well; T-668; Invitrogen). Plates were incubated for 1 h at 37 °C and washed three times with PBS (50 l/well). Fluorescent signals were captured using a fluorescence microscope (Olympus), and analyzed in>500 cells per group. Mitochondria were visualized after the expression of Mito-DsRed (DsRed2 fused to the mitochondrial targeting sequence from subunit VIII of human cytochrome oxidase). Images were captured under a confocal laser scanning microscope (FV10i-w; Olympus), and analyzed in 100 cells per group with the ImageJ software (National Institutes of Health, Bethesda, MD, USA). For intracellular Ca imaging, cells were loaded with the Ca-sensitive dye fluo-4-acetoxymethyl ester (Fluo-4 AM, 5 M; F10471; Invitrogen) at 37 °C for 60 min and washed with PBS to remove extracellular Fluo-4 AM. After drug treatment, fluorescent signals were captured using a fluorescence microscope. The fluorescence intensity reflected [Ca]. Images for 1000 cells per group were analyzed with the ImageJ software. Cytosolic calcium levels ([Ca]) were assessed by ratiometric analysis using fura-2 acetoxymethyl ester (Fura-2 AM; F1221; Molecular Probes). Fura-2 AM was applied in the perfusion system throughout the imaging process. SH-SY5Y cells plated on poly-D-lysine-coated coverslips were loaded with Fura-2 AM (2 M) for 30 min in Normal Tyrode's solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl, 1 mM MgCl, 10 mM glucose, and 10 mM HEPES, pH 7.35), supplemented with 0.01% pluronic acid. Imaging was performed using an inverted microscope (Nikon Ti, Tokyo, Japan) with a × 40 UV objective lens (Nikon, Tokyo, Japan). Fura-2 AM was excited by sequential illuminations at 340 and 380 nm from a Lambda DG-4 illumination system (Sutter, Novato, CA, USA). Image processing was controlled by the Axon Imaging Workbench software 6.0 (AIW; Union City, CA, USA). Emission was detected at a wavelength of 510 nm. Fura-2 emission ratios following excitation at 340 and 380 nm were processed by AIW. Video images were obtained using an intensified CCD camera (LUCA; Andor, Belfast, UK). The analysis and plotting were carried out in the Origen 8.0 software (OriginLab Corp., Northampton, MA, USA). Immunocytochemical staining was performed as described. Briefly, cells were fixed for 15 min in 4% paraformaldehyde/PBS. After blocking, the cells were incubated with primary antibodies overnight at 4 °C. After washing with PBS, the cells were incubated for 1 h at room temperature with fluorescent-labeled secondary antibodies (1 : 500; Invitrogen). Cells were counterstained with DAPI for 10 min. Images were captured with a confocal laser-scanning microscope (FV10i-w). Cells fixed with cold acetone were analyzed using the Duolink Kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer's instructions. Briefly, samples were incubated with anti-ASK1 (rabbit polyclonal), anti-CIB1 (mouse monoclonal), and anti-InsP3R (rabbit polyclonal) antibodies, followed by addition of secondary antibodies conjugated with oligonucleotides (PLA probe MINUS and PLA probe PLUS). Oligonucleotides in hybridization solution will hybridize to two PLA probes if they are in close proximity (<40 nm). A ligase (Ligation Solution), nucleotides, and polymerase were added sequentially, allowing the formation of rolling-circle amplification products, which can be detected via labeled oligonucleotides. Signals visible as distinct dots were analyzed by confocal laser microscopy (FV10i-w). For immunoprecipitation (IP), cell pellets were resuspended in IP buffer (150 mM Tris-HCl, pH 6.8, 10 mM EDTA, 0.25% CHAPS) containing protease inhibitors (Sigma-Aldrich), followed by centrifugation at 13 000 r.p.m. for 15 min. To eliminate non-specific binding, a preclearing step was performed with protein A/G agarose beads (Santa Cruz) for 1 h. Next, samples were centrifuged for 5 min at 2000 × . Equal amounts of protein were precipitated with specific antibodies at 4 °C overnight on a rocker. Protein A/G agarose beads were added to each sample and incubated at 4 °C for 2 h. Immunoprecipitates were collected by centrifugation and washed three times with the same buffer. Finally, agarose beads were resuspended in 50 l of 1 × SDS-PAGE sample buffer and incubated at 55 °C for 10 min to release the proteins. After a pulse spin, supernatants were analyzed by SDS-PAGE. SH-SY5Y cells were fixed overnight in a mixture of cold 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and 2% paraformaldehyde in 0.1 M phosphate or cacodylate buffer (pH 7.2) and then embedded with epoxy resin. Epoxy resin-mixed samples were loaded into capsules and allowed to polymerize at 38 °C for 12 h and 60 °C for 48 h. Thin sections were sliced on an ultramicrotome (RMC MT-XL) and collected on a copper grid. Appropriate areas for thin sectioning were cut at 65 nm and stained with saturated 4% uranyl acetate and 4% lead citrate, followed by examination under a transmission electron microscope (JEM-1400; Tokyo, Japan) at 80 kV. For western blots, protein levels were normalized to pan forms or a housekeeping protein, such as -actin. All data were expressed as means±S.E.M. Student's -test was used for two-group comparisons, and analysis of variance, followed by Fisher's LSD test to compare three or more groups using SigmaStat for Windows Version 3.10 (Systat Software, Inc., Point Richmond, CA, USA). values of <0.05 were considered statistically significant.

Preliminary screening by 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was performed to ascertain the time and dose of the combined treatment of SeC with AF on several human cancer cells and normal cell. The results showed that treatment of human cancer cells with SeC for 48 h or AF for 6 h significantly decreased the cell viability in a dose-dependent manner (). In addition, the cells were pretreated with SeC (0, 3, 6, 12 or 24 h) and then co-treated with AF (6 h), the combined treatment displayed higher anti-proliferative activities on A549 cells than that of single agent (). However, single treatment or combined treatment showed less cytotoxicity toward HK-2 human normal proximal tubular cells (), indicating the selectivity of SeC in combination AF. Specifically, as shown in , treatment of A549 cells with 8 M SeC for 48 h reduced cell viability by 27.8%, and treatment of the cells with 6 M AF for 6 h showed no cytotoxicity. Nevertheless, combined treatment of cells with 8 M SeC pretreatment for 24 h and 6 M AF co-treatment for 6 h significantly decreased the cell viability by 66.3%, implying that SeC pretreatment dramatically enhanced AF-induced growth inhibition against A549 cells. The result showed that the interaction index: =0.56<1, meaning that the combined effects between SeC and AF were strongly synergistic. In the phase-contrast observation, pretreatment of cells with SeC obviously enhanced AF-induced reduction in cell numbers, cell shrinkage and loss of cell-to-cell contact, which further confirm this synergistic effect (figures not shown). To elucidate the mechanisms of combined treatment-induced growth inhibition, we examine the effect of SeC or/and AF on cell apoptosis and cell cycle distribution by flow cytometry in A549 cells. As shown in , treatment of cells with SeC in combination with AF resulted in a marked increase in the proportion of apoptotic cells compared with that of SeC or AF alone, as reflected by the increase in sub-G1 peaks from 14.1% (AF) and 21.9% (SeC) to 64.5% (combination). The results of flow cytometric analysis clearly indicated that SeC pretreatment noticeably enhanced AF-induced apoptosis in A549 cells. These results suggest that SeC can act as an enhancer to sensitize AF-induced cell growth inhibition against A549 cells by induction of apoptosis. Apoptotic signaling can be conducted by two central mechanisms, the extrinsic (death receptor-mediated) and the intrinsic (mitochondrial-mediated) pathways. To investigate the molecular events initiated by SeC or/and AF, we first examined the contribution of caspases for the apoptotic program. As shown in , pretreatment of A549 cells with 8 M SeC distinctly enhanced AF-induced activation of caspase-3, -8, and -9, indicating the activation of both the intrinsic and extrinsic apoptosis pathways. Moreover, activation of caspase-9, as the predominant initiator in mitochondria-mediated apoptotic pathway, is more prominently than that of caspase-8 induced by combined treatment, indicating that combined treatment-induced apoptosis is mainly initiated by the mitochondria-mediated apoptosis pathway. Activated caspases subsequently induced proteolytic cleavage of PARP and finally resulted in cell apoptosis. The hypothesis was further confirmed by western blotting at protein level. As shown in , treatment with SeC and AF in combination resulted in noticeable elevation in activation of caspases-3, -7, -8, -9, and -10, and PARP cleavage. Taken together, these results suggest that SeC as a potent chemosensitizer enhances the AF-induced cell growth inhibition and apoptosis in A549 cells mainly by initiating the mitochondria-mediated apoptosis pathway. Loss of mitochondrial membrane potential (Δ) is catastrophic for cells and leads to the release of cytochrome into the cytosol. Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has been suggested to activate the apoptotic pathway. Therefore, flow cytometric analysis was used to confirm whether combined treatment-induced apoptosis occurred through destroying mitochondrial homeostasis using JC-1 as a molecular probe. As shown in , treatment of cell with 8 M SeC or 6 M AF alone obviously decreased the Δ, as evidenced by the elevation of green fluorescence from the red to green. However, 8 M SeC and 6 M AF in combination resulted in an enhanced depletion of Δ from 27.4% (AF) and 31.2% (SeC) to 72.9% (combination). Bcl-2 family proteins could either promote cell survival (Bcl-2 and Bcl-XL) or induce apoptosis (Bax and Bad). The imbalance of pro-apoptotic and anti-apoptotic Bcl-2 family proteins will lead to the loss of mitochondrial membrane potential and finally result in the induction of apoptosis. Hence, it was of interest to identify the Bcl-2 family members involved in the combined treatment-induced apoptosis. As shown in , combined treatment of SeC with AF remarkably decreased the expression rates of Bcl-2/Bax and Bcl-xL/Bad in treated A549 cells. The result demonstrated that the activation of mitochondrial apoptosis pathway was fulfilled by regulating Bcl-2 family proteins. To further determine the effect of SeC, the time-course effects of SeC on expression of Bcl-2 family proteins was evaluated. The western blotting results showed that SeC treatment for indicated time notably increased expression of Bax, but decreased expression of Bcl-2 in a time-dependent manner (). Taken together, these results all suggest that SeC as a potent regulator of Bcl-2 family protein enhances AF-induced mitochondrial apoptosis pathway by depletion of Δ through regulation of Bcl-2 family protein expression. MAPKs (including JNK, p38, and ERK) and PI3K/Akt are both upstream kinases that have key roles in regulation of cell proliferation, cell growth, and survival. Therefore, it was interested to investigate whether the MAPKs and PI3K/Akt pathways were involved in the induction of apoptosis by combined treatment using specific antibodies against the phosphorylated (activated) forms of the kinases. The results showed that SeC alone induced the obvious downregulation of phosphorylated AKT and phosphorylated ERK (). Moreover, SeC and AF in combination dramatically caused ERK and AKT inactivation. No alteration in phosphorylation status of p38 and JNK was detected in treated A549 cells (), indicating that JNK and p38 MAPK did not have important roles in regulating cell death of A549 cells in response to SeC and AF. To further confirm the role of AKT and ERK, cell viability was detected by MTT assay using specific ERK and AKT inhibitors. As shown in , pretreatment of A549 cells with LY2294002 (an AKT-upstream inhibitor) and U0126 (an ERK inhibitor) for 1 h markedly enhanced combined treatment-induced cell growth inhibition against A549 cells, indicating that combined treatment induced apoptotic cell death of A549 cells with ERK- and AKT-dependent manner. Enhanced activation of caspase-3 further confirmed the effects of ERK and AKT inhibitors on cell apoptosis (). Results in protein level further affirmed this conclusion. For instance, treatment of A549 cells with LY2294002 or U0126 caused significant inactivation of ERK and AKT, respectively. In addition, enhanced PARP cleavages were observed after treatment with these two inhibitors. Taken together, the results above suggest that SeC can act as potent chemosensitizer to enhance AF-induced growth inhibition and apoptosis of A549 cells through inhibition of PI3K/AKT and MEK/ERK pathways. AF can induce ROS-mediated DNA damage by inhibiting TrxR activity. Our previous studies have showed that SeC can trigger DNA damage through ROS overproduction. To examine whether combined treatment induce enhanced DNA damage, two DNA damage markers, phosphorylated p53 (Ser15) and phosphorylation histone (Ser139), were employed to investigate this combined effect. As expected, A549 cells treated with SeC and AF alone activated phosphorylated p53 and phosphorylated histone (). Moreover, SeC pretreatment significantly enhanced AF-induced DNA damage, as convinced by enhanced phosphorylated p53 and phosphorylated histone. DNA damage induced by combined treatment was further confirmed by comet assay and TUNEL-4′,6-diamidino-2-phenyindole (DAPI) co-staining. As shown in , treatment with SeC and AF alone notably triggered DNA damage, and SeC treatment obviously enhanced AF-induced DNA damage, as convinced by tail-DNA in single-cell level (). Furthermore, after TUNEL-DAPI co-staining, A549 cells exhibited enhanced apoptotic features, such as DNA fragmentation and nuclear condensation (). Based on the importance of SeC, a time course of SeC was investigated. As shown in , exposure of A549 cells to 8 M SeC alone resulted in notable elevation of phosphorylated p53 and phosphorylated histone in a time-dependent manner. Taken together, these results indicate that SeC could enhance AF-induced apoptosis through enhancement of AF-induced DNA damage. Furthermore, time-course analysis was carried out to examine the temporal reference of different signaling in response to drug treatments. As shown in , phosphorylated AKT and ERK were detected as a sustained activation after treatment with SeC alone for 4 and 12 h, whereas AF treatment for 3 and 6 h only caused modest decline in phosphorylation of AKT and ERK. In addition, both SeC and AF slightly activated Ser15-p53. Interestingly, addition of AF to SeC-pretreated cells significantly enhanced the phosphorylation of p53 and inactivation of ERK and AKT. Taken together, these results indicate that SeC enhances AF-induced apoptosis through regulation of p53, ERK, and AKT pathways. ROS can cause DNA damage and have an important role in anticancer agents-induced apoptosis. Therefore, combined treatment-induced change of intracellular redox status was evaluated by measure of ROS generation using a fluorescein-labeled probe, DCFH-DA. As shown in , treatment of cell with SeC and AF alone induced obvious ROS accumulation in time-dependent manner, and combined treatment with SeC and AF resulted in enhanced ROS overproduction. For further evaluation of ROS importance, glutathione (GSH) and -acetyl--cysteine (NAC), thiol-reducing antioxidant, were employed to examine the role of intracellular ROS in combined treatment-induced apoptotic cell death. As anticipated, addition of GSH and NAC effectively attenuated combined treatment-induced cell growth inhibition against A549 cells (). For instance, combined treatment with SeC (8 M) and AF (6 M) decreased the cell viability to 44.3%. However, pretreatment of cells with 10 mM GSH for 2 h effectively elevated the cell viability to 66.4%. GSH-ethyl-ester pre-treatment showed the similar protective effect with GSH (), indicating that elimination of intracellular ROS effectively block combined treatment-induced cell killing. The protective mechanism of GSH against the cytotoxic effects of SeC and AF were also investigated in this study. The flow cytometric analysis revealed that elimination of intracellular ROS reversed combined treatment-induced apoptosis (). For instance, combined treatment of SeC and AF triggered cell apoptosis at 57.9%. However, pretreatment with 10 mM GSH for 2 h effectively decreased the sub-G1 peak to 4.6%, suggesting that elimination of intracellular ROS effectively block combined treatment-induced cell apoptosis. Furthermore, both declined caspase-3 activity () and reduced caspase-3 cleavage () detected by western blotting confirmed this protective effect of GSH. For further investigation of underlying mechanism, the Bcl-2 family, DNA damage, and upstream kinases were all examined by western blotting method. Interestingly, addition of ROS scavenger distinctly attenuated combined treatment-induced DNA damage and completely reversed combined treatment-induced the inactivation of ERK and AKT (), indicating that ROS serves as upstream mediator in regulating induction of DNA damage and inactivation of ERK and AKT. Mitochondrial respiratory chain represents the main source of intracellular ROS. Intracellular ROS released from mitochondria was closely regulated by Bcl-2 family. Enhanced Bcl-XL expression after GSH addition was observed in , which indicated the possibility that GSH blocked the ROS-mediated mitochondrial dysfunction by regulating Bcl-2 family proteins. Taken together, these results demonstrate that SeC could sensitize AF-induced apoptosis in A549 cell in ROS-dependent manner. To clarify the mechanism of intracellular ROS accumulation, both TrxR1 activity and TrxR1 expression were evaluated. The TrxR1 activity was measured by 5,5′-dithiobis (2-nitrobenzoic) acid assay with rat liver TrxR as positive control. The results showed that incubation of the cell lysate with SeC or AF alone inhibited the TrxR1 activity in time-dependent manner. However, the TrxR1 activity was more effectively inhibited by the combined treatment of SeC and AF (). The results of western blot analysis revealed that both SeC alone and the combined treatment decreased TrxR1 expression in cell level, but AF alone caused no significant change in TrxR1 expression (). The result indicate that SeC in combination with AF synergistically inhibit TrxR1 . To investigate whether SeC or/and AF target TrxR1 , the therapeutic effect of combined treatment on immuno-deficient nude mice bearing A549 tumor xenografts was evaluated. After 16 days' administration, treatment of SeC and AF alone slightly inhibited xenograft lung tumor growth. However, xenograft lung tumor growth in nude mice was more effectively inhibited by combined treatment with SeC and AF . For instance, combined treatment with SeC and AF significantly inhibited the tumor weight and tumor volume, but not affected body weight of mice (). The mechanism studies revealed that combined treatment inhibited tumor xenografts by induction of mitochondria-mediated apoptosis, as convinced by caspases activities (), cleaved PARP (), and cleaved caspase-3 staining. TrxR1 expression in tumor xenografts detected by western blotting was also evaluated, and the result indicates that SeC alone and combined treatment both reduced TrxR1 expression, but AF treatment alone caused no changes in TrxR1 expression. Furthermore, several cell markers using immunohistochemical (IHC) methods further confirmed that combined treatment with SeC and AF inhibited angiopoiesis (CD31 staining) in tumor xenografts, activated Ser15-p53 expression, and inhibited tumor xenograft cell proliferation (Ki67 staining). Taken together, these data support the conclusion that SeC can synergistically enhance AF-induced tumor growth inhibition by targeting TrxR1. TrxR, overexpressed in many tumors cells, is the central redox protein controlling the cellular thiol redox state. Inhibition of Trx system may cause dramatic imbalance between the formation and the removal of ROS. Consequently, the Trx system has emerged as a novel target for anticancer drug development. In this study, we demonstrated that SeC can target TrxR1 and to enhance AF-mediated lung cancer cell killing through activating mitochondria-mediated apoptosis pathway. And this chemosensitization effect of SeC was achieved by triggering ROS-mediated DNA damage and inactivation of AKT and ERK. To our knowledge, this is the first study to demonstrate that SeC can target TrxR1 and . Our data suggest that the strategy to use SeC and AF in combination could be a highly efficient way to achieve anticancer synergism through synergistically targeting TrxR1 and . Induction of cancer cell apoptosis is an effective method in inhibiting tumor growth . Apoptotic cells exhibit typical apoptotic feature including series of morphology and biochemical changes. Such as cell membrane blebbing, lost of connection in cell to cell, mitochondrial depolarization, caspase activation, apoptotic body formation, chromatin condensation, and DNA fragmentation. In the present study, SeC in combination of AF trigger apoptosis of A549 cells and , and the cells exhibit typical apoptotic feature. For instance, observation by optical microscope shows that combined treatment with SeC and AF dramatically enhanced cell shrinkage, loss of cell-to-cell contact, and cell reduction in cell numbers. Cells stained by TUNEL and DAPI appear enhanced DNA fragmentation and nuclear condensation. Furthermore, A549 cells in tumor xenografts also display apoptotic feature , such as the reduced-angiopoiesis and weaken-cell proliferation were observed after combined treatment. Caspase-3 activation and PARP cleavage both confirmed combined treatment-induced apoptosis and . Mitochondria- and death receptor-mediated apoptosis both contribute to drug-induced cancer cell apoptosis. Caspase, a family of cysteine acid proteases, act important role in induction of apoptosis through the enzymolysis of series of substrates. In this study, SeC and AF in combination synergistically induced mitochondria-mediated apoptosis of A549 cell and because of that the activation of caspase-9 is more obvious than that of caspase-8. Mitochondrial membrane potential (Δ) has a key role in triggering mitochondria-mediated apoptosis and the permeabilization of mitochondria is closely regulated by Bcl-2 family expression, which could be classified into two groups, pro-apoptotic proteins such as Bax and Bad, and anti-apoptotic proteins such as Bcl-2 and Bcl-XL. Bcl-2 family proteins could bind to the membrane of mitochondria to regulate the Δ in response to apoptotic stimulation. In this study, combined treatment with SeC and AF significantly decreased Bcl-2 and Bcl-X expression, but increased Bax and Bad expression. The imbalance of Bcl-2 family expression finally resulted in the mitochondrial dysfunction and induced mitochondria-mediated apoptosis in A549 cells. These results indicate that combined treatment-induced mitochondria-mediated apoptosis of A549 cells was fulfilled by regulation of Bcl-2 family expression. PI3K/AKT and MEK/ERK pathways have pivotal role in fundamental cellular functions such as cell proliferation, cell growth, and survival by phosphorylating a variety of substrates. In recent years, it has been reported that components of PI3K/AKT and MEK/ERK signaling pathways are frequently altered in human tumors, which decisively contribute to the clinic drug resistance. Hence, much effort has been made to develop treatment strategies that target these specific signaling molecules or their downstream effectors. Recently, many evidences support that seleno compounds can induce apoptosis and enhance chemosensitivity of anticancer drugs in cancer cells with involvement of AKT or/and ERK inactivation. However, no information about the importance of MAPK and PI3K/Akt pathways as potential targets for the chemosensitization effects of SeC is available. The present study demonstrated that SeC synergistically enhanced AF-mediated human lung cancer cell killing and apoptosis involving AKT and ERK inactivation. Inhibitors of ERK and AKT significantly enhanced the combined treatment-induced apoptosis in A549 cells, indicating that combined treatment induce apoptosis of A549 cells with ERK- and AKT-dependent manner. Accumulative evidence support that many anticancer drugs exhibit their therapeutic effects on cancers by induction of DNA damage and activation of downstream signaling pathways. Ser139-Histone, a DNA damage marker, can be activated in response to DNA damage. Activated histone can transfer the signal to upstream molecules, such as ataxia telangiectasia-mutated and ataxia telangiectasia and Rad3-related proteins, followed by CHK and p53. p53, a tumor suppressor, is a cell cycle checkpoint protein that contributes to the preservation of genetic stability in response to DNA damage. Activation of p53 in response to DNA damage could trigger downstream signaling molecules, such as Bcl-2 family proteins and p21, to induce cell apoptosis or cell cycle arrest. The present study demonstrated that combined treatment triggered enhanced DNA damage, as convinced by upregulated phosphorylated p53 and phosphorylated histone. In single-cell level, we also observed obvious DNA damage, such as nuclear condensation and DNA fragmentation. These results indicate that induction of DNA damage contributed to combined treatment-induced apoptosis in A549 cells. Intracellular ROS overproduction may injure lipids, proteins, and DNA, and finally caused cell apoptosis. Increasing evidence supports that seleno compounds induce cell apoptosis involving ROS overproduction. In combating cancer cells, selenium acts as pro-oxidant rather than antioxidant by inducing apoptosis through the generation of oxidative stress. In our previous studies, SeC displays broad-spectrum anticancer activity against series of human cancer cells through ROS-mediated apoptosis. And the mechanism studies reveal that inhibition of ROS accumulation can effectively prevent cancer cells from SeC-induced apoptosis. The possibility is that GSH addition plenished intracellular stores of endogenous antioxidants and reversed apoptosis. Our previous studies showed that combined treatment with SeC and 5-fluorouracil can more effectively induce A375 human melanoma cell apoptosis by ROS overproduction. To character the importance of ROS in combined treatment, several antioxidants, such as GSH-ethylene-ester, NAC, diphenyleneiodonium chloride, DMSO, superoxide dismutase, and catalase, were employed to investigate the ROS origin. More importantly, activities of glutathione peroxidase, glutathione reductase, and TrxR were also measured, and the results indicated that SeC can enhance 5-fluorouracil-induced apoptosis in A375 cells by ROS overproduction through regulation of intracellular redox system. Therefore, based on these results, we proposed that SeC enhanced AF-induced A549 cell apoptosis by ROS accumulation through dysregulation of intracellular redox system. The present study demonstrates that SeC can act as a natural inhibitor of TrxR1 and to enhance AF-induced human lung cancer cell killing and apoptosis through ROS-mediated DNA damage and inactivation of ERK and AKT pathways. It is reported that AF could bind to the SeC-containing C-terminal and the N-terminal redox center to inhibit TrxR activity and SeC probably acted as substrates to compete with Trx. We speculate the possibility that SeC inhibits TrxR1 activity by competing with Trx and caused ROS accumulation, which in turn oxidized intracellular thiol-containing antioxidant agents like GSH and Trx, thus sensitized the cancer cells to AF-induced apoptosis. In addition, decreased TrxR1 expression induced by SeC may contributed to combined treatment-induced A549 cell apoptosis. This finding predicts that SeC shows promising implications in improving the therapeutic efficacy when in combination with other anticancer drugs in clinic. In summary, we showed the ability of SeC to enhance AF-induced human lung cancer cell killing and by mitochondria-mediated apoptosis through synergistically targeting TrxR1, and this sensitization can be achieved by triggering ROS-mediated DNA damage and inactivation of ERK and AKT (). Taken together, our results suggest that the strategy to use SeC and AF in combination could be a highly efficient way to achieve anticancer synergism through synergistically targeting TrxR1. SeC, AF, propidium iodide (PI), solid JC-1, DAPI, 2′,7′-dichlorofluorescein diacetate, MTT, bicinchoninic acid kit for protein determination were purchased from Sigma (St. Louis, MO, USA). Reagent kit for single-cell gel electrophoresis assay (Comet Assay) was purchased from Trevigen (Gaithersburg, MD, USA). TrxR1 Assay Kit was bought from Cayman (Ann Arbor, MI, USA). Dulbecco's modified Eagle's medium, fetal bovine serum, and the antibiotic mixture (penicillin-streptomycin) were purchased from Invitrogen (Carlsbad, CA, USA). Caspase-3 substrate (Ac-DEVD-AMC), caspase-9 substrate (Ac-LEHD-AFC), and caspase-8 substrate (IETD-AFC) were purchased from Calbiochem (San Diego, CA, USA). U0126 and LY294002 were obtained from Calbiochem. All of the antibodies used in this study were purchased from Cell Signaling Technology (Beverly, MA, USA). All of the solvents used were of high-performance liquid chromatography grade. The water used for all experiments was supplied by a Milli-Q water purification system from Millipore (Billerica, MA, USA). A549 human lung adenocarcinoma cell line was obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10%), penicillin (100 U/ml) and streptomycin (50 U/ml) at 37 °C in a humidified incubator with 5% CO atmosphere. Preliminary screening was conducted to ascertain the time and dose of the combined treatment of SeC with AF. Briefly, A549 cells seeded in a 96-well microplate at 8 × 10 per well were pretreated with 2.5–40 M SeC (0, 3, 6, 12, or 24 h) and then co-treated with 0.5–20 M AF (0, 1, 2, 4, and 6 h). After treatment, MTT assay was used to ascertain the optimal dose and time of combined treatment. A549 viability was measured by MTT assay and trypan blue staining. A549 cells (8 × 10 per well ) seeded in a 96-well microplate were pre-treated with 8 M SeC for 24 h and co-incubated with 6 M AF for 6 h. After incubation, the cell viability were detected by MTT assay or trypan blue staining. Cell viability was expressed as % of control. For the trypan blue staining assay, the cells after treatment were collected by trypsinization and resuspended in PBS buffer after centrifugation. An aliquot of the cell suspension was mixed with an equal volume of a 0.4% trypan blue solution. Each cell sample was immediately transferred to a hemacytometer for counting in triplicates. Stained (dead) and unstained (viable) cells were counted by phase-contrast microscopy. The cell cycle distribution was analyzed by flow cytometric analysis as previously described. Briefly, cells after treatment with SeC and AF were harvested by centrifugation and washed with PBS. Cells were stained with PI after fixation with 70% ethanol at −20 °C overnight. Labeled cells were washed with PBS and then analyzed by flow cytometer. The cell cycle distribution was analyzed using a MultiCycle software (Phoenix Flow Systems, San Diego, CA, USA). The proportions of cells in G0/G1, S, and G2/M phases were represented as DNA histograms. Apoptotic cells with hypodiploid DNA contents were measured by quantifying the sub-G1 peak. For each experiment, 10 000 events per sample were recorded. The induction of DNA fragmentation and nucleus condensation in A549 cells by SeC and AF were examined by TUNEL-DAPI co-staining assay as previously described. The effects of SeC and AF on the cell mitochondrial membrane potential (Δm) were examined by flow cytometry using JC-1 as specific probe. The percentage of the green fluorescence from JC-1 monomers was used to represent the cells that lost Δ. The effects of SeC and AF on intracellular ROS generation were evaluated by DCF fluorescence assay as previously described. The ROS levels were expressed as percentage of treated cells to that of control. Single-cell gel electrophoresis for detection of DNA damage induced by SeC and AF was performed using Comet assay as previously described. Cellular DNA was stained with SYBR GreenI (Trevigen) and visualized under a fluorescence microscope (Nikon, Eclipse E600, Tokyo, Japan). Activation of caspases in A549 cells exposed to SeC and AF was examined by measuring the activities of caspase-3, -8, and -9 using specific substrates (Ac-DEVD-AMC for caspase-3, Ac-IETD-AMC for caspase-8, and Ac-LEHD-AMC for caspase-9). The effects of SeC and AF on the expression levels of proteins associated with different signaling pathways were examined by western blot analysis. Cytosolic protein was extracted for TrxR1 activity using a Thioredoxin Reductase Assay Kit (Cayman). In this assay, TrxR1 catalyzes the reduction of 5,5′-dithiobis (2-nitrobenzoic) acid with NADPH to 5-thio-2-nitrobenzoic acid (TNB), which generates a strong yellow color (max=412 nm). Briefly, 100 g of cytosolic protein was diluted by assay buffer and incubated with 8 M SeC or/and 6 M AF for 10 min at room temperature. The reaction was started by addition of NADPH and DNTB, and then the TrxR1 activity was detected in 1 h by measurement of TNB quantity at 412 nm. The TrxR1 activity was expressed as % of control. study was administrated in male nude mice. Briefly, about 1 × 10 A549 human lung adenocarcinoma cells in 100 l serum-free medium were subcutaneously injected into the right oxter of mice. When average tumor volume reached about 50–75 mm after 8 days, mice were randomly divided into four groups (eight mice/group): group 1 for PBS as control; group 2 for 5 mg/kg SeC; group 3 for 2 mg/kg AF; group 4 for 5 mg/kg SeC+2 mg/kg AF. Drugs were injected every other day, caudal vein, from the first day until the sixteenth day (eight times). At the termination of the experiments, tumors were collected, photographed, and weighed. Tumor dimensions were measured with calipers and the volume was calculated using the formula: volume= × /2, with being the maximal length and being the width. A portion of the tumors from control and treated animals was used for preparation of tumor lysate used in further analysis. Another portion of tumors were removed, fixed in 10% buffered formalin, embedded with paraffin, and sectioned. The 4-M sections were stained with hematoxylin and eosin staining for histological observation. Protein expression in sections was examined by IHC methods. All animal experiments were approved by the Animal Experimentation Ethics Committee. Experiments were carried out at least in triplicate and repeated three times. All data were expressed as mean±S.D. Statistical analysis was performed using SPSS statistical package (SPSS 13.0 for Windows; SPSS, Inc. Chicago, IL, USA). The difference between two groups was analyzed by two-tailed Student's -test. The difference between three or more groups was analyzed by one-way analysis of variance multiple comparisons. Differences with <0.05 (*) or <0.01 (**) were considered statistically significant. Bars with different characters are statistically different at <0.05 level. The growth inhibitory effects of combination treatment (synergistic, addictive, or antagonistic) was evaluated by the interaction index. The index, denoted by , is defined by the isobolar relation: /+/=, where and are the doses of SeC (alone) and AF (alone), respectively, that give the 50% of cell killing and (, ) is the combination dose that produces the same level. The quantities in equation are obtained from the dose–response curves of drugs , , and the combination. If =1, the interaction is additive; if <1, it is super-additive (synergistic), and if >1, it is sub-additive (antagonistic).

To test whether APN affects hearing impairment, we assessed auditory brainstem response (ABR) thresholds in WT and APN-KO mice at the ages of 2 months and 6 months (=5–7 per group). WT mice did not show any threshold shifts at any frequencies at these ages. At 2 months of age, APN-KO mice exhibited a significant increase in ABR threshold at high frequency (32 kHz) as compared with WT mice=(, <0.01). However, at 2 months of age APN-KO mice exhibited only slight (statistically non-significant) increases in ABR thresholds at 8 kHz and 16 kHz. At 6 months of age, APN-KO mice exhibited significant increases in ABR thresholds at 8, 16, and 32 kHz compared with WT mice at the ages of 2 months and 6 months, and APN-KO mice at the age of 2 months (<0.01). Thus, APN-KO mice exhibit an early onset and progressive course of hearing impairment. Because reduction of blood flow in the cochlea contributes to the progression of hearing impairment, we measured cochlear blood flow (CBF) using a laser-Doppler flowmeter. Quantitative analysis revealed that, at 2 months of age, APN-KO mice showed a marked reduction in CBF (42.1%±4.4%) as compared with WT mice (, =3, <0.05). To investigate the extent of cochlear vessel formation at the microcirculatory level, capillary density was measured in histological sections from the SV of cochleae. shows representative photomicrographs of tissue immunostained for CD31 (arrows: capillaries in the SV). Quantitative analysis revealed that, at 2 months of age, the capillary density in the SV was significantly reduced in APN-KO mice as compared with WT mice (, right panel, =4, <0.05). Apoptosis is responsible for the damage to the organ of Corti caused by hypoxia. We performed TUNEL staining in the organs of Corti from WT and APN-KO mice at 2 months of age. Representative photographs of TUNEL-positive (green) nuclei in the organs of Corti at the basal turn are shown in . Higher proportions of TUNEL-positive hair cells were evident at the basal, middle, and apical turns of the organ of Corti in APN-KO mice as compared with WT mice. Furthermore, we assessed the effect of APN on caspase-3 activation in the organs of Corti from WT and APN-KO mice at 2 months of age by western blot analysis. The expression level of cleaved caspase-3 was greater in APN-KO mice than in WT mice (). To examine the effect of APN on apoptosis at the cellular level, auditory HEI-OC1 cells were subjected to hypoxia under conditions of serum deprivation in the presence of recombinant human APN or vehicle alone. Treatment with a physiological concentration of APN (30 g/ml) significantly reduced the frequency of TUNEL-positive cells under hypoxic conditions (, <0.001). APN treatment also reduced the frequency of TUNEL-positive human umbilical vein endothelial cells (HUVECs), as previously described (<0.01). APN can promote apoptosis through modulation of AMP-activated protein kinase (AMPK) activity in various cell types. It has also been reported that AMPK deficiency increases noise vulnerability after acoustic overstimulation. Thus, we investigated the possible participation of AMPK in the protective actions of APN on hypoxia-induced apoptosis in auditory HEI-OC1 cells (). Treatment of HEI-OC1 cells with APN stimulated the phosphorylation of AMPK. Pretreatment with the AMPK inhibitor ‘compound C' effectively suppressed the inhibitory effects of APN on apoptosis under hypoxic conditions (). Because at 2 months of age the extent of apoptosis was higher in the organs of Corti in APN-KO mice than in those of WT mice, we assessed morphological alterations to the organs of Corti in aged WT and APN-KO mice. Cochlear tissue in the basal turn was stained with hematoxylin and eosin in WT and APN-KO mice at 6 months of age. Hair cells were lost at the basal turn of the cochlea in APN-KO mice, whereas hair cells in WT mice were preserved (, encircled). APN-KO mice also showed severe damage in the regions of spiral ganglion neurons, compared with WT mice (arrow). To further compare the organs of Corti in aged APN-KO and WT mice, we examined them using scanning electron microscopy (SEM). At 6 months of age, in WT mice three rows of outer hair cells (OHCs) and one row of inner hair cells (IHCs) were present in the organ of Corti at the basal turn of the cochlea (, upper panel). However, in APN-KO mice, at 6 months of age, the OHCs in the basal turn of the cochlea were lost in patches (, lower panel). To test whether exogenous APN supplementation prevented the development of hearing impairment, we administered adenoviral vectors via the jugular vein, expressing APN (Ad-APN) or β-galactosidase (Ad-β-gal) as a control, to WT and APN-KO mice aged 1.5 months. We then performed ABR measurements in these mice at 2 months of age. On the sixth day after injection, plasma APN levels were 11.3±1.9 g/ml in WT/Ad-β-gal,<0.05 g/ml in APN-KO/Ad-β-gal, and 10.5±1.6 g/ml in APN-KO/Ad-APN. Ad-β-gal-treated APN-KO mice exhibited an increase in ABR threshold at 32 kHz compared with Ad-β-gal-treated WT mice (, =4, <0.01). Supplementation with APN in APN-KO mice rescued the hearing impairment that was seen in the Ad-β-gal-treated APN-KO mice (=4, <0.05). We also assessed CBF, capillary density, and apoptosis in the organ of Corti. Supplementation with APN in APN-KO mice increased CBF and the capillary density of the SV, and decreased the frequency of TUNEL-positive cells in the organ of Corti (). Finally, we examined whether circulating APN level is associated with hearing threshold in apparently healthy Japanese men. Clinical characteristics of the study population are shown in . Mean BMI, blood pressure, hemoglobin A1c, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and creatinine levels were within the normal range. Pure tone average (PTA)-low and PTA-high were 19.4±0.83 dB and 26.8±1.17 dB, respectively. The average of the Log-APN levels was 0.64±0.02 g/ml. Circulating APN levels correlated negatively with BMI, waist circumference, fasting glucose, and triglycerides and positively with HDL cholesterol (). Plasma APN levels in hearing-impaired subjects (PTA-high>25 dB) were significantly lower than those in normal-hearing subjects (PTA-high ≤25 dB) in the high-frequency range (Log-APN: 0.601±0.025 0.670±0.021 g/ml, <0.05). In contrast, APN levels did not differ significantly between the two groups in the low-frequency range (Log-APN: 0.604±0.038 0.650±0.018 g/ml). Results of logistic regression analysis for hearing threshold are shown in . In single logistic regression analysis, age, fasting glucose, and triglycerides were significantly associated with PTA-low. In multiple logistic regression analysis incorporating age, fasting glucose, and triglycerides, age was significantly correlated with PTA-low. In single regression analysis for PTA-high, age and APN levels were correlated with PTA-high. In multiple regression analysis incorporating age and APN levels, both were significantly associated with PTA-high. Thus, low levels of APN are closely linked with the presence of ARHI, particularly high-frequency sensorineural hearing loss. The present study provides the first evidence that APN deficiency contributes to ARHI. APN-KO mice showed exacerbated hearing impairment, whereas exogenous APN could reduce hearing impairment in these mice. Furthermore, plasma levels of APN, together with age, are independently associated with ARHI in apparently healthy subjects. Thus, these data suggest that approaches aimed at normalizing circulating APN levels could be valuable for the prevention of hearing impairment. Accumulating evidence indicates that APN has favorable effects on vascular responses and endothelial cell functions. APN-KO mice exhibit impaired angiogenesis in response to hindlimb ischemia, whereas overexpression of APN promotes ischemia-mediated angiogenesis. APN supplementation has also been shown to stimulate blood vessel growth in both rabbit corneal angiogenesis and mouse Matrigel plug implantation models. The present study showed that APN deficiency leads to decreased CBF and capillary density of the SV. Recently, it was reported that hypoxia/ischemia in the cochlea causes damage to the organ of Corti and induces hearing loss. It has also been reported that reduced CBF and impaired hearing are observed in diabetic mice exposed to loud noise, and ApoE-KO mice fed a high-fat diet. Thus, it is conceivable that loss of APN leads to hypo-perfusion and subsequent hypoxia/ischemia in the cochlea, thereby contributing to the progression of hearing impairment. It has been suggested that apoptosis of hair cells caused by hypoxia is a key feature of various forms of hearing impairment. In the present study, APN-KO mice showed increased apoptotic activity and loss of hair cells at the organ of Corti. APN also suppressed hypoxia-induced apoptotic activity in cultured auditory HEI-OC1 cells . Similarly, APN deficiency reportedly causes enhanced apoptosis in the heart following ischemia reperfusion. APN prevents hypoxia-induced apoptotic activity in cardiomyocytes. Moreover, APN suppresses apoptosis of endothelial cells under conditions of serum starvation. These data suggest that APN prevents the development of hearing impairment, in part, via suppression of the apoptotic activities of endothelial and auditory sensory hair cells in the cochlea. Collective evidence from numerous clinical studies indicates that low levels of circulating APN are associated with increased prevalence of obesity-linked diseases, including atherosclerosis and ischemic heart disease. Plasma APN levels in apparently healthy subjects are positively correlated with age. However, this correlation does not remain significant when adjusting for obesity-related risk factors. A previous study showed a negative association between plasma APN concentrations and peripheral hearing thresholds in the Taiwanese population. That study evaluated both genders and subjects between the ages of 40 and 86, including healthy adults and subjects with chronic conditions such as heart disease, kidney dysfunction, and diabetes. The present study showed that in apparently healthy middle-aged Japanese men, reduced levels of APN are independently associated with ARHI after adjustment for age. These data suggest that APN levels are predictive of the progression of ARHI. Thus, APN may represent a biomarker not only for metabolic and vascular disorders, but also for ARHI. This study has several limitations. First, we only investigated whether APN affects early sensorineural hearing loss using male mice and assessed its clinical significance in ARHI in apparently healthy males. In our preliminary investigation, female APN-KO mice also developed exacerbation of hearing impairment, particularly in the high-frequency range, compared with female WT mice (data not shown). A previous study also reported a negative association between plasma APN concentrations and peripheral hearing thresholds in males and females in the Taiwanese population. Thus, we believe that disruption of APN results in an early onset and a progressive course of hearing impairment, regardless of gender. Second, we only explored anti-apoptotic effects with regard to the protective action of APN against ARHI. It has been reported that mechanisms contributing to ARHI include hypoxia-induced apoptosis, oxidative stress, and inflammation. As well as pro-survival effects, APN exerts several other biological effects in various cell types, including anti-inflammatory and anti-oxidant activities. Therefore, future experimental studies will be required to determine the APN-mediated intracellular signaling pathways in auditory organs, to gain a better understanding of its protective modes of action. In conclusion, we found that disruption of APN results in reduced blood flow and increased apoptosis in the cochlea, thereby leading to ARHI. Furthermore, we demonstrated that circulating APN levels are reduced in subjects with ARHI. Collectively, these data suggest that APN represents a target molecule for the prevention of ARHI. Recombinant human APN protein generated by a mammalian cell expression system was purchased from BioVendor (Asheville, NC, USA). Adenovirus vectors containing the genes for β-galactosidase (Ad-β-gal) and full-length mouse APN (Ad-APN) were prepared as described previously. Compound C was purchased from Calbiochem (San Diego, CA, USA). Primary antibodies specific for phospho-AMPK (Thr-172), pan-α-AMPK, and cleaved caspase-3 (Asp175) were purchased from Cell Signaling Technology (Danvers, MA, USA). HEI-OC1 cells were a generous gift from the House research institute (Dr. Kalinec F). WT and APN-KO male mice on a C57BL/6 background were used for this study. Study protocols were approved by the Institutional Animal Care and Use Committee of Nagoya University. At the ages of 2 months and 6 months, mice were subjected to ABR measurement under anesthesia with ketamine (100 mg/kg) and xylazine (9 mg/kg) as previously described. In brief, mice were placed inside a quiet room, and generation of acoustic stimuli and subsequent recording of evoked potentials were performed using a data acquisition system (AD Instruments, Bella Vista, NSW, Australia). Acoustic stimuli consisting of tone bursts (0.1 ms cos2 rise/fall and 1 ms plateau) were delivered monaurally through a magnetic speaker (CF1, Tucker-Davis Technologies, Alachua, FL, USA) connected to a funnel fitted into the external auditory meatus. To record bioelectrical potentials, subdermal stainless steel needle electrodes were inserted at the vertex (ground), ventrolateral to the measured ear (active), and contralateral to the measured ear. Stimuli were calibrated against a 1/4-inch condenser microphone (UC-54, RION, Tokyo, Japan) connected to a measuring amplifier (NA-42, RION, Tokyo, Japan). Responses between the vertex and mastoid subcutaneous electrodes were amplified with a differential extracellular amplifier (ER-1, Cygnus Technology, Delaware Water Gap, PA, USA). Thresholds were determined for frequencies of 8, 16, and 32 kHz from a set of responses at varying intensities with 5-dB SPL intervals, and electrical signals were averaged at 512 repetitions. In cases of scale-out, thresholds were defined as the maximum intensity of the tone burst stimuli plus 5 dB SPL, for statistical analysis. In some experiments, 2 × 10 plaque-forming units of Ad-APN or Ad-β-gal were systemically injected into the tail vein of WT and APN-KO mice at the age of 1.5 months, and ABR measurements were performed. CBF was measured in WT and APN-KO mice aged 2 months, using a laser-Doppler flowmeter (OMEGA FLOW, FLO-N1, Neuroscience, Tokyo, Japan), as previously described. The left bulla of each mouse was identified and opened carefully using a ventrolateral approach. After removing the mucosa and periosteum overlying the bone, a probe (CS-N type) from a laser-Doppler flowmeter was placed over the basal turn of the cochlea, where the maximum output of CBF was measured in arbitrary units (AU). CBF values (mean for 20 s after stabilization) were collected using a computer-based chart-recorder. Mice were perfused intracardially with physiological saline, followed by 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS) at pH 7.4. Excised temporal bones were immersed in the same fixative at 4 C for 4 h. The cochleae were dissected from the temporal bones in PBS. Cochlear specimens were then placed into 0.1 M ethylenediaminetetraacetic acid in PBS for decalcification. The samples were incubated with 30% sucrose in PBS at 4 C before embedding in an OCT compound (Tissue-Tek, Sakura Finetechnical, Tokyo, Japan) and frozen at −80 C. Mid-modiolus sections (7 m in thickness) were sliced and stained with hematoxylin and eosin. To determine the capillary density in the SV, we stained tissue sections with an anti-CD31 antibody (Becton Dickinson, Franklin Lakes, NJ, USA). Capillary endothelial cells were quantified by measuring the number of CD31-positive capillaries per mm of SV (capillary density (per mm)=number of capillaries/area of SV (m) × 1 000 000) from four different sections in each tissue block. TUNEL staining of the cochlear was performed using the Cell Death detection kit (Roche, Mannheim, Germany) as described previously. DAPI was used for nuclear staining. Four different sections from each mouse were examined for the presence of TUNEL-positive cells. The number of TUNEL-positive cells and the total cell numbers were analyzed in each cochlea. Mice were perfused intracardially with physiological saline, followed by 2% glutaraldehyde plus 2% PFA in 0.1 M cacodylate buffer at pH 7.4. Temporal bones were then dissected out and perfused with the above-described fixing solution via round and oval windows. The cochlea was further post-fixed in 2% osmium tetroxide and dehydrated in ethanol for electron microscopy. The specimen was freeze-dried and sputter-coated for observation by SEM (S-800, Hitachi, Japan). HEI-OC1 cells, a murine auditory cell line, were cultured in DMEM containing 10% heat-inactivated fetal calf serum (GIBCO-BRL, Gaithersburg, MD, USA) and antibiotics at 33 °C under 5% CO. HUVECs were cultured in endothelial cell growth medium-2. After 12 h of serum starvation, HEI-OC1 cells and HUVECs were treated with the recombinant APN protein (30 g/ml) or the vehicle alone, followed by 24 h under hypoxic conditions. Hypoxic conditions were generated using an AnaeroPack (Mitsubishi GAS Chemical, Tokyo, Japan). TUNEL staining was performed using the Cell Death detection kit as previously described (Roche). TUNEL-positive cells were counted in five randomly selected microscope fields. Each experiment was repeated three times. The organ of Corti and cell samples were prepared in lysis buffer containing 1 m PMSF (Sigma, St. Louis, MO, USA). The protein concentration was calculated using a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of proteins were separated by denaturing SDS-PAGE. Proteins were transferred onto PVDF membrane (Bio Rad, Hercules, CA, USA) and probed with the primary antibody followed by incubation with the horseradish peroxidase-conjugated secondary antibody. The ECL Plus (GE Healthcare, Princeton, NJ, USA) system was used for detection of the protein signal. All data are presented as mean±S.E.M. Statistical analysis was performed using ANOVA followed by the Scheffe's method or Mann–Whitney -test. In the clinical study, the correlations between APN levels and the indicated parameters were examined by single logistic regression analysis. Single and multiple logistic regression analyses were also performed to analyze the correlations of the indicated parameters with hearing impairment (PTA-low>25 dB and PTA-high>25 dB). A -value of <0.05 was considered statistically significant. All analyses were performed using JMP (version 6.03; SAS Institute, Tokyo, Japan).

First, we examined the presence of N-terminal fragment of profilaggrin in nuclear fractions of cultured keratinocytes. Profilaggrin itself was detected at day 5 and strongly expressed at day 7 of prolonged culture after confluency (). Anti-A+B domain antibody (H-300) detected a fragment of 55 kDa in the nuclear extract as well as in the cytoplasmic extract obtained from the day 7 culture (), indicating that a large fragment consisting of the A–B domains together with a truncated domain is translocated into the nucleus after profilaggrin processing. Then, we prepared an expression vector with a 1392 bp insert encoding a 464-amino-acid N-terminal fragment of profilaggrin (FLG-N) (). This N-terminal region was selected based on the observed fragment size and the presence of possible cleavage sites for serine proteases around Arg of profilaggrin, as there is an Arg-rich region located at Arg-Arg. The molecular weight of FLG-N expressed in keratinocytes was similar to that of the fragment detected in nuclear extract (). FLG-N was also recognized by anti-FLG monoclonal antibody (mAb). A time-lapse study using a construct of fluorescent protein Keima-Red (KR) fused to FLG-N (KR-FLG-N) showed that fluorescence emerged from 7 h after transfection and subsequently accumulated in the nucleus ( and ). The transfected cells showed the increase of fluorescence and it gradually disappeared. Immunohistochemical study demonstrated that after FLG-N transfection in the growth phase, only HA-positive and FLG mAb-positive cells exhibited DNA degradation, as judged in terms of TUNEL positivity (). Translocation of FLG-N into the nucleus was confirmed with two different antibodies: anti-A+B domain antibody (H-300) and anti-FLG mAb that recognizes only the FLG repeat. These results show that FLG-N in the nucleus contains not only the A and B domains, but also a truncated FLG domain. Next we tried to identify which domain is responsible for DNA degradation. We constructed expression vectors for A domain (FLG-A), A domain with NLS (FLG-A+nls), B domain (FLG-B) and B domain+truncated FLG repeat (FLG-BT) (). After transfection of FLG-A, only cytoplasm was stained with anti-HA antibody (Ab), and TUNEL staining was completely negative. In contrast, cells expressing FLG-A with nuclear localization signal (NLS) showed accumulation of this fragment in the nucleus and became TUNEL positive. FLG-B- or FLG-BT-transfected cells did not develop TUNEL positivity, although the fragments were translocated into the nucleus. Thus, B domain by itself can enter the nucleus, in accordance with the observation of Pearton However, our results indicate that the A domain is responsible for DNA degradation. To identify molecules interacting with the N-terminal fragment of profilaggrin, we carried out affinity purification-mass spectrometry. An N-terminal fragment consisting of the A and B domains of profilaggrin was prepared as a glutathione--transferase (GST)-fusion protein (GST-N-FLG), following Pearton GST-N-FLG was incubated with five kinds of extracts from keratinocytes and cornified cells, and interacting molecules were subjected to LC/MS/MS analyses. Molecules interacting with GST alone were also identified for exclusion as nonspecific binders. Structural proteins and ribosomal proteins were also excluded (). We finally selected two proteases, trypsinogen 3 and caspase-14, as candidate interactors. These proteases were commonly detected in nuclear extracts of keratinocytes and in cornified cell extracts. Interestingly, the sequence identified by the analysis is identical with one that we have previously cloned, a specific isoform of the PRSS3 gene products that we named trypsinogen 5. After activation, identical mesotrypsin is produced from all three isoforms (trypsinogens 3–5). These proteases are of particular interest because their activation coincides spatiotemporally with denucleation. We constructed expression vectors for constitutively active mesotrypsin, caspase-14, kallikrein-related peptidase-5 (KLK5), and KLK7. KLK5 was chosen as it is suggested to be an initiator protease in desquamation protease cascades, and KLK7 was selected to examine the effect of a chymotrypsin-like enzyme. As caspase-14 is not activated in the monolayer culture system, we used constitutively active caspase-14 generated by reversing the large and small subunits (revC14), prepared in our laboratory. The revC14 showed equivalent enzyme activity to caspase-14 purified from the extract of human cornified cells (). We used two kinds of promoters. The CMV promoter made it possible to express revC14, as well as other active proteases, in proliferating keratinocytes. On the other hand, the involucrin promoter enabled us to express these proteases in differentiated keratinocytes. Keratinocytes were transfected with these expression vectors and continuously cultured after confluency. Expression of the proteases was confirmed by means of western blotting with anti-HA antibody (). Mesotrypsin alone liberated an N-terminal fragment of profilaggrin with a molecular weight of 55 kDa (), to a small extent. However, neither caspase-14 nor KLK5 and KLK7 alone produced the N-terminal fragment. Interestingly, we found that this fragment was efficiently generated when active caspase-14 and mesotrypsin were coexpressed. When constitutively active mesotrypsin was introduced into keratinocytes, the N-terminal fragment was accumulated in nuclei that became TUNEL positive (). Only mesotrypsin-expressing cells showed DNA degradation after confluence had been reached, as judged by colocalization of HA- and TUNEL-positive cells (). We confirmed that expression of active mesotrypsin itself did not induce DNA degradation in the growth phase (). As KLK5 is a trypsin-like protease, it may influence mesotrypsin activity. We tested this possibility, but found that KLK5 had no effect on mesotrypsinogen activation (). Furthermore, we investigated the ratio of TUNEL-positive cells to HA-positive cells. Most of the revC14-expressing cells were TUNEL positive (95%). Active mesotrypsin-expressing cells showed rather lower TUNEL positivity (72%). Coexpression of these proteases resulted in 95% TUNEL positivity. Possible reasons why revC14-expressing cells showed a higher rate of TUNEL positivity would be a higher efficiency of DNA degradation by the ICAD–CAD system and/or a slower rate of degradation by the FLG-N–mesotrypsin system. Proximity ligation assay (PLA) suggested interaction between mesotrypsin and active caspase-14 as well as between mesotrypsin and filaggrin in normal human epidermis (). These results are consistent with a physiological role of epidermal mesotrypsin as an N-terminal-processing enzyme in differentiated keratinocytes. There are four arginines present in 10 residues (Arg, Arg, Arg and Arg) near the C-terminal end of FLG-N. These were changed to Leu (FLGmut), and various expression vectors (FLG-N-KR, AG-FLGmut and FLGmut-KR) were constructed in addition to KR-FLG-N for elucidation of the physiological role of mesotrypsin (). When fluorescent proteins were fused to the N-terminal of FLG-N (KR-FLG-N) or FLGmut (AG-FLGmut), the fusion proteins were translocated into nuclei and the cells became TUNEL positive (). Accumulation of KR-FLG-N as well as AG-FLGmut in nuclei was evident in both cases. In contrast, fusion of the fluorescent proteins to the C-terminal of FLG-N (FLG-N-KR) or FLGmut (FLGmut-KR) completely suppressed the translocation and TUNEL staining was negative. Next, we co-transfected the active mesotrypsin construct into these cells. In the presence of active mesotrypsin, FLG-N-KR-expressing cells became TUNEL-positive, whereas coexpression of FLGmut-KR and active mesotrypsin had no effect (). The results of quantitative analyses are summarized in . Expression of KR-FLG-N and AG-FLGmut gave similar results, showing 88.3% and 91.1% nuclear localization, respectively. More than 95% of these cells were TUNEL positive. In contrast, FLG-N-KR or FLGmut-KR remained almost entirely in cytoplasm, and nuclei showed <1% TUNEL positivity. When FLG-N-KR was co-transfected with mesotrypsin, 63% of FLG-N-KR-expressing cells showed nuclear localization, and 69% of those cells became TUNEL positive. In the case of FLGmut-KR, coexpression caused no observable change. Furthermore, we performed biochemical analysis. Using recombinant FLG-N and FLGmut proteins as substrates, cleavage of the linker region by mesotrypsin was investigated. If the linker region of FLG-N (55 kDa) is cleaved, ∼3 kDa less peptide would be generated. Our results clearly showed that a 52-kDa peptide was generated from the recombinant FLG-N by the incubation with mesotrypsin. The recombinant FLGmut protein, where four Arg residues in the linker region were changed to Leu residues, did not show any changes in the incubation with mesotrypsin, showing insusceptible nature against mesotrypsin digestion (). These results indicate that liberation of the FLG N-terminal is essential for translocation into the nucleus, and is sufficient to initiate DNA degradation. Epidermal mesotrypsin appears to be involved in the cleavage of the Arg-rich linker region. Keratinocytes were transfected with the CMV promoter-driven revC14 vector, and expression of revC14 was monitored by immunostaining with h14D146 (). Although active caspase-14 was expressed in only ∼4.5% of keratinocytes, these cells in the growth phase did not show positive in TUNEL assay (). Control proliferating keratinocytes synthesized little or no caspase-14, as detected with anti-caspase-14 mAb. In another experiment, keratinocytes were transfected with the involucrin-promoter construct and cultured to confluency, then a further differentiation stimulus was applied (addition of 2 mM calcium). shows that involucrin promoter-driven revC14 emerged in the transfected cells after prolonged culture. In a typical experiment, revC14-positive cells amounted to ∼5.3% of the total, as judged by h14D146 antibody staining. Interestingly, most of the cells stained with these antibodies were found to be TUNEL positive (88% of the revC14-positive cells), indicating that DNA degradation occurred in the active caspase-14-expressing differentiated keratinocytes. The results of quantitative analysis are summarized in , confirming that active caspase-14 has the ability to induce DNA degradation in differentiated keratinocytes. Time-lapse study further confirmed that caspase-14-induced changes occurred only in differentiated keratinocytes (). Among proliferating keratinocytes, caspase-14-expressing cells were actively moving, and some even showed cell division. In confluent culture, however, most of the keratinocytes transfected with the involucrin promoter-driven revC14 showed marked morphological changes after the induction of differentiation (). As keratinocytes express many proteases, we further examined the effects of various protease inhibitors on the TUNEL positivity. DNA degradation was significantly suppressed only in the presence of z-VAD-fmk. Quantitative analysis demonstrated that in the presence of z-VAD-fmk, TUNEL-positive cells were markedly decreased to 21.5% of the number in the absence of the inhibitor (). Leupeptin, chymostatin and antipain, which are serine proteinase and lysosomal cysteine proteinase inhibitors, had no significant effect. Furthermore, we tested the effect of the enzyme-dead revC14 (revC14mut: 228Cys to 228Ala) in order to avoid nonspecific cell death. Expression of the inactive revC14 did not cause any significant effect and these expressing cells were TUNEL negative. Thus, DNA degradation was caspase-14 activity dependent. We further examined whether the ICAD–CAD system is involved in this pathway. Purified caspase-14 from human cornified cell extracts caused limited proteolysis of ICAD, releasing three major degradation products of 32, 27 and 11 kDa that appear to be similar to those generated by caspase-3 (). Prolonged incubation (3 h) increased the formation of the cleavage products. Interestingly, similar degradation bands were also observed in extracts of human cornified cells. We also tested the ICAD-degrading activity of revC14, and found that revC14 catalyzed time-dependent ICAD degradation (). Addition of zVAD-fmk strongly suppressed ICAD degradation by revC14. ICAD-degrading activity of caspase-14 was observed only in the presence of kosmotropic salt () that is essential for enzymatic activity of caspase-14. However, in the presence of kosmotropic salt, ICAD degradation by caspase-3 was significantly suppressed compared with that by caspase-14 (). We next addressed the questions of whether CAD is liberated in this reaction and, if so, whether it is translocated into the nucleus. Keratinocytes transfected with revC14 expression vector were cultured for 2 days after confluency. Most CAD-expressing cells showed CAD accumulation in nuclei, and CAD-positive nuclei were also positive for TUNEL reaction (). Expression of revC14 was confirmed with anti-HA antibody (). HA-positive cells were also TUNEL positive. Close association between active caspase-14 and ICAD was demonstrated at the granular layer of normal human epidermis using PLA (). As profilaggrin is neither expressed nor processed in day 2 culture (), DNA degradation was caspase-14 activity dependent. Collectively, these results suggest that caspase-14 has the ability to degrade ICAD, liberating CAD and thereby leading to DNA degradation, although its action is restricted to differentiated keratinocytes. Our results suggested that both the caspase-14-dependent ICAD–CAD pathway and a FLG N-terminal fragment-dependent pathway may be involved in DNA degradation during terminal differentiation of keratinocytes. Thus, we examined the effect of siRNA knockdown of caspase-14 and mesotrypsin in a skin equivalent model. These siRNA treatments efficiently knocked down expression of the corresponding proteases to 9.9% and 24.0% of the control levels, respectively (). We detected considerable numbers of undegraded nuclei in the cornified layer after either caspase-14 knockdown or mesotrypsin knockdown. When both caspase-14 and mesotrypsin were knocked down together, many undigested nuclei were observed in the cornified layer (). Numbers of nuclear remnants were counted in each 450 m field and a total length of 4.5 mm was examined in each model (). Interestingly, the number of remnants was increased significantly in the skin model with mesotrypsin knockdown and further increased in that with caspase-14 knockdown. The greatest effect was consistently observed in the model with double knockdown. Finally, we examined the expression of mesotrypsin and caspase-14 in skin from patients with atopic dermatitis (AD) and psoriasis, as such skin frequently shows abnormal differentiation and parakeratosis. Normal skin showed strong immunostaining for mesotrypsin in the granular layer and for caspase-14 in the spinous to granular layers. Immunostaining clearly demonstrated strong downregulation of procaspase-14 in the lesional skin with psoriasis (). Immunostaining of mesotrypsin was also significantly decreased in the same area. Nuclear staining revealed persistent presence of undigested nuclei in the areas where both enzymes were hardly detectable. Similar results were obtained for skin with AD (). Thus, the FLG-N pathway is likely to be suppressed in parakeratotic areas. We further investigated whether the ICAD–CAD pathway was concomitantly impaired in parakeratotic skin. Skin surface staining for ICAD showed isolated regions of various sizes that were positive for ICAD in AD skin (). Nuclear staining with propidium iodide (PI) showed that parakeratotic nuclei were always present in these patchy islands. Bright field microscopy of the superficial cornified layer showed a bumpy and rough surface compared with the smooth surface of normally keratinized areas. The merged images demonstrate that the parakeratotic areas coincided with ICAD-positive areas. These results indicate that suppression of the two pathways is associated with persistent presence of nuclei in the cornified layer. Loss of nuclei is a critical event in epidermal barrier formation. Our results indicate that at least two pathways are involved in the denucleation process during keratinocyte terminal differentiation, that is, profilaggrin N-terminal fragment-dependent and caspase-14-mediated CAD-dependent DNA degradation pathways. First, we searched for molecules that interact with the N-terminal fragment of profilaggrin by means of LC/MS/MS analyses, and identified epidermal mesotrypsin and caspase-14 as interactors in extracts from keratinocytes and cornified cells (). Identification of these interactions is consistent with the idea that profilaggrin is a natural substrate for these proteases. Mesotrypsin shows a distinct substrate specificity from the PRSS1 gene product, anionic trypsin, or PRSS2 gene product, cationic trypsin. It is not susceptible to intrinsic trypsin inhibitors, but instead has the ability to degrade them. Furthermore, its proteolytic activity is quite limited, and it does not digest large protein substrates into small fragments, as other trypsins do. Mesotrypsin is required for the processing of FLG-N and liberated FLG-N is translocated into the nucleus because of the presence of NLS in the B domain. We also found that the translocated N-terminal of profilaggrin contained not only A and B domains but also a truncated FLG domain. This conclusion was also supported by the results of our mutation study. Our finding is contrary to that of Pearton , who reported that only the A and B domains enter into the nucleus. The reason for this discrepancy is unclear, but it may be because of the difference in cell lines used for assays, that is, human keratinocytes and COS-7, as human keratinocytes express epidermal-specific mesotrypsin. Although several proteases have been reported to be involved in the profilaggrin processing cascade leading to filaggrin, our findings strongly suggest that epidermal mesotrypsin and caspase-14 are key profilaggrin-processing molecules. On the other hand, we also established that caspase-14 has the ability to degrade ICAD, generating peptides similar to those formed by caspase-3. Indeed, expression of constitutively active caspase-14 (revC14) liberated CAD that was accumulated in nuclei. As expected, the resulting cells became TUNEL positive. In addition, we compared the TUNEL-positive rate in revC14-expressing cells and mesotrypsin-expressing cells. Most revC14-expressing cells were TUNEL positive (95%), whereas active mesotrypsin-expressing cells showed lower TUNEL positivity (72%). The reason why revC14-expressing cells showed a higher rate of TUNEL positivity may be higher efficiency of the ICAD–CAD system for DNA degradation and/or slower reactivity of the FLG-N–mesotrypsin system. We recently showed that mesotrypsin plays multiple roles in keratinocyte terminal differentiation. For example, it is secreted to interstitial areas of the cornified layer and is involved in proKLK activation. Thus, its availability may vary at different stages of differentiation. The PLA method demonstrated association of active caspase-14 and ICAD at the boundary area between the granular layer and the cornified layer, where nuclear loss takes place. Involvement of epidermal mesotrypsin and caspase-14 in the denucleation process is also supported by findings in skin equivalent models with siRNA-mediated knockdown of these proteases and in PLA interaction assays using skin samples. Downregulation of both enzymes resulted in a significant increase of undigested nuclei in the cornified layer. Furthermore, it was found that both caspase-14 and mesotrypsin were absent in areas where parakeratotic nuclei were evident. Therefore, these proteases appear to play major roles in denucleation during keratinocyte terminal differentiation. Interestingly, we found that caspase-14 promotes the conversion of mesotrypsinogen to mesotrypsin. This is probably accomplished by cleavage near the enterokinase recognition sequence (DDDDKI), as caspase-14 cleaves only after Asp residues. Indeed, our unpublished data support the idea that active caspase-14 promotes mesotrypsinogen activation, even though enterokinase was several times more potent than caspase-14 as an activator. However, this does not exclude a role of caspase-14 as a possible activator, considering that the catalytic efficiency of enterokinase is 34 000-fold greater than that of trypsin. Collectively, our results indicate the presence of two distinct pathways of epidermal denucleation that appear to work both cooperatively and independently, depending upon the differentiation conditions (). However, a defect of even a single pathway may affect the maintenance of epidermal homeostasis, because it has been shown that a defect of caspase-14 predisposes to the development of parakeratosis. Because the mechanisms of parakeratosis have been unknown, there has been no effective treatment directly targeting parakeratosis. Here, we have shown that at least two pathways are involved in denucleation during keratinocyte terminal differentiation, acting cooperatively for the formation of fully differentiated corneocytes. Simultaneous treatment directed toward both pathways might be effective to improve parakeratosis, as well as barrier disruption. Antibodies used in this study are listed in . Primer sequences used for human cDNA amplification are listed in . Human keratinocytes derived from normal foreskin (Kurabo, Osaka, Japan) were cultured in Humedia KG2 supplemented with epidermal growth factor (0.1 ng/ml), insulin (10 g/ml), hydrocortisone (0.5 g/ml), bovine pituitary extract (0.4%), gentamicin (50 g/ml) and amphotericin B (50 ng/ml). All experiments were carried out on cells at the third or fourth passages. This study was approved by the Ethical Committee of Tokyo Medical University and by the Shiseido Committee on Human Ethics. In total, 7 patients with AD and 14 patients with psoriasis vulgaris were recruited for this study after having given informed consent. Patients with AD (7 men, mean age 32.7 years) and psoriasis (11 men, mean age 44.6 years) had not received topical steroid treatment or UV therapy for at least 4 weeks before participation in the study. The severity of AD was evaluated by using the Eczema Area and Severity Index (EASI) score that ranged from 13.7 to 48 (mean score=29.9). The severity of psoriasis was evaluated in terms of the PASI score (2.0–52.8, mean score=21.1). For all patients, 4 mm punch biopsies were taken from acute or active lesions and nonlesional skin. Tissue samples were fixed in acetone and embedded in paraffin. Keratinocytes (KCs) were collected at 80% confluency (growth phase), 100% confluency (confluency) at and 2, 5 and 7 days after confluency in the presence of 1.2 mM calcium (differentiated phase). Cell pellets were homogenized using a sintered glass homogenizer (Wheaton Science Products, Millville, NJ, USA) with extraction buffer (10 mM Tris HCl (pH 7.5), 50 mM NaCl, 1% NP-40, 5 mM EDTA) and subjected to ultrasonication for 10 s, three times. Supernatant was obtained after centrifugation. Cornified cell extracts were also prepared from scraped heels of healthy individuals as reported previously. Each extract was reacted with GST-fusion N-terminal fragment (A+B domains) and revC14 coupled with GST-Agarose. After extensive washing, bound proteins were eluted with 100 mM glutathione, dialyzed against HO and lyophilized for trypsin digestion. For western blot analysis, cell pellets were solubilized with SDS sample buffer and after centrifugation, supernatants were subjected to SDS-PAGE. Human caspase-14 was purified from cornified cells according to our previous report, using sequential chromatographies. Briefly, revC14 cDNA was inserted into the pET 15b vector at the HI/I sites. Recombinant proteins were purified by means of Ni-NTA-Agarose (Qiagen, Venlo, Netherlands) and Mono Q chromatography. Two kinds of expression vectors for constitutively active proteases were constructed using pCMV-HA vector (Clontech, Mountain View, CA, USA) and pH3700-pL2 vector (kindly given by Dr. LB Taichman (State University of New York, Stony Brook, NY, USA)) that was driven by the involucrin promoter. cDNA of revC14 was cloned into pCMV-HA vector (pCMV-HA-revC14). In addition, a PCR product (HA+revC14) was amplified using the common forward primer with I recognition sequence+HA sequence and the specific reverse primer with I site. It was inserted into pH3700-pL2 vector at the I site (pInv-HA-revC14). Two kinds of expression vectors (pCMV-HA and pInv-HA) for constitutively active KLK5 and active KLK7 were similarly prepared from full-length cDNA clones (OriGene Technologies, Inc., Rockville, MD, USA). Expression vectors for FLG N-terminal domains were also prepared using pCMV-HA. These were A domain construct (FLG-A), A domain with NLS (FLG-A-nls), B domain construct (FLG-B), B domain+truncated FLG repeat (FLG-BT) and N-terminal fragment with A domain+B domain+truncated FLG repeat (FLG-ABT). For time-lapse studies, fluorescent protein, Azami-Green (AG) or KR (Amalgam-MBL, Nagoya, Japan), was inserted after the HA-tag of these vectors (pCMV-HA-AG-revC14, pInv-HA-AG-revC14 and pCMV-HA-KR-FLG-ABT, respectively). These constructs were introduced into keratinocytes during the growth phase using FuGENE (Roche Diagnostics, Tokyo, Japan). As controls, pCMV-HA vector and pH3700-pL2 were similarly transfected. Primers used are listed in . Expression vectors with Arg mutations in the linker region between the FLG-N and the first FLG domain were constructed as described below. This region contains 4 arginines within 10 residues. All four Arg residues were changed to Leu using PCR primers, 5′-GGCAAGCTTCATCTGCAGTCAGCGATCGTGGAC-3′ and 5′-TGCTCACCTGGTAGAGGGAAGACCCTGAAAGTCCAGACAGTTCCCCTGACAGGCCAAGTGTGGACTCTTG-3′. The PCR product was inserted into the dIII/NI site of pCMV-HA-FLG-ABT and named pCMV-HA-FLGmut. pCMV-HA-FLGmut-KR was prepared by inserting KR cDNA into the I/I site of pCMV-HA-FLG-ABT. pCMV-HA-KR-FLGmut was also constructed by inserting KR cDNA into the I/RI site of pCMV-HA-FLGmut. Caspase-14 activity was measured using Ac-WEHD-MCA as a substrate. The assay buffer consisted of 0.1 M HEPES buffer (pH 7.5), 0.06 M NaCl, 0.01% CHAPS ( 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid), 5 mM DTT and 1.2 M sodium citrate (final concentration). revC14 (5 g/ml, 10 l) was preincubated in the assay buffer (80 l) for 15 min at room temperature. After addition of 20 l of 100 M Ac-WEHD-MCA, the mixture was incubated for 30 min. The activity of revC14 was measured using a Fluoroskan Ascent FL (Thermo Electron Co., Wolsam, MA, USA) with excitation at 355 nm and emission at 460 nm. The assay mixture containing 5 l of revC14 or purified caspase-14 from cornified cells in 40 l of the assay buffer was preincubated for 15 min at room temperature, and 5 l of ICAD (0.1 mg/ml) (inhibitor of caspase-activated DNase) was added. At each time point, 3 l of the incubation mixture was removed and diluted with 27 l of HO. SDS-PAGE samples were prepared by adding 10 l of 4 × sample buffer and analyzed by western blotting with the anti-ICAD antibody (FL331; Santa Cruz Biotechnology, Dallas, TX, USA). SDS-PAGE was carried out using a 5–20% gradient gel. After electrophoresis, proteins were transferred onto a polyvinylidine difluoride membrane (Immobilon-P, Millipore, Bedford, MA, USA) and incubated with appropriate primary antibodies. Peroxidase-labeled anti-rabbit IgG (Sigma, St. Louis, MO, USA) or anti-mouse IgG was used as a secondary antibody, and immunoreactive proteins were visualized by chemiluminescence using ECL-plus (Amersham, Little Chalfont, UK). Primary antibodies used are listed in . The mRNA was reverse transcribed with SuperScript II (Invitrogen Corporation, Carlsbad, CA, USA). Real-time quantitative PCR was performed on a LightCycler rapid thermal cycler system using LightCycler FastStart DNA master SYBR green I kit (Roche Diagnostics Ltd) according to the manufacturer's instructions. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a reference gene. Expression of revC14 was detected with anti-HA antibody, anti-caspase-14 mAb and h14D146 antibody. For the detection of epidermal mesotrypsin, we used anti-trypsin Ab (Athens Research & Technology, Athens, GA, USA). As active mesotrypsin and trypsin are ∼90% identical at the amino acid level, we confirmed that this antibody crossreacted with epidermal mesotrypsin without showing any difference in immunostaining or western blot analysis. Alexa Fluor 555 or 488 (Molecular Probes Inc., Eugene, OR, USA) was used as the secondary antibody. DAPI (4′,6′-diamidino-2-phenylindole; Molecular Probes Inc.) was used for visualization of nuclei. The TUNEL reaction was performed using a fluorescein cell death detection kit (Roche Diagnostics Ltd) according to the manufacturer's instructions. After immunostaining, cells with DAPI-stained nuclei were counted. The transfection ratio was calculated from the number of h14D146-positive or anti-HA antibody-positive cells the total cell number. The ratio of TUNEL-positive cells to active caspase-14-expressing cells was calculated similarly. In a typical experiment, over 1500 cells in 7 fields were counted and experiments were repeated three times. Tissue sections from human skin and the skin equivalent models were stained in the same manner. Primary antibodies used are listed in . PLA reaction was performed using two antibody combinations (monoclonal anti-trypsin antibody/h14D146 antibody, rabbit anti-trypsin antibody/monoclonal anti-FLG antibody and h14D146 antibody/anti-ICAD antibody) according to the manufacturer's instructions. For the negative control, normal rabbit IgG was used as the primary antibody. Dilution was 0.5 g/ml for each antibody. For skin surface staining, medical adhesive Aron Alpha A (Sankyo, Tokyo, Japan) was applied on a glass slide and the glass was firmly pressed on the skin for 5 min. It was then carefully removed, and the attached cornified cells were stained for ICAD using anti-ICAD antibody (FL331). PI (1 g/ml) was used for nuclear staining. Observation was done by means of confocal microscopy (LSM5 PASCAL, Carl Zeiss, Oberkochen, Germany). The lens was a Plan APOCHROMAT × 20 magnification. Keratinocytes were treated with control siRNA-A (sc-37007), caspase-14 siRNA (sc-37364) or PRSS3 Stealth RNAi (Invitrogen) and plated on dermal equivalent, Matrex (Toyobo, Osaka, Japan). At 2 days after plating, the cell surface was exposed to air and culture was continued for 10 days. Data are expressed as the mean±S.D. We employed simple pair-wise comparison with Student's test (two-tailed distribution with two-sample equal variance). <0.05 was considered significant.

