The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (ΔIami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of γENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a γENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in γENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.
23 Related JoVE Articles!
Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels
Institutions: AT Still University of Health Sciences.
Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.
Neuroscience, Issue 82, DiI, patch clamp, sensory neurons, muscle afferent neurons, immunocytochemistry, electrophysiology
The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry
Institutions: Washington University in St. Louis.
The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV
1.5), expressed in Xenopus
oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV
1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1
. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.
Developmental Biology, Issue 85, Voltage clamp, Cut-open, Oocyte, Voltage Clamp Fluorometry, Sodium Channels, Ionic Currents, Xenopus laevis
Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples
Institutions: University of Florence, University of Florence.
Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.
Medicine, Issue 86, cardiology, cardiac cells, electrophysiology, excitation-contraction coupling, action potential, calcium, myocardium, hypertrophic cardiomyopathy, cardiac patients, cardiac disease
The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics
), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP
in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
Institutions: Salk Institute for Biological Studies.
We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro
. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.
Cellular Biology, Issue 44, Ion channels, electrophysiology, patch-clamping, site-directed mutagenesis, biophysics, neuroscience
One-channel Cell-attached Patch-clamp Recording
Institutions: University at Buffalo, SUNY, University at Buffalo, SUNY, The Scripps Research Institute, University at Buffalo, SUNY.
Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel’s conductance properties and the temporal sequence of open-closed conformations that make up the channel’s activation mechanism, thus helping to understand their functions in health and disease.
Neuroscience, Issue 88, biophysics, ion channels, single-channel recording, NMDA receptors, gating, electrophysiology, patch-clamp, kinetic analysis
The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33
. To help improve this understanding, proton magnetic resonance spectroscopy (1
H-MRS) can be used as it allows the in vivo
quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41
. In fact, a recent study demonstrated that 1
H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34
. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1
H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31
. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae
Institutions: VU University Amsterdam.
In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo
, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871
, who showed that it was possible to reconstitute these complexes in vitro
starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro
reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g.,
pigment binding sites) or protein domain (e.g.,
protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro
currently used in our laboratory,
and examples describing applications of the method are provided.
Biochemistry, Issue 92, Reconstitution, Photosynthesis, Chlorophyll, Carotenoids, Light Harvesting Protein, Chlamydomonas reinhardtii, Arabidopsis thaliana
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Institutions: KU Leuven.
Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+
-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3
that initiate the propagation of the Ca2+
-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+
-wave propagation are provided by gap junction channels through the direct transfer of IP3
and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+
-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+
-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+
-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.
Cellular Biology, Issue 77, Molecular Biology, Medicine, Biomedical Engineering, Biophysics, Immunology, Ophthalmology, Gap Junctions, Connexins, Connexin 43, Calcium Signaling, Ca2+, Cell Communication, Paracrine Communication, Intercellular communication, calcium wave propagation, gap junctions, hemichannels, endothelial cells, cell signaling, cell, isolation, cell culture
Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells
Institutions: University of Waterloo.
The in vitro
expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28°C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28°C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37°C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28°C for further incubation. We find that the initial incubation at 28°C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.
Neuroscience, Issue 47, brain, invertebrate, calcium channel, electrophysiology, voltage-gated
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+
on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes
Institutions: University of California, Davis.
In T lymphocytes, depletion of Ca2+
from the intracellular Ca2+
store leads to activation of plasmalemmal Ca2+
channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2
. Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3
, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+
currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8
Immunology, Issue 46, human T lymphocytes, CRAC channels, CRAC currents, patch-clamp
Recapitulation of an Ion Channel IV Curve Using Frequency Components
Institutions: University of Utah.
INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously.
METHODS: Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages.
RESULTS AND CONCLUSIONS: In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|<0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.
Biophysics, Issue 48, Ion channel, Kir2.1, impedance spectroscopy, frequency response, voltage clamp, electrophysiology
Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling
Institutions: Worcester Polytechnic Institute.
Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2
, ion pumps3
, and transporters4
. Recent developments have combined site-specific fluorophore labeling alongside TEVC to
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6
. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis
oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5
. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4
. Finally, recent developments
have also enabled the manipulation of select internal ions following co-expression with a second protein9
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (<5%)3
upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of
the membrane protein in order to correlate the conformational dynamics to the function of the protein10
. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.
