Utility and diagnostic accuracy of intraoperative frozen sections in hepato-pancreato-biliary surgical pathology.

BACKGROUND AND PURPOSE: Hepato-pancreato-biliary (HPB) surgeries are one of the most challenging and complex procedures. Intraoperative frozen section (IFS) diagnosis plays a pivotal role in management decisions. Comprehensive large cohort studies evaluating utility of IFS in HPB malignancies are lacking. This study aimed to evaluate the accuracy of frozen section analysis and to analyse discrepancies and impact of IFS on the surgical decisions. PATIENTS AND METHODS: This was a retrospective study of IFS received for the HPB specimens between years 2009 and 2021. The results were compared to the permanent sections to evaluate diagnostic accuracy, sensitivity and specificity. Indications, disagreements and impact on the surgical management were analysed. RESULTS: A total of 1008 specimens were evaluated: bile duct margin (279; 27.7%), gallbladder (203; 20.1%), liver lesions (125 cases; 12.4%), lymph nodes (147; 14.6%), pancreatic margin (120; 11.9%) and deposits (134; 13.3%). IFS were diagnosed as negative for malignancy (805; 79.9%), positive for dysplasia (8; 0.8%), suspicious for malignancy (6; 0.6%) and positive for malignancy (189; 18.8%). The overall diagnostic accuracy was 98.4%, and the discordant rate was 1.6%. The sensitivity, specificity, positive predictive value and negative predictive value were 94.7%, 99.4%, 97.5% and 98.6% respectively. The most important reason of discordant results was technical, followed by interpretational and sampling errors. CONCLUSION: The study demonstrates high diagnostic accuracy (98.4%) of IFS in a large dataset of HPB specimens. This comprehensive analysis apprises of the indications, errors and the impact of IFS diagnosis on subsequent HPB surgical management.

Operational Tolerance after Liver Transplantation: First Report from India.

Operational tolerance after liver transplantation is an ideal goal to avoid long-term morbidities associated with chronic immunosuppressive medication use. It is achievable in a highly selected group of post-transplant recipients but requires long-term follow-up and strict monitoring. We hereby report a post-transplant case who achieved spontaneous operational tolerance after inadvertent immunosuppression withdrawal.

Balloon angioplasty: A promising adjunct to arteriovenous fistula creation compared with hydrostatic dilatation in small-caliber cephalic veins.

BACKGROUND: To maintain the patency and longevity of arteriovenous fistula, the availability of a venous segment with adequate diameter is important. In Indian population, many chronic kidney disease patients have poor caliber veins. The study aimed to evaluate the efficacy of hydrostatic dilatation versus Primary balloon angioplasty of small caliber cephalic veins of (≤2.5 mm) preoperatively in terms of patency rate and maturation time of arteriovenous fistula. METHODS: Patients (n = 80) with an end-stage renal disease requiring arteriovenous access surgery for hemodialysis with small caliber cephalic veins were randomized into two groups, i.e., hydrostatic dilatation and primary balloon angioplasty, each with 40 patients. All patients underwent a thorough clinical examination as well as duplex ultrasound vein mapping of both upper extremities. Patients were followed up for six months and primary patency, maturation time, and complications were noted. RESULTS: Immediate technical success with good palpable thrill was achieved in 97.5% of patients in the primary balloon angioplasty group and 87.5% in the hydrostatic dilatation group. The fistula maturation time in the primary balloon angioplasty group was 34.41 days and 46.18 days in the hydrostatic dilatation group. In the primary balloon angioplasty group, the primary patency of the fistula was 97.5% and 87.5% in the hydrostatic dilatation group, at six months. The arteriovenous fistula functioning rate was 77.5% in the hydrostatic dilatation group as compared to 92.5% in the primary balloon angioplasty group at six months. The incidence of surgical site infection was 5% in the primary balloon angioplasty group as compared to 10% in the hydrostatic dilatation group. CONCLUSION: Primary balloon angioplasty of small caliber cephalic veins (≤2.5 mm) performed prior to arteriovenous fistula creation for hemodialysis is a beneficial procedure.

