Hesperetin suppresses LPS/high glucose-induced inflammatory responses via TLR/MyD88/NF-κB signaling pathways in THP-1 cells.

BACKGROUND/OBJECTIVES: Unregulated inflammatory responses caused by hyperglycemia may induce diabetes complications. Hesperetin, a bioflavonoid, is a glycoside in citrus fruits and is known to have antioxidant and anticarcinogenic properties. However, the effect of inflammation on the diabetic environment has not been reported to date. In this study, we investigated the effect of hesperetin on proinflammatory cytokine secretion and its underlying mechanistic regulation in THP-1 macrophages with co-treatment LPS and hyperglycemic conditions. MATERIALS/METHODS: THP-1 cells differentiated by PMA (1 µM) were cultured for 48 h in the presence or absence of hesperetin under normoglycemic (5.5 mM/L glucose) or hyperglycemic (25 mM/L glucose) conditions and then treated with LPS (100 ng/mL) for 6 h before harvesting. Inflammation-related proteins and mRNA levels were evaluated by enzyme-linked immunosorbent assay, western blot, and quantitative polymerase chain reaction analyses. RESULTS: Hesperetin (0-100 µM, 48 h) treatment did not affect cell viability. The tumor necrosis factor-α and interleukin-6 levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions, and these increases were decreased by hesperetin treatment. The TLR2/4 and MyD88 activity levels increased in cells co-treated with LPS under hyperglycemic conditions compared to normoglycemic conditions; however, hesperetin treatment inhibited the TLR2/4 and MyD88 activity increases. In addition, nuclear factor-κB (NF-κB) and Acetyl-NF-κB levels increased in response to treatment with LPS under hyperglycemic conditions compared to normoglycemic conditions, but those levels were decreased when treated with hesperetin. SIRT3 and SIRT6 expressions were increased by hesperetin treatment. CONCLUSIONS: Our results suggest that hesperetin may be a potential agent for suppressing inflammation in diabetes.

Stabilization of Lipid Lamellar Bilayer Structure of Stratum Corneum Modulated by Poly (2-methacryloyloxyethyl phosphorylcholine) in Relation to Skin Hydration and Skin Protection.

BACKGROUND: One crucial factor in skin tissue engineering is to understand the hydration and barrier property of skin. We investigated the skin hydration and stabilization strategy of inter-lamellar structure of stratum corneum (SC) using poly (2-methacryloyloxyethyl phosphorylcholine) (PMPC). METHODS: The unique hydration and stabilization potency of PMPC on the barrier function of the SC examined using freshly excised hairless mouse skin as a model membrane and the relationship between the stabilization of the lipid lamellar bilayer (LLB) and its enhanced water holding capacity was established. RESULTS: Differential scanning calorimeter based on the phase-transition temperature of lipid domain of SC demonstrated that PMPC stabilized the LLB. The ratio of the heat of lipid phase transition (△H) of SC exposed to water and PMPC for 24 h was 1.51. X-ray crystallography showed the presence of well- organized lipids in intercellular membranes exhibiting short and long periodicity of lamellar phases. The peak at 4.4 nm attributed to the long periodicity phase (LPP) was missing in water-treated SC, where, the presence of 4.2- 4.4 nm peak in PMPC treated SC indicated that PMPC stabilized LPP. Transmission electron microscopy study demonstrated that the LLB structure became more rigid and orderly in PMPC treated SC. CONCLUSION: The unique ion paired structure of PMPC enhances the barrier function of the SC by stabilizing LLB structure and hydration by inducing weakly bound water. The unique hydration state and stabilization effect from extended water exposure could provide a valuable information to prepare reliable artificial skin matrix and skin tissue.

