Arsenic level in bladder tumor of patients from an exposed population: association with progression and prognosis.

Aims: To elucidate the impact of arsenic on progression and prognosis of bladder cancer. Patients & methods: Total arsenic in 145 tumors (80 non-muscle-invasive [NMIBC] and 65 muscle-invasive bladder cancer [MIBC]) was measured and associated with Ki67 expression, tumor-clinicopathological parameters and patient outcome. Results: Tumor arsenic concentration was higher in exposed than unexposed patients (256 µg/kg vs 77 µg/kg; p < 0.0001) and positively correlated (r = 0.65; p < 0.0001) with arsenic content of patient's drinking water. Arsenic concentration showed significant association with Ki67-overexpression (p = 0.001) and advanced tumor stages (NMIBC vs MIBC; p = 0.0009). In NMIBC, high tumor arsenic (>100 µg/kg) and Ki67 overexpression was established as predictors for recurrence (hazard ratio [HR]: 4.68; p = 0.005 and HR: 3.91; p = 0.018) and progression (HR: 6.04; p = 0.023 and HR: 6.87; p = 0.013). In MIBC, association of high arsenic remained significant with increased risk of recurrence (HR: 4.58; p = 0.04). Conclusion: In NMIBC, high arsenic and Ki67 overexpression and in MIBC, only high arsenic showed prognostic importance in predicting poor patient outcome.

Lay abstract Research work suggests arsenic as risk factor for bladder cancer. In developing countries such as India, arsenic contamination of underground drinking water is a major health problem. The present study aimed to evaluate impact of arsenic on parameters of bladder cancer aggressiveness and clinical outcome of patients from West Bengal, India. Our data showed accumulation of arsenic in bladder tumor of patients exposed mainly through contaminated drinking water. Arsenic content in tumor favored aggressive phenotypes in bladder cancer (higher cell proliferation and advanced tumor stages) and was found to be a potential predictor for cases of death and disease recurrence in patients after receiving primary treatment measures. Therefore, arsenic content in bladder tumor may be used to improve existing protocols for better prediction of patient outcomes in populations with a similar type of exposure.

Genomic characterization of Salmonella bacteriophages isolated from India.

Salmonella are a medically important Gram-negative foodborne pathogen. Genomic diversity of Salmonella is increasingly studied but at the same time, we have limited knowledge of Salmonella phage diversity. In this study, we have isolated Salmonella phages from sewage and river water. Genomic characterization of 12 Salmonella phages was carried out using next-generation sequencing platform. Newly sequenced phages were classified based on amino acid sequence phylogenetic analysis. In newly sequenced phages, several virulence genes, DNA metabolism genes, tRNA genes, antibiotic resistance genes and genes not having known role in the life cycle of phages were identified. Annotations of newly sequenced phage genome showed the presence of polymyxin-b resistance gene and penicillin binding protein. Annotation identified number of genes which are involved in DNA metabolism. Results suggest that most of the phages having G + C content different than their host possess DNA metabolism genes. The presence of tRNAs in the genome of Salmonella_phage38-India was identified; however, we did not observe any correlation between tRNA genes and overall codon usage in the phage genome. It is suggested that the phage-encoded tRNAs may increase fitness of phages. In summary, we isolated novel Salmonella phages, determined full genome sequences and provided phylogenetic analysis-based classification.

The ß-prism lectin domain of Vibrio cholerae hemolysin promotes self-assembly of the ß-pore-forming toxin by a carbohydrate-independent mechanism.

