Purified fish skin collagen hydrolysate attenuates TNF-α induced barrier dysfunction in-vitro and DSS induced colitis in-vivo model.

Inflammatory mediators are key components in establishing pathogenesis in inflammatory bowel disease. Balanced expression of anti-inflammatory and pro-inflammatory cytokines is an important cue in maintaining gut native and adaptive immunity. In the present study, purified hydrolysate fraction of fish skin collagen from Clarias batrachus and Pangasius pangasius was evaluated as a treatment agent against TNF-α induced barrier dysfunction in Caco-2 cell line model and DSS induced colitis in mice model. Cell adhesion on purified hydrolysate fraction coated surfaces was found to be enhanced with increasing concentration in both Clarias batrachus and Pangasius pangasius. Alkaline phosphatase activity was enhanced in a concentration-dependent manner. The paracellular permeability assay demonstrated that Pangasius pangasius purified hydrolysate fraction had countered TNF-α induced barrier dysfunction. Analysis of the tight junction proteins (occludin, zonulae occluden, and claudin) by RT PCR, immunofluorescence, and western blot, further confirmed the effectiveness of Pangasius pangasius purified hydrolysate fraction against TNF-α. The Pangasius pangasius purified hydrolysate fraction was further evaluated for efficacy in DSS-induced colitis mice model. Two concentration of Pangasius pangasius purified hydrolysate was chosen based on in-vitro experiments, 80 µg/kg and 200 µg/kg BW of Balb/C male mice administered through intra-rectal route along with fish skin collagen 80 µg/kg BW. Pangasius pangasius purified hydrolysate fraction treatment improved the clinical signs of colitis such as body weight, rectal bleeding, colon length, and stool consistency caused by DSS administration. Immunofluorescence of colon tissue section showed that Pangasius pangasius purified hydrolysate fraction enhanced the expression of occludin protein. This study hints at the use of Pangasius pangasius purified hydrolysate fraction as a potential nutraceutical or treatment agent in healing ulcers of the mucosa.

Biocompatible Films of Collagen-Procyanidin for Wound Healing Applications.

The study investigated the effect of polyphenols present in Cassia auriculata (CA) leaves in enhancing the stability of the collagen protein and the wound healing potential of collagen films. The crude ethanol extract of CA was analyzed for the presence of phytochemicals and purified by column chromatography using solvents with increasing polarity. The ethanol eluted active fractions (EEAF) that precipitated gelatin was characterized using HP-TLC, FTIR spectroscopy, ESI-FT-MS/MS, and 1H NMR spectroscopy. The active compound was identified to be procyanidin B belonging to the proanthocyanidins group. The wound healing property of EEAF and collagen type I extracted from Clarias batrachus fish skin and the bovine tendon was assessed by in vitro scratch assay on L929 mice fibroblast cell lines. The EEAF-treated collagen coating enhanced in vitro wound closure in comparison with the uncoated dish. It was observed that EEAF treatment improved the physical strength of collagen films. The in vivo wound healing of the EEAF-treated collagen film was examined in male Wister rats and the wound site tissues were assessed. In vivo wound examination showed enhanced healing with EEAF incorporated collagen films. Comparatively, the EEAF-treated bovine tendon collagen films showed improved physical properties and better wound healing property than fish collagen films.

Adhesion and proliferation properties of type I collagen-derived peptide for possible use in skin tissue engineering application.

The surface properties of three-dimensional scaffolds are improved by coating or covalently linking certain adhesion-promoting proteins or peptides. In the present study, the effect of type I collagen-derived peptide (GKNGDDGEA) on adhesion and proliferation of HaCaT keratinocytes and NIH3T3 murine fibroblast cell lines was studied to assess its suitability for possible skin tissue engineering applications. Cell adhesion and proliferation of HaCaT and NIH3T3 were found to be enhanced by peptide coating. The optimum peptide coating densities to obtain the best cell adhesion and proliferation were found to be 0.827 µmoles/cm2 and 0.62 µmoles/cm2 for HaCaT and NIH3T3, respectively. Cell adhesion, in the presence of anti-integrin α1 antibody, inhibited attachment of NIH3T3 cells indicating the involvement of integrin α1 receptor. However, the attachment of HaCaT cells was not affected by anti-integrin treatment. The higher expression of paxillin confirmed the effect of the peptide in mediating focal adhesion kinases (FAKs) in cell adhesion and proliferation. Gene expression analysis was performed on cell migration proteins like Rho, Rac, Cdc42, integrin receptor α1, and ß1, and the extracellular matrix modulating proteins like MMP2, TIMP, and COL1A1 to validate their role on the peptide-mediated cell proliferation. Immunofluorescence analysis showed the distribution and localisation of phospho-FAK on cells cultured on the peptide-coated surfaces. Results support the role of peptides in enhancing cell adhesion and proliferation properties.

