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gastroenterologists and surgeons who specialize in the treatment of crohn disease ( cd ) and chronic ulcerative colitis ( cuc ) have become increasingly aware of the implications of different forms of medical therapy on surgical outcomes . this is especially important in the era of top - down and multi - agent therapy for inflammatory bowel disease ( ibd ) . although relatively little literature exists regarding the relationship between pre - operative corticosteroid use and post - operative complications , several authors have found that increasingly higher doses of such immunosuppressive medications may result in an increased rate of post - operative septic complications . similarly , there is increasing concern that combination immunomodulatory therapy with multiple agents may also be associated with an increase in post - operative infectious complications . given reports of the association of monoclonal anti - tumor necrosis factor - alpha ( anti - tnf- ) antibody therapy with pulmonary infections , this concern has specifically been focused on infliximab ( ifx ) , which until recently has been the only fda - approved biologic agent for ibd [ 7 - 11 ] . a recent meta - analysis of the association between immunomodulatory therapy and post - operative complications after surgery for ibd , which included studies of azathioprine , cyclosporine a , and three studies of ifx , found that the available evidence did not support this association . since then , several studies have been published suggesting that ifx is indeed associated with an increased risk of post - operative complications . thus , the potential association between ifx and surgical complications is controversial ; studies both support and refute this potential relationship [ 5,13 - 19 ] . in a study of 270 patients by colombel et al . , 52 patients experienced intra - abdominal septic complications ( iascs ) after bowel resection for cd . analysis did not reveal any increased risk in the ifx - treated subgroup , the moderate - to - high - dose corticosteroid subgroup , or the immunomodulator subgroup . likewise , a matched case - control study from belgium of 31 cd patients also concluded that pre - operative ifx use did not result in a significantly increased rate of post - operative complications , or increased hospital length of stay , as compared to a control group of ifx - nave patients . that study did note , however , a trend towards increased early post - operative infections in the ifx group . more recently , a study of 413 ibd patients ( 156 with cd ) , of which 101 had received ifx within 12 weeks of surgery , also did not find any relationship between pre - operative ifx therapy and post - operative complications . that study , similar to others , was relatively limited by the absolute number of complications . in contrast , a more recent study from appau et al . found an association between pre - operative ifx therapy for cd and post - operative complications . they concluded that ifx therapy within 3 months of surgery was associated with an increased rate of iascs , and of hospital re - admissions . they also found that patients who received a diverting stoma had a lower risk of iascs , thus prompting the observation that a diverting stoma may be prudent when surgeons are faced with cd patients who have recently received ifx . in light of this study , the risks associated with stoma reversal surgery must be weighted against the potential increase in complications associated with ifx for cd . in a study of 151 patients with cuc , 17 ( 10% ) of whom failed ifx therapy and went to surgery and 134 of whom were ifx - nave , no association between ifx and complications was observed . however , the authors observed that ifx patients who had concurrent cyclosporine a treatment were at increased risk for overall and infectious complications . in a larger study by selvasekar et al . in which 47 cuc patients received pre - operative [ ileal pouch - anal anastomosis ( ipaa ) ] ifx therapy and 254 did not , those who received ifx were more likely to have anastomotic leak , and after multivariate adjustment for both disease severity and other medication use , ifx remained independently associated with an increased risk of ileal pouch - related and infectious complications . finally , a recent study of 85 patients with cuc who received ifx pre - operatively also found that patients who receive pre - operative ifx were at increased risk of post - operative septic complications as well as late complications . importantly , the authors also noted that patients who received ifx were more likely to have undergone a 3-stage ipaa , likely due to surgeon reluctance to perform an anastomosis in the setting of pre - operative ifx administration . disease severity determined retrospectively is often inaccurate and surrogate covariates may be inadequate substitutions for validated methods of assessing disease activity . however , it is interesting to note that in the studies of cuc , those that adjusted for disease activity showed a relationship between ifx and complications , while the converse was true for those studies that did not . another major limitation of several of these studies is the relatively long pre - operative ifx window , often 12 weeks or more ( table 1 ) . recent studies of the pharmacokinetics of ifx in ibd suggest that the elimination half - life is between 7 and 18.5 days . by 12 weeks ( 84 days , or 4.5 half - lives ) a window of 12 weeks used by several of the studies published to date may theoretically produce negative results regarding the occurrence of post - operative outcomes if a significant proportion of patients had an interval this long between ifx administration and surgery . ideally , the duration between last infusion and surgery should be included as a continuous variable , but outpatient infusion often makes these data unavailable retrospectively . referral practice patterns may too account in part for the heterogenous findings of these studies , as these may differ substantially in various regions of the world , as demonstrated by at least a 10% variation in the proportion of surgical ibd patients who received ifx . thus , as is usually true of most retrospective studies , results from a single institution may not be broadly generalizable ; the institutions themselves may be considered as a potential confounding factor when comparing results of different studies to each other . in one of the largest studies of the association of ifx and serious infections and mortality , the treat registry study of 6290 patients found that after adjustment for corticosteroid use and disease severity , ifx was not independently associated with increased risk , although both corticosteroids and disease severity were associated with those adverse outcomes . although this study did not include surgical endpoints , the implications are nonetheless important , especially given the lack of large - scale surgical data . in a study of 270 patients by colombel et al . , 52 patients experienced intra - abdominal septic complications ( iascs ) after bowel resection for cd . analysis did not reveal any increased risk in the ifx - treated subgroup , the moderate - to - high - dose corticosteroid subgroup , or the immunomodulator subgroup . likewise , a matched case - control study from belgium of 31 cd patients also concluded that pre - operative ifx use did not result in a significantly increased rate of post - operative complications , or increased hospital length of stay , as compared to a control group of ifx - nave patients . that study did note , however , a trend towards increased early post - operative infections in the ifx group . more recently , a study of 413 ibd patients ( 156 with cd ) , of which 101 had received ifx within 12 weeks of surgery , also did not find any relationship between pre - operative ifx therapy and post - operative complications . that study , similar to others , was relatively limited by the absolute number of complications . in contrast , a more recent study from appau et al . found an association between pre - operative ifx therapy for cd and post - operative complications . they concluded that ifx therapy within 3 months of surgery was associated with an increased rate of iascs , and of hospital re - admissions . they also found that patients who received a diverting stoma had a lower risk of iascs , thus prompting the observation that a diverting stoma may be prudent when surgeons are faced with cd patients who have recently received ifx . in light of this study , the risks associated with stoma reversal surgery must be weighted against the potential increase in complications associated with ifx for cd . in a study of 151 patients with cuc , 17 ( 10% ) of whom failed ifx therapy and went to surgery and 134 of whom were ifx - nave , no association between ifx and complications was observed . however , the authors observed that ifx patients who had concurrent cyclosporine a treatment were at increased risk for overall and infectious complications . in a larger study by selvasekar et al . in which 47 cuc patients received pre - operative [ ileal pouch - anal anastomosis ( ipaa ) ] ifx therapy and 254 did not , those who received ifx were more likely to have anastomotic leak , and after multivariate adjustment for both disease severity and other medication use , ifx remained independently associated with an increased risk of ileal pouch - related and infectious complications . finally , a recent study of 85 patients with cuc who received ifx pre - operatively also found that patients who receive pre - operative ifx were at increased risk of post - operative septic complications as well as late complications . importantly , the authors also noted that patients who received ifx were more likely to have undergone a 3-stage ipaa , likely due to surgeon reluctance to perform an anastomosis in the setting of pre - operative ifx administration . many of these retrospective studies suffer from common limitations . first is lack of adjustment for disease severity ( table 1 ) . disease severity determined retrospectively is often inaccurate and surrogate covariates may be inadequate substitutions for validated methods of assessing disease activity . however , it is interesting to note that in the studies of cuc , those that adjusted for disease activity showed a relationship between ifx and complications , while the converse was true for those studies that did not . another major limitation of several of these studies is the relatively long pre - operative ifx window , often 12 weeks or more ( table 1 ) . recent studies of the pharmacokinetics of ifx in ibd suggest that the elimination half - life is between 7 and 18.5 days . by 12 weeks ( 84 days , or 4.5 half - lives ) a window of 12 weeks used by several of the studies published to date may theoretically produce negative results regarding the occurrence of post - operative outcomes if a significant proportion of patients had an interval this long between ifx administration and surgery . ideally , the duration between last infusion and surgery should be included as a continuous variable , but outpatient infusion often makes these data unavailable retrospectively . referral practice patterns may too account in part for the heterogenous findings of these studies , as these may differ substantially in various regions of the world , as demonstrated by at least a 10% variation in the proportion of surgical ibd patients who received ifx . thus , as is usually true of most retrospective studies , results from a single institution may not be broadly generalizable ; the institutions themselves may be considered as a potential confounding factor when comparing results of different studies to each other . in one of the largest studies of the association of ifx and serious infections and mortality , the treat registry study of 6290 patients found that after adjustment for corticosteroid use and disease severity , ifx was not independently associated with increased risk , although both corticosteroids and disease severity were associated with those adverse outcomes . although this study did not include surgical endpoints , the implications are nonetheless important , especially given the lack of large - scale surgical data . whether pre - operative ifx use is associated with post - operative complications after surgery for ibd remains controversial ; current evidence favors this association after surgery for cuc but does not favor an increased risk after surgery for cd . the potentially practice - changing implications of this debate are occurring now ; increasingly some surgeons are waiting until anti - tnf- agents are washed out before operating or , if that is not possible , performing resections for cd with temporary diverting stomas or advising 3- rather than 2-staged ipaa . larger studies and/or formal meta - analysis of the relationship between ifx and post - operative complications after surgery for ibd are needed before definitive treatment recommendations can be made . the decision of whether or not to perform staged surgery for ibd patients on ifx needs to be made on an individual basis , based on the clinical context and other known risk factors , such as hypoalbuminemia and high - dose corticosteroid treatment .

conflicting data exist regarding the association between pre - operative monoclonal anti - tumor necrosis factor - alpha antibody therapy with infliximab for crohn disease and chronic ulcerative colitis , and the occurrence of post - operative complications . this report reviews the current literature that supports and refutes this association .

brain - derived neurotrophic factor ( bdnf ) is a member of the neurotrophin family , which includes nerve growth factor , neurotrophin-3 , and neurotrophin-4/5 [ 14 ] . the role of bdnf in cell differentiation , neural growth , synaptic connectivity , and maintenance of target neurons is well established ; it has also been implicated in synaptic plasticity of brain function , such as learning and memory [ 5 , 6 ] . in addition to the role of bdnf in neurological disorders , recent studies reported that peripheral injection of bdnf exerts hypophagic and hypoglycemic effects on obese hyperglycemic animals but not on normal animals , pointing to antiobesity and antidiabetic effects [ 710 ] . bdnf mutant mice developed mature onset obesity , characterized by an increase in body weight . in the same study , the mutant mice displayed elevated serum leptin , insulin , glucose , and cholesterol . data from animal experiments and human studies suggested that bdnf may contribute to glucose metabolism and have a pathogenic role in the development of type 2 diabetes mellitus ( t2 dm ) in humans . according to recent studies , bdnf is associated with systemic inflammatory conditions , such as diabetes , acute coronary syndrome , and atherosclerosis [ 13 , 14 ] . the aim of this study was to evaluate the relationship between serum bdnf levels and various metabolic parameters and inflammatory markers in patients with t2 dm . this study was performed at the gop taksim education and research hospital outpatient department of internal medicine . it included 88 patients ( 38 males and 50 females ) and 33 control subjects ( 17 males and 16 females ) . the presence of diabetes was based on a previous diagnosis of t2 dm or a random plasma glucose level of 200 mg / dl or higher , together with classical features of dm , such as polyuria , polydipsia , polyphagia , and weight loss , or a fasting blood glucose level of > 126 mg / dl or higher or a hba1c level of 6.5% or higher . exclusion criteria were the presence of systemic diseases : neoplastic , inflammatory , and infectious diseases . the study protocol was approved by gop taksim research and education hospital ethics committee , istanbul . informed written consent was obtained from all the participants ( patients and controls ) after receiving a full explanation about the study and its purpose . hypertension was defined as antihypertensive drug use or systolic blood pressure 140 mmhg and/or diastolic blood pressure 90 mmhg . body mass index ( bmi ) was obtained using the formula weight ( kg)/height ( m ) . serum cholesterol , triglyceride , and high - density lipoprotein cholesterol ( hdl - c ) were measured by enzymatic colorimetric methods with commercially available kits ( cobas 311 , roche diagnostics gmbh , mannheim , germany ) , and low - density lipoprotein cholesterol c ( ldl - c ) was calculated according to the friedewald formula . serum glucose measures were determined enzymatically using the hexokinase method ( roche diagnostics gmbh , mannheim , germany ) . blood hba1c was determined with a cobas 311 analyzer using the particle - enhanced immunoturbidimetric method ( roche diagnostics , mannheim , germany ) . final results were expressed as percent hba1c of the total hb according to the protocol of the diabetes control and complications trial / national glycohemoglobin standardization program ( dcct / ngsp ) . the particle - enhanced immunoturbidimetric method with a behring nephelometer bn-100 ( behring diagnostic , frankfurt , germany ) was used to measure c - reactive protein ( crp ) . white blood cell ( wbc ) levels were measured with an automatic hematology analyzer ( beckman coulter , brea , ca , usa ) . the erythrocyte sedimentation rate ( esr ) was determined with the westergren method using an established normal range of 020 mm/1 hr . ferritin was measured with an electrochemiluminescence immunoassay ( roche hitachi modular e 170 ) . the insulin level was determined with an electrochemiluminescence immunoassay ( roche diagnostics , mannheim , germany ) on an automated roche cobas e 411 ( roche diagnostics ) . the homeostasis model assessment of insulin resistance ( homa - ir ) index was calculated from the fasting blood glucose and fasting serum insulin concentrations by the formula : homa - ir = fasting serum insulin ( u / ml ) fasting blood glucose ( mmol / l)/22.5 . serum bdnf was quantified using an elisa ( chemikine bdnf sandwich elisa kit , cyt306 ; millipore bioscience research reagents ; usa and canada ) following the instructions of the manufacturer . the intra - assay and interassay coefficients of variation were 3.7 and 8.5% , respectively . number cruncher statistical system ( ncss ) 2007 and power analysis and sample size ( pass ) 2008 statistical software ( utah , usa ) programs were used for the statistical analysis . descriptive statistical methods ( mean , standard deviation , frequency , ratio , minimum , and maximum ) were used to evaluate the study data . mann - whitney u test was used for quantitative parameters that were not normally distributed . values of p < 0.05 , p < 0.01 , and p < 0.001 were accepted as statistically significant . there were no significant differences in the age and gender or triglyceride , total cholesterol , hdl , ldl , wbc , and ferritin levels between the t2 dm patients and control subjects . the t2 dm patients had a significantly higher bmi , waist circumference , systolic pressure , diastolic pressure , fasting plasma glucose , fasting insulin , homa - ir , hba1c , esr , and crp levels than the control subjects ( p < 0.001 ) . among the patients , 64% were using oral antidiabetic drug only , 18% were using oral antidiabetic drug plus insulin , and 18% were using insulin only . on the whole , 30% of patients were taking lipid lowering drug , 61% of patients were taking antihypertension drug , and 9% were taking none . mean diabetes duration for enrolled subjects is 4.03 3.38 ( not shown in table ) . the serum bdnf levels showed a positive correlation with homa - ir ( r = 0.28 ; p < 0.05 ) , the triglyceride level ( r = 0.265 ; p < 0.05 ) , and the wbc level ( r = 0.35 ; p < 0.001 ) in t2 dm as shown in table 2 . in the logistic regression analysis , age ( p < 0.05 ) , bmi ( p < 0.05 ) , crp ( p < 0.05 ) , and bdnf ( p < the serum bdnf levels were significantly higher in the t2 dm patients compared to the healthy controls ( 206.81 107.32 pg / ml versus 130.84 59.81 pg / ml , p < 0.001 ) . a receiver operating characteristic curve ( roc ) analysis and diagnostic screening tests were used to determine the cut - off point for bdnf ( table 4 ) . patients , who had a bdnf level more than 137 pg / ml , were catching study group levels with a sensitivity of 71.79% , a specificity of 68% , a positive predictive value of 87.5% , and a negative predictive value of 43.59% . the area under the roc and the standard deviation were 71.8% and 5.6% , respectively . hba1c was the best predictor , followed by fasting blood glucose and homa - ir ( figure 1 ) . in this study , we investigated changes in the plasma bdnf level and correlations between bdnf and clinical and biochemical parameters in t2 dm patients . we found that the serum bdnf of t2 dm patients was significantly higher than that of control subjects . a previous study showed that serum bdnf levels were elevated in newly diagnosed female t2 dm patients compared to healthy subjects . similarly , a recent study reported that serum bdnf was significantly elevated in t2 dm patients compared to healthy controls . there are various potential mechanisms that link bdnf and development of type 2 diabetes . in animal experiments , it is shown that , by suppressing ppar - alpha and fibroblast growth factor 21 , bdnf might facilitate insulin resistance and dyslipidemia and thus has antidiabetic and lipid lowering effects . furthermore , some researchers claim that bdnf treatment reported to lower blood glucose in diabetic models . similarly , yamanaka et al . demonstrated that treatment with bdnf prevents age - related increase in blood glucose and development of diabetes in prediabetic db / db mice . together with the data derived from animal experiments , authors suggest that exogenous bdnf administration shows its antidiabetic and antilipidemic effects similar to thiazolidinediones . however , the diabetic patients in our study were not newly diagnosed ; thus , the duration of diabetes alone could not explain the elevated level of serum bdnf . on the other hand , fujinami et al . reported that serum bdnf levels were significantly lower in patients with advanced t2 dm compared to control subjects . plasma bdnf levels were decreased in humans with t2 dm and were independent of obesity in a study by krabbe et al . . in the same study , plasma bdnf levels were inversely associated with fasting plasma glucose . . this situation may be the result of ethnic differences . in a previous study , bdnf heterozygous knockout ( bdnf + / ) mice showed obesity and insulin resistance . it is possible that the increase recorded in bdnf in the current study of t2 dm may compensate for hyperinsulinemia and insulin resistance . reported a relationship between fasting blood glucose , triglycerides , and homa - ir and serum bdnf values . a previous study found that serum bdnf was associated with fasting insulin and homa - ir in t2 dm . our study demonstrated that serum bdnf levels were significantly positively correlated with homa - ir and triglyceride . other studies showed that bdnf improved hepatic insulin resistance in diabetic animals and that bdnf was positively correlated with triglyceride , total cholesterol , and ldl - c in humans . some authors observed that bdnf treatment of obese and diabetic animals has a positive effect on glucose and lipid metabolism , with one study demonstrating that subcutaneous administration of bdnf reduces food intake and ameliorates impaired glucose tolerance in diet - induced obese mice . the results of these studies show that bdnf may have a role in the treatment of diabetes and dyslipidemia . previous studies described an association of plasma bdnf with inflammatory conditions [ 25 , 26 ] . some studies demonstrated increased bdnf expression in inflamed bladder tissue and in airway epithelium during allergic airway inflammation [ 27 , 28 ] . shin et al . showed that the plasma bdnf level was positively correlated with inflammatory cytokines in hemodialysis patients , suggesting that plasma bdnf might reflect uremic inflammation in patients undergoing hemodialysis . in our study , inversely , a previous study reported a correlation between bdnf and crp in t2 dm patients . the increase in bdnf levels may be associated with protecting neurons from inflammatory injury . in the logistic regression analysis , bdnf was independently associated with t2 dm , irrespective of age , bmi , and crp . patients who had a serum bdnf level more than 137 pg / ml were a predictive value like hba1c for the diabetes diagnosis with a sensitivity of 71.79% and a specificity of 68% . it had a positive predictive value of 87.5% and a negative predictive value of 43.59% . this novel research suggests that serum bdnf may be used as a prediction data for t2 dm like hga1c in future . more importantly , the standardization and cut - off values of serum bdnf had been controversial in previous studies . for the first time , current study suggests a cut - off point for serum bdnf of type 2 diabetes mellitus . first , this was a cross - sectional study of a relatively small number of patients . third , we measured levels of wbcs and crp to determine the association between bdnf values and inflammation but not those of other serum inflammatory markers , such as interleukin-6 or tumor necrosis factor . in conclusion , serum bdnf level was higher in patients with t2 dm , and the cut - off predictive value of bdnf was 137 pg / ml . the findings of this study suggest that bdnf may contribute to glucose and lipid metabolism and inflammation .

