Differences In Cell Size Research Articles

Abstract 1464: In vitro culturing of CTCs/DTCs in personalized cancer management.

Abstract Subsequent detection, quantification and molecular characterization of CTCs has an enormous potential in numerous of oncology areas. But detection and isolation of CTCs is technically quite challenging. The whole dissemination process encompasses also individual tumor cells which are found in relatively big quantities in peritoneal washings (PW) and pleural effusions (PE), so called disseminated tumor cells (DTCs). Thanks in vitro cultures of separated CTCs/DTCs we were able to introduce additional chemosensitivity testing for the purpose of individualized cancer treatment. We present an isolation and cultivation of CTC/DTC of non-small cell lung cancer and gastrointestinal cancers (esophageal cancer, colorectal cancer, gastric cancer, pancreatic cancer) describing cultivation methodology on patients case studies. The cancer cells were separated from body fluids (PW, PE, blood) using the differences in size of cancer cells if compared to other nucleated cells detected in body cavities. After reaching the confluence the chemosensitivity of cell cultures has been tested by xCELLigence system (RTCA, Roche) for different concentrations of single cis-platinum, gemcitabine and fluorouracyl (incubation time 24-72 hours) or their combinations. The dynamic real-time cell growth monitoring enables parallel or sequential combinations of administered drugs with a direct outcome for patients care. Conclusions: We assume that developing cultivation strategies for CTCs/DTCs could bring us closer to the definition of the CTC/DTC-function within the dissemination process with a perspective to use this knowledge in personalized cancer treatment. Citation Format: Vladimir Bobek. In vitro culturing of CTCs/DTCs in personalized cancer management. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1464. doi:10.1158/1538-7445.AM2013-1464

A survey on osmoregulatory potential of Bream, Abramis brama (Berg, 1949) fry for restocking management programs

Bream ( Abramis brama , Berg, 1949; Family: Cyprinidae) is commercially valuable fish in the Caspian Sea fishing industry. Iranian Fisheries Organization annually produces and release up to 19 million Bream fries size for recruiting of this species. Its fries are mostly released into the Anzali wetland with 4 ppt salinity. Meanwhile, they sometimes are released into Sefidrood River (0.5 ppt), Sefidrood River estuary (8 ppt) and directly into the Caspian Sea (12 ppt). To determine whether these alternative locations are suitable for release, sixty fingerling size Bream (0.5±0.13 g b.w.) were exposed to four salinity levels of these locations i.e. 0.5, 4, 8 and 12 ppt, for 120 hrs. The results displayed that accumulated mortality rate was significantly lower in exposed fish to 4 ppt ( P <0.05) in compare to others. No differences in size of gill chloride cells were observed among treatments; however, chloride cell number was significantly higher in all treatments except for 4 ppt treatment ( P <0.05). Hind-gut epithelium thickness significantly decreased in specimens at 8 and 12 ppt treatments after 120 hrs ( P <0.05). The results do not suggest releasing Bream fries directly into the Caspian Sea and also, the Anzali wetland cwas confirmed as the suitable releasing site.

Changes in the air cell volume of artificially incubated ostrich eggs

A total of 2160 images of candled, incubated ostrich eggs were digitized to determine the percentage of egg volume occupied by the air cell at different stages of incubation. The air cell on average occupied 2.5% of the volume of fresh eggs. For eggs that hatched successfully, this volume increased to an average of 24.4% at 41 days of incubation, just prior to hatching. Air cell volume at 29 days of incubation for infertile eggs (19.3%) was significantly higher when compared to dead-in-shell (DIS) eggs (14.3%) and eggs that hatched (13.8%). There was a significantly larger air cell volume in eggs that hatched normally compared with DIS eggs at 41 days of incubation (28.3% vs. 21.7%, respectively). No differences in air cell volume were observed up to day 17 of incubation for eggs that hatched normally between eggs that exhibited high, average or low rates of water loss, but from 20 days of incubation the air cell volume was significantly larger for high weight loss eggs. However, for the DIS eggs, air cell volume was consistently larger in eggs that exhibited high rates of water loss. Air cell volume was largely independent of adult strain (SA Black or Zimbabwean Blue) or whether chicks were assisted to hatch. Although some subtle differences in air cell size were detected between hatched and DIS chicks during this study, it is unlikely to find useful application in the broader industry.

