MUC1, CK20, and CDX2 immunohistochemical markers can sub-classify periampullary carcinomas into pancreaticobiliary, intestinal, and mixed subtypes.

INTRODUCTION: Pancreaticobiliary subtype of Periampullary carcinoma (PAC) has a poor prognosis in comparison to the intestinal subtype. We assessed the potential of cytokeratins and mucin markers to classify the sub-types of periampullary tumors and compared them with the survival data to identify markers that may predict prognosis. METHODOLOGY: PAC tumor tissues were obtained from 94 patients undergoing Whipples Pancreaticoduodenectomy. Paraffin-embedded tissues were immunostained with cytokeratins CK7, CK20), mucins (MUC1, MUC2, MUC5Ac), and CDX2 antibodies. The survival status of patients was obtained as follow-up up to 5-years of surgery. The Receiver Operating Character Curve (ROC) analysis was used for detecting sensitivity and specificity. The survival data were analyzed using the Kaplan-Meier survival curve. RESULTS: Tumors were initially categorized on the basis of histological classification as pancreaticobiliary (n = 46), intestinal (n = 35) and indeterminate (n = 13). Further, using immunohistochemical markers (MUC1, CK20, and CDX2), we gave systematic classification of IHC-PB (n = 51), IHC-Int (n = 30) and IHC-Mixed (n = 13). The interobserver analysis showed good agreement between histologic and IHC type with a kappa value of 0.554. Combined expression of CK20, MUC1 and CDX2 accurately classify the mixed type of tumor. Overall survival rate and duration were 74.4% and 44.95 ± 2.29 months. Survival analysis for subtypes reveal, pancreaticobiliary tumors have low survival (27.9 ± 1.63 months) than mixed type (35.5 ± 0.45 months) and intestinal-type (52.92 ± 2.18 months). Among these, intestinal-type have better survival. Only TNM Stage III (tumor staging as per American Joint Committee on Cancer classification) and perineural invasion have been associated with predicting poor survival in PAC patients. CONCLUSION: Our results suggest that the combined expression of MUC1, CK20 and CDX2 could serve as markers to diagnose histological inconclusive specimens as mixed subtype tumors.

Efficacy of short-synthetic antifungal peptides on pathogenic Aspergillus flavus.

The efficacies of three short synthetic antifungal peptides were tested for their inhibitory action on pathogenic fungi, Aspergillus flavus. The sequences of the short synthetic peptides are PPD1- FRLHF, 66-10-FRLKFH, 77-3- FRLKFHF, respectively. These test peptides inhibited fungal growth and showed a membranolytic activity. The fungal biomass and ergosterol levels were significantly low in peptides treated samples. Further, the fungal cell wall component chitin was also found to be lower in peptides treated samples. Scanning electron microscopic images also showed highly wrinkled fungal mycelia. Significant membrane permeabilisation as well as potassium ion leakage was also observed in fungal samples treated with peptides. To assess the membrane damage, the uptake of Sytox green dye was employed. At tested concentration, peptides induced fungal membrane damage as evidenced by the green fluorescence. Further, at tested concentration, these peptides induced an oxidative stress in A.flavus as evidenced by an increase in the ROS production, malondialdehyde levels, increase in the antioxidant enzymes - superoxide dismutase, catalase with concomitant decrease in the reduced glutathione content. Additionally, a growth dependent reduction in aflatoxin levels were also observed in peptides treated samples. Docking studies on the interaction of the peptides with a trans-membrane protein calcium ATPase of A. flavus showed that all the peptides were able to bind to the protein with high z rank score. The activity of the calcium ATPase was significantly decreased in peptides treated fungal samples, thereby validating the docking results. Among all the tested peptides, 77-3 peptide exhibited the maximal membrane damage property.

Antiaflatoxigenic effects of selected antifungal peptides.