background and aim . studies have suggested that brain - derived neurotrophic factor ( bdnf ) plays a role in glucose and lipid metabolism and inflammation . the aim of this study was to evaluate the relationship between serum bdnf levels and various metabolic parameters and inflammatory markers in patients with type 2 diabetes mellitus ( t2 dm ) . materials and methods . the study included 88 t2 dm patients and 33 healthy controls . fasting blood samples were obtained from the patients and the control group . the serum levels of bdnf were measured with an elisa kit . the current paper introduces a receiver - operating characteristic ( roc ) generalization curve to identify cut - off for the bdnf values in type 2 diabetes patients . results . the serum levels of bdnf were significantly higher in t2 dm patients than in the healthy controls ( 206.81 107.32 pg / ml versus 130.84 59.81 pg / ml ; p < 0.001 ) . they showed a positive correlation with the homeostasis model assessment of insulin resistance ( homa - ir ) ( r = 0.28 ; p < 0.05 ) , the triglyceride level ( r = 0.265 ; p < 0.05 ) , and white blood cell ( wbc ) count ( r = 0.35 ; p < 0.001 ) . in logistic regression analysis , age ( p < 0.05 ) , body mass index ( bmi ) ( p < 0.05 ) , c - reactive protein ( crp ) ( p < 0.05 ) , and bdnf ( p < 0.01 ) were independently associated with t2 dm . in roc curve analysis , bdnf cut - off was 137 . conclusion . the serum bdnf level was higher in patients with t2 dm . the bdnf had a cut - off value of 137 . the findings suggest that bdnf may contribute to glucose and lipid metabolism and inflammation .

a 48-year - old female patient visited our cardiovascular outpatient department for treatment of a mass - like dilated neck vein as a procedure concomitant with thyroid cancer surgery . we could detect the gross engorgement of the neck mass in the supine position or by using the valsalva maneuver when the patient was in an erect position ( fig . preoperative contrast - enhanced computed tomography ( ct ) of the neck showed a venous dilatation , similar to a cystic mass ( size : 2.52.2 cm ) communicating with the left external jugular vein ( fig . 2 ) . after thyroidectomy under general endotracheal anesthesia , an additional separate skin incision ( length : approximately 2.5 cm ) along the neck dermatome was made because of the distance from the collar incision ( approximately 5 cm ) . we accomplished aneurysmectomy by the division of both ends of the external jugular vein and a tributary of the aneurysm in the subcutaneous layer ( fig . in contrast to focal thinned media with thickened intima by fibrous tissue in a varicose vein , the vascular wall thickness of a venous aneurysm is relatively homogenous with thickened media and localized thickened intima ( fig . acquired venous aneurysm in the neck area is a very rare disease and requires a differential diagnosis including enlarged cervical lymph node , tumor of the adjacent organs , laryngocele , and various cystic formations . according to the incidence rate , the internal jugular vein is a more frequent site of aneurysm development than the external vein , but the anterior jugular vein is the least frequent site . fusiform venous dilatation is frequently diagnosed in children with a congenital etiology and right - side predominance but appears in adults as an acquired form with left - side predominance ; the suggested mechanism in adults is the patient 's hypertensive aorta compressing the left innominate vein , resulting in venous dilatation . in addition , the etiology of an acquired venous aneurysm can involve tumors , inflammation , trauma , or spontaneous development . of the aneurysms resulting from iatrogenic causes , pseudoaneurysm at the internal jugular vein appears most frequently ; a case at the external jugular vein has also been reported . however , the patient in the present case had no previous neck procedures or trauma history . clinically , although painful swelling is associated with intraluminal thrombus , saccular aneurysm appears with painless swelling . the valsalva maneuver , performed by moderately forceful attempted exhalation against a closed airway , usually performed by closing one 's mouth and pinching one 's nose shut while pressing out as if blowing up a balloon , can induce venous engorgement characteristically . however , by manual compression of an engorged neck mass in the case of the external jugular vein , the valsalva maneuver can not make the swelling prominent . because the patient had already undergone contrast - enhanced ct imaging for thyroid cancer , ultrasonography was not required in this case . cosmetic concerns , painful swelling due to intraluminal thrombosis , or phlebitis of the jugular vein are all motives for surgical treatment . otherwise , reassurance and regular follow - up can be a substitute for prompt treatment of an asymptomatic venous aneurysm . although embolic complications have been reported at a lower incidence rate in jugular venous aneurysms , active treatment can not be neglected . a recent report documented a pulmonary thromboembolism derived from an external jugular venous aneurysm , and large - scale studies are needed to overcome the limitations of rare case reports . surgical resection can minimize the risk of pulmonary thromboembolism as well as aneurismal rupture induced by growth and can confirm the histopathological diagnosis . aneurismal resection is accomplished by excision with ligation in the saccular form , and exclusion via bypass in fusiform aneurysms .

saccular aneurysm of the external jugular vein presenting as a neck mass is very rare . we report the surgical treatment of an external jugular venous aneurysm in a 48-year - old female patient due to the cosmetic problem of neck engorgement , concomitant with thyroidectomy for cancer .

