Repeated immunostaining of the same tissue section using alkaline phosphatase as a reporter

One can determine the best dilution of a primary antibody for immunohistochemistry that uses horseradish peroxidase conjugated to a secondary antibody by testing increasing concentrations sequentially on the same tissue section. When the same tissue section is incubated repeatedly with increasing concentrations of primary antibodies to epithelial membrane antigen, smooth muscle α-actin, or vimentin using alkaline phosphatase conjugated to a secondary antibody as the reporter, the best staining was obtained with a less concentrated primary antibody than was optimal for a single staining test. The best concentration of primary antibody for single run staining using an alkaline phosphatase reporting system is usually four times the best concentration for staining with multiple runs. The optimal concentration can be determined by denaturing the residual alkaline phosphatase and extracting residual stain by incubating the section in 4:1 diglyme:phosphate buffered saline for 20 min at 80^o C between tests of primary antibody concentrations. I tested the method for four chromogens from one supplier and one chromogen from a different supplier.     

Quantifying multiple pressure interactions affecting populations of a recreationally and commercially important freshwater fish

The expanding human global footprint and growing demand for freshwater have placed tremendous stress on inland aquatic ecosystems. Aichi Target 10 of the Convention on Biological Diversity aims to minimize anthropogenic pressures affecting vulnerable ecosystems, and pressure interactions are increasingly being incorporated into environmental management and climate change adaptation strategies. In this study, we explore how climate change, overfishing, forest disturbance, and invasive species pressures interact to affect inland lake walleye (Sander vitreus) populations. Walleye support subsistence, recreational, and commercial fisheries and are one of most sought‐after freshwater fish species in North America. Using data from 444 lakes situated across an area of 475 000 km² in Ontario, Canada, we apply a novel statistical tool, R‐INLA, to determine how walleye biomass deficit (carrying capacity—observed biomass) is impacted by multiple pressures. Individually, angling activity and the presence of invasive zebra mussels (Dreissena polymorpha) were positively related to biomass deficits. In combination, zebra mussel presence interacted negatively and antagonistically with angling activity and percentage decrease in watershed mature forest cover. Velocity of climate change in growing degree days above 5°C and decrease in mature forest cover interacted to negatively affect walleye populations. Our study demonstrates how multiple pressure evaluations can be conducted for hundreds of populations to identify influential pressures and vulnerable ecosystems. Understanding pressure interactions is necessary to guide management and climate change adaptation strategies, and achieve global biodiversity targets.     

Barley microgreen incorporation in diet-controlled diabetes and counteracted aflatoxicosis in rats

Increased environmental pollution and unhealthy lifestyle are blamed for escalated chronic diseases. Exposure to aflatoxins was recently suggested to have a role in the increased incidence of type 2 diabetes mellitus. Diet modification and consumption of different functional food are now gaining attention, especially in diabetes management. This study investigates the effect of a diet containing barley microgreen against diabetes induced by streptozotocin with or without aflatoxin administration in rats. Barley microgreen was rich in 3′-Benzyloxy-5,6,7,4′-tetramethoxyflavone (48.8% of total) followed by 5β,7βH,10α-Eudesm-11-en-1α-ol (18.46%). Streptozotocin injection and/or aflatoxin administration significantly elevated glucose level, decreased insulin level, decreased β-cell function, deteriorated liver and kidney function parameters, and induced oxidative stress in the liver. Histopathology revealed irregular small-sized islets and decreased area % of insulin-positive beta cells in the pancreas, hepatic degeneration, nephropathy, and neuropathy in diabetic and/or aflatoxin administered rats compared to control. Barley microgreen diet fed to diabetic rats with or without aflatoxin alleviated all evaluated parameters. Barley microgreen diet also ameliorated the toxic effect of aflatoxin. In conclusion, exposure to aflatoxin aggravated diabetes and its complication. The incorporation of barley microgreen in the diet was able to control type 2 diabetes mellitus and the improved outcomes observed with barley microgreen treatments involved or occurred in conjunction with improved biomarkers of oxidative stress.     

Regulation of Salmonella-induced neutrophil transmigration by epithelial ADP-ribosylation factor 6

Salmonella typhimurium elicits an acute inflammatory response in the host intestinal epithelium, characterized by the movement of polymorphonuclear leukocytes (PMN) across the epithelial monolayer to the intestinal lumen. It was recently shown that SipA, a protein secreted by S. typhimurium, is necessary and sufficient to drive PMN transmigration across model intestinal epithelia (Lee, C. A., Silva, M., Siber, A. M., Kelly, A. J., Galyov, E., and McCormick, B. A. (2000) Proc. Natl. Acad Sci. USA 97, 12283-12288). However, the epithelial factors responsible for this process have not been identified. Here, for the first time, we demonstrate that S. typhimurium-induced PMN transmigration across Madin-Darby canine kidney-polarized monolayers is regulated by the GTPase ARF6. Apically added S. typhimurium promoted the translocation of ARF6 and its exchange factor ARNO to the apical surface. Overexpression of a dominant-negative mutant of ARF6 inhibited Salmonella-induced PMN transmigration, which was due to a reduction in apical release of the PMN chemoattractant PEEC (pathogen-elicited epithelial chemoattractant), without affecting bacterial internalization. Furthermore, ARF6 and its effector phospholipase D (PLD) were both required for bacteria-induced translocation of protein kinase C (PKC) to membranes. These results describe a novel signal transduction pathway, in which Salmonella initiates an ARF6- and PLD-dependent lipid signaling cascade that, in turn, directs activation of PKC, release of PEEC, and subsequent transepithelial PMN movement.     

Signalling networks that cause cancer

Cancer is caused by the stepwise accumulation of mutations that affect growth control, differentiation and survival. The view that mutations affect discrete signalling pathways, each contributing to a specific aspect of the full malignant phenotype, has proved to be too simplistic. We now know that oncogenes and tumour suppressors depend on one another for their selective advantage, and that they affect multiple pathways that intersect and overlap. The interactive nature of each genetic change has important implications for cancer therapy and for the stepwise model of carcinogenesis.     

Phytotoxicity of selected trichothecenes using Chlamydomonas reinhardtii as a model systemt

Trichothecenes are potent inhibitors of cytoplasmic protein synthesis which can affect the severity of plant diseases such as wheat head scab. While many trichothecene-producing fungi share the initial biosynthetic intermediates, Fusarium sp. are unique in the production of trichothecenes containing an oxygen function at C-3. Although the initial trichothecene and the final products have a C-3 hydroxyl group, the intermediate steps are acetylated at C-3. By using Chlamydomonas reinhardtii, a unicellular plant with a well-defined genetic system, we were able to test the proposal that trichothecenes with a C-3 hydroxyl are more toxic to plants, as well as demonstrate that C. reinhardtii is a promising plant trichothecene bioassay system. Seven pairs of trichothecenes with either a C-3 hydroxyl or C-3 acetyl group were assayed. Our results confirm that trichothecenes acetylated at C-3 were far less toxic to Chlamydomonas than those with a C-3 hydroxyl group.