To address the role of autophagy in VSMC foam cell formation, first, we detected the changes of autophagy in oxLDL-treated VSMCs. As shown in , exposure to oxLDL decreased the LC3II/LC3I ratio and beclin-1 level in a time-dependent manner in VSMCs, with an obvious effect at 24 h post oxLDL challenge. Moreover, a marked increase of p62 was observed 24 h after oxLDL exposure (). Next, we examined the effect of autophagy on foam cell formation in oxLDL-treated VSMCs. Rapamycin (Rap, an inhibitor of mTOR) and 3-methyladenine (3-MA, an inhibitor of autophagy) were respectively used to induce and inhibit autophagy in VSMCs. As expected, Rap rescued, whereas 3-MA exacerbated, the impaired autophagy in oxLDL-treated VSMCs, manifested by the changes of LC3-II/LC3-I ratio () and GFP-labeled autophagosomes (). Consistently, both Oil Red O staining and total cholesterol quantification showed that Rap inhibited the oxLDL-induced foam cell formation, whereas 3-MA exerted the opposite effect (). Together, these results show that autophagy protects against foam cell formation in oxLDL-treated VSMCs. A previous study showed that high oxLDL concentration (>60 g/ml) induced VSMC apoptosis. In our study, we also performed apoptosis assays by double staining with Annexin V-FITC/propidium iodide (PI) and detecting caspase 3 activity in cells treated with oxLDL or nLDL. The data indicated early apoptosis induced by 80 g/ml oxLDL. However, late apoptosis and necrosis were not induced (). The induction of capsaicin on autophagy was previously reported in malignant breast epithelial cells and mouse thymocytes. The present study showed that this induction also exists in VSMCs. As shown in , the impaired autophagy by oxLDL was markedly rescued by capsaicin in a dose-dependent manner, with the maximal effect at 1 M, demonstrated by the increased LC3II/LC3I ratio and beclin-1 level. Capsaicin (1 M) significantly increased the LC3II/LC3I ratio, beclin-1 level and GFP-labeled autophagosomes in oxLDL-stimulated VSMCs from wild-type (WT) mice but not TRPV1 knockout (TRPV1) mice (). Furthermore, the promotion of capsaicin on LC3II/LC3I ratio and beclin-1 level were reversed by TRPV1 antagonist 5′-iodo-resiniferatoxin (iRTX), but it was not the case in VSMCs from TRPV1 mice. These data suggest that activation of TRPV1 by capsaicin rescues the impaired autophagy in oxLDL-treated VSMCs. Lysosomes and autophagolysosomes are crucial particles in autophagy-mediated degradation of macromolecules. Therefore, the marker protein of lysosomes, lysosomal-associated membrane protein 1 (LAMP-1), was detected to identify whether activation of TRPV1 by capsaicin activates autophagy–lysosome pathway in oxLDL-treated VSMCs. It was shown that capsaicin significantly increased the expression of LAMP-1 () and the number of lysosomes () in oxLDL-stimulated VSMCs from WT mice that was counteracted by TRPV1 inhibitor iRTX. In oxLDL-stimulated VSMCs from TRPV1 mice, no detectable change in LAMP-1 expression was found. Next, we further investigated the effect of activation of TRPV1 by capsaicin on autophagy–lysosome pathway . Addition of capsaicin significantly increased the expression of LAMP-1 in the area of smooth muscle cells. In contrast, capsaicin exerted no effect on the expression of LAMP-1 in TRPV1 mice (). To investigate whether activation of TRPV1 by capsaicin inhibits VSMC foam cell formation through autophagy induction, we used autophagy-related gene 7 (Atg7) small interfering RNA (siRNA) to knock down Atg7, a key autophagy-related gene. In VSMCs transfected with con siRNA, capsaicin significantly induced autophagy manifested by increased Atg7 () and LC3-II/LC3-I ratio (), and inhibited oxLDL-induced foam cell formation manifested by decreased lipid droplet accumulation () and total cholesterol level (). On the contrary, in Atg7 knockdown VSMCs, capsaicin failed to impede the oxLDL-induced lipid accumulation and cholesterol increase, accompanied by unchanged LC3-II/LC3-I ratio. These data suggest that activation of TRPV1 by capsaicin inhibits foam cell formation through induction of autophagy in oxLDL-treated VSMCs. To further investigate the potential mechanisms of TRPV1 in inducing autophagy in VSMCs, we analyzed the activation status of AMP-activated protein kinase (AMPK) signaling. As shown in , capsaicin significantly increased p-AMPK (Thr-172) expression that was reversed by iRTX in VSMCs from WT mice. In contrast, capsaicin exerted no effect on the expression of p-AMPK in VSMCs from TRPV1 mice. Furthermore, we evaluated the involvement of AMPK activation in TRPV1-induced autophagy by using the AMPK inhibitor compound C. As shown in , compound C significantly inhibited the TRPV1-activated AMPK. Moreover, increase in Atg7 level () and LC3-II/LC3-I ratio () were significantly attenuated by addition of compound C. Being an important origin of foam cells, VSMCs have gained increasing attention. The mechanisms underlying VSMC foam cell formation have become an important area of ongoing investigation. Our study demonstrated that autophagy protected against foam cell formation in oxLDL-treated VSMCs; activation of TRPV1 by capsaicin rescued the autophagy impaired by oxLDL and activated autophagy–lysosome pathway in VSMCs; activation of TRPV1 by capsaicin impeded VSMC foam cell formation through autophagy induction; activation of TRPV1 by capsaicin induced autophagy through AMPK signaling pathway. Autophagy has been implicated in various biological processes, including aging, development, tumorigenesis, cell death and differentiation by breaking down macromolecules and aged/damaged cellular organelles. Autophagy has also been found to play a key role in the development of diseases, including cancer, neurodegenerative disorders, inflammatory bowel disease and atherosclerosis. Reportedly, autophagy exerts both protective and deleterious effects on atherosclerosis. A recent study showed that activation of autophagy reduced levels of total cholesterol and cholesterol esters in macrophages exposed to LDL cholesterol, and thus inhibited the foam cell formation. However, the potential role of autophagy in VSMC foam cell formation is poorly understood. In the present study, we used oxLDL, a well-known atherogenic factor, to induce VSMC foam cell formation. We first detected the effect of oxLDL on autophagy. The data showed that autophagy in VSMCs was significantly inhibited by oxLDL, with decreased LC3II/LC3I ratio and beclin-1 level. The well-established autophagy-negative regulator, mTOR, may mediate the effect of oxLDL on autophagy. It was reported that oxLDL was capable of activating the PI3K/Akt/mTOR pathway in VSMCs. In addition, oxLDL has also been shown to upregulate p38 mitogen-activated protein kinase that can activate mTOR or inhibit autophagy directly. Therefore, we speculated that it was through the mTOR signaling, at least in part, that oxLDL inhibited autophagy. Further studies are warranted to elucidate detailed cellular and molecular mechanisms for this phenomenon. Next, we explored the role of autophagy in VSMC foam cell formation by using Rap (an inhibitor of mTOR) to induce autophagy and 3-MA (an inhibitor of autophagy) to inhibit autophagy. Our results showed that activation of autophagy reduced Oil Red O-positive staining and total cholesterol level in VSMCs exposed to oxLDL, whereas inhibition of autophagy exerted the opposite effect. These data provide evidence that autophagy inhibits oxLDL-induced VSMC foam cell formation, and suggest autophagy as a novel protective mechanism for VSMCs loaded with oxLDL. TRPV1 channel is activated by the specific agonist, capsaicin, that is the major pungent ingredient in hot peppers. TRPV1 channel is highly expressed in sensory neurons but has also been detected in blood vessels. Previous studies indicated that activation of TRPV1 by capsaicin could affect lipid metabolism. In VSMCs treated with oxLDL, TRPV1 activation increased ABCA1 expression and reduced low-density lipoprotein-related protein 1 expression in calcium-dependent and calcineurin- and protein kinase A-dependent manner. The present study showed that TRPV1 activation by capsaicin could rescue the impaired autophagy in oxLDL-treated VSMCs. In WT-derived VSMCs, oxLDL-inhibited autophagy was significantly reversed by TRPV1 activation, and verified by increased LC3II/LC3I ratio, beclin-1 level and GFP-labeled autophagosomes in response to capsaicin treatment that was impeded by TRPV1 antagonist iRTX. However, in TRPV1-derived VSMCs, the capsaicin-induced autophagy amelioration was not shown. These data suggest that induction of autophagy might be another important mechanism of TRPV1 in regulating foam cell formation. For the first time, our study demonstrated that activation of TRPV1 by capsaicin protected against foam cell formation by inducing autophagy in oxLDL-treated VSMCs. We further investigated how autophagy is involved in the activation of TRPV1-regulated VSMC foam cell formation. As we know, autophagosomes fuse with lysosomes to form autolysosomes, and this is an essential step in autophagy that leads to degradation of their cargo. A recent study demonstrated that lipid droplets were delivered to lysosomes via autophagy, where lysosomal acid lipase acted to hydrolyze lipid droplet cholesterol esters to generate free cholesterol mainly for ABCA1-dependent efflux. Another study also showed that autophagy was involved in neutral cholesterol ester hydrolase-mediated degradation of cholesterol esters. In our study, capsaicin increased the level of LAMP-1, the marker protein of lysosomes, whereas TRPV1 antagonist iRTX abolished this effect, suggesting that the lysosome/autophagolysosome pathway mediates the inhibition of TRPV1 on VSMC foam cell formation. Foam cell formation is regulated by multiple mechanisms. Reportedly, change from the contractile to the synthetic phenotype can accelerate the lipid accumulation in VSMCs. Herein, VSMCs in second to sixth passages were used in our study to ensure the contractile phenotype, manifested by high expression of smooth muscle actin (-SMA) and low expression of osteopontin (OPN) (). Furthermore, capsaicin showed inhibitory effect on alteration of VSMCs from contractile to synthetic phenotype ().We therefore exclude the influence of alteration of VSMC phenotype on foam cell formation in the present study. Next, we wondered which mechanism mediates the promotion of autophagy after TRPV1 activation. AMPK plays a key role in the regulation of protein and lipid metabolism, and can be activated by phosphorylating Thr-172 of its subunit. AMPK activation was reported to be required for the induction of autophagy. In this study, activation of TRPV1 by capsaicin significantly increased p-AMPK (Thr-172) expression that was abolished by TRPV1 inhibitor iRTX in WT-derived VSMCs but not in TRPV1-derived VSMCs. Moreover, compound C significantly inhibited the TRPV1-activated AMPK and inhibited autophagy. These results suggest that activation of TRPV1 by capsaicin induces autophagy through activating AMPK signaling pathway. Regarding how the activation of TRPV1 activates AMPK signaling, we speculated it was through the increased cytosolic Ca. Reportedly, AMPK could be phosphorylated and activated by two upstream kinases: the tumor suppressor liver kinase B1 (LKB1) and Ca/calmodulin-dependent protein kinase kinase- (CaMKK). CaMKK mediates the activation of AMPK mainly in response to increased Ca, whereas LKB1 mediates activation primarily in response to energy stress. After phosphorylation, AMPK could activate the uncoordinated-51-like kinase 1 (ULK1) that combines with autophagy-related protein 1 (Atg1) to initiate the autophagic process including receiving signals of cellular nutrient status, recruiting downstream Atg proteins to the autophagosome formation site and governing autophagosome formation. TRPV1 is a well-known cation channel, the activation of which can increase cytosolic Ca significantly. Therefore, we speculated that activation of TRPV1 by capsaicin induced a significant elevation in cytosolic Ca and subsequent CaMKK activation that further activated the AMPK–ULK1–autophagy pathway. Considering that oxLDL can lead to both autophagy and apoptosis concomitantly in atherosclerotic lesions, we also detected the apoptosis in oxLDL-loaded VSMCs. The results showed that 80 g/ml oxLDL induced early apoptosis at a low level, but not late apoptosis and necrosis. This mild apoptotic-induction effect of oxLDL may be correlated to the moderate oxidation extent (22–25 nmol of MDA/mg protein). Reportedly, oxLDL with low oxidation extent was widely used to investigate lipid metabolism, whereas oxLDL with higher oxidation extent was required to induce apoptosis. It would be of interest to investigate the crosstalk between autophagy and apoptosis in oxLDL-loaded VSMCs in future. There is a limitation that should be noted. Because of strong excitability, the dose of capsaicin used could not be as high as that used . The capsaicin plasma concentration of 0.01% oral capsaicin (∼33 nM, reportedly) is lower than the concentration used (1 M), and this may give rise to the possibility of different results between and studies regarding the mechanism we proposed, even though previous studies exhibited the same physiological effect between 0.01% of oral capsaicin and 1 M capsaicin . Further studies are needed to elucidate the above mechanisms. In conclusion, the present study provided evidence that autophagy impeded VSMC foam cell formation induced by oxLDL; activation of TRPV1 by capsaicin rescued the autophagy impaired by oxLDL via activating AMPK signaling pathway, and ultimately inhibited the foam cell formation (). Thus, our study provides novel pathological role of autophagy in VSMC foam cell formation and highlights TRPV1 as a promising therapeutic target in atherosclerosis. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Hycolon (Logan, UT, USA). Opti-MEM medium was from Gibco BRL (Carlsbad, CA, USA). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA, USA). Rap (10 nM), 3-MA (5 mM), iRTX (1 M), capsaicin and compound C (10 M) were obtained from Sigma-Aldrich (St. Louis, MO, USA). GFP-LC3-expressing plasmid (pEGFP-LC3) was from Cell Biolabs (San Diego, CA, USA). Annexin V-FITC Apoptosis Detection Kit was from Beyotime (Beijing, China). Caspase-Glo 3/7 kit was from Promega (Southampton, UK). Antibodies targeting Atg7 (1 : 1000), LAMP-1 (1 : 1000), -actin (1 : 2000), OPN (1 : 1000), Atg7 siRNA (sc-41448) and the scrambled siRNA (sc-37007) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against LC3 (2 g/ml) was from Novus Biologicals (Littleton, CO, USA). Antibody against beclin-1 (1 : 1000) was from Cell Signaling Technology (Beverly, MA, USA). Antibody targeting -SMA (1 : 1000) was from Abcam (Burlingame, CA, USA). The enhanced chemiluminescence (ECL) substrate was purchased from Pierce (San Diego, CA, USA). The C57BL/6 J WT mice and TRPV1 mice were obtained at 8–10 weeks of age from Jackson Laboratory (Bar Harbor, Maine, USA). Both WT and TRPV1 mice were randomly divided into two groups (=6 mice per group): high-fat diet (HFD) and high-fat diet plus 0.01% capsaicin for 16 weeks as previously described. Then, animals were killed for experiments. Animal care and procedures conformed with the Guide for the Care and Use of Laboratory Animals. Protocol approval was obtained from the Animal Research Committee of the Third Military Medical University. VSMCs were isolated from the thoracic aorta of WT or TRPV1 mice using an explant technique using standard methods. Briefly, all the mice were killed by neck breaking and the aortic media was cut into 1 × 1 mm pieces. The pieces were cultured in DMEM supplemented with 20% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin in a humidified, 5% CO/95% air atmosphere at 37°C. When the cells formed a confluent monolayer (10 to 14 days), they were passaged by trypsinization. The cultured VSMCs were verified by using anti--SMA antibody through immunocytochemical staining. To ensure maintenance of the contractile phenotype, we used cells in the second to sixth passages for experiments. Cells were incubated with serum-free medium for 24 h before all following experimental procedures. Human LDL was isolated from fresh human plasma by discontinuous density-gradient ultracentrifugation and was oxidized using 5 M CuSO in PBS at 37°C for 20 h. Oxidation was terminated by adding ethylenediaminetetraacetic acid, and the samples were dialyzed against phosphate buffer at 4°C for 24 h. The extent of modification was determined by measuring thiobarbituric acid-reactive substances (TBARS). TBARS was determined colorimetrically using malondialdehyde (MDA) as standard. The TBARS of starting LDL is 0.10–0.15 nmol of MDA/mg protein and oxLDL is 22–25 nmol of MDA/mg protein. Protein samples were obtained from homogenized cultured cells, and the protein concentration was determined. Protein samples were separated by SDS-PAGE, transferred to a PVDF membrane and blocked with TBS containing 0.05% Tween-20 (TBST) and 5% nonfat milk powder for 2 h. The membranes were then incubated with appropriate primary antibodies overnight at 4°C. After 4 washes with TBST, membranes were incubated with secondary antibodies for 2 h at room temperature. The proteins were detected by ECL and quantified using Labworks 4.6 (UVP, Upland, CA, USA). Western blot quantification was performed by densitometry and normalized to -actin. Apoptosis was determined by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instructions. Briefly, primary VSMCs were grown in six-well plates. After different treatments, cells were trypsinized, harvested, washed twice with cold PBS and centrifuged at 1000 × for 5 min. Cells were resuspended in 195 l binding buffer at a concentration of 1 × 10 cells per ml. Then, 5 l of Annexin V-FITC was added. Cells were gently vortexed and incubated for 10 min in dark at room temperature. After the cells were centrifuged at 1000 × for 5 min, 190 l binding buffer and 5 l of PI were added. Samples were kept on ice and analyzed immediately by flow cytometry (Beckman Coulter, Miami, FL, USA). Normal live cells were represented as Annexin V-FITC negative and PI negative; early apoptotic cells as Annexin V-FITC positive and PI negative; late apoptotic or necrotic cells as Annexin V-FITC positive and PI positive. Apoptosis was also quantified by measuring caspase 3 activation using Caspase-Glo 3/7 Assay on a microplate reader (BioTek, Winooski, VT, USA) reader reading luminometer. The luminescence reading is directly proportional to caspase 3 activity. Foam cells were quantified by Oil Red O staining and intracellular total cholesterol content. Cultured VSMCs were plated on cover slides in six-well plates and treated with noted reagents for 24 h in serum-free DMEM. After that, the cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde and stained with Oil Red O. Foam cells were photographed under a microscope at × 400 magnification. Intracellular total cholesterol content was detected by enzymatic assay as previously described. Briefly, cells after oxLDL treatment were harvested into a centrifuge tube and washed with PBS three times. Isopropylalcohol (100 l) was added to each tube, and intracellular liquid was extracted by ultrasonication. These mixtures were centrifuged for 10 min at 1500 × for the phase separation. The supernatant was used to detect total cholesterol by enzymatic assay. Sediment was then lysed on ice for 30 min in 100 l lysis buffer. After centrifugation, supernatants were collected for determining total cellular protein levels by Bradford assay method. The results were then expressed in g of cholesterol per mg of cellular protein. VSMCs were seeded on coverslips in 24-well plates overnight, and then 0.8 g plasmid/well GFP-LC3 expressing plasmids were transiently transfected into the cells using Lipofectamine 2000 overnight in Opti-MEM medium following the manufacturer's instructions. Before they were treated, VSMCs were maintained in DMEM, supplemented with 10% FBS. Coverslips were mounted on glass slides and all of the cellular images were obtained using a confocal microscope confocal microscope (Leica, Wetzlar, Germany). For quantification of autophagic cells, GFP-LC3 punctated dots were determined from triplicates by counting a total of >60 cells at the end of the treatment. The siRNA transfection was performed using Lipofectamine 2000 according to the manufacturer's protocol. Briefly, the cells were plated in 500 l growth medium without antibiotics in 24-well plates. After cells reached 40–50% confluence, a 20-pmol sample of the siRNA oligomer was diluted in 50 l Opti-MEM; 1 l Lipofectamine 2000 was diluted in 50 l Opti-MEM. The solutions were mixed gently and incubated for 5 min at room temperature. After the 5-min incubation, the diluted oligomer was combined with the diluted Lipofectamine 2000, and the solution was mixed gently and incubated for 20 min at room temperature. The Oligomer-Lipofectamine 2000 complexes were then added to each well. After 6 h of transfection, DMEM complete medium was added. Cells were cultured in complete medium for 24 h before further analysis. Silencing efficiency was measured by western blot analysis. The scrambled siRNA was used as negative control (con siRNA). Serial fresh frozen sections of aorta and cultured VSMCs were fixed with 4% paraformaldehyde. Nonspecific proteins were blocked with 1% bovine serum albumin. After blocking, the sections were then incubated with primary antibody or anti--SMA overnight at 4°C. TRITC-labeled and FITC-labeled secondary antibodies were then added for 1 h at 37°C. Tissue sections or cells were then incubated with 10 mg/ml 40-6-diamidino-2-phenylindole (DAPI; Serva, Heidelberg, Germany) for 5 min at room temperature and analyzed using a confocal microscope (Leica). Data are presented as means±S.D. of at least three independent experiments. Two-group comparison was performed using -test for independent samples. Multiple-group statistical analyses were performed by one-way ANOVA with Bonferroni's multiple comparison test. Statistics were calculated with the GraphPad Prism 5 software package (La Jolla, CA, USA). The significance level was defined as <0.05.

It has been suggested that soluble factors secreted from MSCs have an important role in inhibiting antibody-producing B cells. Thus, we attempted to evaluate the regulatory activity of conditioned medium (CM) of MSCs (MSC-CM) harvested from our clonal MSCs on the Ig production by B cells. When splenic B cells isolated from Balb/c mice were stimulated with lipopolysaccharide (LPS)/interleukin-4 (IL-4), IgE production was observed (). Co-culture of B cells with MSCs almost completely suppressed IgE production by B cells. Similarly, MSCs suppressed the IgE production in transwell cultures as effective as in co-cultures, suggesting that a soluble factor(s) is responsible for the IgE downregulation. When CM harvested from MSCs normally cultured for 3 days was applied to LPS/IL-4-stimulated B cells, it still potently suppressed IgE production, comparable to co-cultures or transwell cultures. Surprisingly, significant IgE downregulation by MSC-CM was observed even at a dilution of 25 600-fold, suggesting that an unknown powerful B-cell inhibitor(s) may be present in the MSC-CM (). In addition, inhibition of Ig production by MSC-CM appeared to be general rather than isotype-specific because the production of other Ig isotypes, including IgG1 and IgM, was also substantially inhibited by MSC-CM (). Our next goal was to identify the soluble factors that are responsible for the IgE downregulation in B cells. During the process, it was unexpectedly found by our routine quality control program that this MSC-CM was contaminated with mycoplasma (). Because a recent study reported that T-cell inhibition by MSCs is markedly affected by mycoplasma infection, we then tried to examine whether B-cell inhibition by MSC-CM is also influenced by mycoplasma infection. In order to eradicate mycoplasmas from infected MSCs, the cells were treated with a commercial antimycotic reagent according to the manufacturer's instruction. Mycoplasma eradication from MSCs was verified by the negative result of the mycoplasma PCR (). Surprisingly, CM harvested from antimycotic-treated MSCs did not downregulate IgE production by B cells. Furthermore, the production of other immunoglobulins, including IgM and IgG1, was not inhibited by the same antimycotic-treated MSC-CM (). These results strongly suggest that MSC-CM-mediated Ig downregulation in B cells is possibly correlated with mycoplasma contamination. Next, we analyzed the DNA present in mycoplasma-infected MSC-CM to identify the infecting mycoplasma strain. DNA sequence analysis strongly indicated that is the infecting strain (; ). To determine whether this strain is specifically responsible for Ig downregulation by MSC-CM, we purchased the identified strain from American Type Culture Collection (ATCC, Manassas, VA, USA). Our approach was to evaluate whether mycoplasma infection explains the MSC-CM-mediated Ig downregulation in B cells by directly infecting healthy MSCs with cultured microbes. Mycoplasma-free MSCs were directly infected with different titers of the mycoplasma strain and PCR analysis was then performed for its detection. -specific PCR detection was verified (). On the basis of semi-quantitative PCR results, we estimated that there are ∼20 colony-forming units per milliliter (cfu/ml) of in MSC-CM (). We then determined the minimal number of required to infect two different cell types, mouse dermal fibroblasts (MDFs) and MSCs. Mycoplasma-free MSCs and MDFs were inoculated with several cfu/ml of and cultured. On the basis of the results of -specific PCR, the minimal numbers of microbes required for successful infection were 20 cfu/ml for MDFs and 40 cfu/ml for MSCs (). When CMs harvested from mycoplasma-free or -infected cells were evaluated for their effect on the IgE production in B cells, only CMs from -infected cells inhibited the IgE production in B cells, regardless of the cell type (). CMs from -free MSCs or MDFs did not influence the IgE production in B cells. On the other hand, we wondered whether itself affects the IgE production in B cells. When was added to LPS/IL-4-stimulated B cells, the IgE production was significantly reduced (). It appeared that just 2 cfu/ml of were sufficient for IgE downregulation (). In addition, other Ig isotypes such as IgG1 and IgM were also significantly downregulated by (). These results suggest that the inhibition of the Ig production in B cells is specifically correlated with the presence of . Intriguingly, we noticed that a highly diluted MSC-CM sample could inhibit IgE production, although mycoplasma was not detectable by PCR (sample 5; 25 600-fold dilution, ). This observation implied the existence of a secreted cellular factor(s) other than mycoplasma particles. Therefore, we hypothesized that a soluble factor(s) secreted from mycoplasma-infected MSCs may mediate Ig downregulation in B cells. To investigate whether a soluble factor(s) present in mycoplasma-contaminated MSC-CM is involved in Ig downregulation, we simply removed mycoplasma particles from MSC-CM by passing through a 0.22-m filter and then used the filtered CM to assay its effect on IgE production. The filtrate inhibited the IgE production in B cells to the same extent as non-filtered CM did (). The absence of mycoplasma in this filtrate was confirmed by PCR as shown in . These results indicate that not only mycoplasma particles but also an unknown soluble cellular factor(s) secreted from mycoplasma-infected MSCs contributes to the immunoregulatory functions of B cells. Next, in order to confirm and characterize a soluble factor(s) that inhibits the IgE production in B cells, the MSC-CM was subjected to heat inactivation, protein transport inhibition, and size fractionation. First, we measured the inhibitory activity of the CM after boiling (or heat inactivation) to examine whether the soluble factor(s) is a protein. Boiling abrogated the inhibitory activity of MSC-CM on IgE production, indicating that the soluble factor(s) is a protein (). Second, we examined the effect of monensin, a protein transport inhibitor, on the inhibitory activity of MSC-CM. IgE downregulation was not observed when monensin-treated MSC-CM was used, suggesting that the soluble protein factor(s) is synthesized intracellularly and secreted extracellularly through a cellular protein transport process (). This result was also consistent with the result from the heat inactivation experiment. Third, we tried to approximate the size of the molecule by fractionating MSC-CM according to the molecular weight. The size of the putative soluble factor(s) was estimated to be over 50 kDa (). Next, we performed fast protein liquid chromatography (FPLC) to separate the putative soluble factor(s) from MSC-CM according to the molecular weight (). IgE downregulation in B cells was evident in only three fractions (fractions 12, 13, and 14) (). The other fractions were excluded because their effect on IgE inhibition was unclear. No inhibition was observed in MDF-CM fractions or the culture medium (). Therefore, these results suggest that -infected MSCs secrete a soluble protein molecule(s) that is able to inhibit the IgE production in B cells. To identify the soluble factor secreted from mycoplasma-infected MSCs responsible for the downregulation of IgE in B cells, we performed liquid chromatography (LC) analysis with fraction 13 of MSC-CM, MDF-CM, and medium. Among the top 100 proteins identified according to peptide score and coverage percentage (), we selected 12 candidate molecules of which the expression was exclusive in MSC-CM according to their potential role in B-cell regulation (). We further selected four proteins, including C3, haptoglobin, translationally-controlled tumor protein (TCTP), and cathepsin L; the protein expression of each molecule in MSC-CM was confirmed by either an enzyme-linked immunosorbent assay (ELISA) or western blot analysis (). Among these four candidate proteins, we investigated the effect of C3, the most potential candidate with the highest peptide score in the LC analysis. When MSCs were infected with , C3 was detected in the CM (). However, it was not detected in CM harvested from B cells after mycoplasma infection (), indicating that infection specifically affects MSCs to secrete C3. Mouse C3 protein by itself downregulated IgE as well as IgG1 and IgM in B cells (). As expected, heat-inactivated C3 treatment of B cells did not reduce the IgE production (). To obtain further evidence of C3 involvement, the downregulation of IgE by mycoplasma-infected MSC-CM was evaluated in the presence of the C3 inhibitor compstatin. Treatment with compstatin reversed the MSC-CM-mediated downregulation of IgE in a dose-dependent manner (). In the presence of compstatin, mycoplasma-infected MSC-CM did not reduce the production of IgG1 and IgM (). The inhibition of IgE production with a size-fractionated sample (fraction 13) of mycoplasma-infected MSC-CM was also abrogated by compstatin treatment (). Taken together, these results suggest that C3 secreted from mycoplasma-infected MSCs may inhibit Ig production in B cells by hampering B-cell differentiation into antibody-producing plasma cells. To investigate this possibility, we examined whether B-cell expression of B-cell-induced maturation protein-1 (Blimp-1), one of the most important regulators in plasma cell differentiation, was influenced by C3 treatment. Blimp-1 expression in B cells was enhanced by LPS/IL-4 stimulation, whereas its expression was completely blocked by either mycoplasma-infected MSC-CM or C3 protein (). Compstatin treatment restored the MSC-CM-induced inhibition of the Blimp-1 expression (). Furthermore, C3, inactivated by boiling, did not block the Blimp-1 expression (). Although it remains unclear at present whether C3 suppresses the Blimp-1 expression directly or indirectly, it is evident that mycoplasma infection-associated abnormal C3 expression from MSCs negatively regulates B-cell differentiation. Collectively, our results showed that mycoplasma infection enhances the MSC-mediated B-cell immunosuppression by altering MSCs to secrete C3, thereby mediating the inhibition of B-cell differentiation. Over the past decade, the finding that -expanded MSCs can modulate immune responses has promoted the clinical translation of stem cell therapy. The MSC-mediated immunomodulation of T cells has been largely investigated, whereas relatively less attention has been paid to B-cell regulation by MSCs. Only few studies reported the effects of MSCs on B cells, showing contradictory results. Some works showed that MSCs induce antibody production in the presence of splenocytes or a TLR9 ligand. In particular, antibody-producing plasma cells were induced via cell–cell contact in the presence of MSCs, when B cells from patients with systemic lupus erythematosus were stimulated with a TLR9 ligand. Similar results were observed in mice; MSCs inhibited the antibody production by B cells in the presence of LPS, splenocytes, or a TLR9 ligand, whereas MSCs increased the production of IgM and IgG in both T-cell-dependent and -independent manners. Contrarily, other studies showed that MSCs suppress the antibody production by human B cells in the presence of activated T cells, plasmacytoid dendritic cells, or a TLR9 ligand. It has been demonstrated that MSCs inhibit the antibody production by B cells in transwell systems as well as in co-culture systems. Considering these results, soluble factors secreted from splenocytes, peripheral blood mononuclear cells, or B cells probably activate MSCs and the activated MSCs inhibit B-cell functions subsequently. However, Rafei reported that MSC-CM alone is sufficient to decrease the number of antibody-producing splenocytes from ovalbumin-immunized mice. The authors suggested that the soluble factors secreted from MSCs are matrix metalloproteinases and chemokine (C-C motif) ligand 2, which suppress the antibody production in B cells. This discrepancy between MSC activation by neighboring cell-secreted soluble factors and MSC-secreted molecules themselves may be owing to different stimulators and conditions, different MSCs, or undefined factors. In this study, we showed for the first time how an unexpected mycoplasma contamination in MSC cultures can affect B-cell functions. Mycoplasma contamination is a well-known problem in cell culture laboratories. The most common contaminants in cell cultures include , , , , and . However, little has been reported on the effects of mycoplasma contamination in MSCs. Zinocker showed that mycoplasma-contaminated MSCs enhance the inhibition of T-cell proliferation . Their MSCs were found to be contaminated with . In our study, was identified as MSC-infecting mycoplasma strain () and found to be significantly correlated with antibody downregulation. Intriguingly, we found that not only directly suppressed the Ig production in B cells but also indirectly downregulated Ig production without affecting the activation status of B cells (). The direct downregulation by appeared to be different from the indirect mechanism with no influence on B-cell activation. Although IgE production was downregulated by direct addition of to B-cell cultures, the mycoplasma rather increased B-cell proliferation (unpublished data). We speculate that hyperactivation of B cells by likely suppressed IgE production. On the other hand, the indirect downregulation occurred through C3, which is induced by -infected MSCs (). In addition, C3 alone could suppress the Ig production in B cells (). C3 has a central role in the activation of the complement system and contributes to innate immunity. C3 convertase catalyzes the proteolytic cleavage of C3 into C3a and C3b during activation. In humans, complement receptor 1 (CR1 or CD35) and 2 (CR2 or CD21) are expressed on B cells. CR1 binds to C3b and prevents B-cell differentiation to plasmablasts and their Ig production, whereas CR2 binds to iC3b, C3dg, or C3d and transduces a positive activation signal upon colligation with surface IgM. The mouse complement system is quite different from the human system. In mice, CR1 and CR2 are generated by alternative splicing of . In (CD21-CD35)-deficient mice, B cells exhibited a reduction in Blimp-1 expression and impaired antibody persistence paralleled by a strongly reduced development of bone marrow plasma cells. These findings suggest that complement receptors maintain antibody responses by delivering differentiation and survival signals to bone marrow plasma cell precursors. Unlike findings, our study showed that C3 directly inhibits Ig production in LPS/IL-4-induced mouse B cells . This inhibition was rescued by compstatin, a peptide inhibitor of the complement system (). Compstatin binds directly to C3 and prevents the cleavage of C3 into C3a and C3b by the C3 convertase. It seems that the cleaved C3 product C3b is required for Ig downregulation in B cells. However, it remains unknown whether mouse CR1 negatively regulates B-cell differentiation, as demonstrated in human B cells. Pappworth generated human CR1 (hCR1) transgenic mice in the presence of mouse CR1/2. In these mice, the number of CD138-positive plasma cells was reduced in spleens of antigen-immunized mice compared with negative control mice, suggesting that hCR1 overexpression may alter B-cell differentiation in mice. On the basis of these data and our results, we speculate that C3 from -infected MSCs can bind to CR1 and inhibit B-cell differentiation via inhibition of Blimp-1 expression. However, the molecular mechanism by which C3/C3b regulates Blimp-1 expression needs to be elucidated. is a mammalian parasite found in a number of animal species. Accumulating evidence in the case reports suggests the pathogenic role of as a new human zoonosis. was first reported to cause a fatal septicemia with pneumonia in a 64-year-old patient with advanced non-Hodgkin's lymphoma. A recent case report provided another evidence of eosinophilic fasciitis associated with infection in a 23-year-old man. was also reported to be isolated from an open femur fracture by an African lion attackin a 56-year-old hunter and from the blood of an immunocompromised Japanese patient with advanced non-Hodgkin's lymphoma. Therefore, these findings suggest the pathogenicity of in humans. The results of our study speculate a potentially new mechanism of the immunomodulatory function of infused MSCs , which is difficult to observe . For example, MSCs transplanted into a patient can encounter pathogens in the blood stream and then get stimulated and secret immunomodulatory factors, such as C3 as demonstrated in this study, to regulate the transcription of genes in immune cells. If this is true, the response of MSC treatment to patients may vary depending on the pre-existing condition of each patient, which may require a pre-examination of patients before MSC transplantation. However, the relationship between a host pathogen and MSCs remains elusive. The immunomodulatory effects of MSCs are being exploited in clinical settings, for example, in patients with autoimmune diseases such as graft--host disease, rheumatoid arthritis, type I diabetes, and Crohn's disease, after allogeneic stem cell transplantation. The findings of this study may initiate the unfolding of unknown mechanisms of immunomodulatory functions of MSCs and provide a scientific basis of why MSCs have different outcomes on a wide range of immune diseases. It is unlikely that mycoplasma contamination deprives MSCs of their stem cell properties, including differentiation potential, proliferation, cytokine secretion, and stem cell marker expression. Thus, mycoplasma contamination in MSC cultures may not be recognized. Because mycoplasma-infected MSCs show remarkably enhanced immunomodulatory activity, more attention and routine examination is required, especially in investigations on the immunomodulation of MSCs. Our present study suggests that C3 secreted from -infected MSCs has an important role in Ig production of B cells via regulation of Blimp-1. However, its role needs to be explored. In addition, further study is required as to whether C3 treatment itself has the same effects as it has in -infected MSCs. MSCs were isolated from C3H HeN (Orient, Seongnam, Korea) bone marrow according to the subfractionation culturing method. MDFs were isolated from C3H HeN dermal tissue. These two cell lines were incubated using Dulbecco's modified Eagle's medium with low glucose (Gibco, Carlsbad, CA, USA) and supplemented with 10% fetal bovine serum (Gibco) and 1 × antibiotic-antimycotic solution (Gibco) at 37 °C in 5% CO. B cells were isolated from Balb/c splenocytes using the EasySep Mouse B Cell Enrichment Kit (StemCell Technologies, Vancouver, BC, Canada) and cultured in RPMI 1640 (Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco), 2-mercaptoethanol (Gibco), and 1 × antibiotic-antimycotic solution (Gibco). MSC- or MDF-derived CM was harvested 3 days post incubation. For some experiments, CM was subjected to some modifications; CM was filtered using a 0.22-m syringe filter (Pall Corporation, Port Washington, NY, USA); filtered CM was heat-inactivated at 100 °C for 5 min; CM was treated with different doses (15–60 M) of compstatin (TOCRIS, Bristol, UK), which is a C3 inhibitor; CM was separated using filter units with a molecular weight cutoff size of 50 or 100 kDa (Millipore, Billerica, MA, USA). For FPLC separation and western blot analysis, 200 ml of filtered CM, which was harvested 3 days post incubation in the absence of serum, were concentrated (200 × ) using filter units with a molecular weight cutoff size of 50 kDa. For ER/Golgi block, cells were incubated for 24 h with 2 M of monensin (Sigma, St. Louis, MO, USA), which blocks the protein transport from the endoplasmic reticulum to the Golgi apparatus. Then, CM was harvested and filtered by using a 0.22-m syringe filter. The experiments were set up using 24-well culture plates (1 ml culture medium per well). Briefly, 10 cells of MSCs in 100 l of MSC culture medium were mixed with 10 B cells for co-culture experiments. For transwell experiments, cells were seeded on the upper side of a transwell chamber (Corning, Tewksbury, MA, USA) on a membrane with a 5.0-m pore size. CM was added at a 1 : 10 (v/v) ratio to B cells (unmodified and filtered, boiled, or size-fractionated, respectively). B cells were stimulated with 10 g/ml LPS (Sigma) and 30 ng/ml mouse IL-4 (Prospec-TanyTechnogene, Rehovot, Israel). Mouse C3 (Kamiya Biomedical Co., Seattle, WA, USA) was treated with B cells at a concentration of 10 ng/ml. The titers of IgE, IgM, and IgG1 in culture medium of B cells were measured by using ELISA (BD Pharmingen, San Diego, CA, USA). Levels of C3 and haptoglobin in CM were measured by using the C3 ELISA kit (Kamiya Biomedical Co.) and the Haptoglobin ELISA kit (Abnova, Taipei, Taiwan). TCTP and cathepsin L in the CMs were detected by performing a western blot analysis using the following antibodies; purified mouse anti-TCTP (BD Biosciences, San Jose, CA, USA), mouse anti-cathepsin L (Pierce, Rockford, IL, USA), and horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA). Mycoplasmas in CM and cell lysates were detected by using a mycoplasma detection kit (e-Myco, iNtRON, Sungnam, Korea) and -detecting PCR using the following primer sequences: Forward: 5′-GATTCCGTTGTGAAAGGAGC-3′, Reverse: 5′-TCAAGCTTTCGCTC ATTGTG-3′. The 16S ribosomal DNA region of the strain with which the cell lines were infected was amplified by PCR and sequenced. These sequences were then compared with those in the NCBI databases using BLAST version 2.2.28+. (ATCC 23243) was obtained from ATCC and propagated in mycoplasma medium. For mycoplasma eradication, mycoplasma-contaminated MSCs were treated with a commercial antimycotic reagent MycoGONE (Genlantis, San Diego, CA, USA) according to the manufacturer's instruction. Mycoplasma eradication was evaluated by PCR. Mycoplasma infection was done by inoculating mycoplasma-free MSCs or MDFs with and cultured for 3 days. Successful infection of the cells was confirmed by PCR (Bio-Rad, Hercules, CA, USA). FPLC was performed on an ÄKTA Prime plus system (GE Healthcare Life Sciences, London, UK). The concentrated CM was loaded onto a superose 12 10/300 GL column (GE Healthcare Life Sciences) equilibrated in phosphate-buffered saline. The column was eluted with two-column volumes of buffer. The fractions were filtered with a 0.22-m syringe filter before using them for treatment of B cells. The FPLC fractions that had an effect on the IgE production of B cells were digested with trypsin (Promega, Madison, WI, USA). Nano-LC-tandem mass spectroscopy (MS/MS) analysis was performed on an Agilent 1100 series nano-LC and LTQ-mass spectrometer (Agilent, Santa Clara, CA, USA). The capillary column used for LC-MS/MS analysis (150 × 0.075 mm) was obtained from Proxeon (Odense M, Denmark) and slurry-packed in house with Magic C18 stationary phase (5 m particle size, 100 Å pore size; MichromBioResources, Auburn, CA, USA). The mobile phase A for LC separation was 0.1% formic acid in deionized water and mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was set up to give a linear increase from 5 B to 35% B in 100 min, from 40 B to 60% B in 10 min, and from 60 B to 80% B in 20 min. The flow rate was maintained at 300 nl/min after splitting. Mass spectra were acquired using data-dependent acquisition with full mass scan (400–1800 /) followed by MS/MS scans. Each acquired MS/MS scan was an average of three microscans on the LTQ. The temperature of the ion transfer tube was controlled at 200 °C and the spray voltage was in the range 1.5–2.0 kV. The normalized collision energy was set at 35% for MS/MS. Mass tolerances of 1.2 and 0.6 Da were used for precursor and fragment ions, respectively. Peptides were allowed to be variably oxidized at methionine residues and to be variably carboxyamidomethylated at cysteine residues. Total RNA was isolated from LPS/IL-4-induced B cells using a total RNA extraction kit, easy-BLUE (iNtRON). RNA was reverse-transcribed using AccuPower RT PreMix (Bioneer, Daejeon, Korea) and OligodT (Bioneer). Blimp-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected by PCR using the following primer sequences: Blimp-1-F: 5′-TGGTATTGTCGGGACTTTGC-3′, Blimp-1-R: 5′-TGGGGACACTCTTTGGGTAG-3′, GAPDH-F: 5′-CCACTGGCGTCTTCACCAC-3′, GAPDH-R: 5′-CCTGCTTCA CCACCTTCTTC-3′. GAPDH was used for loading the control.