The Presence and Distribution of TRPM7 in the Canine Mammary Glands.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) cation channel is a bifunctional ion channel with intrinsic kinase activity and is ubiquitously expressed in the animal/human body. Accumulated knowledge of TRPM7 suggests that it plays an essential role in normal physiological processes, including the development, survival, proliferation, differentiation, and migration of cells. The aim of this study was to demonstrate the presence and expression patterns of TRPM7 in normal canine mammary glands using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. Normal mammary gland tissue samples were obtained from five female beagle dogs. RT-PCR and sequencing of the amplified PCR products demonstrated the presence of TRPM7 mRNA in normal mammary glands, and the presence of TRPM7 protein was confirmed by Western blotting. Immunohistochemical investigations demonstrated the expression of TRPM7 in the apical membrane of acinar and ductal epithelial cells in the canine mammary glands. These results provide the first evidence of the presence and distribution of TRPM7 in the canine mammary gland and could help explain the physiological and pathological roles of TRPM7 in the canine mammary gland; however, additional studies are required to elucidate these roles.

Ectopic Cushing's syndrome associated with a pheochromocytoma in a dog: a case report.

BACKGROUND: Ectopic Cushing's syndrome (ECS) associated with malignant tumors, such as small cell lung carcinoma, bronchial carcinoids, and pheochromocytoma, has been reported in human medicine. However, ECS related to pheochromocytoma has not been reported in dogs. CASE PRESENTATION: An 11-year-old castrated, male Scottish terrier was diagnosed with a left adrenal mass. Cushing's syndrome was suspected based on clinical signs, including pot belly, polyuria, polydipsia, bilateral alopecia, recurrent pyoderma, and calcinosis cutis. Cushing's syndrome was diagnosed on the basis of consistent clinical signs and repeated adrenocorticotropic hormone (ACTH) stimulation tests. In addition, tests for fractionated plasma metanephrine/normetanephrine suggested a pheochromocytoma. Unilateral adrenalectomy was performed after medical management with trilostane and phenoxybenzamine. Histopathology confirmed a diagnosis of pheochromocytoma without cortical lesions. After surgery, fractionated metanephrine/normetanephrine and the findings of low-dose dexamethasone suppression and ACTH stimulation tests were within the normal ranges without any medication. There were no clinical signs or evidence of recurrence and metastasis on thoracic and abdominal X-rays and ultrasonography up to 8 months after surgery. CONCLUSIONS: Pheochromocytoma should be considered a differential diagnosis for dogs with Cushing's syndrome with an adrenal tumor. A good prognosis can be expected with prompt diagnosis and surgical intervention.

CRDS: Consensus Reverse Docking System for target fishing.

MOTIVATION: Identification of putative drug targets is a critical step for explaining the mechanism of drug action against multiple targets, finding new therapeutic indications for existing drugs and unveiling the adverse drug reactions. One important approach is to use the molecular docking. However, its widespread utilization has been hindered by the lack of easy-to-use public servers. Therefore, it is vital to develop a streamlined computational tool for target prediction by molecular docking on a large scale. RESULTS: We present a fully automated web tool named Consensus Reverse Docking System (CRDS), which predicts potential interaction sites for a given drug. To improve hit rates, we developed a strategy of consensus scoring. CRDS carries out reverse docking against 5254 candidate protein structures using three different scoring functions (GoldScore, Vina and LeDock from GOLD version 5.7.1, AutoDock Vina version 1.1.2 and LeDock version 1.0, respectively), and those scores are combined into a single score named Consensus Docking Score (CDS). The web server provides the list of top 50 predicted interaction sites, docking conformations, 10 most significant pathways and the distribution of consensus scores. AVAILABILITY AND IMPLEMENTATION: The web server is available at http://pbil.kaist.ac.kr/CRDS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The relationship between ground reaction force components and peak power according to induced fatigue during 16-km walking.

The previous reviewed studies on inducement of fatigue through long-time walking were not only very confined, but also not cleared on relationship among variables of fatigue inducement active force, decay rate, and power. This study analyzed relationship between power and component of ground reaction force after fatigue being induced through 16-km walking. The fatigue of adult males and females (n=16) was induced through 16-km walking. Then power, measured for pre and post of fatigue inducement, was evaluated by maximal vertical jump on ground reaction force plate. Variables of vertical jump heights, active force, power, and decay rate showed decreased tendency after fatigue inducement, which followed significant difference (P<0.05) and also positive correlation of r=0.628 (R 2=39%) of between vertical jump heights and power and r=0.589 (R 2=34%) of between active force and decay rate respectively. That is, long-time walking for pursuing of exercise rehabilitation, health promotion and leisure activity has been preferred. In the view of this, this study suggested the necessity to understand the relation between fatigue and power to prevent a potential possibility of injury during long-time walking.