the analysis of dna sequence is increasingly being used to guide the discovery of natural product biosynthetic gene clusters capable of encoding for novel metabolites . when studying cultured bacteria , it is possible to fully sequence a bacterial genome and therefore carry out detailed analyses of complete biosynthetic gene clusters to identify those likely to encode for novel metabolites . similar full gene cluster analyses are not usually possible when exploring complex environmental microbiomes because the immense size of most metagenomes precludes full sequencing and assembly of complete gene clusters . to circumvent this limitation , we and others have explored the use of the detailed phylogenetic analysis of individual , highly conserved natural product biosynthetic genes as markers for guiding the discovery of functionally novel natural product biosynthetic gene clusters from complex metagenomes . in this approach , individual biosynthetic genes are pcr - amplified from environmental dna ( edna ) and sequenced in bulk . the resulting amplicon sequences ( i.e. , natural product sequence tags ) are then aligned with reference sequences from functionally characterized gene clusters . in previous studies , we have shown that sequence tags that are only distantly related to any known sequence ( e.g. , sequence tag a in figure 1 ) can guide the identification of gene clusters that encode structurally novel bioactive natural products . we have also shown that sequence tags that are very closely related ( e.g. , sequence tag b in figure 1 ) to known genes can guide the discovery of gene clusters that encode congeners of known molecules . while the utility of sequence tags that reside at the phylogenetic boundaries is now clear , many sequence tags fall at a more ambiguous intermediate distance from known sequences ( e.g. , sequence tag c in figure 1 ) . sequence tags , especially those that fall between clades containing sequences known to encode for structurally distinct subclasses of natural products , might be useful for guiding the discovery of gene clusters that both encode novel bioactive metabolites and provide insights into the evolution of natural products structural diversity . here , we explore these concepts through the comparison of the genotypes ( i.e. , gene content ) and chemotypes ( i.e. , molecules encoded by ) of edna - derived aromatic polyketide ( pk ) pentangular polyphenols ( pps ) . aromatic pks are a large group of secondary metabolites generated by iterative ( i.e. , type ii ) pks biosynthetic systems . the initial step in aromatic pk biosynthesis involves the action of a minimal polyketide synthase ( min - pks ) . the min - pks is composed of the three proteins ks , ks and acp , which together catalyze the iterative condensation of malonyl - coa into polyketides ranging from 16 to 30 carbons in length . pps are constructed from the largest nonreduced pk precursors known ( 2430 carbons in length ) and are , therefore , potentially the most structurally diverse subclass of metabolites in this family . ( a ) phylogeny - guided strategy for the discovery of new natural products . ( b ) az , ab and tx denote amplicon sequences derived from arizona , california and texas desert soil edna libraries , respectively . a number of edna - derived pp ks sequence tags fall between clades associated with pps derived from polyketide precursors of different lengths ( c24 and c26 ) . the specific group of intermediate edna sequence tags used in this study are highlighted in orange . the highly conserved ks and ks genes from the min - pks have proved particularly useful as sequence tags for guiding the discovery of new aromatic polyketides from metagenomes . while edna ks and ks sequencing studies suggested the presence of large numbers of unique pp clusters in the environment , the biosynthetic landscape of pps from culture - based studies remained quite limited . in this study pp gene clusters associated with edna - derived ks and ks sequence tags that fall between well - defined clades containing genes from the biosynthesis of pradimicin and xantholipin type pp metabolites were recovered from archived soil edna libraries . the molecules encoded by these pp gene clusters were accessed through heterologous expression in streptomyces albus and found to encode new bioactive pentangular polyphenols with potent antiproliferative and antibacterial activities . by comparing the gene cluster genotypes and chemotypes across this expanded pentangular polyphenol biosynthetic landscape , we gain specific insights into pentangular polyphenol biosynthesis and general insights into potential limitations of the evolution of natural product structural diversity . to identify novel gene clusters encoding pp pks , archived soil edna libraries were screened with degenerate primers targeting either the ks or ks genes of the min - pks . ks and ks phylogenetic trees were constructed with the resulting edna amplicon sequences and sequences from previously characterized type ii pks gene clusters deposited in genbank . in this phylogenetic analysis we identified two sequence tags of interest ( ks tag ab1692 and ks tag ab1414 ) from our anza borrego desert ( ab ) edna library . both sequence tags fall between well - defined clades containing ks genes from gene clusters that encode structurally distinct pp subfamilies , pradimicins and xantholipin ( figure 1b ) . sequence tags ab1692 and ab1414 were used to guide the recovery of edna cosmid clones containing pp biosynthetic gene clusters associated with these ks sequences . the recovered cosmids were pgm ( personal genome machine)-sequenced and this sequence data was subsequently used to guide the recovery of additional cosmid clones overlapping each end of the two min - pks containing cosmids . sequencing and bioinformatics annotation ( tables s3 and s4 , supporting information ) of each set of three overlapping cosmid clones revealed a biosynthetic gene cluster flanked by collections of genes predicted to encode primary metabolic enzymes , suggesting that both gene clusters were recovered in their entirety on three overlapping cosmids . to access the metabolites encoded by the ab1692 and ab1414 pp gene clusters , each set of three overlapping edna cosmid clones ( ab1692 , ab916 and ab170 ; ab1442 , ab1414 and ab561 ) was assembled into bacterial artificial chromosomes ( bac - ab1692/916/170 and bac - ab1442/1414/561 ) using pathway specific ptara ( e. coli : yeast : streptomyces ) shuttle capture vectors and transformation - associated recombination ( tar ) in yeast ( figure 2 ) . these approximately 90 kb bac constructs were pgm - sequenced to ensure correct assembly and transferred into streptomyces albus by intergenic conjugation and integrated into the s. albus genome using the c31 integrase . for heterologous expression purposes , the resulting strains , s. albus bac - ab1692/916/170 and s. albus bac - ab1442/1414/561 , were grown at 30 c in r5a liquid media for 9 days . lc ms analysis of etoac extracts derived from these cultures showed the presence of one and two major clone - specific peaks , respectively ( figure 2 ) . the three clone - specific metabolites were purified from the extracts of 1 l cultures using , in each case , two rounds of c18 reversed - phase hplc . this gave calixanthomycin a ( 1 ) ( 12 mg / l ) from s. albus bac - ab1692/916/170 , and arenimycins c ( 2 ) ( 4.6 mg / l ) and d ( 3 ) ( 2.5 mg / l ) from s. albus bac - ab1442/1414/561 . heterologous expression and the structures of calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) . two pp gene clusters were recovered from california desert soil edna library on three overlapping edna cosmid clones ( ab1692/916/170 and ab1442/1414/561 ) . they were each reconstructed into bac clones using tar . the heterologous expression of these two pp gene clusters by s. albus led to the production of three clone - specific metabolites , compound 1 from the strain s. albus bac - ab1692/916/170 , and compounds 2 and 3 from the strain s. albus bac - ab1442/1414/561 . detailed hplc trace analysis can be found in the supporting information ( figure s2 ) . key 2d nmr correlations used for determining the structures of calixanthomycin a ( 1 ) and arenimycin c ( 2 ) . the structures of compounds 13 were determined by spectroscopic methods , including hresims , and 1d and 2d nmr ( figure 3 , see supporting information discussions s1 and s2 for structure elucidation details ) . the structure of calixanthomycin a ( 1 ) features a xanthone - containing pp core structure . the polycyclic xanthone core is tailored by oxidation , o - methylation and glycosylation . rare structural features include the f ring lactone , which is more commonly seen as a lactam in xanthone - containing pps , and the ortho - dimethoxy functionality on the a ring . calixanthomycin a ( 1 ) is most closely related to ib-00208 from an unsequenced marine actinomadura sp . they differ by the position of methoxy groups around the a ring , the presence or absence of the double bond in the d ring and the oxidation state of the c ring . the structures of arenimycins c ( 2 ) and d ( 3 ) produced by s. albus bac - ab1442/1414/561 feature a highly oxidized benzo[a]naphthacene quinone core ( rings a through e ) . this core structure is appended with a disaccharide ( ome - l - rhamnose - ome - l - olivose ) in 2 and a monosaccharide ( l - rhamnose ) in 3 via a rare n - glycosidic linkage . these structures share an aglycone with the sf2446s from a soil actinomycete streptomyces sp . sf2446 , and the recently reported arenimycins a and b from a marine actinomycete salinispora sp . calixanthomycin a ( 1 ) is extremely toxic to hct-116 cancer cells ( ic50 0.43 nm ) ; however , it shows no antibacterial activity against mrsa ( methicillin - resistant s. aureus ) and vre ( vancomycin - resistant enterococcus faecium ) at the highest concentrations tested ( 50 g / ml ) , and weak activity against b. subillis ( mic = 3.1 g / ml ) . this is quite interesting as most reported xanthone - containing pps display a broad spectrum of activity with both potent human cell cytotoxicity and antibacterial activities . arenimycin c ( 2 ) shows potent gram - positive antibacterial activity ( mic = 98 ng / ml against mrsa ; 1.5 ng / ml against b. subtilis ) and moderate cytotoxicity against hct-116 cells ( ic50= 0.17 m ) . the biosynthesis of compounds 13 can be rationalized based on the predicted functions of the biosynthetic genes found on each producing bac construct ( figure 4 ) . bac - ab1692/916/170 is predicted to harbor the 50 kb clx ( calixanthomycin ) gene cluster ( genbank km881706 ) containing 47 genes involved in the biosynthesis , regulation and resistance of calixanthomycin a ( 1 ) . in our biosynthetic proposal , the predicted min - pks ( clx911 ) , cyclases ( clx8 , 30 and 31 ) and a ketoreductase ( clx1 ) are responsible for generating a benzo[a]naphthacene quinone intermediate using an acetate starter unit and 12 malonyl coa extension steps . the quinone could then be transformed to a xanthone via an oxidative rearrangement catalyzed by the predicted bayer - villiger oxidase ( bvo ) clx27 ( 51% identity to pnxo4 from the fd-594 gene cluster ) . the final tailoring steps in our proposed calixanthomycin a ( 1 ) biosynthesis scheme involve formation of the ortho - dimethoxy functionality in the a ring and glycosylation in the e ring . formation of the rare ortho - dimethoxy functionality would require reduction at c-13 and hydroxylation at c-12 followed by two o - methylations . the mechanism of c-13 reduction is not clear at this point , but the hydroxylation at c-12 is predicted to be catalyzed by the cytochrome - p450 hydroxylase clx28 due to its sequence similarity to pnxo5 ( 61% sequence identity ) from the fd-594 gene cluster . methylation of the ortho - hydroxyl groups could then occur by the action of the predicted o - methyltransferases clx2 and clx43 . in our proposed biosynthesis , the resulting hexacyclic xanthone aglycone is appended with a d - quinovose sugar moiety by the predicted glycosyltransferase clx40 . proposed biosynthesis of calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) ( in boxes ) based on the predicted gene function and pfam domain analysis . the presence of arenimycin b in the culture extract was confirmed by lc ms . detailed gene annotation tables for the clx and arn gene clusters appear in tables s3 and s4 . bac - ab1442/1414/561 harbors the 40 kb arn ( arenimycin ) gene cluster ( genbank kj440489 ) containing genes predicted to be involved in the biosynthesis , regulation and resistance of arenimycins c ( 2 ) and d ( 3 ) . in our proposed biosynthetic scheme the benzo[a]naphthacene core is synthesized via a min - pks ( arn31 , 32 and 36 ) , an aromatase ( arn21 ) , two cyclases ( arn19 and 20 ) and a ketoreductase ( arn33 ) ( figure 4 ) . on the basis of their high sequence identity to pdmh from the pradimicin gene cluster , arn 22 and 37 are predicted to oxidize c-6 and c-10 to form the two 1,4-benzoquinone moieties ( a and c rings ) seen in 2 and 3 . arn 17 , which shows 54 and 43% sequence identity to grho8 from the griseorhodin gene cluster and xano5 from the xantholipin gene cluster , respectively , is likely to be involved in hydroxylations at the two angular positions ( c-5 and c-18 ) . finally , this aglycone is predicted to be glycosylated by two glycosyltransferases arn11 and arn14 , completing the biosynthesis of 2 and 3 . the mechanism of n - glycosidic bond formation is not clear at this point . different pp chemistries are organized according to the phylogenetic analysis of ks gene sequences ( figure 1 ) . we have designated these scaffolds pp - a , pp - b , pp - c and pp - d . each different pp scaffold and the arn gene cluster harbors all genes predicted to be required for the biosynthesis of the sugar moieties ( rhamnose and olivose ) found in 2 and 3 ( figure 4 ) . interestingly , it also contains three additional predicted sugar biosynthesis genes , whose functions could not be assigned to a specific transformation in the biosynthesis of the sugar moieties found on 2 or 3 . these include genes predicted to encode for an n , n - dimethyltransferase ( arn6 ) , an aminotransferase ( arn7 ) and a 3,4-dehydratase ( arn8 ) . these observations along with the recently reported structure of arenimycin b , which contains the dimethylated amino deoxysugar forosamine in place of the ome - l - olivose seen in 2 , led us to re - examine culture broth extracts obtained from bac - ab1442/1414/561 by lc the selective ion chromatogram for m / z = 809 revealed the presence of a minor clone specific compound with a mass corresponding to that of arenimycin b ( figure s9 ) . it appears that the edna - derived arn gene cluster has the potential to encode a number of different glycosylated compounds , with arenimycin c ( 2 ) being the major product . the annotated genome of salinispora arenicola cnb527 , the arenimycin b producer , was recently made publicly available ( genbank : nz_azxi01000002 ) . a comparison of the arenimycin b and the arn gene clusters reveals that these two gene clusters are very closely related , showing 90 to 95% sequence identity between most biosynthetic genes . in total , 12 pp biosynthetic gene clusters have now been sequenced and functionally characterized , including eight clusters from culture - based studies and four from culture - independent studies . in addition to the edna - derived clx and arn clusters described here , we previously reported edna gene clusters that encode for fasamycin and arixanthomycin type pps . ks and ks sequence tags associated with these four edna clusters were selected by us for detailed analyses because they all fall between well characterized ks clades ( i.e. , intermediate sequence tags ) . in the following analyses , we explore this closely related collection of biosynthetic gene clusters to gain insights into the evolution of natural product structural diversity . the individual proteins that makeup min - pks ( ks , ks , and acp ) are highly conserved across type ii pks gene clusters . ks and ks phylogenetic trees show very similar topologies , both of which correlate closely with differences in the core polyketide structure ( e.g. , chain length and cyclization pattern ) encoded by the gene cluster from which the min - pks arises . ks and , to a lesser extent , ks genes have thus proved to be useful phylogenetic markers for predicting differences in polyketide core structures encoded by type ii pks gene clusters . while pps have so far been considered a single class of type ii polyketides , based on both ks phylogeny and natural product structure , it appears that they arise from four distinct min - pks lineages . these lineages differ in starter unit selectivity ( acetate or hexanoate ) and the number of malonyl coa chain extension steps they carry out ( 11 or 12 ) . pp polyketide precursors range from 24 to 30 carbons in length and generate four unique pp scaffolds that we have designated pp - a ( c24acetate and 11 extensions ) , pp - b ( c26acetate and 12 extensions ) , pp - c ( c28hexanoate and 11 extensions ) and pp - d ( c30hexanoate and 12 extensions ) ( figure 5 ) . the 30-carbon d scaffold is the largest polyketide chain observed in aromatic polyketide biosynthesis . with more than 30 predicted b scaffold - based natural products reported in the literature , this 26-carbon scaffold , which arises from an acetate starter unit and 12 malonyl coa extension steps is responsible for most of the structural diversity seen in known pps ( figure 5 ) . the only reported gene clusters predicted to encode the a , c , and d scaffolds are the pradimicins ( pp - a ) , arenimycins ( pp - a ) , benastatins ( pp - c ) , fd-594s ( pp - c ) and fredericamycins ( pp - d ) . although both ks and ks gene phylogenies indicate that the four pp scaffolds ( a c ) have evolved independently from a common ancestor , similar tailoring modifications are observed across scaffolds from different lineages . this is especially interesting in light of the fact that some of modifications seen in multiple different pp lineages ( e.g. , the xanthone core and the gem - dimethyl functionality ) are in fact quite rare outside of pp biosynthesis . this suggests that functionally successful horizontal transfer of the tailoring genes responsible for these modifications has not occurred globally throughout bacterial secondary metabolism but instead locally within the limited universe of pp biosynthesis . to evaluate evolutionary relationships between the genes responsible for the common modifications found across different pp scaffolds , we carried out detailed phylogenetic analyses of the four common tailoring genes seen in pp gene clusters including baeyer villiger oxidase , geminal bis - methyltransferase , asn - synthetase homologue and glycosyltransferase ( figure 6 ) . the most notable tailoring modification observed in pp biosynthesis is an oxidative rearrangement of the pp scaffold to generate either a xanthone or a spiroketal . these oxidative rearrangements involve the action of fad - dependent baeyer villiger oxidase ( bvo ) . bvos are also found in some angucycline gene clusters ( giloi , jadh and urdm ) and in the mithramycin gene cluster ( mtmoiv ) . to determine the phylogenetic relationship between these genes , a maximum likelihood phylogenetic tree was generated using full - length bvo gene sequences ( figure 6 ) . in this analysis , the pp bvo genes form a monophyletic clade that is distinct from other bvo genes , suggesting that the pp bvo gene was acquired once in pp biosynthesis and diverged to enable the production of distinct xanthone and spiroketal functionalities . interestingly , the bvo genes clx27 and pnxo4 from the calixanthomycin a and fd-594 gene clusters , respectively , show the highest sequence identity even though their ks genes belong to distinct pp lineages ( figure 6 ) . this suggests that once the bvo gene successfully entered the pp biosynthetic universe it was not only passed down vertically through the pp - b lineage , but also horizontally transferred between pp lineages . curiously , while it appears that this bvo gene was capable of horizontal transfer between pp lineages with related core structures , similar , more global transfer to numerous other sequenced type ii pks gene cluster families is not observed , as the bvo genes found in other type ii pks gene clusters appear to be of distinct phylogenetic lineages . geminal bis - methylation is another rare tailoring reaction that is seen in pp biosynthesis . the introduction of this functional group is known to occur through the action of a single , sam - dependent methyltransferase ( geminal bis - methyltransferase , gbm ) via two rounds of c - methylation . known aromatic polyketides with this functionality include two pps , the fasamycins and the benastatins , a pentacyclic polyketide resistomycin , and the tetracyclic quinone tetarimycin . in resistomycin biosynthesis , two methyl transferases are predicted to be required to introduce the dimethyl functionality , neither of which is closely related to the pp gbms . the three tetracyclic structures upon which the pp - like gbms act ( fasamycins , benastatins and tetarimycins ) are closely related ( figure 6 ) , once again suggesting that horizontal transfer of these tailoring genes has been limited to interchange between gene clusters that encode closely related core structures . ( a ) phylogenetic relationships between ks , ks , baeyer villiger oxidase , asn synthase homologue , geminal bis - methyltransferase and glycosyltransferase genes from pp biosynthetic gene clusters are reconstructed from the maximum likelihood phylogenetic trees to highlight the relationships between genes from clusters of different pp lineages . ( b ) the parent maximum likelihood phylogenetic trees for bvo genes , gbm genes and asn synthase homologue genes are shown . for both bvo and gbm tailoring genes , it appears that functionally successful horizontal gene transfer has been limited to gene clusters that encode metabolites with closely related core chemical structures . this would explain their presence in different pp lineages and few other gene clusters outside of pp biosynthesis and suggest that for many tailoring enzymes the challenge of changing substrate specificity ( or limited substrate promiscuity ) may have restricted their more extensive spread throughout natural product biosynthesis by horizontal gene transfer mechanisms . the terminal carboxylate in pp structures undergoes a variety of reactions including o - methylation , esterification / cyclization , alanine addition , serine addition / cyclization and amidation / cyclization . among these , amidation in general and , more specifically , transamination carried out by asn synthetase homologues is the most commonly seen transformation . in the phylogenetic analysis of pp asn transaminase genes , we once again see what appears to be horizontal transfer between different pp lineages . we also see what appears to be horizontal transfer between different polyketide classes as the closest relatives of the llpa and xana genes from the lysolipin and xantholipin gene clusters , respectively , are found in tetracycline - type gene clusters . one of the most common tailoring steps found in aromatic polyketide biosynthesis is glycosylation . to investigate relationships between pp glycosyltransferases , a phylogenetic tree was constructed using 40 glycosyltransferase genes found in sequenced pp gene clusters and additional glycosyltransferase genes found in gene clusters that encode a diverse collection of non - pp aromatic polyketides ( figure s1 ) . this maximum likelihood phylogenetic tree shows that clades do not correlate strongly with aromatic polyketide aglycone substrate structures or donor sugar types . among pp glycosyltransferse genes , clx40 , arx9 , arx3 , llpu and xanp do form a monophyletic clade , suggesting that they have been vertically transferred through pp scaffold b gene cluster evolution . other pp glycosyltransferases , including arn11 , arn14 , pnxgt1 , pnxgt2 , pdms and pdmq , do not clade based on their respective pp min - pks lineages , suggesting that they have likely entered the pp biosynthetic gene clusters in distinct horizontal gene transfer events . for tailoring genes that to date are exclusively found in secondary metabolism ( e.g. , bvo and gbm ) , we see limited introduction of these genes into pp biosynthesis . within the examined set of gene clusters , both bvo and gbm genes appear to have entered pp biosynthesis only once . however , for tailoring gene families that are common across the global metagenome ( e.g. , asn - synthetase homologue and glycosyltransferase genes ) , we see many more introduction events resulting in their more varied use in pp structural diversification . the more frequent introduction of asn - synthetase homologue and glycosyltansferase genes into pp biosynthesis is likely due to both their more frequent appearance in the environment and their more relaxed substrate specificity . soil metagenomes represent the products of nature s ongoing combinatorial biosynthetic efforts . here , we show that sequence tag - based metagenomic screening methods provide an opportunity to identify collections of related natural product gene clusters that not only encode novel metabolites , but can also provide insights into the natural combinatorial biosynthetic process . more specifically , we show that the functional characterization of gene clusters whose sequence tags fall between clades associated with two related but distinct chemotypes ( intermediate sequence tags ) can be used to increase the chemical and the biosynthetic diversity associated with a targeted class of natural products . on the basis of the differences in min - pks gene phylogeny and the pp chemical structures our phylogenetic analysis of pp tailoring genes indicates that the horizontal transfer of at least a subset of pp tailoring genes has likely been restricted to gene clusters that encode closely related chemical structures . if true , this would suggest that nature has sampled only a fraction of the natural product - like chemical space that can theoretically be encoded by secondary metabolite tailoring genes . these observations provide a more detailed picture of the nature s combinatorial biosynthetic process , which can help guide future laboratory - based combinatorial biosynthetic efforts and help ensure the construction of a set of molecules that is orthogonal to those produced naturally . in particular , our study suggests that combinatorial biosynthetic experiments focused on widely distributed enzymes ( e.g. , glyocsyl- or amino - transferases ) are likely to yield metabolites already sampled by evolution . on the other hand , experiments focused on increasing the substrate promiscuity of tailoring genes that are unique to secondary metabolism ( e.g. , bvo or gbm ) would be much more likely to yield metabolites not yet encoded by natural biosynthetic pathways . all recovered cosmids and tar constructs were sequenced using iontorrent personal genome machine ( pgm ) . optical rotations of isolated compounds were measured on a jasco p-1020 polarimeter and ir spectra were recorded on a bruker tensor27 ir spectrometer . all nmr data used for structural characterization were obtained on a bruker avance dmx 600 mhz nmr spectrometer equipped with a cryoprobe . h and c nmr chemical shifts were referenced to the dmso - d6 solvent signals ( h 2.50 and c 39.51 , respectively ) . hresims data was acquired on the lct premier time - of - flight mass spectrometer . our ab ( california ) , az ( arizona ) and tx ( texas ) desert soil edna libraries were used for screening with ks and ks degenerate primers . each library contains > 10 clones , which is predicted to approach saturation of the genetic diversity present in soil microbiomes . these libraries are arrayed as 5000 membered subpools to facilitate screening and gene cluster recovery studies . dna aliquots from the 5000 membered subpools found in each arrayed library were screened using degenerate primers designed to amplify partial ks and full - length ks genes . each 25 l pcr reaction contained 50 ng of cosmid dna , 2.5 m of each primer , 2 mm dntps , 1x thermopol reaction buffer ( new england biolabs ) , 0.5 units taq dna polymerase and 5% dmso . pcr was conducted using the following touchdown protocol : denaturation ( 95 c , 2 min ) , 8 touchdown cycles [ 95 c , 45 s ; 65 c ( 1 c per cycle ) , 1 min ; 72 c , 1 min ] , 35 standard cycles ( 95 c , 45 s ; 58 c , 1 min ; 72 c , 1 min ) and a final extension step ( 72 c , 2 min ) . approximately 500 bp of ks or 600bp of ks gene ( corresponding to nucleotides 5621061 of bena or 25621 of benb ) fragments from each amplicon were aligned using clustalw . the edna clones from which the ab1692 and ab1414 amplicon sequences were amplified were recovered from the ab1692 and ab1414 subpools using serial dilution method . briefly , overnight cultures of the subpools were inoculated into 96-well microtiter plates at a dilution of 50 cells / well . after overnight shaking at 37 c , the diluted cultures were screened by whole - cell pcr using specific primers designed to recognize the ab1692 and ab1414 amplicon sequences . pcr - positive wells from the second round were plated onto lb agar media to give distinct colonies that were screened individually using colony pcr to identify the clones ab1692 and ab1414 . the ab library was screened for overlapping edna clones using clone - specific primers designed to recognize the sequence at each end of the edna clones ab1692 and ab1414 . hits were identified in subpools ab916 for the ab1692 clone , and ab1442 and ab561 for the ab1414 clone . the overlapping edna clones were recovered from these subpools using the serial dilution method outlined above . all three cosmids were pgm - sequenced and annotated using metagenemark , in combination with blast search and pfam domain analysis . annotation results indicated that the biosynthetic gene cluster associated with the ab1414 ks sequence was completely captured in three overlapping clones , ab1442 , ab1414 and ab561 , but the gene cluster associated with ab1692 ks sequence was not complete in the ab916 , therefore the additional overlapping clone was recovered using the same method described above . sequencing of the additional overlapping clone ab170 suggested the complete recovery of the ab1692 ks sequence - associated pp gene cluster over three overlapping edna clones , ab1692 , ab916 and ab170 . the three overlapping edna cosmid clones ( ab1692 , ab916 and ab170 ; ab1442 , ab1414 and ab561 ) that are predicted to contain the ab1692 and ab1414 amplicons - associated pentangular polyphenol gene clusters were assembled into bac clones in yeast using tar . the ab1692 and ab1414 pentangular polyphenol pathway - specific tar capture vectors were constructed using infusion cloning methodology ( clontech ) . five hundred bp upstream ( ups ) and downstream ( dws ) homology arms were amplified from the ab1692 and ab170 cosmids , and the ab1442 and ab561 cosmids , respectively , using the primers listed in the supplementary protocol 1 . gel - purified amplicons ( qiagen ) and bmti / sphi linearized ptara vector were added into a standard infusion cloning reaction ( clontech ) to yield the pathway - specific capture vectors . for tar assembly , 200 ng of each drai - cut cosmid ( ab1692 , ab916 and ab170 ; ab1442 , ab1414 and ab561 ) were mixed with 100 ng of the hpai - digested pathway - specific capture vector and then transformed into 200 l of saccharomyces cerevisiae cry12 spheroplasts prepared according to published protocols . transformed spheroplasts were mixed with synthetic complete ( sc ) top agar ( 1 m sorbitol , 1.92 g / l synthetic complete uracil dropout supplement , 6.7 g / l yeast nitrogen base , 2% glucose and 2.5% agar ) and overlaid onto sc dropout plates without uracil . plates were incubated at 30 c for approximately 72 h , until colonies appeared . twelve yeast colonies were picked and cultured overnight in sc dropout liquid media without uracil . dna was isolated using a zymolyase lysis protocol ( zymo research ) and screened by pcr with primers designed to recognize sequences from each cosmid . a bacterial artificial chromosome ( bac ) clones that amplified with all the primer sets were electroporated into e. coli epi300 ( epicenter ) . dna isolated from the resulting e. coli epi300 was pgm - sequenced to confirm the correct reassembly of the three overlapping cosmids into approximately 90 kb insert bac clones ( bac-1692/916/170 and bac - ab1442/1414/561 ) . bac - ab1692/916/170 and bac - ab1442/1414/561 were transformed into e. coli s17.1 and transferred into s. albus via intergenic conjugation to generate the strains s. albus bac - ab1692/916/170 and s. albus bac - ab1442/1414/561 ( apramycin selection ) . calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) were isolated from 1l cultures ( 200 rpm at 30 c ) of s. albus bac - ab1692/916/170 and s. albus bac - ab1442/1414/561 , respectively , in 125 ml baffled flasks , each containing 50 ml of r5a media . the cultures were harvested after 9 days , extracted with ethyl acetate [ 1:3 ( v : v ) , ethyl acetate to the culture ] and dried in vacuo . calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) were isolated from the resulting ethyl acetate extracts using two rounds of reversed - phase hplc ( c18 column , 10 mm 250 mm , 3.5 ml / min ) . the first round of hplc ( isocratic , 60% acetonitrile for 1 ; 40% acetonitrile for 2 and 3 ) yielded crude samples , from which 13 were purified . the second rounds of hplc used 60% aqueous methanol for calixanthomycin a ( 1 , 6.0 mg / l ) , and 70 and 60% aqueous methanol with 0.1% trifluoroacetic acid for arenimycins c ( 2 , 4.6 mg / l ) and d ( 3 , 2.5 mg / l ) , respectively . yellow brownish powder ; [ ]d 68 ; uv ( meoh ) max 226 , 295 , 329 nm ; ir ( neat ) max 3393 , 2927 , 2736 , 1679 , 1604 , 1557 cm ; h and c nmr , cosy , and hmbc data , see table s1 ; hresims m / z 695.2327 [ m + h ] ( calcd for c36h39o14 , 695.2340 ) . dark reddish powder ; [ ]d 77 ; uv ( meoh ) max 216 , 256 , 421 , 477 nm ; ir ( neat ) max 3384 , 3070 , 2929 , 2856 , 1682 , 1620 , 1511 cm ; h and c nmr , cosy , and hmbc data , see table s2 ; hresims m / z 812.2770 [ m + h ] ( calcd for c40h46 no17 , 812.2766 ) . dark reddish powder ; [ ]d 133 ; uv ( meoh ) max 215 , 254 , 416 , 474 nm ; ir ( neat ) max 3392 , 3072 , 3050 , 2931 , 2858 , 2738 , 1686 , 1641 , 1619 cm ; h and c nmr data , see table s2 ; hresims m / z 654.1826 [ m + h ] ( calcd for c32h32no14 , 654.1823 ) . minimal inhibitory concentrations ( mic ) for calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) against staphylococcus aureus usa-300 ( mrsa ) , bacillus subtilis rm125 , enterococcus faecalis ( vre ) and escherichia coli drc39 were determined by liquid dilution methods . each bacterial strain was grown overnight in either brain heart infusion ( mrsa and vre ) or lb ( b. subtilis rm125 and e. coli drc39 ) liquid media . overnight cultures were diluted 1000-fold and transferred to 96-well microtiter plates ( 75 l / well ) . compounds were dissolved in dmso , added to the media in the first well of row to give a concentration of 50 g / ml and serially diluted 2-fold / well across the plate . these solutions ( 75 l ) were then added to the corresponding well in an assay plate and incubated at 30 c overnight without shaking . bacterial growth was visually inspected and the last well with no bacterial growth was reported as the mic . the antiproliferative activity of calixanthomycin a ( 1 ) , and arenimycins c ( 2 ) and d ( 3 ) was evaluated in duplicate using the colon carcinoma cell line hct-116 ( atcc ; ccl-247 ) . hct-116 cells were grown in mccoy s 5a modified medium ( gibco ) supplemented with 10% ( v / v ) fbs . trypsinized cells were seeded into 96-well plates ( 1000 cells / well ) and incubated overnight at 37 c in the presence of 5% co2 . compounds 13 ( in dmso ) were sequentially diluted in culture media ( 2 or 3-fold dilutions starting at 50 g / ml ) across a 96-well plate and 100 l was transferred to the appropriate well in an assay plate . the plates were incubated at 37 c for 3 days and then evaluated for viability using a crystal violet - based colorimetric assay . cell viability was recorded based on the a590 of stain present in each well relative to no drug dmso control wells .