Cellular Biology, Issue 51, membrane protein, two electrode voltage-clamp, biophysics, site-specific fluorophore labeling, microscopy, conformational dynamics
Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices
Institutions: Purdue University.
Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis
oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as α4 L9′A mice 1
and α6 L9′S mice 2
, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.
Neuroscience, Issue 68, Nicotinic, acetylcholine, neurotransmitter, neuron, patch clamp, brain slice, picospritzer
Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes
Institutions: Charité Medical Faculty and Max-Delbrück Center for Molecular Medicine (MDC), Charité - Universitätsmedizin Berlin, Charité - Universitätsmedizin Berlin.
KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments
. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+
in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+
buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.
Physiology, Issue 73, Medicine, Cellular Biology, Molecular Biology, Genetics, Biomedical Engineering, Anatomy, Cardiology, Cardiac Output, Low, Cardiomyopathies, Heart Failure, Arrhythmias, Cardiac, Ventricular Dysfunction, Cardiomyocytes, Kv channel, cardiac arrythmia, electrophysiology, patch clamp, mouse, animal model
Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica
Institutions: Case Western Reserve University , Case Western Reserve University , Case Western Reserve University .
In animals with large identified neurons (e.g.
mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4
. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5
. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5
, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia
) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7
. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g.
in the suspended buccal mass preparation8
or in vivo9
. This process can also be applied in other motor pools10,11,12
or in other animal systems2,3,13,14
Neuroscience, Issue 73, Physiology, Biomedical Engineering, Anatomy, Behavior, Neurobiology, Animal, Neurosciences, Neurophysiology, Electrophysiology, Aplysia, Aplysia californica, California sea slug, invertebrate, feeding, buccal mass, ganglia, motor neurons, neurons, extracellular stimulation and recordings, extracellular electrodes, animal model
Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
Institutions: Technical University of Berlin, Oregon Health & Science University.
Whereas cation transport by the electrogenic membrane transporter Na+
-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+
-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+
transport by Na+
- or gastric H+
-ATPase in single cells. Using Xenopus
oocytes as expression system, we determine the amount of Rb+
) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+
uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g.
to quantitatively determine the 3Na+
transport stoichiometry of the Na+
-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+
-ATPase. In principle, the assay is not limited to K+
-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.
Biochemistry, Issue 72, Chemistry, Biophysics, Bioengineering, Physiology, Molecular Biology, electrochemical processes, physical chemistry, spectrophotometry (application), spectroscopic chemical analysis (application), life sciences, temperature effects (biological, animal and plant), Life Sciences (General), Na+,K+-ATPase, H+,K+-ATPase, Cation Uptake, P-type ATPases, Atomic Absorption Spectrophotometry (AAS), Two-Electrode Voltage-Clamp, Xenopus Oocytes, Rb+ Flux, Transversely Heated Graphite Atomizer (THGA) Furnace, electrophysiology, animal model
Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies
Institutions: University of Texas Southwestern Medical Center at Dallas.
To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.
Molecular Biology, Issue 77, Biochemistry, Genetics, Cellular Biology, Structural Biology, Biophysics, Membrane Lipids, Phospholipids, Carrier Proteins, Membrane Proteins, Micelles, Molecular Motor Proteins, life sciences, biochemistry, Amino Acids, Peptides, and Proteins, lipid-protein interaction, channel reconstitution, lipid-dependent gating, voltage-gated ion channel, conformation-specific ligands, lipids
Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Institutions: University College London.
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA
Rs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials.
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAA
Rs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other.
To elucidate the underlying molecular mechanisms, a novel in vitro
co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAA
R subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAA
R subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro
model system can be used to reproduce, at least in part, the in vivo
conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAA
Rs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.
Neuroscience, Issue 93, Developmental neuroscience, synaptogenesis, synaptic inhibition, co-culture, stable cell lines, GABAergic, medium spiny neurons, HEK 293 cell line


Research Scientist I - EX (CSL)
The Ben Towne Center for Childhood Cancer Research (BTCCCR) at Seattle Children's Research Institute (SCRI) seeks to develop innovative new cellular therapies and translate these advances to groundbreaking clinical trials for children with the most aggressive forms of cancer. The Center's long-term mission is to eventually replace cancer therapies such as radiation and chemotherapy with "smart" therapies capable of eliminating cancer with precision, leaving the body unharmed.