Aberrant expression of miR-185 has been reported in several types of cancers. To explore the potential role of miR-185 in human gastric cancer, we analyzed 25 pairs of human gastric cancer tissues and matched adjacent noncancerous tissues (). Quantitive analysis revealed that miR-185 levels were significantly reduced in gastric cancer samples, compared with normal gastric tissues (). However, obvious decrease in miR-185 expression in cancer tissues was observed in 20 of 25 patients (). In parallel, miR-185 expression was significantly reduced in all five collected gastric cancer cell lines, compared with that in human gastric epithelium cell line GES-1. The endogenous levels of miR-185 were relatively lower in SGC-7901, BGC-823 and MGC-803 (). When exposed to cisplatin (30 M) or doxorubicin (2 M), markedly elevated miR-185 levels were determined in SGC-7901 () and MGC-803 cells (). The elevation of miR-185 expression induced by cisplatin or doxorubicin treatment led us to consider whether miR-185 was involved in the regulation of anticancer drug-induced apoptosis. To this end, we produced a construct encoding miR-185. Enforced expression of miR-185 was confirmed by quantitative RT-PCR (qRT-PCR) (). Overexpression of miR-185 alone had no significant effect on apoptosis (), which was consistent with the results of previous study. To characterize the function of endogenous miR-185 in the apoptotic signaling pathway, which mediated chemotherapy, miR-185 antagomir was transfected into SGC-7901. MiR-185 levels were reduced by its specific antagomir. Cisplatin- or doxorubicin-induced apoptosis was attenuated by miR-185 antagomir ( and ). A similar result was obtained in MGC-803 cells (). Then, we attempted to investigate the influence of miR-185 on cell susceptibility to chemotherapy. Following low-dose cisplatin (3 M) or doxorubicin (0.2 M) treatment in gastric cancer cells, a limited amount of cells undergoing apoptosis was observed. Whereas when we enforced expression of miR-185, the apoptotic cells were significantly increased in response to the same dose of cisplatin or doxorubicin ( and ). Taken together, these results suggest that miR-185 reduces chemotherapy resistance in gastric cancer. To elucidate the potential mechanisms by which miR-185 regulates apoptosis, we performed a bioinformatic analysis by TargetScan and found that human ARC mRNA 3′-untranslated regions (UTRs) contained two binding sites for miR-185 (). We measured ARC protein levels in GES-1 and gastric cancer cells. Western blot results indicated that ARC was highly expressed in gastric cancer cells, whereas it was not detectable in GES-1. Endogenous levels of ARC were inversely associated with that of miR-185 in these cell lines (). Then, we attempted to evaluate if miR-185 modulated ARC expression. Enforced expression of miR-185 resulted in an obvious reduction of ARC protein levels, but not in ARC mRNA levels in SGC-7901 and MGC-803 cells (). In contrast, administration of miR-185 antagomir attenuated the decrease of ARC protein levels upon cisplatin () or doxorubicin () treatment. Therefore, it seems that miR-185 modulates ARC expression at the post-transcriptional level. To verify whether miR-185 directly targets ARC, we cloned ARC 3′-UTR containing two miR-185 binding sites downstream of the luciferase reporter gene (ARC 3′-UTR-Wt) to examine luciferase translation driven by the 3′-UTR of ARC. Synthesized miR-185 mimic or miRNA mimic control were transfected into HEK-293 cells to perform the luciferase assays. MiR-185 overexpression induced a decrease in the luciferase activity (). Besides, we generated mutated luciferase constructs ARC 3′-UTR-Mut1 and ARC 3′-UTR-Mut2, and mutations were introduced into the two miR-185 binding sites of ARC 3′-UTR, respectively. We found that both the binding sites were responsible for the role of miR-185 in regulating ARC expression (). Simultaneous mutations of two binding sites could nearly abolish the inhibitory effect of miR-185 on luciferase activity (). We further investigated the effect of miR-185 on ARC expression in gastric cancer cells, as shown by luciferase activity. The loss of luciferase expression driven by ARC 3′-UTR upon cisplatin treatment was attenuated by the site-directed mutations in ARC 3′-UTR (). This reduction of luciferase activity was also attenuated when transfecting miR-185 antagomir (). Thus, our data indicate that miR-185 is able to target ARC directly. Abundant studies have reported that ARC was expressed at high levels in cancer cells. There is evidence implicating that ARC may be involved in carcinogenesis and chemotherapy resistance. After cisplatin or doxorubicin treatment, we found a strong reduction of ARC protein levels, which is contrary to the alteration of miR-185 levels. However, ARC mRNA levels were not reduced as much as its protein levels (). We next explored the role of ARC in gastric cancer chemoresistance. High-dose cisplatin or doxorubicin triggers significant apoptosis. Enforced ARC expression diminished chemotherapy-induced apoptosis (). Whereas knockdown of endogenous ARC enhanced the sensitivity of gastric cancer cells to low-dose cisplatin ( and ). We wondered whether miR-185 regulated chemotherapy sensitivity in gastric cancer cells by targeting ARC. Overexpression of miR-185 promoted low-dose cisplatin-induced cell death. ARC without 3′-UTR (ARC) showed a stronger inhibitory effect on cell death than ARC with 3′-UTR (ARC-3′-UTR) in the presence of miR-185 ( and ). These results reveal that ARC contributes to chemotherapy resistance, which is regulated by miR-185. The expression of miRNAs can be regulated by transcriptional factors. To elucidate the mechanism involved in the upregulation of miR-185 upon cisplatin or doxorubicin treatment, we analyzed the promoter sequence of miR-185. Three putative binding sites for RUNX3 were identified (). RUNX3 is a transcriptional factor that has a loss of function in gastric cancer. Our findings that cisplatin or doxorubicin treatment increased RUNX3 expression () led us to consider whether RUNX3 participated in the upregulation of miR-185 expression. To test this hypothesis, the chromatin immunoprecipitation (ChIP) analysis was carried out to test the association of RUNX3 with the promoter of miR-185. It revealed that RUNX3 could directly bind to the binding site 3 (BS3) region but not the binding site 1 (BS1) and 2 (BS2) region in SGC-7901 cells treated with cisplatin or doxorubicin (). We next evaluated the transcriptional activity of miR-185 in response to RUNX3. MiR-185 promoter region was cloned upstream of luciferase reporter gene. Overexpression of RUNX3 induced a significant increase of miR-185 promoter activity, and this elevation was reversed by introducing mutations into the BS3 region of miR-185 promoter (). We determined that cisplatin or doxorubicin treatment increased miR-185 promoter activity in gastric cancer cells, whereas mutations in BS3 region arrested this elevation (). Finally, we found that enforced expression of RUNX3 led to a remarkable increment of miR-185 expression in SGC-7901 and MGC-803 cells ( and ). Collectively, our data indicate that miR-185 is directly upregulated by RUNX3 at the transcriptional level. The involvement of miR-185 and ARC in regulating chemotherapy resistance led us to consider whether RUNX3 controls chemosensitivity and apoptosis in gastric cancer through this pathway. Reduced apoptosis of gastric epithelial cells was found in Runx3-deficient mice. Besides, RUNX3 has been shown to mediate TGF--induced apoptosis. Thus, we first tested whether RUNX3 influenced apoptosis in gastric cancer. An increment of apoptotic cells was observed in SGC-7901 cells overexpressing RUNX3 (). Concomitantly, ARC protein levels, but not its mRNA levels, were strongly reduced (). Silencing RUNX3 by using its siRNA attenuated the increase of miR-185 levels and reduction of ARC protein levels in response to cisplatin or doxorubicin (). Moreover, knockdown of endogenous RUNX3 diminished chemotherapy-induced cell death in SGC-7901 (). Next, we were interested in determining the effect of RUNX3 on cell susceptibility to anticancer drugs. Strikingly, we found that RUNX3 overexpression led to an elevated amount of cells undergoing cell death, as well as an increase of miR-185 levels in response to low-dose cisplatin (). RUNX3 enhanced cisplatin sensitivity of gastric cancer cells. Subsequently, we considered whether miR-185 and ARC contributed to the function of RUNX3 in gastric cancer. Compared with antagomir control, administration of miR-185 antagomir reduced RUNX3-enhanced chemosensitivity ( and ). This reduction was also observed in the presence of the construct encoding ARC. ARC without 3′-UTR exhibited a stronger inhibitory effect on cell death than ARC with 3′-UTR (ARC-3′-UTR) in the presence of RUNX3 ( and ). These results indicate that RUNX3 mediates chemosensitivity and apoptosis in gastric cancer by modulating miR-185/ARC signaling pathway, although RUNX3 can also target other factors involved in apoptosis. It has been reported that miR-185 suppressed the proliferation of human colorectal cells. Having demonstrated that miR-185 significantly increased the susceptibility of gastric cancer cells to chemotherapy , we further investigated the role of miR-185 in xenograft models. First, to test whether miR-185 alone could inhibit gastric cancer tumor growth, SGC-7901 cells stably overexpressing miR-185 or negative control were established. Overexpression of miR-185 was confirmed by qRT-PCR (). The stable cells, SGC-7901-miR-185- or SGC-7901-negative control, were injected subcutaneously into nude mice. Xenograft tumor formation was monitored over a 34-day time course. Compared with controls, miR-185 overexpression resulted in a smaller tumor size (), suggesting that miR-185 suppressed gastric tumor growth . The mice were killed at day 34. As expected, we found significantly increased miR-185 levels in SGC-7901-miR-185-generated tumors, accompanied by the downregulation of ARC (). Next, we attempted to assess miR-185 therapeutic activity against established tumors. SGC-7901 cells were subcutaneously inoculated into nude mice. When the tumor volumes reached 250–300 mm, intraperitoneal delivery of doxorubicin and/or intratumoral injection of miR-185 adenovirus were given every other day. Two different doses of doxorubicin, 2 and 4 mg/kg, were used in this study. During 2 weeks of therapy, we followed subcutaneous tumor growth and body weight of the mice. It was found that high-dose chemotherapy appeared as obvious inhibitors of tumor growth, and clear body weight loss indicated the severe toxicity of chemotherapy ( and ). In comparison with high-dose chemotherapy, miR-185 alone or combined with low-dose doxorubicin did not exhibit obvious adverse effect, which was indicated by slight body weight loss (). Combination therapy using low-dose doxorubicin and miR-185 successfully restrained the growth of established gastric tumor, as effective as high-dose chemotherapy (). Additionally, quantitative analysis of tumors from combination treatment group showed an elevation of miR-185 levels and remarkable reduction of ARC expression (), which may be contributed to enhanced apoptosis, as assessed by the TUNEL assay ( and ) in xenograft models. These findings were consistent with our obtained results . To sum up, our data indicate that miR-185 increases gastric cancer chemosensitivity through suppressing ARC expression . To further explore the potential clinical relevance of miR-185-mediated signaling pathway in human gastric cancer, we determined the expression levels of RUNX3 and ARC in human gastric tissues (). Immunohistochemical staining showed loss of RUNX3 expression and high expression of ARC in tumor tissues (). This observation was further confirmed by immunoblotting of RUNX3 and ARC protein in these clinical samples. Five representative results were shown here (). According to our data, the inverse association between ARC expression and RUNX3 protein levels was indicated. These findings are consistent with the results in gastric cancer cell lines, and suggest that RUNX3–miR-185–ARC axis may be involved in the development of gastric cancer. Chemotherapy has an important role in the treatment of a variety of cancers, including gastric cancer. However, chemoresistance is a major challenge to effective treatment. It is quite urgent to explore potential mechanisms involved in chemotherapy resistance. Our current study identified miR-185 as a regulator in the sensitivity of gastric cancer cells to chemotherapy. We found that miR-185 was expressed at a lower level in several gastric cancer cell lines and human gastric cancer tissues. Enforced expression of miR-185 led to enhanced sensitivity of gastric cancer cells to low-dose chemotherapeutic agents. Furthermore, we discovered an miR-185-controlled apoptotic pathway involving ARC, which was regulated by RUNX3. Our conclusion was also supported by the results in nude mice model and clinical gastric cancer specimens. Aberrant miR-185 expression has been identified in various cancers in previous studies. It has been shown that miR-185 was abundant in hepatocellular carcinoma. However, there were opposite findings that decrease of miR-185 levels occurred in ovarian cancer, glioma and prostate cancer. The function of miR-185 in cancer cells may be cell type-dependent. In gastric cancer, lower expression levels of miR-185 were observed, which was consistent with our findings. However, the exact function of miR-185 in gastric cancer is little known. Our results displayed miR-185 tumor-suppressing activity in gastric cancer xenograft model. Based on our experiments, induction of miR-185 could be a possible therapy strategy for gastric cancer. MiR-185 suppressed the proliferation potential and tumor growth in colorectal cancer, in non-small-cell lung cancer and in ovarian cancer. It inhibited invasion and migration in human prostate cancer xenograft model. Moreover, miR-185 could sensitize high Six1-expressing cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. MiR-152 and miR-185 cocontributed to chemosensitivity through epigenetic regulation in ovarian cancer. These investigations implied that miR-185 was possibly related to apoptosis. However, the mechanism of miR-185 involvement in apoptosis signaling pathway is unclear. Our results showed that miR-185 increased anticancer drug-induced apoptosis by directly targeting ARC. MiR-185 has been shown to target other proteins in different types of cells. For instance, miR-185 regulated HIF-2 in human umbilical vein endothelial cells. Six1, androgen receptor, Cdc42 and RhoA were proven to be downregulated by miR-185 in cancer cells. Interestingly, miR-185 could disrupt DNA methylation by targeting DNA methyltransferase 1 in ovarian cancer and gastric cancer. In this context, we clearly demonstrated that ARC is a direct target of miR-185. To the best of our knowledge, this is the first report on the regulation of ARC expression mediated by an miRNA. ARC was highly expressed in various cancer cells. The function of miR-185 in these ARC-abundant cancer cells needs to be further explored. Because of the limited number of human gastric cancer tissues we collected in this study, we did not analyze the relevance of miR-185 expression levels to clinicopathologic features and prognosis of these patients. The clinical significance of miR-185 during gastric cancer progression needs to be investigated in the further studies. Besides, miR-185 polymorphic nucleotide variations were discovered in breast cancer, chronic lymphocytic leukemia and colorectal cancer. It would be interesting to identify whether miR-185 SNPs exists and functions in gastric cancer. Our present data revealed that RUNX3 enhanced miR-185 expression at the transcriptional level in gastric cancer. It has been reported that RUNX3 increased Bim expression and decreased vascular endothelial growth factor levels owing to its transcriptional activity. Previous researches have reported that methylation modification was possibly related to RUNX3 expression level. MDM2-mediated ubiquitination participated in the regulation of RUNX3 as well. In this study, we observed upregulation of RUNX3 in human stomach cancer cells exposed to chemotherapeutic drugs. The mechanism by which RUNX3 was upregulated in response to anticancer agents remains to be determined. The therapeutic effects of conventional anticancer agents were limited owing to chemoresistance and their toxicity. MiRNAs have been demonstrated to participate in cancer progression and resistance to chemotherapy, thereby acting as a potential candidate for therapeutic intervention. Anti-miR-21 in combination with S-TRAIL resulted in significantly increased apoptosis in murine glioma models. Administration of anti-miR-221 or anti-miR-181b along with tamoxifen led to apparent reduction in breast tumor size. Combination treatment of miR-200c and paclitaxel markedly decreased ovarian tumor burden. Here, we report that restoration of miR-185 alone can inhibit gastric cancer tumor growth. Moreover, combination therapy using enforced miR-185 expression and lower dose chemotherapeutic drugs had an effective therapeutic activity against large established tumors, with decreased host toxicity. Our results are first to implicate that miR-185 can be considered as a target for gastric cancer treatment. It would be quite possible to develop miR-185-based combinatorial strategy for effective cancer therapy. Taken together, we report here that miR-185 increases the chemosensitivity of gastric cancer cells and . It exerts tumor-suppressing function through negatively regulating ARC. Besides, miR-185 upregulation in response to cisplatin or doxorubicin treatment in gastric cancer cells is dependent on RUNX3 transcriptional activity. Our results suggest that miR-185 might be a novel therapeutic target for gastric cancer. Cisplatin, doxorubicin and G418 were purchased from Sigma (St. Louis, MO, USA). Anti-ARC antibody and anti-RUNX3 antibody were obtained from Abcam (Cambridge, UK). Anticleaved caspase-3 antibody was obtained from Cell Signaling Technology (Danvers, MA, USA). Human gastric cancer cell line SGC-7901 was obtained as we described previously. Human gastric epithelium cell, GES-1, and human gastric cancer cells, NCI-N87, MGC-803, BGC-823, AGS, were obtained from Beijing Institute for Cancer Research (Beijing, China). The cells were cultured in Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin in a humidified atmosphere containing 5% CO at 37 °C. Gastric cancer tissues and matched noncancerous gastric tissues from 25 patients (19 males and 6 females; mean age, 59.76 years, range, 44–81 years) with gastric cancer were collected from Beijing Military General Hospital (Beijing, China). These patients were randomly selected from the patient pool of the hospital's Gastrointestinal clinic; none of the patients received chemotherapy or radiotherapy. Human tissues were collected during gastroscopy and immediately frozen in liquid nitrogen. The study was approved by the ethics committee of the Beijing Military General Hospital. Informed consent was obtained from all study subjects. Cell death was determined by trypan blue exclusion. The trypan blue-positive and -negative cells were counted on a hemocytometer. TUNEL assay was performed using a kit from Roche Applied Science (Hamburg, Germany). The procedures were followed as per the instructions in the kit. The samples were imaged using a laser scanning confocal microscope (Zeiss LSM 510 META, Carl Zeiss, Jena, Germany). Adenovirus ARC and adenovirus -galactosidase were as we described previously. Constructions of adenoviral miR-185 and RUNX3 were described in . All adenoviruses were amplified in HEK-293 cells. Adenoviral infection of cancer cells was performed as we described previously. The hsa-miR-185 duplexes were synthesized by GenePharma Co. Ltd (Shanghai, China). MiR-185 mimic sequence was, 5′-UGGAGAGAAAGGCAGUUCCUGA-3′. Mimic control sequence was, 5′-UUGUACUACACAAAAGUACUG-3′. Chemically modified antisense oligonucleotides (antagomirs) were used to inhibit endogenous miR-185 expression. The antagomir sequence was, 5′-UCAGGAACUGCCUUUCUCUCCA-3′. All the bases were 2′-O-methyl-modified (GenePharma Co. Ltd). The antagomir control sequence was, 5′-CAGUACUUUUGUGUAGUACAA-3′ (GenePharma Co. Ltd). Cells were transfected with miRNA duplexes (50 nM) or antagomirs (50 nM) using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. The fragment of ARC 3′-UTR containing two miR-185 binding sites (sites 1 and 2) was amplified by PCR. The forward primer was 5′-ATGCTGCTGGAGCTGAATCGGAT-3′, and the reverse primer was 5′-ATAGAGCGCCTTTTGTGTAAGTC-3′. To generate reporter vector containing miR-185 binding sites, the PCR product was cloned downstream of the stop codon of the luciferase gene of pGL3 vector (Promega, Madison, WI, USA). To generate ARC 3′-UTR-Mut1 and ARC 3′-UTR-Mut2, the mutations (the wild-type ARC 3′-UTR site1: TCCA, ARC 3′-UTR-Mut1: TCCA; the wild-type ARC 3′-UTR site 2: TCCA, ARC 3′-UTR-Mut2: TCCA) were produced using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The two binding sites of miR-185 were mutated simultaneously to generate ARC 3′-UTR-Mut1+2. For luciferase assay performed in HEK-293 cells, cells in 24-well plates were co-transfected with 200 ng per well luciferase reporter constructs, 400 ng per well miR-185 mimic or mimic control using Lipofectamine 2000 (Invitrogen). SV-Renilla luciferase plasmids of 5 ng per well served as the internal control. Cells were harvested at 24 h after transfection and the luciferase activity was detected using the Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer's instructions. A measure of 30 l of protein samples was analyzed in a luminometer. Firefly luciferase activities were normalized to luciferase activity. The similar strategy was used to perform luciferase analysis in SGC-7901 cells. The miR-185 promoter region (1297 bp) was amplified from human genomic DNA to generate wild-type promoter. The fragment containing three RUNX3 putative binding sites was amplified using the forward primer, 5′-GGGCAGAGCAGAGCTACAAATG-3′, and the reverse primer, 5′-AACCCAGCTCTAGCCAGCAGGT-3′. The PCR product was cloned into the reporter vector pGL4.17 (Promega). The introduction of mutations in three potential RUNX3 binding sites (wild-type: TT→mutated: TT) was carried out using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). Luciferase assay was performed as described. Immunoblotting was performed as we described. Briefly, cells were lysed for 1 h at 4 °C in a lysis buffer (20 mmol/l Tris (pH 7.5), 2 mmol/l EDTA, 3 mmol/l EGTA, 2 mmol/l DTT, 250 mmol/l sucrose, 0.1 mmol/l phenylmethylsulfonyl fluoride, 1% Triton X-100) containing a protease inhibitor cocktail. Protein samples were subjected to 12% SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed using corresponding primary antibodies. Then, the horseradish peroxidase-conjugated secondary antibodies were used. Antigen–antibody complexes were tested by enhanced chemiluminescence. Stem-loop qRT-PCR was carried out as described on an Applied Biosystems ABI Prism 7000 sequence detection system (Applied Biosystems, Carlsbad, CA, USA). Total RNA was extracted using Trizol reagent. After DNase I (Takara, Japan) treatment, RNA was reverse transcribed with Reverse Transcriptase Kit (Toyobo, Osaka, Japan). Mature miR-185 levels were measured using SYBR Green Real-Time PCR Master Mix (Toyobo) according to the manufacturer's instructions. The sequences of miR-185 primers were: forward, 5′-CATGGAGAGAAAGGCAGT-3′ reverse, 5′-GTGCAGGGTCCGAGGT-3′. The levels of miR-185 analyzed by qRT-PCR were normalized to that of U6. The sequences of U6 primers were: forward, 5′-CTCGCTTCGGCAGCACA-3′ reverse, 5′-AACGCTTCACGAATTTGCGT-3′. Quantitative detection of ARC and RUNX3 was performed using the same strategy. The primers used for ARC were: forward, 5′-CGAGTCCGAAGATTCCTGA-3′ reverse, 5′-GACCCTCCGGAGTTTATTCA-3′. The primers used for RUNX3 were: forward, 5′-GACAGCCCCAACTTCCTCT-3′ reverse, 5′-CACAGTCACCACCGTACCAT-3′. The mRNAs levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of GAPDH primers were: forward, 5′-GTCGGAGTCAACGGATTTG-3′ reverse, 5′-TGGGTGGAATCATATTGGAA-3′. ChIP was carried out as we described previously. Briefly, cells were washed two times with phosphate-buffered saline (PBS), and then were incubated for 10 min with 4% formaldehyde at room temperature. After washing two times with PBS, cells were lysed in a lysis buffer for 1 h at 4 °C. The cell lysates were sonicated into chromatin fragments (500–800 bp length). The samples were precleared with Protein A-agarose (Roche Applied Science) at 4 °C. The anti-RUNX3 antibody or anti-actin antibody was added and rocked overnight. Immunoprecipitates were captured with 10% (v/v) Protein A-agarose. To analyze RUNX3 binding to the promoter of miR-185, PCRs were carried out using primers that encompass RUNX3 BS1 or BS2 or BS3 of the miR-185 promoter. The primers were: BS1 (forward, 5′-GGACTCTTGAGATGAGATCAC-3′ reverse, 5′-CTCTCCATTCTGTCAACTGG-3′); BS2 (forward, 5′-TCCTGCAGATGTTCAGATGC-3′ reverse, 5′-CAACTCTCAGCATGTCCTTG-3′); BS3 (forward, 5′-CCAGATCAAGATATGGTGACC-3′ reverse, 5′-ACCTGTGACCTTGCCTTTG-3′). For the experiments in , approximately 1 × 10 SGC-7901 cells, established stable SGC-7901-miR-185 or SGC-7901-negative control cells, were injected subcutaneously into the right flanks of female BALB/c nude mice (4–5 weeks old, 5 mice per group).The tumor size was measured with a caliper every 3 days. The tumor volume was calculated using the formula volume=length × width/2. The mice were killed at day 34, and the tumors were separated for further analysis. For the combination therapy experiments in , 1 × 10 SGC-7901 cells were injected subcutaneously into the right flanks of female BALB/c nude mice (4–5 weeks old). When tumors reached an average volume of 250–300 mm, the mice were randomly divided into five groups, six mice in each group. According to the experimental design, intraperitoneal delivery of doxorubicin and/or intratumoral injection of miR-185 adenovirus was administered every other day. Two different doses of doxorubicin (2 and 4 mg/kg) were used. During doxorubicin and/or miR-185 adenovirus treatment in established tumors, the tumor size and body weight of the mice were monitored every other day. At the end of the experiment, the mice were killed and the tumors were separated for further analysis. Animal experiments were reviewed and approved by the Animal Care Committee, Institute of Zoology, Chinese Academy of Sciences. All statistical analyses were performed using the SPSS 13.0 statistical software package. The results are expressed as means±S.D. of at least three independent experiments. The differences among experimental groups were evaluated by one-way analysis of variance. Paired data were determined by two-tailed Student's -test. <0.05 was considered statistically significant. Additional Materials and Methods are available in .

The cytotoxic effect of 5-FU in the SNUC5 cell line and its 5-FU-resistant variant, SNUC5/5-FUR, was compared. The concentration of 5-FU that yielded 50% growth inhibition (IC) was 9 M in SNUC5 cells and 2264 M in SNUC5/5-FUR cells, in which resistance was induced by continuous culture in 140 M 5-FU (). These results were confirmed by a colony formation assay; the colony numbers in both cell lines were decreased by IC concentration of 5-FU compared with the untreated group (). 5-FU treatment induced apoptosis in both cell lines, as shown by annexin V–propidium iodide (PI) staining and TdT-mediated dUTP nick end labeling (TUNEL) staining. The annexin VPI (apoptotic) population showed 29% in SNUC5 and 31% in SNUC5/5-FUR cells (). TUNEL staining data were consistent with annexin V–PI data (). HO-1 expression and activity were higher in SNUC5/5-FUR cells than in SNUC5 cells (). Nuclear Nrf2, a transcription factor for HO-1, and active Nrf2 (phospho Nrf2) expression were higher in SNUC5/5-FUR cells than in SNUC5 cells, and Nrf2 and phospho Nrf2 expression in cytosol were lower in SNUC5/5-FUR cells than in SNUC5 cells (). The enhanced translocation of Nrf2 protein from the cytosol to the nucleus () and its binding to the ARE sequence in the HO-1 promoter () were shown in SNUC5/5-FUR cells. Flow cytometry data showed that ROS levels were higher in SNUC5/5-FUR cells (fluorescence intensity (FI) 342) than in SNUC5 cells (FI 132), and HO treatment in SNUC5 cells (FI 640) was used as positive control (). These results were confirmed by confocal microscopic data; green FI generated by ROS was enhanced in SNUC5/5-FUR cells as compared with SNUC5 cells (). These results suggested that SNUC5/5-FUR cells were exposed to more oxidative stress condition than SNUC5 cells. The putative roles of Nrf2 and HO-1 on the sensitivity of 5-FU in SNUC5/5-FUR cells were investigated by siRNA- or shRNA-mediated silencing and . The siNrf2- or siHO-1-transfected cells significantly lowered cell viability or colony formation numbers than siCon-transfected cells (). 5-FU (140 M) treatment in siNrf2- or siHO-1-transfected cells significantly lowered cell viability or colony formation numbers than 5-FU treatment in siCon-transfected cells or 5-FU-untreated siNrf2- and siHO-1-transfected cells (). In addition, the mice implanted with shNrf2- or shHO-1-transfected SNUC5/5-FUR cells had reduced tumor volume, size, weight, and apoptotic cells than mice implanted with shCon-transfected cells (). 5-FU treatment (20 mg/kg/day) in mice implanted with shNrf2- or shHO-1-transfected cells reduced tumor volume, size, weight, and apoptotic cells than the 5-FU treatment in mice implanted with shCon-transfected cells or phosphate-buffered saline (PBS) treatment in mice implanted with shNrf2- or shHO-1-transfected cells (). Methylation of the Nrf2 promoter was assessed using methylation-specific PCR (MS-PCR) and quantitative MS-PCR. Specific CpG sites in the promoter region of the Nrf2 gene were methylated in SNUC5 cells, but less methylated in SNUC5/5-FUR cells (). These results were confirmed by quantitative MS-PCR; the levels of methylation of the Nrf2 gene in SNUC5 and SNUC5/5-FUR cells were 35% and 5%, respectively (). The expression of epigenetic modification-related proteins in terms of DNA methylation was investigated by assessing the levels of DNMTs DNMT1, DNMT3A, and DNMT3B, and DNA demethylases TET1, TET2, and TET3. DNMT expression did not show significant difference between SNUC5 and SNUC5/5-FUR cells, whereas TET expression was higher in SNUC5/5-FUR cells than in SNUC5 cells (). Also, TET1 expression was confirmed by confocal imaging (). TET1 binding to the Nrf2 promoter was significantly increased in SNUC5/5-FUR cells, as assessed using a chromatin immunoprecipitation (ChIP) assay, whereas DNMT1 did not show significant difference to Nrf2 promoter binding in both cell lines (). Furthermore, knockdown of TET1 decreased expression of Nrf2 mRNA and protein in SNUC5/5-FUR cells, whereas knockdown of DNMT1 did not affect it (). Also, knockdown of TET1 resulted in a reduction of HO-1 protein expression and activity, but knockdown of DNMT1 did not change it (). These results suggest that TET1 can positively regulate Nrf2 activation in SNUC5/5-FUR cells. TET activity was assessed by measuring the levels of 5-mC, 5-hmC, and 5-fC. The level of 5-mC was lower in SNUC5-5FUR cells than in SNUC5 cells, whereas levels of 5-hmC and 5-fC were higher in SNUC5-5FUR cells than in SNUC5 cells, as shown by dot blot analysis (), confocal imaging (), flow cytometry (), and ELISA (). 5-FU treatment increased levels of nuclear Nrf2 and active Nrf2 (phospho Nrf2) in SNUC5 cells (). Methylation of the Nrf2 promoter was decreased in 5-FU-treated SNUC5 cells (). These results were confirmed by real-time QMSP; the methylation levels of the Nrf2 promoter in control and 5-FU treatment in SNUC5 cells were 44% and 29%, respectively (). Furthermore, 5-FU treatment significantly increased TET1 expression (), and TET activity in SNUC5 cells (). Moreover, we investigated the effect of 5-FU on ROS and TET1 in SNUC5 cells. 5-FU promoted the production of ROS in SNUC5 cells, whereas treatment with an antioxidant -acetylcystein (NAC) had the opposite effect (). 5-FU increased TET1 expression () and TET-catalyzed processes (), but NAC treatment downregulated it, suggesting that the effect of 5-FU on TET expression and function is mediated by its induction of ROS. 5-FU remains the standard therapy for the treatment of patients with CRCs, despite the fact it was developed more than 50 years ago. However, most CRC patients develop resistance to 5-FU. Therefore, elucidating the molecular mechanisms underlying 5-FU resistance is important for the development of molecular targeted strategies to overcome drug resistance. Increasing evidence supports the notion that epigenetic alterations play a major role in drug resistance in various cancers, including CRCs. This suggests that epigenetic agents can be used to overcome CRC drug resistance and increase the efficacy of conventional chemotherapeutic agents in the clinic. In the present study, we showed that expression of Nrf2 and the Nrf2-related gene HO-1 was upregulated in a 5-FU-resistant CRC cell line (SNUC5/5-FUR) compared with the parental cell line (SNUC5). Although the protective role of the Nrf2-antioxidant system against carcinogenesis and chemicals has been well documented in normal cells, its prolonged activation in cancer cells might increase resistance to anticancer drugs. Nrf2 controls a battery of genes including HO-1 that protect cells from chemical and oxidative stresses, and are activated in CRCs. Furthermore, Nrf2 is activated by 5-FU, possibly as a result of drug-induced ROS production, in the human colon cancer HT-29 cell line. Consistent with this report, our results showed that ROS levels and Nrf2 and HO-1 expression were elevated in SNUC5/5-FUR cells. Induction of Nrf2-regulated cell defense genes is associated with increased resistance to 5-FU, and is reversed by siNrf2. Furthermore, oncogene (K-Ras, B-Raf, or Myc)-induced Nrf2 transcription promotes ROS detoxification and carcinogenesis, and stably elevates the basal Nrf2 antioxidant program and thereby lowers levels of intracellular ROS. In the present study, SNUC5/5-FUR cells transfected with siRNA or shRNA against Nrf2 or HO-1 showed increased cytotoxicity and decreased cell proliferation and and were more sensitive to 5-FU treatment. Cancer cells that adapt to oxidative stress by upregulating manganese superoxide dismutase, peroxiredoxin I, and Bcl-2 are resistant to 5-FU. Treatment with siRNA against ROS modulator 1 (Romo1) efficiently blocks 5-FU-induced ROS generation, indicating that 5-FU treatment stimulates ROS production through Romo1 induction. ROS may lead to epigenetic alterations that affect the genome and play a key role in human carcinogenesis. More specifically, ROS production is associated with alterations in DNA methylation patterns. In fact, many tumor suppressor genes are inactivated by ROS-mediated aberrant methylation of CpG island-rich promoter regions. For example, when exposed to oxidative stress, the tumor suppressor genes p15 and p16 accrue aberrant methylation patterns, and their expression is ultimately silenced. DNA methylation is arguably the most intensively studied process in epigenetics with regard to carcinogenesis, and it has been the major focus of pharmacological interventions in clinical trials. This modification occurs predominantly at CG dinucleotide pairs and DNMTs transfer a methyl group to the 5-carbon position of the cytosine ring to form 5-mC. The conversion of 5-mC into 5-hmC, 5-fC, and 5-caC was processed by TET proteins. The genomic content of 5-hmC, 5-fC, and 5-caC can be increased or decreased through the overexpression or depletion of TET proteins. The 5-mC oxidative pathway mediated by the TET proteins may be relevant for the activation or repression of gene expression through its association with transcriptional repressors or activation factors. All TET proteins contain a cysteine-rich region, a double-stranded -helix fold, and a CXXC domain, which is a DNA binding domain with CpG-binding motif, and is involved in the recruitment of TET proteins to DNA. TET1 is the primary enzyme for oxidation of 5-mC to 5-fC, although the other TET proteins, TET2 and TET3, compensate for loss of TET1. In addition, it has been reported that TET1 plays an essential oncogenic role in leukemia. Therefore, the present study was focused on TET1 among the members of the TET family. In the present study, the level of 5-mC was lower and the levels of 5-hmC and 5-fC were significantly higher in SNUC5/5-FUR cells than in SNUC5 cells. The induction of TET1 leads to activation of Nrf2 in SNUC5/5-FUR cells. Our results also indicate that the increase in TET1 and TET process proteins by 5-FU is mediated by its enhancement of ROS levels. In conclusion, these results suggest that the development of resistance to 5-FU in CRC cells involves changes in the expression of Nrf2 and HO-1 that are induced by epigenetic modification of DNA demethylation. Therefore, Nrf2 might represent a potential therapeutic target to overcome the resistance of CRCs to 5-FU treatment. The SNUC5 colon cancer cell line was obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured at 37°C in a 5% CO atmosphere in RPMI-1640 medium (Invitrogen, Grand Island, NY, USA) containing 10% heat-inactivated FBS (Sigma-Aldrich Co., St. Louis, MO, USA). The SNUC5/5-FUR cell line was obtained from the Research Center for Resistant Cells of Chosun University (Gwangju, Korea) and subcultured twice per week for more than 6 months until stable cell lines were established in 140 M 5-FU. The effect of 5-FU on cell proliferation was determined using the MTT assay. Cells were seeded in a 96-well plate at a density of 2 × 10 cells/well and treated with 5-FU. After incubating for 48 h, 50 l of the MTT stock solution (2 mg/ml) was added to each well to attain a total reaction volume of 250 l. After 4 h of incubation, the supernatants were aspirated. The formazan crystals in each well were dissolved in 150 l dimethylsulfoxide and absorbance at 540 nm was read on a scanning multi-well spectrophotometer. Cells were seeded at a density of 500 cells per 60 mm dish and cultured for 10 days with either PBS as a control or with 9 M 5-FU in SNUC5 cell and 2264 M 5-FU in SNUC5/5-FUR cells. During colony growth, the culture medium was replaced every 3 days. Colonies with more than 50 cells were counted under microscopic observation using a Diff-Quick staining kit (Sysmex, Kobe, Japan). To identify apoptotic cells by using annexin V and PI staining method, cells were stained with PI and fluorescein isothiocyanate (FITC)-conjugated annexin V using the EzWay Annexin V-FITC apoptosis detection kit (Koma Biotech, Seoul, Korea). Briefly, cells were seeded on a 24-well plate at a density of 1.5 × 10 cells/well. At 16 h after plating, 9 M 5-FU in SNUC5 cell or 2264 M 5-FU in SNUC5/5-FUR cells was treated. After 24 h, cells were washed twice in PBS, resuspended in 100 l of binding buffer supplemented with 5 l of annexin V-FITC and 5 l of PI solution, and incubated in the dark for 15 min at room temperature. After the addition of 450 l of binding buffer, the stained cells were kept on ice and subjected to analysis using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA, USA). FITC fluorescence was read at 515–545 nm and PI fluorescence was read at 564–606 nm. To detect apoptotic cells by using TUNEL assay, it was performed with the cell death detection kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. Briefly, cells were seeded on chamber slides at a density of 1.5 × 10 cells/well. At 16 h after plating, cells were treated with 9 or 2264 M 5-FU. After 24 h, chamber slides were fixed with 4% paraformaldehyde for 1 h at 15−25°C and permeabilized in 0.1% sodium citrate containing 0.1% Triton X-100 for 2 min. After washing in PBS, sections were incubated with the TUNEL reaction mixture for 60 min at 37°C. After washing with PBS, the stained cells were mounted onto a microscope slide in mounting medium (DAKO, Carpinteria, CA, USA). Stained cells were observed under a fluorescence microscope (Olympus IX70, Olympus Optical Co., Japan). Cells were harvested and to extract the total protein, cells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.9, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 0.1 mM orthovanadate, 1% NP-40, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride) and kept at 4°C for 30 min. After centrifugation for 10 min at 10 000 × , the supernatants measured the protein concentration and were stored at −70 °C. In addition, to extract the nuclear protein, cells were harvested and then lysed on ice with 1 ml of lysis buffer (10 mM Tris-HCl, pH 7.9, 10 mM NaCl, 3 mM MgCl, and 1% NP-40) for 4 min. After 10 min of centrifugation at 3000 × , the pellets were resuspended in 50 l extraction buffer (20 mM HEPES, pH 7.9, 20% glycerol, 1.5 mM MgCl, 0.2 mM EDTA, 1 mM DTT, and 1 mM PMSF), incubated on ice for 30 min, and centrifuged at 13 000 × for 5 min. The supernatant was then harvested as nuclear protein extracts and stored at −70°C after determination of protein concentration. Aliquots of the lysates (40 g protein) were boiled for 5 min and electrophoresed on a 10% SDS-polyacrylamide gel. Proteins were transferred onto nitrocellulose membranes that were subsequently incubated with HO-1, Nrf2, TET1, TET2, TET3, DNMT3B, -actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), DNMT1, phospho Nrf2, TATA box binding protein (TBP) antibodies (Abcam, Cambridge, MA, USA), and DNMT3A (Cell Signaling Technology, Beverly, MA, USA). The membranes were further incubated with secondary immunoglobulin-G-horseradish peroxidase conjugates (Pierce, Rockford, IL, USA). TBP was used as loading control for nucleus protein and -actin was used as loading control for total or cytosolic protein. Protein bands were detected using an enhanced chemiluminescence western blotting detection kit (Amersham, Little Chalfont, UK). The protein bands were visualized using a luminescent image analyzer. HO-1 enzyme activity was measured as described previously. Briefly, cells were homogenized in 0.5 ml of ice-cold 0.25 M sucrose solution containing 50 mM potassium phosphate buffer (pH 7.4). Homogenates were centrifuged at 200 × for 10 min, and the supernatants were further centrifuged at 15 000 × for 60 min. The pellet was resuspended in 50 mM potassium phosphate buffer (pH 7.4), and the amount of protein was determined. The reaction mixture (200 l) containing 0.2 mM of the substrate hemin, 500 g/ml cell lysate, 0.5 mg/ml rat liver cytosol as a source of biliverdin reductase, 0.2 mM MgCl, 2 mM glucose-6-phosphate, 1 U/ml glucose-6-phosphate dehydrogenase, 1 mM NADPH, and 50 mM potassium phosphate buffer (pH 7.4) was incubated at 37°C for 2 h. The reaction was stopped by the addition of 0.6 ml chloroform. After extraction, the chloroform layer was measured spectrophotometrically at 464 nm. HO-1 activity was expressed as pmol of bilirubin per mg of protein. Cells plated on chamber slides were fixed with 4% paraformaldehyde for 30 min and permeabilized with PBS containing 0.1% Triton X-100 for 2.5 min. Cells were treated with blocking medium (PBS containing 3% bovine serum albumin) for 1 h and incubated for 2 h with the primary antibody diluted in blocking medium. The primary antibody was detected using a FITC-conjugated secondary antibody (1 : 500; Santa Cruz Biotechnology) for 1 h. After washing with PBS, stained cells were mounted onto microscope slides in mounting medium containing DAPI (Vector, Burlingame, CA, USA) and imaged on a Zeiss confocal microscope using the LSM 510 program (Zeiss, Jena, Germany). The ChIP assay was performed using the simple ChIP enzymatic chromatin IP kit (Cell Signaling Technology) according to the manufacturer's protocol with slight modifications. Briefly, cells were crosslinked by addition of 1% formaldehyde. Chromatin was prepared and digested with nuclease for 12 min at 37°C. ChIP was performed with an antibody against Nrf2 and IgG. Antibodies were added to the chromatin digests and incubated with constant rotation overnight at 4°C. ChIP-grade protein G magnetic beads were then added to capture the immune complexes. The beads were washed and the immunoprecipitates were eluted with ChIP elution buffer. The crosslinks were reversed by incubation at 65°C for 30 min. Proteinase K was added and samples were incubated at 65°C for 2 h. The immunoprecipitated DNA fragments were purified using spin columns. DNA that was recovered from the immunoprecipitated complex was subjected to 35 cycles of PCR. The primers were as follows: sense, 5′-CCAGAAAGTGGGCATCAGCT-3′ and antisense, 5′-GTCACATTTATGCTCGGCGG-3′ for the HO-1 (Nrf2-binding site) gene promoter; and sense, 5′-TGAGATATTTTGCACATCCGATA-3′ and antisense, 5′-ACTCTCAGGGTTCCTTTACACG-3′ for the Nrf2 (DNMT1 and TET1-binding site) gene. The PCR products were separated on 2% agarose gels, and DNA bands were visualized using the Image program (NIH, Bethesda, MD, USA). Cells were seeded in six-well plates at a density of 3 × 10 cells/well. Cells were treated with 25 M dichlorodihydrofluorescein diacetate (DCF-DA) and the fluorescence of 2′,7′-dichlorofluorescein (DCF) was detected using a flow cytometer (Becton Dickinson) and analyzed using CellQuest software. Image analysis for the generation of intracellular ROS was achieved by seeding cells on a coverslip-loaded six-well plate at a density of 2 × 10 cells/well. Then, 100 M DCF-DA was added to each well and cells were incubated for an additional 30 min at 37°C. After washing with PBS, stained cells were mounted onto a microscope slide in mounting medium (DAKO). Images were acquired using the Laser Scanning Microscope 5 PASCAL program (Carl Zeiss) on a confocal microscope. For transfection of siRNA, cells were seeded at a density of 1.5 × 10 cells/well in 24-well plates and grown until ∼50% confluent. Cells were transfected with 10–50 nM siRNA against Nrf2, HO-1, TET1, and DNMT1 (Santa Cruz Biotechnology) by using Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA) based on the manufacturer's instructions. For transfection of shRNA, lentivirus vectors (Addgene, Cambridge, MA, USA) containing shNrf2 or shHO-1 were diluted in OptiMEM containing 6 g/ml polybrene that was then added to SNUC5/5-FUR cells. After 72 h, transfected cells were selected using puromycin (5 g/ml) for 24 h. Isolated individual cells were kept for up to 3 weeks, and silencing of HO-1 and Nrf2 was verified by measuring the protein level of HO-1 and Nrf2. The human shNrf2 sequence was 5′-CCGGCCGGCATTTCACTAAACACAACTCGAGTTGTGTTTAGTGAAATGCCGGTTTTT-3′, the human HO-1 shRNA sequence was 5′-CCGGGCTGAGTTCATGAGGAACTTTCTCGAGAAAGTTCCTCATGAACTCAGCTTTTTG-3′, and the shControl sequence was 5′-TTCTCCGAACGTGTCACGT-3′. Nude mice (4−6 weeks old) were used for xenograft studies. The scrambled shRNA (shCon)-, Nrf2-targeting shRNA (shNrf2)-, or HO-1-targeting shRNA (shHO-1)-transfected SNUC5/5-FUR cells (3 × 10 cells in Matrigel) were injected subcutaneously into the backs of nude mice. After 14 days, the period during which the tumors were established in mice implanted with shCon-transfected SNUC5/5-FUR cells, mice were treated with vehicle (PBS; =6) or 5-FU (20 mg/kg/day; =6) via direct injections into the tumor for every 2 days (9 times). The surface areas of tumors were measured with a vernier caliper at indicated days. The formula (/2) × (/2) × π (where is the maximum diameter of each tumor, and is the length at right angles to ) was used to calculate the tumor surface area as previously described. Mice were killed and tumors were collected at 35 days after tumor cell injection. Bisulfate modification of DNA was performed using the Methylamp DNA modification kit (Epigentek, Pittsburgh, PA, USA) according to the manufacturer's instructions. To analyze methylation of Nrf2 DNA, MS-PCR was performed using an Epitect MSP kit (Qiagen, Valencia, CA, USA). PCR products were separated on 6% nondenaturing polyacrylamide gels, stained with ethidium bromide, and visualized under UV light. The Nrf2 promoter region was interrogated 1176 bp upstream of the translation start site for potential CpG islands using the NCBI database. Two CpG-rich islands were identified within Nrf2 promoter region: −505 to −254 and −252 to +65. PCR primers were designed to the promoter region spanning −479 to −342, containing 11 CpG sites, using methprimer program. The primer sets were as follows: for unmethylated forward, 5′-GGAGGTGTAGTTTTTATATTAATGT-3′ and unmethylated reverse, 5′-ACCAACTAAAATCCCAACAAACA-3′ for methylated forward, 5′-AGGGAGGCGTAGTTTTTATATTAAC-3′ and methylated reverse, 5′-AACTAAAATCCCAACAAACGAA-3′. Real-time QMPCR for Nrf2 was designed to the promoter region spanning −490 to −353, containing 12 CpG sites, using methprimer program. The primer sets were as follows: for unmethylated forward, 5′-ACAAAAAACCTAAAAAAAATCTCCATT-3′ and unmethylated reverse, 5′-GTTTTAAAGTGTTTGAATTTTAGTGA-3′ for methylated forward, 5-GAAAAACCTAAAAAAAATCTCCGTT-3′ and methylated reverse, 5′-GTTTTAAAGCGTTCGAATTTTAGC-3′. As a reference, the following primers specific to the unmethylated promoter region of the -actin gene were used to yield a 133 bp amplicon: forward, 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′ and reverse, 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′. Calibration curves for target and reference genes were constructed using serial dilutions (0.009–90 ng) of a commercially available fully methylated DNA (CpGenome Universal Methylated DNA, Serologicals Corp., Norcross, GA, USA). Amplification reactions were performed in triplicate in a total volume of 20 l that contained 50 ng of bisulphite-modified DNA, 600 nM forward and reverse primers, and 10 l of QuantiTect 2 × SYBR Green PCR mix (Invitrogen, Inc., Rockville, MD, USA). PCR conditions were as follows: 3 min at 95°C for initial denaturation, followed by 50 cycles of 15 s at 95°C, and 1 min at 60–62°C. PCR reactions were performed in 96-well plates in a 7900 sequence detector (Applied Biosystems, Carlsbad, CA, USA) and were analyzed using SDS 2.1.1 software (Applied Biosystems). Total RNA was isolated from cells using Trizol reagent (Gibco BRL, Grand Island, NY, USA). Complementary DNA (cDNA) was amplified in a reverse transcription reaction containing primers, dNTPs, and 0.5 U of Taq DNA polymerase at a final volume of 25 l. The PCR conditions were: 5 min at 94°C for initial denaturation, followed by 35 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C, and a final elongation period of 7 min at 72°C. PCR amplification was carried out in a programmable thermal cycler (Perkin-Elmer Cetus 9600, Roche Molecular Systems Inc., Branchburg, NJ, USA). The primers used to amplify the Nrf2, TET1, and DNMT1 cDNAs were: Nrf2 sense, 5′-GAGAGCCCAGTCTTCATTGC-3′, antisense, 5′-TTGGCTTCTGGACTTGGAAC-3′ TET1 sense, 5′-CCCGAATCAAGCGGAAGAATA-3′, antisense, 5′-TACTTCAGGTTGCACGGT-3′ DNMT1 sense, 5′-CGCTGTATCTAGCAAGGGTCA-3′, antisense, 5′-TCGAATCTCGCGTAGTCTTG-3′ GAPDH sense, 5′-GTGGGCCGCCCTAGGCACCAGG-3′, antisense 5′-GGAGGAAGAGGATGCGGCAGTG-3′. The amplified products were resolved on 1% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light, using Image Quant TL analysis software (Amersham Bioscience, Uppsala, Sweden). For dot blot assay, DNA was isolated using a Puregene core kit A (Qiagen) and twofold serial dilutions of DNA were denatured by adding denaturing buffer (0.5 M NaOH and 1.5 M NaCl) and incubating at 65°C for 45 min. After neutralization (0.5 M Tris-HCl and 1.5 M NaCl), the solution was loaded in duplicate into a well placed on a positively charged nylon membrane (Amersham). The wells were then washed once with 2 × saline sodium citrate buffer (SSC). The membrane was removed and briefly washed with 2 × SCC and then UV crosslinked and dried. Membrane was blocked with 5% nonfat milk for 1 h and incubated with anti-5-mC, anti-5-hmC, and anti-5-fC (1 : 10 000, Active Motif, Carlsbad, CA, USA) detected by HRP-conjugated secondary antibody and enhanced chemiluminescence. For image analysis, fixed cells were incubated for 2 h with anti-5-mC, anti-5-hmC, and anti-5-fC antibodies diluted in blocking medium (PBS containing 3% bovine serum albumin). The primary antibody was detected using a FITC-conjugated secondary antibody (1 : 500, Santa Cruz Biotechnology) for 1 h. After washing with PBS, stained cells were mounted onto microscope slides in mounting medium containing DAPI (Vector) and imaged on a Zeiss confocal microscope using the LSM 510 program. For detection by using flow cytometry, cells were washed with PBS supplemented with 0.1% Tween-20 and 1% bovine serum albumin (PBST-BSA), fixed with 0.25% paraformaldehyde at 37°C for 10 min and 88% methanol at −20 °C for at least 30 min. After two washes with PBST-BSA, the cells were treated with 2 N HCl at 37°C for 30 min and were then neutralized with 0.1 m sodium borate (pH 8.5). The cells were blocked with 10% donkey serum in PBST-BSA for 20 min at 37°C, incubated with anti-5-mC, anti-5-hmC, and anti-5-fC antibodies (1 g/ml) for 45 min at 37°C, followed by staining with secondary antibody conjugated with Rhodamine Red-X (Jackson ImmunoResearch Laboratories, Westgrove, PA, USA). The cells were washed with PBS three times and assessed by using a flow cytometer (Becton Dickinson). For quantification of 5-mC, 5-hmC, and 5-fC, the ELISA-based methylflash methylated DNA quantification kit, the methylflash hydroxymethylated DNA quantification kit, and methylflash 5-formylcytosine DNA quantification kit (Epigentek) were used. A total of 100 ng of total DNA was used for 5-mC quantification, 200 ng for 5-hmC quantification, and 300 ng for 5-fC quantification. The initial incubation time was 90 min and the final developing time was 10 min. The absolute quantification of standard curves was generated by plotting the concentration of the positive control supplied with the assay against the optical density at 450 nm after performing the assay. The levels of 5-mC, 5-hmC, and 5-fC were assessed by absolute quantification. All measurements were performed in triplicate, and values are expressed as the mean±S.E.M. Results were analyzed using analysis of variance and Tukey's test to determine pairwise differences. <0.05 was considered significant.

This study hypothesized that CuE could mediate the survival of primary CRC cell lines, thereby inhibiting proliferation. To explore the antitumor activity of CuE against CRC cells, we initiated an study in which each of the CRC cell lines was exposed to increasing doses of CuE (0, 2.5, 5, and 7.5 M) over a period of 24 h. The proliferation of the CuE-treated cancer cells was then measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The results, outlined in the upper portion of , indicate that cancer cell survival and proliferation rates decreased with the dosage of CuE throughout the 24-h treatment period. This suggests a dose-dependent reduction (=−16.237+108.12, =0.8547). The same results were not obtained in normal skin fibroblasts Hs-68 cells and normal lung fibroblasts MRC-5 cells as control (normal cell) group (data not shown). Moreover, CuE was shown to induce morphological changes in the primary colon cancer cells. Microscopic observation showed that following exposure to CuE (5 M) between 6 and 24 h, the primary colon cancer cells underwent a remarkable change in morphology. In some cases, CuE induced cell death, resulting in the formation of a suspension in the medium (data not shown). In order to determine whether the growth-inhibitory effects of CuE are reversible, we re-cultivated the primary colon cancer cells in fresh culture medium following exposure to CuE for 24 h. We then assessed the recovery of cell proliferation for an additional 24 ( middle) to 48 h ( lower), whereupon an MTT assay was performed. The results in suggest that the cell proliferation ability of the cancer cells remained substantially degraded (=−22.822+104.09, =0.6893) following CuE treatment for 48 and 72 h (=−24.655+102.47, =0.6489). These observations imply that the primary CRC cells underwent an irreversible change, such as apoptosis or cell cycle arrest, at least to a partial extent. In order to identify the role played by CuE in the apoptosis of primary CRC cells, this study used Annexin V-FITC and propidium iodide (PI) staining to reveal the formation of apoptotic cells following 4 h of exposure to CuE. The percentage of apoptotic cells was assessed using flow cytometric analysis (). The dot-plot of Annexin V-FITC fluorescence PI fluorescence indicates a non-significant increase in the percentage of apoptotic cells treated with CuE, compared with untreated cells. No significant increase was observed in the percentage of five CRC cell lines undergoing necrosis, apoptosis (), or caspase 3 activation at CuE concentrations of 2.5–7.5 M (data not shown). Furthermore, the loss of mitochondrial membrane potential (MMP) is a hallmark of apoptosis and an early event that coincides with caspase activation. The percentages of apoptotic CRC cell lines in different CuE-treated groups were determined using flow cytometry. In non-apoptotic cells, JC-1 exists as a monomer in the cytosol (green) and aggregates in the mitochondria, where it appears red. In apoptotic and necrotic cells, JC-1 exists in a monomeric form and stains the cytosol green. CuE-free CRC cell lines with red fluorescing J-aggregates did not undergo apoptosis. Taken together, our observations indicate that treatment with CuE did not lead to a significant reduction in the MMP of CRC cell lines (data not shown). However, the results shown in , indicate that CuE may mediate the survival of primary CRC cells. Thus, we hypothesize that the proliferation of these cells was inhibited by pathways other than apoptosis. Chemotherapy is known to induce cell death in a variety of tumor types, in part by promoting the production of intracellular ROS. To determine whether ROS production is associated with the CuE-induced cell cycle arrest of CRC cells, we assessed the state of ROS at multiple time points following the administration of various dosages of CuE (0, 2.5, 5, and 7.5 M). Flow cytometry () was performed using the Total ROS/Superoxide Detection Kit () to examine the fluorescence intensity of DCHF-DA-incubated cells. Signals produced by peroxides, peroxynitrite, and hydroxyl radicals were detected in the FL1 channel. Superoxide production was detected in the FL2 channel. The cell cycle distribution of CuE-treated cells was analyzed using flow cytometry. Cells were exposed to CuE for 24 h before processing and analysis. As shown in , exposure to CuE increased the number of G2/M phase cells, while simultaneously reducing the number of cells in the S and G1 phases (*<0.05 CuE 0 M). This suggests a dose-dependent induction (=21.343+3.8567, =0.9855) (), and may imply that the CRC cells underwent cell cycle arrest. To differentiate G2 arrest from mitotic arrest, we used an additional marker, MPM-2. This antibody is capable of recognizing proteins, the epitopes of which are exclusively phosphorylated during mitosis, specifically from early prophase to metaphase. MPM-2 is commonly used as an indicator of mitotic disturbance. To provide a positive control, we treated separate groups of primary CRC cells with nocodazole (15 g/ml), an inducer of metaphase arrest. Treating the five CRC cell lines with nocodazole for 24 h synchronized the entire cell populations in the G2/M phase and increased MPM-2 labeling (). That is, in all CuE-treated cells, MPM-2 staining was elevated above the level of the negative controls (48–59% for five CRC cells, respectively; ). illustrates the immunoblotting results for cellular proteins from CRC cells treated with CuE. In this test, CDC2 protein expression was quantified by measuring relative intensities. We found that CDC2 decreased following incubation with CuE (upper portion of and ), and CDC2 levels were significantly lower in cells incubated with CuE concentrations of 2.5, 5, and 7.5 M () (=−6.974+103.12, =0.8300). We also quantified the activity of the cyclin B1/CDC2 complex (important for G2–M transition during the cell cycle) by performing co-immunoprecipitation (Co-IP; lower) and measuring relative band intensities. Through this test, we found that the activity of the cyclin B1/CDC2 complex was significantly suppressed in cells incubated with CuE () (=−6.6879+104.76, =0.9453). These findings indicate that the increase in G2/M phase CRC cells resulted from non-CuE-induced apoptosis of primary CRC cells, the downregulation of CDC2, and the dissociation of the cyclin B1/CDC2 complex following incubation with CuE. Next, we used human genome SurePrint G3 arrays to study the genome-wide gene expression profiles of CRC cells exposed for 4 h to the vehicle (dimethyl sulfoxide (DMSO)) or CuE (5 M). Three sets of the experiments were performed independently to enable the comparative analysis between the CP3, CP4, and CP5 cell lines. Principal component analysis showed that the microarray data derived from CuE-treated cells and DMSO-treated cells constituted two spatially separated planes. This suggests that treatment with CuE had a far greater impact on the gene expression profile than could be reasonably attributed to technical errors. Therefore, as calculated by the expression levels in the CuE-treated cells divided by those in the vehicle-treated cells, we considered changes of>2-fold as substantial upregulation and changes of<0.5-fold as downregulation. To identify biologically relevant molecular networks of these genes, we used distinct pathway analysis tools of bioinformatics, endowed with the comprehensive knowledgebase of the Kyoto Encyclopedia of Genes and Genomes (KEGG) (). In so doing, we identified the KEGG pathway (: CP3; : CP4; : CP5) as well as a battery of downregulated (: CP3; : CP4; : CP5) and upregulated genes (: CP3; : CP4; : CP5). Specifically, CuE treatment reduced the expression of cyclin B1 and CDC2 genes and also elevated the levels of growth arrest and DNA-damage-inducible protein 45 (GADD45)-, -, and - genes (). These findings suggest that common molecular pathways are involved in the induction of cell cycle G2/M arrest. The RT-PCR ( and ) and qPCR analysis further validated microarray analysis findings, which showed substantial cyclin B1 (=−16.699+109.01, =0.8743) and CDC2 (=−11.385+109.72, =0.9841) downregulation, as well as notable upregulation of GADD45 − (=37.573+76.187, =0.8958), − (=78.13+33.907, =0.9821), and −(=571.91−717.67, =0.8028) in CRC cell lines following exposure to CuE (). illustrates the gene expression in five CuE-treated CRC cell lines, revealing an increase in GADD45. Moreover, the activity of the GADD45 /CDC2 complex (important for the blockade of G2–M transition during the cell cycle) was determined by Co-IP () and quantified by measuring the relative band intensities. Our results indicated that the activity of GADD45/CDC2 complex was significantly suppressed in cells incubated at a CuE concentration of 7.5 M () (=7.8819+86.851, =0.7695). These findings indicate that the increase in G2/M phase cells resulted from the downregulation of CDC2 and cyclin B1 as well as the dissociation of the cyclin B1/CDC2 complex by GADD45 following incubation with CuE. Cucurbitacins are a group of naturally tetracyclic triterpenes that have been shown to act as a potent cytotoxic agent in cancer cells. In our previous study, treating human oral squamous cell carcinoma cells of the SAS cell line with CuE led to growth arrest and apoptosis. The most common mode of cell death following treatment with cucurbitacins appears to be apoptosis. Two major apoptotic pathways exist: the death receptor pathway and the mitochondrial pathway. Multiple apoptotic stimuli trigger the activation of proteases called caspases, which in turn initiate and execute the apoptotic program. In a previous study, CuE was shown to activate the caspase-dependent pathway, coinciding with the activation of the mitochondrial pathway in bladder cancer cells. Throughout Asia, CuE has been used in traditional medicine for cancer therapy, and many studies have attempted to elucidate the mechanism underlying its antitumor activity. However, the results summarized in and indicate that CuE may mediate the survival of CRC cells. Thus, the proliferation of these cells was inhibited by a pathway other than apoptosis. Recent studies have shown that CuE can inhibit the growth of tumor precursors and enhance VEGFR2-mediated Jak2–STAT3 pathways. These effects lead to cell cycle arrest, apoptosis, and anti-angiogenesis. In this study, CuE demonstrated anticancer activity as well as the ability to induce cell cycle G2/M arrest. The results collected through our series of tests provide experimental evidence to support the contention that CuE may irreversibly arrest the growth of primary CRC cells. The results of mechanistic analysis further led to the conclusion that both the inhibition of proliferation and the induction of cell cycle G2/M arrest are highly dependent upon the accumulation of CuE in cancer cells. The role of CuE in the inhibition of tumor growth was highlighted by the delay of mitosis through the downregulation of cyclin B1 as well as CDC2 gene expression and dissociation. The arrested GADD45 encodes three highly conserved nuclear proteins, which contribute to cellular homeostasis by responding to stress. Evidence suggests that the GADD45 family fulfills similar functions in survival, cell cycle control, apoptosis, and the repair of DNA damage. Gadd45 has been shown to interact with several key cellular regulators, including cyclin B1 and p21. These interactions result in the proliferation of cell nuclear antigens and mitogen-activated protein kinase. The cellular function of Gadd45 is dependent on the partner with which it interacts. Notably, Gadd45 is able to suppress G2–M progression in response to stress through its ability to interact with and suppress the kinase activity of the cyclin B1/CDC complex. Accordingly, the RNA silencing of Gadd45 expression impairs G2–M checkpoint activity. Whether interactions between Gadd45 and p21 have a role in G1 arrest has yet to be determined. Additionally, the downregulation of Gadd45 is closely associated with the degree of malignancy in cancers. Thus, the Gadd45 gene family may have an important role in carcinogenesis. Unlike the G2 arrest mediated by radiation, the effects of CuE in CRC cells appears to be independent of DNA damage in the Chk1-cdc2-mediated pathway. Rather, these effects predominantly appear to result from metaphase arrest. Interestingly, our findings suggest that cell cycle G2/M arrests occurred primarily at higher CuE doses in the five CRC cell lines (7.5 M), whereas apoptosis and caspase activation were more likely at lower doses (5 M) in SAS and other cancer cells. The results of mechanical analysis led us to conclude that both the inhibition of proliferation and the induction of apoptosis, cell cycle arrest and autophagy are dependent upon the type of cancer cell. However, upon further investigation, our data suggested the existence of a more complex mechanism involving cell cycle deregulation and apoptosis. This mechanisms appears to reflect differences in the degree of CuE-induced toxicity between cancer cell lines. In conclusion, we have demonstrated for the first time that CuE inhibits tumor growth by arresting the cell cycle in the G2/M phase via GADD45 gene expression and the blockage of cyclin B1/CDC2 complex in primary CRC cells (). The role of CuE in the inhibition of tumor growth was highlighted by a delay in mitosis through the upregulation of the GADD45 gene family. These findings suggest the applicability of CuE as an antitumor agent. CuE, DMSO and MTT were obtained from Sigma (St. Louis, MO, USA). Cell culture medium (DMEM), fetal bovine serum, antibiotics, sodium pyruvate, trypsin, and phosphate-buffered saline (PBS) were purchased from Gibco, BRL (Grand Island, NY, USA). Polyvinylidene fluoride (PVDF) membrane was purchased from Merck Millipore (Darmstadt, Germany), and molecular weight markers were purchased from Bio-Rad (Berkeley, CA, USA). All other reagents and compounds were of analytical grades. The five primary cell lines of colon cancer cells were derived, as a gift, from the cell bank maintained in the MedicoGenomics Research Center at KMU. The cells were grown at 37 °C in Dulbecco's Modified Eagle Medium (Gibco, BRL) supplemented with 10% (v/v) fetal bovine serum (HyClone, South Logan, UT, USA) and a combination of antibiotics (penicillin, 200 unit/ml, and streptomycin, 200 g/ml) (HyClone) under an atmosphere of CO/air (5%) for this series of studies. The cells were seeded into 96-well culture plates at 5000 cells/well. The cells were treated with 0, 2.5, 5, and 7.5 M CuE for 1–3 days. MTT dye (1 mg/ml) was added to each well for at least 4 h of treatment. The reaction was stopped by the addition of DMSO, and optical density was measured at 540 nm on a multi-well plate reader. Background absorbance of the medium in the absence of cells was subtracted. All samples were assayed in triplicate, and the mean for each experiment was calculated. Results were expressed as a percentage of control, which was considered as 100%. Each assay was carried out in triplicate, and the results were expressed as the mean (±S.E.M.). The apoptosis was assessed by the ApopNexin FITC apoptosis detection kit (Chemicon, Millipore, Billerica, MA, USA). The cells were treated with 0, 2.5, 5, and 7.5 M CuE for 6 h, and the apoptotic cells were detected by ApopNexin FITC apoptosis detection kit and flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA), and data were analyzed by WinMDI 2.8 free software (BD). The method for the cell cycle analysis was by using PI, that is, using the fluorescent nucleic acid dye PI to identify the proportion of cells that are in one of the three interphase stages of the cell cycle. The cells were treated with 0, 2.5, 5, and 7.5 M CuE for 24 h. Cells were harvested and fixed in 1 ml cold 70% ethanol at least 8 h at −20 °C. DNA was stained in PI/RNaseA solution and the cell cycle (at least 10 000 single cells) was detected by flow cytometry (FACSCalibur, BD). Data were analyzed by the WinMDI 2.8 free software (BD). The cells were first seeded in 24-well plates (Orange Scientific, Braine, Alleud, Belgium). Following the treatment with CuE for 6 h, JC-1 (10 g/ml, Sigma) was added to the culture medium, 50 l per well, and then incubated (at 37 °C for 20 min) for mitochondria staining. After washing twice with warm PBS, the cells were fixed with 2% paraformaldehyde, inspected by Flow cytometry (FACSCalibur, BD) and data were analyzed by the WinMDI 2.8 free software (BD). Intracellular ROS generation was measured using a DCFHDA fluorescent dye (Molecular Probes/Invitrogen, Thermo Fisher Scientific, Grand Island, NY, USA) and a Total ROS/Superoxide Detection Kit (ENZO life Sciences, Plymouth Meeting, PA, USA). The CPs cells were cultured in six-well plates at a density of 1 × 10/well. Following treatment with an appropriate concentration of CuE, the cells were incubated with 10 M DCFH-DA at 37 °C for 30 min and then washed twice with PBS. ROS/superoxide concentration was assessed by staining ROS and superoxide detection reagent according to the protocol provided by the manufacturer (ENZ-51010, ENZO life Sciences). For each experiment, the cells were analyzed for fluorescence using flow cytometry. Data were analyzed using the WinMDI 2.8 free software. The mitotic index was assessed by MPM-2 (anti-phospho-Ser/Thr-Pro) expression. After 24 h of treatment with CuE, cells were harvested and fixed in 70% ethanol overnight. Cells were then washed and suspended in 100 l of IFA-Tx buffer (4% FCS, 150 nM NaCl, 10 nM HEPES, 0.1% sodium azide, 0.1% Triton X-100) with a primary MPM-2 antibody (1 g/ml; Upstate Cell Signaling Solutions, Millipore, Watford, UK) at room temperature for 1 h. Cells were washed and resuspended in IFA-Tx buffer with a rabbit anti-mouse FITC-conjugated secondary antibody (1: 50; Serotec, Oxford, UK) for 1 h at room temperature in darkness. Finally, cells were washed and resuspended in 500 l of PBS with 20 g/ml of PI (Sigma) for 30 min in the dark. MPM-2 expression was analyzed using flow cytometry (FACSCalibur, BD). Data were analyzed using the WinMDI 2.8 free software (BD). A total of 30–50g proteins were separated by SDS-PAGE (10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and transferred to PVDF membranes (Merck Millipore) in a tank blotter (in 25 mM Tris/0.192 M glycine, pH 8.3/20% methanol) at 30 V overnight. The membranes were blocked with 5% non-fat milk (in 10 mM Tris-HCl, pH 8.0/150 mM NaCl/0.05% tween-20) overnight and incubated with anti--actin (AC-15 Sigma), anti-CDC 2 (SC-747, Santa Cruz BioTechnology, Dallas, TX, USA) antibody for 1.5∼2 h. The blots were washed with Tris-HCl (pH 8.0/150 mM NaCl/0.05% Tween-20) for 3 × 10 min and incubated with second antibody (anti-rabbit or anti-mouse immunoglobulins) (IRDye Li-COR, Dallas, TX, USA) at 1/200 dilution for 1 h. The antigen was then visualized and analyzed by Odyssey infrared imaging system (Odyssey LI-COR, Lincoln, NE, USA). Co-IP is an effective means of quantifying protein–protein interaction in cells. Briefly, 500 g of cellular proteins were labeled using anti-cyclin B1 (SC-752, Santa Cruz BioTechnology) and GADD45 (TA505437, OriGene Technologies, Rockville, MD, USA) following overnight incubation at room temperature. The protein-antibody immunoprecipitates were collected by protein A/G plus-agarose (SC-2003, Santa Cruz BioTechnology). Following the final wash, the samples were boiled and centrifuged to pellet the agarose beads. Western blotting analysis of the CDC2 protein in the supernatant was then conducted. Antigens were visualized using a near infrared imaging system (Odyssey LI-COR), and data were analyzed using the Odyssey 2.1 software (Odyssey LI-COR). Briefly, the cells untreated or treated with CuE for 4 h were harvested, and total RNA was isolated utilizing an RNasey kit (Qiagen, Hilden, Germany) as described by the manufacturer. Total RNA was sent to Welgene Company for whole human genome SurePrint G3 arrays GEP analysis (Agilent Technologies, Santa Clara, CA, USA). A reverse transcriptase system (Promega, Southampon, UK) was used to synthesize complementary DNA (cDNA) from 1 g of total RNA. Between 2 and 4 l of cDNA were used for PCR analysis. PCR (50 l) reactions were performed using 100 ng of each primer () and 1 unit of Dynazyme II (Flowgen, Lichfield, UK). Thermal cycling was conducted for 35 cycles at the following temperature/durations: 98 °C for 10 s, 66 °C for 30 s, and 72 °C for 1 min using a Progene thermal cycler (Cambridge, UK). A final extension of 72 °C was performed for 10 min at the end of 35 cycles. The primers used for amplification of the target genes were checked against all other gene sequences for specificity. PCR reactions were analyzed on 1.5% agarose/TAE minigels and stained using 0.5g/ml ethidium bromide. Gels were visualized using an Apligene UV CCD camera system (Oncer & Apligene, Shanghai, China). Real-time PCR was conducted using the SYBR Green PCR MasterMix (Applied Biosystems, Thermo Fisher Scientific, Grand Island, NY, USA) according to the manufacturer's instructions. qRT-PCR was performed using approximately 200 ng of SYBR Green PCR MasterMix and primers () in an ABI 7300 system (Applied Biosystems, Foster City, CA, USA). PCR conditions were 95 °C for 120 s, 60 °C for 30 s, and 72 °C for 30 s for 40 cycles. Sample cells from three plates were run in duplicate, using the threshold suggested by the software for the instrument to calculate Ct. To normalize readings, we used Ct values from 18 s as internal controls for each run, obtaining a delta Ct value for each gene. All data were reported as the means (±S.E.M.) of at least three separate experiments. Statistical analysis was performed by using -test, with the significant differences determined at the level of <0.05.