Vibrio cholerae cytolysin/hemolysin (VCC) is an amphipathic 65-kDa ß-pore-forming toxin with a C-terminal ß-prism lectin domain. Because deletion or point mutation of the lectin domain seriously compromises hemolytic activity, it is thought that carbohydrate-dependent interactions play a critical role in membrane targeting of VCC. To delineate the contributions of the cytolysin and lectin domains in pore formation, we used wild-type VCC, 50-kDa VCC (VCC(50)) without the lectin domain, and mutant VCC(D617A) with no carbohydrate-binding activity. VCC and its two variants with no carbohydrate-binding activity moved to the erythrocyte stroma with apparent association constants on the order of 10(7) M(-1). However, loss of the lectin domain severely reduced the efficiency of self-association of the VCC monomer with the ß-barrel heptamer in the synthetic lipid bilayer from ∼83 to 27%. Notably, inactivation of the carbohydrate-binding activity by the D617A mutation marginally reduced oligomerization to ∼77%. Oligomerization of VCC(50) was temperature-insensitive; by contrast, VCC self-assembly increased with increasing temperature, suggesting that the process is driven by entropy and opposed by enthalpy. Asialofetuin, the ß1-galactosyl-terminated glycoprotein inhibitor of VCC-induced hemolysis, promoted oligomerization of 65-kDa VCC to a species that resembled the membrane-inserted heptamer in stoichiometry and morphology but had reduced global amphipathicity. In conclusion, we propose (i) that the ß-prism lectin domain facilitated toxin assembly by producing entropy during relocation in the heptamer and (ii) that glycoconjugates inhibited VCC by promoting its assembly to a water-soluble, less amphipathic oligomer variant with reduced ability to penetrate the bilayer.

Complex Pathophysiology of Acute Kidney Injury (AKI) in Aging: Epigenetic Regulation, Matrix Remodeling, and the Healing Effects of H2S.

The kidney is an essential excretory organ that works as a filter of toxins and metabolic by-products of the human body and maintains osmotic pressure throughout life. The kidney undergoes several physiological, morphological, and structural changes with age. As life expectancy in humans increases, cell senescence in renal aging is a growing challenge. Identifying age-related kidney disorders and their cause is one of the contemporary public health challenges. While the structural abnormalities to the extracellular matrix (ECM) occur, in part, due to changes in MMPs, EMMPRIN, and Meprin-A, a variety of epigenetic modifiers, such as DNA methylation, histone alterations, changes in small non-coding RNA, and microRNA (miRNA) expressions are proven to play pivotal roles in renal pathology. An aged kidney is vulnerable to acute injury due to ischemia-reperfusion, toxic medications, altered matrix proteins, systemic hemodynamics, etc., non-coding RNA and miRNAs play an important role in renal homeostasis, and alterations of their expressions can be considered as a good marker for AKI. Other epigenetic changes, such as histone modifications and DNA methylation, are also evident in AKI pathophysiology. The endogenous production of gaseous molecule hydrogen sulfide (H2S) was documented in the early 1980s, but its ameliorative effects, especially on kidney injury, still need further research to understand its molecular mode of action in detail. H2S donors heal fibrotic kidney tissues, attenuate oxidative stress, apoptosis, inflammation, and GFR, and also modulate the renin-angiotensin-aldosterone system (RAAS). In this review, we discuss the complex pathophysiological interplay in AKI and its available treatments along with future perspectives. The basic role of H2S in the kidney has been summarized, and recent references and knowledge gaps are also addressed. Finally, the healing effects of H2S in AKI are described with special emphasis on epigenetic regulation and matrix remodeling.

Effectiveness of Oral Cephalexin-Clavulanic Acid, Cefuroxime, and Amoxicillin-Clavulanic Acid in the Management of Dental Infections: A Real-World, Retrospective, Electronic Medical Record-Based Study in India.

BACKGROUND: Despite multiple antibiotics being available to manage dental infections (DI), there is lack of data comparing commonly prescribed antibiotics in India. OBJECTIVES: The aim of this study was to evaluate the real-world effectiveness and tolerability of cephalexin-clavulanic acid fixed-dose combination (cephalexin CV FDC) in contrast with amoxicillin-clavulanic acid (co-amoxiclav FDC) and cefuroxime among patients with dental infections (odontogenic) in India. METHODS: This retrospective, multi-centric, observational, real-world electronic medical record (EMR)-based study was conducted between January 2022 and December 2022. The EMRs of 355 adults with DI receiving oral cephalexin CV, co-amoxiclav, or cefuroxime were categorized into two distinct groups: Group I (Test Group) with patients prescribed cephalexin extended release 375/750 mg along with clavulanic acid 125 mg; and Group II (Comparator Group) with patients prescribed co-amoxiclav 625 mg (500 mg amoxicillin + 125 mg clavulanic acid) or cefuroxime (250 mg/500 mg). RESULTS: Toothache was the most common complaint, reported by 95.5% of patients, followed by swelling (46.8%), tooth sensitivity (35.5%), pus discharge (33.0%), redness and halitosis (30.4% each). Dental caries was observed in 81.1% of patients. Clinical improvement, defined as improvement/partial resolution of infection-related clinical signs and symptoms (composite measure of pain, swelling, fever, requirement of additional antimicrobial therapy) as per dentists' judgment, was recorded in 98.3% of patients with cephalexin CV, 96.8% of patients with co-amoxiclav, and 98.9% of patients treated with cefuroxime within 10 days. Time (days) to clinical improvement was numerically lesser among patients receiving cephalexin CV (4.6 ± 2.0) compared with cefuroxime (4.9 ± 2.1) and co-amoxiclav (5.0 ± 2.6). All treatments were well tolerated. CONCLUSION: Cephalexin CV was as effective as co-amoxiclav and cefuroxime, with faster clinical improvement and better resolution of certain symptoms.

