Occludin regulates macromolecule flux across the intestinal epithelial tight junction barrier

Defective intestinal epithelial tight junction (TJ) barrier has been shown to be an important pathogenic factor contributing to the development of intestinal inflammation. The expression of occludin is markedly decreased in intestinal permeability disorders, including in Crohn's disease, ulcerative colitis, and celiac disease, suggesting that the decrease in occludin expression may play a role in the increase in intestinal permeability. The purpose of this study was to delineate the involvement of occludin in intestinal epithelial TJ barrier by selective knock down of occludin in in vitro (filter-grown Caco-2 monolayers) and in vivo (recycling perfusion of mouse intestine) intestinal epithelial models. Our results indicated that occludin small-interfering RNA (siRNA) transfection causes an increase in transepithelial flux of various-sized probes, including urea, mannitol, inulin, and dextran, across the Caco-2 monolayers, without affecting the transepithelial resistance. The increase in relative flux rate was progressively greater for larger-sized probes, indicating that occludin depletion has the greatest effect on the flux of large macromolecules. siRNA-induced knock down of occludin in mouse intestine in vivo also caused an increase in intestinal permeability to dextran but did not affect intestinal tissue transepithelial resistance. In conclusion, these results show for the first time that occludin depletion in intestinal epithelial cells in vitro and in vivo leads to a selective or preferential increase in macromolecule flux, suggesting that occludin plays a crucial role in the maintenance of TJ barrier through the large-channel TJ pathway, the pathway responsible for the macromolecule flux.     

Gamma radiation induced mutagenesis in <Emphasis Type="Italic">Aspergillus niger</Emphasis> to enhance its microbial fermentation activity for industrial enzyme production

α- and β-Galactosidases find application in food processing, health and nutrition. Aspergillus niger is one of the potent producer of these enzymes and was genotypically improved using gamma-ray induced mutagenesis. The mutant-derivative produced two-fold higher α- and β-galactosidases. For testing genetic variability and its relationship with phenotypic properties of the two organisms, DNA samples of the mutant and parental strains of A. niger were amplified with 28 deca-nucleotide synthetic primers. RAPD analysis showed significantly different pattern between parental and mutant cultures. The mutant derivative yielded homogeneous while parental strain formed heterogeneous amplification patterns. Seven primers identified 42.9% polymorphism in the amplification products, indicating that these primers determined some genetic variability between the two strains. Thus RAPD was found to be an efficient technique to determine genetic variability in the mutant and wild organisms. Both wild and mutant strains were analyzed for their potential to produce galactosidases. Comparison of different carbon sources on enzyme yield revealed that wheat bran is significant (P < 0.01) effective producer and economical source followed by rice bran, rice polishing and lactose. The mutant was significantly better enzyme producer and could be considered for its prospective application in food, nutrition and health and that RAPD can be effectively used to differentiate mutant strain from the parental strain based on the RAPD patterns.     

Comparative analysis of Gossypium and Vitis genomes indicates genome duplication specific to the Gossypium lineage

Genetic mapping studies have suggested that diploid cotton (Gossypium) might be an ancient polyploid. However, further evidence is lacking due to the complexity of the genome and the lack of sequence resources. Here, we used the grape (Vitis vinifera) genome as an out-group in two different approaches to further explore evidence regarding ancient genome duplication (WGD) event(s) in the diploid Gossypium lineage and its (their) effects: a genome-level alignment analysis and a local-level sequence component analysis. Both studies suggest that at least one round of genome duplication occurred in the Gossypium lineage. Also, gene densities in corresponding regions from Gossypium raimondii, V. vinifera, Arabidopsis thaliana and Carica papaya genomes are similar, despite the huge difference in their genome sizes and the different number of WGDs each genome has experienced. These observations fit the model that differences in plant genome sizes are largely explained by transposon insertions into heterochromatic regions.     

Engineered ZnO and TiO(2) nanoparticles induce oxidative stress and DNA damage leading to reduced viability of Escherichia coli

Extensive use of engineered nanoparticle (ENP)-based consumer products and their release into the environment have raised a global concern pertaining to their adverse effects on human and environmental health. The safe production and use of ENPs requires improvement in our understanding of environmental impact and possible ecotoxicity. This study explores the toxicity mechanism of ZnO and TiO(2) ENPs in a gram-negative bacterium, Escherichia coli. Internalization and uniform distribution of characterized bare ENPs in the nano range without agglomeration was observed in E. coli by electron microscopy and flow cytometry. Our data showed a statistically significant concentration-dependent decrease in E. coli cell viability by both conventional plate count method and flow cytometric live-dead discrimination assay. Significant (p<0.05) DNA damage in E. coli cells was also observed after ENP treatment. Glutathione depletion with a concomitant increase in hydroperoxide ions, malondialdehyde levels, reactive oxygen species, and lactate dehydrogenase activity demonstrates that ZnO and TiO(2) ENPs induce oxidative stress leading to genotoxicity and cytotoxicity in E. coli. Our study substantiates the need for reassessment of the safety/toxicity of metal oxide ENPs.     