Role of fish collagen hydrolysate in attenuating inflammation-An in vitro study.

Collagen hydrolysate, an extensively used protein obtained from different sources, has various beneficial effects on human health and diseases. The benefits of collagen hydrolysate are well known and presently varied sources for the preparation of hydrolysate are being investigated. Food as a therapy to combat inflammation is presently a much-focused field of research. The present study aims at screening the anti-inflammatory property of collagen hydrolysate from the skin of Cypselurus melanurus, Catla catla, Indian mackerel, Clarias batrachus (Cb), and Pangasius pangasius (Pp) in activated RAW 264.7 macrophage cells. The fractions, Cb (C2) and Pp (P2) with anti-inflammatory property obtained after two-step chromatographic purification contained peptides in the range of 1-3 kDa molecular weight. The active fractions C2 and P2 showed a reduction in gene expression of TNF-α to 1.6- and 1-fold difference, whereas IL6 expression to 30- and 40-fold difference, respectively, in comparison to LPS treatment. The suppression of inflammatory proteins (TNF-α, IL6, NFκB, and p-IκB) by fractions C2 and P2 confirmed the anti-inflammatory activity. PRACTICAL APPLICATIONS: Collagen hydrolysate and its derived low molecular weight peptides are of great interest in the field of nutraceuticals and biomedical applications. The purified peptide fraction of fish skin hydrolysate displayed a promising anti-inflammatory property. The collagen hydrolysate of Cb and Pp can be a functional food or its purified fraction used as a nutraceutical supplementation due to their anti-inflammatory property in the cellular microenvironment.

Protein Expression Profiling Identifies Key Proteins and Pathways Involved in Growth Inhibitory Effects Exerted by Guggulsterone in Human Colorectal Cancer Cells.

Colorectal cancer (CRC) is a leading killer cancer worldwide and one of the most common malignancies with increasing incidences of mortality. Guggulsterone (GS) is a plant sterol used for treatment of various ailments such as obesity, hyperlipidemia, diabetes, and arthritis. In the current study, anti-cancer effects of GS in human colorectal cancer cell line HCT 116 was tested, potential targets identified using mass spectrometry-based label-free shotgun proteomics approach and key pathways validated by proteome profiler antibody arrays. Comprehensive proteomic profiling identified 14 proteins as significantly dysregulated. Proteins involved in cell proliferation/migration, tumorigenesis, cell growth, metabolism, and DNA replication were downregulated while the protein with functional role in exocytosis/tumor suppression was found to be upregulated. Our study evidenced that GS treatment altered expression of Bcl-2 mediated the mitochondrial release of cytochrome c which triggered the formation of apoptosome as well as activation of caspase-3/7 leading to death of HCT 116 cells via intrinsic apoptosis pathway. GS treatment also induced expression of p53 protein while p21 expression was unaltered with no cell cycle arrest. In addition, GS was found to inhibit NF-kB signaling in colon cancer cells by quelling the expression of its regulated gene products Bcl-2, cIAP-1, and survivin.

Etiology and clinical recommendations to manage the complications following lingual frenectomy: A critical review.

Ankyloglossia, also known as tongue-tie, is an embryological anatomical malformation of the tongue, characterized by an abnormally short and a thick lingual frenum. Tongue-tie restricts the physiologic movements of the tongue and results in various functional, behavioral and speech abnormalities along with the development of frontal and lateral lisps. Ankyloglossia in infants is also linked with the difficulty in breastfeeding difficulty, gagging, choking or vomiting food, delayed development or deterioration of speech and behavioral issues. A lingual frenectomy is a common oral surgical procedure done to correct an ankylosed lingual frenum by severing the abnormal frenal attachment on the ventral surface of the tongue. However, lingual frenectomy is associated with few complications that should be addressed to achieve a good overall prognosis. Though a lot of research is available on the various techniques and rationale to correct ankyloglossia, no paper has yet highlighted the surgical complications associated with lingual frenectomy. Therefore, the present paper for the first time review and highlight the common intraoperative and postoperative complications following lingual frenectomy.

A potential bioactive peptide candidate for biomaterial and tissue engineering applications.