For analyzing the ischemic responses of 14-3-3 proteins in neurons, isoform-specific 14-3-3 antibodies were used. Results of western blot demonstrated that 14-3-3, , , and antibodies recognized the corresponding overexpressed 14-3-3 isoform only (). Then, we measured the expression of 14-3-3 isoforms in primary cultures of rat cerebral cortical neurons subjected to OGD. Results of western blot and statistical analysis demonstrated that but not , , and isoforms of 14-3-3 was significantly increased upon 1, 2, 3 and 4 h of OGD incubation (). The results of fluorescent immunostaining also revealed an elevation of 14-3-3 but not 14-3-3, , and in primary cortical neurons upon 2 h of OGD (). The results of reverse transcription-polymerase chain reaction (RT-PCR) and statistical analysis demonstrated that 14-3-3 transcriptions were significantly increased in primary cortical neurons upon 0.5, 1, 2, 3 and 4 h of OGD (). The OGD responses of 14-3-3 isoforms were then investigated in animal ischemic stroke rats with complexity of the cellular components and ischemic pathology in the brain. On subjecting to 24 h of permanent middle cerebral artery occlusion (pMCAo), the ischemic infarct was prominent (white area in the brain slice, triphenyltetrazolium chloride (TTC) staining, ). Brain cells in the penumbra area of the ipsilateral cortex (Ipsi, indicated by the box, ) of the 1 h-pMCAo rat were injured moderately and could elicit protective responses and were used for analyzing 14-3-3 expression. Fluorescent double immunostaining confirmed that 14-3-3 was expressed in cortical neurons (specific with NeuN) in the Ipsi of rats with 1 h of pMCAo (). Results of histochemical analysis clearly showed that 14-3-3 () but not 14-3-3, , and () was evidently increased in cortical neurons in the Ipsi as compared with its contralateral counterpart (Contra) in rats with 1 h of pMCAo. Statistical analysis demonstrated that the relative level of 14-3-3 but not 14-3-3, , and was significantly increased in the Ipsi as compared with the Contra in rats with 1 h of pMCAo (). Consistently, results of quantitative polymerase chain reaction (qPCR) () demonstrated that only 14-3-3 mRNA but not 14-3-3, , and mRNA was selectively upregulated in the Ipsi as compared with the Contra in rats with 1 h of pMCAo. Further, results of western blot analysis demonstrated the 14-3-3 but not 14-3-3, , and was significantly increased in the Ipsi of 1 h-pMCAo rats as compared with the Contra (). These and data together demonstrated that ischemia selectively upregulated only the -isoform of 14-3-3 in cerebral cortical neurons. Since only 14-3-3 was upregulated, we focused on investigating the functional role of this isoform in OGD-treated neurons. Results of fluorescent double-immunostaining showed that 14-3-3 was elevated in surviving neurons (indicated by conclaved arrowheads, ) but nearly undetectable in dying neurons (with elevated cleaved caspase-3 and highly condensed nuclei, indicated by arrows, ) subjected to 6 h of OGD. In addition, 14-3-3 was elevated in neurons that survived 24 h post 2 h OGD (indicated by conclaved arrowheads, ) but evidently decreased in caspase 3-activated (upper panels, indicated by arrows) or TUNEL-positive (lower panels, indicated by arrows, ) neurons. This evidence supported a positive correlation between 14-3-3 levels and the survival of OGD-treated neurons. To investigate the causative effects of 14-3-3 on the survival or death of OGD-treated neurons, 14-3-3 was overexpressed or knocked down by RNA interfering technique. The average transfection efficiency in primary cortical neurons by using the nucleofector was around 65% (). Overexpression of 14-3-3-shRNA (short hairpin RNA) plasmids for 3 days reduced 14-3-3 to ∼25% of that in scrambled negative control (N-con) in primary cortical neurons, verifying the successful knockdown of endogenous 14-3-3 (). 14-3-3 knockdown in primary cortical neurons at 7 DIV induced significant cell death (0 h, 13.3% 9.7% in N-con, ) under normal culture conditions as measured by PI staining, suggesting that endogenous 14-3-3 alone was important for the survival of cortical neurons. Moreover, knockdown of 14-3-3 significantly exacerbated cell death in primary cortical neurons subjected to 1 h (20.8% 15.1% in N-con) and 3 h (33.8% 27.3% in N-con) of OGD (). On the contrary, enhancing 14-3-3 by ectopic overexpression significantly reduced cell death in primary cortical neurons subjected to 4 h (22% 31% in con, and 6 h (47% 64% in con, ) of OGD. Further, we compared the protective effect of each 14-3-3 isoform (, , , , , and ) in OGD-treated neuroblastoma N2a cells, and the results of LDH and MTT assays demonstrated that the 14-3-3 exerted the maximal protection in N2a cells upon 2 h of OGD (). Taken together, 14-3-3 was an important intrinsic protective factor in cortical neurons during OGD. After demonstrating the protective role of 14-3-3 in OGD-treated neurons, we further investigated the underlying mechanisms. It is well known that 14-3-3 proteins function by interacting with other proteins. In primary cortical neurons, inhibiting the interactions of 14-3-3 proteins with their client proteins by overexpressing the specific 14-3-3 blocking peptide Difopein induced severe cell death in cortical neurons at 3 DIV (day ) under normal conditions () and significantly aggravated the death of cortical neurons at 2 DIV upon 2 or 4 h of OGD as measured by PI staining (), supporting that 14-3-3 proteins exert their protection via protein–protein interactions. We have previously reported that 14-3-3 prevented primary cortical astrocytes from OGD-induced cell death by binding to more p-Bad Ser112 in the cytoplasm. In primary cortical neurons, results of co-immunoprecipitation (co-IP) showed that 14-3-3 also bound to p-Bad Ser112 but their interactions decreased prominently upon 2 and 4 h of OGD incubation (). We then examined the interaction of 14-3-3 with other well-known pro-apoptotic proteins such as Bax, Ask-1 and p53. Results of co-IP showed that 14-3-3 bound to little Bax, p-Ask-1 Ser966 () and p-p53 Ser315 () in primary cultured cortical neurons under normal or OGD incubation. Therefore, it is unlikely that 14-3-3 protected ischemic neurons via binding to these well-known 14-3-3 client proteins in the cytoplasm to suppress their apoptotic effects directly. 14-3-3 has been reported to be presented in the nuclei of OGD-treated astrocytes. Recently, a function of 14-3-3 in gene regulation was also reported. Thus, we investigated the interaction of 14-3-3 with -catenin, an important transcriptional factor involved in cell death or survival. Results of co-IP showed a prominent increase of 14-3-3–p--catenin Ser37 interaction in primary cortical neurons upon 2 and 4 h of OGD incubation (). Further, fluorescence resonance energy transfer (FRET) assay demonstrated that endogenous 14-3-3 bound directly to more p--catenin Ser37 in OGD-treated neurons. In control (0 h) and OGD-treated (2 h) neurons (indicated by arrows, ), bleaching of p--catenin Ser37 fluorescence of the whole neuron (red color in the lower panels of , served as the acceptor in FRET assay) evidently enhanced the fluorescent intensity of 14-3-3 of the same neuron (green color in the upper panels of , served as the donor). Statistical analysis demonstrated that the percentage of 14-3-3/p--catenin Ser37 FRET-positive cells (69% at 2 h OGD 40% at 0 h, ) and the mean 14-3-3/p--catenin Ser37 FRET efficiency (21% at 2 h OGD 10% at 0 h, ) were significantly increased in OGD-treated neurons, while the mean 14-3-3/Bax FRET efficiency was not altered (). This evidence strongly suggested that 14-3-3 protected ischemic neurons by direct binding to more p--catenin Ser37. Consistent with the increase of 14-3-3/p--catenin Ser37 binding, results of western blot demonstrated that p--catenin Ser37 was prominently increased in primary cortical neurons upon 1, 2, 3 and 4 h of OGD (). Results of fluorescent immunostaining showed an evident increase of p--catenin Ser37 but not p--catenin Ser45 in cultured cortical neurons upon 2 h of OGD (), suggesting that -catenin was phosphorylated mainly at Ser37 in cortical neurons upon OGD. In the Contra of rat brains, p--catenin Ser37 was homogenously distributed in cortical neurons as compared with the cytoplasmic distribution of 14-3-3 (). On subjecting to 1 h of pMCAo, both p--catenin Ser37 and 14-3-3 were accumulated in the nuclei of cortical neurons in the Ipsi (indicated by arrows, ). Further, results of fluorescent double-immunostaining clearly showed that p--catenin Ser37 and 14-3-3 were co-translocated into the nuclei of same neuron upon 2 h of OGD (). Therefore, OGD promoted 14-3-3 and p--catenin Ser37 binding in the nuclei of cortical neurons. The binding of 14-3-3 and p--catenin Ser37 in the nuclei suggests that 14-3-3 may regulate gene expression via p--catenin Ser37 in ischemic neurons. Therefore, we further examined the regulatory effects of 14-3-3 on the expression of Bcl-2 families as they are gatekeepers controlling mitochondria-mediated cell death. Results of qPCR demonstrated that 14-3-3 overexpression significantly reduced Bax mRNA, but did not alter the transcripts of other Bcl-2 members such as Bcl-2, Bad, Bcl-x and MCL-1 in cultured neurons at 7 DIV (). Results of western blot analysis demonstrated that doubling 14-3-3 amounts by ectopic overexpression reduced 70% of endogenous Bax as compared with untreated (−) and pcDNA controls in cultured neurons at 7 DIV (). Consistently, knockdown of endogenous 14-3-3 by overexpressing 14-3-3-shRNA for 3 days significantly reduced endogenous 14-3-3 and increased Bax as compared with the N-con in N2a cells (). Further, the results of western blot showed that 14-3-3 had a maximal effect on reducing Bax as compared with the other 14-3-3 isoforms in N2a cells (). These data together demonstrated a critical role of 14-3-3 in reducing Bax. Overexpressing Bax in N2a cells caused ∼80% of cell death in GFP+Bax co-transfected cells as measured by GFP expression level under normal culture conditions (indicated by arrows, ), supporting a critical role of Bax in inducing cell death. In primary cortical neurons, Bax was upregulated prominently upon 4 and 6 h of OGD (). Consistently, 2 h of pMCAo induced Bax in cortical neurons in the Ipsi of rat brain (). Therefore, the elevation of Bax was a key damaging factor in OGD/ischemic neurons. We then analyzed the relationship between 14-3-3 and Bax levels in ischemic neurons . Results of fluorescent double-immunostaining clearly showed that 14-3-3 elevation in the nuclei (indicated by arrows, Ipsi, ) was segregated from Bax elevation in cortical neurons in the Ipsi of rats subjected to 6 h of pMCAo, consistent with the inhibitory effect of 14-3-3 on Bax expression. Finally, we verified the regulatory role of 14-3-3 in Bax expression in OGD-treated neurons. Knockdown of 14-3-3 significantly increased Bax and cleaved caspase-3 in cultured cortical neurons upon 4 h of OGD (). These data together demonstrated that 14-3-3 prevented ischemic death of cortical neurons by reducing Bax. After the key downstream target of 14-3-3's protection was identified, we then investigated whether 14-3-3 reduced Bax and cell death via p--catenin Ser37 interaction or not. Overexpression of wild type -catenin (-catenin) significantly increased the death of cultured cortical neurons upon 2 h of OGD, while abolishing -catenin phosphorylation at Ser37 by overexpressing -catenin completely reversed -catenin enhanced cell death as measured by PI staining (). This evidence demonstrated that p--catenin Ser37 itself was a cell death component of cortical neurons upon OGD. Results of western blot demonstrated that overexpressing -catenin significantly reduced Bax in cultured neurons as compared with -catenin (). In ischemic rat brain, results of double-fluorescent immunostaining showed that higher nuclear -catenin Ser37 levels were correlated well with higher Bax levels in cortical neurons (indicated by arrows, ) in the Ipsi of rats subjected to 2 h of pMCAo. The coefficient () of p--catenin Ser37 and Bax fluorescent intensities in ischemic neurons was 0.86 (). These data together demonstrated a critical role of p--catenin Ser37 in reducing Bax. Finally, we demonstrated that 14-3-3 overexpression significantly reduced -catenin-induced cell death in cultured cortical neurons upon 2 h of OGD as measured by PI staining (). Consistently, -catenin-induced Bax upregulation was completely abolished by co-overexpressing 14-3-3 in cortical neurons upon 2 h of OGD (). Taken together, 14-3-3 reduced the cell death of ischemic neurons by suppressing p--catenin Ser37/Bax signaling. In the present study, we demonstrated that 14-3-3 was an important early ischemia-inducible protective factor in cerebral cortical neurons. Further, we identified a neuronal-specific protective mechanism, that is, 14-3-3/p--catenin Ser37/Bax axis in ischemic neurons, dependent on 14-3-3/p--catenin Ser37 interactions in the nucleus. We tested whether 14-3-3 was an intrinsic survival factor in neurons. In pure cultured cerebral cortical neurons and cortical neurons , only the but not any other isoform of 14-3-3 could be upregulated by OGD or pMCAo. We have previously demonstrated that 14-3-3 is selectively upregulated in pure cultured cerebral cortical astrocytes. These evidences suggest that the ischemic response of 14-3-3 is well conserved in different kinds of brain cells. 14-3-3 elevation was prominent after 1 and 3 h of pMCAo but not 6 h of pMCAo (), suggesting that 14-3-3 was an early ischemia-responsive gene. This property was further supported by using the decapitated ischemic model, in which 14-3-3 elevation in the cerebral cortices of rats was detected within 15 min after decapitation (). Hypoxia (0.1% O) () or glucose deprivation () alone did not elevate 14-3-3 until 6 h of treatment in cultured neurons, suggesting a synergistic effect of hypoxia and glucose deprivation on 14-3-3 induction during OGD. Among all 14-3-3 isoforms, 14-3-3 exerted a greater protective effect in neuronal cells upon OGD. Reducing endogenous 14-3-3 exacerbated neuronal death, while increasing 14-3-3 reduced neuronal cell. Thus, we concluded that 14-3-3 was an early ischemia-inducible protective factor. Our findings narrowed down the protection of 14-3-3 families to a single isoform, making it more practical by targeting 14-3-3 for stroke treatment. Since 14-3-3 was the only isoform induced by OGD/ischemia, we speculated that ischemic pre-conditioning might exert a protection by inducing 14-3-3. To test this possibility, we pretreated mice with CoCl, a well-known chemical ischemic pre-conditioning inducer, and found that CoCl indeed elevated only the isoform of 14-3-3 in mouse cerebral cortex (). This evidence strongly suggested that inducing 14-3-3 by chemical compounds in the brain is a feasible strategy in order to elongate the therapeutic time window for thrombolytic therapy after ischemic stroke. 14-3-3 proteins could prevent cell death by binding to different pro-apoptotic proteins. Under normal conditions, 14-3-3 was distributed predominantly in the cytoplasm and bound mainly to p-Bad Ser112 in cortical neurons. This interaction is well known for the antiapoptotic role of 14-3-3 proteins. Upon OGD, 14-3-3 bound to less p-Bad Ser112 and entered into the nucleus, where it bound to increasing amounts of p--catenin Ser37. -Catenin is a critical transcriptional factor regulating cell death and survival and its regulatory mechanism is not fully understood. p--catenin Ser37 was distributed predominantly in the nucleus of cerebral cortical neurons upon ischemia, suggesting that p--catenin Ser37 itself might be an important transcriptional factor. The direct binding of 14-3-3 with p--catenin Ser37 in the nucleus might suppress the transcriptional function of p--catenin Ser37 in the nucleus and Bax might be an important target of p--catenin Ser37. Bax is a key ischemia-inducible cell death factor and its regulation remains not fully understood. 14-3-3 reduced Bax but did not bind to it directly in cortical neurons. Abolishing -catenin phosphorylation at Ser37 alone by point mutation reduced Bax expression and enhanced neuronal survival during OGD, resembling the effects of 14-3-3 overexpression on reducing Bax and OGD-induced cell death. Therefore, we proposed that 14-3-3 suppressed Bax via p--catenin Ser37 interaction in neurons upon OGD or ischemia. It is reported that the binding of 14-3-3 to p--catenin Ser37 might reduce -catenin degradation and thus increases total -catenin to reduce cell death. In OGD-treated neurons, however, the elevation of total -catenin was not detected. Consistently, total -catenin was not increased by 14-3-3 overexpression. Moreover, overexpression of -catenin enhanced Bax and cell death in cortical neurons upon OGD. These data suggested that 14-3-3 exerted its protection by suppressing p--catenin Ser37 function in the nucleus directly, but not by increasing the total -catenin indirectly. It is well known that ischemic cell death is a continuum of cell death at different stages with the morphological and biochemical features of both necrosis and apoptosis. The early activation of caspases within 3 h of pMCAo might push already injured cells into an inevitable death path via either canonical apoptosis or programmed necrosis as a result of molecular switch depending on energy supply. The early upregulated 14-3-3 might reduce acute ischemic cell death by suppressing either canonical apoptosis via the Bax-caspase 3 apoptotic pathway or necrosis via caspase 3-calpain interplay. The delayed 14-3-3 elevation at 24 h of pMCAo () might protect ischemic neurons from delayed ischemic cell death as indicated by the elevation of 14-3-3 in survived ischemic neurons at 24 h of pMCAo (indicated by conclaved arrowheads, ). In summary, we demonstrated that 14-3-3 was an important inducible protective factor in OGD/ischemic neurons and promoted neuronal survival via a distinct nuclear mechanism, that is, the 14-3-3/p--catenin Ser37/Bax pathway (). In OGD-treated astrocytes, 14-3-3 protects astrocytes via binding to p-Bad Ser112, but neither via p--catenin Ser 37 interaction () nor via Bax downregulation (). Thus, targeting a common protective factor (i.e., 14-3-3) but not a specific damaging factor (e.g., Bax or Bad) is likely to provide a desirable therapeutic effect for ischemic stroke. pcDNA-14-3-3 (, , , , , and ) and pcDNA-difopein (dimeric 14-3-3 peptide inhibitor) plasmids were provided by Dr. Haian Fu (Emory University), and -catenin was provided by Dr. Yasuyuki Fujita (University College London). -catenin S37A was produced by point mutagenesis. 14-3-3-shRNA-1, 2 or 3 targeted to 5′-CGAGCAACTAGTGCAGAAA-3′, 5′-CGGCGAAGGCAACAACTAA-3′ or 5′-CTGCTCCGAGACAACCTAA-3′, respectively. 14-3-3-shRNA was constructed as previously reported. N-con used corresponding scrambled sequences. All plasmids were confirmed by sequencing. Antibodies to 14-3-3, , , , and (Immuno-Biological Laboratories, Takasaki-Shi, Japan), p--catenin Ser37, p-ASK-1 Ser966, p-p53 Ser315, cleaved caspase-3, -catenin, p53 and NeuN (Cell Signaling Technology, Boston, MA, USA), Bax, p-Bad Ser112, green fluorescent protein and -actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p--catenin Ser45 (Signalway Antibody, Baltimore, MD, USA) and Flag (Sigma, Saint Louis, MO, USA) were purchased. Cultured rat cerebral cortical neurons were prepared from 16-day-old Sprague Dawley rat embryos as described. Freshly isolated neurons (5 × 10/35-mm dish) were transfected with plasmids by using the Nucleofector Kit and the Amaxa Nucleofector device (Lonza, Basel, Switzerland) according to the manufacturer's instructions. Cultures were maintained in neurobasal media supplemented with 2% B27 and 2 mM L-glutamine (Invitrogen, Grand Island, NY, USA) 24 h after initial seeding, and half of the media was replenished every 2 days (DIV). The cultures were used for experiments at 7 DIV. Primary neuronal cultures were washed with serum and glucose-free Dulbecco's modified Eagle's medium (DMEM) media (OGD media) (Invitrogen) three times and then incubated with OGD media in an anaerobic chamber (Shanghai CIMO Medical Instrument Manufacturing, Shanghai, China) with 95% N/5% CO mixed gas. Oxygen concentration in the anaerobic chamber was 0.1% as measured with a RSS-5100 (Shanghai Precision & Scientific Instrument, Shanghai, China) as reported previously. Propidium iodide (PI, 1 g/ml) was used to distinguish dead cells in living cultures. Nuclei were stained with Hoechst 33342 (2 g/ml) to identify highly condensed apoptotic nuclei. Total soluble proteins were extracted from cultured cells or brain tissues by using radio-immunoprecipitation assay lysis buffer (Applygen Technologies Incorporation, Beijing, China) containing phenylmethanesulfonyl fluoride (Sigma). Equal amounts of total proteins were subjected to western blotting analysis as previously described. The membranes were blocked with 5% (w/v) nonfat dried milk in Tris-buffered saline and then incubated with primary antibodies. After incubation with IRDye 800CW or IRDye 680CW conjugated goat anti-rabbit or anti-mouse IgG (Licor Biosciences, Lincoln, NE, USA), the blots were visualized and quantified by using the Odyssey Infrared Imaging System (Licor Biosciences). Brain sections or cultured neurons were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 3% bovine serum albumin (BSA), incubated with primary antibodies overnight at 4 °C and then with the corresponding FITC- or rhodamine-conjugated secondary antibodies for 1 h at room temperature. Micrographs were taken with a Zeiss 510 confocal microscope (Carl Zeiss, Oberkochen, Germany). After OGD treatment, cultured neurons were washed once with cold PBS and lysed in 400 l of lysis buffer (20 mM Tris–HCl, pH 7.5, 50 mM NaCl, 50 mM NaF, 0.5 mM EDTA, 0.5% (v/v) Triton X-100 and protease inhibitors) per 35-mm dish. Supernatants were collected by centrifugation at 10 000 × for 10 min at 4 °C. Four hundred micrograms of total soluble proteins in 400 l was incubated with 1 g of mouse anti-14-3-3 antibodies overnight with gentle rotation at 4 °C. Twenty microliters of 50% (v/v) protein G-agarose slurry was added and incubated for another 4 h with gentle rotation at 4 °C. The immunoprecipitates were collected by spin down and washed with ice-cold lysis buffer for five times. Twenty microliters of 2xSDS-PAGE gel loading buffer was used to dissociate proteins from the precipitates by boiling for 5 min. The supernatants were collected and subjected to western blot analysis with the corresponding antibodies. Total RNA was extracted from cells or brain tissues using TRIZOL reagent (Invitrogen). Total RNA (2 g) was used to perform RT by using M-MLV transcriptase (Promega, Fitchburg, WI, USA) and oligo (dT15) primer (Promega) in a total volume of 20 l. Conventional PCR and qPCR were performed as described previously using -actin as the internal control. The primers used in PCR were Bax: 5′-CAGGATGCGTCCACCAAGAA-3′ and 5′-GCAAAGTAGAAGAGGGCAACCAC-3′ Bad: 5′-GCAGCCACCAACAGTCATCAT-3′ and 5′-CAAACTCATCGCTCATCCTTCG-3′ Bcl-2: 5′-CGCTACCGTCGTGACTTCGC-3′ and 5′-CATCCCAGCCTCCGTTATCC-3′ Bcl-x: 5′-TGCGTGGAAAGCGTAGACAAGG-3′ and 5′-TGAAGAGTGAGCCCAGCAGAAC-3′ myeloid cell leukemia sequence-1 (MCL-1): 5′-CTCTTATTTCTTTCGGTGCCTTTG-3′ and 5′-CCAGTCCCGTTTCGTCCTTACA-3′, -actin: 5′-CAGCCTTCCTTCTTGGGTAT-3′ and 5′-GCTCAGTAACAGTCCGCCTA-3′. Triplicate measurements were collected for each sample and 14-3-3 mRNA was normalized to -actin and then expressed as the fold of the corresponding control for each condition. Animal studies were approved by the University Committee on Animal Resources of the University of Huazhong University of Science and Technology (HUST) and conducted in accordance with the NIH guidelines for the care and use of laboratory animals. Adult male SD rats (300 g–350 g) were purchased from the Animal Center of Tongji Medical College, HUST and housed in a 12 : 12 h light–dark cycled room maintained at 22±2 °C with food and water for 5 days prior to the experiments. Focal brain ischemia was induced by MCAo as described previously. Briefly, rats were anesthetized with 4% isoflurane and maintained with 2.5% isoflurane, 30% oxygen and 70% air via a face mask. After the right common carotid artery was exposed and its external branch was ligated, a monofilament nylon suture (0.24–0.26 mm in diameter) with poly-L-lysine was inserted into the internal carotid artery through the external carotid artery stump and advanced up to 18–20 mm or until resistance was felt. Sham-operated rats underwent the same surgical procedure without suture insertion. Rectal temperature was monitored throughout and maintained at 37.0±0.5 °C by a feedback-regulated heating system (Harvard Apparatus, Holliston, MA, USA). Physiological parameters (i.e., arterial pH, PaCO, PaO) were analyzed with 50 l of femoral artery blood by using an AVL OPTI 3 Blood Gas Analyzer (AVL Co., Plymouth, MI, USA) and monitored at three time points, that is, 5 min before MCAo, immediately after MCAo and 45 min after MCAo (). Regional cerebral blood flow (CBF) was monitored at 2 mm posterior and 4 mm lateral to the bregma during MCAo by a Laser Doppler Perfusion Monitor (moorVMS-LDF2, Axminster, UK). After the skull bone was thinned to translucency, the laser-doppler flowmeter probe was placed onto the brain surface of MCA blood supply region. CBF baseline was initially measured and stabilized for 10 min before the ligation of external carotid artery. Then, CBF was monitored throughout the filament insertion and a sharp decrease of CBF to <20% of the baseline was considered as successful MCAo (). The infarct was evaluated from 2-mm-thick coronal sections stained with 2% 2,3,5-TTC (Sigma). Rat brains were perfused and post-fixed for 24 h in 4% paraformaldehyde. Brain slices cut into 20 m with a vibratome (S100, TPI; Leica, Wetzlar, Germany) were collected consecutively in PBS. Brain slices were treated with 0.3% HO/0.5% Triton X-100 in PBS for 10 min, blocked with 5% BSA/PBS for 30 min at room temperature, then incubated with primary antibodies overnight at 4 °C, followed by a biotin-labeled secondary antibody for 1 h. The immunoreaction was detected with a streptavidin-biotin-peroxidase complex and visualized with diaminobenzidine tetrachloride. Negative controls were subjected to the same procedures, but without primary antibody. For statistical analysis, all micrographs were taken with the same parameters (8 micrographs/section). The integral optical density (IOD) of 14-3-3 staining ( × 200 magnifications) was estimated using Image-Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA). Mean IOD was calculated as IOD SUM/area. Relative 14-3-3 level in the Ipsi was compared to its Contra. FRET was conducted by using the acceptor photobleaching method with Zeiss FRET-Plus Version 4.0.4 software incorporated into a Zeiss 510 confocal microscope. Cultured cortical neurons were double-stained with mouse anti-14-3-3 and rabbit anti-p--catenin Ser37 or rabbit anti-Bax antibodies and the corresponding FITC- or TRITC-conjugated secondary antibodies. Selected regions of interest of neurons were bleached using the 543 nm laser at maximum intensity for 20 iterations and the donor/acceptor fluorescent images were collected before and after bleaching. The FRET efficiency was calculated, and an increase of 20% of the donor fluorescence after bleaching was considered as a positive FRET signal. Mouse neuroblastoma N2a cells were cultured in DMEM/Opti-M (V/V=1 : 1) supplemented with 5% fetal bovine serum (Invitrogen). After 24 h, transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For animal experiments, 5–8 animals/group were randomly selected. All experiments were repeated at least three times. All values were expressed as mean±S.E.M. Statistical analysis was performed with a two-way ANOVA followed by the Student–Newman–Keuls test, with <0.05 considered significant.

The levels of XAF1 expression were determined in both the renal tissues of Thy-1N rats () and cultured rat GMCs after sublytic C5b-9 stimulation (). The time course study showed that the expression of XAF1 protein both and increased at 2 h, peaked at 6 h and then reduced at 12 h (). Thus, we examined the expression of XAF1 both and at 6 h time point in the following studies. An immunofluorescent study showed that XAF1 expression was colocalized with C5b-9 deposits in the glomeruli of rats at 6 h after Thy-1N induction (), indicating that a portion of C5b-9-bound cells expressed XAF1 instead of undergoing lysis. Our study also demonstrated that a portion of C5b-9-bound GMCs were TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) positive at 6 h after sublytic C5b-9 stimulation, indicating these cells were undergoing apoptosis (). To further confirm that the above-mentioned effects were in fact because of sublytic C5b-9 complexes, control cells were treated with different media containing 5% Thy-1 antibody (Thy-1 Ab), 5% Thy-1 Ab+4% heat inactive serum (HIS), 5% Thy-1 Ab+4% complement C6-deficient serum (C6DS), 5% Thy-1 Ab+4% C6DS+C6 (0.5 or 2 mg/l) and 5% Thy-1 Ab+4% C6DS+PBS, respectively. The results showed that XAF1 expression was markedly upregulated only in the GMCs induced by sublytic C5b-9 (). Meanwhile, adding C6 back to C6DS could recover its ability to induce XAF1 expression and GMC apoptosis (). These data confirm that XAF1 expression and GMC apoptosis are induced by sublytic C5b-9 complexes. To explore the role of gene in GMC apoptosis in response to sublytic C5b-9, the cultured GMCs were divided into the following six groups: modified Eagle's medium (MEM), sublytic C5b-9, XAF1 short hairpin (sh)RNA (shXAF1)+sublytic C5b-9, control shRNA (shCTR)+sublytic C5b-9, pEGFP-N1/XAF1 and pEGFP-N1 vector. The studies showed that XAF1 overexpression by using the plasmids of pEGFP-N1/XAF1 induced GMC apoptosis, whereas shXAF1 treatment reduced GMC apoptosis induced by sublytic C5b-9 stimulation () but had no effect on the apoptosis of normal GMC without sublytic C5b-9 stimulation (). Moreover, XAF1 overexpression enhanced sublytic C5b-9-induced GMC apoptosis (). These findings suggest that gene expression might contribute to sublytic C5b-9-induced GMC apoptosis. In order to confirm that involvement of XAF1 in regulating GMC apoptosis is related to other stimulation, hydrogen peroxide (HO) was used to induce GMC apoptosis, and the role of XAF1 was explored. The results showed that the expression of XAF1 was also involved in HO-induced GMC apoptosis at least in part (). To determine whether IRF-1 activation of XAF1 is involved in sublytic C5b-9-induced GMC apoptosis, the cultured GMCs were divided into six groups of MEM, sublytic C5b-9, IRF-1 shRNA (shIRF-1)+sublytic C5b-9, control shRNA (shCTR)+sublytic C5b-9, pcDNA3.1/IRF-1 and pcDNA3.1 vector. As presented in , overexpression of IRF-1 by using pcDNA3.1/IRF-1 markedly increased XAF1 expression () and GMC apoptosis (), whereas treatment of GMCs with shIRF-1 inhibited XAF1 expression () and GMC apoptosis () stimulated by sublytic C5b-9. To further test whether sublytic C5b-9 can affect gene transcription through IRF-1 expression, the GMCs were transfected with shIRF-1 or pcDNA3.1/IRF-1 together with pGL3-XAF1-full-length (pGL3-XAF1-FL) for 48 h, and then the GMCs were treated with sublytic C5b-9 for another 6 h. The luciferase analysis showed that the overexpression of IRF-1 significantly enhanced XAF1 promotor activity in the GMCs, whereas silence of gene obviously suppressed XAF1 transcriptional activity in the GMCs exposed to sublytic C5b-9 (). These data implicate that sublytic C5b-9 could upregulate gene expression and further increase GMC apoptosis via enhancing IRF-1 expression. To identify the effective promoter regions required for gene transcription in response to IRF-1 expression and sublytic C5b-9 stimulation, the GMCs were transfected with the luciferase reporter constructs of pGL3-XAF1-FL or different promoter deletion fragments (−554 to +160, −337 to +160, −47 to +160 and −14 to +160 nt) accompanied with pcDNA3.1/IRF-1 for 48 h or sublytic C5b-9 stimulation for 6 h, and the activity of XAF1 promoter was determined subsequently. As presented in , the GMCs transfected with XAF1 promoter (−47 to +160 nt) showed markedly decreased luciferase activity relative to the other groups, suggesting that the region of rat XAF promoter (−337 to −47 nt) might contain an IRF-1-binding element. TFsearch software () predicted that an IRF-E (−65 to −53 nt) is located within the region of rat gene promoter (−337 to −47 nt). Chromatin immunoprecipitation (ChIP) assay revealed that both sublytic C5b-9-induced IRF-1 and overexpressed IRF-1 by using pEGFP-N1/XAF1 could bind to this element (). To further confirm that sublytic C5b-9-induced XAF1 gene transcription could be mediated by the IRF-E, we mutated the IRF-E (pGL3-XAF1). As shown in , −65 to −53 nt region mutation abrogated the activity of gene promoter induced by both IRF-1 overexpression and sublytic C5b-9 stimulation. To further identify the interaction between IRF-1 protein and gene promoter, the plasmids of pGL3-XAF1-FL and pGL3-XAF1 were transfected into 293T cells, respectively, accompanied with pcDNA3.1/IRF-1. Luciferase reporter assay displayed that exogenous IRF-1 markedly promoted the transcription of wild-type gene but not mutated gene in 293T cells (). Collectively, the findings indicate that upregulation of XAF1 promoter activity in sublytic C5b-9-induced GMCs depends on the increase of IRF-1 binding to the IRF-E (−65 to −53 nt) within XAF1 promoter. IRF-1 acetylation emerged in a time-dependent manner, with the maximum level at 3 h after exposure to sublytic C5b-9 (). Reportedly, CREB-binding protein (CBP), p300 and P300/CBP-associated factor (PCAF) are important transcription coactivators that could interact with multiple transcription factors as acetyl transferases. Thus, the expression levels of CBP, p300 and PCAF were further detected, and we found that sublytic C5b-9-induced GMCs exhibited a time-dependent increase of CBP, p300 and PCAF expression that reached the maximum at 3 h (), indicating that the expression of CBP, p300 and PCAF perhaps correlated with IRF-1 acetylation. Co-immunoprecipitation (co-IP) assay further revealed that only p300 markedly interacted with IRF-1 at the protein level in the GMCs induced by sublytic C5b-9 ( and ). Meanwhile, adding C6 back to C6DS could recover its ability to induce the combination of p300 and IRF-1, and IRF-1 acetylation (). Therefore, p300 was chosen for subsequent studies, and the results showed that silencing p300 gene not only obviously inhibited IRF-1 acetylation, but also markedly reduced the binding of IRF-1 to XAF-1 promotor and XAF1 expression as well as GMC apoptosis because of sublytic C5b-9 stimulation (). Further studies were designed to determine whether p300 can bind to gene promotor (−65 to −53 nt) together with IRF-1 as a complex, and p300 itself can directly bind to XAF1 protein and induce the acetylation of XAF1. In , re-ChIP analysis showed that the complex of p300 and IRF-1 bound to the gene promotor through IRF-1 binding to its element (−65 to −53 nt). Meanwhile, co-IP assay showed that p300 did not bind to XAF1 at the protein level, and XAF1 could not be acetylated in sublytic C5b-9-induced GMCs (). To observe whether IRF-1 acetylation may crosstalk with other post-translational modifications such as ubiquitination, IRF-1 ubiquitination was further detected, and the data showed that shp300 had no effect on IRF-1 ubiquitination (). Together, these data support the idea that sublytic C5b-9 complexes could induce GMC apoptosis through p300-regulated IRF-1 acetylation that could enhance gene transcription. To find the IRF-1 acetylation sites, N-terminal Lys-29, Lys-39, Lys-50, Lys-66, Lys-70, Lys-75 and Lys-78 of IRF-1 within the DNA-binding domain were mutated respectively, and the IRF-1 acetylation, XAF1 expression and GMC apoptosis in response to IRF-1 overexpression were detected subsequently. The data showed that site mutation of Lys-39 and Lys-78 not only reduced IRF-1 acetylation () and IRF-1 binding to gene promotor (), but also suppressed XAF1 expression () and subsequent GMC apoptosis (). First, the time course study showed that the expression of p300 and IRF-1 markedly increased at 3 h after Thy-1N induction simultaneously, and then gradually decreased (). Moreover, co-IP assay found marked IRF-1 acetylation and enhanced combination between p300 and IRF-1 protein in the renal tissues of Thy-1N rats at 3 h (), consistent with our above-mentioned results . To further evaluate the roles of p300, IRF-1 and XAF1 expression in GMC apoptosis and secondary proliferation of Thy-1N rats, normal Sprague-Dawley (SD) rats were divided into the following six groups: normal serum (NS), Thy-1N, Lv-shp300+Thy-1N, Lv-shIRF-1+Thy-1N, Lv-shXAF1+Thy-1N and Lv-shCTR+Thy-1N. The protein levels of p300, IRF-1 and XAF1 in rat renal tissues were determined at 3 and 6 h respectively after nephritis induction. The data showed that pretreatments of rats with Lv-shp300, Lv-shIRF-1 and Lv-shXAF1 decreased the expression of p300, IRF-1 and XAF1 in the renal tissues, respectively. On the other hand, the Lv-shp300 pretreatment inhibited IRF-1 acetylation and XAF1 expression, but had no effect on IRF-1 expression. Lv-shIRF-1 pretreatment suppressed XAF1 expression, whereas it had no effect on p300 production, and the Lv-shXAF1 pretreatment also had no effect on p300 and IRF-1 expression as well as IRF-1 acetylation (), consistent with our above-mentioned results . Furthermore, TUNEL staining and electron microscopy (EM) showed that all pretreatments of GMC with Lv-shp300, Lv-shIRF-1 and Lv-shXAF1 decreased the number of glomerular TUNEL-positive cells, and the apoptotic changes including irregular aggregation of chromatin at the periphery of the nucleus and condensation of the nuclear chromatin at 6 h after Thy-1N induction. EM analysis also demonstrated that GMCs were the main part of apoptotic cells (). In addition, HE staining, GMC staining, EM and urinary protein analysis showed that each of the pretreatment by using Lv-shp300, Lv-shIRF-1 and Lv-shXAF1 decreased the numbers of glomerular cells especially GMCs () and urinary protein content (mg per 24 h, ) in the rats with Thy-1N at 7 days. Taken together, the findings suggest that knockdown of , and genes respectively could not only reduce GMC apoptosis, but also suppress the secondary GMC proliferation and ameliorate the renal function of Thy-1N rats. In the process of rat Thy-1N, GMCs exhibited different pathological changes including apoptosis, necrosis and proliferation. There is growing interest in GMC apoptosis as a potential contributor to the renal lesion. Early research demonstrated that administration of cobra venom factor (CVF) to deplete complement significantly attenuates renal injury in Thy-1N rats, indicating that GMC apoptosis in rat Thy-1N is complement dependent. Our previous studies have revealed that although C5b-9 deposits were found on the membrane of glomerular cells in Thy-1N rats, many glomerular cells deposited with C5b-9 still remained intact, indicating that these C5b-9 complexes are sublytic. Our present studies also found that a portion of C5b-9-bound GMCs underwent apoptosis, whereas other C5b-9-bound GMCs did not. These data provide evidences that sublytic C5b-9 is able to trigger GMC apoptosis in the early stage of rat Thy-1N. It has been reported that sublytic C5b-9 is able to induce cell apoptosis in many types of cells; however, sublytic C5b-9 can protect oligodendrocytes from apoptosis and lead to survival, suggesting that the different response to C5b-9 in these cells may be because of different cell types and different biochemical reactions upon sublytic C5b-9 attack. It is worth mentioning that sublytic C5b-9 was also found to be able to induce GMC proliferation at 36 and 54 h after sublytic C5b-9 stimulation in our previous studies. Similarly, our present studies revealed that a portion of C5b-9-bound GMCs underwent apoptosis, whereas other C5b-9-bound GMCs could not be found apoptotic. Further studies need to be done to explore the relationship between GMC apoptosis and proliferation due to sublytic C5b-9 and the underlying mechanisms. Cellular apoptosis can be regulated by the expression of apoptosis-related gene. Our current results manifested that XAF1 expression at the protein level could be induced both and . Furthermore, overexpression of XAF1 induced GMC apoptosis, whereas silencing of XAF1 reduced GMC apoptosis in response to sublytic C5b-9 attack, providing evidence that XAF1 might play an important role in the GMC apoptosis triggered by sublytic C5b-9 stimulation. Our previous studies also revealed that IRF-1 induction was related to the GMC apoptosis triggered by sublytic C5b-9. Wang reported that JNK1 stimulated XAF1 promoter activity through IRF-1 expression in gastrointestinal cancer in which IRF-1 bound to IRF-E within XAF1 promoter. As a consequence, the roles of IRF-1 in promoting gene activation in the GMCs exposed to sublytic C5b-9 were further explored. The results displayed that sublytic C5b-9 could upregulate gene expression and increase GMC apoptosis partially via increasing IRF-1 expression. Subsequently, an IRF-1-binding site (IRF-E element) was found to exist in the −65 to −53 nt region at the 5′ end of XAF1 exon-1, by which IRF-1 could directly bind to gene promoter and further activate gene. It is interesting to note that the truncated XAF1 promoter (−337 to +160 nt) showed higher activity compared with the truncated XAF1 promoter (−554 to +160 nt), suggesting a negative control element in this truncated region (−554 to −337 nt). Further studies need to be done to confirm the possible negative control element and its relationship with IRF-1. Moreover, the truncated XAF promoter (−14 to +160 nt) did decrease luciferase activity relative to the truncated XAF promoter (−47 to +160 nt) in sublytic C5b-9-induced GMCs, indicating there might be a positive control element in this section. However, the truncated XAF promoter (−14 to +160 nt) exhibited the same luciferase activity relative to the truncated XAF promoter (−47 to +160 nt) in GMCs transfected with pcDNA3.1/IRF-1, demonstrating the above-mentioned possible positive control element is not an IRF-1-binding site. Protein acetylation is an important posttranslational modification that regulates a range of cellular processes. It has been reported IRF-1 can undergo acetylation that might be related to the regulation of IRF-1 activation. Our study revealed that the acetylation of IRF-1 progressed in a time-dependent manner in the GMCs upon sublytic C5b-9 attack. Recently, several studies have reported that all of CBP, p300 and PCAF, as an acetyltransferase, can promote protein acetylation. Hence, the interaction between these acetyltransferases and IRF-1 at the protein level was determined, and the results demonstrated that only p300 was able to interact with IRF-1 in sublytic C5b-9-stimulated GMCs. Multiple evidences support that p300 as a transcription coactivator interacts with numerous transcription factors, and then these proteins act synergistically to increase the expression of their target genes. Our current studies showed that the complex of p300 and IRF-1 could bind to the gene promotor through IRF-1 binding to its element (−65 to −53 nt). Subsequent analysis found that p300 gene silence could suppress IRF-1 acetylation and the binding of IRF-1 to XAF1 gene promotor as well as subsequent XAF1 gene transcription and GMC apoptosis due to sublytic C5b-9 stimulation, suggesting that p300 is essential for IRF-1 acetylation and subsequent increase of IRF-1 activity as a transcription factor. Moreover, two acetylation sites of IRF-1 protein (Lys-39 and Lys-78) within the DNA-binding domain were also revealed. Site mutation of Lys-39 and Lys-78 respectively could reduce IRF-1 acetylation and the binding of IRF-1 to XAF1 gene promotor and further suppress XAF1 expression and GMC apoptosis upon sublytic C5b-9 attack. One acetylation site of IRF-1 protein (Lys-78) has been reported by Qi , whereas another acetylation site of IRF-1 protein (Lys-39) is a new one that has not been reported. Besides the studies , our experiments manifested that blockade of p300, IRF-1 and XAF1 gene expression inhibited GMC apoptosis and secondary GMC proliferation, as well as urinary protein secretion in the rats with Thy-1N. Meanwhile, p300 gene knockdown not only suppressed IRF-1 acetylation, but also reduced XAF1 expression, and IRF-1 gene knockdown also inhibited XAF1 production in the renal tissues of Thy-1N rats, in agreement with our findings , indicating that p300-mediated IRF-1-dependent XAF1 gene activation was necessary for the development of Thy-1N. It is worth mentioning that GMC apoptosis induced by sublytic C5b-9 was also found to be associated with the expression of other genes such as () and (). Therefore, GMC apoptosis upon sublytic C5b-9 stimulation in Thy-1N rats might require synergetic activation of these apoptosis-related genes. Further studies need to be carried out to determine the relationship among these genes. In summary, in the present studies, the roles of , and genes in GMC apoptosis as well as their relationships and synergistic regulation were examined both in the GMCs triggered by sublytic C5b-9 () and in the renal tissues of Thy-1N rats (). The results indicated that sublytic C5b-9, as a trigger for GMC lesions, could lead to GMC apoptosis of Thy-1N rats through upregulating these genes. In the process, sublytic C5b-9 could induce p300 and IRF-1 expression simultaneously. The IRF-1 activation of gene and its regulation via p300-dependent IRF-1 acetylation might play a vital role in mediating GMC apoptosis. Monoclonal antibodies (Abs) against IRF-1 (sc-640), C5b-9 (sc-58935), His (sc-53073) and Thy-1 antigen (OX-7, sc-53116) as well as polyclonal Abs against PCAF (sc-8999) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal Abs against XAF1 (ab17204) and CBP (ab32646) and a monoclonal Ab against p300 (ab3164) were purchased from Abcam (Cambridge, UK). Monoclonal Abs against acetylated-Lysine, K63-linkage specific polyubiquitin and -actin and polyclonal Abs against K48-linkage specific polyubiquitin as well as HRP-conjugated anti-rabbit and anti-mouse IgG as well as anti-rabbit IgG (Conformation Specific, 5127) were purchased from Cell Signaling Technology (Danvers, MA, USA). Cy5-linked anti-mouse IgG (115–175–071) was from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Enhanced chemiluminescence (ECL) western blotting substrate and co-IP kit and RIPA lysis buffer were supplied by Thermo (Fremont, CA, USA). The ChIP assay kit was from Millipore (Bedford, MA, USA). The vector of pcDNA3.1, Lipofectamine 2000 and Neon transfection system were purchased from Invitrogen (Carlsbad, CA, USA). The incision enzymes HI, dIII, I and T4 DNA were from NEB (Ipswich, Suffolk, UK). The expression plasmids of pEGFP-N1 were supplied by Clontech (Mountain View, CA, USA). pGL3-basic vector and pRL-SV40 luciferase reporter were purchased from Promega (Madison, WI, USA). The shRNA expression plasmids of pGCsi.U6.neo.GFP were from Genechem (Shanghai, China). A site-directed mutagenesis kit was from Agilent Technologies-Stratagene (Santa Clara, CA, USA). QIAprep spin miniprep kit was obtained from Biomiga (San Diego, CA, USA). An annexin V-APC/PI kit was purchased from Bender MedSystems (Vienna, Austria). A TUNEL staining kit was from Roche (Basel, Switzerland). SD rats were from B&K Universal Ltd (Shanghai, China). An institutional approval for the animal study protocol was obtained. Rat GMC and 293T were provided by China Centre for Type Culture Collection (Wuhan, China) and American Tissue Culture Collection (Manassas, VA, USA), respectively. Normal serum (NS) from 10 healthy adult donors and HIS was obtained by incubating NS at 56°C for 30 min. Human complement C6-deficient serum (C6DS) was obtained from Sigma (St. Louis, MO, USA). Purified human complement C6 was from Sino Biological Inc. (Beijing, China). Rabbit polyclonal Abs against Thy-1 antigen (anti-Thy-1 Ab) were prepared according to previously published procedures. The plasmids of pEGFP-N1/XAF1 (pXAF1) were constructed by inserting the ORF of rat XAF1 (XM_001053682) cDNA into pEGFP-N1. The gene was amplified by polymerase chain reaction (PCR) from cDNA of rat GMCs. The PCR products and pcDNA3.1 vector were digested with HI and dIII, and then ligated using T4 DNA ligase. Here, rattus IRF-1 (NM_012591.1) expression plasmids (pcDNA3.1/IRF-1) have been constructed in our previous study. His-tag was added to the 3′ of IRF-1 by PCR to obtain pcDNA3.1/IRF-1-His. IRF-1 mutants (C29A, C39A, C50A, C66A, C70A, C75A and C78A) were generated by using a site-directed mutagenesis kit according to the manufacturer's procedures with above-mentioned wild-type pcDNA3.1/IRF-1-His as the template. To silence rat and genes, different shRNA sequences against the mRNA of p300 (XM_001076610) and XAF1 (XM_001053682) were designed. The plasmids of p300 shRNA (shp300) and XAF1 shRNA (shXAF1) were constructed by using pGCsi.U6.neo.GFP, and the most effective shRNA expression plasmids were chosen for further experiments. Meanwhile, scrambled control shRNA (shCTR) expression plasmids were produced as a negative control. Here, rattus IRF-1 (NM_012591.1) shRNA expression plasmids (shIRF-1) have been constructed in our previous study. Lentiviral p300 shRNA (Lv-shp300), lentiviral IRF-1 shRNA (Lv-shIRF-1), lentiviral XAF1 shRNA (Lv-shXAF1) and lentiviral control shRNA (Lv-shCTR) were purchased from GenePharma (Shanghai, China). The shRNA oligonucleotide sequences used to knock down the expression of , and genes are the same as the above-mentioned most effective shRNAs against these genes as follows: shp300, 5′-CAGCTCTATAGAGCGAGCTTA-3′ shIRF-1, 5′-CAGCTCTACTCTGCCTGATGA-3′ shXAF1, 5′-CAGCAGACCAAGGAAAGCCAA-3′ and shCTR, 5′-TTCTCCGAACGTGTCACGT-3′. The pGL3-XAF1-full-length (pGL3-XAF1-FL) was constructed by inserting the 1.657 kb rat XAF1 promoter (−1496 to +160 nt) into pGL3-basic vector. This construct contains nucleotides 58, 599, 855−58, 601, 510 from the reference genomic sequence NC_005109.2. To determine the minimal XAF1 promoter sequence required for constitutive and inducible activity, we constructed the following promoter deletion fragments by PCR and cloned them into the same reporter vector:−554 to +160, −337 to +160, −47 to +160 and −14 to +160 nt. Specific primers for full-length and different deletion fragments are listed in . pGL3-XAF1 mutant (ACTTTCGGTTTTG→ACTCTCAGTCTTG, pGL3-XAF1) was also generated using a site-directed mutagenesis kit according to the manufacturer's procedures with above-mentioned wild-type pGL3-XAF1 full-length (pGL3-XAF1-FL) as the template. Rat GMCs were cultured in MEM as previously described. The concentrations of Thy-1 Ab and complement were 5% Thy-1 Ab and 4% NHS respectively, with <5% lactate dehydrogenase (LDH) release () as previously reported. To demonstrate whether the response was due to sublytic C5b-9 (5% Thy-1 Ab+4% NHS), GMCs were treated with 5% Thy-1 Ab, 5% Thy-1 Ab+4% HIS, 5% Thy-1 Ab+4% C6DS, 5% Thy-1 Ab+4% C6DS+C6 (0.5 or 2 mg/l) and 5% Thy-1 Ab+4% C6DS+PBS. Adding C6 back to C6DS could recover its ability to induce LDH release from GMCs (). Moreover, 293T cells were cultured in Dulbecco's MEM (DMEM) supplemented with 10% FBS. GMCs were transfected with corresponding plasmids by using Neon transfection system. The 293T cells were transfected using Lipofectamine 2000 according to the manufacturer's instructions. The transfection efficiency was examined by the fluorescence of green fluorescence protein (GFP; ) or the expression of corresponding protein (). First, male SD rats (180–200 g) were randomly divided into two groups (=6 in each time point/group). Thy-1N group: rats were given Thy-1 Ab (0.75 ml per 100 g) by a single i.p. injection. NS group: rats were injected i.p. with normal rabbit serum (0.75 ml per 100 g). Rat renal cortexes were obtained by killing at 0, 1, 2, 3, 6 and 12 h, and were then examined for the expression of p300, IRF-1 and XAF1, the interaction between p300 and IRF-1, as well as the acetylation of IRF-1 by immunoblot (IB) and co-IP. To confirm the roles of p300, IRF-1 and XAF1 in apoptotic and proliferative changes of Thy-1N rats, male SD rats (180–200 g) were divided into six groups (=6–8 in each time point/group), namely: NS, Thy-1N, Lv-shp300+Thy-1N, Lv-shIRF-1+Thy-1N, Lv-shXAF1+Thy-1N and Lv-shCTR+Thy-1N. The rats belonging to NS and Thy-1N groups were treated with the above-mentioned methods. For 3–6 groups, the lentiviral shRNA (Lv-shRNA) was transfected into rat kidney via renal artery perfusion followed by renal veins occlusion for 10 min immediately. After 4 days, Thy-1N was induced as mentioned above. Rat renal cortexes were then collected by killing at 3 h and 6 h and on day 7 after Thy-1 Ab or NS injection. GFP expression was observed to define the efficiency of transferring Lv-shRNA into rat kidneys (), and the efficiency of gene silencing was evaluated by IB assay. The pathological changes of GMC were determined by light microscopy (LM) and EM. Parts of renal tissues were also examined by using IB and co-IP analysis for the expression of corresponding genes including their interaction at protein level. Cultured GMCs or 293T and rat renal tissues were lysed using RIPA lysis buffer. Equal amounts (40 g/lane) of protein were subjected to SDS-PAGE gel respectively. IB analysis was performed as described previously. -Actin was used as an internal control of protein loading and the relative protein level in each group was expressed relative to control group. Each sample was assayed in triplicate. A total of 400 g of extract prepared from GMCs, 293T or rat renal tissues was mixed with 40 l protein G-Sepharose beads in co-IP assay buffer, incubated for 2 h and centrifuged for 2 min. The recovered supernatant was incubated with the corresponding Abs (2 g, preimmune IgG as a control reaction) at 4°C overnight. Then, 40 l of protein G-Sepharose beads was added, and the incubation was continued for 2 h. Protein G-precipitated protein complex was recovered by centrifugation and harvested beads resuspended in 30 l of 2 × SDS PAGE sample buffer were boiled for 5 min. The samples were then analyzed by IB assay with specific Abs. A 40 g aliquot of whole-cell extract (WCE) was used as an input control. 5 × 10 GMCs were resuspended in binding buffer containing Annexin V–APC and propidium iodide. The samples were analyzed on a FACScan flow cytometer (BD, Franklin Lakes, NJ, USA). The percentage of apoptotic cells in a 10 000 cell cohort was determined by flow cytometry. TUNEL staining (TMR-labeled) was used to label the cells (5 × 10 GMCs) at 6 h after sublytic C5b-9 stimulation. Subsequently, flow cytometry analysis was performed to detect the cellular apoptosis. In addition, both TUNEL staining (FITC-labeled) and the Ab against C5b-9 (adding Cy3-labeled secondary Ab) were used to label the cells (5 × 10 GMCs) at 6 h after sublytic C5b-9 stimulation. Flow cytometry analysis was then performed to detect the cellular apoptosis and C5b-9 deposits on GMC membrane. The activity of full-length, different deletion fragments and mutant gene promotor in the GMCs stimulated with sublytic C5b-9 or overexpression of IRF-1 was detected by luciferase reporter assay. The luciferase activity was measured as mentioned previously. ChIP was done by using Abs against IRF-1 and His, and preimmune mouse IgG, respectively, as mentioned previously. A proximal region in promotor (−169 to −19 nt) was amplified from the immunoprecipitated chromatin by PCR using a pair of primers (sense, 5′-GAATGGCCTTCTGGGAGTAT-3′ and antisense, 5′-CTTCGGTGGAGTTTCTGTTT-3′). Re-ChIP was performed by using an Ab against p300 and preimmune mouse IgG respectively. The proximal region in gene promotor (−169 to −19 nt) was amplified from the products of re-ChIP through PCR by using the above-mentioned primers, and the results were normalized to input. For LM, paraffin-embedded renal histological sections (4 m) of rats on day 7 after nephritis induction were stained with H&E, and 100 glomerular cross-sections from each rat were examined according to the same method. For EM, ultrathin sections of renal tissues were stained with uranyl acetate and lead citrate, and the ultrastructural changes were observed. To detect GMC proliferation, frozen sections (4 m) of rat renal tissues were stained with an Ab to Thy-1 antigen (OX-7, as a marker of GMCs). Then, the sections were incubated with corresponding Cy5-conjugated secondary Ab. In addition, the number of glomerular OX-7-positive cells was counted in a double-blinded manner by two independent observers counting 100 glomerular cross-sections from each rat as described previously. Frozen sections (4 m) of rat renal tissues were treated with proteinase K, and incubated with 50 l reaction mixture of TUNEL for 60 min. The number of TUNEL-positive nuclei in 100 glomerular cross-sections was counted in a double-blinded manner under fluorescence microscopy. Rat urine in different groups was collected on day 7 after the corresponding treatments. The contents of urinary protein (mg per 24 h) were measured by the total protein UC FS (DiaSys Diagnostic Systems, Holzheim, Germany). Each sample was assayed in triplicate. Data are presented as means±S.E. One-way ANOVA was used to determine significant differences among groups. Where significant differences were found, individual comparisons were made between groups using the -statistic and adjusting the critical value according to the Bonferroni method. <0.05 was considered significant.

An NIH3T3 mouse fibroblast cell line expressing GFP-tagged histone H3 was generated to facilitate the imaging of mitosis in living cells. The NIH3T3/H2B-GFP cells were transfected with an siRNA library targeting the mouse kinome complement (also included some phosphotransferases and protein kinase regulators). Each of the 588 genes in the library was targeted by three siRNAs (). We used a strategy that involved addition of a DNA-damaging agent (Adriamycin) at 2.5 h before analysis. Activation of the G DNA damage checkpoint prevented entry into mitosis but not the progression of cells already in mitosis. Therefore, while the mitotic population in siRNA transfection not affecting mitosis was effectively reduced to nil, it remained elevated for siRNAs that resulted in a delay in mitotic progression. Using this approach, 20 candidates were found to increase the mitotic index above 2 × S.D. of the mean (). Although the screen should also reveal kinases important for the DNA damage checkpoint, we found that most of the candidate siRNAs increased the mitotic index even in the absence of DNA damage (), suggesting that these kinases were involved in regulating mitosis instead of the DNA damage checkpoint. Intriguingly, the protein kinases identified in our screen has only limited overlap with those identified in a pioneering kinome RNAi screen using S2 cells. Four protein kinases were found to be common targets for mitotic regulation in both mouse and cells (). As expected, the PLK1 (polo in ) was a potent mitotic regulator in both mouse and models. Other common candidates include Sik3 (CG15072), Scyl1 (CG1951), and Tbk1 (ik2) ( proteins are indicated in the brackets). Given that Sik3 was found to be important for mitosis in both mouse and cells, and that other AMPK-related kinases (such as Brsk2; ) were also identified in both screens, we further characterized the role of Sik3 in mitosis in this study. SIK3 (salt-inducible kinase 3, also called QSK) is a member of AMPK family. As the original screen involved the use of a mixture of siRNAs against each kinase, we first verified the results for Sik3 using the different siRNAs individually. shows that transfection with the three Sik3 siRNAs increased the mitotic index in NIH3T3 fibroblasts irrespective of the presence or absence of Adriamycin-mediated DNA damage, supporting the specificity of the mitotic effects for Sik3. We next investigated if the mitotic effects of SIK3 are conserved in human cells. shows that SIK3 was expressed in most cell lines we examined, including several relatively normal (fibroblasts and immortalized normal epithelial cells from liver and nasopharynx) and cancer cell lines (from liver and nasopharynx, except C666-1). To evaluate if the expression of SIK3 affects mitosis in human cells, HeLa cells were transfected with three siRNAs targeting different regions of human . All three siRNAs were able to downregulate SIK3 protein effectively (). While the results from siSIK3 no. 1 were mainly used in this paper, similar results were obtained with at least one other siSIK3s. As in mouse fibroblasts, knockdown of SIK3 in HeLa cells increased the mitotic index by 1.5–2-fold (). Similar results were obtained using H1299 cells (), indicating that the effect was not only specific for HeLa cells. Although the increase in mitotic index was less impressive than after depletion of the classic mitotic kinase PLK1 (), it was nevertheless significant and reproducible. We further analyzed the mitotic population using bivariate flow cytometry to measure DNA contents and histone H3 phosphorylation together. shows that more siSIK3-transfected cells displayed phosphorylated histone H3 than in control cells, verifying that depletion of SIK3 increased the mitotic population. To demonstrate more directly the increase in mitotic duration, HeLa cells expressing histone H2B-GFP were transfected with siSIK3 and followed at single-cell levels with time-lapse microscopy. The average duration of mitosis was increased following siSIK3 transfection () (the complete fates of individual cells are shown in ). The increase in mitotic duration was reproducible with two siSIK3s (), both of which were able to reduce the expression of endogenous SIK3 (). Finally, siSIK3 also extended mitosis in another cell line (Hep3B), excluding the possibility that the effect was only specific for HeLa cells (). As SIK3 was originally identified in our screen in the presence of DNA damage (), we also verified that siSIK3 did not abrogate the DNA damage checkpoint in human cells. Exposing HeLa cells to ionizing radiation abolished mitotic entry (). While incubation with a CHK1 inhibitor (UCN-01) overrode the checkpoint and triggered precocious mitotic entry as expected, depletion of SIK3 did not affect the cell cycle arrest. Collectively, these data show that downregulation of SIK3 resulted in an increase in mitotic duration in both mouse and human cells but did not affect the DNA damage checkpoint. More detailed analysis revealed that metaphase plate could form normally after SIK3 depletion. However, onset of anaphase was significantly delayed. The metaphase plate typically flipped and rotated for an extensive period of time before anaphase (). Another example is shown in . To examine the mitotic defects in more synchronized population, cells were trapped at prometaphase with nocodazole after they were first released from a double thymidine block. Flow cytometry revealed that after they were released from the nocodazole-mediated block, control cells were in at =2 h. In contrast, entry into G was delayed in siSIK3-expressing cells (). In agreement with this, degradation of several anaphase-promoting complex/cyclosome (APC/C) targets, including cyclin B1 and securin, as well as dephosphorylation of histone H3, was delayed in siSIK3-transfected cells (). Finer temporal resolution using time-lapse microscopy confirmed the delay of anaphase onset (). To determine if mitotic entry is also affected by siSIK3, cells were synchronized at the G phase with the CDK1 inhibitor RO3306 and released into mitosis. We found that while the timing of entry into mitosis was not affected, anaphase onset was delayed in the absence of SIK3 (). Taken together, these results indicate that mitotic exit but not mitotic entry was delayed in the absence of SIK3. Given that depletion of SIK3 results in aberrant mitotic exit, we next investigated if the efficacy of antimitotic chemicals can be altered by SIK3. Classic spindle poisons such as nocodazole (an inhibitor of microtubule polymerization) activate the spindle-assembly checkpoint and trap cells in mitosis. A relatively low concentration of nocodazole was used in these experiments with the aim of delaying mitosis without inducing extensive mitotic cell death. As expected, mitosis was extended after treatment with siSIK3 or nocodazole individually (). When siSIK3 and nocodazole were applied together, the cells were trapped in mitosis for an extensive period of time before undergoing apoptosis. Similar to siSIK3 alone, the metaphase–anaphase transition was markedly delayed when siSIK3 and nocodazole were added together ( and ). The stimulation of spindle poison-mediated cell death by siSIK3 was not limited to HeLa cells, as similar results were found with the colon carcinoma HCT116 cells (). Depletion of SIK3 also enhanced mitotic cell death following challenge with Taxol (an inhibitor of microtubule depolymerization), indicating that siSIK3 enhanced the effects of multiple spindle poisons (). Taken together, these results suggest that downregulation of SIK3 can sensitize cells to mitotic arrest and cell death induced by spindle poisons. The shortcomings of spindle poisons as chemotherapeutic agents include neuropathy and drug resistance. Newer generations of antimitotic drugs are developed to target specific mitotic components such as Aurora kinases. Aurora A (AURKA) is a centrosomal protein that regulates the maturation and separation of centrosomes and formation of bipolar spindle; Aurora B (AURKB) is a component of the chromosomal passenger complex that mainly regulates late mitotic events. We used a small-molecule inhibitor Alisertib (also called MLN8237) that inhibits both AURKA and AURKB, as well as newer generations of specific inhibitors of AURKA (MK-5108, also called VX-689) and AURKB (Barasertib, also called AZD1152-HQPA). As complete pharmacological inhibition of AURKA and AURKB triggers an early mitotic arrest and mitotic slippage, respectively, cells were treated with relatively low concentrations of the drugs that were insufficient to trigger these effects on their own. shows that depletion of SIK3 significantly enhanced the G/M delay induced by these Aurora inhibitors. The main effect of AURKB inhibition is mitotic slippage, encompassing of premature mitotic exit without sister chromatid separation. Live-cell imaging indicated that the increase in G/M population after codepletion of SIK3 was caused by an exacerbation of mitotic slippage ( and ). In contrast to inhibition of Aurora kinases, inhibition of the PLK1 induces a metaphase arrest. We found that siSIK3 collaborated with the PLK1 inhibitor BI-2536 in promoting G/M delay (). Live-cell imaging indicated that the cells were trapped in mitosis before undergoing apoptosis (). As both siSIK3 and BI-2536 individually delayed metaphase–anaphase transition, it is not surprising that cells treated with both reagents were arrested at metaphase ( and ). Collectively, these data indicate that the mitotic defects and cytotoxicity induced by small-molecule inhibitors of AURKA, AURKB, and PLK1 are substantially enhanced in the absence of SIK3. Eg5 (also called kinesin family member 11, KIF11) is a plus-end-directed microtubule motor of the kinesin-5 family. Eg5 is implicated in various mitotic microtubule functions, including microtubule crosslinking, antiparallel microtubule sliding, and bipolar spindle formation, ensuring the fidelity of chromosome segregation. We found that the effects of pharmacological inhibition of Eg5 could be enhanced in the absence of SIK3. While depletion of SIK3 or a relatively low concentration of the Eg5 inhibitor SB743921 alone did not affect cell cycle distribution, treatment of siSIK3 and SB743921 together triggered a significant G/M delay (). Live-cell imaging revealed that the cells were arrested in mitosis before undergoing apoptosis (). Notably, inhibition of Eg5 or SIK3 produced rather different effects on mitosis. While SB743921 delayed early mitosis with monopolar spindle, siSIK3 delayed mitosis mainly after the formation of metaphase plate. Interestingly, when both SB743921 and siSIK3 were added together, the cells were trapped in early mitosis without forming the metaphase plate ( and ). This suggested that the effect of Eg5 inhibition was exacerbated in the absence of SIK3. Development of drug resistance is one of the major obstacles of anticancer therapies. To generate SB743921-resistant cells, we propagated HeLa cells in medium containing progressive increasing concentrations of SB743921. shows that while the parental HeLa cells could be arrested at mitosis with 10 nM of SB743921, the SB743921-resistant cells were unaffected by the same concentration of SB743921 (two independent clones are shown). Significantly, after SIK3 was depleted with siSIK3, both drug-resistant clones could be arrested at mitosis with SB743921. As only a few substrates of SIK3 are known, the molecular basis of how SIK3 regulates mitosis remains to be defined. As class IIa HDACs can be phosphorylated by SIK3, one possibility is that SIK3 exerts its effects on mitosis indirectly through HDACs. For example, downregulation of HDAC4 with RNAi in HeLa cells was shown to induce mitotic arrest; and small chemical inhibitors of HDACs were found to cause a variety of chromosome segregation defects. In this connection, we have tested if SIK3 depletion enhances the mitotic defects of HDAC inhibition. SAHA (also called Vorinostat or Zolinza or MK0683) is an FDA-approved pan-HDAC inhibitor for cutaneous T-cell lymphoma. Incubation of HeLa cells with 1 nM of SAHA resulted in a delay of mitotic exit. However, depletion of SIK3 did not further delay mitosis induced by SAHA (), indicating that depletion of SIK3 did not act synergistically with all antimitotic drugs. These data are also consistent with the model that SIK3 may function in the same pathway as HDAC in disrupting mitosis. Using a simple kinome-wide RNAi screen, we found that downregulation of Sik3 increased the mitotic population in mouse fibroblasts (). We characterized Sik3 in particular because downregulation of the homolog (CG15072) was also reported to affect mitosis, in particular on spindle morphology, suggesting a possible conservation of mitotic functions for this kinase. Another determining factor is that downregulation of several AMPK-related proteins, including SNF1A in and BRSK2 in mouse () and human cells (data not shown), also affected mitosis. Interestingly, downregulation of another AMPK-related protein BRSK2 also increased the mitotic duration in mouse and human cells (). Given that both SIK3 and BRSK2 are downstream targets of LKB1, it is possible that LKB1-deficient cells are also sensitized to antimitotic drugs. However, the effect will probably be complicated by the fact that LKB1 is a master regulator of 13 AMPK-related kinases. We first confirmed that SIK3 could be effectively downregulated with siRNAs in human cells (). Several methods including mitotic index count (), histone H3 phosphorylation (), and live-cell imaging () showed that depletion of SIK3 increased the mitotic duration in human cell lines. Live-cell imaging analysis also indicated that mitotic exit was delayed in the absence of SIK3 (). A broad implication of this study is that if specific inhibitors of SIK3 can be developed, they may be able to act synergistically with conventional antimitotic drugs. Inactivation of SIK3 may allow lower doses of antimitotic drugs to be used, and thus may benefit patients by lowering the side effects of the drugs. As a test of principle, depletion of SIK3 enhanced mitotic arrest and cell death induced by classic spindle poisons, including nocodazole and Taxol (). Downregulation of SIK3 also promoted mitotic arrest induced by newer classes of antimitotic drugs, including those targeting AURKA, AURKB, PLK1 (), and Eg5 (). Remarkably, siSIK3 could also restore drug sensitivity of cells resistant to the Eg5 inhibitor SB743921 (). Another possibility is that cancer cells with low expression of SIK3 may be more sensitive to antimitotic drugs. It is interesting that while siSIK3 delayed mitosis mainly after the metaphase was formed, treatment with siSIK3 and antimitotic chemicals together did not necessarily delay cells after metaphase. For example, treatment of cells with siSIK3 and the Eg5 inhibitor SB743921 together arrested cells in early mitosis similar to treatment with SB743921 alone (), suggesting that SIK3 may have multiple roles in mitosis. Interestingly, siSIK3 did not act synergistically with HDAC inhibitors including SAHA () and an HDAC6-specific inhibitor (data not shown). Although this is consistent with a model that SIK3 may act through HDACs to regulate mitosis, more refined approaches will be required in the future to test this hypothesis. As repression of SIK3 alone resulted in a mild extension of mitosis () rather than the extensive cell death induced by inhibition of classic mitotic kinases such as PLK1 (), SIK3 probably may not be a very effective stand-alone target. On the other hand, it can be argued that inhibition of essential mitotic kinases such as PLK1 is too toxic for normal cells. Less critical mitotic kinases such as SIK3 may actually provide better opportunities for combined anticancer therapies. One surprising finding of the present study is the small number of genes that overlap with the mitotic kinome using cells. Of the 60 genes that affect some aspects of mitosis in cells, only four homologs were also found to affect the mitotic index in mouse cells (). Bettencourt-Dias also specifically examined the mitotic index and found six candidates (polo, for, fray, gw1, CG7156, inaC) that increase the mitotic index to outside 90% CI. Among these, only the polo kinase homolog was also found in our screen. It is reasonable to surmise a difference of mitotic regulators between and mammalian cells. While the mouse kinome (540 genes) is similar in size to the human kinome (518 genes), the kinome is considerably smaller (228 genes). Furthermore, many of the mouse kinome siRNAs probably affected aspects of mitotic regulation but did not significantly influence mitotic duration (which was the only parameter designed to be addressed in this study). Another more trivial explanation that is inherited in most RNAi screens is the unpredictable differences in the efficiency and specificity of RNAi. Indeed, although several established mitotic kinases, such as PLK1, was identified in our screen, some notable ones including AURKA was missing. As we have subsequently found using human cell lines, knockdown of AURKA also only marginally extended mitosis (our unpublished data), suggesting that it is perhaps challenging to deplete AURKA to a level that induces mitotic delays. However, knockdown of other established mitotic kinases is expected not to increase the mitotic index, as they would either prevent entry into mitosis (e.g. CDK1) or promote mitotic slippage (e.g. AURKB). Finally, several candidates we identified have already been linked to some aspects of mitotic control, including Erbb3, Lats2, Pim1, Scyl1, and Stk38l. It would be important to corroborate these and other novel candidates and establish if they are promising antimitotic targets. In conclusion, our whole-kinome screening revealed a number of potentially interesting mitotic kinases in mammalian cells. Downregulation of SIK3, one of the conserved mitotic kinases in mammalian and cells, delayed mitotic exit and increased the overall mitotic duration. Furthermore, depletion of SIK3 enhanced the mitotic delay and cytotoxicity induced by a number of antimitotic chemicals, highlighting the potential of SIK3 as an antimitotic drug target. All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless stated otherwise. Mouse kinase RNAi collection was obtained from Life Technologies (Carlsbad, CA, USA). Each gene in the library was targeted by three different Stealth siRNAs. The Stealth siRNA was diluted 1 : 10 in Opti-MEM Reduced Serum Medium and transferred to 96-well plates (10 l each well). The Stealth siRNA was mixed with 0.2 l of Lipofectamine RNAiMAX reagent and 10 l of Opti-MEM Reduced Serum Medium, and incubated at 25 °C for 20 min. NIH3T3/H2B-GFP cells were then added to the wells (5 × 10 cells in 100 l of medium). The plates were incubated at 37 °C in a humidified incubator at 37 °C in 5% CO for 24 h. The cells were then incubated with 0.2 g/ml of Adriamycin for 2.5 h. Images of the cells were then captured automatically using a fluorescent microscopy. For each well, bright-field and fluorescent images from nine random positions were captured. The mitotic index of each transfection was scored independently by three individuals. GFP-HA-SIK3 in pEGFP-C1 was obtained from the University of Dundee (Cat no. DU1338). It was cut with I and HI and ligated into pGEX-KG (GE Healthcare, Amersham, UK) to generate GST-HA-SIK3 in pGEX-KG. GFP-HA-SIK3 in pEGFP-C1 was cut with HI and ligated into HI-cut and phosphatase-treated pUHD-P3T/PUR to generate FLAG-HA-SIK3 in pUHD-P3T/PUR. Hep3B, HepG2, and IMR90 were obtained from the American Type Culture Collection (Manassas, VA, USA). The HeLa used in this study was a clone that expressed the tTA tetracycline repressor chimera. Nasopharyngeal carcinoma cell lines C666-1, CNE2, HNE1, and HONE1 were obtained from NPC AoE Cell Line Repository (The University of Hong Kong, Pokfulam, Hong Kong). Normal liver cells LO2 and MIHA were gifts from Irene Ng (The University of Hong Kong). Cells were propagated in RPMI1640 (for C666-1) or Dulbecco's modified Eagle's medium (for other cell lines) supplemented with 10% (v/v) calf serum (Life Technologies) (for HeLa) or fetal bovine serum (Life Technologies) (for other cell lines) and 50 U/ml penicillin streptomycin (Life Technologies). IMR90 cells were cultured in minimum essential medium (Eagle's) (Life Technologies) supplemented with 2 × essential and non-essential amino acid and 15% uninactivated fetal bovine serum. Telomerase-immortalized nasopharyngeal epithelial cell lines NP361, NP460, and NP550 (gifts from George Tsao, The University of Hong Kong) were propagated in keratinocyte serum-free medium (Life Technologies) supplemented with 50% (v/v) Epilife. Cells were cultured in humidified incubators at 37 °C in 5% CO. H1299, HCT116, HeLa, Hep3B, NIH3T3 that stably expressed histone H2B-GFP were used for live-cell imaging. Unless stated otherwise, cells were treated with the following reagents at the indicated final concentration: Adriamycin (0.2 g/ml), Alisertib (Selleck Chemicals, Houston, TX, USA) (62.5 nM), Barasertib (Selleck Chemicals) (12.5 nM), MK-5108 (Selleck Chemicals) (125 nM), BI-2536 (1.25 nM), nocodazole (0.1 g/ml), RO-3306 (Alexis) (10 M), SB743921 (Selleck Chemicals) (10 nM), SAHA (Selleck Chemicals) (1 nM), Taxol (20 ng/ml), thymidine (2 mM), and UCN-01 (100 nM). Synchronization at mitosis was performed by first releasing cells from a double thymidine block for 6 h before adding nocodazole for another 6 h. Mitotic cells were then collected by mechanical shake off and released by washing three times with PBS before replating in fresh medium. To release cells from the G phase, HeLa were first treated with 10 M of RO-3306 for 18 h. The cells were washed two times with PBS and replated in fresh medium. Cell-free extracts were prepared as described previously. HeLa cells were cultured in the presence of 2.5 nM of SB743921 for 2 weeks. The cells were then subcultured with limited dilution in 5 nM of SB743921 for three weeks. Individual colonies were isolated and tested for resistance to 10 nM of SB743921-induced G/M arrest with flow cytometry. The cells were subsequently propagated in the absence of SB743921. IR was delivered with a caesium source from an MDS Nordion (Ottawa, ON, Canada) Gammacell 1000 Elite Irradiator. Cells were transfected with siRNA by Lipofectamine RNAiMAX (Life Technologies). The following siRNAs were obtained from the indicated suppliers: 5′-UUCUUCGAACGUGUCACGUTT-3′ or 5′-ACGUGACACGUUCGGAGAATT-3′ (control siRNA; GenePharma, Shanghai, China); 5′-GCUGCACAAGAGGAGGAAATT-3′ (siPLK1; RiboBio, Guangzhou, China); 5′-CCACAGAAUGUGAGCAUUUTT-3′ (siSIK3 no. 1; RiboBio); 5′-CCUGAAGCAUUGGUGCGCUAUUUGU-3′ (siSIK3 no. 2; Life Technologies; Stealth siRNA); CCAAAUUGCCCUUGUGUUUTT (siSIK3 no. 3; RiboBio). Unless stated otherwise, siSIK3 no. 1 was used. Flow cytometry analysis after propidium iodide staining was performed as described previously. Bivariate analysis of DNA content and histone H3 phosphorylation was performed as described previously. The setup and conditions of time-lapse microscopy of living cells were as described previously. Antibodies against -actin, CDK1, cyclin B1, and FLAG tag were obtained from sources as described previously. Antibodies against phospho-histone H3 and securin (sc-56207) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GST-HA-tagged SIK3 was expressed in bacteria and purified with GSH-agarose chromatography. Rabbit antibodies against SIK3 were raised against a bacterially expressed GST-HA-tagged SIK3 protein. Immunoblotting and immunoprecipitation were performed as described.