Odor enrichment sculpts the abundance of olfactory bulb mitral cells

Mitral cells are the primary output cell from the olfactory bulb conveying olfactory sensory information to higher cortical areas. Gene-targeted deletion of the Shaker potassium channel Kv1.3 alters voltage-dependence and inactivation kinetics of mitral cell current properties, which contribute to the “Super-smeller” phenotype observed in Kv1.3-null mice. The goal of the current study was to determine if morphology and density are influenced by mitral cell excitability, olfactory environment, and stage of development. Wildtype (WT) and Kv1.3-null (KO) mice were exposed to a single odorant (peppermint or citralva) for 30 days. Under unstimulated conditions, postnatal day 20 KO mice had more mitral cells than their WT counterparts, but no difference in cell size. Odor-enrichment with peppermint, an olfactory and trigeminal stimulus, decreased the number of mitral cells in three month and one year old mice of both genotypes. Mitral cell density was most sensitive to odor-stimulation in three month WT mice. Enrichment at the same age with citralva, a purely olfactory stimulus, decreased cell density regardless of genotype. There were no significant changes in cell body shape in response to citralva exposure, but the cell area was greater in WT mice and selectively greater in the ventral region of the OB in KO mice. This suggests that trigeminal or olfactory stimulation may modify mitral cell area and density while not impacting cell body shape. Mitral cell density can therefore be modulated by the voltage and sensory environment to alter information processing or olfactory perception.

Ontogeny of erythroid gene expression

AbstractErythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H2O2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H2O2 exposure. We have created a user-friendly Web site (http://www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community.

Mitochondrial Transmembrane Potential (ψm) Assay Using TMRM

During cellular respiration, nutrients are oxidized to generate energy through a mechanism called oxidative phosphorylation, which occurs in the mitochondria. During oxidative phosphorylation, the gradual degradation of molecules through the TCA cycle releases electrons from the covalent bonds that are broken. These electrons are captured by NAD+ through its reduction into NADH. Finally, NADH transports the electrons to the complexes of the electron chain in the internal membrane of mitochondria. These complexes use the energy released by the electrons to pump protons into the intermembrane space, generating an electrochemical gradient across the internal membrane of mitochondria, which provides energy for the ATP-synthase complex, ultimately producing adenosine triphosphate (ATP). We assessed the mitochondrial membrane potential (ψm) using tetramethylrhodamine methyl ester (TMRM), a cell-permeant, cationic, red fluorescent dye. To measure specifically the mitochondrial membrane potential (ψm) we quantified the fluorescence intensity before and after applying FCCP, a mitochondrial electron chain uncoupler. The difference of intensity before and after applying FCCP corresponds specifically to the mitochondrial membrane potential. We analyzed mitochondrial membrane potential (ψm) by cytofluorimetry. The ratio between the total level of signal and the signal generated after uncoupling provided a normalized value for the difference in cell size. Furthermore, to normalize for the different size of cells that we were analyzing we have analyzed TMRM in live imaging using IN Cell Analyzer, so that the level of mitochondrial membrane potential could be detected per unit of mitochondrial membrane area measured. Thus, our protocol can also be used to compare the mitochondrial membrane potential of cells that are different in size.

Isolation of Rare Cells through their Dielectrophoretic Signature

The isolation of rare cells using their dielectrophoretic signature is becoming a popular tool for medical diagnostics, individualized medicine, and anti-bioterrorism. This unique property varies between cell types due to differences in cell size and structure, including membrane morphology and composition.

Body site matters: an evaluation and application of a novel histological methodology on the quantification of mucous cells in the skin of Atlantic salmon, Salmo salar L.

Mucous cell size and distribution were investigated in the skin of five salmon using a novel stereology-based methodology: one (48 cm) fish to test 15 tissue treatment combinations on measures of cell area and density on the dorsolateral region and, using the most suitable treatment, we mapped mucous cell differences between body regions on four (52 cm) salmon, comprising a male and a female on each of two diets. The section site, decalcification, embedding medium and plane of sectioning all impacted significantly on mucous cell size, whereas mucous cell density is more robust. There were highly significant differences in both mucosal density and mean mucous cell size depending on body site: the dorsolateral skin of the four salmon had significantly denser (about 8% of skin area) and larger (mean about 160 μm(2)) mucous cells, whereas the lowest mean density (about 4%) and smallest mean area (115 μm(2)) were found on the head. We found that 100 random measurements may be sufficient to distinguish differences >7 μm(2) in mean mucous cell areas. The results further suggest that salmon exhibit a dynamic repeatable pattern of mucous cell development influenced by sex, diet and possibly strain and season.