The surface modification of biomaterials with matrikines for tissue engineering application is one of the recent approaches to improve their biocompatibility. In an earlier study, a peptide containing 21 amino acid isolated from bovine tendon collagen was shown to promote good cell adhesion in HeLa cell, and a smaller region in the peptide was identified using bioinformatics tool to mediate cell-peptide interaction. Hence, the present study was undertaken to validate the cell adhesion property of the smaller region of the peptide and elucidate probable peptide-cell interaction pathway. Cell adhesion and proliferation properties of the peptide were studied on cells cultured on surfaces coated with varying concentrations of peptide. Expression of focal adhesion related proteins like paxillin and pFAK Tyr397 was confirmed by immunoblotting and immunofluorescence microscopy respectively. The anti-pFAK Tyr 397 stained confocal micrographs and mRNA transcription levels of Cdc42 and Rho further confirmed peptide mediated cell spreading. The change in the expression levels of integrin α1 and ß1 indicates an integrin mediated cell-peptide interaction for cell survival and proliferation. Integrin mediated adhesion was further confirmed by anti-integrin blocking assay. The modulation of ECM components by the peptide was assessed by expression of COL1A1, TIMP mRNA levels and gelatin zymography for MMPs. The results of the study confirm the role of the small region of the larger collagen peptide in cell adhesion and proliferation and hint at the possible use of such small peptides as biocompatible surface modifiers for tissue scaffolds.

Matrikines for therapeutic and biomedical applications.

Matrikines, peptides originating from the fragmentation of extracellular matrix proteins are identified to play important role in both health and disease. They possess biological activities, much different from their parent protein. Identification of such bioactive cryptic regions in the extracellular matrix proteins has attracted the researchers all over the world in the recent decade. These bioactive peptides could find use in preparation of biomaterials and tissue engineering applications. Matrikines identified in major extracellular matrix (ECM) proteins like collagen, elastin, fibronectin, and laminin are being extensively studied for use in tissue engineering and regenerative medicine. They are identified to modulate cellular activity like cell growth, proliferation, migration and may induce apoptosis. RGD, a well-known peptide identified in fibronectin with cell adhesive property is being investigated in designing biomaterials. Collagen hexapeptide GFOGER was found to promote cell adhesion and differentiation. Laminin also possesses regions with strong cell adhesion property. Recently, cell-penetrating peptides from elastin are used as a targeted delivery system for therapeutic drugs. The continued search for cryptic sequences in the extracellular matrix proteins along with advanced peptide coupling chemistries would lead to biomaterials with improved surface properties. This review article outlines the peptides derived from extracellular matrix and some of the possible applications of these peptides in therapeutics and tissue engineering applications.

Serum activin B concentration as predictive biomarker for ectopic pregnancy.

We evaluated the diagnostic accuracy of activin B in discriminating tubal ectopic pregnancy (tEP) from intrauterine miscarriages (IUM), and normal viable intrauterine pregnancy (IUP). We included 28 women with tEP, 31 women with IUM, and 29 normal IUP, confirmed both by clinical examination and ultrasonography. Serum activin B concentration was measured at the time of admission using the ELISA kit. The median serum activin B concentration was found to be significantly decreased in both tEP (p=0.004) and IUM (p=0.022) compared to normal IUP. When compared between tEP and IUM, activin B concentrations did not differ significantly. ROC analysis of activin B and free ß-hCG demonstrated AUC of 0.722 and 0.805, respectively to discriminate tEP from viable IUP. The model including both activin B and free ß-hCG improved the discriminating potential with greater AUC (0.824), and specificity (93%) than individual one. To discriminate tEP from IUM, activin B, free ß-hCG and combination of both performed poorly. We conclude that serum activin B concentration is lower in tubal ectopic pregnancy, and can discriminate it from normal pregnancy with moderate accuracy. It also shows improved diagnostic potential along with free ß-hCG, but cannot distinguish tEP from IUM reliably.

Clinical importance of median mandibular flexure in oral rehabilitation: a review.

The mandible has a property to flex inwards around the mandibular symphysis with change in shape and decrease in mandibular arch width during opening and protrusion of the mandible. The mandibular deformation may range from a few micrometres to more than 1 mm. The movement occurs because of the contraction of lateral pterygoid muscles that pulls mandibular condyles medially and causes a sagittal movement of the posterior segments. This movement of mandible can have a profound influence on prognosis and treatment outcome for various restorative, endodontics, fixed, removable and implant-related prosthesis. The review unfolds the causes, importance and clinical implications of median mandibular flexure in oral rehabilitation. This review also highlights the appropriate preventive measures and techniques that should be adopted by clinicians to minimise the effect of flexural movement of the jaw during oral rehabilitation. This would not only help clinicians to achieve a good prosthesis with accurate fit and longevity but also maintain the health of the surrounding periodontal or periimplant gingival tissues and bone.