Survival outcomes with 12 weeks of adjuvant or neoadjuvant trastuzumab in breast cancer.

Background: There is limited access to 1 year of adjuvant trastuzumab in resource-constrained settings. Most randomized studies have failed to prove non-inferiority of shorter durations of adjuvant trastuzumab compared to 1 year However, shorter durations are often used when 1 year is not financially viable. We report the outcomes with 12 weeks of trastuzumab administered as part of curative-intent treatment. Methods: This is a retrospective analysis of patients treated at Tata Memorial Centre, Mumbai, a tertiary care cancer center in India. Patients with human epidermal growth factor receptor (HER2)-positive early or locally advanced breast cancer who received 12 weeks of adjuvant or neoadjuvant trastuzumab with paclitaxel and four cycles of an anthracycline-based regimen in either sequence, through a patient assistance program between January 2011 and December 2012, were analyzed for disease-free survival (DFS), overall survival (OS), and toxicity. Results: A total of 102 patients were analyzed with a data cutoff in September 2019. The median follow-up was 72 months (range 6-90 months), the median age was 46 (24-65) years, 51 (50%) were postmenopausal, 37 (36%) were hormone receptor-positive, and 61 (60%) had stage-III disease. There were 37 DFS events and 26 had OS events. The 5-year DFS was 66% (95% Confidence Interval [CI] 56-75%) and the OS was 76% (95% CI 67-85%), respectively. Cardiac dysfunction developed in 11 (10.7%) patients. Conclusion: The use of neoadjuvant or adjuvant 12-week trastuzumab-paclitaxel in sequence with four anthracycline-based regimens resulted in acceptable long-term outcomes in a group of patients, most of whom had advanced-stage nonmetastatic breast cancer.

A Study on Diagnostic Evaluation of Two Different Rapid DNA Polymerase Chain Reaction Techniques Namely Gene Xpert Mycobacterium Tuberculosis/Rifampin (MTB/RIF) and Mycoreal Polymerase Chain Reaction in the Diagnosis of Endometrial Tuberculosis Considering Culture as Gold Standard.

CONTEXT: The study involves the evaluation of two polymerase chain reaction (PCR) techniques one of which has been endorsed by the WHO for their diagnostic capabilities. AIMS: The aim of this study is to evaluate the diagnostic accuracy of GeneXpert mycobacterium tuberculosis/Rifampin (MTB/RIF) and mycoreal PCR techniques in the diagnosis of endometrial tuberculosis (TB) considering culture as the gold standard. SETTINGS AND DESIGN: A retrospective study conducted at Gunasheela surgical and maternity hospital. Patients who attended the outpatient department between January 2013 and August 2016, satisfying the eligibility criteria, were included in the study. METHODOLOGY: Women included in the study underwent endometrial pipelle sampling premenstrually after ruling out pregnancy in that cycle. Endometrial samples were tested for TB by Mycoreal PCR, Gene Xpert and BACTEC culture. STATISTICAL ANALYSIS USED: Statistical analysis was done using the R software version 3.6.1. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of test were calculated. RESULTS: A total of 3229 samples were analyzed, of which 1754 were evaluated by Mycoreal TB PCR and 1475 were evaluated by Gene Xpert MTB/RIF assay. The sensitivity of mycoreal TB PCR technique was 34.78%, specificity was 99.08%, PPV was 33.33%, NPV was 99.13%, and accuracy was 98.23%. The sensitivity of GeneXpert MTB/RIF technique was 6.90%, specificity was 99.79%, PPV was 40.00%, NPV was 98.16%, and accuracy was 97.97%. CONCLUSIONS: MYCOREAL seemed to be more sensitive than Gene Xpert (MTB/RIF) considering culture as the gold standard in the diagnosis of endometrial TB.

Densitometric quantification for the validation of decolorization of Disperse Orange ERL by lichen Parmelia sp.