Effect of muscle mass asymmetric between upper and lower limbs on the postural stability and shock attenuation during landing.

The aim of the study was to analyze the effect of muscle mass asymmetric between upper and lower limbs on postural stability and shock attenuation during landing. Twenty adults (without lower limb disorders and who could land from a 35-cm height) participated in this study (mean age, 21.85±2.97 years; mean height, 1.68±0.10 m; mean weight: 68.64±17.36 kg). Subjects performed one-leg landing from 36-cm vertical heights. Ground reaction force components and medial-lateral, anterior-posterior, vertical and dynamic postural stability index were obtained from force platform recordings. We found that muscle mass in right limbs more increased than that of left limbs. Medial-lateral force, vertical force, vertical stability index, and dynamic postural stability index in left leg showed higher value than that of right leg during landing. The asymmetry of muscle mass (%) and ground reaction force variables showed a similar correlation, including dynamic postural stability index (r=0.316). These findings allow us to conclude that the factor of muscle mass asymmetric is a contributor to impulse control and dynamic postural stability index asymmetry. Therefore, knowledge of bilateral limbs asymmetry may provide insights into exercise rehabilitation and performance.

Changes in pre- and postoperative serum leptin concentrations in dogs with gallbladder mucocele and cholelithiasis.

BACKGROUND: Leptin has been shown to have various physiological and pathological roles in the canine gallbladder. In this study, we performed pre- and postoperative short-term follow-up analyses to confirm changes in serum leptin levels before and after cholecystectomy due to gallbladder mucocele (GBM) or cholelithiasis in dogs. RESULTS: Twenty-six cholecystectomized dogs (GBM: n = 14; cholelithiasis: n = 12) for prophylactic or clinical symptom relief were enrolled in the present study. Dogs were subgrouped according to clinical symptoms and prognosis after surgery as follows: 1) asymptomatic group (n = 13), 2) recovery group (n = 8), and 3) death group (n = 5). Liver enzymes, total bilirubin, lipid profiles, and leptin concentrations were determined from sera on the pre-operative day and at 1, 3, and 7 days postoperation. Serum leptin concentrations were gradually but significantly decreased in the asymptomatic group (p = 0.008, 0.004, and 0.004 on days 1, 3, and 7, respectively, compared with that before surgery) and the recovery group (p = 0.048 and 0.048 on days 3 and 7, respectively, compared with that before surgery). However, in the death group, leptin concentrations did not differ significantly over time (p = 0.564). Additionally, serum leptin levels in the recovery group (p = 0.006) and death group (p = 0.021) were significantly higher than those in the asymptomatic group. Liver enzymes and total bilirubin (T-Bil) were significantly decreased only in the recovery group, particularly on day 7. In the asymptomatic group, liver enzymes and T-Bil were not changed significantly over time, and in the death group, only T-Bil was significantly decreased on day 7. Total cholesterol and triglyceride levels were not significantly decreased over time in all groups. CONCLUSIONS: These results indicate that leptin is a potential biomarker reflecting the severity and prognosis of GBM and cholelithiasis both before and after cholecystectomy in dogs.

Alteration of microRNA 340-5p and Arginase-1 Expression in Peripheral Blood Cells during Acute Ischemic Stroke.