This is an opportunity for a Research Scientist to join the Correlative Studies Laboratory, focused on elucidating the T cell intrinsic factors that determine the success of immunotherapy. The successful candidate will process incoming clinical samples and perform flow cytometry and other assays. Responsibilities will include operating standardized scientific procedures and techniques in a highly regulated environment (GLP compliant) for clinical research applications; contributing to the planning, design, execution and interpretation of process optimization experiments (process development and/or assay development); making detailed observations, recording data, as well as assessing the processes and results for reliability/efficacy/compliance. The candidate should have excellent oral, writing and organizational skills, careful attention to detail, integrity, credibility and a positive attitude in daily performance.
Required Education/Experience: - Bachelor's Degree in an engineering or scientific discipline . - Prior experience in a laboratory setting; may include relevant educational programs. Preferred: - Significant experience conducting research in area of specialization. - Some experience with laboratory management.
- This posting is for a Research Scientist I or Research Scientist II; final position will be based on the final candidate's qualifications.*
Meet Some of Seattle Children's's Employees
Clinical Research Associate
Shannon works with sites across the nation that conduct cystic fibrosis trials to ensure that they have everything they need to perform their life-saving clinical research successfully.
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Dr. Marika Stone was originally from Alaska, where she grew up and attended high school. While in Ketchikan, Alaska, she and her dentist discovered they both liked tying flies for fly fishing. Her dentist suggested she pursue a career in dentistry, as she had an eye for detail. She attended dental school at the University of Minnesota: School of Dentistry, where she was awarded her Doctor of Dental Surgery degree. She went on to complete a hospital-based residency in San Antonio, Texas.
After completion of her residency, she moved to Bend, Oregon, and purchased Mill Point Dental Center in 2008. She owned Mill Point Dental Center for over 10 years, during which time she was a very active member of the local community. She became a name in the local cycling community, as she had a great talent for endurance. She developed relationships with her patients that were meaningful and long-lasting. As a dentist, she pursued education and knowledge to further her ability to provide the highest quality of care possible to those who sat in her chair.
On December 30, 2017, she was tragically killed by an impaired motorist while out riding her bicycle in Bend, Oregon. She left behind a family of two children, Kiptyn and Kinley; her husband, Jerry; and several relatives and family members.
As Dr. Stone made going to the dentist easier for her patients, this page is a memorial to her legacy. As the Mill Point Dental team moves forward with Dr. Chelsea Longlet and Dr. Michael Longlet, we will keep Dr. Stone’s legacy near and dear to our hearts.


The 2011 Cancer Diagnostics Market: New Product Development Opportunities, Market Penetration Strategies, Entry Barriers and Risks16 Feb 2011 • by Natalie Aster
New York - The report “The 2011 Cancer Diagnostics Market: New Product Development Opportunities, Market Penetration Strategies, Entry Barriers and Risks” by Venture Planning Group identifies major opportunities emerging in the cancer diagnostics market during the next ten years; proposes “ideal” product models with tentative prices and operating characteristics; and suggests alternative business expansion strategies for suppliers.
Published: October 2010
Price: US$ 850.00
Report Sample Abstract
Matritech's serum based assay reacts with PC-1. Matritech was granted an exclusive worldwide license to prostate cancer-specific nuclear matrix proteins, including PC-1 developed by Johns Hopkins. The specificity of this antibody for cancerous nuclei, as well as its potential ability to identify pre-cancerous conditions on tissue biopsy, may offer an advantage over existing prostate cancer diagnostic products. The company researcher found six nuclear matrix proteins which differ in healthy and cancerous colon cells. When these six proteins are present in the nuclear matrix, it is highly likely that the cell is cancerous. Matrix proteins appear to initiate the process by which genes are activated. In cancer cells, the proteins may physically stabilize DNA and trigger cancer.
Researchers at Columbia-Presbyterian Medical Center (New York, NY) have developed PCR-based amplification/hybridization assay to detect PSA mRNA. The test is sensitive to detect one cancer cell in a million blood cells. Researchers at the College of Physicians and Surgeons of Columbia University (New York, NY) have detected the PTI-1 gene in samples of The BRCA syndrome is typified by mutations of BRCA1 gene which codes for a protein expressed in different tissues.