Densitometric high performance thin layer chromatography (HPTLC) quantification method was developed to validate the decolorization/biotransformation of Disperse Orange ERL and dye mixture by lichen Parmelia sp. which release several colored compounds during decolorization process, hence unable to use colorimetric estimation. Percent decolorization of Disperse Orange ERL and dye mixture by lichen Parmelia sp. was observed when estimated using developed HPTLC method. Limit of detection and limit of quantification for both dyes in mixture were obtained as 0.3 and 1 µg/µl, respectively. Area of peak of control Disperse Orange ERL was reduced by 43% after 12 h, 71% after 48 h and upto 82% after 72 h of incubation. Precision and repeatability of data elucidated the % relative standard deviation less than 3 for all the values thus indicating statistically acceptable. Biodegradation of dye and mixture was confirmed with Fourier transform infrared spectroscopy analysis, i.e., altered fingerprinting spectral pattern.

Decolorization and detoxification of dye mixture and textile effluent by lichen Dermatocarpon vellereceum in fixed bed upflow bioreactor with subsequent oxidative stress study.

Navy Blue HE22 (NBHE22), dye mixture and real textile effluent were decolorized and degraded by lichen Dermatocarpon vellereceum. Up-flow bioreactor showed about 80%, 70%, 80% and 65% removal of American dye manufacturer index (ADMI), biological oxygen demand (BOD), total suspended solids (TSS) and total dissolved solids (TDS), respectively of dye mixture at flow rate of 25mlh-1. The removal of ADMI, BOD, TSS and TDS of real textile effluent were 75%, 65%, 82% and 70%, respectively at flow rate of 30mlh-1. Significant induction of extracellular enzymes such as manganese peroxidase and lignin peroxidase was observed up to 46% and 36% during decolorization of dye mixture, while 43% and 24% during effluent treatment, respectively. Exponential enhancement in the activities of stress enzymes such as catalase (CAT) and guaiacol peroxidase (GPX) was observed after exposure to NBHE22 (116% and 125%, respectively), dye mixture (150% and 300%, respectively) and effluent (400% and 350%, respectively) endorsing the stress tolerance ability of model lichen. Phytotoxicity and genotoxicity studies demonstrated less toxic nature of metabolites resulted from biodegradation.

IFN-γ promotes transendothelial migration of CD4+ T cells across the blood-brain barrier.

Transendothelial migration (TEM) of Th1 and Th17 cells across the blood-brain barrier (BBB) has a critical role in the development of experimental autoimmune encephalomyelitis (EAE). How cytokines produced by inflammatory Th1 and Th17 cells damage the endothelial BBB and promote transendothelial migration of immune cells into the central nervous system (CNS) during autoimmunity is not understood. We therefore investigated the effect of various cytokines on brain endothelial cells. Among the various cytokines tested, such as Th1 (IFN-γ, IL-1α, IL-1ß, TNF-α, IL-12), Th2 (IL-3, IL-4, IL-6 and IL-13), Th17 (IL-17A, IL-17F, IL-21, IL-22, IL-23, GM-CSF) and Treg-specific cytokines (IL-10 and TGF-ß), IFN-γ predominantly showed increased expression of ICAM-1, VCAM-1, MAdCAM-1, H2-Kb and I-Ab molecules on brain endothelial cells. Furthermore, IFN-γ induced transendothelial migration of CD4+ T cells from the apical (luminal side) to the basal side (abluminal side) of the endothelial monolayer to chemokine CCL21 in a STAT-1-dependent manner. IFN-γ also favored the transcellular route of TEM of CD4+ T cells. Multicolor immunofluorescence and confocal microscopic analysis showed that IFN-γ induced relocalization of ICAM-1, PECAM-1, ZO-1 and VE-cadherin in the endothelial cells, which affected the migration of CD4+ T cells. These findings reveal that the IFN-γ produced during inflammation could contribute towards disrupting the BBB and promoting TEM of CD4+ T cells. Our findings also indicate that strategies that interfere with the activation of CNS endothelial cells may help in controlling neuroinflammation and autoimmunity.

Corrigendum: Agro-Industrial Wastes for Production of Biosurfactant by Bacillus subtilis ANR 88 and Its Application in Synthesis of Silver and Gold Nanoparticles.