To assess the role of Met in cardiac protection from hypoxia injury, we studied the H9c2 rat cardiomyoblasts. These cells express cardiac markers Connexin43 and Troponin-I (). First, we demonstrated using flow cytometry that the endogenous Met receptor is present at the cell surface (). Moreover, immunofluorescence (IF) analysis showed that Met is localized both at the plasma membrane and intracellularly (). Second, we stimulated the Met tyrosine kinase activity by using the HGF ligand or two mAbs, DN30 and DO24, which we previously showed to be partial and full agonist of the Met receptor, respectively. All three Met agonists induced phosphorylation of Met (), activation of its principal effector Gab1 () and stimulation of the main signalling pathways downstream Met (that is, Akt, Erk and mTOR, data not shown). In a classical ‘wound-healing' assay, treatment with either mAb induced cells to migrate and to cover the wounded area at an extent similar to that induced by HGF (). As a control, treatment with an irrelevant (anti-VSV-G) antibody had no effect on cell migration (). CoCl mimics the hypoxic/ischaemic condition and is a simple and validated tool to study the molecular mechanisms driven by hypoxia. Indeed, after CoCl treatment, HIF-1 protein expression significantly increased, starting from 3 h (), whereas mRNA level was super-induced at later time (starting from 12 h; ). To further assess the involvement of HIF-1 in CoCl response, we analysed the expression of known typical HIF-1 target genes, such as , , and itself. In time course experiments, a significant increase in mRNA levels was observed at 3 h for Glut1 and at 12 h for CAXII and Met (). Induction of GAPDH protein was detected at 24 h (). To investigate the CoCl effect on cell viability, we performed the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cardiomyoblasts treated with CoCl showed a significant reduction in cell survival in a dose- and time-dependent manner (). Moreover, CoCl decreased prosurvival pathways (that is, Akt, Erk and mTOR), which are stimulated by HGF-activated Met receptor (). Consistently, CoCl treatment significantly impaired the signalling response to HGF survival factor (). As it is known that hypoxia produces cell death through mitochondrial membrane permeabilization, we analysed the apoptotic response. We found that treatment with CoCl leads to a significant increase in the number of pyknotic nuclei, as compared with untreated cells (); this apoptotic event was mediated by the caspase-dependent pathway of cell death, as it was affected by the specific inhibitor zVAD (). We also analysed the ratio between Bax and Bcl-2 protein levels, known to be crucial in caspase-mediated apoptosis. The treatment with CoCl increased the pro-apoptotic Bax and decreased the anti-apoptotic Bcl-2 at early (12 h) and late (48 h) time points, resulting in a significant enhancement of Bax/Bcl-2 ratio (). Consistent with the activation of the canonical mitochondrial pathway of apoptosis, the ratio between the cleaved and the total caspase-3 protein was significantly raised in cells treated for 48 h (). Finally, the ratio between cleaved and uncleaved PARP, one of the substrates of caspase-3, significantly increased at 72 and 96 h (). To restore the prosurvival signalling and protect cardiomyoblasts from apoptosis, we concomitantly treated cells with CoCl and Met agonist antibodies or HGF. Met stimulation by either mAb or HGF fully prevented the loss of cell viability in the MTT assay (). The PHA-665752 (PHA), a specific inhibitor of the Met kinase, abolished the prosurvival activity of Met agonist antibodies and HGF; thus, protection was mediated specifically by the Met receptor (). We also demonstrated that the two mAbs, as well as HGF, protected from CoCl-induced reduction of cell number (). As a control the treatment with anti-VSV-G antibody did not produce any effect (). Importantly, DN30, DO24 and HGF prevented the CoCl-induced rise in the number of pyknotic nuclei (). Accordingly, all above Met agonists prevented caspase-3 activation induced by CoCl (). In our experiments, the apoptosis inhibitor zVAD failed to fully inhibit cell death (). This observation suggested that the caspase-dependent apoptosis only partially contributes to the loss of cell viability induced by CoCl. It has been reported that prolonged stimulation of the HIF-1 pathway produces excess autophagy, which may also result in cell death. Thus, we evaluated the induction of pro-autophagic mechanisms in cells treated for different lengths of time with CoCl. We performed an mRNA expression analysis of Redd1 and Bnip3, known transcriptional targets of HIF-1, which promote autophagy by inhibition of mTOR. Both genes were significantly induced by CoCl treatment starting from 12 h and were maintained at high levels for 48 h (). Redd1 and Bnip3 protein levels were also increased in presence of CoCl (). Another HIF-1-dependent mechanism of autophagy induction is mediated by AMPK phosphorylation and activation. We found that CoCl treatment induced a significant increase in AMPK phosphorylation, relative to untreated cells (). It is known that P-AMPK is also an inhibitory molecule of the mTOR pathway; thus, we analysed the phosporylation state of p70S6K and 4EBP1, the two main substrates of mTOR. Phosphorylation levels of both proteins were significantly decreased in a time-dependent manner by CoCl treatment, starting from 24 h (). This result indicates that CoCl treatment induces the inhibition of the mTOR pathway. As negative regulation of mTOR signalling promotes autophagy we analysed some key proteins of different phases of autophagosome formation. Autophagy is positively regulated by Beclin-1, which binds the class III PI3K Vps34, thereby facilitating autophagosome formation. Another phase of autophagy is the elongation of the autophagosome, with generation of the lipidated form of LC3 called LC3II. Finally, p62/SQSTM1 protein is an important marker of the autophagic machinery, as it recognizes ubiquitin and determines the cargo of ubiquitinated substrates in the autophagic–lysosomal pathway. Accordingly, we observed that both Beclin-1 and p62 protein levels were induced in CoCl-treated cells () and that LC3 was processed from the inactive (LC3I) to the active form (LC3II, ) in a time-dependent manner. In addition, the mRNAs of these autophagic proteins were induced in the presence of CoCl for 48 h (). Moreover, IF analysis revealed that CoCl induced LC3 puncta formation, proof of autophagosome maturation (). Finally, we asked whether increased autophagic response could participate into the CoCl-induced loss of cell viability. We co-treated cardiomyoblasts with CoCl and 3-Methyladenine (3-MA), a selective inhibitor of the autophagic initiator PI3K type III. The inhibitor decreased the LC3 puncta formation (B and C) and, importantly, prevented the loss of cell viability and recovered as much as 80% of the basal value (). This last result indicates that, besides apoptosis, the autophagic process is involved in cell death induced by CoCl. The increase in pro-autophagic proteins by hypoxic injury may be due to either enhanced formation or reduced degradation of autophagosomes. Treatment with bafilomycin A1 (Baf), an inhibitor of the vacuolar H ATPase of lysosomes, significantly increased the LC3II/LC3I ratio (), indicating a block on the basal lysosomal-dependent degradation of the autophagosome cargo. H9c2 cells concomitantly treated with CoCl and Baf showed LC3II/LC3I protein levels and LC3 immunofluorescent puncta significantly higher than those observed in cells treated with CoCl alone ( and ). Altogether, these data suggest that CoCl-induced death results from enhanced flux of autophagosome formation rather than from reduced clearance. Treatment with DN30, DO24 or HGF prevented the increase of both LC3II/LC3I ratio and p62 protein levels (). These results demonstrate that Met agonists inhibit the autophagy induced by CoCl. To investigate the effect of HGF stimulation on the autophagic flux we again used the Baf inhibitor. In cell cultures exposed to Baf or Baf+CoCl, the addition of HGF resulted in a significantly lower LC3II/LC3I ratio as compared with the corresponding treatment in the absence of the cytokine (). Lysosomal proteolysis was completely blocked by Baf; thus, an LC3II decrease below the Baf-induced levels indicates that HGF reduces the flux of autophagosome formation. The two Met agonist antibodies also decreased the LC3II/LC3I ratio in cells cultured in the presence of CoCl and Baf (). To identify the protective signalling involved in Met agonist action, we blocked the mTOR pathway known to be the main inhibitor of autophagy. In the presence of temsirolimus, a specific inhibitor of the mTOR complex, the Met agonist-mediated recovery of cell viability upon hypoxic injury was abrogated (). The level of mTOR activation in cultures exposed to CoCl in combination with Met agonists was higher than in cells treated with CoCl alone (). In contrast, the Met agonist antibodies- or HGF-mediated mTOR activation was completely blunted by temsirolimus (). Accordingly, the Met agonist-mediated recovery of LC3II/LC3I ratio from CoCl-induced autophagy was reduced by mTOR pathway inhibition (). Altogether, these results indicate that mTOR activation has a key role in the Met-stimulated protection from autophagy. The cardioprotective action of the two Met agonist antibodies as well as HGF was evaluated under hypoxic environment at 1% of oxygen tension (). First, we showed that hypoxic treatment produced reduction of cell viability (), increase of pycnotic nuclei () and induction of pro-autophagic proteins Redd1 and Bnip3 (). Next, we demonstrated that Met stimulation by the two mAbs as well as HGF under hypoxic conditions ameliorates cell viability (), reduces the number of pyknotic nuclei () and decreases the level of autophagic ratio LC3II/LC3I and p62 (). In conclusion, these results show that Met agonists have anti-apoptotic and anti-autophagic effects also in low oxygen tension conditions. The effect of hypoxia in cardiomyocytes relies on duration and intensity of hypoxic stimulus. Short-term induction of HIF-1 is beneficial, as the injured myocardium metabolically adapts to hypoxic condition. In contrast, long-term stabilization of HIF-1 may be detrimental. Accordingly, we found that sustained hypoxic injury is followed by cardiomyoblast grief. It is known that HIF-1 stabilization sensitizes tumour and neuronal cells to programmed cell death. The mechanisms at the basis of HIF-1 apoptotic effect are not yet well understood but presumably involve Bnip3, p53 and increased production of ROS. All these processes converge on the activation of the intrinsic pathway of apoptosis. We found that treatment with CoCl induced a significant rise of pyknotic nuclei with concomitant increase in the Bax/Bcl-2 ratio followed by activation of caspase-3 cleavage. Blocking caspases through a chemical inhibitor significantly improved cell viability, validating the apoptotic pathway as a relevant factor in the CoCl hypoxic mimicking model. Here we show that CoCl-induced hypoxia – besides apoptosis – is also a strong inducer of autophagy. In fact, chemical hypoxia increases known markers of the autophagic–lysosomal pathway: Beclin-1 (an indicator of autophagy induction), conversion of LC3I to lipidated LC3II (an index of autophagosome abundance) and p62 (a marker of ubiquitinated substrate sequestration and degradation). Moreover, cell death induced by CoCl was blocked by 3-MA, an inhibitor of the class III PI3K Vps34, a canonical initiator of autophagy. The latter experiment indicates that autophagy causes uncontrolled ‘self-cannibalism' in cardiomyoblasts and contributes to cell death. We also enlightened the cross-talk between apoptotic and autophagic pathways, as CoCl decreases Bcl-2 and concomitantly induces Bnip3 and Beclin-1, known triggers of autophagy. In fact, it is reported in the literature that Beclin-1 is inhibited by Bcl-2 and, in turn, Bcl-2 is associated with and inhibited by Bnip3. The main regulator of autophagy is mTOR, a potent sensor and integrator of signals stimulated by growth factors, nutrients and energy status. Under conditions of nutrient availability and normoxia, mTOR is active and phosphorylates ATG13, preventing its association with ULK and formation of the autophagosome. We found that both CoCl and real hypoxia promote upregulation of Bnip3 and Redd1, two different hypoxia-inducible target genes. It is known from the literature that these two proteins inhibit Rheb, a Ras-related small GTPase, and, as a consequence, lead to a negative regulation of mTOR. Bnip3 directly binds and inhibits Rheb, while Redd1 acts through a not-yet-defined mechanism involving TSC1/2, an inhibitor of Rheb. We also demonstrated that CoCl induces phosphorylation and activation of AMPK, another negative regulator of mTOR. HGF has been proposed as a cardioprotective factor. Patient administration of biologically active HGF may however be difficult as it is a large molecule containing a heparin-binding domain sequestered by low affinity–high avidity sites widespread among the extracellular matrix proteoglycans. Moreover, HGF is produced as an inactive precursor that has to be activated after cleavage by specific convertases. Compared with the natural HGF ligand, the mAbs have a major advantage of being easier to be produced and more manageable for the therapeutic formulation. For agonist antibodies, their divalent nature is a benefit as they dimerize and activate the receptors. This property has been scrutinized in detail in the case of anti-Met antibodies in the effort of generating monovalent fully inhibitory Fabs, successfully employed in preclinical cancer therapy. Here we show that either HGF or the Met agonist antibodies DN30 and DO24 protect from caspase-dependent apoptosis as well as autophagy. The protection from autophagy is mediated by activation of mTOR. Consistently, the increase in phospho-p70S6K and phospho-4EBP1 levels and the inhibition of autophagic LC3 lipidation were abrogated by the mTOR inhibitor. It is likely that activation of mTOR is triggered by the PI3K/Akt pathway at different levels, such as the regulation of mTOR/Raptor complex or phosphorylation of TSC2. In addition, Erk inhibits the TSC1/2 complex. Met agonist antibodies represent a new important tool enriching the therapeutic arsenal to manage cardiac diseases. Apoptosis is harmful in the reperfusion phase of myocardial infarction. Furthermore, apoptosis is involved also in a wide spectrum of inherited cardiomyopathies, myocarditis, transplant rejection and heart failure. Met agonist antibodies prove to be effective in inhibiting autophagy as well, a less considered mechanism of cell damage in heart diseases. While several studies have reported that basal levels of autophagy are required for cardiac homoeostasis, induction of autophagic flux in response to ischaemia/reperfusion insult is detrimental; thus, new tools against autophagic damage are desirable. This work provides a novel therapeutic approach to cardiac diseases that offers molecules displaying a dual mechanism of prosurvival activity. The rat cardiomyoblasts cell line H9c2 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The two Met agonist mAbs (DN30 and DO24) were produced by one of us (RA) from hybridomas obtained with the fusion between the immune spleen cells from Balb/c mice (Charles River Laboratories, Wilmington, MA, USA) immunized with GTL-16 cells, where the proto-oncogene is amplified and overexpressed, and the P3.X63.Ag8.653 myeloma cells. The purification and selection of the antibodies were performed as described in Prat HGF was acquired from Tebu-Bio (Le-Perray-en-Yvelines, FR), whereas CoCl and anti-VSV-G mAb were from Sigma (St. Louis, MO, USA). CoCl was dissolved in water. Met tyrosine kinase inhibitor PHA-665752 (PHA, 500 nM) was supplied by Pfizer (New York, NY, USA). mTor inhibitor Temsirolimus was used at 1 M and purchased from Sigma. The following inhibitors were purchased from Sigma: zVAD (caspase inhibitor, 50 M), 3-MA (inhibitor of class III PI3K Vps34, 1 mM) and Baf (inhibitor of the lysosomal vacuolar H ATPase, 50 nM). H9c2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma), 1% penicillin (Sigma), 1% streptomycin (Sigma) and 1% L-Glutamine (Sigma) and were incubated under 5% CO at 37 °C. Cells were passed regularly and subcultured to ∼80/90% of confluence before all the experiments. To mimic hypoxic conditions, the cell culture was starved with 0.5% FBS and then treated with CoCl at various concentrations and for different lengths of time. In real hypoxia experiments, cells were cultured at low oxygen tension (1%) in a HeraCell (AHSI, Bernareggio, MB, Italy) incubator. HGF (0.5 nM) and the two Met agonist antibodies (DN30 and DO24, 100 nM) were administrated alone or concomitantly with the hypoxic treatment. The mAb against the VSV-G (100 or 300 nM) was used as control. Cells were lysed in two types of ice-cold lysis buffers: the not denaturant Dim Buffer for IP analysis and the RIPA Buffer for direct WB experiments, both added with protease inhibitor cocktail (Sigma). Cell lysates were subsequently sonicated and centrifuged at 14 000 r.p.m. (20 min at +4 °C). The protein concentration was evaluated through the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). For the IP analysis, 1.2 mg of cell lysates were incubated overnight at +4 °C with 5 g DN30 and DO24 for Met IP or with 5 g anti-Gab1 antibody (Millipore, Billerica, MA, USA) for Gab1 IP. Then, 20 l of protein G sepharose (GE-healthcare, Little Chalfont, BKM, UK) were added and incubated for 2 h at +4 °C. The WB and IP lysates were separated using SDS-PAGE and transferred to Hybond-P pvdf membrane (Amersham plc, Amersham, BKM, UK). After incubation in blocking solution (10% bovine serum albumin, BSA, Sigma) at room temperature (RT), membranes were incubated overnight at +4 °C with the primary antibodies (see ). Membranes were washed and then incubated with specific horseradish peroxidase-conjugated secondary antibodies (Amersham) for 1 h at RT. The proteins were revealed by enhanced chemiluminescence of the ECL Prime detection kit (GE-healthcare) and quantified with the Image Lab software (Bio-Rad Laboratories). To observe Met expression on cellular surface, ∼2 × 10 cells, resuspended in 100 l of PBS added with 1% FBS, were stained 20 min at RT, in the dark, with DN30 and DO24 mAbs previously conjugated with PE-Cy7 fluorochrome (using Lightning-Link PE-Cy7 Tandem Conjugation Kit, Innova Biosciences, Cambridge, UK). After wash, samples were analysed on a CyAn ADP LX nine-color analyser (Beckman Coulter, Brea, CA, USA). Dead cells were eliminated by excluding DAPI-positive population. Cells were plated in 24-well plates and maintained in DMEM 10% FBS until confluence, and then were incubated in DMEM 0.5% FBS for 18 h. The monolayers were wounded with a plastic pipette and a cross between two lines was created. Then cells were washed with PBS and incubated for 24, 48 and 72 h in medium with or without HGF, Met agonist mAbs or anti-VSV-G mAb. Images of wound at the start moment and after the treatment were taken with DMRI Leica inverted microscope. Migration was quantified by evaluating the area of wound at time zero (A0) and at time after the treatment (Ay, =24 h, 48 or 72 h). Normalization and quantification on the basis of three independent experiments were obtained by the formula (A0−Ay)/A0. Total RNA was extracted with Trizol reagent, following the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). Then RNA was quantified with the NanoDrop ND-1000 and the reverse transcription was performed using High Capacity cDNA Reverse Transcription Kit according to the manufacturer's recommendation (Applied Biosystem, Foster City, CA, USA). The cDNA was used for real-time PCR analysis using the SsoFast EvaGreen supermix and the MiniOpticon Thermal Cycler (Bio-Rad Laboratories) and for semi-quantitative RT-PCR using various primers (see ). Control samples were prepared without adding the RT enzyme to the reaction, and -actin was used as control. Cells plated in 24-well plates were fixed with ice-cold 4% paraformaldehyde (PAF, Sigma) dissolved in PBS for 10 min and then washed with PBS. In all the experiments, except for the membrane fluorescence of , left panel, the fixed cells were permeabilized with 0.1% Triton X-100 (Sigma). Then the cells were saturated with BSA (1%, Sigma) and incubated with the primary antibody (See ) for 1 h at RT. Secondary antibody incubation was performed with the Alexa Fluor 488-conjugated goat anti-mouse or anti-rabbit antibody or with the Alexa Fluor 546-conjugated goat anti-mouse antibody (Molecular Probes/Invitrogen) for 1 h at RT. DAPI was added at the end of secondary antibody incubation for 5 min at RT. Images were taken through the DMRE Leica fluorescence microscope and processed with LAS AF software (Leica Microsystems, Wetzlar, Germany). All data are expressed as the mean±S.D. The significant difference between the means taken from different samples was analysed through independent two-tailed -tests. -value <0.05 was considered statistically significant. For experimental details see each figure legend.

Akt phosphorylation is widely established to be one of the key-stone events involved in the regulation of prostate cancer cell survival. Since ML-9 has been suggested to be the potent inhibitor of Akt phosphorylation in other cell models, we first checked for this function of ML-9 in prostate cancer cells. In lymph node carcinoma of the prostate (LNCaP) cells, ML-9 (30 M) reduced the phosphorylation of Akt kinase suggesting its inhibition (). Since mTOR kinase acts downstream of Akt kinase, we further examined the effect of ML-9 on mTOR activity. We found that phosphorylation of mTOR was significantly reduced by ML-9 in a dose-dependent manner, which suggests the inhibition of mTOR kinase activity (). As mTOR kinase represents a central player in autophagy regulation, we next tested the effect of ML-9 on autophagy by analyzing the levels of endogenous LC3-II protein, the most widely used marker for autophagosomes. ML-9 significantly increased LC3-II levels in LNCaP cells in a dose- and time-dependent manner (). In addition, in LNCaP cells stably expressing eGFP-LC3 (LNCaP-eGFP-LC3), treatment with ML-9 resulted in accumulation of eGFP-LC3 puncta (). Further, transmission electron microscopy (TEM) analysis revealed the increased number of large autophagosome-like and autolysosome-like vacuoles in ML-9-treated cells compared with the control ones (). Moreover, the accumulation of multi-lamellar membrane structures, small vesicles, cytoplasmic material and organelles at different stages of degradation has been detected within the lumen of the numerous expanded vacuoles after ML-9 treatment (). Importantly, we have noted that apparent autolysosomes were significantly greater in number than autophagosomes (; ). In addition, immuno-TEM analysis showed the presence of immunogold-labeled lysosomal protein lysosomal-associated membrane protein 2 (Lamp2) in these vacuoles (). These findings suggest autophagic/lysosomal origin of these vacuoles. ML-9 also caused an increase in LC3-II levels in prostate cancer PC-3 cells, human embryonic kidney 293 (HEK-293) cells, pancreatic cancer ASPC1 and BxPC3 cells, but not in DU-145 cells lacking full-length autophagy-related gene 5 (ATG5) (), suggesting the necessity of the functional autophagic pathway. An accumulation of autophagic vacuoles can be the consequence of two different processes: increased autophagosome formation and/or block in autophagosome maturation/degradation. Therefore, we analyzed autophagic flux in LNCaP cells using the lysosome inhibitor chloroquine (CQ). We showed that at concentrations up to 20 M ML-9 induced LC3-II accumulation without inhibiting autophagic flux, as clear increase in LC3-II levels was detected upon addition of CQ (50 M for 1 h). In contrast, CQ failed to increase LC3-II levels induced by higher concentrated ML-9 (30–50 M) (). In addition, combined treatment of LNCaP cells with ML-9 (30 M) and bafilomycin A1 (another inhibitor of lysosomal acidification) did not elevate LC3-II levels compared with the treatment with ML-9 alone (). These results suggest that at concentrations >30 M ML-9 effectively inhibits autophagic flux. Consistent with immunoblotting experiments CQ failed to enhance ML-9-induced eGFP-LC3 puncta accumulation in LNCaP-eGFP-LC3 cells, while a clear increase in eGFP-LC3 puncta has been observed under basal and serum-starved conditions (). We next confirmed these results by analyzing the levels of autophagy substrate protein p62—another widely used autophagy marker, which is degraded by autophagy. ML-9 induced p62 accumulation in LNCaP cells in a dose-dependent manner, once again supporting the inhibitory role for ML-9 in autophagy (). Interestingly, we found that ML-9 blocked serum starvation-induced autophagic flux. Addition of CQ to LNCaP cells treated with ML-9+ starvation caused no further increase in LC3-II and p62 levels (). Thus, these results demonstrate that ML-9 arrests basal and induced autophagic flux. Next, to confirm these results we utilized a tandem mCherry-GFP reporter fluorescence assay designed to monitor autophagic flux (see Materials and methods). LNCaP cells were transiently transfected with mCherry-GFP-LC3B and treated with normal, serum-starved or ML-9 (30 M) containing medium for 12 h. The colocalization of GFP and mCherry signals was analyzed. Under starvation conditions both the number of puncta and fraction of red-only puncta increased, indicating that fusion with acidic endosomes/lysosomes occurs rapidly after autophagosome formation. In contrast, ML-9 treatment caused a dramatic decrease in the proportion of red-only puncta, thus the majority of the puncta were double positive mCherry+/GFP+ (). Thus, the accumulation of enlarged double positive mCherry+/GFP+ LC3-puncta (as it follows from confocal microscopy) together with the accumulation of autolysosomes (as it goes from TEM) following ML-9 treatment suggest that ML-9 inhibits autophagic flux mainly through the increase in endosomal/lysosomal pH and impairment of proteolytic degradation of autophagy substrates inside the autolysosomes, but not through the prevention of fusion between autophagosomes and endosomes/lysosomes. To test this assumption we utilized acridine orange, an acidotropic dye that is widely used as an indicator of acidification. LNCaP cells stained with acridine orange exhibited green fluorescence with a few red puncta in basal medium. Serum starvation induced accumulation of red puncta, indicating an increase in autolysosomes number. In contrast, ML-9-treated cells showed an accumulation of enlarged vesicles emitting in both green and red, indicating an increase in intraorganellar pH (). This effect of ML-9 could be explained by its weak base properties (pKa=8.04, calculated using the Marvin software by ChemAxon, Budapest, Hungary). Thus, it seems that ML-9 behaves like a lysosomotropic agent which is accumulated in the lysosomes and leads to an increase in lysosomal pH. We confirmed these results by assessing Lamp1 immunofluorescence in LNCaP cells transfected with mCherry-GFP-LC3B. ML-9 visibly increased the size of Lamp1-positive vesicles/puncta (). In addition, ML-9 induced accumulation of mCherry-GFP-LC3B puncta, which were largely colocalized with Lamp1-positive vesicles/puncta (). Next, we assessed the levels of another important autophagy protein Beclin1 upon treatment with ML-9. Western blot analysis showed that ML-9 induced an increase in Beclin1 levels (). To test whether Vps34-Beclin1 complex is important in ML-9-induced accumulation of autophagic vacuoles, we treated LNCaP cells with ML-9 or serum-starved medium, followed by the addition of 100 nM Wortmannin (an inhibitor of Vps34) for 1.5 h. Wortmannin effectively reduced the LC3-II levels in LNCaP cells treated with low concentrations of ML-9 (up to 20 M). In contrast, Wortmannin showed no significant effect when cells were treated with higher concentrations of ML-9 (). This could be due to the inability of Wortmannin to influence autophagosome degradation. To test this possibility, we used another Vps34 inhibitor 3-methyladenine (3-MA) and pretreated our cells with 5 mM 3-MA for 1 h before treatments with full, serum-starved or ML-9 containing medium for 6 h in the continuous presence of 3-MA. 3-MA effectively reduced LC3-II levels under basal and serum-starved conditions as well as in LNCaP cells treated with 20 M ML-9. In contrast, 30 M ML-9 induced LC3-II accumulation even following pretreatment with 3-MA (). In line with this, ML-9 (30 M) was effective in increasing the number of eGFP-LC3-positive puncta in LNCaP-eGFP-LC3 cells pretreated with Wortmannin (). Considering the previously reported data, suggesting that combination of Akt inhibitors with late-stage autophagy inhibitors promotes cancer cell death, we next assessed the effect of ML-9 on prostate cancer cell viability. MTS and trypan blue staining assays revealed that ML-9 reduced the viability of LNCaP cells in a concentration-dependent manner. Moreover, this cell-killing effect of ML-9 was strikingly potentiated in a serum-free medium (). Next, we assessed whether ML-9 induces apoptosis in LNCaP cells. First, we checked whether ML-9 could induce cleavage of poly (ADP-ribose) polymerase (PARP). Treatment of LNCaP cells with ML-9 (30 M) for 24 h resulted in the appearance of a cleaved PARP 89-kD fragment characteristic of apoptosis. PARP cleavage was significantly higher when cells were treated with ML-9 under serum-starved conditions (). These results indicate that ML-9 induces apoptosis in LNCaP cells. To confirm this, we determined the level of apoptosis by Annexin V/Propidium iodide double staining. Treatment with ML-9 (30 M) for 24 h significantly increased the percentage of Annexin V-positive LNCaP cells and serum-free medium greatly potentiated this effect (). In addition to LNCaP cells ML-9 effectively reduced the viability of PC-3, DU-145 as well as HEK-293 cells, although these cell lines exhibited greater resistance to ML-9-induced cell death compared with LNCaP cells, with DU-145 being the most resistant (). At high concentrations (50–100 M) ML-9 induced pronounced rounding up and detachment of LNCaP cells within the first 2 h of treatment. Again, this effect was intensified by the removal of serum from medium (data not shown). Of note, after 4 h of treatment with ML-9 (30–50 M) we detected vacuolization of the majority of LNCaP cells under the light microscope (). This effect correlates well with the weak base properties of ML-9 and the accumulation of autophagic vacuoles (as revealed by TEM and fluorescence microscopy). Given that ML-9 effectively induces cell death in prostate cancer cells, our next step was to see whether ML-9 could be useful as an adjuvant to anticancer chemotherapy. LNCaP, PC3 and DU-145 cells were treated with docetaxel (5 nM), ML-9 (30 M) or combination of docetaxel+ML-9 for 48 h. As shown in , cell viability was significantly reduced when docetaxel was combined with ML-9 compared with either drug alone. These results suggest that ML-9 could potentially be considered as an adjuvant to existing anticancer chemotherapy. Previous reports demonstrated that ML-9 could influence cellular calcium homeostasis. As calcium was shown to be an important autophagy regulator, we next investigated whether calcium-related mechanisms are involved in ML-9-induced autophagy. First, we checked whether ML-9 influences cytosolic calcium levels in LNCaP cells using a Ca ionophore ionomycin and an inhibitor of sarco/endoplasmic reticulum Ca ATPase (SERCA) thapsigargin (TG). Application of 2 M ionomycin in the absence of extracellular Ca caused a transient increase in cytosolic calcium, which was significantly less when cells were pretreated with 30 M ML-9 for 6 h (). Consistent with this, ML-9 treatment significantly reduced both TG-induced calcium release and store operated calcium entry (SOCE) (), suggesting that ML-9 reduces both calcium content of intracellular calcium stores and SOCE in LNCaP cells. It is well known that depletion of ER calcium content can trigger ER stress. ER stress has been linked to both autophagy and cell death. Given that ML-9 induces Ca release from intracellular stores and slows down its refilling by the inhibition of SOCE, we hypothesized that in prolonged treatments ML-9 could induce ER stress. To test this hypothesis, we assessed the GRP78 (a widely used ER stress marker) level and phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK), a protein kinase representing a major sensor of unfolded protein response. Treatment with ML-9 (50 M) induced an increase in both the GRP78 level and the phosphorylation level of PERK, suggesting ER stress induction (), which could potentially contribute to ML-9-induced autophagy and cell death. It is known that Ca mobilizing agents, such as ionomycin and TG, regulate autophagy in a calcium-dependent manner. We therefore tested whether ML-9-induced autophagy is calcium dependent using an intracellular calcium chelator BAPTA/AM (1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis/acetoxymethyl ester). Addition of 20 M BAPTA/AM together with 30 M ML-9 to LNCaP cells for 3 h completely abolished the ML-9-induced increase in LC3-II levels (), indicating that Ca is required for ML-9-induced autophagosome formation. Recent reports demonstrated that the inhibitory effect of ML-9 on SOCE is attributable to its inhibition of Stim1 redistribution. We therefore tested whether the effect of ML-9 on autophagy is mediated by the inhibition of Stim1. Silencing of Stim1 by small interfering RNA (siRNA) did not induce accumulation of LC3-II in LNCaP cells (). In line with this result, treatment of LNCaP cells with BTP2, another inhibitor of SOCE, was without effect on LC3-II (). These results indicate that ML-9 exerts its effects on autophagy independently of Stim1 and SOCE inhibition. In this study, we provide evidence that ML-9, a widely used inhibitor of Akt kinase, MLCK and STIM1, represents the ‘two-in-one' compound which stimulates autophagosome formation and inhibits their degradation. We also demonstrate that cytosolic calcium is essential for ML-9-induced autophagosome formation; however, neither STIM1 nor SOCE is necessary. Further, we show that ML-9 as a monotherapy effectively induces prostate cancer cell death associated with the accumulation of autophagic vacuoles. In addition, ML-9 enhances the anticancer activity of docetaxel, suggesting its potential application as an adjuvant to existing anticancer chemotherapy. We also demonstrate that ML-9 significantly reduces ER calcium content and induces ER stress, which could contribute to its cytotoxicity. Altogether our results revealed the complex effect of ML-9 on autophagy and indentified ML-9 as an attractive tool for targeting autophagy in cancer therapy through dual inhibition of both the Akt pathway and the autophagy. Recently, Zhang performed high-throughput image-based screening for small-molecule regulators of autophagy and among others identified ML-9 as a compound that increases GFP-LC3 vesicles in human glioblastoma H4 cells. In another study ML-7, a structurally related analog of ML-9, was shown to induce accumulation of vesicle-like structures in Schwann cells. The evidence presented here clearly demonstrates that the reported increase in GFP-LC3 vesicles by ML-9 is in fact the consequence of both the stimulation of autophagosome formation and the inhibition of their degradation. To reach this conclusion, we used a number of approaches including LC3 protein detection, electron microscopy for direct visualization of autophagic process, fluorescence and confocal microscopy for GFP-LC3+ and mCherry-GFP-LC3+ puncta analysis as well as endogenous autophagy substrate (p62) degradation analysis. We demonstrated that ML-9 stimulates autophagy through inhibition of mTOR kinase. Several mechanisms may contribute to the inhibition of mTOR by ML-9. First, as mTOR acts downstream of Akt and Akt is known to positively regulate mTOR, inhibition of Akt by ML-9 could consequently induce mTOR inhibition and activation of autophagy. Second mechanism is based on the fact that during autophagy the nutrients generated by degradation of cargo in the autolysosomes stimulate mTOR, representing a feedback regulatory loop. Accordingly, ML-9-induced lysosomal dysfunction leads to a decrease in autophagic flux thereby decreasing nutrients availability and promoting mTOR inhibition. Intriguingly, we discovered that ML-9 being an autophagy activator inhibits autophagy in the late stages. This effect of ML-9 could be attributed to its weak base properties. We propose that ML-9 as a lipophilic weak base enters the cell by simple diffusion. At neutral pH, ML-9 is in its non-protonated form. When ML-9 enters acidic vacuoles (such as lysosomes and amphisomes) it becomes positively charged through protonation and trapped in these vacuoles. Then, the increased osmotic pressure in these vacuoles stimulates water influx and vacuoles enlargement. All these events result in the increased intravacuolar pH, subsequent inhibition of lysosomal enzymes and block in degradation of the autolysosomal content. Based on the immuno-TEM and immunofluorescence analysis, we concluded that the majority of the vacuoles represent autolysosomes favoring the hypothesis that ML-9 does not prevent fusion between autophagosomes and lysosomes. Surprisingly, the early-stage autophagy inhibitors 3-MA and Wortmannin did not prevent accumulation of autophagic vacuoles induced by high doses of ML-9 (>30 M). This result could indicate that ML-9 could at once induce both canonical and non-canonical autophagy via different pathways. A number of studies reported the modulation of cellular Ca homeostasis by ML-9. ML-9 has been shown to inhibit redistribution of STIM1 and it is widely used as an SOCE inhibitor. However, our data demonstrate that STIM1 and SOCE do not contribute to ML-9-induced autophagy. In contrast, ML-9 induces an increase in GRP78 level and PERK activation (suggesting ER-stress induction) presumably through the decrease in ER calcium content. Interestingly, ER stress has been shown to negatively regulate Akt/mTOR pathway and as such stimulate autophagy. Of note, it was proposed that ML-9 promotes calcium release through the IP3 receptor. The accumulated data suggested a complex role for IP3R in autophagy regulation. Thus, IP3R pathway as well as PERK pathway could potentially contribute to both ML-9-induced autophagy and cell death. In addition, we demonstrated that cytosolic calcium is essential factor for ML-9-induced autophagy, as its removal by chelation blocks autophagy at an early stage before autophagosome formation. A graphic model for ML-9's mode of action is represented in . Recently, it was proposed that dual inhibition of Akt kinase and autophagy represents a prospective strategy in anticancer therapy. Indeed, one of the most important functions of Akt kinase is cell survival. This kinase is often upregulated in cancer and promotes cell proliferation, cell growth and resistance to apoptosis. Thus, inhibiting Akt kinase appears to be an effective approach in cancer treatment. Similarly, autophagy is thought to be predominantly a cell-survival mechanism. Elevated autophagy is often detected in cancer cells in response to radiation and chemotherapy. Furthermore, autophagy seems to contribute to the therapeutic resistance of some cancers. Thus, autophagy inhibition also seems to represent a promising therapeutic strategy in the treatment of cancer. The combination of these two approaches shows additive efficacy in anticancer therapy. Our results suggest that ML-9 represents a dual inhibitor of both Akt pathway and autophagy and thus is potentially interesting for cancer treatment. Indeed, ML-9 as a monotherapy effectively induces prostate cancer cell death as well as enhances the anticancer activity of docetaxel, indicating its potential application as an adjuvant to existing anticancer chemotherapy. These data are in line with the previously published studies showing that ML-9 and its structurally related analog ML-7 promote apoptotic cell death in a variety of cell lines, although these effects have been linked to MLCK inhibition. Moreover, ML-7 was proposed as a promising candidate for treating cancer. Interestingly, we found that cell-killing effect of ML-9 was strikingly potentiated in a serum-free medium. This result can be explained by the dual inhibition of androgen receptor (AR) and Akt/mTOR signaling pathways in these conditions. Indeed, serum-free medium mimics steroid-deprived conditions and thus inhibits AR signaling pathway. It should be noted that interaction between AR and Akt/mTOR pathways has been reported to be involved in tumorigenesis. Moreover, the use of Akt/mTOR inhibitors in combination with AR antagonists has been demonstrated to improve anticancer efficacy. Thus, combining inhibitors of Akt, AR and autophagy could potentially constitute a novel strategy in prostate cancer therapy. Overall, our data suggest that ML-9 represents an attractive tool for targeting autophagy in cancer through dual inhibition of AKT pathway and autophagy. Further, the chemical structure of ML-9 could serve as a ‘template' for the synthesis of improved structurally related and more selective compounds, which could potentially be used for cancer treatment. The pDest-mCherry-eGFP-LC3B plasmid was kindly provided by Prof. Terje Johansen (Institute of Medical Biology, University of Tromso, Tromso, Norway). The pcDNA3.1(-)-GFP-LC3 plasmid was a kind gift from Prof. Geert Bultynck (KU Leuven, Leuven, Belgium). Prostate cancer cell lines LNCaP, PC3 and DU-145 were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium (31870; Gibco-Life Technologies, Grand Island, NY, USA) supplemented with 5 mM -glutamine (25030; Gibco-Life Technologies) and 10% fetal bovine serum (F7524; Sigma-Aldrich). HEK-293 cells were cultured in Dulbecco's minimal essential medium (DMEM)+GlutaMAX (31966, Invitrogen, Life Technologies) supplemented with 10% fetal bovine serum (F7524; Sigma-Aldrich). Pancreatic cancer cell lines ASPC1 and BxPC3 were purchased from the ATCC and cultured in RPMI-1640 medium (31870; Gibco-Life Technologies) supplemented with 5 mM -glutamine (25030; Gibco-Life Technologies) and 10% FCS Gold (PAA Laboratories GmbH, Colbe, Germany). LNCaP cells stably expressing eGFP-LC3 (LNCaP-eGFP-LC3) were generated by stable transfection with eGFP-LC3 plasmid using Xtremegene HP transfection reagent (Qiagen, Courtaboeuf, France) and selection with 500 g/mL G418 (Sigma-Aldrich). LNCaP cells were transiently transfected with mCherry-eGFP-LC3B construct using Nucleofector Technology (Lonza, Basel, Switzerland) as described by the manufacturer. Briefly, one million of cells were transfected with 2 g of mCherry-eGFP-LC3B plasmid and seeded on tissue culture dishes with cover glass bottom (FluoroDish, FD35; World Presicion Instruments, Inc., Hertfordshire, UK). Two days after plating, cells were used for treatments and subsequent confocal imaging. Tandem mCherry-GFP reporter fluorescence assay is based on the use of the pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive GFP. Under neutral pH, both mCherry and GFP fluoresce and colocalize indicating an autophagosome which is not fused with acidic lysosome. Alternatively, colocalization of mCherry and GFP signals could point on an amphisome or autolysosome with decreased acidification and/or impaired proteolytic degradation. In contrast, mCherry signal without GFP corresponds to an amphisome or an autolysosome with physiologically acidic interior. LNCaP cells were transfected with 40 nM of siRNA against STIM1 (Eurogentec, Angers, France) using 6 l Hyperfect transfection reagent (Qiagen), following the manufacturer's instructions. The level of apoptosis was determined by Alexa Fluor 488 Annexin V/Propidium iodide double staining. At the end of the treatments, both floating and attached cells were collected by trypsinization, centrifuged, washed with PBS and stained with Alexa Fluor 488 Annexin V and Propidium iodide according to the manufacturer instructions (Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit; Life Technologies). Cells were examined by fluorescence microscopy on Zeiss Axio Imager A1 microscope (Carl Zeiss S.A.S., Marly-le-Roi, France). The percentage of Alexa Fluor 488 Annexin V-positive cells was determined by counting at least 500 cells in random fields. Cells were washed with cold PBS and lysed in ice-cold buffer containing: 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 1% Sodium deoxycholate, 10 mM PONa/K buffer, a protease inhibitor cocktail (Sigma-Aldrich) and a phosphatase inhibitor cocktail PhosSTOP (Roche, Basel, Switzerland). The lysates were centrifuged at 15 000 × at 4°C for 15 min to remove cell debris and supernatant protein concentration was determined by the BCA protein assay kit (Thermo Fisher Scientific, Courtaboeuf, France). In all, 30 g of total protein was subjected to SDS-PAGE followed by transfer onto PVDF membranes using the Trans-Blot SD semi-dry transfer cell (Bio-Rad, Marnes-la-Coquette, France). The membranes were blocked in a 5% fat-free milk containing TNT buffer (Tris-HCl, pH 7.5, 140 mM NaCl and 0.05% Tween-20) for 1 h at room temperature. The membranes were next incubated overnight at 4°C with primary antibodies, and then for 1 h at room temperature with secondary antibodies conjugated to horseradish peroxidase. After washing, the membranes were processed for chemiluminescence detection using Luminata Western HRP substrate (Millipore, Billerica, MA, USA). Image J software (NIH, Bethesda, MD, USA) was employed for quantitative analysis. LNCaP-GFP-LC3 cells were grown on glass coverslips. Following treatments cells were rinsed with PBS, fixed with 4% paraformaldehyde-1 × PBS for 15 min. After three washes with PBS the slides were mounted with Mowiol (81381, Sigma-Aldrich) on glass slides and subjected to subsequent fluorescence analysis using Zeiss Axiovert microscope (Carl Zeiss S.A.S.). LNCaP cells were seeded on tissue culture dishes with cover glass bottom (FluoroDish, FD35; World Presicion Instruments, Inc.). Two days after plating, cells were treated with normal, serum-starved or ML-9 (30 M) containing medium for 12 h. At the end of treatments, acridine orange was added to the cells (1 g/ml final concentration) for 15 min in 37°C. Then, the cells were washed two times with appropriate medium and subjected to confocal imaging. Upon excitation by blue light acridine orange emits at 525 nm (green). Due to its weak base properties acridine orange accumulates in acidic organelles, such as lysosomes and autolysosomes, where it precipitates and emits at around 650 nm (red). Thus, healthy acidic vesicles appear as red puncta in green cytoplasm. When the pH inside the acidic organelles increases, acridine orange fluorescence switches from red to green. Live-cell images were obtained using confocal laser scanning microscope (LSM 700, Carl Zeiss MicroImaging GmbH, Jena, Germany) with a Plan Apochromat 40 × /1.3 numerical aperture oil immersion objective and equipped with a CO and thermocontrolled chamber. The images were analyzed in Zeiss LSM Image Browser software (Carl Zeiss MicroImaging GmbH) and prepared for publication in Adobe Photoshop. Ratiometric dye Fura-2/AM was used as a Ca indicator. LNCaP cells were loaded with 2 M Fura-2/AM for 45 min at 37°C and 5% CO in RPMI medium and subsequently washed three times with external solution containing (in mM): 140 NaCl, 5KCl, 1 MgCl, 2 CaCl, 5 Glucose, 10 Hepes (pH 7.4). The coverslip was then transferred in a perfusion chamber on the stage of Nikon Eclipse Ti microscope (Nikon, Champigny-sur-Marne, France). Fluorescence was alternatively excited at 340 and 380 nm with a monochromator (Polychrome IV, TILL Photonics GmbH, Gräfelfing, Germany) and captured at 510 nm by a QImaging CCD camera (QImaging, Surrey, BC, Canada). Acquisition and analysis were performed with the MetaFluor 7.7.5.0 software (Molecular Devices Corp.). Data were analyzed using Origin 7.0 (Microcal Software Inc., Northampton, MA, USA). Statistical analysis was performed using Student's -test, and <0.05 was considered as significant. Asterisks denote *<0.05, **<0.01 and ***<0.001.

In a systems biological approach, we have previously generated gene expression profiles from 48 normal and 47 tumor prostate tumor tissue samples. Here, gene expression analyses of BRD4 revealed a significant increase of in our prostate tumor samples (). To investigate which BRD4 target genes are most significantly de-regulated in cancer, we first aimed to identify BRD4 target genes. We therefore generated RNA-Seq expression data of two knockdown experiments in HEK293T cells and integrated it with BRD4 DNA-binding results gained by two BRD4 chromatin immunoprecipitation (ChIP)-Seq experiments (). In gene expression analyses, we identified 1844 genes significantly differentially regulated (log2FC>±0.4) in both RNA-Seqs with mean log2FC>±0.5 (). Of these, 970 were upregulated – and 887 were downregulated. We focused our further analyses on the 887 downregulated genes to elucidate a potential direct transcriptional activating role of BRD4. With BRD4 ChIP experiments, we identified 1885 significant BRD4-binding site peaks (FDR<5%) when combining enriched peaks of our two ChIP-Seq experiments. We annotated genes with a BRD4-binding site within 0.5 kb of the transcription start site (TSS) (). Integration of both gene lists – the data obtained by the RNA-Seq and ChIP-Seq experiments – resulted in 52 commonly activated genes of BRD4, now considered as our top candidate target genes (). Further, we selected genes from our prostate tissues gene expression profiles with correlations to expression>0.4 (). Out of our 52 BRD4 target genes, we identified 21 genes with positive correlations to in prostate cancer (). We termed them cancer-relevant BRD4 target genes. Interestingly, among these, three were involved in the maintenance of physiological concentrations of intracellular ROS: (sestrin 3), (histone deacetylase 6), and . These and two additional genes ( and ) were validated in independent ChIP-quantitative PCR (qPCR) experiments in DU-145 prostate cancer cell lines (). The KEAP1/NRF2 interplay, mediator of one of the major oxidative stress response pathways, is frequently disturbed by somatic mutations in many cancer entities. We found upregulated in prostate cancer and highly correlated with ( and ). To see whether the oxidative stress response pathway in general is affected by BRD4, we used a list of oxidative stress responsive genes (Gene Ontology, GO:0034599, ‘cellular response to oxidative stress') and performed an enrichment analysis in the knockdown experiment (). Indeed, we found a significant enrichment of these genes indicating a functional role of BRD4 in the defense against oxidative stress (OR=3.9; -value=2.7 × 10, ). Besides the KEAP1/NRF2 pathway, we also found other stress-responsive pathways affected by knockdown, albeit to a lower degree (). Next, we asked which genes of the oxidative stress response were most significantly de-regulated by depletion. Besides transcriptional regulators, such as , and , we found genes coding for enzymes of the antioxidant system, including (catalase), (superoxide dismutase 2) and (glutathione peroxidase 1). These genes are predominant targets of NRF2 and were all significantly downregulated upon knockdown. This was a somewhat unexpected result, because knock down of decreased KEAP1 and should result in an increase of NRF2 and its target genes. Besides CAT, SOD2 and GPX1, the inducible enzyme HMOX1 is one of the best investigated targets of the NRF2/KEAP1 pathway. Although due to incongruent results not part of our initial RNA-Seq candidate list, we also found de-regulated after knockdown by western blotting () and qPCR () experiments. Thus, both – as well as (and other NRF2 target genes) – were significantly downregulated after BRD4 knockdown, demonstrating their dependence on BRD4. Interestingly, when we investigated the expression levels of , , and in three prostate cancer cell lines (DU-145, LNCAP and PC3), we found a significant upregulation of and and a downregulation of in all cell lines (). In contrast, was upregulated in DU-145, downregulated in PC3 and not altered in LNCAP cells, again showing that a downregulation of not necessarily results in a low expression of HMOX1. Thus, we were now wondering whether this is also consistent after HMOX1 induction. expression is inducible by various stimuli, including metalloporphyrins, such as CoPP. Having observed that knockdown in unstressed cells significantly repressed and expression, we asked whether BRD4 influences the CoPP-mediated induction of . Indeed, knockdown experiments in combination with CoPP induction showed that, under these conditions, a reduced level resulted in an elevated expression in a time-dependent manner of CoPP stimulation (). To get further insight into the effect of BRD4 on the CoPP-mediated induction of HMOX1, we transfected cells with a BRD4 overexpression plasmid and analyzed the expression after stimulation with varying concentrations of CoPP. In this case, the overexpression of BRD4 led to an attenuated activation of RNA expression compared with the corresponding mock control (). To explore whether the enhanced induction of by knockdown after stimulation was due to an increase in NRF2 caused by a decreased KEAP1 protein level (as already seen after BRD4 knockdown), we treated HEK293T cells (+/− knockdown) with 20 M CoPP and analyzed the endogenous NRF2 protein level at different time points. NRF2 increased in a time-dependent manner after CoPP induction in the control BRD4 cells, but, interestingly, NRF2 was even more increased in the knockdown cells after CoPP treatment (). The increased NRF2 level was not due to an increased transcriptional activity (). As expected, we found downregulated with and without stress (). In addition, we found an increased nuclear accumulation of NRF2 in knockdown cells under CoPP treatment compared with the control cells (). To further investigate whether the increased NRF2 accumulation also resulted in an enhanced DNA binding of NRF2, we performed ChIP experiments in CoPP-treated and -untreated knockdown cells. NRF2 binds to antioxidant-responsive elements and NF-E2/Maf-recognition elements, respectively, that are mainly found in the enhancer regions E1 and E2, located ∼3 kb and ∼10 kb upstream of the transcription-initiation site. We tested the NRF2 enrichment on both enhancers as well as on the promoter region, which was used as negative control. Actually, reduction significantly increased NRF2 binding to both enhancers after CoPP induction (). Further functional experiments demonstrated a low level of BRD4 to be advantageous for cells under stress: We treated control cells as well as cells with knockdown with hydrogen peroxide (HO) and measured cell survival over 72 h. Accordingly, cells with diminished expression displayed an increased cell survival upon HO treatment compared with control cells (). The upregulation of HMOX1 protects cells against increased levels of ROS and consequently against oxidative stress-mediated cell death. Hence, we tested whether the increased HMOX1 induction in knockdown resulted in a decreased ROS level and an enhanced cell survival. For these experiments, we used the bromodomain and extra-terminal (BET) inhibitor JQ1 and measured the expression level of . Similar to the knockdown experiments, we found a downregulation of in a dose-dependent manner upon JQ1 treatment (). We also measured the cell viability of cells treated with and without JQ1 under oxidative stress conditions. These experiments confirmed our observation of a decreased cell death after oxidative stress induction in cells with an inhibited function (). Next, we investigated the level of ROS in cells with diminished activity with and without HO addition. We used a dihydrorhodamine 123 (DHR) flow cytometry assay and observed, after HO treatment, a significant reduced number of cells with high levels of ROS in JQ1-treated cells compared with the dimethyl sulfoxide (DMSO) control (). Thus, the inhibition of BRD4 lowers intracellular ROS levels after exposure to stress, which might be the explanation for the increased cell viability (). Furthermore, we also measured the amount of ROS in the prostate cancer cell line DU-145. As we had observed a significant upregulation of in DU-145 cells (), we expected an increased ROS production in these cells upon HO treatment. Indeed, we detected a slight increase in ROS levels in the DU-145 cell line in comparison to the cell line RWPE-1, a cell line derived from normal prostate tissue (data not shown). Nevertheless, a treatment with JQ1 inhibitors in DU-145 cells did not result in a shift towards lower ROS concentrations. This was also mirrored by cell viability assays upon JQ1 treatment. In these experiments, the prostate cancer cell line DU-145 seemed to be resistant against JQ1, further supporting a diminished KEAP/NRF2 axis in DU-145 cells. During our effort to clarify the regulatory role of BRD4 in the oxidative stress KEAP1/NRF2 response pathway, we have observed that a diminution of BRD4 leads to an increased production of the inducible HMOX1 after stimulation with CoPP. The situation in an uninduced state was different: A knockdown resulted in a decreased level. To further analyze this discrepancy, we performed luciferase reporter assays in the absence of stress. expression is regulated over several transcriptional regulatory elements and transcription factor binding sites and enhancer regions E1 and E2 located upstream of the promoter region. We generated different luciferase constructs that either contained the enhancer region 1 in addition to a 2-kb region of the promoter (E1-HMOX1-WT (wild type)) or fragments of different sizes of the promoter region alone (HMOX1-WT, HMOX1-367, HMOX1-228) (). An overexpression of NRF2 showed the expected increase of the luciferase signal with E1-HMOX1-WT but not with HMOX1-WT, confirming the transcriptional enhancement by NRF2 through the enhancer regions (). Interestingly, a co-transfection with BRD4 only marginally increased the reporter activity of the E1-HMOX1-WT construct but instead showed an enhanced luciferase activity with the HMOX1-WT promoter construct. This suggests an additional transcriptional regulation mechanism of HMOX1 through BRD4 besides the KEAP1/NRF2 pathway. A knockdown significantly decreased the promoter activity of the HMOX1-WT construct to nearly 60% (). To investigate whether BRD4 directly activates the transcription by direct association to the promoter, we performed a ChIP experiment with an -BRD4 antibody. QPCR analyses showed that the promoter, but not the NCR (non coding region), was>2-fold increased in the BRD4 ChIP (). To elucidate how BRD4 may regulate the HMOX1 promoter, we shortened the promoter to curtail the regulatory elements. Using 367- and 228-bp long reporter constructs, we found the activating function of BRD4 independent of the NF-B site (230–250 bp upstream of the TSS) (). To identify BRD4 consensus-binding motifs, we used the binding regions identified from our ChIP-Seq experiments and performed bioinformatics analyses. This resulted in the SP1-binding motif as one of our top candidates for BRD4 binding (, ). We inserted SP1-binding site point mutations in the HMOX1-WT luciferase reporter plasmid and tested the promoter activity following BRD4 overexpression (). The mutation significantly abolished the transcriptional activation function of BRD4 by 50%. BRD4, an acetylated histone-binding protein, has been identified as an important factor for the regulation of primary response and interferon-stimulated gene expression. Furthermore, BRD4 seems to have a complex role in the transcriptional program of different tumor entities. On the one hand, studies in colorectal and breast tumors suggest BRD4 to act as a tumor suppressor. On the other hand, an overexpression of the short BRD4 isoform results in increased metastasis formation in breast tumors. Becoming aware of BRD4 as a turnstile in tumor pathogenesis, understanding the molecular mechanism of BRD4 action can help to shed light on the complex de-regulated transcription machinery and to identify central cancer pathways. Here we used an integrated analysis of data from ChIP-Seq, RNA-Seq and gene expression correlation analyses of primary prostate normal and tumor tissue samples to nominate 21 key BRD4 target genes in cancer. Searching for possible common biological functions among these genes, we compared our list with the regions published by Loven and found for eight genes an enhancer region within the TSS. Interestingly, three of these genes are involved in the maintenance of physiological concentrations of ROS: , and . De-regulation of the intracellular ROS homeostasis can have fatal consequences for the organism, including the development of cancer, neurodegenerative diseases, rheumatoid arthritis, diabetes and inherited syndromes, such as Fanconi anemia. In the absence of stress, we noticed that BRD4 activates the promoter and regulates the expression over the SP1 promoter-binding sites (). Thus, BRD4 acts as a positive transcriptional activator of , whereas BRD4 silencing results in decreased mRNA and protein expression as well as a reduced promoter activity (). This may be a mechanism for a fast fine-tuning of the cellular reactions towards small ROS deviations that do not activate the KEAP1/NRF2 oxidative stress response. However, we also identified as one of our top 21 BRD4 target genes. BRD4 binds to the promoter not only in normal but also in prostate cancer cells, highlighting as activating direct target for BRD4 in cancer. Under CoPP treatment, and thus exertion of stress, NRF2 is significantly increased, which drives expression (). An additional knockdown further enhances this mechanism by a downregulation of KEAP1 and a lack of NRF2 degradation (). The increase in goes along with an increased cell viability and a decreased amount of intracellular ROS under HO stress and a decreased BRD4 function. Thus, under exertion of stress, BRD4's regulation of HMOX1 is mediated via the KEAP1/NRF2 pathway resulting in a counteraction of BRD4 and HMOX1 levels. The KEAP1/NRF2 pathway is frequently disrupted by somatic mutations in tumors. NRF2-activating somatic mutations have been described in lung, head and neck, esophagus and skin cancers. Mutations in NRF2 and KEAP1 are clustered within the KEAP1–NRF2-binding surface but are mutually exclusive. KEAP1 mutations are found, next to abnormally increased DNA methylation, to impair KEAP1 function, thus liberating NRF2 to the nucleus. The increased activity of NRF2 in tumors, in turn, can promote ROS detoxification and tumorigenesis by conferring a more reducing intracellular environment. HMOX1, the rate-limiting enzyme in the heme catabolism and part of the cellular defense against oxidative stress, is, similar to NRF2, recognized as resistance mechanism against tumor necrosis factor-induced cell death as shown in acute myeloic leukemia (AML). Interestingly, in prostate cancer the block of the KEAP1/NRF2 pathway seems to be located even downstream of NRF2: We showed that in prostate cancer tissues, as well as in a subset of prostate cancer cell lines, is upregulated irrespective of NRF2. In this case, a treatment with JQ1 did not decrease ROS levels and did not lower cell viability as one would have expected owing to the high BRD4 levels in these cells. Similarly, Wyce found that IC values for the BET inhibitor I-BET762 for DU-145 cells were>3 M, whereas for LNCaP cells<30 nM, supporting a therapy resistance of DU-145 cells. As our measurement of was done without exogenous stress, BRD4 might have bypassed the KEAP1/NRF2 pathway and directly regulated the promoter, as we have shown it in a cell culture model in absence of stress. As far as HMOX1 is concerned, Alaoui-Jamali described a significant elevation of HMOX1 levels in hormone-refractory prostate cancer and silencing of the gene or exposure to a small molecular HMOX1 inhibitor (OB-24) reduced cell proliferation, tumor growth and metastatic invasion. Having this all in mind, one may speculate that a high BRD4 level in combination with a high amount of HMOX1 might be an indicator for BET inhibitor resistance. , the classic fields of application for BET inhibitors (e.g., JQ1, I-BET) so far are AML, MLL, MM and melanoma. Abrogation of BRD4's function results in a delayed tumor progression, survival benefits, loss of stem cell characteristics and a reduction of MYC oncogene expression. Searching the Oncomine database, we found for AML and MM significantly decreased expressions. Thus, the effective treatment of AML and MM patients with BET inhibitors may – at least partly – be supported by low HMOX1 levels. Taken together, our data provide new insight into the transcriptional regulatory network of BRD4 as it nominates BRD4 as key mediator of KEAP1 in the oxidative stress response and to directly target SP1-binding sites in the promoter (). In prostate tumors, these regulatory mechanisms appear to be disturbed. The increased and expression in prostate cancer does not go along with decreased levels. In contrast, seems to be upregulated independently of the KEAP1–NRF2 pathway (), suggesting that the strong increase in may, over a direct regulation of the promoter, counteract the downregulation of HMOX1 via KEAP1-NRF2. The two-sided regulatory mechanism of BRD4 may prevent tumor cells from a loss of HMOX1, an increase of ROS and promote cell survival. As outlined above, further studies of the BRD4 transcriptional network and the cooperation between BRD4, KEAP1, NRF2 and NRF2 target genes as transcriptional regulators in prostate cancer have the potential to elucidate important druggable oncogenic dependencies. The BRD4 shRNA knockdown vectors (shBRD4-1, shBRD4-2) and the control vector have been described previously in Schweiger The siRNA-BRD4-pool (siBRD4) as well as the non-targeting control pool (siControl) were purchased from Thermo Scientific (Schwerte, Germany). The pcDNA4c-plasmid containing the full-length human BRD4 has been described previously in You NRF2 and SP1 were cloned into the pTLFlagC vector system using the following primers: The luciferase construct or the corresponding negative control (pGL3-basic vector) were transfected together with a BRD4, NRF2 or SP1 expression construct or shRNA plasmids. Luciferase activities were measured 24 h after transfection for overexpression or 72 h after knockdown approaches according to the manufacturer's protocol (Dual-Luciferase Reporter Assay System from Promega). Luciferase activities were normalized to the luciferase activity of a co-transfected expression plasmid. Cells were cultivated at 37 °C with 5% CO in their specific cell culture medium (Hek293T and DU-145: Dulbecco's Modified Eagle's Medium, WI-38: Minimal essential Medium containing 10% fetal calf serum, 2 mM L-glutamine and 100 U penicillin/streptomycin and RWPE-1: keratinocyte serum-free medium supplemented with EGF and BPE). Cells were treated with 20–100 M CoPP (dissolved in DMSO) for 4–14 h and with various concentrations of JQ1 (Cayman Chemical: 1268524-70-4, Ann Arbor, MI, USA) for 72 h. Plasmids were transfected into the cells using X-treme gene 9 transfection reagent (Roche, Penzberg, Germany) and siRNA using HiPerFect transfection reagent (Qiagen, Hilden, Germany). Cell viability was measured using the Alamar Blue reagent from Life Technologies (Darmstadt, Germany). Cells were seeded in 96-well plates and treated with siRNAs or JQ1 inhibitor. After incubation for 72 h at 37 °C, 10 l of Alamar Blue cell viability reagent was added according to manufacturer's instructions, and the resulting fluorescence intensity was read on the fluorescence spectrometer LS55 (Perkin Elmer, Waltham, MA, USA). Cells were seeded in 12-well plates and treated with siRNAs or JQ1 inhibitor at various concentrations. After incubation for 72 h, 10 M DHR or 10 M DHR+1 mM HO was added for additional 4 h to determine the levels of oxidative stress products. The absorbance of the green fluorescent rhodamine 123 was measured using an ACCURI C6 cytometer and analyzed with the Flowing Software 2 (BD Bioscience, San Jose, CA, USA). Cells were harvested and resuspended in 300 l lysis buffer A (10 mM Hepes-KOH (pH 7.4), 10 mM NaCl, 1 mM DTT, 3 mM MgCl and protease inhibitor cocktail (Roche)). Cells were incubated for 10 min on ice before passaging through a needle 10 times. NaCl was added to a final concentration of 300 mM, and the lysate was rotated at 4 °C for 20 min. After centrifugation for 5 min at 2500 × at 4 °C, the supernatant was transferred to a new tube. The pellet containing insoluble proteins was resuspended in 300-l lysis buffer B (10 mM Hepes-KOH (pH 7.4), 300 mM NaCl, 1 mM DTT, 20 mM MgCl, 0.2 U DNase and Protease Inhibitor cocktail (Roche)) and incubated for 30 min at 37 °C. The extract was centrifuged at 2500 × at 4 °C for 5 min, and the supernatant was combined with the soluble protein fraction. Protein extracts (30 g) were separated on a 10% SDS gel, transferred to a polyvinylidene difluoride (PVDF) membrane and immunoblot analyses were performed with the aforementioned antibodies. ChIP was done according to the protocol of Dahl Briefly, immediately before harvesting the cells sodium butyrate (Sigma-Aldrich) was added to the cell culture medium to a final concentration of 20 mM and mixed gently. The cells were harvested by trypsinization and cross-linked with 1% (v/v) formaldehyde for 10 min at room temperature. The cross-link reaction was stopped by adding glycine to a final concentration of 125 mM for 5 min. Cells were lysed with lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 1% SDS, protease inhibitor cocktail and 20 mM sodium butyrate). The chromatin was sheared by sonication to a DNA fragment size of 200–600 bp and precipitated by centrifugation at 12 000 × at 4 °C for 10 min. For immunoprecipitations, 5 g of each antibody or of a rabbit immunglobulin G (IgG) control were incubated overnight with the sheared DNA. The next day, 50 l protein G Dynabeads (Life Technologies) were added and incubated for 4 h at 4 °C. The antibody/chromatin/beads complexes were washed four times with RIPA buffer (10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton-X100, 0,1% SDS, 0,1% sodium-deoxycholate and 140 mM NaCl) and once with TE-buffer (10 mM Tris-HCl, 10 mM EDTA). DNA was incubated with elution buffer (20 mM Tris-HCl, 5 mM EDTA, 20 mM sodium butyrate, 50 mM NaCl) containing 50 g/ml proteinase K at 68 °C for 2 h. DNA was purified using the Qiagen MinElute columns (Qiagen). In all, 300 pg of ChIP and input DNA were analyzed using qPCR with the following primers: The relative enrichment towards the IgG control was calculated using the %Input method previously described. Sequencing libraries were prepared according to the Illumina's ChIP-Seq Sample Prep Kit and analyzed on the Illumina Genome Analyzer IIx (Illumina, San Diego, CA, USA) (). Library preparations were performed following the instructions of the TrueSeq RNA Sample Preparation Kit (Illumina), and sequencing was performed on the Illumina HighSeq 2500 (). Validation of the RNA-Seq data was performed with qPCR experiments and resulted in Pearson's correlation coefficients of 0.86 (). #text R e a d s w e r e m a p p e d a g a i n s t t h e h u m a n g e n o m e G R C h 3 7 / h g 1 9 u s i n g b w a v 0 . 5 . 9 - r 1 6 w i t h d e f a u l t p a r a m e t e r s . Exon read coverages were obtained with coverageBed v2.17.0 using exon coordinates of the Ensembl database v63. Transcript counts were generated by summing read counts across all exons of a given transcript. Expression changes were calculated as log2(ratios) of the read counts+1 read per transcript for two investigated conditions, and ratios were normalized against the median ratio of each condition. italic #text italic sup #text

