Inhibition of Clostridioides difficile Toxins TcdA and TcdB by Ambroxol

Clostridioides (C.) difficile produces the exotoxins TcdA and TcdB, which are the predominant virulence factors causing C. difficile associated disease (CDAD). TcdA and TcdB bind to target cells and are internalized via receptor-mediated endocytosis. Translocation of the toxins’ enzyme subunits from early endosomes into the cytosol depends on acidification of endosomal vesicles, which is a prerequisite for the formation of transmembrane channels. The enzyme subunits of the toxins translocate into the cytosol via these channels where they are released after auto-proteolytic cleavage. Once in the cytosol, both toxins target small GTPases of the Rho/Ras-family and inactivate them by mono-glucosylation. This in turn interferes with actin-dependent processes and ultimately leads to the breakdown of the intestinal epithelial barrier and inflammation. So far, therapeutic approaches to treat CDAD are insufficient, since conventional antibiotic therapy does not target the bacterial protein toxins, which are the causative agents for the clinical symptoms. Thus, directly targeting the exotoxins represents a promising approach for the treatment of CDAD. Lately, it was shown that ambroxol (Ax) prevents acidification of intracellular organelles. Therefore, we investigated the effect of Ax on the cytotoxic activities of TcdA and TcdB. Ax significantly reduced toxin-induced morphological changes as well as the glucosylation of Rac1 upon intoxication with TcdA and TcdB. Most surprisingly, Ax, independent of its effects on endosomal acidification, decreased the toxins’ intracellular enzyme activity, which is mediated by a catalytic glucosyltransferase domain. Considering its undoubted safety profile, Ax might be taken into account as therapeutic option in the context of CDAD.

Treatment of Acute Kidney Injury Using a Dual Enzyme Embedded Zeolitic Imidazolate Frameworks Cascade That Catalyzes In Vivo Reactive Oxygen Species Scavenging

Significant advances have been made in recent years for the utilization of natural enzymes with antioxidant properties to treat acute kidney injury (AKI). However, these enzymes have been of limited clinical utility because of their limited cellular uptake, poor pharmacokinetic properties, and suboptimal stability. We employed a novel biomimetic mineralization approach to encapsulate catalase (CAT) and superoxide dismutase (SOD) in a zeolitic imidazolate framework-8 (ZIF-8). Next, this SOD@CAT@ZIF-8 complex was anchored with MPEG2000-COOH to yield an MPEG2000-SOD@CAT@ZIF-8 (PSCZ) composite. The composite was then used as a stable tool with antioxidant properties for the integrated cascade-based treatment of AKI, remarkably improved intracellular enzyme delivery. This dual-enzyme-embedded metal-organic framework could effectively scavenge reactive oxygen species. In conclusion, the ZIF-8-based “armor plating” represents an effective means of shielding enzymes with improved therapeutic utility to guide the precision medicine-based treatment of AKI.

ADP-ribosyl transferase activity and gamma radiation cytotoxicity of Pseudomonas aeruginosa exotoxin A

This work explores the ADP-ribosyltransferase activity of Pseudomonas (P.) aeruginosa exotoxin A using the guanyl hydrazone derivative, nitrobenzylidine aminoguanidine (NBAG) and the impact of gamma radiation on its efficacy. Unlike the conventional detection methods, NBAG was used as the acceptor of ADP ribose moiety instead of wheat germ extract elongation factor 2. Exotoxin A was extracted from P. aeruginosa clinical isolates and screened for toxA gene using standard PCR. NBAG was synthesized using aminoguanidine bicarbonate and 4-nitrobenzaldehyde and its identity has been confirmed by UV, FTIR, Mass and 13C-NMR spectroscopy. The ADP-ribosyl transferase activity of exotoxin A on NBAG in the presence of Nicotinamide adenine dinucleotide (NAD+) was recorded using UV spectroscopy and HPLC. In vitro ADP-ribosyl transferase activity of exotoxin A protein extract was also explored by monitoring its cytotoxicity on Hep-2 cells using sulforhodamine B cytotoxicity assay. Bacterial broths were irradiated at 5, 10, 15, 24 Gy and exotoxin A protein extract activity were assessed post exposure. Exotoxin A extract exerted an ADP-ribosyltransferase ability which was depicted by the appearance of a new ʎmax after the addition of exotoxin A to NBAG/NAD+ mixture, fragmentation of NAD+ and development of new peaks in HPLC chromatograms. Intracellular enzyme activity was confirmed by the prominent cytotoxic effects of exotoxin A extract on cultured cells. In conclusion, the activity of Exotoxin A can be monitored via its ADP-ribosyltransferase activity and low doses of gamma radiation reduced its activity. Therefore, coupling radiotherapy with exotoxin A in cancer therapy should be carefully monitored.