A genetics-based description of Symbiodinium minutum sp. nov. and S. psygmophilum sp. nov. (Dinophyceae), two dinoflagellates symbiotic with cnidaria.

Traditional approaches for describing species of morphologically cryptic and often unculturable forms of endosymbiotic dinoflagellates are problematic. Two new species in the genus Symbiodinium Freudenthal 1962 are described using an integrative evolutionary genetics approach: Symbiodinium minutum sp. nov. are harbored by widespread tropical anemones in the genus Aiptasia; and Symbiodinium psygmophilum sp. nov. are harbored by subtropical and temperate stony corals (e.g., Astrangia, Cladocora, and Oculina) from the Atlantic Ocean and Mediterranean Sea. Both new species are readily distinguished from each other by phylogenetic disparity and reciprocal monophyly of several nucleic acid sequences including nuclear ribosomal internal transcribed spacers 1 and 2, single copy microsatellite flanker Sym15, mitochondrial cytochrome b, and the chloroplast 23S rRNA gene. Such molecular evidence, combined with well-defined differences in cell size, physiology (thermal tolerance), and ecology (host compatibility) establishes these organisms as distinct species. Future descriptions of Symbiodinium spp. will need to emphasize genetics-based descriptions because significant morphological overlap in this group obscures large differences in ecology and evolutionary divergence. By using molecular evidence based on conserved and rapidly evolving genes analyzed from a variety of samples, species boundaries are defined under the precepts of Evolutionary and Biological Species Concepts without reliance on an arbitrary genetic distance metric. Because ecological specialization arises through genetic adaptations, the Ecological Species Concept can also serve to delimit many host-specific Symbiodinium spp.

Rice phytochrome-interacting factor-like protein OsPIL1 functions as a key regulator of internode elongation and induces a morphological response to drought stress

The mechanisms for plant growth restriction during stress conditions remains unclear. Here, we demonstrate that a phytochrome-interacting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator of reduced internode elongation in rice under drought conditions. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period. Under drought stress conditions, OsPIL1 expression was inhibited during the light period. We found that OsPIL1 was highly expressed in the node portions of the stem using promoter-glucuronidase analysis. Overexpression of OsPIL1 in transgenic rice plants promoted internode elongation. In contrast, transgenic rice plants with a chimeric repressor resulted in short internode sections. Alteration of internode cell size was observed in OsPIL1 transgenic plants, indicating that differences in cell size cause the change in internode length. Oligoarray analysis revealed OsPIL1 downstream genes, which were enriched for cell wall-related genes responsible for cell elongation. These data suggest that OsPIL1 functions as a key regulatory factor of reduced plant height via cell wall-related genes in response to drought stress. This regulatory system may be important for morphological stress adaptation in rice under drought conditions.

Microfluidic flow fractionation device for label-free isolation of circulating tumor cells (CTCs) from breast cancer patients

Circulating tumor cells (CTCs) are dissociated from primary tumor and circulate in peripheral blood. They are regarded as the genesis of metastasis. Isolation and enumeration of CTCs serve as valuable tools for cancer prognosis and diagnosis. However, the rarity and heterogeneity of CTCs in blood makes it difficult to separate intact CTCs without loss. In this paper, we introduce a parallel multi-orifice flow fractionation (p-MOFF) device in which a series of contraction/expansion microchannels are placed parallel on a chip forming four identical channels. CTCs were continuously isolated from the whole blood of breast cancer patients by hydrodynamic forces and cell size differences. Blood samples from 24 breast cancer patients were analyzed (half were from metastatic breast cancer patients and the rest were from adjuvant breast cancer patients). The number of isolated CTCs varied from 0 to 21 in 7.5ml of blood. Because our devices do not require any labeling processes (e.g., EpCAM antibody), heterogeneous CTCs can be isolated regardless of EpCAM expression.