sequence - guided mining of metagenomic libraries provides a means of recovering specific natural product gene clusters of interest from the environment . in this study , we use ketosynthase gene ( ks ) pcr amplicon sequences ( sequence tags ) to explore the structural and biosynthetic diversities of pentangular polyphenols ( pp ) . in phylogenetic analyses , edna - derived sequence tags often fall between closely related clades that are associated with gene clusters known to encode distinct chemotypes . we show that these common intermediate sequence tags are useful for guiding the discovery of not only novel bioactive metabolites but also collections of closely related gene clusters that can provide new insights into the evolution of natural product structural diversity . gene clusters corresponding to two edna - derived ks sequence tags that reside between well - defined ks clades associated with the biosynthesis of ( c24)-pradimicin and ( c26)-xantholipin type metabolites were recovered from archived soil edna libraries . heterologous expression of these gene clusters in streptomyces albus led to the isolation of three new pps ( compounds 13 ) . calixanthomycin a ( 1 ) shows potent antiproliferative activity against hct-116 cells , whereas arenimycins c ( 2 ) and d ( 3 ) display potent antibacterial activity . by comparing genotypes and chemotypes across all known pp gene clusters , we define four pp subfamilies , and also observe that the horizontal transfer of pp tailoring genes has likely been restricted to gene clusters that encode closely related chemical structures , suggesting that only a fraction of the natural product - like chemical space that can theoretically be encoded by these secondary metabolite tailoring genes has likely been sampled naturally .

the study began in december 2004 and terminated on february 17 , 2007 . a notice sent to all physicians in maritime canada described the study and asked the physicians to submit serum samples from patients with suspected q fever ( febrile illness or pneumonia after exposure to parturient cats or other animals ; outbreaks of pneumonia in a family ) . all samples were sent to the nova scotia public health laboratory for testing for antibodies to coxiella burnetii . physicians of patients with a positive test result were contacted , and they in turn contacted their patients to ask if they would participate in the study . this study was approved by the university of alberta research ethics review board and the capital health research ethics board . during the study period , serum samples from 210 patients suspected of having c. burnetii infection were tested by using commercially available immunoglobulin ( ig ) m and igg elisas ( panbio , brisbane , queensland , australia ) . convalescent - phase samples were collected from those who agreed ; further testing for igg antibodies to phase i and phase ii c. burnetii antigen was conducted by using an indirect immunofluorescence test as previously described ( 3 ) . of the 210 patients , 35 had antibodies to c. burnetii and 13 met the criteria for acute q fever ( positive igm elisa result and a > 4-fold rise in antibody to phase ii antigen between the acute- and convalescent - phase samples ) . phase i titers > 512 , suggestive of chronic q fever , were found for 3 patients . of the 13 patients who fit the case definition of acute infection , 11 agreed to participate in the study , 1 declined , and 1 moved to another country . of the 11 participating patients , 7 were from nova scotia , 2 were from new brunswick , and 2 were from prince edward island ; 6 were male ; and mean age was 54.6 years ( table ) . one case occurred in december 2004 ; 6 in 2005 ; 4 in 2006 , and no cases in the first 6 weeks of 2007 . cases occurred in every month except august , september , october , and february . * within 2 weeks of onset of symptoms , this patient had occupied a small space ( automatic bank teller area ) with 2 farmers who smelled of manure . one patient ( patient 8) was a sheep farmer who had recently had 60 lambs born on his farm , several of which were stillborn in the 2 weeks before the farmer became ill ; 7 patients had > 1 cat as a pet ; and only 2 ( patients 5 and 8) had no pets . in terms of clinical signs , all 11 patients had sweats , fever , and myalgia ; 9 had chills ; 8 had a cough ; 7 were short of breath ; 5 each had nausea , diarrhea , sputum production , and confusion ; 4 had chest pain , which was pleuritic for 2 ; and 2 had abdominal pain and vomiting . of the 7 patients for whom chest radiographs were taken , 6 had acute opacities compatible with pneumonia . patient 11 had diffuse bilateral pneumonia , which required him to be admitted to an intensive care unit to receive ventilatory support ( figure ) . chest radiograph of patient 11 at time of admission to hospital , before intubation , demonstrating extensive bilateral airspace disease . only 4 received initial empiric therapy that would be considered effective against c. burnetii , e.g. , doxycycline ( n = 2 ) , ciprofloxacin ( n = 1 ) , or levofloxacin ( n = 1 ) . four other patients received azithromycin , which may have been effective but has suboptimal in vitro activity against c. burnetii ( 4 ) . acute q fever is still present in maritime canada ; however , the number of cases has diminished considerably from the 1980s and early 1990s . since 2004 , only 45 cases have been reported each year . the passive design of our study may have underestimated the number of cases . however , in the 1980s , a number of q fever outbreaks involved entire families . a typical scenario was exposure to the parturient family cat and her newborn kittens , after which everyone in the family became ill ( 2 ) . some outbreaks involved poker players ( 5 ) or most of the employees of a factory ( 6 ) . for our study , we carefully asked whether family members were ill ; only 2 patients mentioned such illness , and for each , it was a spouse . pneumonia seems to still be the dominant form of acute q fever in maritime canada . major differences in the manifestations of q fever occur in different regions . in maritime canada and in the basque region of spain , the predominant manifestation is pneumonia ( 7,8 ) ; in newfoundland and australia , fever with no apparent localization of infection ( 9,10 ) ; in the canary islands , fever and hepatitis ; and in southern france , hepatitis and pneumonia , although hepatitis is more frequent than pneumonia ( 11,12 ) . the factors responsible for these disparate manifestations are not known . when isolates of c. burnetii from different geographic areas were typed by using multisequence typing , all 7 isolates from nova scotia were identical and shared this type with 2 isolates from france and 1 from the united states ( 13 ) . the reservoirs for human infection with c. burnetii in nova scotia have likely spread from cats and dogs ( 14 ) to the more traditional reservoirs of sheep and cattle ( 12 ) . patient 8 , a sheep farmer , had pneumonia that appeared on radiographs as a rounded opacity in the right middle lobe . rounded opacities are very common in cat - associated cases of q fever but may not be specific for cat - associated infection ( 15 ) . our findings indicate that after c. burnetii is established in an area , it is likely to persist , although the incidence may fluctuate .

since the 1990s , reports of q fever in nova scotia , canada , have declined . passive surveillance for q fever in nova scotia and its neighboring provinces in eastern canada indicates that the clinical manifestation of q fever in the maritime provinces is pneumonia and that incidence of the disease may fluctuate .

accurate diagnosis is the most critical assignment in planning orthognathic surgery , and conventional lateral cephalometry has been the standard for evaluation and orthodontic diagnosis of maxillofacial deformity since the early 1930s1 . various methodologies have been introduced , and normal average value ( norm ) for corresponding analysis has been reported2 . analysis of lateral cephalometry includes the analysis of angle and length of hard tissues and soft tissues of the face and is the most commonly used method3 . however , two - dimensional ( 2d ) lateral cephalometry is associated with many problems . second , it is difficult to clearly reflect the difference between the right and left sides of the face . third , there is a fundamental problem in that a three - dimensional ( 3d ) subject is illustrated in two dimensions7 . in addition , weaknesses of 2d lateral cephalometry have been reported including low concordance with results of actual clinical evaluation8,9 . conventional lateral cephalometry has another limitation in that deficiency in the paranasal and infraorbital areas can not be accurately demonstrated . 3d facial analysis has been presented to solve such shortcomings and can be used to achieve simulation treatment for 3d operation and orthodontic processes10,11,12,13 . systems have been established for normal average values , landmarks , reference lines , and reference planes , all of which can serve as standards of analysis14 . if a landmark is defined for 3d analysis , such landmarks should be used for 2d analysis in most cases . this is because lateral cephalometry analysis has been widely performed , and the massive amount of data accumulated can be effectively used for 3d analysis15 . however , few studies have analyzed whether 3d analysis agrees with the large amount of 2d data . in this regard , this study analyzed the concordance and patterns between eight angles measured using lateral cephalometry as well as 3d computed tomography ( ct ) images . in addition , it also aims to identify a 3d frankfort horizontal ( fh ) plane with a high degree of concordance to the 2d plane by studying four fh planes . the study population consisted of 20 patients who visited the department of oral and maxillofacial surgery , samsung medical center ( seoul , korea ) in 2012 who underwent lateral cephalometry ( planmeca promax ; planmeca oy , helsinki , finland ) and 3d ct ( ge lightspeed vct xt ; general electronic medical system , milwaukee , wi , usa ) imaging . , 20 patients were selected among whom there were 12 males and eight females with an average age of 23.2 years . the area from the nasal bone to the mandible was included in the image , and the axial slice thickness was 1 mm . during ct scanning , the jaw and teeth were maintained in centric occlusion . after completion of ct scanning , the images were saved as digital imaging and communication in medicine ( dicom ) files . ct images were also reconstructed to produce 3d images using simplant pro 2011 ( materialize dental nv , leuven , belgium ) . the following patients were excluded : those with severe congenital malformation , such as hemifacial microsomia , those with pathological lesions of the jaw , and those with severe inflammation . in the reconstructed 3d image , lines , planes , and angles were set as follows , and the fh plane was the horizontal reference plane in this study . the fh plane on lateral cephalometry was defined with the lines passing the orbitale ( or ) and porion ( po ) , and the upper occlusal plane angle ( uopa ) was measured . four fh planes ( table 1 ) were established in the 3d system , and the uopa of each point was measured . the angles measured in 3d and lateral cephalometry were evaluated with regard to intraclass correlation coefficient ( icc ) with ibm spss statistics 21.0 ( ibm co. , armonk , ny , usa ) . ( 1 ) gonial angle ( ga ) , ( 2 ) palatal angle , ( 3 ) mandibular plane angle ( mpa ) , ( 4 ) uopa , ( 5 ) u1 to occlusal plane angle ( u1opa ) , ( 6 ) u1 to fh plane angle ( u1fhpa ) , ( 7 ) sna , ( 8) snb were measured both in lateral cephalometry and reconstructed 3d ct ( table 1 ) and were evaluated with regard to icc . the study population consisted of 20 patients who visited the department of oral and maxillofacial surgery , samsung medical center ( seoul , korea ) in 2012 who underwent lateral cephalometry ( planmeca promax ; planmeca oy , helsinki , finland ) and 3d ct ( ge lightspeed vct xt ; general electronic medical system , milwaukee , wi , usa ) imaging . , 20 patients were selected among whom there were 12 males and eight females with an average age of 23.2 years . the area from the nasal bone to the mandible was included in the image , and the axial slice thickness was 1 mm . during ct scanning , the jaw and teeth were maintained in centric occlusion . after completion of ct scanning , the images were saved as digital imaging and communication in medicine ( dicom ) files . ct images were also reconstructed to produce 3d images using simplant pro 2011 ( materialize dental nv , leuven , belgium ) . the following patients were excluded : those with severe congenital malformation , such as hemifacial microsomia , those with pathological lesions of the jaw , and those with severe inflammation . in the reconstructed 3d image , lines , planes , and angles were set as follows , and the fh plane was the horizontal reference plane in this study . the fh plane on lateral cephalometry was defined with the lines passing the orbitale ( or ) and porion ( po ) , and the upper occlusal plane angle ( uopa ) was measured . four fh planes ( table 1 ) were established in the 3d system , and the uopa of each point was measured . the angles measured in 3d and lateral cephalometry were evaluated with regard to intraclass correlation coefficient ( icc ) with ibm spss statistics 21.0 ( ibm co. , armonk , ny , usa ) . ( 1 ) gonial angle ( ga ) , ( 2 ) palatal angle , ( 3 ) mandibular plane angle ( mpa ) , ( 4 ) uopa , ( 5 ) u1 to occlusal plane angle ( u1opa ) , ( 6 ) u1 to fh plane angle ( u1fhpa ) , ( 7 ) sna , ( 8) snb were measured both in lateral cephalometry and reconstructed 3d ct ( table 1 ) and were evaluated with regard to icc . on lateral cephalometry , the average uopa was 11.38. when the concordance of uopa against the four fh planes on 3d was evaluated , the iccs showed high concordance with 0.745 , 0.751 , 0.755 , and 0.754 , respectively . the icc differences among the four angles acquired in 3d were also not significant different.(table 2 ) concordance of the average of eight angles was verified.(table 3 ) iccs of ga , palatal angle , u1opa , and u1fhpa were low at 0.583 , 0.287 , 0.404 , and 0.617 , respectively . thus demonstrating a low concordance between lateral cephalometry and 3d ct . on the other hand , iccs of mpa , uopa , sna , and snb were high at 0.752 , 0.745 , 0.798 , and 0.869 , respectively . additionally , ga and mpa acquired in 2d were larger than those in 3d for all 20 patients included in this study.(table 4 ) on lateral cephalometry , the average uopa was 11.38. when the concordance of uopa against the four fh planes on 3d was evaluated , the iccs showed high concordance with 0.745 , 0.751 , 0.755 , and 0.754 , respectively . the icc differences among the four angles acquired in 3d were also not significant different.(table 2 ) concordance of the average of eight angles was verified.(table 3 ) iccs of ga , palatal angle , u1opa , and u1fhpa were low at 0.583 , 0.287 , 0.404 , and 0.617 , respectively . thus demonstrating a low concordance between lateral cephalometry and 3d ct . on the other hand , iccs of mpa , uopa , sna , and snb were high at 0.752 , 0.745 , 0.798 , and 0.869 , respectively . additionally , ga and mpa acquired in 2d were larger than those in 3d for all 20 patients included in this study.(table 4 ) two - dimensional cephalometry has been widely used for diagnosis and operation in craniofacial skeletal development and orthodontic and orthognathic surgeries13,16 . the measured values from the patients can be compared with generally known normal values to judge the asymmetry17 . in lateral cephalometry , however , it is difficult to distinguish the overlapping structures ; in patients with an asymmetric face , the accuracy of analysis is low . consequently , it is desirable to also use other radiographic images in order to achieve more accurate analysis . to evaluate the asymmetry of a face , panorama radiography18 and posteroanterior cephalometry panorama radiography is the most familiar technique to most dentists and orthodontists , and it is the representative examination for dental diagnosis and treatment planning . this type of imaging enables dentists to detect cysts and tumors , supernumerary teeth , missing teeth , and bony deformities and to evaluate the asymmetry of the lower jawbone19,20 . updegrave21 , however , reported that an incorrect treatment plan may results from serious asymmetry of the mandible ramus and condyle and coronoid processes on panorama radiography . posteroanterior cephalometry has the same problem in that precise perception is difficult because of many landmark overlaps13 . as such however , no overlapping landmarks are produced in 3d imaging , minimizing the confusion of left- and right - side structures . therefore , with 3d ct imaging , clinicians can diagnose all patients accurately including those with facial asymmetry , easily identify the shapes of craniofacial structures , and recognize the shapes of bones and soft tissues from various angles . according to previous studies measuring length on volume - rendered 3d ct images and in cadavers23 , the difference in length between two groups was minimal , confirming the accuracy of 3d - ct images . in addition , 3d lateral cephalometry analysis abstracted from 3d ct was introduced , and its accuracy has been reported . van vlijmen et al.1 performed comparative analysis on various angles and distances between 3d lateral cephalometry and conventional lateral cephalometry and reported that there was no statistically significant difference in many parts . it is true that lateral cephalometry analysis has been the major method to evaluate facial types for tens of years15,25 . 3d ct analysis , a very useful method in diagnosis , uses the same landmarks as conventional lateral cephalometry15 . however , the concordance between the measurements of lateral cephalometry and 3d ct has not been sufficiently studied . using the relationship and patterns of measurements acquired from two analysis methods , as well as their concordance , 3d ct values the uopa abstracted from the fh plane defined in 3d ct and that from lateral cephalometry were found to be very similar.(table 2 ) similarly , the four 3d fh planes did not show differences in concordance with those of 2d analysis . in such cases , it is desirable to choose landmarks with higher representation and reliability . in a study by yu et al.26 , two observers marked lateral cephalometry landmarks three times and calculated the concordance with respect to the x - axis and y - axis . in this experiment , po showed the lowest concordance among 17 landmarks on the x - axis , while or did not show a significant difference in concordance among observers or among trials of the same observer . additionally , when defining po on 3d ct , a wide curve is produced , making repetition and reproduction difficult.(fig . 1 ) based on these results , it is reasonable to define the fh plane in 3d using two or and one po values . according to the results of this study , ga measured on 2d is larger than that measured on 3d in all patients.(table 4 ) there are three possible explanations . first , the difference may be because of error in menton ( me ) . when marking me on 3d ct ( fig . 2b ) and observing the resulting 3d lateral cephalometry , me marked on the superior position that me on conventional lateral cephalometry.(fig . 2c ) this occurs because anatomic structures near the me are extended in the lower direction on 3d ct.(fig . 2a ) in patients with such structures , the 3d values may be different from those on 2d even when me is accurately marked on 3d ct . additionally , when me is marked at the front of skull , it may be marked in a little superior position ( fig . thus , when me is marked on 3d ct , it is recommended to approach from the bottom of the lower jawbone . second , articulare ( ar ) may have larger x - axis error on lateral cephalometry compared with 3d ct . trpkova et al.7 confirmed through meta - analysis that ar and basion showed the largest x - axis errors on lateral cephalometry . the possibility of lower accuracy of ga can not be excluded because of lower accuracy of the reference points . ar on lateral cephalometry is defined as the point of overlap between the skull base and posterior surface of the lower jawbone condyle , while ar in the present study is defined as the most posterior point of the mandibular condyle . thus , it is difficult to assert that ar in this study is absolutely concordant with ar on 2d . however , it is impossible to define ar in 3d using the same definition of that in 2d . therefore , if ar is defined using the method of this study , ga in 2d is slightly larger than that in 3d ct . mpa , just like ga , was larger in 2d than in 3d in all patients . the angles showing low concordance were palatal angle , u1opa , and u1fhpa.(table 3 ) an angle is made by two lines ; if the lines are short , the measurement is more difficult , and the risk of error increases , which lowers the probability of repetition and reproduction and consequently the probability of concordance . the straight palatal line and u1 line used to define the aforementioned three angles had short distances , which resulted in their lower concordance values . on the other hand , mpa , uopa , sna , and snb were formed by two long lines and so showed higher concordance . for measurement of angles made up of two long lines , the concordance between two methods is high . in this study when me was included in the analysis , it was marked in a different location from that of 2d analysis because of the nature of the measurement points . additionally , the fh plane on 3d ct showed concordance with the fh plane on lateral cephalometry , indicating no difference in concordance no matter what is selected among four fh planes defined on 3d ct . however , it is desirable to define the fh plane on 3d ct with two or and one po values considering the reproduction of or itself .