Roche Elecsys Series
The Elecsys 1010 immunoassay system is designed to process small volumes, while the Elecsys 2010 is designed for larger laboratories. For greater testing capacity, two Elecsys analyzers with sample rack modules can be linked with a conveyor system and an LSM. After the bar codes on the sample containers are read, the samples are automatically directed to an analyzer by the LSM, which controls the rational distribution of samples to the analyzers, and checks the analyzer systems and components. The test menu includes third generation TSH, Free T4, Free T3, T4, T3, TUptake (18 minute incubation time) and STAT (9 minute incubation time) Troponin T, CKMB and HCG. By the end of 1997, the company expects to add LH, FSH, Estradiol, Prolactin, Testosterone, Progesterone, CA-125, CA 19-9, CA 15-3, free PSA, Cyfra, NSE, CA 72-4, folic acid, B12 and ferritin. The Elecsys uses electro chemiluminescence detection and streptavidin-coated paramagnetic microparticles. The Elecsys 1010 is a stand-alone, sequential batch analyzer with STAT capability. It offers six on-board reagents, 66-sample capacity, and throughput of 45 tests per hour. The Elecsys 2010 is a random access analyzer. It holds 15 on-board reagents and offers nearly twice the throughput of the Elecsys 1010.
More information can be found in the report “The 2011 Cancer Diagnostics Market: New Product Development Opportunities, Market Penetration Strategies, Entry Barriers and Risks” by Venture Planning Group .
To order the report or ask for sample pages contact firstname.lastname@example.org


Would you like to get back in good shape fast and fast?
One of the better ways to shed weight is by using an excellent and natural garcinia cambogia on dr oz show.
Natural weight loss supplements use a come a long way and some of the best ones are now clinically proven to assist weight reduction even without diet or exercise.
Garcinia cambogia is one such fat loss supplement which includes taken the entire world from a big storm. Garcinia cambogia is actually a small fruit that looks such as a miniature pumpkin and might help lose weight with least amount of changes in your daily diet or exercise routine.
Garcinia cambogia contains hydroxycitric acid or HCA that is certainly successful in ensuring effortless weight-loss. It ensures a 3-fold effect within your body.
To begin with, hydroxycitric acid helps boost metabolism within your body. By improving your metabolic process, HCA encourages the body to get rid of accumulated body fat faster.
Secondly, it prevents further fat production inside your body. This is the most important benefit of forever garcinia plus. HCA inhibits enzyme ATP citrate lyase from converting sugar into fat inside your body. Thus, it acts as a powerful fat blocker.
Thirdly, it will help increase serotonin inside your body. This assists in curbing your appetite so that you are able to lessen the food intake. Diet control is really important to make certain weight loss.
Garcinia cambogia is really a powerful weight-loss agent which can help you lose fat even without diet or exercise. However, it is recommended to combine a great garcinia pill having a dieting and exercise to obtain faster fat loss results.
Garcinia pills are really popular and the best ones include other 100 % natural ingredients like raspberry ketones.
Raspberry ketone has been around in drogarcinia news for quite a while. It is a powerful fat burner that will burn fat even from the most stubborn areas within your body including your love handles etc.
Raspberry ketones aid in increasing a protein hormone in your body, called adiponectin. This hormone is inversely linked to fat in your body. It may help shrink fat cells in your body and allow you to lose fat faster.
Garcinia cambogia pills are successful. Combining it having a detox supplement, further enhances its fat reduction effects. By getting rid of toxins from your body, what are the side effects to garcinia cambogia will help increase your metabolism so your body may help you slim down faster.


Virtual Counseling Services
TeleHealth and Online Therapy
Imagine receiving counseling services from the comfort and privacy of your own home. Citrine Mental Health offers online therapy sessions which offer a convenient, easy way for clients to access counseling services care via a secure video-conferencing venue. Clients receive the same psychological care offered in our physical offices via secure, HIPAA-compliant video-conferencing with our highly qualified clinical team. The video-conferencing system we use is Doxy.me; a secure, HIPAA-compliant platform that is designed specifically for TeleHealth and privacy requirements. It is far more secure than standard video-conference platforms for general use. The only technology requirements a patient needs to access TeleHealth services are a computer or laptop, with a web camera and microphone, and reliable high-speed internet access.