Acute stroke alters the systemic immune response as can be observed in peripheral blood; however, the molecular mechanism by which microRNA (miRNA) regulates target gene expression in response to acute stroke is unknown. We performed a miRNA microarray on the peripheral blood of 10 patients with acute ischemic stroke and 11 control subjects. Selected miRNAs were quantified using a TaqMan assay. After searching for putative targets from the selected miRNAs using bioinformatic analysis, functional studies including binding capacity and protein expression of the targets of the selected miRNAs were performed. The results reveal a total of 30 miRNAs that were differentially expressed (16 miRNAs were upregulated and 14 miRNAs were downregulated) during the acute phase of stroke. Using prediction analysis, we found that miR-340-5p was predicted to bind to the 3'-untranslated region of the arginase-1 (ARG1) gene; a luciferase reporter assay confirmed the binding of miR-340-5p to ARG1. miR-340-5p was downregulated whereas ARG1 mRNA was upregulated in peripheral blood in patients experiencing acute stroke. Overexpression of miR-340-5p in human neutrophil and mouse macrophage cell lines induced downregulation of the ARG1 protein. Transfection with miR-340-5p increased nitric oxide production after LPS treatment in a mouse macrophage cell line. Our results suggest that several miRNAs are dynamically altered in the peripheral blood during the acute phase of ischemic stroke, including miR-340-5p. Acute stroke induces the downregulation of miR-340-5p, which subsequently upregulates ARG1 protein expression.

KRDS: a web server for evaluating drug resistance mutations in kinases by molecular docking.

Kinases are major targets of anti-cancer therapies owing to their importance in signaling processes that regulate cell growth and proliferation. However, drug resistance has emerged as a major obstacle to cancer therapy. Resistance to drugs has various underlying mechanisms, including the acquisition of mutations at drug binding sites and the resulting reduction in drug binding affinity. Therefore, the identification of mutations that are relevant to drug resistance may be useful to overcome this issue. We hypothesized that these mutations can be identified by combining recent advances in computational methods for protein structure modeling and ligand docking simulation. Hence, we developed a web-based tool named the Kinase Resistance Docking System (KRDS) that enables the assessment of the effects of mutations on kinase-ligand interactions. KRDS receives a list of mutations in kinases, generates structural models of the mutants, performs docking simulations, and reports the results to users. The changes in docking scores and docking conformations can be analyzed to infer the effects of mutations on drug binding and drug resistance. We expect our tool to improve our understanding of drug binding mechanisms and facilitate the development of effective new drugs to overcome resistance related to kinases; it may be particularly useful for biomedical researchers who are not familiar with computational environments. Our tool is available at http://bcbl.kaist.ac.kr/KRDS/ .

The effect of serum types on Chondrogenic differentiation of adipose-derived stem cells.

BACKGROUND: Fetal bovine serum (FBS) is the most essential supplement in culture media for cellular proliferation, metabolism, and differentiation. However, due to a limited supply and subsequently rising prices, a series of studies have investigated a biological feasibility of replaceable serums to substitute FBS. Along with the increasing interests to manufacture stem cell-based cellular products, optimizing the composition of culture media including serums and exogenous growth factors (GFs) is of importance. In this experiment, the effect of bovine serum (BS) and newborn calf serum (NCS) on proliferation and chondrogenic differentiation capacity of human adipose derived stem cells (ADSCs) was evaluated, especially in the chondrogenically supplemented culture condition. METHODS: ADSCs were chondrogenically cultured with FBS, BS, and NCS for 14 days. For the acceleration of in vitro chondrogenesis, exogenous insulin-like growth factor and transforming growth factor-ß3 were added. Viability and proliferation of ADSCs were evaluated using Live/Dead fluorescence staining and DNA amount, respectively. To investigate a chondrogenic differentiation, a series of assays were performed including a quantification of glycosaminoglycan deposition, alcian blue staining, and RT-PCR analysis for type II collagen, aggrecan and Sox-9 genes. RESULTS: The results demonstrated that proliferation of ADSCs was facilitated in FBS condition as compared with other serum types. For chondrogenic marker gene expression, serum substitutes enhanced Sox-9 expression level on day 14. The deposition of glycosaminoglycan was more facilitated in BS condition regardless of additional chondrogenic GFs. CONCLUSION: It could be presumably speculated that serum types and exogenous supplements of GFs could also be important parameters to optimize culture media composition, especially in order to maintain the enhanced levels of both proliferation and chondrogenic differentiation of ADSCs during expansion.

Local Silencing of Connective Tissue Growth Factor by siRNA/Peptide Improves Dermal Collagen Arrangements.