Mechanisms of Intracellular Scaling

Cell size varies widely among different organisms as well as within the same organism in different tissue types and during development, which places variable metabolic and functional demands on organelles and internal structures. A fundamental question is how essential subcellular components scale to accommodate cell size differences. Nuclear transport has emerged as a conserved means of scaling nuclear size. A meiotic spindle scaling factor has been identified as the microtubule-severing protein katanin, which is differentially regulated by phosphorylation in two different-sized frog species. Anaphase mechanisms and levels of chromatin compaction both act to coordinate cell size with spindle and chromosome dimensions to ensure accurate genome distribution during cell division. Scaling relationships and mechanisms for many membrane-bound compartments remain largely unknown and are complicated by their heterogeneity and dynamic nature. This review summarizes cell and organelle size relationships and the experimental approaches that have elucidated mechanisms of intracellular scaling.

Quantitative Approaches to Detect Donor and Passage Differences in Adipogenic Potential and Clonogenicity in Human Bone Marrow-Derived Mesenchymal Stem Cells

Bone marrow-derived multipotent stromal cells (MSCs), also known as mesenchymal stem cells, have great promise due to their capacity for tri-lineage differentiation and immunosuppressive properties, which allows for their allogeneic use and ultimately may allow for treatment of many diseases. MSCs will require extensive expansion and passaging to obtain cells in sufficient numbers necessary for cell therapies. MSCs from many donors could potentially be used. Because of this, there is a need to understand the role of passaging and donor differences on differentiation capacity using quantitative approaches. Here, we evaluated MSCs from two donors (noted as PCBM1632 and PCBM1641 by the manufacturer) at tissue culture passages 3, 5, and 7. We used a colony forming unit (CFU) assay and limiting dilution to quantify clonogenicity and precursor frequency during adipogenesis, and quantitative real-time-polymerase chain reaction for adipogenic markers to evaluate changes on a gene expression level. Further, we observed changes in cell size, and we sorted small and large populations to evaluate size-related adipogenic potential. While the adipogenic precursor frequency of ∼1 in 76 cells remained similar through passages for cells from PCBM1641, we found a large decrease in the adipogenic potential of MSCs from PCBM1632, with 1 in 2035 cells being capable of differentiating into an adipocyte at passage 7. MSCs from both donors showed an increase in cell diameter with increasing passage, which correlates with a decrease in clonogenicity by CFU analysis. We also measured adipose lineage gene expression following induction of adipocyte differentiation. Expression of these genes decreased with passage number for MSCs from PCBM1632 and correlated with the decrease in adipogenic potential by passage 7. In contrast, MSCs from PCBM1641 showed increased expression of these genes with increasing passage. We have shown that several quantitative assays can detect differences in MSC differentiation capacity, clonogenicity, and cell size between donors and passages. These quantitative methods are useful to assess the quality of MSCs.

NITROGEN LIMITATION INCREASES BREVETOXINS IN KARENIA BREVIS (DINOPHYCEAE): IMPLICATIONS FOR BLOOM TOXICITY(1).

Laboratory and field measurements of the toxin content in Karenia brevis cells vary by >4-fold. These differences have been largely attributed to genotypic variations in toxin production among strains. We hypothesized that nutrient limitation of growth rate is equally or more important in controlling the toxicity of K. brevis, as has been documented for other toxic algae. To test this hypothesis, we measured cellular growth rate, chlorophyll a, cellular carbon and nitrogen, cell volume, and brevetoxins in four strains of K. brevis grown in nutrient-replete and nitrogen (N)-limited semi-continuous cultures. N-limitation resulted in reductions of chlorophyll a, growth rate, volume per cell and nirtogen:carbon (N:C) ratios as well as a two-fold increase (1%-4% to 5%-9%) in the percentage of cellular carbon present as brevetoxins. The increase in cellular brevetoxin concentrations was consistent among genetically distinct strains. Normalizing brevetoxins to cellular volume instead of per cell eliminated much of the commonly reported toxin variability among strains. These results suggest that genetically linked differences in cellular volume may affect the toxin content of K. brevis cells as much or more than innate genotypic differences in cellular toxin content per unit of biomass. Our data suggest at least some of the >4-fold difference in toxicity per cell reported from field studies can be explained by limitation by nitrogen or other nutrients and by differences in cell size. The observed increase in brevetoxins in nitrogen limited cells is consistent with the carbon:nutrient balance hypothesis for increases in toxins and other plant defenses under nutrient limitation.

Artificial polyploidy improves bacterial single cell genome recovery.