Two liver diseases that affect dogs are chronic hepatitis and copper-associated hepatopathy. These illnesses impair the dog liver's ability to function properly and are life-threatening if not treated.
Always take your pet to a veterinarian if you suspect they are experiencing liver problems.
If pharmaceutical drugs such as steroids are necessary, ask your vet if your pet would benefit from natural treatments such as milk thistle or antioxidants to combat the harsh effects of NSAID and steroids.
Chronic Hepatitis in dogsChronic hepatitis is an inflammation of the liver caused by a virus, bacterial infection, kidney disease or another unknown source. Affected dogs develop a slowly progressive liver disease.
Diagnosis of Dogs with Chronic Hepatitis
Dogs suffering from chronic hepatitis experience vague and non-specific symptoms. These symptoms can include a loss of appetite, vomiting, weight loss, depression, lethargy and increased or decreased drinking and urination. As the disease worsens and the liver continues to fail, causing a build up of toxins in the body, jaundice, blood thinning, the accumulation of fluid, and neurological symptoms such as confusion or convulsions, become evident.Blood tests to check liver function are used to diagnose the disease, although a definite diagnosis can only be obtained through a liver biopsy.
Conventional Treatment for Canine Hepatitis
Traditional treatment for chronic hepatitis in dogs includes trying to remove the source of the problem, such as a toxin in the environment or a primary illness that triggered the hepatitis. Veterinarians also prescribe steroids as well as a low-protein diet to help the liver return to normal function.
With long term use, steroids can cause problems in dogs such as diabetis and immune suppression. Some owners turn to natural products to help support the liver in a safer way. These herbs aid the body in healing and in regenerating the damaged liver.
Natural remedies for chronic hepatitis in dogs include extracts of herbs such as milk thistle, dandelion and phyllanthus. Anecdotal evidence has shown that milk thisle and dandelion reduce inflammation of the liver and encourage new cell growth, while phyllanthus has anti-viral properties effective against hepatitis.
Copper-associated Hepatopathy in Dogs
Copper-associated hepatopathy is a condition caused by an excess of copper in the liver. Copper can enter the body through diets high in this mineral, or through copper in the
environment. Some breeds of dogs, dalmations and labradors seem more predisposed to develop this condition.
The symptoms of copper-associated hepatopathy include an unhealthy looking coat, depression, and frequent drinking and urinating. The problem is diagnosed through blood tests, which include toxicity testing to determine if there is an excess of copper in the blood stream.


Ayurvedic Solutions for Low Testosterone: Balancing Hormones the Natural Way
Ayurveda, an ancient Indian system of medicine, has been employed for centuries to address a wide array of health concerns. Rooted in the philosophy that the human body comprises mind, body, and spirit, Ayurveda focuses on harmonizing all three components to achieve optimal well-being.
Low testosterone, a common condition, can manifest in various symptoms like decreased sex drive, fatigue, mood swings, and even erectile dysfunction. Ayurveda offers an array of treatments for low testosterone, aiming to improve both symptoms and overall quality of life.
Prominent Ayurvedic Treatments for Low Testosterone
- Ashwagandha: This herb has been a cornerstone of Ayurveda for centuries, recognized for enhancing vitality and overall health. Ashwagandha is known to increase testosterone levels, thereby improving sexual function and fertility.
- Shilajit: Derived from the Himalayas, Shilajit is a mineral-rich substance. Studies have shown its capacity to boost testosterone levels and enhance sperm quality.
- Tribulus Terrestris: A renowned herb in Ayurveda for male sexual function, Tribulus terrestris can elevate testosterone levels and libido.
- Shatavari: Though primarily recognized for its benefits in female fertility and hormonal balance, Shatavari may also be beneficial for men with low testosterone.
Ayurveda Embraces Lifestyle Changes
In addition to herbal treatments, Ayurveda encourages various lifestyle changes to naturally elevate testosterone levels:
- Maintaining a Healthy Weight: Obesity is a significant risk factor for low testosterone. Shedding excess weight can improve testosterone levels and overall health.