We previously showed that HSF1 controls the stability of mutp53 protein in human cancer cells via activation of Hsp90, which strongly stabilizes mutp53 (Li and ). Conversely, here we show that mutp53 also induces HSF1 in all human cancer cell lines and mouse primary cells tested. First, RNAi-mediated depletion of mutp53 in SKBr3 breast cancer cells results in downregulation of the HSF1 protein (). Next, we generated stable isogenic lines of mutp53 MDA231 breast cancer cells that express either Tet-inducible shp53RNA or excess ectopic mutp53 R280K protein matching its endogenous p53 mutation. Similar to SKBr3 cells, shp53RNA downregulates HSF1 and its transcriptional target Hsp70 in MDA231 cells. Importantly, this effect is further enhanced by HSF1 activation via heat shock (HS), a strong inducer of HSF1 transcriptional activity (). Conversely, excess ectopic native mutp53 R280K in MDA231 cells further increases the HSF1 targets Hsp70/Hsp27 upon HS (). Likewise, expression of mutp53R175H in p53 null H1299 cells induces HSF1 and Hsp70/Hsp27 (), further enhanced by HS at the protein () and mRNA levels. Upregulation of HSF1 by mutp53 appears to be generic, as it is observed in response to different p53 mutants (). To confirm these results in mice we generated a novel breast cancer model by introducing the well-characterized p53 R172H allele (H thereafter) into MMTV-ErbB2 transgenic mice. p53−/−ErbB2 littermates served as controls. These mice spontaneously develop mammary tumors. Importantly, compared with their p53−/− littermates, primary cultures derived from H/H;ErbB2 mammary epithelial cells (MECs; Figure 5c) and −/+ErbB2 H/+ErbB2 mammary tumors both show increased levels of the HSF1 protein and its targets (Hsp70, Hsp27; ). qRT-PCR analysis demonstrated that in contrast to Hsp70, HSF1 transcripts are not affected by ectopic expression of mutp53 in H1299 cells, indicating that mutp53 upregulates HSF1 at the post-transcriptional level. In unstressed cells, HSF1 shuttles between the nucleus and cytoplasm, but localizes predominantly in the cytoplasm, due to sequestration by Hsp90. Upon HS, HSF1 is phosphorylated, liberated from Hsp90, undergoes trimerization and translocates to the nucleus to activate target gene expression by binding to specific heat-shock elements (HSE) in target promoters. Importantly, phosphorylation of HSF1 at Serine 326 (p-Ser326) is pivotal to render HSF1 transcriptionally competent. Furthermore, Ser326 phosphorylation protects HSF1 from polyubiquitination and proteosomal degradation, causing HSF1 to stabilize. Thus, we looked for a correlation between levels of p-Ser326 HSF1 and mutp53. Indeed, HS significantly elevated p-Ser326 HSF1 in total cell lysates of MDA231R280K control MDA231 cells (). Moreover, total HSF1 was also increased in the cytoplasm of unstressed MDA231R280K cells, implying that mutp53 affects basal levels of HSF1 even in the absence of proteotoxic stress (). HS induced nuclear translocation and p-Ser326-phosphorylation of HSF1, which was augmented in MDA231R280K compared with control cells (). Conversely, shRNA-mediated downregulation of mutp53 decreased (mainly cytoplasmic) total HSF1 of unstressed cells and decreased HS-activated nuclear p-Ser326 HSF1 (). Notably, HSF1 was specifically upregulated by mutant but not wtp53. No increase in HSF1 levels or activation was seen in HCT116 p53−/− p53+/+ cells (). The best-described mechanism of mutp53 gain-of-function relates to its ability to interact with other transcription factors and modulate their target gene expression. To test whether this mechanism is also engaged in the mutp53-mediated regulation of the HSF1 transcriptional program, we performed co-immunoprecipitations. Indeed, HS induced a specific mutp53-HSF1 complex in MDA231R280K cells, which contained total and activated p-Ser326 HSF1 (). Importantly, in reciprocal co-immunoprecipitations from MDA231 cells an endogenous nuclear mutp53-HSF1 complex, enhanced by HS, was confirmed (). To directly establish that nuclear mutp53 mainly interacts with the transcriptionally active form of HSF1, nuclear fractions from MDA231 cells after HS were immunoprecipitated with pan-HSF1- or p-Ser326 HSF-specific antibodies and loading normalized for similar amounts of immunoprecipitated total HSF1. Indeed, mutp53 was predominantly complexed with p-Ser326 HSF1 (). We failed to detect the wtp53-HSF1 complex in HCT116 p53+/+ cells despite HS, indicating that HSF1 interaction is a mutp53-specific trait (). The fact that mutp53 preferably interacts with activated HSF1 () suggests that it may modulate the transcriptional activity of HSF1. Therefore, we examined the mRNA levels of Hsp70 after shRNA-mediated mutp53 knockdown. As expected, HS-mediated proteotoxic stress strongly induced Hsp70 mRNA () and protein () in MDA231 cells. Importantly, in mutp53-depleted cells mRNA induction was suppressed by 50%, especially upon HS with maximum HSF1 activity (). These results were confirmed by HSF1 reporter assays using HSE-luciferase (). Conversely, in H1299 cells expressing mutp53R175H Hsp70, mRNA () and protein () were induced compared with vector controls, whereas the HSF1 transcript level itself was unresponsive to mutp53 (). In support, HSE-Luc reporter activation was stimulated by 40% in H1299 cells expressing mutp53R175H compared with controls (). Likewise, the elevated levels of mutp53 in MDA231R280K cells increased HSE-Luc activity upon HS (). Thus, mutp53 is an important enhancer of the HSF1 transcriptional activity. Next, we asked whether mutp53 proteins directly contribute to HSF1's physical recruitment to HSF1-binding sites. Chromatin immunoprecipitation (ChIP) analyses in heat-shocked MDA231 and MDA468 cells showed that mutp53 is bound with similar efficiency to Hsp27, Hsp90 and Hsp70 promoters as HSF1 (). Moreover, HSF1 binding to HSE is mutp53-dependent, as HSF1 recruitment to HSE was greatly enhanced in the MDA231R280K compared with MDA231 control cells (, lanes 7, 8). On contrary, HSF1 binding to HSE was reduced in mutp53-depleted cells before (, lanes 9, 11) and after HS (, lanes 10, 12). Hence, mutp53 stimulates its Ser326 HSF1 phosphoactivation, binds to activated p-Ser326 HSF1 and enhances its transcriptional activity via HSF1 recruitment to its HSE elements. As a result, mutp53 significantly amplifies the heat-shock response. Next, we asked whether mutp53-mediated HSF1 activation provides a survival advantage to cancer cells by bestowing increased tolerance to proteotoxic stress. First, we tested the thermotolerance of MDA231R280K cells with elevated levels of mutp53 compared with vector-transfected cells. Indeed, HS-stressed MDA231R280K cells exhibited higher levels of activated HSF1 (), with subsequent upregulation of Hsp70/Hsp27 (). Importantly, MDA231R280K acquired higher thermotolerance, indicated by higher cell viability, compared with MDA231 control cells (). Whereas downregulation of mutp53 by siRNA decreases levels of activated HSF1 and its transcriptional target Hsp27 and confers increased sensitivity to HS (). Notable, immortalized mutp53 H/H;ErbB2 mouse MECs also exhibited elevated levels of HSF1 and its targets Hsp70/Hsp27 compared to p53−/−ErbB2 MECs. As a consequence, mutp53 MECs developed higher resistance to proteotoxic stress induced by HS or proteasome inhibition than p53 null MECs (). Importantly, that in addition to upregulation of pro-survival HSPs, HSF1 also coordinates a broad pro-tumorigenic transcriptional network in cancer cells including the inhibition of pro-apoptotic genes. Thus, we tested whether elevated mutp53 levels in MDA231R280K cells promotes chemoresistance compared to control cells. High levels of mutp53 rendered MDA231R280K cells chemoresistant to the chemotherapeutic Camptothecin, as indicated by increased viability and lack of PARP cleavage (). To date signaling pathways leading to HSF1 activation by Ser326 phosphorylation are not well understood. To identify how mutp53 enhances Ser326 HSF1 phosphorylation in breast cancer cells, we screened a panel of kinase inhibitors. We identified the selective MEK 1/2 inhibitor UO126 and the PI3K inhibitor LY294002 as potent suppressors of HSF1 Ser326 phosphorylation (). The levels of total and active HSF1 dramatically decreased in MDA231 cells after UO126 treatment in the absence and presence of HS, causing a parallel decline in Hsp70 and Hsp27 (). Notably, mutp53 depletion sensitizes cells for UO126's ability to suppress HSF1 activation (, lanes 4–8, p-Ser326 HSF1), indicating that mutp53 promotes HSF1 activation via MAPK signaling. Likewise, PI3K inhibitor LY294002 suppressed p-Ser326 and total HSF1 levels (). Moreover, dual inhibition of PI3K and MAPK cascades by combined drug treatment further impeded HSF1 levels and expression of its target genes upon HS (, lanes 2, 3). Conversely, the inhibition of the HSF1 response imparted by these drugs was largely rescued by overexpression of mutp53 (), underlining the importance of mutp53 in stimulating MAPK and PI3K signaling to regulate HSF1 activity. Interestingly, levels of mutp53 were also reduced by UO126 and LY294002 in MDA231 cells ( and ). Mutp53 levels also decreased upon LY294002/UO126 treatment in primary mutp53 H/H;ErbB2 MECs (). This effect is likely due to decreased transcriptional activity of HSF1, as our previous studies showed that HSF1 ablation destabilizes mutp53 via Hsp90 inhibition (). Indeed, as a result of HSF1 inhibition, LY294002/UO126 treatment significantly reduced the levels of Hsp90 in mutp53 MECs compared with its minor reduction in p53 null MECs, further supporting the notion that regulation of HSF1 is wired through mutp53 when it is present (). The transcriptional program involved in mammary tumorigenesis is often modulated by the Epidermal Growth Factor Receptor family including EGFR and ErbB2. As both PI3K and MAPK cascades induce HSF1 activation (), we hypothesized that HSF1 Ser326-phosphorylation could be regulated by upstream EGFR and/or ErbB2 signaling in a mutp53-dependent manner. To test this notion, we treated SKBr3 cells with dual EGFR/ErbB2 tyrosine kinase inhibitor CP724714. As expected, CP724714 inhibited phosphorylation of ERK/pERK and AKT/pAKT (). Similar to the effect of the PI3K/MAPK blockade (), EGFR/ErbB2 inhibition by CP724714 also reduced levels of activated p-Ser326 HSF1, leading to a decline in mutp53 levels (). To further test our hypothesis, we stimulated EGFR signaling by adding EGF into the medium of serum-starved MDA231 cells. Indeed, EGFR activation by Tyr845 phosphorylation not only induces AKT and ERK phosphorylation but also enhances Ser326 HSF1 phosphorylation (), confirming that HSF1 activation is mediated via EGFR signaling. As EGFR- and/or ErbB2-mediated downstream signaling are involved in HSF1 phosphorylation, we reasoned that mutp53 might enhance HSF1 activation via stimulating the EGFR/ErbB2 pathways. Thus, we tested the effect of differential mutp53 expression on EGFR and ErbB2 signaling in breast cancer cells. Indeed, mutp53 overexpression in MDA231R280K cells potentiated EGFR signaling, as indicated by enhanced EGFR-Tyr845 phosphorylation after EGF stimulation, compared with control MDA231 cells (). This effect was concomitant with induction of p-Ser326 HSF1 even in the absence of proteotoxic stress (), further confirming that HSF1 activation is mediated by EGFR signaling in a mutp53-dependent manner. Likewise, stable overexpression of native p53R175H in SKBr3 increased the level of ErbB2 and pAKT (). Consistently, siRNA-mediated depletion of mutp53 in SKBr3 cells reduced both ErbB2 () and EGFR levels and was accompanied by decreased p-Ser326 HSF1 (). Similar to p53 depletion, HSF1 knockdown also reduced the level of ErbB2 and EGFR ( and ), consistent with ErbB2 and EGFR being well-established HSPs clients. In agreement with the effects seen in human cancer cells, the presence of the mutp53 allele in MECs from H/+ ErbB2 mice correlated with increased levels of ErbB2 and higher HSF1 activity, indicated by elevated Hsp70/Hsp27 (, left). Moreover, H/H MECs, even in the absence of the ErbB2 transgene, showed detectable amounts of ErbB2 and higher HSP levels compared with p53−/− control mice (, right). In addition, H/H;ErbB2 established mammary tumor cell lines, showed elevated levels of ErbB2, EGFR and activated HSF1 (indicated by increased Hsp70) compared with p53−/−ErbB2 littermate (). Importantly, dual inhibition of PI3K and MAPK signaling not only affected HSF1 activation ( and ) but also decreased EGFR levels in MDA231 cells (). The fact that this dual inhibition was significantly less pronounced in mutp53-overexpressing MDA231R280K cells () implies that mutp53 positively regulates the tyrosine kinase receptors ErbB2 and EGFR, and via their downstream signaling affects HSF1 activation. Together, our studies imply that mutp53 cancer cells enhance HSF1 activation in a feed forward mechanism by deregulating EGFR and ErbB2 receptors. To support this notion in human tumors, we examined tissue microarray of 150 breast cancer biopsies with known molecular status (ER+, PR+, Her2+ or triple negative) and correlated the intensity of p53 staining with the localization/level of activated HSF1. Consistent with our model, we found a clear correlation between p53 and nuclear p-Ser326 HSF1 staining only in strongly (3+) Her2-positive tumors (rho=0.213, =0.008). No correlation between p53 and p-Ser326 HSF1 staining was found in Her2-negative, ER+, PR+ tumors (rho=−0.243, =0.932; , ). Thus, the mutp53-HSF1 circuit constitutes a novel mutp53 gain-of-function, whereby mutp53 initiates a feed forward loop that enhances EGFR/ErbB2 signaling and amplifies HSF1-induced transcriptional program, imparting an enhanced proteotoxic defense (). Inherent to malignant transformation is massive perennial proteotoxic stress due to aneuploidy, ROS, hypoxia and acidosis. To overcome proteotoxic stress, cancer cells mount a wide range of adaptive mechanisms in which the HSF1-orchestrated response has central role. Aside from maintaining cellular homeostasis by stress-mediated induction of HSPs, the master transcription factor HSF1 coordinates a wide range of fundamental cellular processes that are critical for malignancy including cell cycle control, metastases and inhibition of apoptosis. Not surprisingly, HSF1 is upregulated in 80% of breast cancers and is associated with high histologic grade and increased mortality. Although interception of pathways leading to activation of the HSF1-mediated adaptive mechanisms will likely have high therapeutic potential, the molecular mechanisms causing HSF1 activation remain poorly defined. p53 mutations are the most frequent genetic alterations in breast cancer, such as in ErbB2+ (72%) and triple negative (80%), and correlate with high rates of metastatic recurrence, chemoresistance and poor overall survival. A critical cancer specific phenotype of mutp53 is increased its protein stability causing mutp53 accumulation in tumors, but not in normal tissues. We and others showed that cancer-specific accumulation of mutp53 is critical for many aspects of tumorigenesis, and is the key determinant of mutp53's gain-of-function. Due to its high translational impact, the question of what causes tumor-specific mutp53 stabilization has recently attracted a lot of attention. Previously we showed that HSF1, by transactivation of the inducible Hsp90, stabilizes mutp53 via Hsp90 complex formation that protects mutp53 from E3 ubiquitin ligase degradation by Mdm2 and Chip. Thus, upregulation of HSF1 in cancer cells can mechanistically underlie the cancer-cell-specific stabilization of mutp53. Conversely, here we show that mutp53 is also an important determinant of HSF1 function, constituting a positive feedback loop. Mutp53 promotes phosphoactivation and stabilization of HSF1 by stimulation of the EGFR/ErbB2/MAPK/PI3K signaling cascades. Moreover, mutp53 directly interacts with p-Ser326 HSF1 to recruit HSF1 to its specific DNA-binding sites in target gene promoters and enhances its transcriptional program (). As a consequence, mutp53 endows cancer cells with superior resistance to proteotoxic stress and broadly promotes oncogenic signaling via HSF1. These observations may have significant clinical impact, as conventional chemotherapeutics further stabilize already elevated mutp53 levels (), which inadvertently may stimulate adaptation to adverse environments and promote cancer cell survival and chemoresistance. Thus, the notorious chemoresistance of mutp53 cancer cells can at least in part be attributed to the positive mutp53-HSF1 circuit (). In contrast, mutp53-destabilizing therapies may impede or at least offset the adaptive responses, or even sensitize tumors to conventional cytotoxic therapies. Interestingly, contrary to mutp53, wtp53 has been shown to negatively regulate cytoprotective function of HSF1 and heat-shock response, which at least in part is mediated by SIRT1, NAD+-dependent deacetylase. How exactly does mutp53 stimulate HSF1 activation? Recently, Dai demonstrated that HSF1 activation by Ser326 phosporylation depends on deregulated MAPK signaling. On the other hand, using ectopically expressed mutp53 in H1299 cells, Muller established that mutp53 drives enhanced EGFR recycling to the cell surface of cancer cells. This trafficking activity of mutp53 protein depends on Rab-coupling protein (RCP) and results in constitutive mutp53-driven EGFR signaling. Sustained EGFR signaling launches PI3K and MAPK intracellular signaling cascades. Indeed, we identified mutp53-driven deregulated MAPK/ERK and PI3K signaling to be important effectors of HSF1 activation (), whereas inhibition of the upstream EGFR and ErbB2 receptors prevented HSF1 activation (). Importantly, the modulation of mutp53 levels not only affects EGFR activation upon EGF stimulation but also the total amounts of EGFR, suggesting that in addition to the RCP-mediated receptor recycling mechanism, mutp53 may also regulate protein stability of the EGFR () and ErbB2 ( and ). Conversely, HSF1 knockdown destabilizes both EGFR and ErbB2 ( and ), consistent with the facts that EGFR is an established Hsp90 client and that ErbB2 is stabilized by Hsp90, Hsp70/Hsc70 and Hsp27. In aggregate, our results strongly imply that the EGFR and ErbB2 signaling, at least in part, is mediated by mutp53 in an HSF1-dependent manner. In support, we found a correlation between mutp53 and nuclear p-HSF1 levels only in strongly (3+) HER2-positive, but not in HER2-negative, ER/PR-positive human breast cancers (). Together, our data indicate a strong oncogenic cooperation between mutp53 and EGFR/ErbB2 signaling, and implicating the latter as an important determinant of mutp53 gain-of-function activity. Thus, we delineate a novel gain-of-function of mutp53 defined by a mechanistic feed forward link between HSF1 and mutp53. We propose that mutp53, through enhanced recycling and/or stability of EGFR and ErbB2, augments MAPK and PI3K signaling, causing phosphoactivation of HSF1. Concurrently, mutp53 via direct interaction with activated HSF1 facilitates binding of HSF1 to its DNA-binding sites and stimulates transcription of HSPs that further stabilize EGFR, ErbB2, mutp53 and other oncogenes, reinforcing tumorigenesis (). Hence, mutp53 initiates a feed forward loop that endows cancer cells more resistant to proteotoxic stress, providing a distinct survival advantage. Human mutp53 breast cancer cells MDA231 (p53R280K), MDA468 (p53R273K), T47D (p53L194F), SKBr3 (p53R175H), colon cancer cells SW480 (p53 R273H/P309S) and isogenic HCT116 p53−/− wtp53+/+ cells were used. Generation of stable Tet-On shp53 MDA231 was described. MDA231 and SKBr3 stably overexpressing native ectopic p53 (R280K and R175H) or vector only were generated by transfection followed by selection in G418. All cells were cultured in 10% FCS/DMEM and where indicated treated with CP724714 (Selleckchem, Huston, TX, USA), UO126, LY294002 (LC Labs, Woburn, MA, USA) and Camptothecin (Sigma, St. Louis, MO, USA). Viability was determined using CellTiter-Blue Assay, and HSF1 activity using Dual-Luciferase Reporter Assay (Promega, Madison, WI, USA) with the Hsp70 HSE-Luc reporter (Qiagen, Valencia, CA, USA). Pools of four different siRNA duplexes specific for p53 or HSF1, or scrambled controls, were transfected with Lipofectamine (RNAiTM/RNAiMAX, Invitrogen, Carlsbad, CA, USA). Cells were harvested 48 h later and analyzed. ChIP assays were performed as described in Denissov Antibodies were the following: p53: (DO-1, BD Pharmingen, San Jose, CA , USA), HSF1 (sc9144X, Santa Cruz Biotechnology, Dallas, TX, USA) and IgG (ab46540, Abcam, Cambridge, MA, USA). After purification of the DNA with the Mini Elute Kit (Qiagen), the relative binding of HSF1 and p53 to HSP promoter sites was analyzed using gene-specific primers: Hsp90F: 5′-TTTAAGGCGGAGGGATCTAC-3′, Hsp90R: 5′-TACCCAGACAGTCCCAACAC-3′, Hsp27F: 5′-AGTTTCTGAGAGCCCAGACC-3′, Hsp27R: 5′-GCAGGCTGGTAGGGATTAAC-3′ and Hsp70F: 5′-CTGTCAATTAGGCGCTGAAG-3′, Hsp70R: 5′-TCTTCTGGGATTCACTGGAG-3′ and Real-time qPCR analysis. Analysis of the myoglobulin promoter (myoF: 5′-CTCATGATGCCCCTTCTTCT-3′ myoR: 5′-GAAGGCGTCTGAGGACTTAAA-3′ served as an internal negative control. The primers to detect Hsp70-specific HSE were F:5′-GAAGACTCTGGAGAGTTCTG-3′ and R:5′-CCCTGGGCTTTTATAAGTCG-3′. For immunoblots, equal total protein of cell lysates (2.5–20 g) were detected with antibodies to mouse p53 (FL393), human p53 (PAb1801; Santa Cruz Biotechnology), HSF1, p-Ser326 HSF1, AKT, pAKT, Erk, pErk, Hsp70, Hsp90, Hsp90, EGFR, EGRF-Tyr845P (all Cell Signaling, Danvers, MA, USA), ErbB2, actin, GAPDH, GTS, GFP, HSc70 and HDAC1 (all Neomarkers, Fremont, CA, USA). SDS-PAAG gels (6%) were used to detect slower migrating HS-activated HSF1 used in experiments presented in and . SDS PAAG (10%) gels were used in all other experiments. Co-immunoprecipitations were performed with 1 g of antibody overnight. Beads were washed with SNNTE and RIPA buffers 3 × each, followed by blotting. Cells were harvested, rinsed, pelleted, resuspended in 5 vol of cold CARSB buffer (10 mM Tris pH 7.5, 1.5 mM CaCl, 10 mM NaCl, protease inhibitor cocktail, 10 mM sodium orthovanadate) and allowed to swell on ice for 15 min, after which Triton X-100 was added to a final concentration of 0.3%. The homogenate was spun for 10 min at 1000 × . The supernatant (cytoplasmic fraction) was adjusted to 200 mM NaCl. The crude nuclear pellet was suspended in buffer C (10 mM Tris pH 7.9, 1.5 mM MgCl, 10 mM KCl, 400 mM NaCl, 0.5% Triton X-100, protease inhibitor cocktail, 10 mM sodium orthovanadate) and sonicated. The homogenate was centrifuged for 15 min at 16 000 × . This final supernatant comprises the nuclear fraction. Mammary glands were dissected from 8-week-old virgin female mice and sequentially digested at 37 °C for 2 h in collagenase/hyaluronidase, 0.05% Trypsin, DNAse I and Dispase (Stem Cell Technology, Tewksbury, MA, USA). The ensuing cell suspension was treated with red blood cell lysis buffer, rinsed with PBS and passed through a 40-m mesh after resuspension in Opti-Mem medium (Gibco, Grand Island, NY, USA). Cells were plated on gelatin-coated plates and grown in CnT-BM1 medium (Cell-N-Tec, Bern, Switzerland). MMTV-ErbB2 mice harboring activated ErbB2 (strain FVBN-Tg(MMTV-ErbB2)NK1Mul/J) were from Jackson Labs (Bar Harbor, ME, USA). p53 R172 (called p53H/H) and control p53 null (p53−/−) mice (C57Bl6J background) were a gift from G. Lozano. p53 mice were interbred to generate H/− mice. Compound p53H/−ErbB2 mice were generated by crossing ErbB2 into the p53−/− background and then breeding the p53+/−ErbB2 progeny with p53H/H animals. H/−ErbB2 mice were then crossed to generate p53H/H;ErbB2 and p53−/−ErbB2 females for analysis. These F2 mice were of mixed background. Littermates were used for all analyses. Mice were treated according to the guidelines approved by the Institutional Animal Care and Use Committee. Tissue arrays of breast invasive ductal carcinomas (75 cases/150 cores; BR1504, Biomax, Rockville, MD, USA) with known TNM, pathologic grade and markers (ER, PR, Her2 and Ki67) were stained with antibodies to p53 (DO-1, Santa Cruz Biotechnology) or p326-HSF1 (Epitomics, Burlingame, CA, USA). Staining intensity was scored blindly as absent, weak, moderate or strong. Histoscores were analyzed using Spearman's rank correlation. Significance was calculated using Fisher's exact test.

We used a wound healing assay to measure the migration rate of 4T1 cells with and without GC treatment (100 g/ml of GC for 24 h). GC significantly reduced the motility of 4T1 cells compared with the untreated control for up to 6 h (). Cell migration rates were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) (). Subsequently, we performed an invasion assay using Matrigel-coated transwells, which showed that GC also reduced the invasion of 4T1 cells compared with untreated controls (). Reduced cell migration and invasion were also detected in human MDA-MB-231 breast cancer cells treated by GC (). The dose and treatment duration have been described in the Materials and Methods. The 3-[4,5-dimethylthiazol-2-yl]-2,5diphenylterazoliumbromide (MTT) assays showed that viability of both 4T1 cells and MDA-MB-231 cells was not significantly affected by GC up to a concentration of 250 g/ml (), suggesting that operational levels of GC were non-cytotoxic. A new 4T1_PB3R line of cells was created as described in the Materials and Methods. These cells exhibited high luciferase activity as measured by luminescent assay (). The expression of monomeric red fluorescence protein (mRFP) in 4T1_PB3R cells was pronounced compared with that of parental 4T1 cell, as visualized under fluorescence microscope (). The growth rate () and invasion rate () of 4T1_PB3R cells were the same as that of the parent 4T1 cells. In addition, the viability of 4T1_PB3R cells was not affected by GC, even at a concentration up to 250 g/ml (). These results indicate that it is appropriate to use the 4T1_PB3R cell line as a surrogate for monitoring the behavior of 4T1 tumor cells via reporter gene imaging. 4T1_PB3R cells were subcutaneously implanted into Balb/C mice. After tumors reached a size of 100 mm, tumor-bearing mice were injected with microbubbles via tail vein and treated with HIFU, followed by intratumoral injection of GC (, see Materials and Methods). Tumor-bearing mice were also treated with GC only or HIFU only. The bioluminescent images showed that in untreated mice, tumors metastasized to various locations within 28 days of implantation (). Tumor metastasis was also detectable in mice treated with GC only or HIFU only, but was suppressed in GC-HIFU-treated mice (). Bioluminescent signals from outside the primary tumor in mice of different experimental groups were detected and compared with that of the untreated control group. It appears that GC-HIFU-treated mice showed reduced bioluminescent signals in non-primary tumor sites, suggesting inhibited metastasis (). Although 4T1 cells metastasize to various primary organs, including the lung, liver, bone and brain, we focused on lung metastases in this study because they occurred in all untreated mice (data not shown). We collected lung tissue from tumor-bearing mice for histological analysis 4 weeks after each treatment. Lung metastases were noticeably reduced in mice treated by GC-HIFU compared with all other groups (). The size and the number of nodules in affected lungs were visualized and measured (). These findings strongly suggest that GC and HIFU have a synergistic effect on the suppression of lung metastasis of breast cancer. We measured macrophage activity to demonstrate the immunomodulatory effect of GC-HIFU treatment. Tumor sections were collected 2 weeks after GC, HIFU or GC-HIFU treatment. Immunohistochemical (IHC) staining analysis was used to detect antibodies against the F4/80 protein, a macrophage marker, on each tumor section. There was an accumulation of F4/80 markers in primary tumors treated by GC-HIFU (). The results of IHC were also quantified by counting the number of F4/80-stained cells in tumor sections (). Therefore, GC-HIFU treatment may enhance the innate immune response. Whether this effect leads to modulation of metastasis is unknown and needs to be further investigated. Blood plasma was extracted from tumor-bearing mice 2 weeks after GC, HIFU or GC-HIFU treatment. The plasma was diluted (1 : 10) in culture medium and added to cultured 4T1_PB3R cells in a 96-well plate. Cytotoxicity was then determined using the MTT assay (). shows that plasma obtained from GC-HIFU-treated mice markedly inhibited the viability of 4T1_PB3R cells compared with plasma from any other treatment group (). On the contrary, the viability of non-tumorigenic NIH-3T3 fibroblasts was not affected by any of these treatments (). These results suggest that GC-HIFU treatment may induce tumor-specific immunity. Epithelial–mesenchymal transition (EMT) is an important mechanism for promoting metastasis. According to previous reports, Twist-1, Snail, and Slug are upregulated, whereas epithelial cadherin (E-cadherin) is downregulated during EMT. We compared the protein levels of Twist-1, Snail, Slug, and E-cadherin in 4T1_PB3R cells before and after GC treatment using western blot analysis. Twist-1 levels were slightly reduced after 10 and 100 g/ml of GC treatment for 24 h; further suppression of Twist-1 and Slug was found up to 48 h, but the expression of Snail was not significantly reduced by GC treatment (). The level of E-cadherin was also upregulated by GC; however, lower concentrations of GC showed a greater impact than higher concentrations (). Densitometric quantification of immunoblots further confirmed this observation (). These results suggest that GC may influence the expression of EMT-related makers, at least in part. GC is an effective nonspecific immunoadjuvant for cancer treatment in combination with noninvasive laser photothermal therapy. HIFU can increase the permeability of blood vessels and mediate antitumor immune responses. Thus, we investigated the effect of GC, HIFU, and GC-HIFU on the motility, invasion, and metastatic potential of the 4T1 tumor line. Our wound healing assay and cell migration test show that GC alone reduces cell motility and invasion, despite its lack of direct toxicity toward 4T1 and other cells (). To monitor effectively tumor progression , we created a new 4T1_PB3R cell line. These cells emit both bioluminescent signals (by luciferase expression) and fluorescent signals (by mRFP expression) that can be used for cell tracking , as shown in , while maintaining the properties of their parent 4T1 line, such as growth (), migration (), and GC treatment response (). Our bioluminescent imaging results indicate that GC-HIFU treatment can reduce the metastatic ratio of 4T1_PB3R breast cancer in mice. It is noteworthy that neither treatment by HIFU nor GC alone had any significant effect on the primary tumor or lung metastases (). A recent report proposed that low pressure-pulsed focused ultrasound with microbubbles could promote antitumor immunological responses in a xenograft CT-26 colon tumor animal model. Additionally, pulsatile HIFU has been reported to be beneficial for drug delivery in tumor treatment. Whether this mode can directly stimulate immune responses or enhance immune responses in the presence of an immunostimulator (GC in our case) is unknown. We thus decided to use pulsatile HIFU in combination with GC in our studies. Although our results did not completely agree with previous findings, a different tumor model, ultrasound apparatus, and treatment parameters, may have led to this discrepancy. Because continuous HIFU provides better thermal therapeutic effects on tumor, it may be used in combination with GC in the future as a comparator to this study. Future investigations could correlate HIFU parameters and tumor growth, as well as address the auxiliary role of GC on pulsatile HIFU delivery. Furthermore, HIFU delivery parameters should be optimized to maximize primary tumor eradication. In this study, the GC-HIFU combination was successful in inducing accumulation and activation of macrophages in treated tumors compared with treatment by GC or HIFU alone (). Although this study demonstrated that GC-HIFU could induce significant immune responses in tumors, it does not exclude the possibility that intratumoral injection using needles may cause enough physical damage to induce immune responses. The use of intratumoral injection of GC is based on previous studies; however, other administration routes may be investigated to avoid potent side effects. We have demonstrated that combined GC-HIFU treatment exhibits a synergistic effect in reducing metastases (). However, it is unclear whether GC-HIFU-induced accumulation of macrophages in primary tumors is directly related to reduced lung metastasis . Because tumor growth at the primary site was not suppressed (), macrophage accumulation may be insufficient to inhibit tumor proliferation. Most interesting is our discovery that plasma extracted from mice treated by GC-HIFU reduces the viability of cultured 4T1 cells but not NIH-3T3 fibroblasts (), suggesting a tumor-specific immunity induced by GC-HIFU. The antimetastatic effects of GC-HIFU may be associated with an increase in cytokine release. However, we used enzyme-linked immunosorbent assay (ELISA) to analyze the level change of tumor necrosis factor- (TNF-), and showed no significant difference among all experimental groups (data not shown). Therefore, use of cytokine-based protein array analysis in the future will help us understand which cytokines are implicated in GC-HIFU-mediated systemic immune responses. We found that GC displays remarkable efficacy in reducing mobility and invasion of 4T1 cells and MDA-MB-231 cells, despite the fact that GC is non-toxic to tumor cells. Thus, we postulate that this GC-mediated suppression is not caused by direct cytotoxicity. GC suppressed the Twist-1 and the Slug transcription factors, and induced E-cadherin expression, but did not suppress the expression of the Snail transcription factor that is also influential in EMT. Twist-1 and Snail have been reported to be essential for the maintenance of late EMT and initiation of EMT, respectively. Because 4T1 breast cancer cells are highly metastatic cells, we speculate that this cell type has entered late EMT so that the expression of Twist-1, but not Snail, was more treatment-susceptible. However, further studies are required to better understand the discrepancy between Snail and other EMT-related marker expression after GC treatment. Although more EMT-related markers should be examined, our current data suggest that GC at least partially inhibits EMT. Moreover, whether GC influences the change of other migration- and invasion-related molecules, such as matrix metalloproteinases and tissue inhibitor of metalloproteinases family proteins would be of great interest for further investigation. A microarray assay may be helpful to better understand the underlying mechanisms of GC-mediated inhibition of tumor motility and invasion. Although GC exhibited antimigratory effects on cultured cells, it appeared that GC alone was insufficient to suppress tumor metastasis in animal models. The discrepancies between and effects have been reported in various studies. As shown in our earlier studies, GC may be effective in stimulating a systemic immune response through its interactions with tumor cells at treatment sites. GC's antitumor effect appeared to be mediated by activation of immune cells , rather than direct inhibition of tumor cell migration, although it is difficult to measure the inhibition effect on tumor cells . Furthermore, GC was administered about 7 days after tumor cell seeding in mice. Because the antimigratory effects of GC on cell culture were only investigated in an hourly manner (see the wound healing assay), it seems impossible to reflect completely the results of GC treatment because tumor progression was monitored for several weeks after early treatment of GC. In summary, our results demonstrate that GC, as an immunoadjuvant, is able to reduce the migration of 4T1 breast cancer cells. This effect is likely associated with suppression of EMT-related molecules. The GC-HIFU-induced immune response was demonstrated through the accumulation of macrophages at tumor lesions as well as potent plasma immunity. Combining GC and HIFU results in a synergistic effect on the reduction of the lung metastatic ratio of 4T1 breast cancer cells , which may be related to HIFU-mediated direct tumor destruction and GC-mediated antitumor immunity. This combination may become the foundation for a feasible cancer treatment modality, particularly for metastatic cancers. 4T1 cells are triple-negative (lacking the expression of estrogen, progesterone, and Her2/neu receptors) murine breast carcinoma cells that closely mimic human breast cancer in both tumor growth and metastasis. These cells were cultured in RPMI1640 medium (Gibco; Invitrogen Inc., Carlsbad, CA, USA) with 10% fetal bovine serum (HyClone; Thermo, Waltham, MA, USA), 1% penicillin–streptomycin solution (100 × ) (Caisson Laboratories Inc., North Logan, UT, USA), and 1% -glutamine (200 mM) (Sigma-Aldrich Co., St. Louis, MO, USA). Human triple-negative MDA-MB-231 breast cancer cells and NIH-3T3 cells were maintained in Dulbecco's modified Eagle medium (Gibco; Invitrogen Inc.) with 10% fetal bovine serum, 1% penicillin–streptomycin solution, and 1% -glutamine (200 mM). Cells were maintained in an incubator containing 5% CO at 37 °C and were passaged every 2 days. We previously established a stable murine 4T1-PB-2R/PBase breast cancer cell line containing monomeric red fluorescent protein (mRFP) and herpes simplex virus type 1-thymidine kinase reporter genes using the transposon system for tumor imaging. We modified this system by adding a firefly luciferase () gene to the original dual-cistronic PB-2R construct to create a new PB-3R construct for two reasons. First, we wanted to overcome the low sensitivity of mRFP for optical imaging. Second, has a high sensitivity for bioluminescent imaging of metastases. 4T1 cells were stably transfected with this multicistronic reporter gene system to create a new 4T1_PB3R cell line. The PB-3R-puro plasmid was constructed from PB-2R plasmid that was provided by Dr. Yu Kang and Dr. Congjian Xu. The 4T1_PB3R cells were cultured in RPMI medium as described above. GC (10 mg/ml, dissolved in deionized distilled water) was prepared as described previously. GC was stored at 4 °C and was transferred to room temperature before usage. The dosage of GC administration was dependent on the experimental purposes as described below. 4T1 cells (2 × 10 cells per well) were seeded in a 6-well plate. After cells reached 80% confluence, 100 g/ml of GC was added to the wells for 24 h. Phosphate-buffered saline (PBS) of the same volume was added to the cells in the control groups. The wounds were then incised using a 200 l Pipetman tip in three random positions in each well. Detached cells were washed with PBS and fresh medium was then added. Wound healing was visualized at 3 and 6 h under a light microscope to confirm gap width consistency. The wound healing rate was determined by visualizing cell migration to the scraped area using light microscopy, and images were acquired and analyzed using ImageJ software (version 1.46). Matrigel (BD Biosciences, San Jose, CA, USA) was mixed with 25 l serum-free medium at a ratio of 1 : 4, added into transwells (24 Well Millicell 8.0 μm; Millipore Co., Billerica, MA, USA), and then incubated at 37 °C overnight. Cultured 4T1 cells grown in exponential phase were treated with 100 g/ml of GC or left untreated for 24 h and then trypsinized. Cells (1 × 10) were then mixed with 200 l serum-free medium and added into each transwell. Each transwell was then placed into a 24-well plate containing 400 l normal medium in each well. After 24 h of incubation, each transwell was washed with 1 × phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde (Sigma-Aldrich Co., St. Louis, MO, USA) for 10 min. The transwells were then incubated in 1.25% crystal violet solution (Sigma-Aldrich Co.) for 2 min. After PBS destaining, the membranes embedded in the transwells were cut and placed onto slides for microscopic visualization. Crystal violet-stained cells on the membrane were then counted. 4T1 cells were seeded in a 96-well plate (800 cells per well) with GC of various concentrations (0, 50, 100, 150, 200, and 250 g/l, and incubated at 37 °C for 4 days. After removal of the supernatant, 1 mg/ml MTT solution (Sigma-Aldrich Co.) was mixed with serum-free medium and added to each well. The cells were then incubated at 37 °C for 3 h. After removal of the MTT solution, 100-l dimethyl sulfoxide was added to dissolve crystals. The plate was then placed in an ELISA reader (ELISA plate reader; Bio-Tek Instruments, Winooski, VT, USA) and cell viability was determined by light absorption at 570 nm. 4T1 and 4T1_PB3R tumor cells (1 × 10 in 100 l serum-free medium) were subcutaneously injected into the upper backs of ∼4- to 5-week-old female Balb/c mice (=6 for each experimental group) (National Laboratory Animal Center, Taipei, Taiwan). Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang-Ming University (Taipei, Taiwan; approval number: 1011103). When the tumors became palpable, their dimensions were measured via caliper two times a week. We estimated tumor volume with the equation volume=length (mm) × width (mm)/2. Before HIFU treatment, the skin overlying the tumor was epilated and covered with ultrasound transmission gel (Pharmaceutical Innovations, Newark, NJ, USA). The ultrasound contrast agent used in this procedure contained phospholipid-coated microbubbles with a mean diameter of 2.5 m, at a concentration of 1 × 10–5 × 10 bubbles per ml. The tumor-bearing mice were then injected with microbubbles (300 l/kg) via tail vein. HIFU was subsequently supplied using a single-element focused ultrasound transducer (A392S; Panametrics, Waltham, MA, USA) with a diameter of 38 mm, a radius of curvature of 63.5 mm, and a center frequency of 1 MHz. The transducer delivered HIFU at an acoustic power of 1.43 W, a pulse repetition frequency of 1 Hz, and a duty cycle of 5% for 2 min. The setup for the transducer-driving system was the same as that of our previous work. After HIFU, a 100-l GC solution (10 mg/ml) was injected into the center and three additional random locations in each tumor using a 27 G needle as described in previous reports, with slight modifications. 4T1_PB3R tumor cells expressing a firefly luciferase gene were subcutaneously injected into the upper backs of Balb/c mice. Each tumor-bearing mouse (=6 for each experimental group) then received an intraperitoneal injection of 150 mg/kg -luciferin (Caliper Co., Hopkinton, MA, USA). After 15 min, animals were anesthetized with 2% isofluorane, and luminescent signals were detected via IVIS50 system (Xenogen Co., Alameda, CA, USA). Region of interest tracings were drawn around each tumor site and luminescent signal was quantified by the number of photons detected per second. Lung tissue samples were excised from three random tumor-bearing mice after different treatments for 28 days and immediately fixed in 4% paraformaldehyde (Sigma-Aldrich Co.) overnight and then embedded with paraffin. Ten tissue sections (each 5 m) were collected for analysis. They were deparaffinized using xylene immersion (Sigma-Aldrich Co.) for 30 min, rehydrated for 5 min in 70, 80, or 90% ethanol, and then stained with hematoxylin and eosin. We also performed IHC analysis of macrophage markers in primary tumors. Tumor sections were treated with antigen retrieval solution (100 °C 2,2′,2′′,2′′′-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), pH=8) for 25 min and cooled on ice for 30 min. The sections were subsequently blocked with 3% HO and protein blocking reagent (Biogenex Laboratories, San Ramon, CA, USA) in the dark and then incubated with anti-mouse F4/80 antibody (eBioscience Inc., San Diego, CA, USA) for 2 h, followed by horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. Finally, tissue sections were incubated with Liquid DAB+Substrate Chromogen System (Dako North America Inc., Carpinteria, VA, USA) until a brown color developed, and then counterstained with hematoxylin. The slides were visualized under light microscope, entire tissue sections were examined, and three random regions were selected and photographed using a digital camera. Mice (=6 for each experimental group) were treated with GC and HIFU as described above and killed using cervical dislocation. Blood was immediately collected via cardiac puncture using a 26 G needle and a syringe filled with 120 mg/ml EDTA. Extracted blood was centrifuged at 1000 r.p.m. at 4 °C for 10 min. The supernatant was then collected and centrifuged two more times. The plasma was then sterilized with a 0.2 m Super Membrane Low Protein Binding filter (PALL Inc., Port Washington, NY, USA). Plasma was diluted (1 : 10) in culture medium and added into a 96-well plate seeded with 4T1 cells for MTT analysis. 4T1 cells were incubated in GC solution at different concentrations (0, 10, and 100 g/ml) for 24 and 48 h. Protein was then extracted from cells using protein lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 0.5% NP-40) with 2% PMSF and quantified using the Bio-Rad Protein Assay (Bio-Rad Laboratories Inc., Hercules, CA, USA). Protein was mixed with sampling buffer (250 mM Tris-HCl (pH 6.8), 10% sodium dodecyl sulfate (SDS), 30% glycerol, 5% -mercaptoethanol, 0.02% bromophenol blue), denatured with heating, and subjected to SDS-polyacrylamide gel electrophoresis Gel was electrotransferred to a nitrocellulose membrane (BioTraceTM NT; Pall, Port Washington, NY, USA) after electrophoresis, and the membrane was blocked with 4% milk in Tris-buffered saline with Tween-20 buffer (150 mM NaCl, 10 mM Tris-HCl (pH 8.0), and 0.1% Tween-20) for 2 h. The membrane was incubated with primary antibody overnight, and followed by HRP-conjugated secondary antibody. The membrane was rinsed with Western lightning plus-ECL (Perkin-Elmer Inc., Waltham, MA, USA) and the chemoluminescent signals were detected using the LAS-4000 gel imaging system (GE Healthcare Inc., Wauwatosa, WI, USA). The band densities were quantified using ImageJ software (version 1.46). The primary antibodies used in this study included anti-Twist-1, anti-Slug, anti-Snail and anti-E-cadherin (kindly provided by Dr. Muh-Hwa Yang at National Yang-Ming University, Taipei, Taiwan). Anti-glyceraldehyde 3-phosphate dehydrogenase antibodies were used as a control and were purchased from GeneTex Inc. (Irvine, CA, USA). Experimental data were represented as the mean of three independent experiments±S.D. Data were analyzed with -test or Mann–Whitney test (for animal experiments), with <0.05 indicating statistical significance.