[This corrects the article on p. 492 in vol. 8, PMID: 28392783.].

Agro-Industrial Wastes for Production of Biosurfactant by Bacillus subtilis ANR 88 and Its Application in Synthesis of Silver and Gold Nanoparticles.

Biosurfactants, surface-active amphiphilic compounds, despite having a wide range of applications, have a high cost of production, which severely restricts their use. For cheaper production of biosurfactant, we investigated the potential of the indigenously isolated biosurfactant producing organism, Bacillus subtilis ANR 88, to grow on different cheap carbon sources (molasses, whey, and extracts of potato peels, orange peels, banana peels, and bagasse). We found that, B. subtilis ANR 88 used significant amounts of total sugar to produce cell biomass and biosurfactant. The biosurfactant production in minimal medium containing glucose as sole source of carbon was 0.207 g/l and the same with molasses as carbon source was 0.241 g/l. With whey as carbon source, isolate failed to produce biosurfactant. Amongst the extracts of the agro-wastes, the extracts of bagasse and orange peels gave 0.127 and 0.089 g/l of biosurfactant respectively. One-variable-at-a-time (OVAT) studies carried out to optimize the production of biosurfactant by B. subtilis ANR 88 resulted into maximum biosurfactant yield of 0.513 g/l in medium: molasses 4%, ammonium ferric citrate 0.25%, pH 7. Plackett-Burman design based statistical method for optimization increased the production of biosurfactant to 0.746 g/l, which is 3.6-fold of that produced on glucose. The biosurfactant produced by B. subtilis ANR 88 was analyzed by Fourier Transform Infrared Spectroscopy (FT-IR); it showed that the biosurfactant contained alkyl as well as peptide groups. The biosurfactant of B. subtilis ANR 88 was found effective in the synthesis of silver as well as gold nanoparticles in the total absence of conventional chemical reducing agents. Interestingly, nanoparticles produced were almost uniform in their size and shapes i.e., spherical silver (4-18 nm) and hexagonal gold nanoparticles (40-60 nm), as evident in TEM images.

Comparative study of epidural bupivacaine with butorphanol and bupivacaine with tramadol for postoperative pain relief in abdominal surgeries.

INTRODUCTION: To compare the efficacy of combination of epidural local anesthetic with tramadol and butorphanol in major abdominal surgeries. AIMS: To evaluate duration of analgesia, analgesic efficacy, and safety profile of two groups of drugs-epidural butorphanol with bupivacaine and epidural tramadol with bupivacaine. MATERIALS AND METHODS: A prospective, randomized controlled, double-blinded study was undertaken in 50 patients scheduled for major abdominal surgeries. Group B received epidural butorphanol 2 mg + bupivacaine 0.125% first dose and subsequent doses, butorphanol 1 mg + bupivacaine 0.125% (total volume 10 ml). Group T received epidural tramadol 2 mg/kg + bupivacaine 0.125% first dose and subsequent doses, tramadol 1 mg/kg + bupivacaine 0.125% (total volume 10 ml). Observed parameters were the quality of analgesia, sedation, and hemodynamic parameters in the intra and post-operative period. Time for request of rescue analgesia was noted in all the patients. Continuous data are analyzed by Student's t-test using IBM SPSS software version 20. P ≤0.05 was considered to be statistically significant. P ≤ 0.001 was considered to be statistically highly significant. RESULTS: Visual analog scale better with butorphanol group than tramadol (0.12 ± 0.332 and 0.84 ± 0.746 for Group B and Group T) at 30 min after first dose. Onset of action (8.44 ± 1.158 min in Group B and 12.80 ± 1.354 min in Group T) faster with butorphanol but duration of analgesia longer with tramadol (5.92 ± 0.76 h in Group B vs. 7.68 ± 0.76 h in Group T). Sedation was seen in patients with butorphanol group. Nausea and vomiting more frequent with tramadol group. CONCLUSIONS: Epidural tramadol with antiemetic is better than butorphanol for its longer duration in ambulatory surgery, elderly patients, obese patients, and suitable high-risk patients.