objectivesthe aim of this study was to verify the concordance of the measurement values when the same cephalometric analysis method was used for two - dimensional ( 2d ) cephalometric radiography and three - dimensional computed tomography ( 3d ct ) , and to identify which 3d frankfort horizontal ( fh ) plane was the most concordant with fh plane used for cephalometric radiography.materials and methodsreference horizontal plane was fh plane . palatal angle and occlusal plane angle was evaluated with fh plane . gonial angle ( ga ) , palatal angle , upper occlusal plane angle ( uopa ) , mandibular plane angle ( mpa ) , u1 to occlusal plane angle , u1 to fh plane angle , sna and snb were obtained on 2d cephalmetries and reconstructed 3d ct . the values measured eight angles in 2d lateral cephalometry and reconstructed 3d ct were evaluated by intraclass correlation coefficiency ( icc ) . it also was evaluated to identify 3d fh plane with high degree of concordance to 2d one by studying which one in four fh planes shows the highest degree of concordance with 2d fh plane.resultsiccs of mpa ( 0.752 ) , uopa ( 0.745 ) , sna ( 0.798 ) and snb ( 0.869 ) were high . on the other hand , iccs of gonial angle ( 0.583 ) , palatal angle ( 0.287 ) , u1 to occlusal plane ( 0.404 ) , u1 to fh plane ( 0.617 ) were low respectively . additionally ga and mpa acquired from 2d were bigger than those on 3d in all 20 patients included in this study . concordance between one uopa from 2d and four uopas from 3d ct were evaluated by icc values . results showed no significant difference among four fh planes defined on 3d ct.conclusionfh plane that can be set on 3d ct does not have difference in concordance from fh plane on lateral cephalometry . however , it is desirable to define fh plane on 3d ct with two orbitales and one porion considering the reproduction of orbitale itself .

the herpes family of viruses includes 8 separate species that infect humans : herpes simplex virus 1 ( hsv-1 ) , herpes simplex virus 2 ( hsv-2 ) , varicella zoster virus , epstein - barr virus , cytomegalovirus , human herpesvirus 6 ( hhv-6 ) , human herpesvirus 7 ( hhv-7 ) and human herpesvirus 8 ( hhv-8 ) . these viruses account for a significant proportion of human cutaneous diseases and they cause similar histological and clinical findings . although herpes labialis , herpes vulvovaginitis , herpetic whitlow and herpes gladiatorum are the most well - known clinical features of the hsv-1 and hsv-2 infections , there are various descriptions of herpesvirus involvement in erythema multiforme , atopic dermatitis , lichenoid dermatitis and seborrheic keratosis [ 2 , 3 , 4 , 5 , 6 ] . lichen simplex chronicus ( lsc ) and acute bilateral carpal tunnel syndrome ( cts ) are unusual complications of herpesviruses , and apparently a few cases of adult patients have been reported previously [ 7 , 8 , 9 ] . we describe an 11-year - old girl with lsc and acute bilateral cts following hsv-1 infection . an 11-year - old girl was admitted with recurrent fever , pain and swelling of her right wrist and proximal phalanx , and relapsing painful , itchy lesions consisting of erythematous papules with excoriations and crusting on the skin with a central hyperpigmentation , mostly distributed on the neck , face , legs and hips . her medical history was significant for a 6-month period of recurrent lesions , always involving the same parts of her body and preceded by severe pain and hyperesthesia on 1 side of her entire body ( sometimes right , sometimes left ) , with a frequency of 3 times a week . she described that both of her wrists , especially the right one and proximal phalanx had become tender and swollen at the same time as the appearance of the skin lesions . up to this time , she had been diagnosed with atopic dermatitis and eczema and treated with various medicines such as systemic antihistamines , local and systemic corticosteroids and/or analgesics and antipsychotics but showed no improvement . on physical examination , she had both old and new skin lesions at the same time on different parts of her body , particularly on the cheeks , neck , extremities and hips . laboratory investigations revealed no abnormality of counter blood cell , erythrocyte sedimentation rate , c - reactive protein and fibrinogen . immunoglobulin levels of iga , igg and ige were within the normal range , but igm showed an elevated pattern . viral hepatitis ( a , b , c ) , epstein - barr virus , cytomegalovirus , hsv-2 , human immunodeficiency virus , parvovirus b19 , lyme serologies , salmonella and brucella agglutinations , organ- and tissue - specific autoimmune antibodies ( especially antinuclear antibodies ) , rheumatoid factor , cryoglobulins and ena test were all negative . antineutrophil cytoplasmic antibodies assay ( anca ) showed a negative pattern for both p - anca ( perinuclear staining ) and c - anca ( cytoplasmic staining ) . the c3 level was 107 mg / dl ( normal 80150 ) and c4 was within the normal range at 22 mg / dl ( normal 2050 ) . specific ige tests involving milk , egg , mold allergy , grass allergy , dog epithelium , house dust allergy , cat - specific ige and weed panel were also negative . cranial mri was performed in order to reveal the reason for her neurologic symptoms but showed no abnormality . u / l ( normal 22240 ) and an electroneuromyographic investigation revealed moderate delay in distal sensory - motor latencies of both median nerves , with delayed median nerve f - responses on both sides . serological tests showed positive hsv-1 specific igm and hsv-1 dna was detected in the patient 's serum by using pcr . a punch skin biopsy specimen from the right upper extremity showed hyperkeratosis with areas of parakeratosis , overlying an acanthotic epidermis with irregular elongation of the rete ridges . chronic perivascular inflammatory infiltrate and fibrosis were also seen in the papillary dermis , but immunofluorescence studies were all negative ( fig . 1 , fig . postherpetic lsc and acute bilateral cts associated with hsv-1 infection were considered and high - dose acyclovir treatment ( 30 mg / kg / day i.v . for 5 days ) , 10 mg / kg / day naproxen sodium and high doses of vitamin b1 , b6 and b12 combinations were started immediately . a dramatic improvement was observed on her neurologic symptoms and skin lesions within a few weeks . during her follow - up , neither adverse effects due to high - dose acyclovir treatment nor any other new lesions were observed . a control pcr was negative and serological tests showed a negative pattern for hsv-1 igm and a positive pattern for hsv-1 igg in the first month of her follow - up . forty - five days after the initial acyclovir treatment she went to the seaside for summer holiday and a few days later she was readmitted with swelling of the proximal phalanx of her right hand and a new skin lesion on her right hip ( fig . 3 ) ; these symptoms were preceded by severe pain and hyperesthesia in the right half of her entire body . a new pcr showed a positive pattern again , but serological tests revealed a negative pattern for hsv-1 igm and a positive pattern for hsv-1 igg . a new viral shedding was thought to be responsible and oral acyclovir ( 30 mg / kg / day ) treatment was given for 2 weeks . afterwards , she had continued the therapy at a dose of 10 mg / kg / day for 2 weeks . the skin lesion on her right hip and swelling of the joints resolved within a few weeks and pcr became negative again . we discontinued the therapy and follow up the patient monthly . thus far , 10 months have passed without any new skin lesions . lsc ( circumscribed neurodermatitis ) is an idiopathic disorder characterized by the presence of 1 or more erythematous , scaling , lichenified plaques with varying degrees of overlying excoriation . in cases of long - standing duration the most common sites are the neck ( sides ) , ankles , scalp , vulva , pubis , scrotum and extensor forearms . the peak of incidence is between 35 and 50 years of age , and women are more affected than men ( f : m = 2:1 ) . severe and intractable itch is the predominant symptom in nearly all patients and is characterized by paroxystical attacks [ 10 , 11 , 12 ] . acute clinical presentation of herpesviridae is often secondary to active virus replication and host immune response , but resolution of symptoms does not herald clearance of the virus . lsc is an unusual complication of herpesviruses ; however , there are a few reports of postherpetic pruritus in adult patients . liddel reported a patient with severe pruritus after a herpes zoster infection , gerritsen et al . reported a patient with lsc following herpes zoster infection of the scalp and darsow et al . described a patient with circumscribed pruritus which started 5 years after herpes zoster in the same dermatome . either clinically or pathologically there were no classical skin lesions of the hsv-1 infection in our patient . but positivity of the serological tests and detection of hsv-1 dna by pcr and the patient 's response to the acyclovir treatment suggested that lsc developed in this patient as a complication of recurrent viral shedding of hsv-1 . it was difficult to distinguish whether the exact cause of the pain was postherpetic neuralgia or increasing stimulation that may produce itch and pain , as these sensations share the same neuroanatomical substrate . we thought that the biopsy taken from the margin of the main lesion and differentiation of herpetic lesions to lsc may be related to the rubbing and scratching . on the other hand , it is known that there is a potential relationship between ultraviolet light and recurrent herpes simplex infections . viruses can reactivate from the latent state in neurons to form recrudescent lesions due to exposure to sunlight [ 14 , 15 ] . the occurrence of a new lesion after the summer holiday in this patient was thought to be the result of exposure to ultraviolet irradiation which led to a new viral shedding . cts is one of the most common peripheral compression neuropathies , but it is rarely seen in children . many aspects of its etiology are not at all clear , and it is often termed idiopathic ; however , it has also been attributed to a variety of underlying disorders and processes and secondary to infectious diseases [ 17 , 18 , 19 ] . childhood cts often has an unusual presentation , with modest complaints and children are often too young to communicate their problem . in our patient , pain and swelling of the wrists and proximal phalanx was thought to be associated with the mechanical entrapment of the median nerves in relation to acute arthritis during the viral shedding of hsv-1 . the rapid response to non - steroid anti - inflammatory drugs and high doses of vitamin b1 , b6 and b12 combinations , tends to support this hypothesis . in conclusion , this is an unusual childhood case of hsv type-1 infection complicated by lsc and acute bilateral cts during viral shedding . the experience with this patient emphasizes the importance of serological tests and pcr as well as the other laboratory techniques for the accurate diagnosis and management of the disease .

we describe an 11-year - old girl presenting with lichen simplex chronicus ( lsc ) and acute bilateral carpal tunnel syndrome ( cts ) following herpes simplex virus type 1 ( hsv-1 ) infection as evidenced by serological data and by detection of hsv-1 dna in the blood with the use of pcr . based on the literature search , this case represents the first childhood case of lsc and acute bilateral cts following hsv-1 infection . the experience with this patient emphasizes the importance of serological tests and pcr as well as the other laboratory techniques for the accurate diagnosis and management of the disease .

cerebral palsy ( cp ) is a non - progressive disease with symptoms that include neurological disorders or developmental disabilities . children with cp have spasticity , musculoskeletal problems , mobility disturbances , and decreased pelvic movements that lead to awkward movement and sitting posture1 , 2 . symptoms also induce unstable posture control , imbalance , and aberrant control of movement . postural control is the ability to control the body position in space , and it has a relationship with the sense of balance3 . hippotherapy and horseback riding have been suggested as interventions for correcting the balance problems of children with cp4 . two types of horseback riding are available : hippotherapy and therapeutic horseback riding ( thr ) . hippotherapy provides better pelvic , hip , and trunk movement and influences childrens posture , balance , and coordination5 . hippotherapy has short - term beneficial effects on the muscle symmetry of the trunk and hip , whereas thr has no effect on muscle tone6 . the effects of these two types of horseback riding have been studied in children with cp , and both types have been demonstrated to improve gross motor function and promote better standing and bipedal balance7 , 8 . a horseback riding simulator ( joba , panasonic inc . , it was developed to overcome the primary limitations of hippotherapy , such as unavailability and high cost . this device offers an indoor experience of horseback riding and mimics the rhythmic movement of horseback riding , thereby promoting muscular strength and improving sense of balance . the objective of this study was to compare hippotherapy to the use of a horseback riding simulator ( joba , panasonic inc . , japan ) with respect to their effects on the static and dynamic balance of children with cp . our results indicate that a horseback riding simulator has beneficial effects as an intervention for children with cp . this study included 26 children with cp who were receiving physical therapy at the h horseback riding center and the n horseback riding center in kyung - ki in korea . the selection criteria for the subjects were a modified ashworth scale ( mas ) grade less than + 1 . children who could perform more than 10 m independent walking and were available for more than 30 min training per day were selected . the parents or guardians of all the participants provided their written informed consent in accordance with the ethical principles of the declaration of helsinki ( table 1table 1 . general characteristics of subjectshippotherapyhrsgender ( m / f)8/59/4age ( year)10.81.610.02.2height ( cm)125.812.6122.614.3weight ( kg)25.26.425.55.7meansd , hrs : horseback riding simulator ) . meansd , hrs : horseback riding simulator the children were randomly divided into two groups : a hippotherapy group that included 13 children , and a horseback riding simulator ( joba , panasonic inc . the two groups participated in 1 hour of exercise per day , 3 times a week , for 12 weeks . the hippotherapy group carried out anterior - sitting , posterior - sitting , and side - sitting exercises for 10 min each while horseback riding at a walking pace ( 6 km / h ) . the course leader held the reins of the horse and two side - walkers held the child s legs to assist the child in sitting in the saddle in order to prevent the child from falling and to help the child to exercise during the horseback riding walk . the horseback riding simulator group exercised with the same protective equipment and were assisted by the same leader and side - walkers . both groups received 20 min of conventional physical therapy before hippotherapy and performed stretching on the horse or horseback riding simulator for 5 min before and after the exercise . three physical therapists , who had more than 3 years of experience in pediatric physical therapy and had received education in the experimental method , participated in this study . the subjects static balance ability was measured using bpm ( software 5.3 , sms healthcare inc . , uk ) as the center of pressure sway length while standing for 30 seconds with the eyes open and looking to the front . statistically significant differences between the measurements obtained before and after the intervention in each group were determined using the independent t - test . the children were divided into two groups and evaluated pre- and post - test using bpm ( software 5.3 , sms healthcare inc . , uk ) and the pbs ( pediatric balance scale ) ( table 2table 2 . comparison of measurement values at pre - test and post - testvariablegroupprepostpbs ( score)hippotherapy35.63.841.24.7*hrs35.84.738.55.3*sway length ( mm)hippotherapy220.127.6135.014.3*hrs219.931.7142.818.8**p<0.05 meansd , pbs : pediatric balance scale , hrs : horseback riding simulator ) . * p<0.05 meansd , pbs : pediatric balance scale , hrs : horseback riding simulator the hippotherapy and horseback riding simulator groups showed significantly decreased sway lengths in the static balance test . comparison of the pre- and post - test sway lengths showed that the hippotherapy group and the horseback riding simulator group showed significant decreases in sway length of 85 mm and 80 mm , respectively ( p<0.05 ) . both groups showed improved static balance , but no significant difference was found between the two groups . dynamic balance was also evaluated pre- and post - test using the pbs ( pediatric balance scale ) . the hippotherapy group showed an increase in score of about 4 points , while the horseback riding simulator group showed an increase of 3 points . both groups showed significant improvements in dynamic balance , but no significant difference was found between the two groups ( p<0.05 ) . balance control is very important for children with cp , and maintaining balance requires three distinct sensory system inputs : visual , somatosensory , and vestibular9 . generally , the dynamic sense of balance depends primarily on vestibular input on unstable surfaces ; however , static balance primarily depends on somatosensory input . many studies have investigated balance and clinical tools for children with cp . a systematic review identified 22 tools that can assess balance10 . hippotherapy and thr are types of exercises that affect balance , coordination , and posture , contributing to the development of sensory and perceptual motor skills6 , 11 . a horse s rhythmic movement , velocity , and variations can facilitate righting and equilibrium actions . during hippotherapy and thr , these facilitations improve muscular co - contraction and postural balance resulting in improvements in the gross motor function of children with cp12,13,14,15 . many studies of hippotherapy and thr in children with cp have demonstrated they have positive effects on postural control and balance . the horseback riding simulator has also been reported to be beneficial for children with cp , improving their postural control and global function , and providing enjoyment16 . haehl et al . found no significant improvement of in hippotherapy and thr but indicated that hippotherapy had positive effects on the postural control and balance of children with cp17 . other studies confirmed that hippotherapy and thr are suitable interventions for children with cp . the horseback riding simulator used in the present study offers several advantages over hippotherapy . the simulator is free from space limitations , has a low price , is easy to handle , and is non - affected by weather conditions16 . use of the horseback riding simulator resulted in improved muscle strength and contraction in elderly people18 . a previous study of children with cp reported that use of a horseback riding simulator significantly improved their postural control and balance16 . our data indicate that both hippotherapy and use of the horseback riding simulator improved the static and dynamic balance of children with cp . however , due to the fast learning characteristics of children , the long - term 12-week exercise program might have been responsible for the similar results seen in balance improvement . many factors remain to be considered regarding exercise for children with cp . therefore , if other tools or exercises were utilized , the results might not match the results presented here . overall , our results indicate that children with cp may respond equally well to the use of a horseback riding simulator as they do to hippotherapy in terms of balance improvement . first , the number of subjects was small , and second , only static and dynamic balance were evaluated . in spite of these limitations , our results show that the use of a horseback riding simulator can be a good intervention for children with cp . further studies incorporating larger sample sizes , various cp types , and full utilization of the programs of the horseback riding simulator might be needed . in conclusion , the horseback riding simulator could be a useful alternative to hippotherapy for improving of static and dynamic balance of children with cp .