BACKGROUND: Collagen organization within tissues has a critical role in wound regeneration. Collagen fibril diameter, arrangements and maturity between connective tissue growth factor (CTGF) small interfering RNA (siRNA) and mismatch scrambled siRNA-treated wound were compared to evaluate the efficacy of CTGF siRNA as a future implement for scar preventive medicine. METHODS: Nanocomplexes of CTGF small interfering RNA (CTGF siRNA) with cell penetrating peptides (KALA and MPG∆NLS) were formulated and their effects on CTGF downregulation, collagen fibril diameter and arrangement were investigated. Various ratios of CTGF siRNA and peptide complexes were prepared and down-regulation were evaluated by immunoblot analysis. Control and CTGF siRNA modified cells-populated collagen lattices were prepared and rates of contraction measured. Collagen organization in rabbit ear 8 mm biopsy punch wound at 1 day to 8 wks post injury time were investigated by transmission electron microscopy and histology was investigated with Olympus System and TS-Auto software. CONCLUSION: CTGF expression was down-regulated to 40% of control by CTGF siRNA/KALA (1:24) complexes (p < 0.01) and collagen lattice contraction was inhibited. However, down-regulated of CTGF by CTGF siRNA/MPG∆NLS complexes was not statistically significant. CTGF KALA-treated wound appeared with well formed-basket weave pattern of collagen fibrils with mean diameter of 128 ± 22 nm (n = 821). Mismatch siRNA/KALA-treated wound showed a high frequency of parallel small diameter fibrils (mean 90 ± 20 nm, n = 563). CONCLUSION: Controlling over-expression of CTGF by peptide-mediated siRNA delivery could improve the collagen orientation and tissue remodeling in full thickness rabbit ear wound.

Bilateral medial iliac lymph node excision by a ventral laparoscopic approach: technique description.

The aim of this study was to describe a ventral laparoscopic technique for bilateral medial iliac lymphadenectomy in dogs. Twelve intact male purpose-bred research dogs, weighing less than 15 kg, were positioned in dorsal recumbency, and a 3-portal technique was used. Bilateral dissection was performed with vessel-sealing devices while tilting the surgical table by up to 30° towards the contralateral side of the target medial iliac lymph node (MILN) without changing the surgeon's position. Using a ventral laparoscopic approach, bilateral MILNs were identified and excised in all dogs. The mean times for unilateral and bilateral MILN dissections were 9.7 ± 3.8 and 21.0 ± 6.0 min, respectively. The mean times for the right and left MILN dissections were 10.8 ± 4.3 and 9.8 ± 2.5 min, respectively. The mean total surgery time was 43.7 ± 7.7 min. In total, 26 MILNs were dissected. Several complications, including mild to moderate capillary hemorrhage from perinodal fat and vessels (controlled laparoscopically), mild spleen trauma caused by the first trocar insertion and capsular damage of MILNs, were observed. However, there were no other major complications. All MILN samples were evaluated and deemed suitable for histopathologic diagnosis. Laparoscopic excision of MILNs is a useful method of excisional biopsy for histopathologic diagnosis. Using this ventral laparoscopic approach with the 3-portal technique, bilateral MILN dissection suitable for obtaining histopathologic samples could be achieved in a short time in dogs weighing less than 15 kg.

Associations of Caffeinated Beverage Consumption and Screen Time with Excessive Daytime Sleepiness in Korean High School Students.

The present study investigated caffeinated beverage consumption and screen time in the association with excessive daytime sleepiness (EDS) and sleep duration. We conducted a cross-sectional study including 249 Korean male high school students. These participants responded to a questionnaire inquiring the information on lifestyle factors, consumption of caffeinated beverages, time spent for screen media, and sleep duration as well as to the Epworth Sleepiness Scale (ESS) questionnaire. EDS was defined as ESS scores of 9 or greater. Students with EDS consumed greater amount of chocolate/cocoa drinks and spent longer time for a TV and a mobile phone than those without EDS (p < 0.05). In addition, students with short sleep (≤ 6 hours) consumed greater amount of coffee than others whereas students with long sleep (> 8 hours) consumed greater amount of chocolate/cocoa drinks than others (p < 0.05). Screen time did not differ according to the categories of sleep duration. Although these findings do not support causal relationships, they suggest that screen time is associated with EDS, but not with sleep duration, and that consumption of certain types of caffeinated beverages is associated with EDS and sleep duration. Adolescents may need to reduce screen time and caffeine consumption to improve sleep quality and avoid daytime sleepiness.