We previously identified PDGFR cells in the interstitial space of mouse skeletal muscle and showed that these cells represent a unique mesenchymal progenitor population distinct from satellite cells. Because PDGFR mesenchymal progenitors have been demonstrated to contribute to pathologic changes of skeletal muscle such as fatty degeneration and fibrosis in a mouse model, it is clinically important to characterize such cells in humans. Therefore, we analyzed human skeletal muscle using PDGFR as a marker for mesenchymal progenitors. We first identified satellite cells on human muscle sections. M-cadherin, Pax7 and CD56 have been used as markers for human satellite cell identification, but it was also reported that basal lamina staining was necessary for reliable detection of human satellite cells. When human muscle sections were stained with antibodies against M-cadherin, Pax7 and laminin, M-cadherinPax7 satellite cells locating beneath the basal lamina were identified (). We observed 434 M-cadherin sublaminar satellite cells on muscle sections from 10 different patients, and found 99.5% of them were also positive for CD56 (). Thus, these markers in combination with basal lamina staining were useful for the identification of human satellite cells. We next examined the relationship between satellite cells and PDGFR cells. PDGFR cells were found in the interstitial space of human muscle, whereas satellite cells (stained with Pax7 or CD56 antibody) were localized beneath the basal lamina, as described above (). We carefully examined muscle sections from 24 different patients, but never observed PDGFR cells that satisfy the criteria for satellite cells (markers and localization). These results indicate that PDGFR cells and satellite cells represent discrete cell populations in humans, similar to the results obtained from mice. PDGFR cells were more frequently observed in the perimysium than in the endomysium and were conspicuous in the perivascular space (). We next sought to isolate PDGFR cells from human muscle. Because the starting volume of human muscle obtained from biopsy was limited (typically 0.1–0.3 g), we enzymatically digested human muscle and first cultured dissociated cells. Because human muscle-derived cells grew vigorously in a hypoxic condition (3% O) compared with a normoxic condition (see ), we used the hypoxic condition to expand or maintain the cells in the following experiments. However, cells in a normoxic condition showed more efficient differentiation (data not shown), and therefore a normoxic condition was used in differentiation experiments. When cells reached ∼70% confluence, human muscle-derived cells were analyzed for PDGFR and CD56 expression by flow cytometry. Populations positive for these markers were clearly observed in varying percentages in 30 different preparations (PDGFR cells ranging from 10 to 73% and CD56 cells ranging from 6 to 68% ). Importantly, PDGFR cells and CD56 cells represented discrete single-positive populations and never overlapped each other. Because CD56 cells was reported to give rise to CD56 cells and CD56 cells lose CD56 expression during long-term culture, we reanalyzed PDGFR and CD56 expression after two passages (totally, three passages). Almost all PDGFR cells maintained their PDGFR single-positive state, and so did CD56 cells in our culture condition (). This was also confirmed by immunofluorescent staining of cultured cells (see ). The cell surface phenotype of PDGFR cells differs from that of CD56 cells in the expression of CD90 and CD166, but is similar to that of bone marrow-derived mesenchymal stromal cells (). It has been reported that CD166, also known as ALCAM, is expressed on human bone marrow-derived mesenchymal stem cells. Three-color fluorescence-activated cell sorting (FACS) analysis revealed that CD56PDGFR cells are almost identical to CD56CD166 cells (see ). Therefore, CD166 can be an additional marker to identify mesenchymal progenitors in human muscle. The three human muscle-derived cell populations were sorted by FACS, and gene expression was examined by RT-PCR. Myogenic genes were detected only in CD56 cells, indicating that satellite cell-derived myogenic cells were exclusively sorted in this population (). After culturing in the growth condition, the myogenic markers MyoD and Pax7 were again detected only in CD56 cells, and other populations did not become positive for these markers (). The three human muscle-derived cell populations were induced to differentiate into adipocytes. PDGFR cells efficiently differentiated into adipocytes, adopting a spherical shape, accumulating lipid, and expressing the adipogenic transcription factors C/EBP and PPAR (). After adipogenic differentiation, PDGFR cells were no longer positive for PDGFR (data not shown). CD56 cells did not show any adipogenic activity, but a few CD56PDGFR cells differentiated into C/EBPPPAR adipocytes (). However, PDGFR cells were stained significantly more by Oil Red O than the other two populations (), indicating that adipogenic differentiation potential is highly enriched in the PDGFR population of human muscle-derived cells. PDGFR cell-derived adipocytes express both white and brown adipocyte-specific genes, and , indicating that PDGFR cells possess the capacity to differentiate into both white and brown adipocytes (). When myogenic differentiation was evaluated after adipogenic induction, we did not see any myogenic activity in PDGFR cells and CD56PDGFR cells (). However, even on adipogenic induction, CD56 cells differentiated into large well-developed myotubes expressing myogenic proteins such as MyoD and myosin heavy chain (MyHC) (). In light of the results that CD56 cells cannot undergo adipogenesis (), CD56 cells appear to be committed to a myogenic lineage and cannot convert their fate into other lineages. In the mouse, PDGFR cells not only differentiate into adipocytes but also produce fibrogenic cells and contribute to fibrosis of the skeletal muscle. We therefore investigated the fibrogenic property of human muscle-derived PDGFR cells. We performed clonal culture and found that 71 out of 576 PDGFR cells formed colonies and that 29.6% of these colonies were adipogenic. Interestingly, all adipogenic colonies consisted not only of adipocytes but always contained collagen I or -smooth muscle actin-expressing fibrogenic cells (). These results suggest that a single human muscle-derived PDGFR cell can produce both adipocytes and fibrogenic cells. Transforming growth factor-1 (TGF-1) is known as potent profibrogenic cytokine, and we previously reported that this cytokine induces the expression of fibrosis markers in mouse PDGFR cells. Human muscle-derived PDGFR cells expressed much higher levels of fibrosis-related molecules compared with CD56 cells, and these expressions were further upregulated in response to TGF-1 stimulation (). TGF-1 treatment did not affect MMP activity and TIMP-1 expression (), suggesting that upregulation of collagens and -SMA does not merely reflect an increase in remodeling. These results suggest the possibility that fibrosis observed in human skeletal muscle is mainly attributable to PDGFR cells rather than CD56 myogenic cells. Constitutively active PDGFR knock-in mice displayed connective tissue hyperplasia and developed systemic fibrosis including the skeletal muscle, and stimulation of PDGFR signaling promoted proliferation of mouse PDGFR cells. These reports prompted us to examine the effect of PDGF-PDGFR signaling on human muscle-derived PDGFR cells. Addition of PDGF-AA to serum-free medium (SFM) activated PDGFR on human PDGFR cells, and this activation was inhibited by imatinib, a tyrosine kinase inhibitor, or by a specific neutralizing antibody (). The effect of PDGFR signaling on the proliferation of PDGFR cells was evaluated by measuring 5-bromo-2′-deoxyuridine (BrdU) incorporation. PDGFR stimulation promoted the proliferation of PDGFR cells, and imatinib or neutralizing antibody completely inhibited this growth-stimulating activity (). To further investigate the signaling pathway through which PDGFR promotes the proliferation of PDGFR cells, we used inhibitors of PI3K-Akt and Ras-MAPK pathways, which are known to be downstream signaling pathways of PDGFR. The inhibitors did not affect the viability of PDGFR cells at the concentrations used in these experiments (see ). LY294002 (PI3K inhibitor) and U0126 (MEK1/2 inhibitor) completely inhibited the stimulatory effect of PDGF-AA, but PD98059 (MEK1 inhibitor) had no effect (), suggesting that PDGFR exerts its proliferative effect through PI3K-Akt and MEK2-MAPK pathways. LY294002 strongly inhibited Akt phosphorylation induced by PDGF-AA but had no effect on MAPK activation, whereas U0126 strongly inhibited p44/42 MAPK phosphorylation induced by PDGF-AA but had no effect on Akt activation (). Thus, both PI3K-Akt and MEK2-MAPK pathways are necessary for PDGFR-driven proliferation. To establish the clinical relevance of PDGFR cells, we examined skeletal muscles of DMD patients. In DMD, dystrophin deficiency evokes repeated regeneration–degeneration cycles and eventually leads to the development of fatty and fibrous connective tissue in the skeletal muscle. Many PDGFR cells were observed around ectopically formed fat cells in DMD muscles (). Although fat cells themselves were negative for PDGFR, we found some PDGFR cells expressing PPAR in proximity to perilipin ectopic fat cells (). When we looked at fibrotic areas of DMD muscles, a striking increase in the number of PDGFR cells coincided with increased fibrosis (). Considerable accumulation of PDGFR cells was prominent, particularly around blood vessels, but they never overlap with vascular components such as endothelial cells or mural cells (). Such aberrant behavior of PDGFR cells was observed in all the patients we investigated. In contrast to PDGFR cells, CD56 cells were not observed around ectopic fat and in fibrotic areas where many PDGFR cells were localized, but they were located on the inside of the basal lamina (). We next investigated skeletal muscles of a patient who suffered from Volkmann's contracture. Volkmann's contracture is caused by acute ischemia and necrosis of the muscles of the forearm and results in fatty and fibrous degeneration of affected muscles. Thus, this condition represents a non-genetic disorder of skeletal muscle. We found severe fatty and fibrous degeneration with the increased number of PDGFR cells in the patient's muscle (). We next explored the relation between the extent of PDGFR staining and severity of fibrosis. Muscles from 11 DMD patients with a range of symptoms and 3 normal muscle samples were analysed. HE staining revealed a broad range of dystrophic phenotype of the patients ( and ). Muscles with severe dystrophic phenotype typically showed more intense PDGFR staining ( and ). Many PDGFR cells were localized to collagen-deposited fibrotic areas in DMD muscles, but only a few PDGFR cells could be encountered in the interstitial space of normal muscles where weak collagen expression was observed ( and ). The extent of PDGFR staining was graded on a scale of 1–5 (grade 1 represents muscles with occasional PDGFR cells in the interstitium and grade 5 represents muscles with numerous PDGFR cells in the extended interstitial space, see ), and the degree of fibrosis was quantified by measuring hydroxyproline (HOP) content. Statistical analysis indicate significant correlation between the extent of PDGFR staining and severity of fibrosis (). Taken together, these results strongly suggest that PDGFR mesenchymal progenitors are involved in the pathogenesis of human skeletal muscle. Fatty and fibrous degeneration is a hallmark of diseased skeletal muscle and is very prominent in pathological conditions such as muscular dystrophy or aging. Because fatty and fibrous connective tissue lacks contractile ability, it becomes an aggravating factor in muscle weakness. We previously identified mesenchymal progenitors residing in mouse muscle interstitium using PDGFR as a specific marker and demonstrated that these cells directly contribute to fatty and fibrous connective tissue development in the skeletal muscle. A human counterpart to these progenitors would be a therapeutic target for muscle diseases accompanied by fat infiltration and fibrosis. Other groups also identified progenitors with adipogenic potential in mouse skeletal muscle using Sca-1 as a marker. However, there is no human ortholog of Sca-1; in fact, a 500-kb region of mouse chromosome 15 encoding several Ly6 family members including Sca-1 was deleted between mouse and rat speciation. Thus, this marker is not applicable to human study. In this study, we used PDGFR, which is conserved in humans, and demonstrated that cells equivalent to mouse PDGFR mesenchymal progenitors can be identified in human muscle using the same marker. PDGFR was also used for the prospective isolation of mesenchymal stem cells from murine embryos and bone marrow of adult mice. Furthermore, a recent study succeeded in purifying mesenchymal stem cells possessing hematopoietic stem cell niche activity from human bone marrow by using PDGFR in combination with CD51. Because mesenchymal stem/progenitor cells are reported to exist in almost all organs of both mice and humans, it will be interesting to see whether PDGFR can serve as a universal marker to identify mesenchymal stem/progenitor cells in diverse organs of various species. Although the functional significance of PDGFR in development is evident as PDGFR-null mice die , its role in the adult physiological context has not yet been completely understood. However, several studies, including ours, revealed a link between PDGFR and fibrogenesis. Olson and Soriano generated mice in which mutant PDGFR with increased kinase activity was knocked into a PDGFR locus and thus increased PDGFR signaling is operative only in cells that express PDGFR endogenously. Importantly, the engineered mice showed progressive fibrosis in multiple organs including the skeletal muscle. A connection between PDGFR and the pathogenesis of fibrosis has also been revealed by studies using imatinib, an inhibitor of several tyrosine kinases, including PDGFR, c-kit, and bcr-abl oncogene. Imatinib was shown to ameliorate the muscle dystrophy of mdx mice. We also showed that imatinib is effective for improving dystrophic symptoms of DBA/2-mdx, a more severe mouse model of muscular dystrophy. Imatinib-treated mdx muscle showed decreased PDGFR phosphorylation, and treatment of PDGFR mesenchymal progenitors with imatinib resulted in inhibition of PDGFR-induced proliferation and expression of fibrotic genes, indicating that the therapeutic effect of imatinib was achieved at least in part through targeting PDGFR mesenchymal progenitors. The antifibrotic effect of imatinib associated with inhibition of PDGFR activity was also seen in a rat model of myocardial fibrosis. In the present study, we showed that imatinib or a specific neutralizing antibody effectively inhibited PDGFR phosphorylation and proliferation of human PDGFR mesenchymal progenitors. The growth-stimulating activity of PDGFR depends on both PI3K-Akt and MEK2-MAPK signaling pathways. Importantly, increased accumulation of PDGFR cells was conspicuous in the skeletal muscle of patients with both genetic and non-genetic muscle diseases, whereas this aberrant increase in the number of PDGFR cells was never found in healthy muscle. Consequently, PDGFR may have great value not merely as a good marker but also as a molecular therapeutic target for muscular disorders associated with fibrosis. Wosczyna demonstrated that Tie2PDGFRSca-1 progenitors are the cellular origin of ectopic bone in a BMP2-induced mouse model of heterotopic ossification (HO). The progenitor cells identified by Wosczyna appear identical to the mesenchymal progenitors described by us because both of them are located in the muscle interstitium and showed similar expression profiles of cell surface antigens. We further showed that human muscle-derived PDGFR mesenchymal progenitors formed bone-like tissue after subcutaneous transplantation into immunodeficient mice, and that the number of PDGFR cells was increased in proximity to ectopically formed bone in the muscle of a patient who developed HO. Therefore, it is highly probable that PDGFR mesenchymal progenitors contribute to HO in addition to fatty and fibrous connective tissue formation. In addition to their roles in pathogenesis of skeletal muscle, PDGFR mesenchymal progenitors have been shown to regulate positively muscle regeneration in mouse. Joe showed that mesenchymal progenitors promote myogenic differentiation of cocultured satellite cells. Their support function in muscle regeneration has also been demonstrated by using genetically engineered mice that permit conditional ablation of Tcf4 connective tissue fibroblasts. Tcf4 cell-ablated mice showed impaired muscle regeneration with premature satellite cell differentiation, depletion of the early pool of satellite cells and smaller regenerated myofibers. Although precise relationship between Tcf4 cells and mesenchymal progenitors remains to be demonstrated, Tcf4 cells express PDGFR. Thus, these cells appear to be largely overlapping. Given the pathophysiological importance of PDGFR mesenchymal progenitors, isolation of these progenitors from human muscle could be of considerable clinical value. Our method for isolating mesenchymal progenitors is based on the expression of PDGFR, which is specific and functionally important, and therefore reliable and highly reproducible. Thus, techniques described in this study provide a platform for studying muscle pathophysiology and developing a therapeutic strategy to treat muscle diseases. Experiments using human samples were approved by the Ethical Review Board for Clinical Studies at Fujita Health University. Non-dystrophic muscle samples were obtained from gluteus medius muscles of patients undergoing total hip arthroplasty. Muscle samples for histological analysis were obtained from 26 patients ranging in age from 32 to 85 years. Muscle samples for cell culture were obtained from 35 patients ranging in age from 35 to 78 years. DMD muscle samples were obtained from muscle (rectus femoris or biceps brachii) biopsies performed on 16 DMD patients ranging in age from 9 months to 16 years for diagnostic purposes. Dystrophin deficiency was confirmed by immunohistochemistry and western blotting. For Volkmann's contracture muscle samples, forearm muscle tissues were obtained from a 54-year-old patient diagnosed with Volkmann's contracture after trauma to the axillary brachial artery. All patients or their parents gave written informed consent. Muscle samples for histochemistry were rapidly frozen in isopentane cooled with liquid nitrogen. Frozen muscle sections (7 m thick) were cut by cryostat and fixed with 4% paraformaldehyde (PFA) for 5 min. The Volkmann's contracture sample was fixed with 10% formaldehyde, and then embedded in paraffin. Paraffin-embedded sections (5 m thick) were deparaffinized and treated with Antigen Retrieval Reagent (R&D, Minneapolis, MN, USA). Cultured cells were rinsed with PBS and then fixed with 4% PFA for 5 min. Specimens were blocked with protein-block serum-free reagent (Dako, Glostrup, Denmark) for 15 min, and incubated with primary antibodies at 4 °C overnight, followed by secondary staining. Primary antibodies used were anti-PDGFR (2.5 g/ml; R&D; cat. no. AF-307-NA), anti-M-cadherin (1 : 200; R&D), anti-Pax7 (1 : 2; DSHB, Iowa City, IA, USA), anti-CD56 (1 : 20; Miltenyi Biotec, Bergisch Gladbach, Germany), anti-laminin (1 : 200; Sigma, St. Louis, MO, USA), anti-MyoD (1 : 400; Santa Cruz, Dallas, TX, USA), anti-C/EBP (1 : 400; Santa Cruz), anti-PPAR (1 : 100; Santa Cruz), anti-MyHC (1 : 2; DSHB), anti-collagen I (1 : 250; Abcam, Cambridge, MA, USA), anti-FABP4 (1 : 50; R&D), anti--SMA (1 : 200; Sigma), anti-phospho-Akt (1 : 100; Cell Signaling, Danvers, MA, USA), anti-phospho-p44/42 MAPK (1 : 200; Cell Signaling), and anti-perilipin (1 : 250; Sigma). Secondary antibodies used with a dilution of 1 : 1000 were Alexa Fluor 488- or 568-conjugated anti-goat IgG (Molecular Probes, Carlsbad, CA, USA), CF488A-conjugated anti-sheep IgG (Biotium, Hayward, CA, USA), Cy3-conjugated anti-mouse IgG (Jackson, West Grove, PA, USA), and Alexa Fluor 488- or 647-conjugated anti-rabbit IgG (Molecular Probes). Specimens were counterstained with DAPI (Invitrogen, Carlsbad, CA, USA) and mounted with SlowFade Gold anti-fade reagent (Invitrogen). For sequential immunohistochemistry and HE staining, immunofluorescent staining was performed first and immunofluorescent images were captured. Slides were immersed in PBS to remove the cover glass and subjected to HE staining. Then, images of the corresponding HE-stained fields were captured. To stain lipids, cells were fixed in 10% formalin, rinsed in water, and then 60% isopropanol, stained with Oil Red O in 60% isopropanol, and rinsed in water. Stained cells and tissues were photographed using a fluorescence microscope BX51 (Olympus) equipped with a DP70 CCD camera (Olympus, Tokyo, Japan) or an inverted fluorescence microscope DMI4000B (Leica, Wetzlar, Germany) equipped with a DFC350FX CCD camera (Leica). Confocal images of muscle sections were taken using the confocal laser scanning microscope system LSM700 (Carl Zeiss, Oberkochen, Germany). Muscles were transferred to PBS. Nerves, blood vessels, tendons, and fat tissues were carefully removed under a dissection microscope. Trimmed muscles were minced and digested with 0.2% type II collagenase (Worthington, Lakewood, NJ, USA) for 30 min at 37 °C. Digested muscles were passed through an 18 G needle several times and further digested for 10 min at 37 °C. Muscle slurries were filtered through a 100-m cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) and then through a 40-m cell strainer (BD Biosciences). Erythrocytes were eliminated by treating the cells with Tris-buffered 0.8% NHCl. Cells were resuspended in the growth medium (GM) consisting of DMEM supplemented with 20% FBS, 1% penicillin–streptomycin, and 2.5 ng/ml bFGF (Invitrogen), and maintained at 37 °C in 5% CO and 3% O. Human bone marrow mesenchymal stromal cells (Lonza, Basel, Switzerland) were maintained in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in 5% CO and 21% O. Cells were trypsinized and resuspended in washing buffer consisting of PBS with 2.5% FBS, and stained with primary antibodies for 30 min at 4 °C. Cells were then stained with streptavidin-PE/Cy5 (1 : 200; BD Pharmingen, Franklin Lakes, NJ, USA) for 30 min at 4 °C in the dark. Primary antibodies used for cell staining were PE-conjugated anti-CD56 (1 : 20; Miltenyi Biotec), biotinylated anti-PDGFR (2.5 g/ml; R&D; cat. no. BAF322), FITC-conjugated anti-CD34 (1 : 10; BD Pharmingen), FITC-conjugated anti-CD45 (1 : 10; BD Pharmingen), FITC-conjugated anti-CD90 (1 : 200; BD Pharmingen), FITC-conjugated anti-CD105 (1 : 10; BioLegend, San Diego, CA, USA), and FITC-conjugated anti-CD166 (1 : 20; MBL, Aichi, Japan). Stained cells were analyzed by FACSCalibur or FACSVantage SE (BD Biosciences). Cell sorting was performed on a FACSVantage SE. For adipogenic differentiation, sorted cells were cultured on Matrigel-coated (BD Biosciences) eight-well chamber slides (Nalge Nunc, Waltham, MA, USA) in GM at 37 °C in 5% CO and 3% O. Ten thousands cells were plated per well. After 3 days, cells were treated with an adipogenic induction medium consisting of DMEM with 10% FBS, 0.5 mM IBMX (Sigma), 0.25 M dexamethasone (Sigma), and 10 g/ml insulin (Sigma) for 3 days, followed by adipogenic maintenance medium consisting of DMEM with 10% FBS and 10 g/ml insulin for 1 day at 37 °C in 5% CO and 21% O. This treatment was repeated three times. For clonal analysis, sorted PDGFR cells were seeded onto a Matrigel-coated 96-well plate at a density of 0.8 cell per well and incubated for 7 days in GM at 37 °C in 5% CO and 3% O. After 7 days, adipogenic induction was carried out as described above. For TGF-1 stimulation, cells were cultured in 10% FBS-DMEM with or without 5 ng/ml TGF-1 (R&D) for 3 days. For PDGF-AA stimulation, cells were serum-starved for 24 h by culturing in SFM consisting of DMEM supplemented with 2% Serum Replacement 1 reagent (Sigma). Then, cells were cultured in SFM with or without 10 ng/ml PDGF-AA (R&D) for 2 days. Imatinib (10 M; Alexis, Farmingdale, NY, USA), anti-PDGFR (5 g/ml; R&D; cat. no. AF-307-NA), LY294002 (25 M; Cell Signaling), PD98059 (50 M; Cell Signaling), or U0126 (10 M; Cell Signaling) were added 1 h before PDGF-AA stimulation. Cell viability was examined using ProstoBlue reagent (Invitrogen). Total RNA was extracted using an RNeasy Micro Kit (Qiagen, Hilden, Germany), and equal amounts of RNA were reverse transcribed into cDNA using a QuantiTect Reverse Transcription Kit (Qiagen). The PCR reactions were performed with 0.5 l cDNA product under the following cycling conditions: 94 °C for 30 s, followed by 30 cycles of amplification (94 °C for 5 s, 60 °C for 20 s, 72 °C for 10 s), and a final incubation at 72 °C for 5 min. Real-time quantitative PCR was performed on a Thermal Cycler Dice Real Time System (Takara, Shiga, Japan) with SYBR Premix Ex Taq (Takara) under the following cycling conditions: 95 °C for 10 s, followed by 40 cycles of amplification (95 °C for 5 s, 60 °C for 15 s, 72 °C for 10 s) and dissociation curve analysis. Specific primer sequences used in this study were: 5′-TGGCTTTCAACCATCTCATTC-3′ and 5′-GTGCTGCTGGGATCTGACAC-3′ for Pax3, 5′-GACCCCTGCCTAACCACATC-3′ and 5′-GTCTCCTGGTAGCGGCAAAG-3′ for Pax7, 5′-GAGGTGTACCACGACCAACC-3′ and 5′-CCTGCTCTCTCAGCAACTCC-3′ for MYF5, 5′-GCCACAACGGACGACTTCTATG-3′ and 5′-TGCTCTTCGGGTTTCAGGAG-3′ for MYOD1, 5′-TCCTCTGCCTGACATTGACC-3′ and 5′-TGAAGGTGGAACTGCTGGAAC-3′ for PDGFRA, 5′-AACCCTGTGCGGATTCTTG-3′ and 5′-GGAGACTGACTGCGTGTGTG-3′ for LEP, 5′-AAATCAGCTCCGCCTCTCTC-3′ and 5′-GGGTTGCCCAATGAATACTG-3′ for UCP1, 5′-GCAAGGTGTTGTGCGATGAC-3′ and 5′-TTGGTCGGTGGGTGACTCTG-3′ for COL1A1, 5′-AACCAGGAGCTAACGGTCTCA-3′ and 5′-CCAGGGTTTCCATCTCTTCC-3′ for COL3A1, 5′-CAATGGCTCTGGGCTCTGT-3′ and 5′-TGCTCTGTGCTTCGTCACC-3′ for ACTA2, and 5′-ACCCACTCCTCCACCTTTGA-3′ and 5′-TTGCTGTAGCCAAATTCGTTG-3′ for GAPDH. To measure the extent of differentiation of cultured cells, five to seven randomly selected fields were photographed per well. Images were collected and pooled from three independent experiments. The percentage of positive cells was determined by dividing the number of marker cells by the number of DAPI nuclei using the Win ROOF software (Mitani Corp., Fukui, Japan). To assess the efficiency of adipogenic differentiation, Oil Red O-stained cells were extracted with 100 l of 4% NP-40 in isopropanol for 5 min, and the absorbance of the dye–triglyceride complex was measured at 520 nm. Cells were lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM NaF, 1% NP-40, and protease inhibitor cocktail (Roche, Basel, Switzerland). For the detection of phosphorylated proteins, lysis buffer was supplemented with phosphatase inhibitor cocktail (Roche). Cell lysates were centrifuged at 1000 × for 10 min at 4 °C and the supernatants were recovered. Aliquots of the lysates containing 10 g of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were probed with anti-collagen I (1 : 1000; Abcam), anti-collagen III (1 : 2000; Abcam), anti-GAPDH (1 : 2000; Cell Signaling), anti-phospho-Akt (1 : 2000; Cell Signaling), anti-Akt (1 : 1000; Cell Signaling), anti-phospho-p44/42 MAPK (1 : 2000; Cell Signaling), and anti-p44/42 MAPK (1 : 1000; Cell Signaling). After incubation with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence reactions, images of the developed immunoblots were captured using a Light-Capture imaging system (Atto, Tokyo, Japan). TGF-1 stimulation was carried out as described above. Cell supernatants were collected and centrifuged at 1000 × for 10 min at 4 °C. The supernatants were recovered and MMP activity in the supernatants was measured by SensoLyte Generic MMP Fluorimetric Assay Kit (AnaSpec, Fremont, CA, USA). TGF-1 stimulation was carried out as described above. Cell supernatants were collected and centrifuged at 1000 × for 10 min at 4 °C. The supernatants were recovered and TIMP-1 concentration in the supernatants was measured by Human TIMP-1 Quantikine ELISA Kit (R&D). PDGFR cells were seeded onto 100 mm dishes. PDGF-AA stimulation was carried out as described above. Cells were rinsed with PBS two times and then lysed in 250 l of lysis buffer consisting of 1% NP-40 Alternative, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM activated sodium orthovanadate, 10 g/ml aprotinin, and 10 g/ml leupeptin for 15 min on ice. Samples were centrifuged at 10 000 × for 5 min at 4 °C, and supernatants were recovered. Protein concentrations were quantified using DC protein assay (Bio-Rad, Hercules, CA, USA). PDGFR phosphorylation was measured using equal amount of proteins by Human Phospho-PDGFR DuoSet IC Kit (R&D). PDGFR cells were seeded onto 96-well plate. PDGF-AA stimulation was carried out as described above. One day after adding PDGF-AA, 10 M BrdU was added to each well. After 2 days stimulation, BrdU incorporation was measured using Cell Proliferation ELISA Kit (Roche). Frozen muscle tissues of 10–30 mg were homogenized in HO by Shakeman homogenizer (BMS, Tokyo, Japan) using 100 l HO for every 10 mg of tissue. HOP content in the homogenates was measured using HOP Colorimetric Assay Kit (BioVision, Milpitas, CA, USA). Significance of the difference between two groups was assessed by Student's -test. For comparisons of more than two groups, one-way analysis of variance (ANOVA) followed by Tukey's test or Dunnett's test was used for statistical analysis. To assess correlation between the extent of PDGFR staining and severity of fibrosis, Spearman's rank correlation coefficient was used. A probability of <5% (<0.05) was considered statistically significant.

Features of NL (normal liver) individuals and NAFLD patients studied are detailed in . All groups were well matched in terms of age and sex distribution. In the NAFLD cohort, NAS patients had significantly higher body mass index (BMI) mean (=0.017), insulin (=0.007), homeostasis model assessment of insulin resistance (HOMA-IR) score (=0.009), triglycerides (=0.031) and -GT (<0.001), as well as lower HDL-cholesterol mean (=0.039) than subjects with NL. In NASH patients, the BMI mean, insulin, HOMA-IR score, triglycerides, ALT, AST and -GT were significantly higher than in NL individuals (<0.001 for all). In addition, HDL-cholesterol mean was significantly lower in NASH patients than in NL subjects (=0.013). As expected, NASH patients had significantly higher serum ALT than NAS patients (<0.001). First, we analyzed ER stress markers in human liver biopsies from patients with NAFLD. Messenger RNA (mRNA) levels of ATF4 decreased in NAS patients compared with the other two groups (). Moreover, GRP78 and CHOP mRNA levels were increased in NASH patients compared with NAS patients or NL individuals. When protein analysis was carried out, an increase in CHOP was detected in NAS and NASH patients, whereas GRP78 only increased in the NASH group (). Next, we analyzed mRNA levels of autophagy mediators. Hepatic mRNA levels of BECN1 were significantly lower in patients with NAS as compared with individuals with NL (). However, hepatic mRNA levels of the autophagic substrate SQSTM1 (p62) increased in both NAS and NASH, although no statistical significance was found when these patients were compared with NL individuals. As lipidation of LC3 and its association with autophagosome membranes has been established as useful sign for autophagy, we detected LC3 by immunoblotting and fluorescence microscopy. Importantly, a significant increase in LC3-II/LC3-I ratio was detected in both NAS and NASH patients compared with NL subjects (). Moreover, LC3-II punctuate was clearly visible by immunofluorescence in biopsies from both patient groups, whereas diffuse staining was visualized in NL samples (). However, these techniques should be analyzed carefully, as positive results clearly indicate increased numbers of autophagosomes, but do not always mean upregulation of the autophagic flux. True autophagic function can be measured by the change in LC3-II levels in the presence versus absence of lysosomal inhibitors, which measures the rate of LC3-II degradation. As these approaches were not possible in human liver samples, we measured protein levels of p62, a selective substrate of autophagy, because activation of the autophagic flux leads to a decline in p62 expression, and . As shows, a significant increase in p62 was detected in both NAS and NASH patients compared with individuals with NL, indicating impairment in the autophagic flux. Furthermore, p62 was significantly elevated in NASH compared with NAS patients. Fibrogenic markers (h-COL1A1 and h-PAI1) were significantly elevated in NASH patients compared with NL and NAS groups. Likewise, mRNA levels of DAPK1 were exclusively elevated in the NASH group (). The histological analysis by hematoxylin/eosin (H&E) and Masson's trichrome staining of these representative samples is shown in . The analysis of cell death by TUNEL previously reported revealed the presence of apoptosis only in the NASH group. We next examined whether activation of the ER stress signaling was accompanied by changes in the autophagic flux during the progression of experimental NAFLD in mice. For this purpose, male C57BL6 mice were fed with high fat diet (HFD) for 30 weeks to generate NAS or with methionine-choline-deficient diet (MCD) for 4 weeks as a well-established model of NASH. Control mice were fed with chow diet (CHD). Upon histological examination, the livers of mice fed with HFD exhibited steatosis (). Phosphorylation of PERK, eIF2 and JNK, the latter activated via IRE, and CHOP protein levels were significantly increased in mice with hepatic steatosis compared with the control group (). However, protein levels of GRP78 were similar in both groups. These results agree with other studies. As nutrient-mediated activation of mTOR suppresses autophagy, we evaluated the phosphorylation of mTOR and its downstream target S6K1. shows significant increases in both phosphorylated proteins in HFD-fed mice compared with mice receiving CHD. Consequently, the autophagic flux was blocked as revealed by significant increase in levels of polyubiquitinated proteins, accumulation of p62 and increased LC3-II/LC3-I ratio in mice fed with HFD that was also evidenced by the increased LC3-II punctuate in liver sections (). Electron microscopy (EM) revealed accumulation of double membrane structures (autophagosomes) in HFD-fed mice compared with control mice (CHD). Moreover, TUNEL analysis showed the presence of apoptotic hepatic cells only in HFD-fed mice (). Mice fed with MCD diet developed NASH manifested by fat accumulation, ballooning and infiltration of inflammatory cells in the histological evaluation () and also in the elevation of serum ALT (CHD, 30.1±2.6 U/l and MCD, 206.3±10.5 U/l). As observed in HFD-fed mice, mice fed with MCD showed elevated phosphorylation of ER stress markers, as well as increased GRP78 and CHOP protein expression compared with the control group (). Moreover, mTOR and S6K1 phosphorylations were elevated in livers of MCD-fed mice () in parallel to an increase in the amount of polyubiquitinated proteins, an accumulation of p62, higher LC3-II/LC3-I ratio and visualization of punctuate LC3-II staining and autophagosomes compared with CHD controls (). These effects were coincident with increases in apoptosis in the MCD group as assessed by TUNEL (). Altogether, these results indicate that increased ER stress, blockade of the autophagic flux and apoptosis are hallmarks during NAFLD. We next investigated the possibility to overcome the blockade of the autophagic flux by fatty acid overload by performing experiments in Huh7 human hepatic cells treated with palmitic acid (PA) combined with rapamycin, a well-known inducer of autophagy. Surprisingly, autophagic flux was not impaired after a short-term (8 h) treatment with PA, as assessed by reduced phosphorylation of mTOR and S6K1 in parallel to a decrease in p62 levels and increase in the LC3-II/LC3-I ratio. This effect was even more pronounced than that of rapamycin, which also activated the authophagic flux compared with untreated cells (). Additionally, we monitored the autophagic flux analyzing LC3 turnover assay using bafilomycin A1, which inhibits lysosomal function and the late degradation stage of autophagy, thereby triggering the accumulation of autophagosomes. The combination of PA and bafilomycin A1 increased p62 expression and also induced a higher accumulation of LC3-II compared with PA-treated cells (). These data demonstrated that LC3-II accumulation induced by PA results from the activation of autophagosome formation, but not from an inhibition of the autophagosome degradation steps. Regarding ER stress, PA treatment (8 h) induced PERK, eIF2 and JNK phosphorylations in Huh7 cells compared with the untreated controls (). This effect was accompanied by increased expression of GRP78. Cotreatment of PA and rapamycin reduced these events induced by PA and significantly blocked JNK phosphorylation. These results showed that activation of the UPR concurs with an active authophagic flux at early time periods of saturated fatty acid loading. Notably, cell death was not observed in Huh7 cells treated with PA for 8 h (results not shown). When we evaluated the effect of prolonged treatment (24 h) with PA in Huh7 cells, we found that accumulation of p62 was paralleled to an increase in the LC3-II/LC3-I ratio, reflecting a loss of the autophagic flux (). This effect was similar to the blockade of the autophagic flux induced by bafilomycin A1 or chloroquine (CQ). To verify these data, we performed LC3-II immunostaining and EM. Punctuate LC3-II was observed in Huh7 cells treated with PA for 24 h, but not in vehicle-treated cells. In addition, a marked accumulation of double-membrane structures containing cellular organelles was visualized by EM in PA-treated Huh7 cells (). This effect was higher in CQ-treated cells as compared with PA, indicating a more potent inhibition of the authophagic flux by this chemical compound. Under these experimental conditions, rapamycin decreased p62 and LC3-II/LC3-I ratio in PA-treated cells for 24 h as compared with cells treated with PA alone, indicating a recovery of the authophagic flux (). This effect was parallel to decreased LC3-II punctuate and accumulation of autophagosomes as visualized in the EM images (). Similar effects of PA and rapamycin were found in p62 levels and LC3-II/LC-I ratio in primary cultures of human hepatocytes (). During the prolonged treatment of Huh7 cells with PA, the phosphorylation of mTOR and S6K1 remained similar to untreated cells in contrast to the effect observed at 8 h (). The ongoing ER stress activation was assessed by the expression of GRP78 and this effect was ameliorated in the presence of rapamycin (). In addition, we evaluated the impact of the restoration of the autophagic flux by rapamycin in apoptotic cell death and lipid accumulation. For this purpose, Huh7 cells were treated with PA, rapamycin or the combination of both for 24 h, and 4, 6-diamidino-2-phenylindole (DAPI) staining of nuclei and Nile Red staining of lipid droplets was analyzed. As depicted in , 24 h of PA treatment increased the number of apoptotic nuclei as described previously. Treatment with rapamycin alone did not alter the number of apoptotic nuclei; meanwhile, cotreatment with rapamycin and PA induced a significant decrease as compared with the effect of PA alone. Conversely, treatment with rapamycin did not modify PA-induced lipid droplets formation (). To clarify if elevated ER stress triggers disruption of the autophagy flux, we performed experiments inhibiting ER stress by reduction of CHOP by siRNA. As shows, CHOP mRNA levels were reduced by almost 60% in Huh7 cells treated with CHOP siRNA (siCHOP) compared with those cells treated with a control oligo (siControl). When we evaluated the effect of prolonged treatment with PA in Huh7 cells, we observed that silencing of CHOP reduced p62 together with LC3-II accumulation (). Interestingly, addition of siCHOP to PA-treated cells decreased apoptosis (). During the past recent years, it has been established that defective autophagy promotes ER stress and insulin resistance in the liver, whereas degradation of damaged organelles by autophagy ameliorates liver diseases such as ethanol-induced steatosis and HCC, suggesting that autophagy could have a relevant pathogenic role in liver diseases. Previously, one study in human liver samples has found that the progression from NAS to NASH was accompanied by increased phosphorylation of JNK and increased ER stress reflected by elevated mRNA. Lake recently reported increased nuclear accumulation of total XBP-1 protein in NASH human livers. This paper also reported an enrichment of upregulated genes of autophagy by performing microarrays, although their validation was not shown. Thus, in the present study, we have analyzed for the first time the relationship between specific markers of ER stress and autophagy in the liver of NAS and NASH patients in comparison with individuals with normal liver. We found that protein amount of p62 was accumulated within the liver of patients with NAS or NASH in parallel with an increase in the LC3-II/LC3-I ratio. Of note, levels of p62 were significantly higher in NASH compared with NAS patients, suggesting that this effect is related with the disease progression in humans. Moreover, NASH patients displayed more elevated ER stress markers, such as and mRNAs, as well as protein levels, reinforcing the notion that enhanced ER stress within liver cells may be relevant in the progression from NAS to NASH. It has been shown that activation of the autophagic flux leads to a decline in p62 expression and, contrarily, an accumulation of p62 reflects a decrease in the autophagic flux. When autophagy is off, LC3-II distributes diffusely throughout the cytoplasm. However, upon induction of autophagy, LC3-II is recruited via its lipid moiety to the inner and outer surfaces of autophagosome membranes forming LC3-autophagic punctuate. Therefore, accumulation of LC3-II and autophagosomes, event previously interpreted as induction of autophagy, is in fact a consequence of a blockade of the autophagic flux. Therefore, it is conceivable that the combination of elevated p62 and punctuate LC3-II, which was observed in liver samples from both NAS and NASH patients, could be reflecting an inhibition of autophagosome degradation. Considering findings mentioned so far, a potential mechanistic explanation for the pattern of p62 and LC3-II/LC3-I ratio during the progression of NAFLD would be that elevated ER stress due to lipid and nutrient overload might increase accumulation of unfolded proteins and/or damaged organelles, triggering liver injury through the disruption of the autophagic flux and leading to apoptotic cell death. In this regard, our previous results demonstrated that apoptosis was evident in NASH patients. In the liver, persistent UPR has been described in HFD-fed mice and in ob/ob mice that display hepatic steatosis, and also in MCD fed mice. In the present study, we found an accumulation of polyubiquitinated proteins in hepatocytes of HFD- and MCD-fed mice, which probably lead to chronic activation of the UPR, as demonstrated by elevated PERK and eIF2 phosphorylation and increased GRP78 and CHOP protein expression, overall reflecting ER overload. Whereas polyubiquitinated proteins and p62 levels are commonly increased in HFD-fed mice showing defective autophagic function, there are studies showing both decreased or increased LC3-II (Singh and our study). Since, as stated above, the LC3-II/LC3-I ratio by itself does not assess the activation of the autophagic flux, it is possible that LC3-II levels might vary depending on the conditions of the HFD intervention. Nevertheless, our data clearly show that the autophagic flux is decreased in mice fed with HFD as well as in the MCD model, leading to the accumulation of autophagosomes, indicating for the first time that all the machinery that connects ER stress with the blockade of the autophagic flux is already activated in mice with hepatic steatosis. These results may have relevant clinical implications, suggesting that therapies aimed to restore the autophagic flux at early stages of NAFLD, such as NAS, might ameliorate progression to more advanced and irreversible forms of liver disease, such as NASH. Free fatty acid (FFA)-induced lipotoxicity has a critical role in the pathogenesis of NAFLD (reviewed by Komatsu and Ichimura and Malhi and Gores), being saturated FFAs the more toxic lipid species. In the present study, we found that UPR was triggered in Huh7 human hepatic cells loaded with PA for 8 h concomitantly with the activation of the autophagic flux that was reflected by reduced phosphorylation of mTOR and S6K1, decreased p62, increased LC3-II/LC3-I ratio and by the effect of bafilomycin A1. These results suggest that this early response conferred protection against cell death as no apoptotic cells were detected at this time period. In contrast, a more prolonged exposure of Huh7 cells to ER stress by long PA treatment (24 h) induced cell death manifested by the abundance of apoptotic nuclei as compared with untreated cells. Notably, this later effect was coincident with the visualization of a high number of autophagosomes. These data clearly indicate that prolonged PA treatment induced a switch to the inhibition of the autophagic flux. To our knowledge, this is the first study that shows this transition monitored by the sequential accumulation of the autophagic substrate p62 between 8 and 24 h of PA exposure that was parallel to an increase in LC3-II punctuate and, importantly, to the induction of cell death. The fact that the reduction of the autophagic flux in Huh7 cells and primary human hepatocytes loaded with PA for 24 h was not accompanied by increased mTOR/S6K1 phosphorylation is intriguing and contrasts with our study in mice and data from Khamzina One possible explanation for such difference is the more complex regulation of mTOR-mediated signaling pathway in liver tissue that integrates a number of signals emerging from different non-parenchymal cell types. In addition, an mTOR-independent regulation of autophagy and PA-mediated cell death through the activation of protein kinase C has also been described. Despite the lack of induction of mTOR phosphorylation by long-term treatment with PA in Huh7 cells, its inhibition by rapamycin reactivated the autophagic flux by decreasing p62 and the accumulation of autophagosomes (also observed in primary human hepatocytes) and ameliorated PA-induced increase in apoptotic cells. Reduction of ER stress by decreasing CHOP levels also prevented the blockade of autophagic flux by long-term treatment with PA and partly reduced cell death. The involvement of CHOP in ER stress-induced autophagy and cell death agrees with the previous data. Thus, the prosurvival function of autophagy suggests a potential therapeutic strategy in the protection of hepatic cells against lipotoxicity. In conclusion, we have demonstrated that autophagic flux is impaired in livers from both patients and murine models of NAFLD, as well as in long-term PA-loaded human hepatocytes, providing evidences that disruption of autophagy could be due to elevated ER stress leading to apoptosis. Our results strongly suggest that therapies aimed to restore the autophagic flux might prevent or attenuate the progression of NAFLD. Fetal bovine serum (FBS), culture media DMEM, TRIzol reagent, SuperScript III First-Strand Synthesis System and DAPI (D1306) were obtained from Invitrogen (Grand Island, NY, USA). PA (P0500), rapamycin, CQ, bafilomycin A1 and bovine serum albumin (BSA) were from Sigma-Aldrich (St. Louis, MO, USA). H&E and Masson's trichrome solutions were from Panreac Química (Barcelona, Spain). Bradford reagent, acrylamide and Immunoblot PVDF membrane were from Bio-Rad (München, Germany). Immobilon Western Chemiluminescent HRP Substrate was purchased from Millipore (Billerica, MA, USA). This study comprised 49 non-diabetic patients with a clinical diagnosis of NAFLD who underwent a liver biopsy by a percutaneous route during programmed cholecystectomy. Inclusion criteria for NAFLD patients were based on the absence of alcohol intake (<20 g per day), the presence of biopsy-proven steatosis with/without necroinflammation and/or fibrosis, and no evidence of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. Patients with other causes of chronic liver disease or those receiving potentially hepatotoxic drugs were excluded. We further studied 34 patients to whom a liver biopsy was taken during programmed laparoscopic cholecystectomy and were diagnosed of histologically normal liver (NL). All these NL patients had normal fasting glucose, cholesterol and triglycerides, normal serum ALT levels, and negative anti-HBV, anti-HCV and anti-HIV tests. In addition, all of them drank <20 g of alcohol per day and none used potentially hepatotoxic drugs. This study was performed in agreement with the Declaration of Helsinki, and with local and national laws. The Institution's Human Ethics Committee approved the study procedures, and written informed consent was obtained from all patients before inclusion in the study. Male 8-week-old C57BL/6 mice purchased to Charles River Laboratories (Charles River, Barcelona, Spain) were maintained in light/dark (12 h light/12 h dark), temperature (22 °C) and humidity-controlled rooms with free access to drinking water. Mice were fed with CHD (SAFE A04-10 Panlab, Barcelona, Spain), HFD (TD-88137; Harland-Teklad, Indianapolis, IN, USA) for 30 weeks or MCD (TD-90262; Harland-Tecklad) for 4 weeks. All animal experimentation was controlled following the recommendations of the Federation of European Laboratory Animal Science Associations (FELASA) on health monitoring, whereas use of animals in experimental procedures was approved by the Consejo Superior de Investigaciones Científicas (CSIC, Madrid, Spain) Animal Care and Use Committee. Serum ALT activity was determined using Reflotron strips (Roche Diagnostics, Barcelona, Spain), in accordance with the manual instructions. For EM, liver biopsies were fixed in a glutaraldehyde (2%) plus -formaldehyde (4%) for 60 min. Samples were postfixed in 1% osmium tetroxide for 60 min at 25 °C, stained with uranyl acetate (5 mg/ml) for 1 h at 25 °C, dehydrated in acetone and embedded in Epon 812 (EMbed 812; Electron Microscopy Science, Hatfield, PA, USA). Ultra-thin sections, unstained or poststained with uranyl acetate and lead hydroxide, were examined under a Morgagni 268D transmission electron microscope (FEI, Hillsboro, OR, USA) equipped with a Mega View II charge-coupled device camera (SIS, Soft Imaging System GmbH, Munster, Germany) and analyzed with AnalySIS software (SIS). For LC3 immunostaining, paraffin-embedded liver biopsy sections (5 m) were stained with anti-LC3 antibody and anti-rabbit conjugate Alexa 546. For cell death detection staining, paraffin-embedded liver biopsy sections (5 m) were stained using DeadEnd Fluorimetric TUNEL system (Promega, Madison, WI, USA), in accordance with the manual instructions. After corresponding inmunostaining, liver sections were mounted with fluorescent mounting medium and images were taken using a confocal microscope LSM710 (Zeiss, Barcelona, Spain). Human hepatocytes were prepared from liver biopsies obtained from 12 patients (eight males, four females aged 58±4.0 years) submitted to a surgical resection for liver tumors after obtaining patients' written consent. Hepatocytes isolation was based on the two-step collagenase procedure. Huh7 cell line was kindly provided by Dr. Kern (Department of General Pathology, University Hospital Heidelberg, Heidelberg, Germany). Huh7 cell line authentication and intraspecies cell line cross-contamination were analyzed using Promega's StemElite ID System (Madison, WI, USA) for the following short tandem repeats (STRs): D21S11, TH01, TPOX, vWA, CSF1PO, D16S539, D7S820, D13S317 and D5S818; Amelogenin was also analyzed for gender identification together with a specific marker for mouse DNA. STRs were analyzed with the Applied Biosystems 3130xl Genetic Analyzer in the Genomics Core Facility of the Instituto de Investigaciones Biomédicas Alberto Sols CSIC-UAM. Cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS at 37 °C with 5% CO. PA was dissolved in isopropyl alcohol at a stock concentration of 80 mM and added to DMEM containing 1% BSA to assure a physiological ratio between bound and unbound PA in the medium. The concentration of PA used in the experiments (800 M) was similar to the fasting PA plasma concentrations observed in human non-alcoholic steatohepatitis and was used before for mimicking hepatic lipoapoptosis. Confluent cells were treated with PA (800 M) for the indicated time periods in the absence or presence of rapamycin (25 ng/ml). Bafilomycin A1 (10 nM) and CQ (50 nM) were used as positive control of autophagosome accumulation. siRNA oligonucleotides were synthesized by Dharmacon RNAi Technologies (Fisher Scientific-USA, Pittsburgh, PA, USA) for gene silencing of human CHOP. Huh7 cells were transfected with control or siCHOP (5 nM) following DharmaFECT General Transfection Protocol (Dharmacon RNAi Technologies). After 36 h, cells were used for experiments. For EM, Huh7 cells were grown in chamber slides and treated as described above. Then, cells were washed two times with PBS, fixed in a glutaraldehyde (2%) plus -formaldehyde (4%) for 60 min and processed similar to liver biopsies. For LC3 immunostaining, Huh7 cells were grown in glass coverslips and treated with PA as described in Results and Figure legends. Then, cells were washed two times with PBS, fixed in -formaldehyde (4%) for 10 min and processed for LC3 immunofluorescence as described above. Huh7 cells were grown in glass coverslips and treated as described above. Then, cells were washed two times with PBS, fixed in -formaldehyde (4%) for 10 min and characteristic morphological changes of apoptosis were assessed by staining nuclei with DAPI, followed by the analysis by fluorescence microscopy. After treatment, Huh7 cells were fixed in -formaldehyde (4%) and resuspended in Nile Red working solution (0.4 g/ml). The fluorescence was determined using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). Total RNA of liver biopsy samples was extracted by using TRIzol reagent and was reverse transcribed using a SuperScript III First-Strand Synthesis System for quantitative real-time polymerase chain reaction (qPCR) following manufacturer's indications. qPCR was performed with an ABI 7900 sequence detector (Life Technologies, Carlsbad, CA, USA) using the SYBR Green method and d(N) random hexamer with primers purchased from Invitrogen. PCR thermocycling parameters were 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Each sample was run in triplicate and was normalized to 18S RNA. Fold changes were determined using the ΔΔCt method. Primer sequences are available upon request. Liver biopsy samples were homogenized in a medium containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl, 1 mM EGTA, 10% glycerol, 0.5% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), 1 mM -mercaptoethanol and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). To obtain total cell lysates, attached cells were scraped off and incubated for 10 min on ice with lysis buffer (25 mM HEPES, 2.5 nM EDTA, 0.1% Triton X-100, 1 mM PMSF and 5 g/ml leupeptin). After protein content determination with Bradford reagent, total protein were boiled in Laemmli sample buffer and submitted to 8–15% SDS-PAGE. Proteins were transferred to Inmunoblot PVDF membrane, and after blocking with 3% BSA or 5% non-fat dry milk, incubated overnight with several antibodies as indicated. Immunoreactive bands were visualized using the ECL Western blotting protocol (Millipore). Densitometric analysis of the bands was performed using the Image J software (NIH, Bethesda, MD, USA). Anti-phospho-PERK (Thr 980) for mouse samples (no. 3179), anti-phospho-eIF2 (Ser 51) (no. 9721), anti-LC3 (no. 4108), anti-phospho-mTOR (Ser 2448) (no. 2971), anti-phospho-JNK (no. 4668), anti-S6K1 (no. 9202) and anti-ubiquitin (no. 3936) antibodies were from Cell Signaling Technology (Boston, MA, USA). Anti-p62 (SQSTM1) antibody was from MBL International (Woburn, MA, USA). The anti-phospho-S6K1 (Thr 389) (sc-11759), anti-JNK (sc-571), anti-IR (sc-711), Beclin (sc-11427), anti-phospho-PERK (Thr 980) for human samples (sc-32577), anti-PERK (sc-13073), anti-eIF2 (sc-11386), anti-GRP78 (sc-376768), anti-CHOP for human samples (sc-575) and anti-CHOP for mouse samples (sc-7351) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-GAPDH antibody (AM4300) was from Ambion (Grand Island, NY, USA). Anti--actin antibody (A-5441) was from Sigma Aldrich. Anti-rabbit-conjugated Alexa 546 (A11035) was from Life Technologies (Grand Island, NY, USA). Anti-Atg5 antibody was a gift from Dr. Patricia Boya (CSIC). Categorical variables are presented as frequency and percentage. Continuous variables are shown as median and range. The baseline characteristics of the patients studied were compared by the Pearson test for categorical variables and the unpaired -test or Mann–Whitney -test for continuous variables. Data from qPCR and western blotting are presented as mean±S.E.M., and were compared by using the Bonferroni ANOVA test. All statistical analyses were performed using the IBM SPSS Statistics 21.0 (SPSS Inc., IBM, Armonk, NY, USA) software with two-sided tests, with a -value of <0.05 considered as statistically significant.

We commenced this study by evaluating the subcellular expression and radioinducibility of APPL1 and APPL2 in the pancreatic carcinoma cell lines MiaPaCa2 and Capan-1. Both APPL1 and APPL2 proteins were localized in the cytoplasm as well as in the nucleus (). Moreover, we observed colocalization of about 45% of APPL2 vesicles to APPL1, whereas only 30% of APPL1 vesicles localized to APPL2 vesicles (). Irradiation did not change the subcellular localization of APPL1 and APPL2 but resulted in a fast and significant increase of APPL2 expression in both MiaPaCa2 and Capan-1 cell lines relative to unirradiated controls as measured by western blot (). Whereas APPL1 expression was not affected, the radiogenic APPL2 induction maintained at higher levels over the 24-h observation period. These data indicate a differential role of these proteins in both normal cell physiology and cellular radiation response. Next, we analyzed whether APPL proteins are essential in the radiation survival response by silencing APPL1 and/or APPL2 using small interfering RNA (siRNA) technology. We found that depletion of APPL proteins significantly enhanced the radiosensitivity of both MiaPaCa2 and Capan-1 cells, whereas basal cell survival was similar to siRNA controls (, ). Supporting this, stable overexpression of APPL1 in Capan-1 cells resulted in enhanced radioresistance (). In addition to the pancreatic cancer cell lines, we assessed the effects of APPL1 knockdown in cancer cell lines originating from the lung, colorectum, head and neck, stomach and cervix and found no modification of their cellular radiosensitivity (). As cellular radiation survival is strongly influenced by DNA damage repair, we next examined the number of residual DSBs dependent on APPL1 and APPL2 expression. As shown in , APPL knockdown cultures had significantly more radiation-induced 53BP1 foci compared with controls (, ). The effect of a combined downregulation of APPL1 and APPL2 on radiation survival and DNA damage was only slightly more pronounced than that caused by single knockdown of either APPL1 or APPL2 (). Interestingly, there was no effect on radiation-induced caspase-3 cleavage (, ), indicating that the observed differences in cell survival are not due to increased apoptosis. Nevertheless, these data suggest that APPL1 and APPL2 are both essential regulators of the DNA damage survival and repair response and that loss of one protein cannot be fully compensated by the other. The increased and similar induction of 53BP1-positive foci after irradiation resulting from APPL protein depletion () is suggestive of a function of these proteins in DNA damage repair. Therefore, we evaluated the effect of APPL knockdown on ATM, a serine/threonine kinase well known to be a key regulator of DSB repair. After depletion of APPL1 and APPL2, the number of radiation-induced phosphorylated ATM S1981 foci—indicative of the ATM activity status—decreased when compared with control cultures (). Accordingly, downregulation of APPL1 alone and in combination with APPL2, but not APPL2 alone, led to a significant reduction of ATM S1981 phosphorylation as measured by western blotting (). In contrast, proteins associated with ATM such as radiogenic phosphorylation of Chk2 and the expression of meiotic recombination 11(Mre11), Rad50 and NBS1 were not robustly affected upon APPL protein silencing (). To evaluate whether the observed APPL1-related reduction in ATM phosphorylation upon irradiation is critically involved in APPL knockdown-mediated radiosensitization, we performed ATM siRNA transfections alone or in combination with APPL depletion. As shown in , efficient downregulation of ATM radiosensitized the pancreatic carcinoma cells and resulted in an increase in residual 53BP1-positive foci (, ). Additional knockdown of APPL proteins revealed no further effect on clonogenic radiation survival and radiogenic foci (), indicating that ATM, APPL1 and APPL2 are part of the same signaling axis. In addition, depletion of ATM in Capan-1 transfectants diminished the radioprotective effect of APPL1 overexpression (), suggesting that ATM is the key mediator of APPL1-mediated radioresistance. These findings provide evidence that APPL proteins modulate DNA repair processes by regulating ATM, thereby having an impact on cell survival after irradiation. To detect a differential function of APPL proteins in the early late phase of the DNA repair process, we next explored ATM phosphorylation and foci removal kinetics at time points ranging from 30 min to 24 h after irradiation in cells silenced for APPL1, APPL2 and ATM. In addition, we observed strongly diminished levels of phosphorylated ATM S1981 between 30 min and 2 h after 6 Gy in APPL1+2 knockdown cultures compared with controls (). This time interval also comprised the maximum of ATM activation, as after 24 h ATM phosphorylation was decreased to the control level (). In parallel, removal of 53BP1-positive foci was significantly delayed in APPL1+2, ATM and APPL1+2/ATM knockdown cultures already 2 h after irradiation (). The early time points, that is, 30 min and 2 h, indicated no significant difference in foci number of APPL1- or APPL2-depleted cells relative to controls (). These data show a major impact of APPL proteins on the DNA repair process conducted between 2 and 24 h, which is marginally altered by additional ATM inhibition at earlier time points than 24 h (). To explore mechanistically how APPL1 modulates ATM phosphorylation, we analyzed the localization of ATM upon irradiation and additionally utilized a proximity ligation assay to detect a possible interaction between the two proteins. After irradiation, the nuclear expression of ATM and phosphorylated ATM strongly increased (). Additionally, we found a significantly enhanced interaction of phospho-ATM with APPL1 30 min after irradiation, which was mainly found in the nucleus (). In contrast, radiation-induced colocalization of total ATM and APPL1 occurred in the cytoplasm as well as in the nucleus (, ).These data suggest that the observed APPL1/ATM interaction might be important for ATM activation and ATM-mediated DNA repair processes. To develop strategies to overcome resistance to therapy, tumor cell biology requires further unraveling. Although DNA repair mechanisms seem to be well understood, the cellular response to genotoxic injury induced by conventional chemotherapies and radiotherapy appears to be more complex. In this study, we investigated the endosomal APPL proteins and report on their essential function in ATM-dependent DNA repair and radiation survival of human pancreatic cancer cells. Our results provide evidence that the endosomal APPL proteins regulate ATM activity and thereby influence radioresistance and DNA repair of pancreatic adenocarcinoma cells, revealing a new link between endocytic pathways and DNA repair mechanisms. Endocytic proteins control essential cell functions like proliferation and cell survival. In addition to Caveolin-1 being a key factor in caveolae-mediated endocytosis and its modulatory role in the radiation survival and DNA damage response, APPL proteins also seem to be essential in this context. The first line of evidence is based on the observation that total APPL2 but not APPL1 expression is inducible by X-ray radiation, while the subcellular localization of APPL proteins remains unchanged in the pancreatic adenocarcinoma cells. Interestingly, there did not exist a 100% overlap of APPL1- and APPL2-positive vesicles either. In cervix carcinoma cells, however, both APPL proteins largely colocalized, particularly in defined structures near the plasma membrane, leading to the assumption that APPL-mediated endocytosis is differentially regulated in these two tumor entities. On the basis of these findings, we hypothesize a differential role of APPL1 and APPL2 in radiation survival and DSB repair response. Both proteins significantly enhanced radiosensitivity to a similar degree in the tested pancreatic cancer cell lines when depleted using siRNA. Combined silencing of APPL1 and APPL2 conferred no further radiosensitization compared with either single knockdown. These results are in line with several studies showing a role of APPL proteins in the regulation of survival mechanisms including apoptosis. In line with cellular radiosensitization, MiaPaCa2 and Capan-1 cells showed a higher number of 53BP1-positive foci 24 h after irradiation, especially under concomitant APPL protein depletion. This observation corroborates data from others that 53BP1 foci numbers correlate with radiation survival. Despite the fact that APPL proteins interact with a variety of promitotic and prosurvival protein kinases such as EGFR and AKT, a recently published protein sequence analysis suggested a possibility for APPL1/ATM interactions, which we confirm with the PLA experiments. Our experiments, based on significant downregulation of ATM S1981 autophosphorylation under APPL1 silencing, point to a possible regulatory association of both proteins. Intriguingly, the MRN complex, although undoubtedly an important factor in DNA repair processes, was unaffected by both irradiation and APPL knockdown. One possible explanation for these observations could be the R248W mutation within the p53 protein sequence, which is present in MiaPaCa2 cells. Song and Hollstein found that this specific p53 mutation results in diminished binding of Mre11-Rad50-NBS1 to DSBs causing reduced activation of the DNA repair machinery. Taking current knowledge into account, it was expected to see an increase in radiosensitivity and 53BP1 foci in the pancreatic carcinoma cells after ATM knockdown. Not only do data from patients with ataxia telangiectasia but also a large amount of and studies give evidence for ATM's central function in the cellular radiation response. As additional depletion of APPL proteins had equal effects as with ATM knockdown alone, we concluded that ATM and APPL proteins are part of the same signaling axis. The strongest induction of ATM phosphorylation could be observed between 30 min and 2 h after irradiation, whereas after 24 h phospho-ATM expression declined to the level of unirradiated cells. Similar findings were previously obtained in cells of other tumor entities such as lung cancer. A further indication for an interrelation between APPL proteins and ATM was given by the analysis of the DNA repair kinetics. Single depletion of APPL1, APPL2 and ATM modulated early and late DSB repair phases and displayed analogous time kinetics of foci removal, and this was even more pronounced upon the combined knockdown of APPL1/ATM and APPL2/ATM. In summary, our study shows a critical function of the endosomal APPL proteins on radiation survival and DNA repair mechanisms of pancreatic carcinoma cells. The presented data indicate a regulatory interaction of APPL proteins with the DNA repair kinase ATM, thus providing novel insights into molecular processes controlling cell fate and tumor cell resistance. Antibodies against APPL1 (western blot), ATM S1981 (immunofluorescence, western blot), ATM (western blot), Chk2, Chk2 Th68, Mre11, NBS1/p95, Rad50, cleaved caspase-3 (Cell Signaling, Frankfurt, Germany), 53BP1 (Novus Biologicals, Herford, Germany), ATM (immunofluorescence) (GeneTex, Irvine, CA, USA), APPL2, -Actin (Sigma, Taufkirchen, Germany), ATM S1981 (PLA) (Rockland, Gilbertsville, PA, USA), APPL1 (immunofluorescence) (Zerial lab) and horseradish peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse (Amersham, Freiburg, Germany), AlexaFluor 594 anti-mouse and AlexaFluor 488 anti-rabbit (Invitrogen, Darmstadt, Germany) antibodies were purchased as indicated. Complete protease inhibitor cocktail was from Roche (Mannheim, Germany), SuperSignal West Dura Extended Duration Substrate was from Thermo Scientific (Bonn, Germany), nitrocellulose membranes were from Schleicher and Schuell (Dassel, Germany), Vectashield/DAPI mounting medium from Alexis (Gruenberg, Germany), oligofectamine from Invitrogen (Karlsruhe, Germany), dimethyl sulfoxide (DMSO) from AppliChem (Darmstadt, Germany) BSA from Serva (Heidelberg, Germany). Pancreatic carcinoma cell line MiaPaCa2 was purchased from DMSZ (Braunschweig, Germany). The pancreatic cancer cell line Capan-1 was kindly provided by C. Pilarsky (Department of Surgery, University Hospital, Dresden, Germany). Cells were cultured in DMEM GlutaMAX 1 medium (Gibco, Karlsruhe, Germany) supplemented with 10% (MiaPaCa2) or 20% (Capan-1) FCS (PAA, Cölbe, Germany) and 1% nonessential amino acids (PAA) at 37 °C in a humidified atmosphere containing 7% CO. In all experiments, asynchronously growing cell cultures were used. Irradiation was delivered at room temperature using single doses of 200 kV X-rays (Yxlon Y.TU 320; Yxlon, Copenhagen, Denmark) filtered with 0.5 mm Cu. The absorbed dose was measured using a Duplex dosimeter (PTW, Freiburg, Germany). The dose rate was ∼1.3 Gy/min at 20 mA and applied doses ranged from 0 to 6 Gy. APPL1 siRNA (sequence: 5′-GGGUGGAAAUUUAAUGAGUtt-3′), APPL2 siRNA (sequence: 5′-GGAUCUCACAGAAGUAAGCtt-3′), ATM siRNA (sequence: 5′-GGCACAAAAUGUGAAAUUCtt-3′) and the nonspecific control siRNA (sequence: 5′-GCAGCUAUAUGAAUGUUGUtt-3′) were obtained from MWG (Ebersberg, Germany). Cells were transfected as recently published using oligofectamine and 20 nM siRNA under serum-free cell culture conditions. At 48 h after transfection, cells were irradiated as indicated. Colony formation assay, western blotting or immunofluorescence staining was performed. Capan-1 cells were stably transfected with pCherry empty vector control or pCherry-APPL1 using Lipofectamine 2000 as previously published. Selection of stable transfectants was performed with G418 (Calbiochem, Darmstadt, Germany) for 6 weeks. Expression of Cherry-APPL1 or Cherry was confirmed by western blotting or fluorescence microscopy. This assay was applied for measurement of clonogenic cell survival as published. Cells were plated 24 h after siRNA transfection. After another 24 h, that is, 48 h after siRNA transfection, the time of maximum silencing of protein expression, cells were irradiated with 2–6 Gy or left unirradiated. Cell colonies were stained with Coomassie blue and colonies of >50 cells were counted 9 days (Capan-1, MiaPaCa2) after plating. Plating efficiencies were calculated as follows: numbers of colonies formed/numbers of cells plated. Surviving fractions (SF) were calculated as follows: numbers of colonies formed/(numbers of cells plated (irradiated) × plating efficiency (unirradiated)). Each point on survival curves represents the mean surviving fraction from at least three independent experiments. Adherent cells were rinsed with ice-cold 1XPBS before harvesting total proteins by scraping using modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet-P40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, Complete protease inhibitor cocktail (Roche, Mannheim, Germany), 1 mM NaVO, 2 mM NaF). Samples were stored at −80 °C. Amounts of total protein extracts were determined using BCA assay (Pierce, Bonn, Germany). After SDS-PAGE and transfer of proteins onto nitrocellulose membranes (Schleicher and Schuell), probing and detection of specific proteins was accomplished with indicated antibodies and SuperSignal West Dura Extended Duration Substrate as published. A total number of 5 × 10 cells were plated. After 24 h, cells were irradiated with 6 Gy and fixed, by using 3% formaldehyde/PBS, 15 min or 1 h after X-ray irradiation. For control, unirradiated cells were used. Permeabilization was carried out with 0.25% Triton X-100/PBS. Immunostaining was performed using anti-APPL1 or -APPL2 antibodies and secondary Alexa Fluor594 anti-mouse or Alexa Fluor488 anti-rabbit antibodies. Fluorescence images were obtained using a laser scanning microscope (LSM) equipped with Axiovert 200 M, LSM 510 Meta from Carl Zeiss (Jena, Germany). To provide further mechanistic insight into the enhanced radiosensitivity after APPL1/APPL2/APPL1+2 and ATM silencing, we measured DNA DSBs by using the foci assay. For detection of DSBs, p53 binding protein 1 (53BP1) foci assay was performed as described. 53BP1-positive nuclear foci of at least 150 cells were counted microscopically with an Axiscope 2 plus fluorescence microscope (Carl Zeiss) and defined as marker for residual DSBs. Fluorescence images were taken using a Zeiss Duoscan Laser Scanning Microscope (63X oil, 1.4, 1 Airy unit). These images were then processed using MotionTracking. Briefly, this first involved vesicle identification and then statistical analysis. For colocalization, the threshold was set at 0.35 using the volume of the vesicles, and for each condition >15 000 vesicles were analyzed per experiment. Second, to determine the intensity of APPL1 in the ATM channel, the cells were first masked to show only the nuclei using the DAPI channel as a reference. Following this, the median intensity of APPL1 was found only within the ATM intensity and vice versa. This was also performed to measure the amount of APPL1 in the cytoplasm in ATM by using a mask for the inverse nucleus (i.e. masking the nuclei) and again measuring ATM in the APPL1 channel. In each experiment, the median intensity was normalized to the value at 0 Gy. After siRNA-mediated knockdown and irradiation, cells were fixed in 4% PFA, permeabilized with Triton X-100, blocked in 10% fFCS and then incubated with the cleaved caspase-3 or pATM S1981 antibody in 5% FCS. DAPI was included during the secondary antibody incubation. Fluorescence images were taken using the Zeiss Duoscan Laser Scanning Microscope (63X oil, 1.4, 1 Airy unit). Visual inspection and number of cells positive for pATM foci were manually counted for the 0 Gy condition at both time point 0 and time point 24 due to the low number. At the 24 h-6 Gy condition, the nuclei were outlined in ImageJ using the DAPI channel, and the number of foci/nuclei was manually determined in the pATM channel. At the 1 h-6 Gy condition, nuclei were also outlined in ImageJ using the DAPI channel. Then using the 3D Objects Counter, foci/‘objects' were identified in each image by using the same threshold conditions and size settings (10–100 voxel). The number of foci/nuclei was then counted. Incomplete nuclei were not analyzed. Approximately 100 cells per condition were examined. To analyze APPL1/ATM interactions, we performed a Proximity ligation assay using the Duolink Detection Kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer's protocol and as previously described. Cells were plated 24 h before irradiation with 6 Gy. Unirradiated cells were used as control. After 30 min, cells were fixed, permeabilized and stained with primary antibodies for APPL1 and ATM. After incubation with PLA probes, ligation and amplification steps, samples were analyzed with an LSM 510 meta (Carl Zeiss). Interactions of at least 34 cells per experiment were counted. Mean±S.D. of at least three independent experiments was calculated with reference to untreated controls defined in a 1.0 scale. To test statistical significance, Student's -test was performed using Microsoft Excel 2003 (Redmond, WA, USA). Results were considered statistically significant if a -value of <0.05 was reached. Densitometry of western blots was performed by scanning of the exposed film and using ImageJ analysis software ().