Sequential Percutaneous LAA Ligation and Pulmonary Vein Isolation in Patients with Persistent AF: Initial Results of a Feasibility Study.

INTRODUCTION: Left atrial appendage (LAA) ligation results in LAA electrical isolation and a decrease in atrial fibrillation (AF) burden. This study assessed the feasibility of combined percutaneous LAA ligation and pulmonary vein isolation (PVI) in patients with persistent AF. METHODS AND RESULTS: A total of 22 patients with persistent AF underwent LAA ligation with the LARIAT device followed by PVI. PVI was confirmed with the demonstration of both entrance and exit block. Patients (n = 10) in sinus rhythm pre- and post-LAA ligation underwent P-wave analysis. Monitoring for AF was performed at 1, 3, and 6 months postablation. LAA ligation was successful in 21 of 22 (95%) patients. The procedure was aborted in one patient due to pericardial adhesions. PVI was performed in 20 of 21 patients. One patient converted to atrial flutter with a controlled ventricular response after LAA ligation and refused subsequent PVI. Demonstration of entrance and exit block was achieved in 19 of 20 patients. At 3 months, 13 of 19 (68.4%) patients were in sinus rhythm. Four patients underwent a second PVI. At 6 months, 15 of 20 (75%) patients were in sinus rhythm. There was a significant decrease in P-wave duration and P-wave dispersion after LAA ligation. Complications with LAA ligation included pericarditis, a delayed pleural effusion, and a late pericardial effusion. CONCLUSIONS: Staged LAA ligation and PVI is feasible and decreases P-wave dispersion. Randomized studies are needed to assess the efficacy of LAA ligation as adjunctive therapy to PVI for maintaining sinus rhythm in patients with persistent AF.

Electroplated porous polypyrrole nanostructures patterned by colloidal lithography for drug-delivery applications.

Porous nanostructures of polypyrrole (Ppy) were fabricated using colloidal lithography and electrochemical techniques for potential applications in drug delivery. A sequential fabrication method was developed and optimized to maximize the coverage of the Ppy nanostructures and to obtain a homogeneous layer over the substrate. This was realized by masking with electrophoretically-assembled polystyrene (PS) nanospheres and then electroplating. Drug/biomolecule adsorption and the release characteristics for the porous nanostructures of Ppy were investigated using rhodamine B (Rh-B). Rh-B is an easily detectable small hydrophobic molecule that is used as a model for many drugs or biological substances. The porous Ppy nanostructures with an enhanced surface area exhibited higher Rh-B loading capacity than bulk planar films of Ppy. Moreover, tunability of surface morphology for further applications (e.g., sensing, cell adhesion) was demonstrated.

Sunitinib in urothelial cancer: clinical, pharmacokinetic, and immunohistochemical study of predictors of response.

BACKGROUND: Sunitinib has activity in patients with metastatic urothelial cancer (UC), but most patients do not respond. OBJECTIVE: To identify predictors of response to sunitinib. DESIGN, SETTING, AND PARTICIPANTS: Seventy-seven patients with advanced UC received sunitinib on one of two schedules at a single institution. Blood pressure (BP), immunohistochemistry (IHC), and pharmacokinetic (PK) results were correlated with response to sunitinib. MEASUREMENTS: BP was assessed on day 1 and 28 of each cycle and on day 14 of cycle 1. IHC was performed on 55 samples from 38 cases using mammalian target of rapamycin and hypoxia-inducible factor (HIF) pathway marker antibodies. Blood samples for PK analysis were collected from 15 patients at three time points. Response was assessed using Response Evaluation Criteria in Solid Tumors criteria. RESULTS AND LIMITATIONS: Sunitinib-induced hypertension predicted improved response when hypertension was categorized as a discrete (p = 0.02) or continuous variable (p = 0.005 [systolic BP] and p = 0.007 [diastolic BP]). The odds ratio of response was 12.5 (95% confidence interval, 1.95-246.8) for grade 3/4 hypertension compared with grade 0. Response was associated with low HIF-1α expression in primary (p = 0.07) tissue. A nonstatistically significant trend was seen for an association between greater drug concentration and best response. A correlation between expression markers within the same pathways was identified, phosphorylated-4EBP1 and phosphorylated-S6 (p = 6.5 × 10(-9)), and vascular endothelial growth factor receptor 2 and HIF-1α (p = 0.008). Results are limited by small numbers. CONCLUSIONS: Clinical and molecular biomarkers of response to sunitinib may have clinical relevance and require prospective validation. There is an urgent need for predictive biomarkers to guide the management of UC.