Hair growth promoting effect of dermal papilla like tissues from canine adipose-derived mesenchymal stem cells through vascular endothelial growth factor.

The purpose of this study was to investigate the protein expression pattern and the in vivo trichogenicity of dermal papilla-like tissues (DPLTs) made from canine adipose-derived mesenchymal stem cells (ASCs) in athymic nude mice. Canine ASCs were isolated and cultured from adipose tissue, and differentiation was induced by culturing ASCs in dermal papilla forming media. DPLTs were embedded in collagen gel, and their structural characteristics and protein expression were evaluated by hematoxylin and eosin stain and immunohistochemistry. Athymic nude mice were divided into two groups (control and DPLTs groups), and DPLTs were injected in skin wounds of mice in the DPLTs group. The trichogenicity of DPLTs was assessed by gross and histological evaluations for 30 days. The fate and the growth factor-secretion effect of DPLTs were evaluated by immunohistochemistry and Western blotting. DPLTs have a compact aggregated structure, form extracellular matrix and highly express the protein specific for dermal papillae, including ALP and versican. New hair follicle formation was remarkable in nude mice of the DPLTs group in gross findings and H&E stain. Vascularization was increased in the DPLTs group, which was the effect of vascular endothelial growth factor secreted by DPLTs in vitro and in vivo. These data suggest that engineered canine DPLTs have characteristics of dermal papillae and have a positive effect on hair regeneration by secreting growth factors.

Effect of Palmitoyl-Pentapeptide (Pal-KTTKS) on Wound Contractile Process in Relation with Connective Tissue Growth Factor and α-Smooth Muscle Actin Expression.

To evaluate whether Palmitoyl-pentapeptide (Pal-KTTKS), a lipidated subfragment of type 1 pro-collagen (residues 212-216), plays a role in fibroblast contractility, the effect of Pal-KTTKS on the expression of pro-fibrotic mediators in hypertropic scarring were investigated in relation with trans-differentiation of fibroblast to myofibroblast, an icon of scar formation. α-SMA was visualized by immunofluorescence confocal microscopy with a Cy-3-conjugated monoclonal antibody. The extent of α-SMA-positive fibroblasts was determined in collagen lattices and in cell culture study. Pal-KTTKS (0-0.5 µM) induced CTGF and α-SMA protein levels were determined by western blot analysis and fibroblast contractility was assessed in three-dimensional collagen lattice contraction assay. In confocal analysis, fibroblasts were observed as elongated and spindle shapes while myofibroblast observed as squamous, enlarged cells with pronounced stress fibers. Without Pal-KTTKS treatment, three quarters of the fibroblasts differentiates into the myofibroblast; α-SMA-positive stress fibers per field decreased twofold with 0.1 µM Pal-KTTKS treatment (75 ± 7.1 vs 38.6 ± 16.1%, n = 3, p < 0.05). The inhibitory effect was not significant in 0.5 µM Pal-KTTKS treatment. Stress fiber level and collagen contractility correlates with α-SMA expression level. In conclusion, Pal-KTTKS (0.1 µM) reduces α-SMA expression and trans-differentiation of fibroblasts to myofibroblast. The degree of reduction is dose-dependent. An abundance of myofibroblast and fibrotic scarring is correlated with excessive levels of α-SMA and collagen contractility. Delicate balance between the wound healing properties and pro-fibrotic abilities of pentapeptide KTTKS should be considered for selecting therapeutic dose for scar prevention.

Presence and distribution of leptin and leptin receptor in the canine gallbladder.