- Eating a Balanced Diet: A diet rich in fruits, vegetables, and whole grains is essential for improving testosterone levels and overall health.
- Regular Exercise: Physical activity plays a vital role in sustaining a healthy weight and boosting testosterone levels.
- Adequate Sleep: Quality sleep is crucial for overall health and hormone production. Aim for 7-8 hours of restful sleep each night.
- Stress Management: Chronic stress can lower testosterone levels. Discover healthy strategies to manage stress, such as exercise, yoga, or meditation.
Before embracing Ayurvedic treatments for low testosterone, consulting with a healthcare professional is essential. Ayurveda is a holistic system of medicine, and it is crucial to ensure that Ayurvedic treatments are suitable for your specific needs and interact safely with any medications you may be taking.
Please be aware that the information in this blog post is for educational purposes and should not be considered as a substitute for professional medical advice. If you are experiencing symptoms of low testosterone, it is imperative to consult a healthcare provider for proper diagnosis and treatment.


Medicating with Kava is NOT Worth the Risk
Kava is a fairly well known and utilized herbal remedy for several disorders from anxiety to cancer prevention. The challenge with this herbal is that it also has a very strong tendency to wreak havoc in multiple systems throughout your body. In addition, there are a host of drug interactions that necessitate extreme caution and vigilance when in use. Conditions from chronic fatigue to Hypertension to Diabetes to high cholesterol can each be adversely affected when Kava and medication are in use simultaneously. This is one herbal that I strongly recommend that the average American NOT use. By the way, Kava has already been banned in a few countries abroad.
First, let’s review what Kava is and how people use it. Kava is a plant whose leaves are made into capsules and teas to treat anxiety and various related conditions.
Orally, it is used to calm the following:
Benzodiazepine Drug Withdrawal
What makes kava effective is the kava lactones, which affect the brain and other parts of the nervous system.
When Kava Hurts
Serious conditions including liver damage have occurred, even with short-term use at normal doses. Use of Kava for even just 1 to 3 months has caused the need for liver treatment and has even caused death.
Large amounts of Kava tea can lead people to drive erratically leading to traffic tickets issued under the category of “driving under the influence”.
In other instances, Kava may actually:
- Worsen depression
- Make Parkinson’s disease worse
- Intensify the effects of anesthesia and other meds used during surgery
Stop using Kava at least 2 weeks before scheduled surgery.
Pregnancy and breastfeeding
Kava may also have the following consequences for nursing mothers:
- May affect the uterus.
- Some chemicals in Kava can pass into breast milk and might hurt breast fed infants.
Drug interactions and Kava
Below is only a short list of drug interactions to keep in mind IF you are using Kava:
When taking Xanax, Klnopin, Lorazepam or donnatal, kava may cause excessive sleepiness.
Also, Kava can produce the following outcomes when taken with these drugs:
- Levadopa: Reduced effectiveness (Parkinson’s Disease )
- Medications that are detoxified by the liver: Elavil, Valium, Celebrex, Glucotrol, Dilantin, Coumadin-decreased liver processing
- Medications that harm the liver: Tylenol, Diflucan, Erythromycin, Dicantin and Stantins-become intensified.
Please note that this is nowhere near an exhaustive list of drug interactions with Kava or side effects. All in all, it is not worth the risk in our assessment to use this herbal element. There are several other natural remedies with far fewer toxic side effects ( i.e.-not likely to contribute to death ) that can be used for any of the conditions of which Kava has been tried.


Ogdensburg DeFelsko employees who dyed their hair pink donate $200 to Claxton-Hepburn radiology department
OGDENSBURG -- Employees from DeFelsko Corporation in Ogdensburg recently presented a $200 check to Debbie King, radiology manager at Claxton-Hepburn Medical Center.A few of the ladies from DeFelsko each paid $10 to have a section of their hair dyed pink in honor of Breast Cancer Month.
The ladies raised $100 and DeFelsko matched the donation.
The money will be used to spruce up the patient waiting area outside the breast biopsy room in the radiology department, which is part of the new comprehensive Breath Health Center at Claxton-Hepburn.
Shown in the photo are King and Cindy Carr from DeFelsko.