Capsicum annuum proteinase inhibitor ingestion negatively impacts the growth of sorghum pest Chilo partellus and promotes differential protease expression.

BACKGROUND: Chilo partellus is an important insect pest infesting sorghum and maize. The larvae internalize in the stem, rendering difficulties in pest management. We investigated the effects of Capsicum annuum proteinase inhibitors (CanPIs) on C. partellus larvae by in-vitro and in-vivo experiments. METHODS: Recombinant CanPI-7 (with four-Inhibitory Repeat Domains, IRDs), -22 (two-IRDs) and insect proteinase activities were estimated by proteinase assays, dot blot assays and in gel activity assays. Feeding bioassays of lab reared C. partellus with CanPI-7 and -22 were performed. C. partellus proteinase gene expression was done by RT-PCR. In-silico structure prediction of proteinases and CanPI IRDs was carried out, their validation and molecular docking was done for estimating the interaction strength. RESULTS: Larval proteinases of C. partellus showed higher activity at alkaline pH and expressed few proteinase isoforms. Both CanPIs showed strong inhibition of C. partellus larval proteinases. Feeding bioassays of C. partellus with CanPIs revealed a dose dependent retardation of larval growth, reduction of pupal mass and fecundity, while larval and pupal periods increased significantly. Ingestion of CanPIs resulted in differential up-regulation of C. partellus proteinase isoforms, which were sensitive to CanPI-7 but were insensitive to CanPI-22. In-silico interaction studies indicated the strong interaction of IRD-9 (of CanPI-22) with Chilo proteinases tested. CONCLUSIONS: Of the two PIs tested, CanPI-7 prevents induction of inhibitor insensitive proteinases in C. partellus so it can be explored for developing C. partellus tolerance in sorghum. GENERAL SIGNIFICANCE: Ingestion of CanPIs, effectively retards C. partellus growth; while differentially regulating the proteinases.

Ways and Means for the Infusion of Bioactive Constituents in Solid Foods.

The development and consumption of functional food, or foods that promote health not merely basic nutrition, is on rise. In recent years, industrial and consumer interests have focused on developing foods supplemented with bioactive constituents that provide greater physiological benefits. The direct addition of these components to liquid or fabricated solid foods has led to a wide range of new products appearing on the market. Osmotic dehydration, an operation in which food stuff is soaked in solution of low water activity, has been reported as a suitable technology for formulating new products because of the twofold effect that it has on food where it partially removes water and impregnates the food pieces (solid food matrix) with solutes from the osmotic solution. The article focuses on the impregnation of bioactive constituents having added advantage to human health such as antioxidants, minerals, vitamins, and probiotics. The infusion of enzymes and aroma also has been discussed. Application of ultrasound, vacuum, high pressure, and/or atmospheric impregnation techniques appears to be the feasible technologies for impregnation of solid food matrix for the incorporation of bioactive ingredients.

Investigation of selective sensing of a diamine for aldehyde by experimental and simulation studies.

An organic molecule--o-phenylene diamine (OPD)--is selected as an aldehyde sensing material. It is studied for selectivity to aldehyde vapours both by experiment and simulation. A chemiresistor based sensor for detection of aldehyde vapours is fabricated. An o-phenylene diamine-carbon black composite is used as the sensing element. The amine groups in the OPD would interact with the carbonyl groups of the aldehydes. The selectivity and cross-sensitivity of the OPD-CB sensor to VOCs--aldehyde, ketone and alcohol--are studied. The sensor shows good response to aldehydes compared to other VOCs. The higher response for aldehydes is attributed to the interaction of the carbonyl oxygen of aldehydes with -NH2 groups of OPD. The surface morphology of the sensing element is studied by scanning electron microscopy. The OPD-CB sensor is responsive to 10 ppm of formaldehyde. The interaction of the VOCs with the OPD-CB nanocomposite is investigated by molecular dynamics studies. The interaction energies of the analyte with the OPD-CB nanocomposite were calculated. It is observed that the interaction energies for aldehydes are higher than those for other analytes. Thus the OPD-CB sensor shows selectivity to aldehydes. The simulated radial distribution function is calculated for the O-H pair of analyte and OPD which further supports the finding that the amine groups are involved in the interaction. These results suggest that it is important and easy to identify appropriate sensing materials based on the understanding of analyte interaction properties.