Serum lactate dehydrogenase as a biochemical marker for maternal outcome in pre-eclampsia

Background: LDH is a cytoplasmic intracellular enzyme present in the heart, kidney, muscle, leukocytes and erythrocytes, of all major organ systems. The presence of LDH in extracellular space points towards cellular damage, endothelial dysfunction. Preeclampsia is a multisystem disorder during pregnancy causing cellular damage or death. Hence, serum LDH levels can be helpful in determining the extent of cell damage and the seriousness of this disease. The present study aimed to correlate the maternal serum lactate dehydrogenase levels with maternal and perinatal outcomes in women with pre-eclampsia.Methods: It was a prospective observational study. A total of 120 antenatal patients diagnosed with hypertensive disorder of pregnancy were included in this study. Serum LDH levels were estimated by enzymatic method on the autoanalyzer. Patients were grouped into 3 categories according to serum LDH levels: a) <600 IU/l, b) 600-800 IU/l c) >800 IU/l. Clinical manifestation of development of complications of hypertensive disease and its relation with serum LDH in respective patients were analyzed.Results: An LDH level of more than 800 IU/l was seen in 19.2% while between 600 to 800 IU/l was seen in 16.7% cases. A significant association was observed between incidence of maternal complications with high LDH levels (p<0.01). High LDH levels were observed to be associated with development of ante-partum haemorrhage, eclampsia and requirement of ICU admission. No significant association was found between different categories of LDH and deep tendon reflexes, levels of proteinuria at the time of admission in these preeclamptic women.Conclusions: Close monitoring and early intervention of the preeclampsia patients with elevated serum LDH levels can help avoid adverse effects of the disease and thereby help improve maternal and perinatal outcomes in pregnant women with preeclampsia.

SERUM LACTATE DEHYDROGENASE AS A BIOCHEMIAL MARKER FOR MATERNAL AND PERINATAL OUTCOME IN PRE-ECLAMPSIA

Background: Pre-eclampsia affects about 5-10% of all pregnancies and is a major cause of maternal, fetal and neonatal morbidity and mortality, particularly in developing countries. FOGSI and other studies show the incidence of pre eclampsia in India ranges between 11-13%. Lactate Dehydrogenase (LDH) is mainly an intracellular enzyme. LDH is present in many body tissues, especially heart, liver, kidney, skeletal muscle, brain, blood cells and lungs. Its level is increased in the scenario of increased cell injury, hemolysis and cell death. Cellular enzymes in the extracellular space although of no further metabolic function in this space, are still of benet because they serve as indicators suggestive of disturbance of cellular integrity induced by pathological conditions and is used to detect cell damage or cell death.This can be further used as help in making decision, regarding the management strategies to improve the maternal and foetal outcome. Objectives: To compare serum LDH levels in the normal pregnant women and in women with preeclampsia and eclampsia in ante-partum period and to correlate the severity of the disease, maternal and perinatal outcome with Lactic Dehydrogenase (LDH) levels in serum in patients of pre-eclampsia and eclampsia. Material and Methods: A prospective comparative study was conducted in the department of Obstetrics and Gynaecology, Ispat General Hospital, Rourkela. Out of 150 women studied, 50 were normal pregnant women, 32 were of mild preeclampsia, 35 were of severe preeclampsia and 33 of eclampsia. The statistical analysis was done by Chi-square test, Fischer Exact test, analysis of variance and student ''t'' test (two tailed and independent). Results: LDH levels were signicantly elevated in women with preeclampsia and eclampsia (p value <0.001). LDH levels had signicant direct correlation with increasing blood pressure (p value <0.001) as well as poor maternal and perinatal outcome. Conclusion: High serum LDH levels correlate well with the severity of the disease and poor maternal and fetal outcomes in patients of preeclampsia and eclampsia and can be considered as a supportive biochemical and prognostic marker from early third trimester.

Strategies of Detecting Bacteria Using Fluorescence-Based Dyes

Identification of bacterial strains is critical for the theranostics of bacterial infections and the development of antibiotics. Many organic fluorescent probes have been developed to overcome the limitations of conventional detection methods. These probes can detect bacteria with “off-on” fluorescence change, which enables the real-time imaging and quantitative analysis of bacteria in vitro and in vivo. In this review, we outline recent advances in the development of fluorescence-based dyes capable of detecting bacteria. Detection strategies are described, including specific interactions with bacterial cell wall components, bacterial and intracellular enzyme reactions, and peptidoglycan synthesis reactions. These include theranostic probes that allow simultaneous bacterial detection and photodynamic antimicrobial effects. Some examples of other miscellaneous detections in bacteria have also been described. In addition, this review demonstrates the validation of these fluorescent probes using a variety of biological models such as gram-negative and -positive bacteria, antibiotic-resistant bacteria, infected cancer cells, tumor-bearing, and infected mice. Prospects for future research are outlined by presenting the importance of effective in vitro and in vivo detection of bacteria and development of antimicrobial agents.

Isolation and Screening of Pectinase Producing Bacteria from Soil Sample

The vast majority of the industrial use of enzymes is covered from microorganisms. Microorganisms are favoured in industry because of their several advantages for example rapid growth, short life expectancy and simplicity in doing genetic alterations. Microbial enzymes are thus amply provided, very much standardized and promoted by many companies. Among various enzymes, Pectinases hold an exceptional place because of its different uses in various sectors like food, textile and biofuel industries.A total of 25% of total enzyme market is being shared by Pectinase alone.The current study was carried out to evaluate the pectinase activity of the pectinolytic bacteria. 40 Bacterial strains were isolated from different soil samples and screened for Pectinase production. Primary and Secondary screening showed 3 potential isolates I38 , I39 and I40 showing pectin degradation on Vincent’s media. Further, extracellular pectinase was partially purified by ammonium sulphate precipitation and dialysis. Sequential ammonium sulphate saturations from 20-80% i.e. (20, 40, 60 and 80%) showed 60% ammonium sulphate was optimum for precipitation of intracellular enzyme whereas 80% was optimum for extracellular enzyme.

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 Spike Glycoprotein

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both SARS-CoV-2 infection severity and long-term immunity, either after recovery or vaccination. A hallmark of COVID-19 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides by 10 common allotypes of ER aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for MHC class I molecules, including known SARS-CoV-2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses to COVID-19.