Structural and magnetic characterization of batch-fabricated nickel encapsulated multi-walled carbon nanotubes.

We report on the growth and fabrication of Ni-filled multi-walled carbon nanotubes (Ni-MWNTs) with an average diameter of 115 nm and variable length of 400 nm-1 µm. The Ni-MWNTs were grown using template-assisted electrodeposition and low pressure chemical vapor deposition (LPCVD) techniques. Anodized alumina oxide (AAO) templates were fabricated on Si using a current controlled process. This was followed by the electrodeposition of Ni nanowires (NWs) using galvanostatic pulsed current (PC) electrodeposition. Ni NWs served as the catalyst to grow Ni-MWNTs in an atmosphere of H2/C2H2 at a temperature of 700 °C. Time dependent depositions were carried out to understand the diffusion and growth mechanism of Ni-MWNTs. Characterization was carried out using scanning electron microscopy (SEM), focused ion beam (FIB) milling, transmission electron microscopy (TEM), Raman spectroscopy and energy dispersive x-ray spectroscopy (EDX). TEM analysis revealed that the Ni nanowires possess a fcc structure. To understand the effects of the electrodeposition parameters, and also the effects of the high temperatures encountered during MWNT growth on the magnetic properties of the Ni-MWNTs, vibrating sample magnetometer (VSM) measurements were performed. The template-based fabrication method is repeatable, efficient, enables batch fabrication and provides good control on the dimensions of the Ni-MWNTs.

Cognition in type 2 diabetes: Association with vascular risk factors, complications of diabetes and depression.

BACKGROUND: The role of variables like duration of diabetes, diabetic control and microvascular complications in the causation of cognitive decline in patients with type 2 diabetes is not well studied. The contribution of hypertension to the cognitive decline in nondemented diabetic patients is unclear. AIMS: We wanted to see if cognition in patients with type 2 diabetes is associated with the duration of diabetes, control of diabetes, complications of diabetes, vascular risk factors, or depression. We also looked at association of noncompliance with cognition, and depression. SETTINGS AND DESIGN: We recruited ambulant patients with type 2 diabetes who are 55 years or more in age from the weekly diabetic clinic. We excluded patients with past history of stroke. METHODS AND MATERIAL: We selected the time taken for the Trial A test, delayed recall on ten-word list from Consortium to Establish a Registry for Alzheimer's Disease (CERAD), Rowland Universal Dementia Assessment Scale (RUDAS) and Centre for Epidemiologic Studies Depression scale (CES-D) screening instrument to assess these patients. STATISTICAL ANALYSIS USED: We utilized mean, standard deviation, Chi-square test and Pearson's correlation for statistical analysis. We considered P < 0.05 to be significant. RESULTS: RUDAS scores inversely correlated (r = -0.360) with CES-D scores (P = 0.002). Scores of the screening instrument for depression, the CES-D was associated with the duration of diabetes mellitus (P = 0.018), fasting blood glucose (P = 0.029) as well as with 2-hour post prandial blood glucose (P = 0.017). CONCLUSIONS: There is correlation between depression and global cognitive score. Depression seems to be associated with duration of diabetes and control of diabetes.

Ethnobotanical investigations among tribes in Madurai District of Tamil Nadu (India).

BACKGROUND: An ethnobotanical survey was carried out to collect information on the use of medicinal plants in Southern Western Ghats of India (Madurai district, Tamil Nadu). Information presented in this paper was gathered from the paliyar tribes using an integrated approach of botanical collections, group discussions and interviews with questionnaires in the years 1998 - 1999. The informants interviewed were 12 among whom 4 were tribal practitioners. RESULTS: A total of 60 ethnomedicinal plant species distributed in 32 families are documented in this study. The medicinal plants used by paliyars are listed with Latin name, family, local name, parts used, mode of preparation and medicinal uses. Generally, fresh part of the plant was used for the preparation of medicine. CONCLUSION: We observed that the documented ethnomedicinal plants were mostly used to cure skin diseases, poison bites, stomachache and nervous disorders. The results of this study showed that these tribal people still depend on medicinal plants in Madurai district forest areas.