The hormone leptin is produced by mature adipocytes and plays an important role in regulating food intake and energy metabolism through its interaction with the leptin receptor. In addition to roles in obesity and obesity-related diseases, leptin has been reported to affect the components and secretion of bile in leptin-deficient mice. Furthermore, gallbladder diseases such as cholelithiasis are known to be associated with serum leptin concentrations in humans. We hypothesized that the canine gallbladder is a source of leptin and that the leptin receptor may be localized in the gallbladder, where it plays a role in regulating the function of this organ. The aim of this study was to demonstrate the presence and expression patterns of leptin and its receptors in normal canine gallbladders using reverse transcriptase-PCR (RT-PCR) and immunohistochemistry. Clinically normal gallbladder tissue samples were obtained from four healthy beagle dogs with similar body condition scores. RT-PCR and sequencing of the amplified PCR products revealed the presence of leptin mRNA and its receptors in the gallbladder. Immunohistochemical investigations demonstrated the expression of leptin and its receptors in the luminal single columnar and tubuloalveolar glandular epithelial cells. In conclusion, the results of this study demonstrated the presence of leptin and its receptors in the gallbladders of dogs. Leptin and its receptor were both localized throughout the cytoplasm of luminal and glandular epithelial cells. These results suggested that the gallbladder is not only a source of leptin, but also a target of leptin though autocrine/paracrine mechanisms. The results of this study could increase the understanding of both the normal physiological functions of the gallbladder and the pathophysiological mechanisms of gallbladder diseases characterized by leptin system dysfunction.

Using reverse docking for target identification and its applications for drug discovery.

INTRODUCTION: In contrast to traditional molecular docking, inverse or reverse docking is used for identifying receptors for a given ligand among a large number of receptors. Reverse docking can be used to discover new targets for existing drugs and natural compounds, explain polypharmacology and the molecular mechanism of a substance, find alternative indications of drugs through drug repositioning, and detecting adverse drug reactions and drug toxicity. AREAS COVERED: In this review, the authors examine how reverse docking methods have evolved over the past fifteen years and how they have been used for target identification and related applications for drug discovery. They discuss various aspects of target databases, reverse docking tools and servers. EXPERT OPINION: There are several issues related to reverse docking methods such as target structure dataset construction, computational efficiency, how to include receptor flexibility, and most importantly, how to properly normalize the docking scores. In order for reverse docking to become a truly useful tool for the drug discovery, these issues need to be adequately resolved.

Inhibition of fibrotic contraction by C-phycocyanin through modulation of connective tissue growth factor and α-smooth muscle actin expression.

The effects of C-phycocyanin (C-pc), a phycobiliprotein, on the expression of pro-fibrotic mediators in hyper-tropic scarring such as connective tissue growth factor (CTGF) and α-smooth muscle actins (α-SMA) were investigated in relation to trans-differentiation of fibroblast to myo-fibroblast, an icon of scar formation. C-pc was isolated from Spirulina Platensis extract using sonication method and C-pc concentration was determined by Bennet and Bogorad equation. α-SMA and CTGF levels in wounded primary human dermal fibroblasts were determined by western blot analysis and immuno-fluorescence confocal microscope was employed. Fibroblast contractility was examined by three-dimensional collagen lattice contraction assay. There was an elevation of α-SMA (121%) and CTGF (143%) levels in wound cells as compared with non-wound cells. The does-response profiles of down regulation demonstrated that the maximum inhibitions of α-SMA by 63% (p<0.05) and CTGF by 50% (p<0.1) were achieved by C-pc (6 nM) treated cells. In confocal assay, non-wound fibroblasts exhibited basal level of α-SMA staining, while wounded cells without C-pc treatment showed strong up-regulation of α-SMA by 147% (p<0.05). C-pc (6 nM) inhibited α-SMA expression by 70% (p<0.05) and reduced collagen contraction by 29% (p<0.05). C-pc seemed to lessen the over expression of CTGF, α-SMA, subsequently alleviating the fibrotic contracture. This study suggests the potential application of C-pc to regulation of the expression of pro-fibrotic mediators in scarring process and its potential usage as an efficient means for anti-fibrosis therapy.