Effect of cholesterol on the ion-membrane interaction: Zeta potential and dynamic light scattering study.

Cholesterol in a bio-membrane plays a significant role in many cellular event and is known to regulate the functional activity of protein and ion channel. In this study we report a significant effect of cholesterol on the ion-membrane interaction. We prepare large unilamellar vesicles, composed of zwitterionic lipid DOPC and anionic lipid DOPG with different cholesterol concentration. Electrostatics of anionic membranes containing cholesterol in the presence of NaCl has systematically been explored using dynamic light scattering and zeta potential. Negative zeta potential of the membrane decreases its negative value with increasing ion concentration for all cholesterol concentrations. However, zeta potential itself decreases with increasing cholesterol content even in the absence of monovalent ions. Electrostatic behaviour of the membrane is determined from well-known Gouy Chapmann model. Negative surface charge density of the membrane decreases with increasing cholesterol content. Binding constant, estimated from the electrostatic double layer theory, is found to increase significantly in the presence of cholesterol. Comparison of electrostatic parameters of the membrane in the presence and absence of cholesterol suggests that cholesterol significantly alter the electrostatic behaviour of the membrane.

Role of Physiotherapy in HPV Proven Cases of Oral Submucous Fibrosis.

Background: To assess the role of physiotherapy in human papillomavirus (HPV) proven cases of oral submucous fibrosis (OSMF). Materials and Methods: Overall, 100 patients got recruited. Only histopathologic confirmed cases of OSMF were enrolled. Purified DNA of tissue blocks was quantified by spectrophotometry. Prevalence of HPV was evaluated. The participants got randomized into 2 cohorts: HPV positive cases and HPV negative cases. Physiotherapy was done and outcome was done and outcome was assessed and compared. Assessment of results was done by SPSS software followed by statistical evaluation. Results: HPV was seen in 80% of the patients. Mean mouth opening pretreatment and postphysiotherapy among patients with HPV positive status was 26.31 mm and 30.12 mm, respectively. Mean mouth opening pretreatment and postphysiotherapy among patients with HPV negative status was 25.11 mm and 29.74 mm, respectively. Nonsignificant results were obtained while comparing the outcome of physiotherapy among HPV positive and negative groups. Conclusion: Outcome of physiotherapy among OSMF patients is independent of HPV status.

Hemolysin induces Toll-like receptor (TLR)-independent apoptosis and multiple TLR-associated parallel activation of macrophages.

Vibrio cholerae hemolysin (HlyA) displays bipartite property while supervising macrophages (MΦ). The pore-forming toxin causes profound apoptosis within 3 h of exposure and in parallel supports activation of the defying MΦ. HlyA-induced apoptosis of MΦ remains steady for 24 h, is Toll-like receptor (TLR)-independent, and is driven by caspase-9 and caspase-7, thus involving the mitochondrial or intrinsic pathway. Cell activation is carried forward by time dependent up-regulation of varying TLRs. The promiscuous TLR association of HlyA prompted investigation, which revealed the ß-prism lectin domain of HlyA simulated TLR4 up-regulation by jacalin, a plant lectin homologue besides expressing CD86 and type I cytokines TNF-α and IL-12. However, HlyA cytolytic protein domain up-regulated TLR2, which controlled CD40 for continuity of cell activation. Expression of TOLLIP before TLR2 and TLR6 abrogated TLR4, CD40, and CD86. We show that the transient expression of TOLLIP leading to curbing of activation-associated capabilities is a plausible feedback mechanism of MΦ to deploy TLR2 and prolong activation involving CD40 to encounter the HlyA cytolysin domain.