For more information about Claxton-Hepburn’s Breast Health Center call 1-888-908-2462


Antifertility and Antioxidant Activities of Ethanolic Extract of Leonotis nepetifolia in Male Albino Rats
B Kadalmani, R Niranjana, Uma A Natarajan, I Sarath Chandiran
Citation Information :
Kadalmani B, Niranjana R, Natarajan UA, Chandiran IS. Antifertility and Antioxidant Activities of Ethanolic Extract of Leonotis nepetifolia in Male Albino Rats. 2020; 3 (3):115-121.
Background and objective: This study based on the antifertility action of Leonotis nepetifolia ethanol extract (whole plant) in the animal model male Wistar Albino rats. Leonotis nepetifolia is reported to have antioxidant activity, antiproliferative potential, hypotensive potential, anti-inflammatory, antiplasmodial, antibacterial, antifungal, analgesic, anticancer, laxative and narcotic activities. The plant aids in the recovery of malaria, diarrhea, bronchial asthma, common cold, cough, and fever, particularly used during menstrual pain. Materials and methods: The animals were grouped into four with five rats each. Control group (group I) received normal saline. The other three groups of rats were treated with the specified dose (group II-100 mg/kg, group III-150 mg/kg, and group III-200 mg/kg) of Leonotis nepetifolia ethanolic extract for a period of 55 days. Results: A marked reduction in the weight of accessory sex organs/testis, sperm count, and sperm motility were observed in the treated groups. Serum hormonal levels and few biochemical parameters also showed variation compared to the control group. The reports revealed that the treated adult male rats had decreased the counts of female's impregnation. Conclusion: Hence, this current research concluded that L. nepetifolia ethanolic extract (whole plant) repressed sperm motility, concentration, and the testosterone proving dose-dependent antifertility effect.
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|Malignant mesothelioma is an asbestos cancer form that has an impact on the thin tissue layer that is there in the internal organs of the body http://www.maillotsdefootbelgique.be/maillot-jordan-lukaku.html , commonly known as mesothelium. The main reason responsible for malignant mesothelioma is asbestos exposure.Health care providers have divided malignant mesothelioma into various kinds on the basis of mesothelium area. It includes pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma and testicular mesothelioma.Pleural Mesothelioma: is the most common cancer form that has an impact on the lungs lining, commonly known as pleura.Peritoneal Mesothelioma: is the second most common cancer form that makes its presence felt in the abdomen lining, commonly known as peritoneum.Pericardial Mesothelioma: makes its presence felt in the membrane that is there around the heart, commonly known as pericardium.Testicular Mesothelioma: is the rarest cancer form that makes its presence felt in the membranous lining surrounding the testicles, commonly known as tunica vaginalis.Malignant Mesothelioma SymptomsPeople suffering from malignant mesothelioma normally do not show any symptoms until 15 to 30 years after asbestos exposure. This happens because of the long latency period that is associated with mesothelioma. The mesothelioma symptoms are quite general and often do not cause any serious condition, which make the whole diagnosis process lot tougher.You will find quite a bit of variation in the malignant mesothelioma symptoms on the basis of mesothelioma type. Some of the common symptoms associated with malignant mesothelioma are breath shortness, persistent cough and chest pain. People suffering from peritoneal mesothelioma may show symptoms like development of lumps, abdominal swelling and modifications in bowel movement. People suffering from pericardial mesothelioma may experience chest pain, fever and heart palpitations. People suffering from testicular mesothelioma may experience testicular lumps.Malignant Mesothelioma TreatmentsIrrespective of the kind of malignant mesothelioma you are suffering from, there are plenty of treatment options available. However, how you are going to react with different treatment options will depend entirely on your overall health condition. Your health care provider will set treatment on the basis of your disease stage and past medical record.To cure mesothelioma and get rid of cancer cells, health care providers normally employ surgery, radiation and chemotherapy. The main objective of radiation and chemotherapy treatments is to get rid of the cancerous cells and stop the growth of cell division. During treatment, main emphasis is on harmful cells but there is a strong possibility that it can also have an impact on the healthy cells. After treatment, you mat experience various side effects. Palliative treatments which give people suffering from malignant mesothelioma comfort can also be used by your health care provider.Malignant Mesothelioma CausesMesothelioma makes its presence felt when asbestos fibers are ingested and make a mark in the body organs. With the passage of time, fibers cause inflammation and scarring, which often results in malignant mesothelioma development. The asbestos fibers also have ability to damage cellular cells. Bad spots Scar tissue Cure
If you're intending to now have break-outs frighten treatment or maybe procedure, be sure to do not have any established acne breakouts. The physician or possibly dermatological doctor will not care for acne breakouts scar tissue for people with productive acne breakouts simply because it can make individual frustrated and can in fact trigger bulge.