Aflatoxins are potent carcinogenic mycotoxins produced as secondary metabolites mainly by the fungi Aspergillus flavus and Aspergillus parasiticus. Control measures to curtail the contamination of aflatoxin in food products is still a challenge. Although there are several reports on the antifungal peptides, there is no specific study on the action of antifungal peptides on aflatoxin synthesis. This work details the effect of four antimicrobial peptides (AMPs) - PPD1 (FRLHF), 66-10 (FRLKFH), 77-3 (FRLKFHF) and D4E1 (FKLRAKIKVRLRAKIKL) on the aflatoxin production by A. flavus and A. parasiticus. Results of the investigations suggests that AMPs at near minimum inhibitory concentrations (MIC) were effectively inhibiting aflatoxins, without hindering the growth of the fungi. These AMPs, at concentrations near MIC, induced membrane permeabilisation, without inducing cellular leakage. The involvement of oxidative stress for the aflatoxin synthesis was reversed by the antioxidant nature of the peptides as evidenced by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay, reactive oxygen species production, malondialdehyde and antioxidant enzymes analysis. Quantitative real time polymerase chain reaction (RT-qPCR) analysis of the aflatoxin gene cluster showed that 'aflR' and its downstream genes expressions were significantly down regulated. Conidiation of the fungi were negatively influenced by the peptides as evidenced by scanning electron microscopy analysis and RT-qPCR. mRNA levels of Manganese-superoxide dismutase (Mn-SOD) showed a decrease in the expression in RT-qPCR. The effect of these peptides on aflatoxin inhibition provides insight into their use as novel antiaflatoxigenic molecules.

Biosorption of nickel and total chromium from aqueous solution by gum kondagogu (Cochlospermum gossypium): A carbohydrate biopolymer.

Gum kondagogu (Cochlospermum gossypium), an exudates tree gum from India was explored for its potential to decontaminate toxic metal ions in aqueous solution. The toxic metal ions nickel and total chromium biosorption capacity of the gum kondagogu were studied in the batch experimental mode. The optimum conditions of biosorption were determined by investigating pH, contact time, and initial metal ion and biosorbent concentrations. The Freundlich and Langmuir adsorption models were used for the mathematical description of biosorption equilibrium and the data were analyzed on the basis of pseudo-second-order kinetic model. The maximum biosorption capacity of gum kondagogu as calculated by Langmuir model were found to be 50.5 mg g(-1) for nickel at pH 5.0+/-0.1 and 129.8 mg g(-1) for total chromium at pH 2.0+/-0.1, respectively. FTIR, SEM-EDXA and XPS analysis were used to evaluate the binding characteristics of gum kondagogu with metals. The experimental results demonstrate that the metal-ion interaction occurs through ion-exchange, adsorption and precipitation mechanisms.

Epidemiological profile of snake-bite cases from Andhra Pradesh using immunoanalytical approach.

BACKGROUND & OBJECTIVE: Snake-bites are the common cause of morbidity and mortality in tropical countries. In India, there are 216 species of snakes, of which only four are venomous snakes (cobra, krait, Russell's viper and saw scaled viper). This study was undertaken to find out the epidemiological profile of snake-bite incidences in the State of Andhra Pradesh, based on the data collected from State Forensic Science Laboratory, Hyderabad. METHODS: Data from 1379 snake-bite cases were collected from case reports for a 5 yr period (1999- 2003) that included age and sex of the victim, district, month of incidence, time of incident, death of a victim and the time point of analysis. On the basis of the forensic data, specimens were collected from forensic medicine department, during rainy season and were analysed for the venom antigens (cobra and krait) by ELISA method. RESULTS: The peak number of snake-bite cases were seen during June-September. Majority of the cases were observed in the age group 21-50 yr (71%). Higher incidence of snake-bite was recorded in males (76%). Of the 22 cases analysed by the ELISA, 6 tested positive for cobra venom, while 8 cases tested positive for krait venom, the remaining specimens tested negative for both cobra and krait venom. INTERPRETATION & CONCLUSION: Evaluation of forensic specimens (autopsy & biopsy) of human snakebite victims based on specific molecular epidemiological tool like ELISA gives a true estimate of the incidence supplementing clinical and circumstantial evidence.