To investigate whether OPT can preferentially inhibit cell proliferation and induce cell death in cancer cells, we compared the effect of OPT on MDA-MB231 breast cancer and MCF10A untransformed human breast epithelial cells. The viability of cells treated with different concentrations of OPT was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. As shown in , a significant portion of the MDA-MB231 cells lost viability with 3 M OPT treatment while a majority of MCF10A cells survived at much higher concentrations of OPT (40 M). To further characterize the effect of OPT, we stained MCF10A and MDA-MB231 cells with Annexin-V and PI. OPT treatment significantly increased Annexin-V and PI staining of the MDA-MB231 but not the MCF10A cells (). Additionally, OPT preferentially killed MLL-AF9 mouse leukemia cells but not the mouse bone marrow cells () and induced a significant level of cell death in HCT116 colorectal cancer cells as well as in KOPN1 lymphoblastic leukemia cells (). These data indicate that OPT preferentially kills cancer cells. We used a caspase-3 activity indicator (C3AI), which shows fluorescence only after cleavage by caspase-3 like proteases, to further characterize whether OPT-induced cell death is correlated with increased caspase-3 activity. Indeed, OPT treatment significantly increased C3AI fluorescence (). These observations suggest that OPT-induced cell death is mostly mediated by caspase-dependent apoptosis. A xenograft model of HCT-116 human colorectal cancer cells was used to determine whether OPT can inhibit cancer growth . Exponentially growing firefly luciferase-tagged HCT-116 cells were inoculated into the flanks of athymic nude mice. Beginning on Day 1, animals were also administered with OPT or vehicle at 15 mg/kg/day. Tumor growth was measured by Xenogen bioluminescence imaging every week. Treatment with OPT significantly decreased the Xenogen imaging signal (). Quantitative analysis of the imaging data () revealed that treatment with 15 mg/kg of OPT significantly inhibited xenograft tumor growth starting from week 3 (* and ** indicate <0.05 or <0.01, respectively). These results demonstrate that OPT can inhibit human tumor growth in a xenograft model. To investigate the mechanisms by which OPT induces cancer cell death, we characterized the ability of OPT to induce ER stress, ROS, and autophagy. OPT treatment induced significant levels of XBP1 splicing, CHOP expression, as well as GRP78 protein accumulation in MDA-MB231 cancer cells (), and a similar effect was observed by the ER stress inducer Tunicamycin (). Interestingly, as shown in , the XBP1 splicing and CHOP mRNA induction was detectable as early as 2 h and persistently induced at 24 h after OPT treatment. However, GRP78 mRNA was not induced 2 h after OPT treatment () and significantly increased GRP78 protein levels were observed after 8 h of OPT treatment (), significantly later than the induction of CHOP and XBP1 splicing. As CHOP functions to promote ER stress-induced cell death while GRP78 protects cells from ER stress-induced cell death, the early induction of proapoptotic genes such as CHOP and the relatively late induction of protective mechanisms such as GRP78 potentially contribute to the cell death induced by OPT treatment. To determine whether ER stress induction correlates with OPT-induced cell death, we determined the level of XBP1 splicing in the OPT-sensitive as well as resistant cells. MDA-MB231 and HCT116 cancer cells, which are sensitive to OPT treatment, exhibited a high level of XBP1 splicing after OPT treatment while MCF10A cells, which are resistant to OPT treatment, did not exhibit OPT-induced XBP1 splicing (). Therefore, the ability of OPT to induce ER stress correlates nicely with OPT-induced cell death. We also investigated the ability of OPT to induce oxidative stress and autophagy in cancer cells. Interestingly, OPT treatment induced significantly increased ROS levels in HCT116 colorectal cancer cells () but not in MDA-MB231 cells (), suggesting that OPT-induced ROS production is cell type dependent. Similarly, OPT induced a significant Microtubule-associated protein light chain 3 (LC3)-GFP puncta formation in HCT116 but not in MDA-MB231 cells (), indicating OPT induced autophagy in HCT116 but not in MDA-MB231 cells. Therefore, OPT can induce ROS and autophagy only in a subset of OPT-sensitive cancer cells. Since OPT-induced ER stress is correlated with cell death induction in all the cells examined, we further characterized the contribution of ER stress to OPT-induced cell death. ER chaperone protein GRP78 is an important regulator of ER stress. Overexpression of ER chaperone protein GRP78 using a myc-tagged GRP78 construct () significantly decreased OPT-induced ER stress as shown by decreased levels of XBP1 splicing () and decreased OPT-induced cell death in MDA-MB231 cells (). In contrast, knockdown of GRP78 using shRNA constructs against GRP78 decreased GRP78 levels (), increased ER stress (), and significantly increased OPT-induced cell death (). To further confirm that ER stress contributes to OPT-induced cell death, we determined the effect of knocking out components of the UPR pathway, PERK and XBP1, on OPT-induced cell death using established mouse embryonic fibroblast (MEF) cells. XBP1 MEFs showed an increased basal level of XBP1 splicing, but significantly reduced OPT-induced ER stress, as shown by reduced induction of GRP78 expression and XBP1 splicing (). The reduced ER stress induction is correlated with reduced OPT-induced cell death in XBP1 MEFs (). Similarly, PERK MEFs also significantly decreased OPT-induced ER stress as shown by reduced GFP78 induction (), which correlated with decreased OPT-induced cell death (). Therefore, OPT-induced ER stress contributes to its induction of cell death. Inhibition of protein synthesis will decrease protein influx to the ER, which can potentially help to restore ER homeostasis. Indeed, blocking protein synthesis can block the accumulation of unfolded/misfolded proteins in ER and therefore decrease ER stress and ER stress-induced cell death. To further characterize the molecular mechanism of OPT-induced ER stress and cell death, we tested the effect of blocking protein synthesis by cycloheximide (CHX). Pre-treatment with CHX reduced OPT-induced ER stress and inhibited the induction of CHOP in both MDA-MB231 cells and HCT116 cells (). The reduced ER stress and CHOP induction correlated with significantly inhibited OPT-induced cell death () in these cancer cells. These data further confirm that OPT-induced cell death is associated with its induction of ER stress. ERAD has an important role in maintaining the homeostasis of ER through delivering of misfolded protein to the cytosol for degradation by the proteasome-ubiquitin pathway. Interestingly, OPT treatment induced significantly increased accumulation of ubiquitinated proteins in both MDA-MB231 and HCT116 cancer cells () but not in untransformed MCF10A breast epithelial cells (). The accumulation of ubiquitinated protein correlated with the ability of OPT to induce cell death and ER stress (, ). Furthermore, the inhibition of ER stress and cell death by CHX correlated with significantly decreased accumulation of ubiquitinated proteins (). These observations suggest that OPT may interfere with the degradation of ubiquitinated proteins, causing the accumulation of misfolded proteins resulting in the induction of ER stress. To further test the possibility that OPT may interfere the ubiquitin/proteasome-mediated protein degradation, we determined the effect of OPT on c-Myc protein, which is an unstable protein degraded by the ubiquitin/proteasome pathway. Proteasome inhibitor MG132 treatment was used as a positive control, and led to increased c-Myc protein accumulation (). Interestingly, OPT treatment induced similar accumulation of c-Myc protein in a time-dependent manner (). These results further support the hypothesis that OPT interferes with the ubiquitin/proteasome pathway. In addition to ER stress, OPT also induced ROS and autophagy in HCT116 cells (). To investigate whether OPT-induced ROS and autophagy affect OPT-induced cell death, we determined the effect of antioxidants, -acetyl cysteine (NAC), and autophagy inhibitor Chloroquine (CQ). NAC treatment partially decreased OPT-induced cell death in HCT116 cells (), suggesting that OPT-induced ROS contributes to cell death induction in HCT116 cells. In contrast, inhibition of autophagy by CQ significantly increased cell death in HCT116 cells (), indicating that OPT-induced autophagy protects HCT116 cells from apoptosis induction. As expected, neither NAC nor CQ affects OPT-induced cell death in MDA-MB231 cells (), which do not show ROS or autophagy induction by OPT (). Activation of the proapoptotic proteins, BAX or BAK, is required for cell death in response to diverse stimuli, including ER stress. Activation of BAX and BAK proteins is controlled by the BH3-only proapoptotic proteins, which are induced by diverse stimuli to induce apoptosis. Indeed previous studies demonstrated that BH3-only proteins have an important role during ER stress and proteasome inhibitor-mediated cell death. To determine whether apoptotic regulators contribute to OPT-induced cell death, we investigated the expression of BH3-only proteins that are induced by OPT. We found the levels of apoptotic regulators Bim and Noxa to be significantly induced by OPT treatment as determined by qRT-PCR in both MDA-MB231 and HCT116 cells (). The induction of Bim by OPT can also be detected at the protein level (). To further investigate whether the observed Bim and Noxa induction contributes to OPT-induced cell death, we used lentivirus to introduce shRNA to specifically knock down Noxa or Bim expression. Knockdown of Bim significantly decreased OPT-induced Bim levels () and significantly decreased OPT-induced cell death in both MDA-MB231 () and HCT116 cells (). Similarly, knockdown of Noxa significantly blocked OPT-induced Noxa expression () and significantly reduced OPT-induced cell death in both MDA-MB231 and HCT116 cells (). Furthermore, knockdown of both Noxa and Bim largely blocks OPT-induced cell death (). Therefore, both Noxa and Bim contribute to OPT-induced cancer cell death. MCL1 is an unstable protein with a half-life of around 40 min. Interestingly, in addition to inducing the expression of proapoptotic regulators Noxa and Bim, OPT also induced accumulation of the antiapoptotic MCL1 protein (). The effect of OPT on MCL1 is likely due to its ability to block protein degradation since the MCL1 mRNA was not significantly affected by OPT treatment () and proteasome inhibitor MG132 also induced MCL1 accumulation (). Therefore, OPT-induced cell death is likely determined by the relative increase in the activities of the proapoptotic Noxa and Bim the antiapoptotic MCL1. Consistent with this idea, knockdown of MCL1 () significantly increased OPT-induced cell death in MDA-MB231 and HCT116 cancer cells (). These observations suggest that the anticancer effects of OPT can potentially be enhanced by simultaneously inhibiting MCL1. Our results show that OPT preferentially kills cancer cells and inhibits tumor growth through inducing excessive ER stress. Decreasing the level of ER stress by inactivating components of the UPR pathway or by overexpressing GRP78 significantly inhibited OPT-induced cell death. We show that OPT-induced ER stress is correlated with an accumulation of ubiquitinated protein and the stabilization of unstable proteins. These observations suggest that OPT functions, at least in part, by interfering with the ubiquitin/proteasome degradation pathway. In support of this idea, inhibition of protein synthesis decreased accumulation of ubiquitinated proteins, reduced OPT-induced ER stress, and significantly inhibited OPT-induced cell death. The ubiquitin/proteasome system regulates many cellular functions, including the level of key regulators of the cell proliferation and survival systems, as well as removal of unfolded/misfolded proteins via ERAD, to maintain ER homeostasis. The ubiquitin/proteasome system is a potential target for cancer therapeutic agents. Indeed, proteasome inhibitor Bortezomib has been approved for treating multiple myeloma and mantle cell lymphoma. Although it is traditionally thought that the stabilization of a particular subset of proteins might be responsible for the antitumorigenic activity of bortezomib, it is equally likely that its efficacy results from enhanced proteotoxic stress. The Bcl2 family of proteins includes the BH3-only type of proapoptotic regulators that are induced by different cell death signals, as well as the antiapoptotic regulators such as Bcl-2 and MCL1. The balance between the proapoptotic and the antiapoptotic regulators is critical in determining whether a cell will survive or die. We show that Bim and Noxa are induced by OPT and that knockdown of both Bim and Noxa significantly blocked OPT-induced cell death. Therefore, Bim and Noxa are critical mediators of OPT-induced cell death. These results are consistent with previous reports on the involvement of Bim and Noxa in mediating ER stress or proteasome inhibition-induced apoptosis. Interestingly, OPT treatment also significantly increased the level of MCL1, which is also a highly unstable protein. The observed MCL1 stabilization is likely related to the role of OPT in blocking protein degradation and potentially protects cells from undergoing apoptosis. Decreasing the level of MCL1 significantly sensitized cancer cells to OPT-induced cell death, suggesting that OPT-induced cancer cell death can be further enhanced by inhibitors of MCL1. Natural products are a valuable source of drugs for disease intervention and prevention. For example, current important anticancer drugs such as paclitaxel and vinblastine were initially discovered from natural products. Similarly, both salicylate, the active metabolite of aspirin, and the antidiabetic drug metformin are originally derived from herbal medicines and have been linked with reduced cancer incidence. Our demonstration that OPT preferentially kills cancer cells and inhibits tumor growth by interfering the ubiquitin/proteasome pathway and inducing the expression of Noxa and Bim suggest OPT can potentially be used to in cancer prevention or design new combinatorial therapies based on complementary mechanisms of action of different available anticancer drugs. The root bark of (Sm.) Miq., from Oregon, USA, was obtained from Pacific Botanicals (Grants Pass, OR, USA) and authenticated by Dr. Chong-Zhi Wang. The voucher specimens were deposited in the Tang Center for Herbal Medical Research at the University of Chicago. Air-dried, powdered root bark of was extracted with 80% ethanol under refluxing, suspended in water, then extracted with petroleum ether (60–90°C), ethyl acetate, and -butanol successively to give three fractions. The ethyl acetate fraction was separated by silica gel, RP-C silica gel, and preparative HPLC to afford the test compound, OPT. The structure of this isolated compound was determined by a combination of spectroscopic analyses, including IR, H and C NMR, hydrogen-hydrogen correlation spectroscopy (H-H COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond coherence (HMBC), mass spectroscopic data, and chemical methods. Based on the spectroscopic analysis, this compound was identified as OPT. MG132 was obtained from Fisher Scientific (Chicago, IL, USA). Annexin-V apoptosis kit was obtained from BD Biosciences (San Jose, CA, USA). CHX, NAC CQ. and Tunicamycin were obtained from Sigma (St. Louis, MO, USA). Anti-GRP78, anti-Bim, and anti-PERK antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-Ubiquitin, anti--actin, anti-c-Myc and anti-MCL1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MCF10A untransformed human mammary epithelial cells, MDA-MB231 human breast cancer cells, and HCT116 human colorectal cancer cells were obtained from ATCC. The MEFs used were established by SV40 large T antigen and were described previously. MCF10A cells were maintained in the ATCC-suggested complete growth medium. HCT116 and MDA-MB231 cells were maintained in DMEM supplemented with 10% fetal bovine serum, mouse bone marrow and MLL-AF9 mouse leukemia cell were maintained in IMEM supplemented with 10% fetal bovine serum containing 10 ng/ml IL-3, 10 ng/ml IL-6 and 100 ng/ml SCF, and KOPN-1 were maintained in RPMI medium supplemented with 10% fetal bovine serum. All cells were cultured in a humidified chamber with 5% CO at 37°C. Cell viability was determined by the MTT assay (Sigma-Aldrich, St. Louis, MO, USA). MTT was dissolved in PBS at 5 mg/ml as a stock solution and sterilized using 0.2 mm filter. MDA-MB231 cells and MCF10A cells were exposed to the indicated concentrations of OPT in a 96-well plate for 48 h followed by the addition of MTT solution (0.5 mg/ml) to the cells, and then the solution was incubated at 37°C for 4 h. The colored metabolite was dissolved in dimethyl sulfoxide (DMSO). Optical densities were determined on a microplate reader (Molecular Devices, Sunnyvale, CA, USA). For the FACS analysis, 2 × 10 cells/well were seeded into 6-well plates. Samples were prepared based on the instructions provided together with the Annexin-V Apoptosis Kit. Briefly, after treatment as indicated in Section, the adherent and detached cells were collected and washed twice with binding buffer containing 10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl, and then 1 × 10 cells were resuspended in 100 l of binding buffer. In all, 5 l of Annexin V-FITC and 10 l of propidium iodide (50 g/ml, stocking concentration) were added to the cell suspension. After gently mixing, the cells were incubated for 15 min at room temperature, and then 400 l of binding buffer was added to get the sample ready. Quantification of cell death was performed using a FACScan (BD Biosciences). Annexin V-positive and/or PI-positive cells were considered as cell death. Intracellular ROS production was monitored by the permeable fluorescence dye, H2DCFDA. H2DCFDA can readily react with ROS to form the fluorescent product 2,7-dichlorofluorescein (DCF). The intracellular fluorescence intensity of DCF is proportional to the amount of ROS generated by the cells. After the indicated treatment, the cells were incubated with 10 M of H2DCFDA for 30 min and then cells were harvested and resuspended in PBS (10 cells/ml). The fluorescence intensity of intracellular DCF (excitation 488 nm, emission 530 nm) was measured using FACScan. All the data analyses were performed using the FlowJo analysis software, version 6.0 (Tree Star, Ashland, OR, USA). Total RNA was extracted with the RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Valencia, CA, USA). cDNA was synthesized using M-MLV reverse transcriptase from Promega (Madison, WI, USA). Primer pairs used for RT-PCR are human XBP1, 5′-GGAGTTAAGACAGCGCTTGG-3′ (sense) and 5′-ACTGGGTCCAAGTTGTCCAG-3′ (antisense); human CHOP, 5′-TGGAAGCCTGGTATGAGGAC-3′ (sense) and 5′-TGTGACCTCTGCTGGTTCTG-3′ (antisense); human Noxa, 5′-GCTCCAGCAGAGCTGGAAGT-3′ (sense) and 5′-GCAGTCAGGTTCCTGAGCAG-3′ (antisense); human Bim, 5′-GACTCTGACTCTCGGACTGA-3′ (sense) and 5′-GCAGGCTGCAATTGTCTACC-3′ (antisense); human GRP78, 5′-TGAAGAGCTCAACATGGATCTGTT-3′ (sense) and 5′-CTACAGCTTCATCTGGGTTTATGC-3′ (antisense); human BCL2, 5′-ATGTGTGTGGAGAGCGTCAA-3′ (sense) and 5′-CGTACAGTTCCACAAAGGCA-3′ (antisense); human BCL-XL, 5′-CCTTTGCCTAAGGCGGATTT-3′ (sense) and 5′-GCTCACTCACTGAGTCTCGT-3′ (antisense); human MCL1, 5′-AGAAAGCTGCATCGAACCATT-3′ (sense) and 5′-CAGCTCCTACTCCAGCAAC-3′ (antisense); human GAPDH, 5′-CTCTGACTTCAACAG CGACAC-3′ (sense) and 5′-CATACCAGGAAATGAGCTTGACAA-3′ (antisense); mouse XBP1, 5′-ACACGCTTGGGAATGGACAC-3′ (sense), mouse GAPDH, 5′-GCACAGTCAAGGCCGAGAAT-3′ (sense) and 5′-GCCTTCTCCA TGGTGGTGAA-3′ (antisense). Human GRP78 cDNA was subcloned into the lentiviral expression vector pCDH-CMV-EF1-puro (System Biosciences, Mountain View, CA, USA). The pLKO.1 lentiviral sh-RNA expression system was used to generate shRNA constructs (sh-GRP78, 5′-CTTGTTGGTGGCTCGACTCGA-3′), (sh-Noxa, 5′-GTAATTATTGACACATTTCTT-3′), (sh-Bim, 5′-CCTTCTGATGTAAGTTCTGTT-3′), and (sh-MCL1, 5′-CGGGACTGGCTAGTTAAAC-3′). Female athymic nude mice (=10/group, 4–6 weeks of age; Harlan, Indianapolis, IN, USA) were used. The use and care of animals was carried out under the guidelines approved by the Institutional Animal Care and Use Committee. Subconfluent HCT-116-Luc cells were harvested and resuspended in PBS. Before inoculation, cell viability was tested by 0.4% trypan blue exclusion assay (viable cells>90%). For subcutaneous injection, ∼1 × 10 HCT-116-Luc cells in 50 l PBS were injected into both flanks of each mouse for each point. Starting the same day, OPT was intraperitoneally (IP) administered at dose of 15 mg/kg/day. Control mice were injected with the vehicle. Animal whole body optical imaging was carried out as described previously. Briefly, animals were subjected to Xenogen IVIS 200 imaging system (Caliper Life Sciences, Hopkinton, MA, USA) for imaging weekly after tumor cell inoculation. -Luciferin sodium salt (Gold Biotechnology, St. Louis, MO, USA) at 100 mg/kg body weight in 0.1 ml sterile PBS was administered IP as a substrate before imaging. The acquired pseudo images were collected by superimposing the emitted light over the grayscale photographs of the animal. Quantitative image analysis was performed with Xenogen's Living Image V2.6.1 software (Xenogen Corp, Alameda, CA, USA). Unless otherwise indicated, at least three independent assays were carried out and the values are presented as mean±S.D. Statistical significance was assessed by Student's two-tailed test and <0.05 was considered as statistically significant.

To evaluate whether MB prevents noise-induced injury to the cochlea, we designed the experiments as shown in . At first, in order to confirm the efficacy of MB pretreatment, threshold shifts were calculated based on the difference between auditory brainstem response (ABR) thresholds before and after noise exposure in each animal. The baseline ABR thresholds (−1 day) did not differ among the experimental groups (). The compound threshold shift (CTS) was measured at 1 day after noise exposure, whereas the permanent threshold shift (PTS) was measured at 14 days after noise exposure. show that the patterns of the threshold shift at 16 and 32 kHz in both the noise-only group and the MB pretreatment (pre-MB) group were similar. In the noise-only group, the mean CTS was >40 dB at both 16 and 32 kHz. In contrast, the pre-MB group demonstrated a significantly decreased mean CTS compared with the noise-only group (). When we measured the PTS in the pre-MB group, there was approximately a 50% reduction in the threshold shift compared with that in the noise-only group (). In addition, to evaluate whether MB posttreatment after noise exposure also has a protective effect, we treated animals with MB for 3 consecutive days after noise exposure (the post-MB group). Although posttreatment with MB attenuated PTS by approximately 20%, we did not observe additional attenuation of noise-induced threshold shift compared with that in the pre-MB group (), suggesting that pretreatment with MB is more effective than posttreatment with MB. We tested the efficacy of the combination regimen of pretreatment with MB and posttreatment with MB for 7 consecutive days before and after noise exposure (the pre+post-MB group). Further, we did not observe any additional reduction in the threshold shift compared with that after MB pretreatment. These results suggest that MB treatment attenuated a noise-induced threshold shift, and moreover, the preventive effect of MB against a noise-induced threshold shift was greater than its rescue effect. Accordingly, we performed pretreatment with MB for further experiments. To evaluate whether MB pretreatment has a protective effect on hair cell death after noise exposure, we performed two types of hair cell morphology assays. First, at 14 days after noise exposure, surface preparation (SP) was used to detect and quantify OHC loss in the basal turn, which is the most vulnerable site for acoustic injury. Confocal microscopic analysis revealed that a number of stereocilia and nuclei were lost in the noise-only group and more numbers of stereocilia and nuclei were preserved in the pre-MB group than in the noise-only group, indicating that the survival rate of OHC in the noise-only group was significantly decreased. However, MB pretreatment significantly increased the survival rate of OHC by approximately 40%. The three rows of OHCs were equally protected by MB (). Second, scanning electron microscopy (SEM) analysis was performed in the basal turn areas. SEM images revealed a significant damage to the stereocilia of OHC in the noise-only group compared with those of the control group; further, the damage to the stereocilia of OHC in the pre-MB group was decreased compared with that in the noise-only group (). These results are in agreement with the results of SP, thereby suggesting that MB pretreatment effectively attenuated hair cell loss after acoustic overstimulation. It is known that excessive ROS and RNS generation after acoustic overstimulation is associated with hair cell loss. 4-Hydroxynonenal (4-HNE) is a membrane peroxidation product that is generated by the reaction of free radicals in the plasma membrane; it is also known to be produced in the cochleae that is damaged by sound-induced trauma. To evaluate whether MB pretreatment attenuates ROS generation after acoustic overstimulation, serial immunohistochemistry for 4-HNE was performed on samples with or without MB pretreatment. After the acoustic overstimulation, 4-HNE expression was strongly detected in the stria vascularis, spiral ligament and OC in the noise-only group compared with the control group. In the case of MB pretreatment, 4-HNE expression was slightly increased compared with that of the control group; however, there was a significant reduction of 4-HNE expression in both the lateral wall and OC compared with that in the noise-only group (). We also examined the levels of 4-HNE protein adducts in mouse cochlear homogenates by the western blotting. Indeed, the elevation of 4-HNE after noise injury was significantly decreased in the pre-MB group (), thereby indicating that lipid peroxidation was attenuated by MB pretreatment. 3-Nitrotyrosine (3-NT) can be used as a marker for oxidation and nitration products, which reflect the reaction with peroxynitrite (ONOO-). The serial immunohistochemistry for 3-NT indicated that it was strongly detected in the lateral wall and OC in the noise-only group compared with the control group (). 3-NT expression was attenuated in the pre-MB group compared with the noise-only group (), which was similar to the result for 4-HNE. Hence, these results suggest that MB pretreatment effectively alleviated oxidative stress in the cochlea after acoustic overstimulation. The blockade of mitochondrial complexes by selective inhibitors can induce the leakage of electrons, thereby accounting for ROS generation and resultant adenosine triphosphate (ATP) depletion-induced cytotoxicity. In order to elucidate the molecular mechanism underlying the protective effects of MB on noise-induced cochlear injury, we used UB-OC1 cells, an organ of Corti (OC) cell line. UB-OC1 cells have been used to investigate the mechanisms of sensorineural hearing loss. Mitochondrial damage induced by mitochondrial complex inhibition has also been used as an model of NIHL. Therefore, we used rotenone, antimycin A and oligomycin to inhibit complex I, III and V, respectively, with or without MB treatment of UB-OC1 cells. Cell viability was significantly increased in rotenone and MB co-treated cells compared with that in rotenone-only treated cells (). This protective effect was also observed in antimycin A and MB co-treated cells (). In contrast to these results obtained with complex I and III inhibitors, MB failed to protect cells from complex V inhibition by oligomycin (). Next, we examined the effect of rotenone, antimycin A and oligomycin with or without MB on mitochondrial ATP generation. After 3 h of rotenone, antimycin A and oligomycin treatment, a significant decrease in cellular ATP concentrations was observed. However, MB co-treatment significantly restored ATP concentrations reduced by both rotenone and antimycin A; yet, MB co-treatment did not restore ATP concentrations reduced by oligomycin (). Neither cell viability nor mitochondrial ATP generation was affected solely by MB treatment (). Taken together, the data from the cell line experiment indicate that the amelioration of impaired mitochondrial function by MB is due to the reduction of electron leakage specifically in complex I/III. Recent studies have found that MB has been reported to improve the activity of complex IV. Therefore, we next tested whether MB pretreatment improved the cochlear mitochondrial complex IV activity Complex IV activity in the mouse cochlea was suppressed by approximately 20% immediately after noise exposure in the noise-only group. On the other hand, MB significantly prevented the reduction of complex IV activity after noise exposure (). However, only MB pretreatment had no effect on the complex IV activity. MB treatment has been reported to promote an increase in the level of brain-derived neurotrophic factor (BDNF) , which may contribute to improved behavior in Huntington's disease model. Acoustic overexposure is associated with the loss of trophic support to the central auditory pathway as well as with the alteration of afferent nerve endings in the IHC. Therefore, we also examined the expression of two representative cochlear neurotrophic factors (NTFs), NT-3 and BDNF. Western blotting data indicate that MB pretreatment without noise exposure had no effect on the expression of both BDNF and NT-3 (). However, MB pretreatment significantly increased NT-3 expression in the acute phase of noise-induced cochlear damage (). In contrast, neither noise exposure nor MB pretreatment affected BDNF expression (). We further examined the neuroprotective effect of MB on cochlear synaptic structures. Synapsin-1, a synaptic vesicle membrane protein, was used to identify the putative efferent nerve endings that innervate OHC and IHC. On day 2, we found that synapsin-1 expression was significantly diminished in the noise-only group compared with the control group. However, the impairment of synapsin-1 expression was reversed by MB pretreatment (). Further, we also observed postsynaptic density protein 95 (PSD-95) expression to detect the changes in postsynaptic afferent nerve endings between IHCs and spiral ganglion neurons (SGNs). On day 2, the distribution area of PSD-95 positive puncta on the synaptic region between IHCs and first-order synaptic neurons was reduced in the noise-only group (). On the other hand, MB conserved the distribution area of afferent synapses around IHCs compared with the noise-only group. In addition, the average number of PSD-95 puncta per IHC was quantified (). MB pretreatment effectively conserved PSD-95 puncta compared with the noise-only group. These results indicate that the possible mechanisms underlying the protective effect of MB may not only be related to the improvement in the mitochondrial function of hair cells but also to the conservation and/or regeneration of cochlear synaptic components. As an old drug, MB has been used in various areas of medicine and biology, because it is inexpensive, well tolerated and relatively effective. Recently, this agent has attracted great attention because of its neuroprotective effects in a variety of mitochondria-targeted cytotoxicity paradigms, such as ROS overproduction; therefore, it is potentially useful in treating many neurological disorders. As in neurodegenerative diseases, the overproduction of ROS and RNS has been known to contribute significantly to the progression of NIHL, which can be an effective therapeutic target of MB. It has been reported that a single infusion of MB could rapidly reach a higher concentration in various organs, such as the brain and liver, compared with the plasma. Furthermore, MB contains a central aromatic thiazine ring system that confers a high lipophilicity. Therefore MB could cross the blood-cochlear barrier as well as the blood-brain barrier easily and be distributed to the cochlear tissue. Our data indicate that MB reduces noise-induced ROS and RNS formations in the cochlea. It is noteworthy that both 4-HNE and 3-NT formations were reduced not only in the OC but also in the stria vascularis by MB pretreatment, indicating that MB could augment the mitochondrial antioxidant function in the stria vascularis, which is highly metabolically active and is most commonly affected by mitochondrial dysfunction. Mitochondrial complexes I and III are responsible for the non-specific transfer of electrons to O2 and the resultant free radical generation. On the other hand, complex IV, also referred to as cytochrome oxidase, is the key enzyme in the production of ATP and carries electrons from cytochrome to molecular oxygen in the final step of the mitochondrial ETC. As MB has an auto-oxidizing capacity, it can enter a reversible redox cycle and interact with oxygen in order to form water, which would decrease ROS generated by high energy demands or mitochondrial failure. It has also been reported that MB has cytoprotective effects against mitochondrial damage. In agreement with previous reports, we observed the involvement of MB in mitochondrial complexes I-III . In UB-OC1 cells, MB selectively reduced ATP depletion and cytotoxicity induced by complexes I and III inhibitors, such as rotenone and antimycin A, respectively but not that induced by complex V inhibitor, oligomycin, suggesting that MB may function as an alternative electron carrier between complex I and complex III in auditory hair cells. As these inhibitors and MB participate in distinct parts of ETC, these effects might not be originated from direct interaction between these inhibitors and MB. In addition, MB has also been reported to enhance complex IV activity. We also observed that noise exposure attenuated mitochondrial complex IV activity, whereas MB pretreatment ameliorated the decrease of complex IV activity induced by noise exposure . A possible mechanism by which MB could enhance complex IV activity is through the increased expression of complex IV subunits, such as subunit I and subunit II. Therefore, these results indicate that improvement in both complexes I-III and complex IV activity by MB may reduce the mitochondria-generated oxidative stress in the cochlea and result in an increased ATP production, which reflects the improved energy status in hair cells. In agreement with our speculation, it was demonstrated that the supplementation of coenzyme Q10 analog in noise-exposed rats attenuated cochlear injury, which was partly a consequence of sustained respiratory enzyme activation by exogenous quinones. Several studies have demonstrated the role of NO in the noise-exposed cochlea. Intense noise exposure or ischemia/reperfusion injury engendered an upregulation of inducible nitric oxide synthase (iNOS) in the cochlea, altering the NO concentration in hair cells. It affects cellular calcium homeostasis and also increases nitrotyrosine, a harmful byproduct of NO-mediated reactions, which causes irreversible mitochondrial respiratory damage. Nitric oxide (NO)-induced elevation of intracellular calcium is mediated by soluble guanylyl cyclase (sGC), which acts as a receptor for NO and is considered as an inhibitory target of MB, independent of mitochondrial ETC. Therefore, our result, that MB decreases noise-induced 3-NT formation, is possibly not only due to the indirect reduction of RNS generation by MB following the reduction of mitochondrial ROS but also due to the direct reduction of RNS generation following the inhibition of sGCs in hair cells as well as NOS in SGNs. In addition, NOS is also activated by excitatory glutamatergic receptors in the postsynaptic neurons and could be an effective modulatory target of MB. We also observed that MB selectively increased NT-3 expression in the noise-exposed cochlea. However, unlike the previous finding, that MB increases BDNF expression in Huntington's disease mouse model, MB did not affect BDNF expression in the present study. NTFs, including nerve growth factor, BDNF, NT-3 and neurotrophin-4/5, have an essential role in neuronal survival, differentiation, axonal growth and neural plasticity. BDNF and NT-3 are predominantly expressed in the cochlea. BDNF is vital for the survival of vestibular sensory neurons, whereas SGNs depend on NT-3 during development. These NTFs have also been reported to scavenge free radicals, interrupt cell death pathways and, further, modulate calcium homeostasis, any of which may attenuate NIHL . Therefore, the delivery of exogenous NTFs, such as NT-3, fibroblast growth factor 2 and glial cell-derived NTF, into the cochlea has been reported to prevent noise-induced hair cell death. In addition to preserving hair cell survival, NTFs have also been reported to preserve neuronal survival in the absence of surviving hair cells. Moreover, NT-3 and BDNF have also been reported to protect the inner ear from cisplatin- or aminoglycoside-induced ototoxicity and Accordingly, MB-induced NT-3 expression may contribute to the protective role of MB in noise-induced cochlear injury. Synaptic efficiency between hair cells and SGNs in the cochlea can be affected by noise overexposure. Intense noise can induce the release of toxic concentrations of glutamate, which causes the alteration of synaptic connection between hair cells and SGNs and, ultimately, lead to the degeneration of SGNs. In addition to excitotoxicity induced by glutamate, these synaptic damages can also be attributed to glutamate-driven oxidative cell death pathways. In this study, we observed that MB reversed the change in the cochlear synaptic structure induced by intense noise exposure. Exposure to intense noise resulted in the loss of PSD-95 positive puncta on the synaptic region. However, it increased the PSD-95 expression in the region of the first-order synaptic neurons, which is in agreement with a previous report, which demonstrates that PSD-95 is increased in SGNs by sound stimuli and, further, the increase in PSD-95 expression is dependent on the synaptic activity. An increase in PSD-95 expression was also observed in the MB pretreatment group, suggesting that MB may not reduce the glutamate release induced by noise exposure, but instead, it may inhibit glutamate-driven cell death pathways by attenuating ROS and RNS formation and/or by enhancing NT-3 expression. Chemical-induced hearing loss, such as that caused by aminoglycoside and cisplatin, and presbycusis share pathophysiological mechanisms with NIHL to some extent, particularly in the context of protective effects of MB demonstrated in this study. Accordingly, the results obtained in the present study indicate that MB might protect the cochlea from hearing loss caused by chemicals and senescence. MB has already been used in humans for other indications, and therefore, it can be translated into clinical trials more quickly; such factor could be an additional advantage of MB. This study provides and evidences for the therapeutic use of MB in NIHL, and moreover, they serve as a basis for the investigation of sensorineural hearing loss caused by impaired mitochondrial ETC and neurotrophic signaling. The protocol used in this study was approved by the animal protocols and guidelines established by Ajou University School of Medicine Ethics Review Committee for Animal Experiments. All animal work was approved by the Ethical Committee for Animal Research of Ajou University (AMC-55). BALB/c male mice, 7–8-week old, were used in this study. The mice, which were obtained from Orient Bio Inc. (Seoul, Korea), were examined and maintained according to the experimental schedule after noise exposure (). The MB pretreatment group was treated with 2 mg/kg of MB (66720, Sigma Aldrich, St. Louis, MO, USA) via oral gavage for 4 days before noise exposure. Weight loss or activity impairment was not observed in the treated animals. The noise-only group was treated with distilled water by gavage at the same time. The control animals were neither exposed to noise nor given MB. A total of 118 animals were used for the study. Exposure to noise was performed in a sound chamber. The ventilated chamber was fitted with a high-frequency driver (DH-7, Electro-Voice, Burnsville, MN, USA) driven by a function/arbitrary waveform generator (33210A, Agilent Technologies, Santa Clara, CA, USA), generating a 0.1 Hz–15 MHz range sound wave, and power amplifier (AX5505, Inkel, Incheon, Korea). The driver was located directly above the animals. Animals were exposed to a 112-dB sound pressure level of broad-band white noise (1–20 kHz) measured by a dual channel real time frequency analyzer (type 2144, Brüel and Kjær, Nærum, Denmark) for 3 h. Each animal was separated by a wire mesh cage with a diameter of 10 cm in order to abolish the attenuation of sound pressure by the adjacent animals. ABR analysis was performed in order to measure the auditory thresholds in all mice as well as to exclude animals with hearing problems. ABR measurements were carried out as previously reported. The hearing status was evaluated with a Biosig 32 ABR system (Tucker-Davis Technologies, Gainesville, FL, USA). Briefly, each mouse was anesthetized with an intraperitoneal injection of chloral hydrate (00672, Sigma Aldrich). ABRs were recorded transcutaneously with sterile electrode needles. The reference electrode was inserted beneath the pinna of the measured ear and the ground beneath the opposite ear; the active electrode was inserted beneath the skin on top of the head. The stimuli were presented through an earphone, which was placed directly in the external auditory canal. After selecting the hearing frequency, precalibrated stimuli of 16 or 32 kHz were presented from 75 dB and were then automatically attenuated to 10 dB in five steps until the ABR waves disappeared. An average of 1000 stimuli was gathered at each frequency, and each result was recorded and saved. The hearing thresholds were determined by the lowest intensity that is capable of eliciting replicable, visually detectable waves. ABRs for the 16 and 32 kHz stimuli were recorded before the noise exposure as well as at 1 and 14 days after noise exposure. Animals were anesthetized and transcardially perfused with saline solution containing 0.5% sodium nitrate and heparin (10 U/ml), followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), pH 7.2, for tissue fixation. Cochleae were dissected from both sides of the temporal bone and postfixed overnight at 4 °C in 4% PFA. The fixed cochleae were washed three times with a phosphate-buffered saline (PBS) for 10 min and then decalcified with a 5% EDTA solution for 3 days. After decalcification, the samples were fixed in paraffin. Four series of 5-m sections were obtained with a sliding microtome (Leica, Wetzlar, Germany). The serial sections were then mounted on gelatin-coated slides, rinsed three times with PBS, treated with 3% HO for 5 min and, finally, rinsed with PBS containing 0.2% Triton X-100 (PBST). A non-specific binding was blocked with 1% bovine serum albumin in PBST. The sections were incubated overnight at 4 °C, with the primary antibody against 4-HNE (ab46545, 1 : 100 dilution; Abcam, Cambridge, UK) and 3-NT(ALX-804-505, 1 : 400 dilution; Enzo life science, Farmingdale, NY, USA). After rinsing in PBST, the sections were incubated with a biotinylated secondary antibody (BA-1000, BA-2001, Vector Laboratories, Burlingame, CA, USA) for 1 h and with the avidin/biotin system (Vector Laboratories) for 1 h; they were then visualized using a 3, 3'-diaminobenzidine (D5637, DAB, Sigma Aldrich) solution (0.05% DAB and 0.003% hydrogen peroxide in 0.1 M PB). Subsequently, the nuclei were counterstained with hematoxylin QS (H-3404, Vector Laboratories). Bright field images were obtained using the PictureFrame Application 2.3 software (Optronics, Goleta, CA, USA). The pictures were taken only from the midmodiolar sections, from which only the basal turn area was shown in this study. Three independent experiments were performed for the immunohistochemical analysis. Phalloidin staining was performed in order to detect the changes in hair cells. After the completion of ABR analysis at day 14, temporal bones were dissected. The dissected cochleae were perfused with 4% PFA in 0.1 M PB and kept overnight at 4 °C. The cochleae were washed three times with PBS for 10 min and then decalcified with 5% EDTA solution for 3 days. After decalcification, the otic capsule was removed, followed by the removal of the lateral wall, Reissner's membrane and the tectorial membrane under a light microscope. The OC was stained with Texas Red VR-X phalloidin, which was used for observing hair cell stereocilia (T7471; 200 U/ml methanol diluted 1 : 100 in PBS; Invitrogen, Eugene, OR, USA) for 1 h. Thereafter, the whole mount was rinsed in PBS and further dissected into a SP (microdissected into individual turns). Then, it was mounted on glass slides using a mounting solution containing 4′,6-diamidino-2-phenylindole (DAPI), which was used for observing hair cell nuclei (H-1200; 1.5 g/ml; Vector Laboratories). The slides were incubated for at least 15 min. Hair cells in the OC were visualized under a confocal microscope (LSM510, Carl Zeiss, Jena, Germany). Based on the study by Viberg and Canlon, the examined areas and cytocochleograms were assessed by measuring from the hook area. Each region comprised 200 m of OC corresponding to 32 kHz. The criterion for hair cell loss was missing OHCs using both DAPI and Texas Red VR-X phalloidin staining. Missing OHCs appeared as a dark spot in the spaces previously filled by OHCs due to the complete disappearance of the nuclei and the stereocilia. For immunofluorescence, anti-synapsin-1 (SC-55774; 1 : 100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used in order to label the efferent nerve endings; the anti-PSD-95 (75-028, 1 : 400 dilution; UC Davis/NIH NeuroMab Facility, Davis, CA, USA) was used to label the postsynaptic densities in the first-order cochlear neurons. The dissected OCs were incubated overnight with primary antibodies. After rinsing in PBST, the specimens were incubated with Alexa Fluor 568-conjugated secondary antibody (A11057, A11031, Invitrogen) for 1 h. We quantified the fluorescent intensity of synapsin-1 staining. Images used for quantification were taken with identical microscope settings and analyzed using the ImageJ software (NIH, Bethesda, MD, USA) based on the study by Cerpa Each region comprised 100 m of OC corresponding to 32 kHz. In addition, we counted the number of PSD-95 puncta in the dendrites having direct contact with IHC. Each IHC and PSD-95 punctum was counted within the region corresponding to 32 kHz. Further, each region comprised of 12–15 IHCs. In order to avoid undercounting the puncta due to the superimposition of the stack images, three-dimensional-rendered images were produced by the Zeiss LSM Image Browser (Carl Zeiss) and then rotated in order to make accurate estimates in the x-y plane projection images. SEM was performed for the morphological evaluation of hair cells in the cochlea. Mice were anesthetized with an intraperitoneal injection of urethane (Sigma Aldrich) and then decapitated quickly. Next, temporal bones were carefully dissected. The round and oval windows of the cochlea were perforated with a sharp pick. The perilymphatic space was then perfused with 2% glutaraldehyde in PBS by placing a Pasteur pipette with a modified butterfly catheter over the round window. Each specimen was then placed in glutaraldehyde solution overnight. The cochlear surface and perilymphatic space were rinsed three times with PBS. Then the specimens were perfused with 1% osmium tetroxide and placed on a tissue rotator for 15 min. The samples were then rinsed three times in PBS. Under a dissecting microscope, the bony capsule of the cochlea was carefully removed and the lateral wall was cut away in order to expose the OC. The tissue was dehydrated serially in 50, 70, 90, 95 and 100% acetone. Each specimen was treated with hexamethyldisilazane and then air dried; subsequently, it was placed on a stub for sputter-coating with platinum. The tissue was then viewed and photographs were taken with a JSM-6380 (Jeol, Tokyo, Japan). Hair cell damage was analyzed in the basal turn areas corresponding to 32 kHz and was determined by the loss of the stereocilia. The animals were killed immediately after ABR test at day 14. At days 0, 1, 3 and 7, animals were euthanized with a urethane intraperitoneal injection (1 g/kg), and both temporal bones were immediately removed. Two cochleae were homogenized in a tube containing ice-cold lysis buffer (100 mM Tris, pH 7.4, 200 mM NaCl, 1% NP-40, 10 mM MgCl2) with protease inhibitors. Western blotting was performed, as described previously. In brief, lysates were centrifuged for 20 min at 13 000 r.p.m. at 4 °C; in addition, proteins in the supernatant were separated using 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were incubated overnight with the primary antibodies against 4-HNE (1 : 1000 dilution), BDNF (ab6201, 1 : 1000 dilution; Abcam) and NT-3 (ab65804, 1 : 1000 dilution; Abcam). After rinsing in TBST, the membranes were incubated with the HRP-conjugated secondary antibody (81–1620, A16104, G21040; 1 : 5000; Invitrogen) for 1 h. Bands were detected by chemiluminescence (LF-QC0103, Ab Frontier, Seoul, Korea). Further, blots were stripped and reprobed with actin (SC-1616, Santa Cruz Biotechnology), thereby serving as a protein loading control. Band intensities were quantified using the ImageJ software (NIH). UB-OC1 cells were cultured (1.1 × 10 cells per well of six-well plate) and maintained for 24 h under permissive conditions: 33 °C, 5% CO in DMEM supplemented with 10% FBS and 50 U/ml interferon-γ. Cells were further incubated in glucose-free DMEM (11966–025, Invitrogen) supplemented with 25 mM galactose for 48 h in order to force the cells to use mitochondrial oxidative phosphorylation. Then the cells were treated with vehicles (DMSO, ethyl alcohol), rotenone (557368; 0.2 M in DMSO; Millipore, Billerica, MA, USA), antimycin A (A8674; 1 M in ethyl alcohol; Sigma Aldrich) and oligomycin (O4876; 5 M in DMSO; Sigma Aldrich) in the presence or absence of 2 M of MB for 12 h. At the end of 12 h, the medium was replaced by a maintenance medium supplemented with 5% water soluble tetrazolium salt (EZ3000, Daeil Lab Service, Seoul, Korea) for 30 min in order to determine the cell viability. Then the optical density (OD) of each culture well was measured using a microplate reader at 540 nm. The OD of the control cells was taken as 100% viability. UB-OC1 cells were plated in a six-well plate, as described above. The same doses of rotenone, antimycin A and oligomycin with or without MB were added to the culture medium and incubated for 3 h. The level of ATP was determined by the ATP bioluminescence assay kit according to the manufacturer's instructions (11699709001, Roche, Mannheim, Germany). In brief, the treated cells were rapidly rinsed with ice-cold PBS. Next, they were scraped from the bottom of the dishes with a dilution buffer and then moved to conical tubes. The same volume of the cell lysis reagent was added; further, the lysates were incubated at room temperature for 5 min. After transferring the lysates to a black microtiter plate, measurements were performed immediately after the addition of the luciferase reagent to the samples and standards at 562 nm. A standard curve was generated on the same plate. Cells treated with neither MB nor mitochondrial complex inhibitors were used as the control. The activity of mitochondrial complex IV was determined by measuring the cytochrome oxidase activity using the mitochondrial complex IV (mouse) activity assay kit according to the manufacturer's instructions (AAMT006, Merck, Darmstadt, Germany). Both cochleae from each mouse were homogenized in a tube containing ice-cold dilution buffer, and a detergent was added; then, the samples were centrifuged for 20 min at 16 000 r.p.m. at 4 °C. Finally, the supernatant was incubated in an assay solution, and the reduced state of cytochrome was added. In all, 50 g of protein was added, and then the OD of each culture well was monitored using a microplate reader at 550 nm for 120 min at 30 °C. The rate of activity was determined by calculating the slope between two points within the linear region. The OriginPro software (Ver.8; OriginLab, Northampton, MA, USA) was used for all statistical analyses. Group comparisons between the noise-only group and various MB treatment groups were performed using a one-way ANOVA or two tailed Student's -test. Whenever a significant difference was found with ANOVA, a Tukey's test was performed in order to identify the specific difference between groups. The data are presented as the mean±S.E.M. A -value of<0.05 was deemed to indicate statistical significance.

transcripts were detected at high levels in testis, while at low levels in heart and kidney (). To detect the global level of ESET and H3K9me3 in spermatogenesis, qRT-PCR and western blot using multiple tissues from testis at different developmental stages were performed. The expression of ESET was gradually increasing during testis development, in parallel to the global level of H3K9me3 (). We further examined the distribution of ESET and H3K9me3 in SSCs by co-immunofluorescence staining. The result showed both ESET and H3K9me3 were presented in SSCs (positive for PLZF or Thy1, and ). Interestingly, distinct distribution of H3K9me3 was observed in mouse testis. H3K9me3 displayed an exclusively perinuclear distribution in SSCs while localized to punctate foci in differentiated spermatogonia (positive for KIT, ), which is consistent with previous reports. As a heterochromatin marker, this unique distribution of H3K9me3 may be used to distinguish SSCs and the differentiated spermatogonia in mouse testis. As the population of SSCs in the testis is very low, MACS using anti-Thy1 IgG-conjugated microbeads was performed to enrich SSCs. The purity of the isolated cells was 92% as indicated by immunocytochemistry for Thy1 (). To reveal the role of ESET in SSCs, RNA interference using lentiviral vectors coding for short hairpin (sh) RNAs directed against was performed in primary SSCs. The expression of ESET in -small hairpin RNA (shRNA) lentiviral transduced SSCs was significantly reduced examined by qRT-PCR () and western blot (). In addition, we found that depletion of ESET resulted in deduction of H3K9me3 (), suggesting that ESET is required for the maintenance of H3K9me3 in SSCs. SSCs were enriched through MACS, transduced with -shRNA lentiviral particles and cultured on feeder layer of Sertoli cells. Following 2-week cultivation , the number of SSCs in ESET-knockdown (KD) group (transduced with -shRNA lentivirus) was significantly reduced compared with the control (transduced with scrambled shRNA lentivirus or no lentivirus infection) (). Immunofluorescence for Lin28 was performed to further confirm the SSCs number post culture (). It has reported that Lin28, as a pluripotency factor, was specifically expressed in As, Apr and Aal spermatogonia, which were widely considered as SSCs in mouse. Lin28 was used as a SSCs marker in the previous studies and has been shown to be associated with stemness of SSCs. In our experiments, Lin28 was used to distinguish the undifferentiated spermatogonia from other types of cells(such as Sertoli cell and differentiated spermatogonia).The result showed that Lin28-positive cells in ESET-KD group were significantly reduced (), suggesting ESET deficiency led to decrease of the number of SSCs probably because of apoptosis or differentiation. To test whether ESET regulates the maintenance of SSCs , we applied the assay of SSC transplantation. SSCs were enriched through MACS, seeded on laminin-coated plates and transduced with -shRNA lentiviral particles. Two days later, cells were transplanted into recipient mouse testis that was previously treated with busulfan to deplete endogenous germ cells (). At 10 weeks post surgery, the weight of testes from recipient mice transplanted with ESET-KD SSCs was less than that of those transplanted with NC SSCs or Mock SSCs (no lentivirus transduction) (<) (). We examined further the spermatogenesis in the recipient mice. Seminiferous tubules showing spermatogenesis were analyzed according to the previous reports. The number of seminiferous tubule with (at least two layers of germ cells) or without spermatogenesis was counted for measuring the recovery of spermatogenesis. The result showed the number of seminiferous tubules showing spermatogenesis in ESET-KD group was lower than that in the control groups (). These data suggested that in the control groups, SSCs could home in the basement membrane of seminiferous tubules and initiate spermatogenesis, but SSCs in the ESET-KD group could not do so. Since the lentiviral backbone contained an EF-1/GFP expression cassette, GFP expression would mirror the transplanted SSCs that were tranduced with lentiviral vectors successfully and their progeny cells in the seminiferous tubules. Expression of GFP and PLZF significantly reduced in the ESET-KD group, indicating that the number of transplanted SSCs in recipient mice testis was reduced upon depletion of ESET (). These observations were consistent with the experiments (), indicating that ESET was essential for the survival of SSC. SSCs were enriched through MACS and transduced with lentiviral shRNA expression construct as above. Following 1-week cultivation , terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was applied to detect apoptotic cells. The results showed that the number of apoptotic cells in ESET-KD group was significantly increased (),suggesting ESET regulated the apoptosis of SSCs. The above data also suggested that apoptosis might be one of key reasons in reduction of the number of SSCs after ESET depletion both and . cDNA microarray analysis was further performed to screen the genes modulated by ESET. RNA was isolated from the 1-week cultured cells. Interestingly, the expression of apoptosis-associated genes such as apoptotic protease activating factor 1 ( was upregulated, whereas that of apoptosis -suppressed genes such as X-linked inhibitor of apoptosis protein was downregulated. The changes in expression of the aforementioned genes were further validated by qRT-PCR (). In addition, western blot assay showed that the suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase (PARP) (). These data suggested the scenario for SSC apoptosis by ESET depletion might initiate from upregulating apoptosis inducers such as Apaf1, Caspase9 and downregulating apoptosis suppressors such as Bcl2l1, X-linked inhibitor of apoptosis protein to promote assembly of the apoptosome complex, and in turn activating the effector Caspase3 and further causing the cleavage of PARP. To further understand the mechanisms by which ESET regulates SSC apoptosis, we conducted chromatin immunoprecipitation (ChIP) analysis. As it was hard to collect a large number of the primary SSCs for ChIP assay, a spermatogonial stem/progenitor cell line (C18-4 cells) was used in this study. We selected 16 of the upregulated genes from the microarray data and analyzed their proximal promoter within 4 kb upstream from the transcriptional start site. ChIP analysis using anti-ESET IgG showed that ESET bound to the promoter region of five genes ( spermatogenesis and oogenesis specific basic helix-loop-helix 2 () and deleted in azoospermia-like ()) (), the expression of which increased when ESET was knocked down (), indicating that these five genes were regulated by ESET. However, we did not find that ESET bound to promoter loci of apoptotic inducer and examined by ChIP assay using ESET antibody, although the transcription of these two genes was upregulated in ESET-KD group. Previous studies have shown that CoxIV enrichment were early events preceding the onset of apoptosis and that oxidation of cytochrome by cytochrome oxidase-stimulated caspase activation. Therefore, among the five ESET-targeting genes, Cox4i2 (also named as CoxIV-2, belongs to the cytochrome oxidase IV family) may be a mediator for ESET to regulate apoptosis of SSCs. To further address whether KD of Cox4i2 rescues the cell death phenotype of ESET-KD cells, both siRNA of ESET and Cox4i2 were transfected to C18-4 cells simultaneously. TUNEL assay showed that apoptosis was reduced in cells co-transfected with both siRNA of ESET and Cox4i2 compared with that of only transfected with siRNA (). qRT-PCR assay showed that the level of Caspase9 was decreased in co-transfection group (), which is consistent with the TUNEL assay. These observations further suggest that KD of Cox4i2 partially rescues the cell death phenotype of ESET-KD cells and ESET may indirectly regulate Caspase9 via Cox4i2 and influence SSC apoptosis. Interestingly, ChIP experiment using H3K9me3 antibody revealed that H3K9me3 in the promoter regions of the regulated genes decreased in ESET-KD treatment (), suggesting that ESET binds to these genes and represses their expression via increased H3K9me3 marks. To further elucidate whether depletion of ESET lead to change of DNA methylation, we performed bisulfite sequencing PCR. DNA methylation at promoter regions of ESET-target genes and (co-targeted by ESET and H3K9me3) was analyzed. The results showed that DNA methylation at the promoter decreased, but did not change at the promoter of Dazl, in ESET-KD cells (), suggesting ESET also regulates and influence SSC apoptosis by increasing DNA methylation. SSCs maintain sperm production in the testis throughout adult life. In rodents, SSCs are considered to be a subpopulation of the most undifferentiated spermatogonia: the single type A (As) spermatogona. The study of the mechanisms regulating SSC fate is challenging because of the rare number and difficulty in genetic manipulation of SSCs. To solve this problem, in this study MACS was applied to enrich SSCs and lentiviral vectors were used to manipulate the purified SSCs. MACS with anti-Thy1 IgG has been successfully enriched SSCs in our laboratory and the other groups. The Thy1-positive (as a marker of SSCs) cells were as high as 92% in the MACS-sorted cells ().Short hairpin RNAs can be expressed from lentiviruses, allowing for high transfection efficiency of a variety of cell types, including non-dividing cells and stem cells. It has been reported that lentivirus showed high transduction (about 53.7%) with an optimized protocol in SSCs. In this experiment, SSC transplantation was used to study the function of SSCs. SSCs were purified from fertile donors and transplanted into the testes that was previously treated with busulfan to deplete endogenous germ cells. SSCs are able to migrate into the basement membrane of the seminiferous tubules, form colonies and produce sperm cells. SSC transplantation has become a golden standard for detecting functional activities of SSCs and was used for elucidating the molecular mechanisms involved in self-renewal and differentiation of SSCs. We acquired the purified SSCs from 6–8 days old mice. After lentiviral transduction, SSCs were transplanted into recipient mouse testis. ESET-KD SSCs were under apoptosis and lost the SSC activity, while the control SSCs were able to homing and initiate spermatogenesis. Ten weeks after transplantation, the number of seminferous tubules with spermatogenesis in the control was significant higher than that in the ESET-KD group. The more the seminiferous tubules with spermatogenesis, the more SSCs survived and differentiated. Since the transduction efficiency of lentivirus was moderate, SSCs, which were not transduced with lentiviral particles in ESET-KD group, were also able to homing and initiate spermatogenesis. Therefore, it would produce background when counting the colonies. To avoid this problem, western blot of GFP were used in this study. Because the lentiviral backbone contained an EF-1/GFP expression cassette, only lentiviral transduced SSCs and their progeny cells would show GFP expression. Therefore, the level of GFP in recipient mouse testis can reflect the number of transplanted SSCs with lentiviral transduction. Expression of GFP significantly reduced in the ESET-KD group (), indicating that the number of SSCs in recipient mice testis was reduced upon depletion of ESET. As well known, once cytochrome is released into the cytoplasm it binds to Apaf1, which then binds to Caspase9 to form a protein complex known as an apoptosome. Apoptosome is central to the induction of apoptosis through activating the effector Caspase3 and further causing the cleavage of PARP. In SSCs, as a negative regulator of apoptosis, ESET inhibits assembly of the apoptosome complex through suppression of and , and thus influence activation of and the cleavage of PARP. We noticed that Caspase9 and the number of apoptotic cells in co-transfection group were also higher than those in the control group, although they were significant lower than those in -siRNA group in the rescue experiment. We speculated that other apoptosis regulatory pathways regulated by ESET may exist in SSCs. On the one hand, ESET may upregulate Caspase9 expression by other target gene(s) in addition to , because depletion of expression in ESET-KD cell was not able to restore the Caspase9 expression to normal level (). On the other hand, ESET may modulate apoptosis via other pathways. The focus of our present work was to provide a regulation mode of ESET including H3K9me3 and DNA methylation, and the integral mechanisms of apoptosis regulation require further study. In mammalian cells, H3K9me3 is a hallmark of heterochromatin and is important for silencing of genes and retroelements. In the present study, ChIP assays showed that ESET directly bd to the promoters of the and . Furthermore, in the depletion of ESET, the H3K9me3 mark on these gene loci was reduced, leading to upregulation gene expression of and . It was reported that the genes upregulated after deletion of ESET in mouse embryonic stem cells (ESCs) are distinct from those derepressed in ESC deficient in the DNA methyltransferases, with the exception of a small number of primarily germline-specific genes (such as and ). In SSCs, we found and showed high level of H3K9me3 and DNA methylation in their promoter region, suggesting these genes were suppressed by H3K9me3 and DNA methylation simultaneously. Interestingly, the DNA methylation in the promoter loci of , but not of , was influenced by ESET, indicating that ESET also regulate gene expression through DNA methylation in addition to histone methylation. In conclusion, we showed that in SSCs, expression was suppressed by ESET, which was recruited to the promoter of and increased the level of H3K9me3 and DNA methylation which highly correlated with constitutive heterochromatin. SSC apoptosis caused by ESET depletion through derepressed Cox4i2 and further upregulated which in turn activates effector and cleavage of PARP (). Wild-type C57BL/6J and Kunming mice were used in our experiments. All animals were housed in a barrier facility under normal light and dark conditions with free access to food and water. All experimental procedures involving animals were approved by the Northwest A&F University's Institutional Animal Care and Use Committee. SSCs were cultured on plate with laminin-coated or Sertoli cell feeders. Primary Sertoli cells cultured using DMEM/F12 (Gibco, Grand Island, NY, USA) were supplemented with 10% FBS (Gibco). When Sertoli cells reached about 90% confluency, the cells were treated with mitomycin C (10 mg/l, Sigma, St. Louis, MO, USA) for 3 h and washed five times with PBS. SSCs culture medium consisted of DMEM/F12 (Gibco) supplemented with 1% FBS (Gibco), 30 ng/ml -estradiol (Gibco), 100 U/ml penicillin, 100 g/ml streptomycin, 1 × MEM non-essential amino acids, 20 ng/ml GDNF, 10 ng/ml mouse EGF, 10 ng/ml bFGF. The medium was changed every 2–3 days. The C18-4 cell line was established from type A spermatogonia isolated from 6-day-old mouse testes. The cells were maintained in DMEM medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, 2 mM glutamine, 50 U/ml penicillin, 50 g/ml streptomycin and 100 mM non-essential amino acids. The siRNA sequences targeting mouse ESET and Cox4i2 mRNA were designed and synthesized by Genepharma Company (Shanghai, China). The siRNAs were transfected into C18-4 cells using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Testes from 3-month-old C57BL/6J mice were used for immunohistochemistry. The following primary antibodies were used: rabbit anti-ESET (1 : 50; Proteintech, Chicago, IL, USA), rabbit anti-H3K9me3 (1 : 500; Millipore, Billerica, MA, USA), goat anti-PLZF (1 : 100; Santa Cruz, Dallas, TX, USA), goat anti-KIT (1 : 100, Santa Cruz) and goat anti-Thy1 (1 : 150, Santa Cruz). The following secondary antibodies were used: Alexa 488-conjugated donkey anti-goat IgG and Alexa 594-conjugated donkey anti-rabbit IgG (1 : 400, Invitrogen). Immunofluorescence images were obtained with a Nikon i90 microscope (Nikon, Tokyo, Japan). The U6 RNAi cassette fragment from pSilencer 2.1-U6 hygro (Life Technologies, Carlsbad, CA, USA, AM5760) was amplified and cloned into pCDH-CMV-MCS-EF1-GreenPuro (CD513B-1, SBI, Mountain View, CA, USA) to generate pCDH-U6-MCS-EF1-GreenPuro Lentivector. A sequence specific to the mouse cDNA used in the experiment was 5′-GGTGATGAGTACTTTGCAAAT-3′. A scramble sequence (5′-GATGAAATGGGTAAGTACA-3′) was used as a negative control. HEK 293T cells were transfected with pCDH-U6-ESET/NC-shRNA and the other three plasmids (pGag/Pol, pRev, pVSV-G). Lentivirus-containing supernatants were collected and stored at −80 °C. Lentivirus titers were identified by infecting NIH3T3 cells with viral supernatant for 16 h. After incubation in fresh medium for an additional 48 h, stably infected colonies were selected with puromycin (2 g/ml) for 3 days, and viral titer was calculated by counting the TurboGFP-positive colonies (Life Technologies). Testicular germ cell suspensions were obtained from Kunming mice at 6–8 days after birth using a two-step enzymatic digestion protocol. Sixty to eighty male mice were used in each experiment. Briefly, after removal of the tunica albuginea, the seminiferous tubules were digested with collagenase IV (1 mg/ml, Gibco) followed by digestion with 0.25% trypsin-EDTA (Hyclone, Erembodegem, Belgium) and DNase I (1 mg/ml, Gibco). Fetal bovine serum (Hyclone) was added to stop enzymatic digestion. The resulting cell suspension was filtered with a strainer (pore size: 40 m, BD biosciences) and centrifuged at 600 × for 10 min. Cells were preliminarily purified by differential plating. Magnetic microbeads conjugated to anti-Thy1 antibody (30-H12; Miltenyi Biotec, Bergisch Gladbach, Germany) were used for MACS to enrich Thy1-positive cells. Briefly, the single-cell suspension (1 × 10 cells in 90 l of MACS buffer) was incubated with 10 l of Thy1 microbeads for 20 min at 4 °C. After rinsing with MACS buffer, Thy1-negative cells were selected by passing through an MS separation column (Miltenyi Biotec) that was placed in a magnetic field. After removal of the column from the magnetic field, the magnetically retained Thy1-positive cells were eluted. The fraction of Thy1-positive cells was passed over a new prepared column for further purification. Thy1-positive cells (2 × 10) were re-plated onto laminin-treated (20 g/ml, Sigma) 24-well plates in DMEM/F12 (Gibco) with 5% FBS (Gibco). The next day, SSCs (Thy1-positive cells) were transduced with -shRNA lentivirus or NC-shRNA lentivirus (MOI=10), with polybrene (4 g/ml, Sigma). After 12 h of culture, the medium was replaced with fresh SSCs culture medium. After 1 week of culture, cells were used for RNA and protein extraction, TUNEL analysis or microarray. The cells were fixed in 4% paraformaldehyde for 40 min and incubate with 0.2% Triton X-100 for 30 min at room temperature. Cells were labeled using a One Step TUNEL Apoptosis Assay Kit (Beyotime, Jiangsu, China), according to the manufacturer's protocol. The nuclei were counterstained with Hoechst 33342 (Beyotime) to determine the percentage of TUNEL-positive nuclei relative to the total number of Hoechst-stained nuclei. Mice were anesthetized by intraperitoneal injection of Avertin (Sigma). Donor lentivirus-infected SSCs (1 × 10 cells/ml) were transplanted into the recipient mouse testis that was treated with busulfan to deplete endogenous germ cell 4 weeks before surgery (three replicates). About 10 weeks after transplantation, the recipient mouse testes were collected and used for protein extraction and immunohistochemistry. Testes of transplanted mice were fixed and embedded in paraffin. Sections were stained with hematoxylin and eosin and observed under a light microscope. The number of seminiferous tubule with (at least two layers of germ cells) or without spermatogenesis was counted and the proportion of the sections positive for spermatogenesis was recorded. The values for each transplantation group were determined in three replicates, in each of which at least five sections and an average of 80 tubules/section was examined. SSCs (Thy1-positive cells), which were enriched through magnetic-activated cell sorting and transduced with -shRNA or NC-shRNA lentiviral particles, were subjected to RNA extraction. Samples were RNA pool from three independent experiments. mRNA were reverse-transcribed, labeled and analyzed using the Roche NimbleScan microarray platform (Mouse 12 × 135K Gene Expression Array, NimbleGen Systems, Madison, WI, USA). Array was processed as per the manufacturer's instruction. RNA was extracted from the cell or tissues with Trizol (Invitrogen) according to the manufacturer's protocol. RNA samples were subjected to reverse transcription using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Real-time PCR was performed with SYBR Green II PCR Mix (Takara) using an IQ5 (Bio-Rad, Berkeley, CA, USA). Reactions were run in triplicate in three independent experiments. The primer sequences are provided in . Expression data were normalized to the geometric mean of housekeeping gene to control the variability in expression levels and were analyzed using the 2 method. Protein concentration of the cell lysates was determined using a Bradford assay (Thermo Scientific, Rockford, IL, USA). Cell lysates were separated by SDS-PAGE, and transferred to PVDF membranes (Millipore). Membranes were probed using the following primary antibodies: anti-ESET (Proteintech, 1 : 1500), anti-Caspase9 (Proteintech; 1 : 1000), anti-H3K9me3 (Millipore, 1 : 2000), anti-Caspase3 (Santa Cruz and Proteintech, 1 : 1000), anti-PLZF (Santa Cruz, 1 : 1000), anti-GFP (Beyotime, 1 : 1000) and anti-PARP (Cell Signaling Technology, Danvers, MA, USA; 1 : 1000). Secondary antibodies were horseradish peroxidase-linked anti-rabbit or anti-goat antibody (Abcam, Cambridge, UK; 1 : 5000). Protein bands were visualized on a Bio-Rad Chemidoc XRS using a Western Bright ECL Kit (Advansta, Menlo Park, CA, USA). ChIP analysis was carried out using EZ-ChIP Kit (Upstate, Lake Placid, NY, USA) following the manufacturer's protocol. Formaldehyde-treated C18-4 cells were re-suspended in SDS lysis buffer, and the cell lysates were sheared by sonication. The chromatin fragments were immunoprecipitated with an antibody against ESET (Proteintech), H3K9me3 (Millipore), H4K20me3 (Millipore) and the purified DNA was analyzed by qPCR. The primer sequences are provided in . For bisulfite sequencing, cells were directly subjected to bisulfite conversion by using an EZ DNA Methylation Direct kit (Zymo Research, Orange, CA, USA). Bisulfite-modified DNAs were amplified (). For sequence analysis, the PCR products obtained after bisulfite conversion were cloned into a pBackZero-T Vector (Takara), and six (Dazl) or nine (Cox4i2) individual clones were sequenced. Data were expressed as the mean±S.E.M. Differences between groups were assessed using ANOVA with a Duncan's multiple range test (SPSS 12 for Windows; Chicago, IL, USA). A difference of was considered significant.