[ purpose ] we with respect to their effects on the compared hippotherapy with a horseback riding simulator ( joba , panasonic inc . jp ) static and dynamic balance of children with cerebral palsy ( cp ) . [ subjects and methods ] twenty - six children were randomly divided into two groups : a hippotherapy group that included 13 children , and a horseback riding simulator ( joba , panasonic inc . , japan ) group , which was also composed of 13 children . the two groups participated in 1 hour of exercise per day , 3 times a week , for 12 weeks . the subjects static balance ability was measured using bpm ( software 5.3 , sms healthcare inc . , uk ) as the center of pressure sway length while standing for 30 seconds with their eyes open and looking to the front . dynamic balance ability was measured using the pbs ( pediatric balance scale ) . [ results ] both groups showed significant improvements in static and dynamic balance but significant differences between the two groups were not found . [ conclusion ] the horseback riding simulator could be a useful alternative to hippotherapy for the improvement of static and dynamic balance of children with cp .

it is known that 9095% of all cancers are caused by or closely associated with environmental factors and lifestyle . this includes diet ( 3035% ) , cigarette smoking ( 2530% ) , and alcohol consumption ( 46% ) . cigarette smoking is an important risk factor for heart disease , chronic obstructive pulmonary disease , stroke , and acute respiratory diseases . in addition to all these noncancer diseases , it is also highly associated with human cancer development . the international agency for research on cancer ( iarc ) identified cigarette smoking as the cause of cancer in more organ sites than any other human carcinogens . these include cancers of the lungs , oral cavity , larynx , nasal cavity , esophagus , stomach , pancreas , liver , kidney , urinary bladder , uterine cervix , and bone marrow . there are over 5000 chemical compounds identified in tobacco and 62 of these have been evaluated by iarc as showing sufficient evidence for carcinogenicity in either animals or in humans [ 2 , 3 ] . the major carcinogenic compounds include , but not limited to , radioactive polonium , n - nitrosamines such as 4-(methylnitrosaminao)-1-(3-pyridyl)-1-butanone ( nnk ) , polycyclic aromatic hydrocarbons ( pahs ) ( e.g. , benzo[a]pyrene ( bap ) ) , and benzene . multistep processes of genetic and molecular defects have taken place before the manifestation of cancer . traditionally , there are three basic stages of carcinogenesis : initiation , promotion , and progression . carcinogenesis process is usually accompanied by changes in structure and function of central genomic information coded in the dna leading to various oncogene activations and tumor suppressor gene inactivations . in addition , multiple signaling pathways may also be deregulated during the process of cancer development . cancer growth also requires molecular changes that either affect the tumor cells themselves or alter the interaction between tumor cells and their surrounding stromal environment or the immune system . cigarette smoke components have been reported to promote tumorigenesis by several mechanisms involving all three stages of carcinogenesis . genotoxic agents in cigarette smoke induce dna damage through several mechanisms including gene point mutation , deletions , insertions , recombinations , rearrangements , and chromosomal aberrations . in addition to genotoxic effects , nongenotoxic effects of cigarette smoke are also extremely important . these effects can also act as modulators which alter cellular functions including cell proliferation and cell death . while synergistic effects of genotoxic carcinogens are known to occur , interaction between non - genotoxic ( epigenetic ) factors and genotoxic agents may also synergistically increase the risk for carcinogenesis . the genotoxicity leading to carcinogenesis has been extensively reviewed in recent years [ 911 ] . in this present review , aside from a brief overview on the genotoxic effects of cigarette smoke components , we will provide a more extensive review on the non - genotoxic mechanisms of carcinogenesis by cigarette smoke or its components . step 1 ( initiation of carcinogenesis)carcinogenesis may be the result of chemical or biological insults to normal cells through multistep processes that involves genomic changes ( initiation of cancer development ) . some of the cigarette smoke components can act directly on dna , but many require enzyme conversion before becoming carcinogenic [ 10 , 11 ] . most of such conversions involve metabolic changes via cytochrome p450s ( p450s ) such as p450s 1a2 , 2a13 , 2e1 , and 3a4 to form the electrophilic entities that can bind to dna to form dna adducts . such adduct formation is usually at the adenine or guanine sites of the dna and lead to mutations such as those observed in the kras oncogene in lung cancer or those in the tp53 gene in a variety of cigarette smoke - induced cancers [ 13 , 14 ] . 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) and n-nitrosonornicotine ( nnn ) are the most potent tobacco - specific nitrosamines in tobacco products and cigarette smoke . these compounds are formed from tobacco alkaloids like nicotine during the curing process of tobacco and are important tobacco carcinogens that can affect different tissues depending on the specific nitrosamines or their metabolites involved [ 5 , 10 ] . nnn has been shown to be carcinogenic to esophagus , nasal cavity , and respiratory tract in laboratory animals . in humans , metabolites derived from nnk and the metabolites of nnk can also be identified in the smoker 's urine .benzo[a]pyrene ( bap ) , one of the pahs , is classified as a group 1 carcinogen to humans . it has been shown to have strong association and tumor - induction potentials in lungs , trachea , and mammary glands . the carcinogenic potency of bap has been demonstrated to be related to its metabolites which form dna adducts with site - specific hotspot mutation in the p53 tumor suppressor gene . positive correlations of such adduct formation and tumor are indeed found in the lung cancer tissues of cigarette smokers . these findings indicate that dna mutations are increased in both tumor and nontumor bearing tissues of smokers . however , it must be pointed out that dna adduct formations induced by cigarette smoke still can not fully represent all the risk factors for cancer development in cigarette smokers . for example , while there is higher incidence of pancreatic cancer in cigarette smokers than nonsmokers . assays for nnk metabolites in pancreatic cancer tissues in humans showed no significant difference between smokers and nonsmokers . thus , it is apparent that nnk - induced dna adducts alone are not solely responsible for the pancreatic cancers in cigarette smokers . nevertheless , nnk and its metabolite , nnal ( 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol ) , are the only environmental carcinogens known to induce pancreatic cancer in animal models . thus , the contribution of nnk to pancreatic cancer in cigarette smokers still can not be ignored . furthermore , it is suggested that , in addition to dna damage , synergistic interactions between dna reactivity and epigenetic actions such as increased cell proliferation induced by nnk or by other chemicals in cigarette smoke may be needed for actual cancer development in such patients [ 23 , 24 ] . there is indication that cigarette smoke carcinogens or cocarcinogen , such as nicotine , may also play a direct role to enhance cancer promotion and progression in human cancers after cancer development . such genotoxic mechanisms for cancer initiation and carcinogenesis by cigarette smoke components are well covered and discussed in several excellent reviews [ 5 , 10 , 11 , 2628 ] . , we will provide more information on the non - genotoxic ( epigenetic ) mechanisms involved in cancer promotion and progression via cigarette smoke . carcinogenesis may be the result of chemical or biological insults to normal cells through multistep processes that involves genomic changes ( initiation of cancer development ) . some of the cigarette smoke components can act directly on dna , but many require enzyme conversion before becoming carcinogenic [ 10 , 11 ] . most of such conversions involve metabolic changes via cytochrome p450s ( p450s ) such as p450s 1a2 , 2a13 , 2e1 , and 3a4 to form the electrophilic entities that can bind to dna to form dna adducts . such adduct formation is usually at the adenine or guanine sites of the dna and lead to mutations such as those observed in the kras oncogene in lung cancer or those in the tp53 gene in a variety of cigarette smoke - induced cancers [ 13 , 14 ] . 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) and n-nitrosonornicotine ( nnn ) are the most potent tobacco - specific nitrosamines in tobacco products and cigarette smoke . these compounds are formed from tobacco alkaloids like nicotine during the curing process of tobacco and are important tobacco carcinogens that can affect different tissues depending on the specific nitrosamines or their metabolites involved [ 5 , 10 ] . nnn has been shown to be carcinogenic to esophagus , nasal cavity , and respiratory tract in laboratory animals . in humans , metabolites derived from nnk and the metabolites of nnk benzo[a]pyrene ( bap ) , one of the pahs , is classified as a group 1 carcinogen to humans . it has been shown to have strong association and tumor - induction potentials in lungs , trachea , and mammary glands . the carcinogenic potency of bap has been demonstrated to be related to its metabolites which form dna adducts with site - specific hotspot mutation in the p53 tumor suppressor gene . positive correlations of such adduct formation and tumor are indeed found in the lung cancer tissues of cigarette smokers . these findings indicate that dna mutations are increased in both tumor and nontumor bearing tissues of smokers . however , it must be pointed out that dna adduct formations induced by cigarette smoke still can not fully represent all the risk factors for cancer development in cigarette smokers . for example , while there is higher incidence of pancreatic cancer in cigarette smokers than nonsmokers . assays for nnk metabolites in pancreatic cancer tissues in humans showed no significant difference between smokers and nonsmokers . thus , it is apparent that nnk - induced dna adducts alone are not solely responsible for the pancreatic cancers in cigarette smokers . nevertheless , nnk and its metabolite , nnal ( 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol ) , are the only environmental carcinogens known to induce pancreatic cancer in animal models . thus , the contribution of nnk to pancreatic cancer in cigarette smokers still can not be ignored . furthermore , it is suggested that , in addition to dna damage , synergistic interactions between dna reactivity and epigenetic actions such as increased cell proliferation induced by nnk or by other chemicals in cigarette smoke may be needed for actual cancer development in such patients [ 23 , 24 ] . there is indication that cigarette smoke carcinogens or cocarcinogen , such as nicotine , may also play a direct role to enhance cancer promotion and progression in human cancers after cancer development . such genotoxic mechanisms for cancer initiation and carcinogenesis by cigarette smoke components are well covered and discussed in several excellent reviews [ 5 , 10 , 11 , 2628 ] . , we will provide more information on the non - genotoxic ( epigenetic ) mechanisms involved in cancer promotion and progression via cigarette smoke . step 2 ( cancer promotion)cancer promotion is characterized by deregulation of signaling pathways which control cell proliferation , apoptosis , and so forth , . it is believed that although there are various genetic pathways which may lead to cancer development or cancer behaviors , there are certain hallmark capabilities or mechanisms which are commonly shared by all tumors . in the following discussion cancer promotion is characterized by deregulation of signaling pathways which control cell proliferation , apoptosis , and so forth , . it is believed that although there are various genetic pathways which may lead to cancer development or cancer behaviors , there are certain hallmark capabilities or mechanisms which are commonly shared by all tumors . in the following discussion these signals are transmitted into cells by receptors that bind distinct signaling molecules . in cancer cells , receptor overexpression allows cancer cells to become hyper - responsive to low levels of growth factors that generally are not sufficient to trigger proliferation in normal cells . nicotine , a major component of cigarette smoke , is known to be a chemical that plays an important role in carcinogenesis in cigarette smokers . nicotine behaves like those growth factors which exert their biological functions mainly through the nicotinic acetylcholine receptors ( nachr ) , -adrenoceptors ( -ar ) or epidermal growth factor receptor ( egfr ) . the functions of these receptors are cell - type specific and the expression level and receptor sensitivity can be modified by nicotine . obviously , alterations in either the receptor expressions or sensitivity play an important role in cigarette smoke - induced carcinogenesis [ 3436 ] . recent study by lee et al . reported that 9 nachr expression in human breast tumors is elevated in advanced stages of breast cancer and plays important roles in human breast carcinogenesis . nicotine has been shown to mediate 9 nachr signaling and upregulate cyclin d3 expression in breast caner cells and breast cancer tissues . furthermore , it is also found that activation of the expression of 9 nachr by nicotine is through akt signaling and activation of 9 nachr signaling would elevate the phosphorylation status of adhesion molecule which plays a role in cancer metastasis . this enhancement has been shown to be via 7 nachr with activation of erk1/2 cascade as well as induction of nf-b and bad phosphorylation . all these events eventually lead to inhibition of apoptosis and increase of cancer risk . these findings were further supported by wada et al . who observed that nicotine promoted cell proliferation via 7 nachr mediated p44/p42-mapk activation . moreover , in our own study , we also reported that nicotine induced human bladder cells proliferation through erk1/2 and stat3 signaling downstream of 7 nachr and -adrenoceptors ( -ar ) . in sum , all these studies indicate that nicotine , an important ingredient of cigarette smoke , promotes cellular proliferation which plays a critical role in carcinogenesis . other than nicotine , nitrosamines , such as nnk and nnn , also induced cancer cells growth through nachr . nnk induced carcinogenesis by binding to nachr especially for 7 nachr , whereas the biological impact of nnn is mainly modulated by 4/2 nachr [ 8 , 4446 ] . it has been demonstrated that nicotine or nnk stimulated lung cancer cell proliferation via 7 nachr with activations of pkc , raf1 , akt , erk1/2 , and transcription factors such as jun , fos , and myc [ 4749 ] . question has been raised concerning the possibility that specific nachr subunit upregulated by nicotine or nnk may be tissue specific or dependent . for instance , with nicotine or nnk , 7 nachr is the primary nachr subunit which mediates tumorigenesis in lungs giving rise to pulmonary squamous cell carcinoma and mesothelioma . on the other hand , thus , the specific types of nachr expressed in cancer cells may be considered as useful molecular targets for potential clinical therapy . however , most of the nachr present in cancer cells are still not functionally characterized yet . future study will be needed to understand the functions of different nachr subtype in cancer cells and the downstream signal pathways involved in tumorigenesis . in addition to nachr , a number of studies indicated that nicotine and nnk might also exert their biological activities through activation of receptors such as -adrenoceptors ( -ar ) , egfr , or insulin - like growth factor receptor ( igfr ) or transactivation by nachr signaling . for example , nnk can stimulate ht-29 cell proliferation through -ar followed by cyclin amp elevation and cox-2 expression . consistently , nnk stimulates the growth of pulmonary adenocarcinoma in vitro and in vivo via the release of arachidonic acid through cox-2 and 5-lipoxygenase ( 5-lox ) pathways that are mainly regulated by -ar . in another study by schuller and cekanova , nnk is reported to stimulate 2-ar receptor pathway ( including pka , camp , creb ) and transactivate egfr pathway ( such as raf-1/erk1/2 signaling ) in the development of lung cancer . it has also been reported that antagonists of -ar can inhibit the development of nnk - induced lung adenocarcinoma . such antagonists are also found to be effective in reducing the stimulatory effects of nicotine on pkc , erk1/2 activations , cox-2 expression , and gastric cancer cell proliferation . elevation of noradrenaline by nicotine via 7 nachr up - regulation leading to significantly enhanced growth and angiogenesis in both gastric cancer and colon cancer has also been demonstrated . various investigators have also shown increases in neurotransmitters lead to -ar activation , transactivation of egfr , and the release of egf [ 32 , 54 , 56 ] . indeed , our recent investigation provided compelling evidence that chronic nicotine exposure induced release of noradrenaline via 4/2 nachr activation followed by -ar transactivation . our study further demonstrated that blocking of -ar with antagonist reversed the nicotine - induced cellular proliferative and chemoresistance . al - wadei et al . first reported that nicotine contributes to the development of smoking - related pancreatic ductal adenocarcinoma ( pdac ) with elevated levels of stress neurotransmitters ( adrenaline and noradrenaline ) and induction of camp , pcreb , and perk1/2 , and inhibition of -aminobutyric acid ( gaba ) . gaba has been reported to possess tumor suppressor function suppressing both -ar stimulated pdac growth and migration in vitro . however , while gaba is suppressed in pdacs , noradrenaline , pka , p - creb , and perk1/2 in these tissues are overexpressed . these authors suggested that nicotine and nnk may contribute to the development of pdac in smokers by suppression of gaba with induction of stress neurotransmitters . further proposed that nicotine induces the release of stress neurotransmitters through activation of 7 nachr and inhibits release of gaba via inhibition of 4/2 nachr . it is now believed that the stress neurotransmitter released via nachr activation plays an important role in smoking - associated tumorigenesis . however , the precise mechanisms involved in the regulation and the function of neurotransmitter released by nicotine and nnk are still uncertain . future research on this area is encouraged . it has also been shown that nnk can promote -ar - mediated transactivation of egfr followed by erk1/2 phosphorylation leading to an increased proliferation in pancreatic cancer cells . nnk is also reported to induce endogenous igfr which is associated with the development of lung tumors . huang et al . also indicated that both activation of thromboxane a2 ( txa2 ) receptor and synthesis of txa2 play critical roles in nnk - promoted lung cancer cell proliferation . txa2 activates the transcriptional factor creb through both erk and pi3k / akt pathways , which may also lead to pcna and bcl-2 overexpressions and cell proliferation . these studies provide valuable information on the mechanisms which involve in proliferative signaling stimulated by nicotine and nnk through activation of nachr , -ar and other growth factor receptors in cancer cells . triggering such receptors by cigarette smoke would further lead to rapid cell proliferation , cellular migration , invasion , and metastasis . in short , these investigations on the nachr , and nachr transactivated with other receptors represent the pivotal role in regulating multiple cellular cascades in general cell functions and in carcinogenesis . nicotine is also known to influence signal transducers and activators of transcription 3 ( stat 3 ) which is an important signal transducer mediating signaling by numerous cytokines , growth factors , and oncoproteins . findings from our own laboratories indicate that nicotine induces bladder cancer cells proliferation through 7 nachr , 42 nachr , and -ar followed by activation of erk1/2 and stat 3 . stat3 signaling further enhanced nf-b activation , cyclin d1 overexpression , and cell cycle progression . moreover , we also revealed that prolonged stimulation by nicotine upregulated 4/2 nachr and -ar followed with activation of stat 3 leading to significant increase in chemoresistance in cells from bladder cancers . in recent years the biological effects of pahs are mainly mediated via aryl hydrocarbone receptor ( ahr ) . through ahr , other pahs , such as benz(a)anthracene ( baa ) , has also been found to increase dna synthesis and promote g1-s progression in serum deprived mcf-7 cells . bap has been shown to increase incidence of tumors in estrogen - responsive rodents , suggesting that it may also affect er - mediated signaling . some investigators believed that the estrogenic property of pahs may be responsible for the induction of cell proliferation . bap and baa have been reported to act as estrogens that stimulate and initiate the er - mediated transcription and cell cycle progression and enhance er phosphorylation . on the other hand , there is also indication that the estradiol - dependent cell growth of mcf-7 cells can be inhibited by bap and baa [ 71 , 72 ] . thus , the actions of pahs on estrogen - dependent cell proliferation are still controversial . further studies are needed to elucidate more on the roles of pahs in carcinogenicity . in normal tissues , antigrowth signals can block proliferation by forcing the cell cycle progression into the quiescent ( g0 ) state . the cell cycle transition from g1 to s phase is the key regulatory step in the cell cycle and is mainly regulated by cdk4/6-cyclin d and cdk2-cyclin e complexes . nicotine has been reported to induce binding of raf-1 to rb with activation of cyclins and cdks as well as inactivation of rb . via activations of nachr and -ar , nicotine and nnk both exhibit mitogenic properties by inducing cyclin d1 overexpression leading to g1/s transition and increasing cell cycle progression [ 49 , 75 , 76 ] . nnk can also stimulate normal human lung epithelial cells proliferation through nf-b and cyclin d1 upregulation in an erk1/2-dependent pathway . in our own laboratory , we have also demonstrated that nicotine - induced cyclin d1 overexpression is regulated via stat3 , erk1/2 , and nf-b - dependent pathways in bladder cancer cells . other study also shows that pi3k / akt - dependent cellular proliferation is also enhanced in response to nnk . the pi3k / akt pathway is critical in cancer cells because it influences tumorigenesis , tumor growth , and therapeutic resistance . the pi3k / akt activation is documented in both nnk - treated a / j mice and in human lung cancers from smokers . it also plays a role in nnk - induced cell transformation , proliferation , and metastasis . it has been suggested that akt and nf-b may serve as key targets for nicotine or nnk stimulation in the development of lung cancer . west et al . also reported that beas2b cells treated with nnk for eight - week period increased cellular proliferation through activation of pi3k / akt pathways . however , pi3k / akt activation does not always occur in all cancer cells induced by nicotine . our previous study indicates that nicotine induced bladder cancer cell proliferation through stat3 and erk1/2 signalings instead of via akt pathway . all these investigations suggest that nicotine or nnk