The role of C-terminus carbohydrate-binding domain of Vibrio cholerae haemolysin/cytolysin in the conversion of the pre-pore ß-barrel oligomer to a functional diffusion channel.

BACKGROUND & OBJECTIVES: Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that ß1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus ß-prism lectin domain contributed to pore formation in erthrocytes. METHODS: VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of ß-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. RESULTS: Proteolytic truncation of the C-terminus ß-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. INTERPRETATION & CONCLUSIONS: Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

Three-dimensional structure of different functional forms of the Vibrio cholerae hemolysin oligomer: a cryo-electron microscopic study.

Vibrio cholerae hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable beta-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa beta-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an approximately 1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.

Sudden death due to ruptured oesophageal varices - autopsy-based case report.

Acute haemorrhage from ruptured oesophageal varices is a serious consequence of portal hypertension in cirrhotic patients. It represents a medical emergency with a high morbidity and mortality rate. Studies over the years have shown a direct link with chronic alcoholism in the development of such complications. Although the gastrointestinal system accounts for a few numbers of sudden deaths, bleeding through ruptured varices represent a life-threatening condition. The role of forensic pathologist is vital in dealing with sudden deaths. Here, we report a case of a 46-year-old man who died suddenly following the rupture of oesophageal varices.

Memory T-cell responses to Vibrio cholerae O1 infection.

Vibrio cholerae O1 can cause diarrheal disease that may be life-threatening without treatment. Natural infection results in long-lasting protective immunity, but the role of T cells in this immune response has not been well characterized. In contrast, robust B-cell responses to V. cholerae infection have been observed. In particular, memory B-cell responses to T-cell-dependent antigens persist for at least 1 year, whereas responses to lipopolysaccharide, a T-cell-independent antigen, wane more rapidly after infection. We hypothesize that protective immunity is mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue, and T-cell responses may be required to generate and maintain durable memory B-cell responses. In this study, we examined B- and T-cell responses in patients with severe V. cholerae infection. Using the flow cytometric assay of the specific cell-mediated immune response in activated whole blood, we measured antigen-specific T-cell responses using V. cholerae antigens, including the toxin-coregulated pilus (TcpA), a V. cholerae membrane preparation, and the V. cholerae cytolysin/hemolysin (VCC) protein. Our results show that memory T-cell responses develop by day 7 after infection, a time prior to and concurrent with the development of B-cell responses. This suggests that T-cell responses to V. cholerae antigens may be important for the generation and stability of memory B-cell responses. The T-cell proliferative response to VCC was of a higher magnitude than responses observed to other V. cholerae antigens.

Vibrio cholerae hemolysin is apoptogenic to peritoneal B-1a cells but its oligomer shepherd the cells for IgA response.

Vibrio cholerae hemolysin (HlyA) can exist as a monomer with hemolytic activity and an oligomer that agglutinates erythrocytes. Biochemical differences accompanying the change in state of aggregation led us to weigh possible differences between the two forms from mucosal immunoregulation perspective. HlyA oligomer-treated murine B-1a cells up-regulated TLR2 and involved the signaling molecules MyD88, TRAF6 and NF-kappaB. The cells subsequently expressed IgM and IgA. HlyA monomer treatment although resulted in TLR2 up-regulation, could not induce these effects. Apoptosis was detected in majority of the monomer-treated cells that involved caspase-9 and caspase-3. This study shows for the first time that two forms of the same protein could drive the host immune cell to two different outcomes, one of death and the other towards activation.

Oligomerization of Vibrio cholerae hemolysin induces CXCR3 upregulation and activation of B-1a cell.

The hemolysin oligomer promotes the proliferation of B-1a cells and the expression of CD25, which is indicative of cell activation, on B-1a cells. The upregulation of CD86 induced by the oligomer showed its selective bias for the B7-2 member of B7 family while the monomer failed to induce these effects. The oligomer induced the expression of CXCR3, associated with B cell activation, while the monomer induced the expression of CXCL4, a powerful angiostatic chemokine. In conclusion, we found that B-1a cells responded to the apoptogenic monomer by expressing CXCL4, whereas oligomerization of the immunogen induced CXCR3 to shift the response towards activation.

Vibrio cholerae hemolysin: The ß-trefoil domain is required for folding to the native conformation.

Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa ß-pore-forming toxin causing lysis and death of eukaryotic cells. Apart from the core cytolysin domain, VCC has two lectin domains with ß-trefoil and ß-prism folds. The ß-prism domain binds to cell surface carbohydrate receptors; the role of the ß-trefoil domain is unknown. Here, we show that the pro-VCC mutant without the ß-trefoil domain formed aggregates highly susceptible to proteolysis, suggesting lack of a properly folded compact structure. The VCC variants with Trp532Ala or Trp534Ala mutation in the ß-trefoil domain formed hemolytically inactive, protease-resistant, ring-shaped SDS-labile oligomers with diameters of ~19 nm. The Trp mutation induced a dramatic change in the global conformation of VCC, as indicated by: (a) the change in surface polarity from hydrophobic to hydrophilic; (b) movement of core Trp residues to the protein-water interface; and (c) decrease in reactivity to the anti-VCC antibody by >100-fold. In fact, the mutant VCC had little similarity to the wild toxin. However, the association constant for the carbohydrate-dependent interaction mediated by the ß-prism domain decreased marginally from ~3×108 to ~5×107 M-1. We interpret the observations by proposing: (a) the ß-trefoil domain is critical to the folding of the cytolysin domain to its active conformation; (b) the ß-prism domain is an autonomous folding unit.

Macrophage distinguishes Vibrio cholerae hemolysin from its protease insensitive oligomer by time dependent and selective expression of CD80-CD86.

The monomeric and oligomeric forms of Vibrio cholerae hemolysin (HlyA), a membrane damaging toxin that forms transmembrane pentameric diffusion channels in target eukaryotic membrane, show a pronounced difference in protease susceptibility, presumably due to masking of sensitive peptide bonds during oligomerization. In this work, we examined if resistance of a protein to proteolytic processing affects the expression of costimulatory molecules, CD80 and CD86, on macrophage exposed to the same antigen. The murine peritoneal cavity macrophages expressed both CD80 and CD86 after 24 h of incubation with HlyA monomer but failed to express the costimulatory molecules when treated with the HlyA oligomer. The expression of CD80 molecule on macrophage after 48 h by the HlyA oligomer that failed to express the costimulatory molecules after 24 h indicates that proteolytic processing plays a decisive role in expression of CD80 and CD86 on cell surface.

Cryo-electron microscopy reveals the membrane insertion mechanism of V. cholerae hemolysin.

Vibrio cholerae hemolysin (HlyA) is a 65 kDa pore-forming toxin which causes lysis of target eukaryotic cells by forming heptameric channels in the plasma membrane. Deletion of the 15 kDa C-terminus ß-prism carbohydrate-binding domain generates a 50 kDa truncated variant (HlyA50) with 1000-fold-reduced pore-forming activity. Previously, we showed by cryo-electron microscopy that the two toxin oligomers have central channels, but the 65 kDa toxin oligomer is a seven-fold symmetric structure with bowl-, ring-, and arm-like domains, whereas the 50 kDa oligomer is an asymmetric jar-like heptamer. In the present study, we determined three-dimensional(3D) structures of HlyA and HlyA50 in presence of erythrocyte stroma and observed that interaction of the 65 kDa toxin with the stroma induced a significant decrease in the height of the ß-barrel oligomer with a change in conformation of the ring- and arm-like domains of HlyA. These features were absent in interaction of HlyA50 with stroma. We propose that this conformational transition is critical for membrane-insertion of the toxin.

Management of erythema nodosum leprosum by mycophenolate mofetil.

Mycophenolate mofetil has been tried in 20 cases of chronic relapsing erythema nodosum leprosum reaction where long use of systemic steroid produce complications or are contraindicated. Excellent results have been observed in all the cases to arrest the reaction followed for a period of six to eight months duration.

Condom leukoderma.

Contact dermatitis from natural latex of condom has been reported and is attributed to latex sensitivity. Chemical leukoderma from rubber condom is probably not reported. Here we present a case of chemical leukoderma in a 32-year-old male who developed depigmentation around the shaft of the penis in a circumferential pattern. Since the lesion was solitary and the site corresponded to the point of maximum contact of the condom, a diagnosis of contact leukoderma due to latex condom was thought of. Patch testing was done with mercaptobenzothiazole (MBT), dusting powder present in the condom and condom latex as such. The patient tested positive (3+) with mercaptobenzothiazole and the condom latex. On discontinuation of condom use and with UVB phototherapy, lesions repigmented in eight weeks.