BackgroundSingle cell genomics (SCG) is a combination of methods whose goal is to decipher the complete genomic sequence from a single cell and has been applied mostly to organisms with smaller genomes, such as bacteria and archaea. Prior single cell studies showed that a significant portion of a genome could be obtained. However, breakages of genomic DNA and amplification bias have made it very challenging to acquire a complete genome with single cells. We investigated an artificial method to induce polyploidy in Bacillus subtilis ATCC 6633 by blocking cell division and have shown that we can significantly improve the performance of genomic sequencing from a single cell.Methodology/Principal FindingsWe inhibited the bacterial cytoskeleton protein FtsZ in B. subtilis with an FtsZ-inhibiting compound, PC190723, resulting in larger undivided single cells with multiple copies of its genome. qPCR assays of these larger, sorted cells showed higher DNA content, have less amplification bias, and greater genomic recovery than untreated cells.SignificanceThe method presented here shows the potential to obtain a nearly complete genome sequence from a single bacterial cell. With millions of uncultured bacterial species in nature, this method holds tremendous promise to provide insight into the genomic novelty of yet-to-be discovered species, and given the temporary effects of artificial polyploidy coupled with the ability to sort and distinguish differences in cell size and genomic DNA content, may allow recovery of specific organisms in addition to their genomes.

Viable circulating tumour cell detection using multiplex RNA in situ hybridisation predicts progression-free survival in metastatic breast cancer patients

Background:Current approaches for detecting circulating tumour cells (CTCs) in blood are dependent on CTC enrichment and are based either on surface epithelial markers on CTCs or on cell size differences. The objectives of this study were to develop and characterise an ultrasensitive multiplex fluorescent RNA in situ hybridisation (ISH)-based CTC detection system called CTCscope. This method detects a multitude of tumour-specific markers at single-cell level in blood.Methods:Healthy blood samples spiked with tumour cell lines were used as a model system for the development and initial characterisation of CTCscope. To demonstrate the feasibility of CTC detection in patient blood, duplicate blood samples were drawn from 45 metastatic breast cancer patients for analysis by CTCscope and the CellSearch system. The association of CTCs with the tumour marker CA15-3 and progression-free survival (PFS) were assessed.Results:CTCscope detected CTC transcripts of eight epithelial markers and three epithelial-mesenchymal-transition (EMT) markers for increased sensitivity. CTCscope was used to detect CTCs with minimal enrichment, and did not detect apoptotic or dead cells. In patient blood samples, CTCs detected by CellSearch, but not CTCscope, were positively correlated with CA15-3 levels. Circulating tumour cells detected by either CTCscope or CellSearch predicted PFS (CTCscope, HR (hazard ratio) 2.26, 95% CI 1.18–4.35, P=0.014; CellSearch, HR 2.50, 95% CI 1.27–4.90, P=0.008).Conclusion:CTCscope offers unique advantages over existing CTC detection approaches. By enumerating and characterising only viable CTCs, CTCscope provides additional prognostic and predictive information in therapy monitoring.

ANTI‐OBESITY EFFECT OF SHK‐186, A K+ CHANNEL BLOCKER

We describe the anti‐obesity effects of ShK‐186, a Kv1.3‐selective peptide inhibitor. C57BL/J mice fed a diet containing high fat (21%) diet and fructose solution (60%) were administered ShK‐186 (500 μg/kg) or vehicle on alternate days by subcutaneous injection. ShK‐186‐treatment caused a dose‐dependent reduction in weight gain over the 45‐day trial, the maximal weight difference being 30%. ShK‐186‐treatment reduced plasma levels of free fatty acids without affecting cholesterol, HDL, LDL or glucose. CT scan revealed significant reduction in volume of white adipose tissue in ShK‐186‐treated mice compared to controls. However, histology revealed no difference in cell size and fat content of white fat cells. In metabolic cage experiments, ShK‐186‐treatment increased cumulative intake of food and fluid compared to vehicle‐treated mice, especially during the light‐cycle. Furthermore, their respiratory exchange ratio and energy expenditure were also higher. In PET experiments, ShK‐186 treatment increased uptake of 18‐fluoro‐deoxyglucose (18FDG) by brown adipose tissue (BAT) and skeletal muscle compared to vehicle‐controls; pre‐administration of the beta‐adrenoceptor blocker propranolol (5mg/kg) inhibited 18FDG uptake by BAT but not skeletal muscle. ShK‐186's anti‐obesity effect appears to be mediated by activation of BAT and/or skeletal muscle, two important thermogenic tissues in the body

Do fathead minnows, Pimephales promelas Rafinesque, alter their club cell investment in responses to variable risk of infection from Saprolegnia?