Whiteheads Space Treatment plan
You need to comprehend of which immediately acne treatment just short-term correct, however it is not a fixed term answer. In fact, pimple require 3 weeks to break out and about, e . g natural look for a whole facial foundation cleaning method, not merely a short term pimples spot solution.
A great cleanse if you suffer from critical acne pimples is often a delicate remedy in salt water. Salt water definitely will fresh the fats not to mention earth off of your skin layer likewise a good cleaning solution may, but yet devoid of the disadvantageous dehydrating outcomes of a number of projects skin cleansers that will be suitable for acne remedy.
You need to use over-the-counter bad acne applications that will with each of your an acne breakout. These are generally mostly applicable and customarily can include well known acne breakout substances for example benzoyl hydrogen peroxide and salicylic plaque created by sugar. You've got to be cautious with one of these whilst and follow the instructions about how exactly considerably to employ and also how again and again to work with these people. In addition, these may always be as well extreme for a few, so when you need questions, you can ask a doctor as well as skin specialist.
Best Complexion Applications
Garlic has become the greatest acne treatments there may be. It is stuffed with herbal antioxidants to help ensure that your body looking great, in addition to beans even offers anti-inflammatory together with antibacterial added benefits. Only slash all of the clove amenable together with chafe it directly on the actual skin everywhere a pimples fast is certainly forming.
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In the event various at-home pimple remedies are unsuccessful, it is chance to schedulae an appoitment with a real doctor. This is certainly immensely important for all having sore cystic or even nodular complexion. Should agitated or cared for poorly, these could lead to scars as well as irreversible scar tissue. Your personal health-care professional will attempt to work together with you to check the regime custom-tailored for one's skins different needs.
Normal Acne Treatment
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As per studies and reports , plasma therapy may work only when obtained from recently recovered Covid-19 patient .
Donating plasma is similar to giving blood or platelets.Plasma regeneration varies in individuals .It takes about 48 hours on an average for it completely regenerate plasma after donation.There are no side effects or weakness after donating plasma.the donor can go back home after the procedure.
Plasma donation is a safe and comfortable experience, and hospitals are ensuring proper safety for donors .So if you have recently recovered from COVID-19 do come forward to donate your plasma as plasma therapy can help save a life
Here’s the database of organisations that can be contacted for both requirement and donation of plasma for plasma therapy
|KAB Welfare Foundation
|Team Step One (Dr.Monica Bhagat)
|Rotary Blood Bank (Gurgaon)
|COVID-19 Plasma Donor-Recipient Help Group India
|Panthak Saanjh Initiative
|Blood Donors India
|Team SOS India
|Give Red India
For more information and update on COVID 19 , please click here
Disclaimer: Kindly note that some of these numbers have been crowd-sourced from our community and a few from outside the community.


Reliability and validity of an objective structured clinical examination for assessing the clinical performance of residents.Arch Intern Med. 1990 Mar; 150(3):573-7.AI
Clinical performance of residents should be assessed as reliably and validly as possible. This study investigated the reliability and validity of an objective structured clinical examination (OSCE) for assessing clinical performance of internal medicine residents. Residents were required to take a 17-patient OSCE in their first and second year. Reliability of the OSCE was 0.40. Validity studies indicated second-year students were significantly better than third-year students for five of six OSCE skill scores; first-year students were significantly better for three scores. Resident's scores for diagnosis, plan, and total significantly increased on their second OSCE. Generally faculty overall ratings of residents' clinical performance did not correlate with OSCE scores. American Board of Internal Medicine certifying examination scores were consistently positively correlated only with diagnosis. This 17-case OSCE is a feasible method for obtaining moderately reliable, valid data not available from other sources about the clinical performance of residents. More cases should be added to increase its reliability.