can activate erk1/2 , stat3 , or akt signaling to interrupt the antigrowth signals leading to enhanced cell cycle progression and cancer promotion . it is important to remember that cigarette smoke components other than nicotine or nnk may also impede on antigrowth mechanisms enhancing cancer development and promotion . apoptosis plays an important role in controlling normal development , homeostasis , and immune defense via elimination of redundant or abnormal cells in the organism . failure in cell elimination ( reduction of apoptosis ) may lead to undesirable cell survival and unchecked cell growths . resistance to apoptosis is often seen in cancers where cancer cells tend to lose their proapoptotic potentials because of gene mutations . nicotine has been shown to inhibit apoptosis induced by tumor necrosis factor ( tnf ) , by ultraviolet ( uv ) , radiation , or by chemotherapeutic drugs such as cisplatin , vinblastine , paclitaxel , and doxorubicin . this antiapoptotic action has been shown to be via pi3k / akt , raf / mekk / erk1/2 , nf-b , bcl-2 , bax , bad , or surviving [ 23 , 8082 ] . west et al . demonstrated inhibition of apoptosis and promotion of proliferation in human bronchial epithelium cells by nnk are induced via activation of 3/4 nachr followed by upregulation of akt , mitogen - activated protein kinase ( mapk ) , and pkc pathways . similar results are also observed by xu and coworkers showing that both akt and survivin pathways are involved in anticisplatin - induced apoptosis by nicotine . indeed , drug - induced enhancements of p53 and p21 expressions are shown to be suppressed by nicotine . our recent study also indicated that long - term nicotine treatment activated 4/2 nachr and -ar leading to reduction of apoptosis induced by cisplatin or paclitaxol . consistently , zhao et al . also reported that nicotine induced up - regulation of mcl-1 phosphorylation though erk1/2 via -ar activation with increased chemoresistance ( anti - apoptosis ) of human lung cancer cells . other investigators also indicate that nnk can prevent cell apoptosis by modulating the anti - apoptotic bcl-2 and c - myc proteins . it is found to be associated with cellular proliferation and is elevated during the developments of certain malignant tumors such as gastric and thyroid cancers [ 1113 ] . comparing the ho-1 in lung tissues of smokers and nonsmokers , li et al . noticed that the expression of ho-1 is significantly increased in both tumor and nontumor tissues of smokers . these studies further revealed that nnk or its metabolites probably induce oxidative stress in lung tissues with elevation on stimulates the expression of ho-1 . such event is through erk and nf-b activation and bad phosphorylation induction leading to eventual apoptosis inhibition [ 11 , 85 ] . cell proliferation and apoptosis can also be modulated by the peroxisome proliferator - activated receptors ( ppars ) . ppar/ is expressed in most tissues and has been reported to be associated with cancer growths , especially those in liver , colon , breast and lungs [ 8789 ] . sun et al . reported a novel mechanism that nicotine increases ppar/ expression through 7 nachr follow by pi3k / mtor activation leading to enhanced lung tumor cells proliferation . in contrast to ppar/ , activation of ppar by its ligands induces apoptosis and inhibits cell proliferation . thus , an intact ppar levels or its activation is needed to reduce cancer risk ( anti - apoptosis and cell proliferation ) . furthermore , a significant reduction in the transcriptional activity of ppar and its endogenous ligands , including 15-s - hydroxyeicosatertraenoic acid ( 15(s)-hete ) and 3-s - hydroxyocatadecadienoic acid ( 13(s)-hode ) , are found reduced in lung tissues of nnk - treated mice . indeed , lung tumors developed in these mice later . further suggested that the reduction of 15(s)-hete and 13(s)-hode may enable lung cells to be more resistant to apoptosis by nnk and facilitate tumor development in the animals . in contrast to nicotine or nnk , pahs induce either apoptosis or antiapoptosis in mammalian cells [ 94 , 95 ] . for instance , bap is known to induce signaling through igfr and increases cell survival through pi3k activation in human mammary epithelial cells . reported that both akt and erk1/2 act as anti - apoptosis signals leading to bad phosphorylation . however , bap can also induce apoptosis through p53 and p21 signaling in the same model . the results suggest that bap is capable in stimulating both apoptosis and anti - apoptosis signals . reported that light - irradiated bap ( lbap ) inhibited apoptosis through production of ros from degraded bap . this anti - apoptotic signal induced by bap in combination with dna damage would increase the possibility of cell survival and producing mutations . thus , while the apoptotic signal of bap induces cell death ( cytotoxicity ) , the anti - apoptotic signals of bap play an important role in cell proliferation and carcinogenesis . the anti - apoptosis mechanisms induced by components of cigarette smoke are obviously quite complex . it is evident that evading apoptosis plays a critical role in cigarette smoke - induced tumorigenesis and chemoresistance . new understandings on the molecular target regulating the apoptotic and anti - apoptosis machineries by cigarette smoke could provide novel strategies for drug development with substantial therapeutic benefits . when a cell population has progressed through a certain number of doublings ( replications ) , they would normally stop growing and enter into a process called senescence . tumor cells , however , appeared to have limitless replicative potentials ( immortalization ) during tumor progression . telomeres , which define the end segments of chromosomes , consist of short , tandemly repeated dna sequences ( ttaggg)n together with associated proteins . small amount of these end dna sequences may be lost during each cell cycle as a result of incomplete dna replication . however , de novo additions of ttaggg repeats by the enzyme telomerase may compensate for this loss . thus , telomerase plays an important role in the maintenance of the telomere ends in normal cells . ectopic expression of telomerase would immortalize the cells . by using human tissue samples , yim et al . reported that there are different distributions of the telomerase activity between smokers or ever - smokers and non - smoker . a strong correlation between telomerase activity and the number of packs years smoked can be established among these subjects indicating that there is an association between tobacco exposure and telomerase activity in the human bronchial epithelium . increased telomerase activity would extend the lifespan of cells and put these cells to be at higher risks for malignant transformation and carcinogenesis . similar finding is reported by targowski et al . that extensiveness of tobacco smoking correlated positively with increases in telomerase activity in tumor cells from patients with non small cell carcinoma of the lungs . all these studies point to the fact that enhancement of the telomerase activity by cigarette smoke certainly underlies the cancer promotion potentials of cigarette smoke . however , which components in cigarette smoke altered telomerase activity are still not known . indeed , there are indications that certain protein synthesis and mitochondria play central roles in neoplastic transformation . it is well known that mtor and map kinase signaling pathways modulate the phosphorylation of transcriptional factors , stability of mrnas , and protein synthesis . jin et al . reported that both nicotine and its metabolite nnk can induce survivin mrna expression through akt - mtor and mediated de novo synthesis of survivin protein in normal lung epithelial cell hbe cells . this induced survivin expression has been claimed to play a role in the malignant transformation of hbe cells by stimulating the survival pathways . cigarette smoke may damage respiratory chain function in mitochondria enhancing oxidative stress leading to mitochondria dysfunction [ 103 , 104 ] . it has also been reported that nicotine exposure resulted in reduced pancreatic mitochondrial enzyme activity , degranulation of beta cells , elevated islet oxidative stress , and impaired glucose stimulated insulin secretion in rats . continued exposure to ros and free radicals from such mitochondrial stress may lead to mitochondria dna ( mtdna ) mutation which may play an important role in carcinogenesis . analyzing clinical samples , . also showed that tumor cells with mtdna mutations grow faster then cells without mitochondrial mutation . hence , it is apparent that cigarette smoke would induce oxidative damage to the mtdna leading to more aggressive tumor growths . step 3 ( cancer progression)the malignancy of a tumor is usually evaluated by its ability in invasion and metastasis as well as in the associated angiogenesis . there are ample evidence which indicate that cigarette smoke participates in the processes of angiogenesis , invasion , and tumor metastasis . the malignancy of a tumor is usually evaluated by its ability in invasion and metastasis as well as in the associated angiogenesis . there are ample evidence which indicate that cigarette smoke participates in the processes of angiogenesis , invasion , and tumor metastasis . angiogenesis , the development of new blood vessels from endothelial cells ( ecs ) , is a critical event which allows the cancer cells to receive adequate nutrients and oxygen . angiogenesis involves mature vascular changes , including detachment of pericytes , degradation of extracellular matrix , endothelial cells remodeling , proliferation , migration , and formation of new endothelial cells into tubular structures . survival and proliferation of vascular endothelial cells are often stimulated by tumor - derived mitogens , and vice versa . tumor cells are known to activate angiogenesis by changing the balance of angiogenic inducers such as vegf ( vascular endothelial growth factor ) and bfgf ( basic fibroblast growth factor ) , and by countervailing inhibitors such as thrombospondin-1 . it has been shown that nicotine can induce angiogenesis both in vitro and in vivo and contributes to the growth of tumors [ 30 , 110 ] . similar to the fgf , nicotine is found to have the ability to promote migration , proliferation , tube formation and nitric oxide ( no ) production of endothelial cells . no is a well - known vasodilator and angiogenesis mediator , and nicotine has been reported to enhance the expression of endothelial nitric oxide synthetase and promote no release . nicotine is also found to induce expression of endothelial growth factors such as vegf , bfgf , pdgf , tgf- , and tgf- in endothelial cells and smooth muscle cells [ 112 , 113 ] . enhanced bfgf release and increases in metalloproteinase expression with degradation of ecm have been demonstrated with nicotine [ 114 , 115 ] . moreover , nicotine is found to induce secretion of prostacyclin which is a vasodilating molecule associated with endothelial cell proliferation , survival and migration . these effects are believed to be associated with cigarette smoke - induced hyperplasia of the intima in the blood vessels and other vascular wall lesions . 7 nachr is important in both physiological and pathological angiogenesis [ 110 , 118 ] . 7 nachr in endothelial cells needs to be sensitized or activated by hypoxia or ischemia in order to induce angiogenesis . indeed , specific antagonist of the 7 nachr ( -bungaratoxin ) is shown to inhibit nicotine - induced angiogenesis ( new vascular tube formation from endothelial cells ) [ 25 , 114 ] . interestingly enough , it is apparent that the akt pathway is found to be not involved in either angiogenesis or vegf release induced by nicotine . suggested that inhibition of erk1/2 , p38 mapk , and pi3k / akt can completely block and prevent endothelial tubule formation induced by nicotine - triggered 7 nachr activation . consistent with heeschen 's study , zhang and coworkers reported that nicotine apparently increases angiogenesis and invasion by activating pkc , pi3k / akt , erk1/2 , mtor , and src in human nsclc . excellent reviews on angiogenesis induced by nicotine were recently published [ 120 , 121 ] and will not be further discussed here . interaction between nachr and the growth factor - mediated angiogenesis occurs at signaling and transcription levels . nicotine - induced expression of vegf has been shown to be regulated by egfr transactivation and via the erk1/2 pathway in smooth muscle cells . phosphorylation of the vegf receptor kdr by nicotine activates vegf and increases its activity . additionally , nicotine can also upregulate the expression of vegf receptor vegfr2 during angiogenesis in certain cancer cells . recent study further indicated that nicotine can synergistically promote the proangiogenic effect of estradiol in nonsmall lung cancer . induction of angiogenesis in colon cancer by nicotine via -ar followed by arachidonic acid pathway has also been reported [ 32 , 125 ] . in sum , 7 nachr subtype has been linked to angiogenic process induced by nicotine leading to tumor vascularity , inflammation , and ischemia . nevertheless , whether nicotine or nnk acts specifically via nachr or -ar receptors or both or whether it is controlled in a cell - specific manner needs further study . other components present in cigarette smoke that may also contribute to angiogenesis remain to be identified . several excellent reviews on the roles of nicotine and nachr in angiogenesis exist [ 117 , 120 , 121 , 126 ] . the ability of invasion and metastasis allows cancer cells to escape from the primary tumor mass to new terrains in the body . the genetic and biochemical determinants as well as the molecular mechanisms involved are still poorly understood . many evidence indicate that cigarette smoking not only increases proliferation of cancer cells but also promotes metastasis . clinical and epidemiological studies suggest that smokers have more rapidly progressing tumors and cancer metastasis than non - smokers . these processes are now known to be dependent on cellular and stromal interactions and on extracellular matrix degradation . e - cadherin is a cell - to - cell interaction molecule expressed on epithelial cells . a loss of e - cadherin is seen in epithelial to mesenchymal transition ( emt ) , which is a major pathologic event in cancer metastasis . chronic treatment of nicotine downregulated the expression of ecm proteins such as e - cadherin and -catenin with concomitant increases of fibronectin and vimentin in lung cancer cells . wei et al . also indicated that nnk enhanced colon cancer cell migration with downregulation of e - cadherin . this author also found that the expressions of snail and zeb1 , 2 major transcription repressors of e - cadherin , were also induced by nnk in colon cancer cell cultures . its upregulation is significantly linked with tumor progression , metastasis and poor prognosis in lung cancer patients . it has been shown that nnk can upregulate contactin-1 via 7 nachr / erk activation and enhances invasiveness of lung cancer cells . breakdown of the extracellular matrix ( ecm ) through a family of enzyme called matrix metalloproteinases ( mmps ) is needed for tumor cells to invade adjacent tissue and to metastasize . reported that nicotine enhanced the invasiveness of esophageal squamous carcinoma cells ( te-13 ) by up - regulating the expressions and activity of mmp-2 , and cox-2 . nicotine is found to enhance the activity of mmp-2 , and mmp-9 as well as activation of plasminogen activators in a cox-2 and vegf - dependent manner . it serves as a good marker for pdac ( pancreatic ductal adenocarcinoma ) metastasis especially in cigarette smoking population . in a recent investigation , lazar et al . demonstrated that nicotine contributes to pdac metastasis through the induction of mmp-9 and vegf mediated by opn . pahs , including bap , are also found to play a role in the promotion of cancer metastasis . through augmented cox-2 expression and pge2 production via activated ahr pathway , bap induces breast cancer cell invasions . bap and pahs mixture has also been demonstrated to induce cancer cell invasions and metastasis through upregulating the expressions of mmps , proteinase - activated receptor-2 , fibronectin , migration stimulating factor , and bcl-2 protein in lung adenocarcinoma . the importance of fgf-9 and its up - regulation by bap in lung cancer invasion and metastasis has been proposed . indeed , recent study by ueng et al . demonstrated that bap increases the invasive potential of lung cancer cells in vitro . such process involves the up - regulation of fgf-9 mrna expression via the p38 and erk1/2 pathways . during metastasis , the cancer cells co - opt signals that control leukocyte trafficking and chemokines - mediated cell migration . among these chemokines , cxcr4 and its natural ligand cxcl12 serve as key mediators for tumor migration and metastasis . nicotine has been shown to increase the expressions of several cxc chemokines receptors such as cxcr2 , cxcr3 , and cxcr4 as well as ccl12 in sclc cells suggesting the nicotine would stimulate cancer cell migration and eventual metastasis . although epidemiology studies have long demonstrated the relationship between smoking and cancer metastasis , the molecular mechanisms of metastasis influenced by cigarette smoke or its components remain very limited . in this paper , we have reviewed the recent investigations concerning cigarette smoke and cancer development , promotion and progression . while chemicals with carcinogenic potentials in cigarette smoke are many ( over 62 ) , most research efforts have been devoted to three components of cigarette smoke : nicotine , nnk , and pahs . while pahs are common chemicals in the environment , nicotine and nnk are considered to be tobacco specific . these three important components of cigarette smoke , especially nicotine and nnk , therefore , are targeted as the major compounds of focus in this review . many previous reviews have devoted to the interrelationship between cigarette smoke and lung carcinogenesis or the genotoxicity of cigarette smoke or its components . in this review , we are focused on the mechanistic information on tumorigenesis , especially those involving epigenetic or non - gentoxic effects . aside from lung cancer , it is our hope that this review will summarize the vast information cumulated in the literature and provide valuable reference resource for those who are interested in tobacco - related carcinogenesis . the overall mechanisms on carcinogenesis cancer promotion and progression are complex involving many molecular targets which include receptors , cell cycle regulators , mitogen - activated protein kinases , apoptosis mediators , angiogenic factors , and invasion , and metastasis mediators . among the receptors , nachr , -ar , and ahr probably are the most important and have the closest association with cigarette smoke - induced carcinogenesis . overexpression or activation of these receptors may result in the release of neurotransmitters and growth factors that participate in apoptosis inhibition , cell proliferation , angiogenesis , cancer cell invasion and metastasis . it should be noted that the importance of nachr in cancer may be cell - type - dependent or specific and their sensitivity and expression can be also be modified by various environmental factors such as insecticide organophosphates . as shown in figure 1 , signaling pathways , pi3k / akt , stat3 , and erk1/2 play important roles in the carcinogenesis processes . they are also common paths affected by the cigarette smoke components , including nicotine , nnk , and pahs . in addition , pkc , akt , erk , and cox-2 signaling pathways are involved in both promotion and progression stages by cigarette smoke . it is suggested that these molecules could be utilized the potential targets for future developments in cancer diagnoses or therapies . avoidance of cigarette smoke remains to be the best way of prevention for cigarette - related cancer . however , in view that tobacco smoke is legalized and smokers are still abundant , understanding on the health impacts by tobacco smoking still constitutes important public health concern . understanding the disease process and the mechanisms involved is the first step to solution . the emerging understanding on the molecular mechanisms in the development and progression of caners induced by cigarette smoke provides novel inspirations and approaches for potential measures on early diagnosis , reduction in progression and metastasis , and therapy of cancers . many dietary supplements , foods , or herbal medicines might significantly attenuate the proliferative effects by cigarette smoke . , the natural compound pterostilbene could induce apoptosis and autophagy in chemoresistant bladder cancer cells derived from nicotine exposure . future research on natural compounds may help to provide additional novel chemopreventive or chemotherapeutic possibilities in reducing cancer risks or other health impacts of cigarette smoke . this review has also discussed the various molecular mechanisms and paths involved in carcinogenesis induced by cigarette smoke . however , there are still many mysteries in the carcinogenic process by cigarette smoke . , most research efforts were focused on the proliferative and antiapoptosis mechanisms induced by cigarette smoking . as tumors are the results of multiple and interactive genetic abnormalities , study of cancers induced by cigarette smoke should assess more than one or two acquired alterations or paths . explorations of other paths or mechanism other than those popular ones are needed . those molecular pathways which are significantly activated by cigarette smoke are probably the most important ones involved in cigarette smoke - induced tumorigenesis . these pathways include nachr signaling ( such as 7 nachr , 9 nachr , or 4/2 nachr ) , -ar signaling , pi3k / akt signaling , erk1/2 signaling , stat3 signaling , vegf , and mmps pathways , and so on . targeting to modulate these pathways via dietary factors or therapeutic drugs may reduce cigarette smoking induced tumorigenesis significantly . studies on the non - genotoxic ( epigenetic ) effects of cigarette smoke components are few and need more efforts . the epigenetic effects of cigarette component must be evaluated to include both upstream and downstream pathways . carcinogenesis is often species or cell - type specific and can be influenced by many factors or cofactors . the same factor which is highly oncogenic to certain cell type or individuals may not be oncogenic to others . moreover , some cell type may become susceptible to a carcinogen only in the presence of certain factor(s ) , co - factor(s ) , genetic predisposition , or immune depression . specific mechanism for carcinogenesis for the same carcinogen may also vary in different tissues . synergistic interaction between cigarette smoke components and other environmental toxicants or carcinogens , such as arsenic or dioxin , on cancer development has been demonstrated both epidemiologically and in animal studies [ 143145 ] . traditionally , most past investigations focused only on single compound or one cigarette smoke component . the synergistic interaction between otherwise safe level of environmental chemical and low level of cigarette smoke or its component ( via either active or secondhand smoking ) for carcinogenesis raised novel public health concerns and challenging questions . we have provided an overview on the major concepts and insights on the molecular mechanisms involved in cigarette smoke - induced cancers . it is hoped that these mechanistic insights can be translated into practical applications for the prevention and treatment of cigarette smoke - related cancers . we also hope that these suggestions will be helpful to those who are interested in this area of cancer research .