Lichen Permelia perlata: a novel system for biodegradation and detoxification of disperse dye Solvent Red 24.

Lichen is a self-supporting symbiotic association of fungi and algae which was not yet explored for its bioremediation potential. Lichen Permelia perlata showed potential of decolorization and biodegradation of Solvent Red 24 (SR24). Optimum pH and temperature for decolorization was found to be 8 and 50°C, respectively. Induction in the activity of laccase in P. perlata during biodegradation of SR24 showed their involvement. HPTLC, FTIR and GC-HRMS analysis confirmed biodegradation of SR24 in to metabolites such as naphthalen-1-yldiazene, naphthalene, 1-(2-methylphenyl)-2-phenyldiazene and diphenyldiazene. Phytotoxicity and genotoxicity analysis revealed the reduction in toxicity of SR24 after its biodegradation.

Potentiality of Bacillus subtilis as biocontrol agent for management of anthracnose disease of chilli caused by Colletotrichum gloeosporioides OGC1.

A soil bacterium, Bacillus subtilis, isolated from the rhizosphere of Chilli, showed high antagonistic activity against Colletotrichum gloeosporioides OGC1. A clear inhibition zone of 0.5-1 cm was observed in dual plate assay. Microscopic observations showed a clear hyphal lysis and degradation of fungal cell wall. In dual liquid cultures, the B. subtilis strain inhibited the C. gloeosporioides up to 100 % in terms of dry weight. This strain also produced a clear halo region on chitin agar medium plates containing 0.5 % colloidal chitin, indicating that it excretes chitinase. The strain also produced other mycolytic enzymes-glucanase and cellulase, demonstrated by a clear zone of hydrolysis of yeast cell wall glucan (YCW 0.1 % v/v) and carboxymethylcellulose (CMC 0.1 % v/v). In liquid cultures, the strain showed appreciable levels of chitinase, glucanase and cellulase activities and hydrolytic activity with C. gloeosporioides OGC1 mycelia as the substrate. The role of the B. subtilis strain in suppressing the fungal growth in vitro was studied in comparison with a UV mutant of that strain, which lacked both antagonistic and hydrolytic activity. The mycolytic enzyme mediated antagonism of B. subtilis was further demonstrated by heat inactivation (70-100 °C), treatment with trypsin and TCA of the crude enzyme extract which lacked antifungal property also. Treatment of the chilli seeds with Bacillus sp. culture showed 100 % germination index similar to the untreated seeds. The treatment of the seed with co-inoculation of the pathogen with Bacillus sp. culture showed 65 % reduction in disease incidence by the treatment as compared to the seed treated with pathogen alone (77.5 %).

Simultaneous imaging of microRNA or mRNA territories with protein territory in mammalian cells at single cell resolution.

Cellular mechanisms that inhibit mRNA translation by regulatory molecules involving microRNAs (miRNAs), a class of noncoding RNAs (ncRNAs), are well recognized in recent days. However, methodologies that measure these changes in cell populations lack the capabilities to observe such effects at single cell resolution. This is mostly due to the low level of transcript abundance and the heterogeneity of cell populations, together with the inability to measure transcripts and proteins at the same time. Here, we combine an in situ TaqMan PCR method with immunostaining so as to amplify low abundance transcripts in cellular compartments and image these efficiently at single cell resolution. The method offers flexibility to end-users for further fine-tuning of this optimized protocol based on the number of PCR cycles for individual genes in any cell type. After immunostaining, confocal microscopy is performed to detect the fluorescence of TaqMan probes (representing amplified transcripts/miRNA) and fluorophores tagged to antibodies (representing proteins) simultaneously. The presented technique offers an important tool to understand functional genomics as well as molecular mechanism of transcriptional and translational regulation so as to map these at single cell resolution.