Movement mechanics as a determinate of muscle structure, recruitment and coordination

During muscle contractions, the muscle fascicles may shorten at a rate different from the muscle-tendon unit, and the ratio of these velocities is its gearing. Appropriate gearing allows fascicles to reduce their shortening velocities and allows them to operate at effective shortening velocities across a range of movements. Gearing of the muscle fascicles within the muscle belly is the result of rotations of the fascicles and bulging of the belly. Variable gearing can also occur as a result of tendon length changes that can be caused by changes in the relative timing of muscle activity for different mechanical tasks. Recruitment patterns of slow and fast fibres are crucial for achieving optimal muscle performance, and coordination between muscles is related to whole limb performance. Poor coordination leads to inefficiencies and loss of power, and optimal coordination is required for high power outputs and high mechanical efficiencies from the limb. This paper summarizes key studies in these areas of neuromuscular mechanics and results from studies where we have tested these phenomena on a cycle ergometer are presented to highlight novel insights. The studies show how muscle structure and neural activation interact to generate smooth and effective motion of the body.     

Imidazo[1,5-a]quinoxalines as irreversible BTK inhibitors for the treatment of rheumatoid arthritis

Imidazo[1,5-a]quinoxalines were synthesized that function as irreversible Bruton's tyrosine kinase (BTK) inhibitors. The syntheses and SAR of this series of compounds are presented as well as the X-ray crystal structure of the lead compound 36 in complex with a gate-keeper variant of ITK enzyme. The lead compound showed good in vivo efficacy in preclinical RA models.     

2-Anilinonicotinyl linked 2-aminobenzothiazoles and [1,2,4]triazolo[1,5-b] [1,2,4]benzothiadiazine conjugates as potential mitochondrial apoptotic inducers

A series of N-(2-anilino-pyridyl) linked 2-amino benzothiazoles (4a-n) and [1,2,4]triazolo [1,5-b]benzothiadiazine conjugates (5a-j) have been designed, synthesized and evaluated for their antiproliferative activity. Some of these compounds (4h-k, 4n, and 5e) have exhibited potent cytotoxicity specifically against human leukemia HL-60 cell lines with IC(50) values in the range of 0.08-0.70 μM. All these compounds were tested for their effects on the cell cycle perturbations and induction of apoptosis. Morphological evidences of apoptosis, including fragmentation of nuclei and inter nucleosomal DNA laddering formation were clearly observed after 24h exposure to compound 4i. Flow cytometry analysis revealed that compound 4i showed drastic cell cycle perturbations due to concentration dependant increase in the sub-G0 region which comprises of both the apoptotic and debris fraction, thus implying the extent of cell death. These compounds trigger the mitochondrial apoptotic pathway that results in the loss of mitochondrial membrane potential through activation of multiple caspases followed by activation of caspase-3, and finally cleavage of PARP. Further the mechanism of cell death was analysed by fluorescent microscopic analysis and also by scanning electron microscopy. The cytotoxicity of 4i correlated with induction of apoptosis, caspases activation and DNA damage and thus indicating the apoptotic pathway of anticancer effect of these compounds.     

Prey bacteria shape the community structure of their predators

Although predator–prey interactions among higher organisms have been studied extensively, only few examples are known for microbes other than protists and viruses. Among the bacteria, the most studied obligate predators are the Bdellovibrio and like organisms (BALOs) that prey on many other bacteria. In the macroscopical world, both predator and prey influence the population size of the other''s community, and may have a role in selection. However, selective pressures among prey and predatory bacteria have been rarely investigated. In this study, Bacteriovorax, a predator within the group of BALOs, in environmental waters were fed two prey bacteria, Vibrio vulnificus and Vibrio parahaemolyticus. The two prey species yielded distinct Bacteriovorax populations, evidence that selective pressures shaped the predator community and diversity. The results of laboratory experiments confirmed the differential predation of Bacteriovorax phylotypes on the two bacteria species. Not only did Bacteriovorax Cluster IX exhibit the versatility to be the exclusive efficient predator on Vibrio vulnificus, thereby, behaving as a specialist, but was also able to prey with similar efficiency on Vibrio parahaemolyticus, indicative of a generalist. Therefore, we proposed a designation of versatilist for this predator. This initiative should provide a basis for further efforts to characterize the predatory patterns of bacterial predators. The results of this study have revealed impacts of the prey on Bacteriovorax predation and in structuring the predator community, and advanced understanding of predation behavior in the microbial world.