Influence of extremely low frequency magnetic fields on Ca2+ signaling and NMDA receptor functions in rat hippocampus.

Extremely low frequency (ELF<300Hz) electromagnetic fields affect several neuronal activities including memory. Because ELF magnetic fields cause altered Ca(2+) homeostasis in neural tissues, we examined their influence on Ca(2+) signaling enzymes in hippocampus and related them with NMDA receptor functions. Hippocampal regions were obtained from brains of 21-day-old rats that were exposed for 90 days to 50Hz magnetic fields at 50 and 100 microT intensities. In comparison to controls, ELF exposure caused increased intracellular Ca(2+) levels concomitant with increased activities of Ca(2+)-dependent protein kinase C (PKC), cAMP-dependent protein kinase and calcineurin as well as decreased activity of Ca(2+)-calmodulin-dependent protein kinase in hippocampal regions. Simultaneous ligand-binding studies revealed decreased binding to N-methyl-D-aspartic acid (NMDA) receptors. The combined results suggest that perturbed neuronal functions caused by ELF exposure may involve altered Ca(2+) signaling events contributing to aberrant NMDA receptor activities.

Use of egg yolk antibody (IgY) as an immunoanalytical tool in the detection of Indian cobra (Naja naja naja) venom in biological samples of forensic origin.

An immunoglobulin Y (IgY) based indirect double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed for the detection of Indian cobra (Naja naja naja) venom in the biological samples of forensic origin. Polyclonal antibodies were raised and purified from chick egg yolk and rabbit serum. The cobra venom was sandwiched between immobilized affinity purified IgY and the rabbit IgG. The detection concentration of cobra venom was in the range of 0.1 to 300ng. The calibration plot was based on linear regression analysis (y=0.2581x+0.4375, r(2)=0.9886). The limit of detection of the assay was found to be 0.1ng. The coefficient of variation (CV) of different concentrations of working range in inter (n=6) and intra-assay (n=6) was observed to be less than 10%. The recovery of venom was found to be in the range of 80-99%, when different concentrations (0.002, 0.1, 0.2, 1, and 2microg) of cobra venom were spiked to pooled normal human serum (ml(-1)). No cross reactivity was observed with krait and viper venom in the immunoassay system in the concentration range of 0.1-1000ng. The method was initially, validated by analyzing specimens (autopsy) of experimental rats injected with cobra venom (1.2mgkg(-1) body mass). Further, human specimens (autopsy and biopsy) of snake bite victims of forensic origin were also analyzed. The methodology developed may find diagnostic application in forensic laboratories.

Role of mycoferritin from Aspergillus parasiticus (255) in secondary metabolism (aflatoxin production).

Aspergillus parasiticus (255), a non-toxigenic isolate showed the presence of secondary metabolites-aflatoxins (B1, B2, G1, G2) when grown in yeast extract sucrose media but not in basal media, thus demonstrating its toxigenic potential. Native PAGE of the crude protein isolated at different growth periods of A. parasiticus in yeast extract sucrose media containing iron showed prominent expression of mycoferritin from day four onwards. The production of aflatoxins was also maximal on day four, both in the presence and absence of iron. Indicators of oxidative stress metabolites such as reactive oxygen species, thiobarbituric acid reactive species, reduced and oxidized glutathione and antioxidant enzymes like superoxide dismutase and glutathione peroxidase were analyzed both in the presence and absence of iron and the experimental data suggest oxidative stress as a pre-requisite for aflatoxin production. The pro-oxidant role of iron was minimized by induction of mycoferritin and the concomitant alterations in oxidative stress parameters imply an antioxidant role to mycoferritin in secondary metabolism, a finding of significance that has not been reported previously in fungal systems.

Substrate suitability of different genotypes of sorghum in relation to Aspergillus infection and aflatoxin production.