Cellular detection of multiple antigens at single cell resolution using antibodies generated from the same species.

Biology at a cellular level comes with a great amount of heterogeneity. It is now evident that even clonally propagated cells in an in vitro population do not express the same set of cellular epitopes. The vascular endothelial as well as blood cells show a very high degree of heterogeneity in expression of specific proteins. Although several methods exist for identification of genome or transcriptome from a single cell, there is still limited advancement in detection of multiple cellular antigens in a single cell. This has been mainly due to the limited availability of different antibodies. Single-cell detection methods involving the use of multiple monoclonal antibodies generated in the same species would therefore provide with an important tool for cellular detection of antigens. Here, we describe a method to assess multiple proteins in a cell using different antibodies generated in the same species.

Lactobacillus plantarum (VR1) isolated from an ayurvedic medicine (Kutajarista) ameliorates in vitro cellular damage caused by Aeromonas veronii.

BACKGROUND: Lactobacillus plantarum is considered as a safe and effective probiotic microorganism. Among various sources of isolation, traditionally fermented foods are considered to be rich in Lactobacillus spp., which can be exploited for their probiotic attribute. Antibacterial property of L. plantarum has been demonstrated against various enteric pathogens in both in vitro and in vivo systems. This study was aimed at characterizing L. plantarum isolated from Kutajarista, an ayurvedic fermented biomedicine, and assessing its antagonistic property against a common enteropathogen Aeromonas veronii. RESULTS: We report the isolation of L. plantarum (VR1) from Kutajarista, and efficacy of its cell free supernatant (CFS) in amelioration of cytotoxicity caused by Aeromonas veronii. On the part of probiotic attributes, VR1 was tolerant to pH 2, 0.3% bile salts and simulated gastric juice. Additionally, VR1 also exhibited adhesive property to human intestinal HT-29 cell line. Furthermore, CFS of VR1 was antibacterial to enteric pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Aeromonas veronii and clinical isolates of P. aeruginosa and E. coli. Detailed study regarding the effect of VR1 CFS on A. veronii cytotoxicity showed a significant decrease in vacuole formation and detrimental cellular changes in Vero cells. On the other hand, A. veronii CFS caused disruption of tight junction proteins ZO-1 and actin in MDCK cell line, which was prevented by pre-incubation with CFS of VR1. CONCLUSIONS: This is the first study to report isolation of L. plantarum (VR1) from Kutajarista and characterisation for its probiotic attributes. Our study demonstrates the antagonistic property of VR1 to A. veronii and effect of VR1 CFS in reduction of cellular damage caused by A. veronii in both Vero and MDCK cell lines.

Association of small Rho GTPases and actin ring formation in epithelial cells during the invasion by Candida albicans.

Invasion of epithelial cells is a major virulence determinant of Candida albicans; however, the molecular events that occur during invasion are not discerned. This study is aimed to elucidate the role of the host's actin remodeling and involvement of small GTPases during invasion. Actin filaments formed a rigid ring-like structure in the rabbit corneal epithelial cell line SIRC after C. albicans invasion. During invasion, an increase in the mRNA content of Cdc42, Rac1 and RhoA GTPase was observed in SIRC cells. Immunochemical staining and expression of chimeric green fluorescent protein (GFP)-GTPases showed that all three GTPases colocalize at invasion and actin polymerization sites. This colocalization was not seen in SIRC cells expressing a GFP-tagged dominant-negative mutant of GTPases. Inhibition of invasion was observed in SIRC cells expressing dominant-negative mutants of Rac1 and RhoA GTPases. Involvement of zonula occludens-1 (ZO-1) was observed in the process of actin-mediated endocytosis of C. albicans. Actin, GTPases and ZO-1 were colocalized in epithelial cells during uptake of polymethylmethacrylate beads coated with spent medium from a C. albicans culture. The results indicate that host actin remodeling and recruitment of small GTPases occur during invasion and molecules that are shed or secreted by C. albicans are probably responsible for cytoskeletal reorganization.