Enterotoxigenic Escherichia coli CS6 gene products and their roles in CS6 structural protein assembly and cellular adherence

Enterotoxigenic Escherichia coli (ETEC) produces a variety of colonization factors necessary for attachment to the host cell, among which CS6 is one of the most prevalent in ETEC isolates from developing countries. The CS6 operon is composed of 4 genes, cssA, cssB, cssC, and cssD. The molecular mechanism of CS6 assembly and cell surface presentation, and the contribution of each protein to the attachment of the bacterium to intestinal cells remain unclear. In the present study, a series of css gene-deletion mutants of the CS6 operon were constructed in the ETEC genetic background, and their effect on adhesion to host cells and CS6 assembly was studied. Each subunit deletion resulted in a reduction in the adhesion to intestinal cells to the same level of laboratory E. coli strains, and this effect was restored by complementary plasmids, suggesting that the 4 proteins are necessary for CS6 expression. Bacterial cell fractionation and western blotting of the mutant strains suggested that the formation of a CssA–CssB–CssC complex is necessary for recognition by CssD and transport of CssA–CssB to the outer membrane as a colonization factor

Transcriptional regulation of a gene encoding 67 kDa glycoprotein (p67)

A 67 kDa glycoprotein (p\sp{67}) protects the key peptide chain initiation factor eIF-2 $\alpha$-subunit from inhibitory phosphorylation in animal cells. This promotes protein synthesis even in presence of active eIF 2 kinases. In several cases studied, the p\sp{67} level changes under different growth conditions in rat hepatoma (KRC-7) cells in culture. Results suggest that p\sp{67 } is regulated at the mRNA level. The p\sp{67} mRNA was present in confluent cells but disappeared almost completely from serum starved cells. However, when phorbol 13-myristate 12-acetate (PMA) was added to the same serum starved cells, p\sp{67} mRNA appeared in increasing quantities. Further studies involving the expression of p\sp{67} sense or antisense DNA in KRC-7 cells have provided evidence that the presence or absence of p\sp{67} mRNA is the critical factor in regulating protein synthesis activity in these cells. To gain a better understanding of the mechanism how p\sp{67} gene is regulated, its promoter has been cloned and characterized. Sequence analysis of this 1.7 kilobase DNA fragment showed that the 898 base pairs at the 5\sp\prime-end of the promoter was identical to several LINE sequences. 148 base pairs at the 3\sp\prime-end contained the beginning of the first exon including the ATG initiator codon. The remaining 652 base pairs in between contained two AT-rich regions and several regulatory sequences. The mRNA initiation site was identified at 89 base pairs upstream from the ATG codon. Transcriptional regulation of the p\sp{67} gene was also analyzed by transient transfection. When linked to a firefly luciferase reporter gene, this fragment enhanced transcription in KRC-7 cells. Using a series of deletions in the promoter, the minimum essential promoter region from $-$177 to $-$60 was identified. The promoter activity was also enhanced by treatment with PMA. This enhancement required an AP-1 sequence from $-$298 to $-$292 and a similar sequence form $-$97 to $-$88. Deletion of either of these sequences significantly reduced PMA enhancement. Deletion of both of these sequences almost completely eliminated PMA enhancement

A review of venous thromboembolism in India

Venous thromboembolism (VTE), which entails the formation of a thrombus (blood clot) in a vein, has a significant disease burden worldwide. While VTE has traditionally been considered to predominantly affect Caucasian populations, recent studies have indicated a gradual shift in the disease burden towards Asian populations, with added significance of it being a key driver of post-operative mortality. It is imperative to develop a sound understanding of the various factors that affect VTE in stratified local populations. However, there is a glaring paucity of quality data on VTE and its ramifications among Indians - both in terms of quality of life and cost of healthcare. This review aims to throw light on the disease burden, epidemiology, risk factors, environmental factors, food and nutrition that plays a key role in VTE. We also explored the association of VTE with coronavirus disease 2019 to grasp the interplay between the two most significant public health crises of our time. It is vital to place a special emphasis on future research on VTE in India to plug the gaps, which exist in our current knowledge of the disease, particularly with respect to Indian population

Smartphone camera‐based analysis of ELISA using artificial neural network

The proposed method indicates an inexpensive, portable, and easily accessible method for the quantitative analysis of medical samples for the detection of disease in the enzyme‐linked immunosorbent assay (ELISA). The procedure follows a point‐of‐care diagnostic model and attends to the several challenges in healthcare system in rural settings. The proposed technique will alleviate the inconveniences faced by the average citizen of a country with insufficient resources to implement an affordable healthcare administration for its entire population. A smartphone is used to procure images of an ELISA containing para‐nitrophenol samples which is then fed into a machine learning algorithm, specifically artificial neural network. The introduction of two relatively new technologies in medical aid – the smartphone and machine learning not only reduces cost and time of detection, but also presents ample possibility for further development. The predictions result in highly accurate diagnostic labels. The same method can be used for blood samples for the prediction of presence of any disease, provided adequate training set has been deployed

PCR-Based Identification of Common Colonization Factor Antigens of Enterotoxigenic Escherichia coli▿

Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli (ETEC) have been classified into several groups based on their distinct antigenicity. We describe here a PCR-based method to detect common CFAs of ETEC, which were characterized using conventional serology. This PCR assay is simple and sensitive for the detection of expressed CFA genes

The <i>Vibrio cholerae</i> Extracellular Chitinase ChiA2 Is Important for Survival and Pathogenesis in the Host Intestine

<div><p>In aquatic environments, <i>Vibrio cholerae</i> colonizes mainly on the chitinous surface of copepods and utilizes chitin as the sole carbon and nitrogen source. Of the two extracellular chitinases essential for chitin utilization, the expression of <i>chiA2</i> is maximally up-regulated in host intestine. Recent studies indicate that several bacterial chitinases may be involved in host pathogenesis. However, the role of <i>V. cholerae</i> chitinases in host infection is not yet known. In this study, we provide evidence to show that ChiA2 is important for <i>V. cholerae</i> survival in intestine as well as in pathogenesis. We demonstrate that ChiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers. Deglycosylation of mucin corroborated with reduced uptake of alcian blue stain by ChiA2 treated mucin. Next, we show that <i>V. cholerae</i> could utilize mucin as a nutrient source. In comparison to the wild type strain, Δ<i>chiA2</i> mutant was 60-fold less efficient in growth in mucin supplemented minimal media and was also ∼6-fold less competent to survive when grown in the presence of mucin-secreting human intestinal HT29 epithelial cells. Similar results were also obtained when the strains were infected in mice intestine. Infection with the Δ<i>chiA2</i> mutant caused ∼50-fold less fluid accumulation in infant mice as well as in rabbit ileal loop compared to the wild type strain. To see if the difference in survival of the Δ<i>chiA2</i> mutant and wild type <i>V. cholerae</i> was due to reduced adhesion of the mutant, we monitored binding of the strains on HT29 cells. The initial binding of the wild type and mutant strain was similar. Collectively these data suggest that ChiA2 secreted by <i>V. cholerae</i> in the intestine hydrolyzed intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by <i>V. cholerae</i> for growth and survival in the host intestine.</p></div