Fish in the Superorder Ostariophysi possess large epidermal club cells that release chemical cues warning nearby conspecifics of danger. Despite the long-held assumption that such club cells evolved under the selective force of predation, recent studies demonstrated that predation has no effect on club cell investment. Rather, club cells have an immune function and cell production may be stimulated by skin-penetrating pathogens and parasites. The current work investigates whether fathead minnows, Pimephales promelas, alter their club cell characteristics based on variation in infection risk. In a 2 × 3 design, we exposed minnows to infective cysts of two oomycete species (Saprolegnia ferax and S. parasitica) at three different concentrations (2, 20 or 200 cysts L(-1)). Club cell characteristics (number and size) were quantified 12 days after exposure. Saprolegnia parasitica is thought to be more pathogenic than S. ferax, hence we predicted greater club cell investment and a larger turnover rate of cells by minnows exposed to S. parasitica than S. ferax. We also predicted that minnows exposed to higher numbers of cysts should invest more in club cells and have a higher turnover rate of cells. We found no difference in club cell density or size between fish exposed to the two Saprolegnia species; however, fish exposed to high concentrations of pathogens had smaller club cells than those exposed to low concentrations, indicating a higher rate of turnover of cells in the epidermis.

Cytometric comparisons between circulating tumor cells from prostate cancer patients and the prostate-tumor-derived LNCaP cell line

Many important experiments in cancer research are initiated with cell line data analysis due to the ease of accessibility and utilization. Recently, the ability to capture and characterize circulating tumor cells (CTCs) has become more prevalent in the research setting. This ability to detect, isolate and analyze CTCs allows us to directly compare specific protein expression levels found in patient CTCs to cell lines. In this study, we use immunocytochemistry to compare the protein expression levels of total cytokeratin (CK) and androgen receptor (AR) in CTCs and cell lines from patients with prostate cancer to determine what translational insights might be gained through the use of cell line data. A non-enrichment CTC detection assay enables us to compare cytometric features and relative expression levels of CK and AR by indirect immunofluorescence from prostate cancer patients against the prostate cancer cell line LNCaP. We measured physical characteristics of these two groups and observed significant differences in cell size, fluorescence intensity and nuclear to cytoplasmic ratio. We hope that these experiments will initiate a foundation to allow cell line data to be compared against characteristics of primary cells from patients.

Anti-Obesity Effect of Shk-186, a K+ Channel Blocker

Obesity is a global health problem and there is a pressing need for safe therapeutics. Here, we describe the anti-obesity effects of ShK-186, a Kv1.3-selective peptide inhibitor with picomolar potency and an excellent safety profile in rodents and monkeys. C57BL/J mice fed a diet containing high fat (21%) diet and fructose solution (60%) were administered ShK-186 (20, 100 or 500 μg/kg) or vehicle on alternate days by subcutaneous injection. ShK-186-treatment caused a dose-dependent reduction in weight gain over the 45-day trial, the maximal weight difference being 30%. ShK-186-treatment reduced plasma levels of triglyceride and free fatty acids without affecting cholesterol, HDL, LDL or glucose. CT scan revealed significant reduction in volume of white adipose tissue (WAT) in ShK-186-treated mice compared to vehicle-controls. However, histology revealed no difference in cell size and fat content of white fat cells. In metabolic cage experiments, ShK-186-treatment increased cumulative intake of food and fluid compared to vehicle-treated mice, especially during the light-cycle. Furthermore, their respiratory exchange ratio and energy expenditure were also higher. In PET experiments, ShK-186 treatment increased uptake of 18-fluoro-deoxyglucose (18FDG) by brown adipose tissue (BAT) and skeletal muscle compared to vehicle-controls; pre-administration of the beta-adrenoceptor blocker propranolol (5mg/kg) inhibited 18FDG uptake by BAT but not skeletal muscle. ShK-186's anti-obesity effect appears to be mediated by activation of BAT and/or skeletal muscle, two important thermogenic tissues in the body.