cigarette smoking is one of the major causes of carcinogenesis . direct genotoxicity induced by cigarette smoke leads to initiation of carcinogenesis . nongenotoxic ( epigenetic ) effects of cigarette smoke also act as modulators altering cellular functions . these two effects underlie the mechanisms of tumor promotion and progression . while there is no lack of general reviews on the genotoxic and carcinogenic potentials of cigarette smoke in lung carcinogenesis , updated review on the epigenetic effects and molecular mechanisms of cigarette smoke and carcinogenesis , not limited to lung , is lacking . we are presenting a comprehensive review of recent investigations on cigarette smoke , with special attentions to nicotine , nnk , and pahs . the current understanding on their molecular mechanisms include ( 1 ) receptors , ( 2 ) cell cycle regulators , ( 3 ) signaling pathways , ( 4 ) apoptosis mediators , ( 5 ) angiogenic factors , and ( 6 ) invasive and metastasis mediators . this review highlighted the complexity biological responses to cigarette smoke components and their involvements in tumorigenesis .

many benign tumors can affect the orbit and , if symptomatic or cosmetically disfiguring , most can be removed via various cutaneous and bony approaches . many of these approaches involve removal of the lateral orbital wall , with replacement after tumor excision [ 17 ] . this approach can produce excellent operative exposure of the lesion and allow for removal with minimal manipulation of the orbital contents . with interest in minimizing surgical risk , postoperative recovery and scarring , recent surgical trends favor minimally invasive techniques and local anesthesia where possible [ 1 , 2 ] . traditionally , orbital tumors located in the middle to posterior orbit are approached from a variety of soft - tissue incisions incorporating a bony lateral orbitotomy ( marginotomy ) of the zygoma [ 17 ] . the bony flap increases exposure of the deep orbit and provides the surgeon with added maneuverability for tumor removal [ 6 , 7 ] . however , the bony marginotomy can be associated with longer operative time , increased postoperative pain , recovery time , temporalis muscle wasting , and external scar . using modern imaging modalities in conjunction with minimal incision surgical techniques , we have found that bony marginotomy is rarely needed in order to access presumed benign tumors of the middle and deep orbit . in this study , we present our surgical experience in the removal of orbital tumors using a combination of soft - tissue approaches , without bony marginotomy , under monitored local anesthesia and general anesthesia . in this retrospective case series , electronic medical records of all patients with orbital tumors removed through the oculoplastics clinic of the jules stein eye institute between 1992 and 2013 were reviewed . the institutional review board at the university of california , los angeles approved the study protocol , and the tenets of the declaration of helsinki were followed . data including snellen visual acuity , ocular motility , tumor size and type , follow - up duration , recurrence , and intra- and postoperative complications were recorded . imaging was used to classify tumor location as posterior or anterior based on confinement to the anterior half of the orbit or extension into the posterior half , respectively . surgeries were performed under general anesthesia . however , if the case was amenable to completion under local anesthesia , an array of orbital and regional nerve blocks was utilized . after adequate anesthesia is achieved , the appropriate incision is chosen based on location of the tumor within the orbit . tumors inferior to the optic nerve are generally approached with a conjunctival incision 4 mm below the inferior tarsal margin through the lower eyelid retractors and into orbital fat ( figures 1 and 2 ) . the incision can be extended medially or laterally as necessary . when extending the incision laterally , the lateral canthal tendon can be loosened slightly by blunt spreading with scissors , avoiding complete release from the orbital rim . for tumors located superiorly , an incision is made through the upper eyelid crease 810 mm above the lid margin . the incisions were customized medially or laterally based on the location of tumor and the site needs to be exposed . an incision through the septum with medial or lateral displacement of the levator fibers allows blunt dissection through the intermuscular septum . the levator can be safely distracted medially or laterally and we have not found a vertical lid splitting procedure necessary to access the superior orbit . working towards the anticipated 3-dimensional location of the tumor predicted by imaging studies , stevens scissors and peanut or cotton tipped applicators can be used to dissect fatty soft tissue off the face of the tumor . once the face of the tumor is within view , the surgeon can assess the gross surgical pathology and confirm the diagnosis of a benign lesion . a half - circle needle can now be passed through the face of the tumor if appropriate , which if compressible will cause partial exsanguination and a decrease in tumor size . an additional pass of the needle is utilized to create a whip suture that will provide better control and forward traction . in the case of cavernous malformations , this stitch also aids in exsanguination of the lesion , decreasing its overall size for delivery anteriorly . some tumors such as dermoid cysts or schwannomas can be additionally decompressed by suctioning the contents through a small anterior incision into the tumor . with the tumor partly exsanguinated or decompressed ( if possible ) and suture in place , blunt dissection ( e.g. , with a freer elevator ) can now be carried back toward the posterior edge of the tumor with simultaneous forward traction . as the attachments are dissected , the tumor will move towards the surgical opening , revealing posterior attachments , which can be gently lysed with blunt and sharp dissection ( figure 3 ) . there are usually no major vascular stalks associated with cavernous hemangiomas , but any bleeding vessels are controlled with bipolar cautery ( figure 4 ) . small conjunctival or eyelid crease incisions allow a relatively quick recovery with minimal ecchymosis and minimal visible scar ( figure 5 ) . median age was 48.5 ( range 3187 ) years old and median follow - up was 24.5 ( range 4375 ) weeks ( table 1 ) . the average standard deviation tumor size , measured in its longest dimension based on preoperative imaging , was 22.6 6.5 mm ( range 1051 ) . twenty - nine ( out of 30 ) tumors were located in the posterior half of the orbit . surgeries were performed through eyelid crease ( 17 eyes ) , conjunctival ( 9 eyes ) , lateral canthal ( 2 eyes ) , and transcaruncular ( 2eyes ) approaches . six cases were performed with monitored anesthesia care and local block , and 24 were performed under general anesthesia . two patients ( # 16&29 ) had mild decrease in visual acuity at the last follow - up , which was related to ocular surface changes . confirmed surgical pathology revealed several tumor types including 15 cavernous hemangiomas , 5 pleomorphic adenomas , 4 solitary fibrous tumors , 2 neurofibro- mas , 2 schwannomas , and 1 orbital varix . middle to deep orbital tumors are most commonly removed through a bony lateral marginotomy [ 17 ] . this technique was first described by kronlein in 1889 for the removal of dermoid cysts , providing a relatively wide field of view to search for retrobulbar tumors . with the advent of modern imaging modalities and ability to distinguish tumor character and location preoperatively , other less invasive soft - tissue approaches have been described . all documented series have reported surgical excision under general anesthesia and typically involve a lateral canthotomy or rectus resection for exposure . both kiratli et al . and cheng et al . presented series of intraconal orbital cavernous hemangiomas removed via transconjunctival approach [ 10 , 11 ] . their series were limited to tumors touching or near the globe and necessitated removal of the medial rectus for larger tumors . another 2004 paper by yan and wu presented results of removing 139 ( out of 214 ) orbital cavernomas through an anterior orbitotomy . the remaining 75 tumors ( out of 214 ) were removed successfully by standard lateral orbitotomy with bony marginotomy . the authors stated that traditional lateral orbitotomy must be used in cases where tumor dimensions exceed 3 cm , or the tumor extends to the orbital apex , or has imaging inconsistent with cavernous hemangioma . in another series limited to cavernous hemangiomas , pelton and patel excised 5 medial intraconal cavernomas through a superomedial eyelid crease . the authors concluded that the superomedial lid crease approach to the medial intraconal space has a number of advantages over the medial transconjunctival and lateral orbital approaches , including ease of dissection , incision - to - nerve distance , and angle of approach to the optic nerve . with appropriate screening and intraoperative flexibility , we believe the most benign intraconal tumors can be removed through one of many soft - tissue approaches , often under local anesthesia . if the lesion is compressible , for example , cavernous hemangioma or dermoid cyst , large size is not a contraindication to a small incision approach . . the anticipated pathology should be consistent with a benign tumor . additionally ct or mri imaging modalities should demonstrate a well - defined mass , without tethering , infiltration into surrounding tissue or growth into bone or the sinuses . these features suggest that the tumor is likely benign in nature and distinct from the surrounding tissues making it possible to be completely resected . tumors located in the inferior orbit are best approached through an inferior conjunctival incision . most often a canthotomy is not required , but for more exposure , the canthus can be loosened with a small internal incision , leaving the skin and orbicularis intact . occasionally a deep medial orbital tumor adjacent to the periosteum may be more easily accessed using a caruncular incision . superiorly positioned orbital tumors are removed through an upper eyelid crease incision with appropriate medial or lateral orbital entry ( working around the levator aponeurosis ) . most benign tumors of the deep orbit can be safely reached through careful dissection with any of these incisional approaches , although tumors at the deepest apex , for example , those extending through the superior orbital fissure , generally require bone removal for optimal exposure . this is achieved through careful examination of fine cut orbital ct or mri images preoperatively in combination with adequate lighting and the use of intraoperative landmarks such as the bony rim , globe , and rectus muscles . using this spatial knowledge along with palpation and ballottement of the tumor during dissection allows the surgeon to accurately focus the blunt and sharp dissection through the relatively small opening . once the dissection has reached the face of the tumor , the gross pathologic appearance should be assessed . if visual inspection and palpation suggest an infiltrative process , the surgeon must be flexible enough to change the operative plan , for example , to biopsy the lesion or convert to a larger access procedure under general anesthesia . none of our cases necessitated conversion from monitored anesthesia care to general anesthesia or bone removal . the next critical point is the pass of the half - circle needle through the face of the tumor . in the case of a cavernous venous malformation ( cavernous hemangioma ) , the resulting exsanguination of the tumor , which can be easily suctioned , shrinks the tumor in size and allows more space to maneuver . additional passes of the needle create a whip suture , allowing forward traction for adequate dissection and expression of the tumor through the small soft - tissue incision . local anesthesia is preferred by many patients as it shortens the operative time , avoids the discomfort and risks of general anesthesia , and allows the patient to return home sooner . we have found that benign orbital tumors can be removed under monitored local anesthesia in some patients . patients who are overly anxious will do better under general anesthesia although moderate anxiety will respond to common anxiolytic agents used for monitored anesthesia [ 6 , 7 ] . in our series , those who underwent monitored anesthesia did well with common anxiolytic agents . intraoperatively , the orbit should become adequately anesthetized superficially and deep with blocks of the zygomaticotemporal , zygomaticofacial , infraorbital , and supraorbital nerves as well as along the orbital floor back to the level of the superior orbital fissure . it is important to maintain communication with the patient and provide more local anesthetic when necessary . more local anesthetic is often necessary in the central and medial orbit when dissecting down to the orbital rim , as there are many sensory nerves in this region . the retrobulbar block may dampen or eliminate the physiologic pupillary reaction of the ipsilateral eye . this should not affect operative decision - making : the surgeon always uses maximal care to minimize traction and pressure on nerves in the orbit . pupillary dilation is more often related to the efferent nerves to the pupil and is not a reliable sign of optic nerve compression . optic nerve function can be better assessed , if needed , by looking for a reverse afferent papillary defect in the opposite pupil . this includes a detailed history , physical exam , and careful study of appropriate imaging modalities . combining this data with a thoughtful orbital differential diagnosis , appreciation for nuances of intraoperative gross surgical pathology , and readiness to convert to an open procedure if necessary allows the surgeon to safely approach most orbital tumors with the techniques described above . the desire to perform minimally invasive surgery should not be pursued to the extent that adequate exposure or patient safety is compromised in any way . however , within the constraints of good judgment and safety , it is appropriate to try to minimize the invasiveness of orbital surgery . in our experience , with appropriate preoperative evaluation and a creative flexible surgical approach , many benign orbital tumors can be safely approached through a minimally invasive soft - tissue approach , avoiding a bony marginotomy .

to present our experience of removing middle to deep orbital tumors using a combination of minimally invasive soft tissue approaches , sometimes under local anesthesia . methods . in this retrospective case series , 30 patients ( 13 males and 17 females ) underwent tumor removal through eyelid crease ( 17 eyes ) , conjunctival ( nine eyes ) , lateral canthal ( two eyes ) , and transcaruncular ( two eyes ) approaches . all tumors were located in the posterior half of the orbit . six cases were removed under monitored anesthesia care with local block , and 24 were under general anesthesia . results . the median ( range ) age and follow - up duration were 48.5 ( 3187 ) years old and 24.5 ( 4375 ) weeks , respectively . visual acuity and ocular motility showed improvement or no significant change in all but one patient at the latest followup . confirmed pathologies revealed cavernous hemangioma ( 15 cases ) , pleomorphic adenoma ( 5 cases ) , solitary fibrous tumor ( 4 cases ) , neurofibroma ( 2 cases ) , schwannoma ( 2 cases ) , and orbital varix ( 1 case ) . none of the patients experienced recurrence . conclusions . creating a bony marginotomy increases intraoperative exposure of the deep orbit but adds substantial time and morbidity . benign orbital tumors can often be removed safely through small soft - tissue incisions , without bone removal and under local anesthesia .