Grain sorghum is often damaged by rain in the field and severely infected by grain mold, which includes Aspergillus infection and aflatoxin production. The objective of the study is to investigate the extent of aflatoxin production with Aspergillus infection in vitro in different sorghum genotypes with different pericarps, red, yellow, and white, the physical and chemical characteristics of grain during infection, and the changes in grain polyphenols and phytic acid in comparison to maize and groundnut. The physical characters and biochemical composition of sorghum grain contribute to make it less susceptible to Aspergillus infection and aflatoxin contamination compared to maize and groundnut. The lowest amounts of aflatoxin and ergosterol were observed in genotypes with red pericarp, whereas higher amounts of aflatoxin and ergosterol were found in white genotypes followed by maize and groundnut. All of the red genotypes differ in polyphenol composition and aflatoxin produced, showing resistance to mold damage. Another indication of resistance in red genotypes was the delayed peaking of aflatoxin production (9 days after infection). In red sorghum genotypes there was a significant, positive correlation existing between polyphenol content and aflatoxin produced at 3 and 6 days after infection, the r values being 0.589 and 0.513, respectively. The starch content decreased whereas the protein content in all sorghum genotypes increased during infection. Maximum phytic acid was observed in white sorghum genotypes. Phytic acid in yellow genotypes was found to have a significant negative correlation (r = -0.569) with aflatoxin produced.

Determination of aflatoxin B1-DNA adduct in rat liver by enzyme immunoassay.

A simple, rapid and highly sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (AFB1)-DNA adducts is reported. Polyclonal antibodies specific to the aflatoxin B1-N7-guanine adduct were produced using a novel synthetic antigen, bovine serum albumin (BSA)-guanine-AFB1. The antibodies were characterized by the Ouchterlony double diffusion technique and by antibody capture assay. The working range of the indirect competitive assay developed was between 0.45 and 330 ng of the analyte [calf thymus (CT)-DNA-AFB1]. A 50% inhibition was attained at 15 ng of the analyte (CT-DNA-AFB1). The antibody capture assay indicated that the antibody produced cross-reacted 100, 92 and 110% with BSA-guanine-AFB1, CT-DNA-AFB1 and CT-DNA-formamidopyrimidine-AFB1, respectively. When free AFB1 and guanine were used as competing analytes, the antibodies showed < or = 5% and zero cross-reactivity at the 50% inhibition level. Spiking studies indicated a recovery in the range 96-97 and 74-78% when standard CT-DNA-AFB1 was added to 10 mM phosphate buffer (pH 7.2) and control rat liver tissue, respectively. Rats exposed to a single oral dose of 1 mg kg-1 body mass of pure AFB1 were used to validate the method. The AFB1-DNA adduct formed in the liver tissue after 48 h of exposure was determined using the ELISA method developed. The liver AFB1-DNA adduct ranged between 6.06 and 7.94 micrograms mg-1 DNA. The proposed method may find application in the biological monitoring of aflatoxin B1 in molecular epidemiological studies to assess the dietary exposure of aflatoxins.

Dip-strip method for monitoring environmental contamination of aflatoxin in food and feed: use of a portable aflatoxin detection kit.

Aflatoxin contamination of food and feed have gained global significance due to its deleterious effect on human and animal health and its importance in the international trade. The potential of aflatoxin as a carcinogen, mutagen, teratogen, and immunosuppressive agent is well documented. The problem of aflatoxin contamination of food and feed has led to the enactment of various legislation. However, meaningful strategies for implementation of this legislation is limited by nonavailability of simple, cost-effective method for screening and detection of aflatoxin under field conditions. Keeping in mind the analytical constraints in developing countries, a simple-to-operate, rapid, reliable, and cost-effective portable aflatoxin detection kit has been developed. The important components of the kit include a hand-held UV lamp (365 nm, 4 W output), a solvent blender (12,000 rpm) for toxin extraction, and adsorbent-coated dip-strips (polyester film) for detecting and quantifying aflatoxin. Analysis of variance indicates that there were no significant differences between various batches of dip-strips (p > 0.05). The minimum detection limit for aflatoxin B1 was 10 ppb per spot. The kit may find wide application as a research tool in public health laboratories, environmental monitoring agencies, and in the poultry industry.