Underwater Adhesives from Redox-Responsive Polyplexes of Thiolated Polyamide Polyelectrolytes.

We report the fabrication of optically clear underwater adhesives using polyplexes of oppositely charged partially-thiolated polyamide polyelectrolytes (TPEs). The thiol content of the constituent PEs was varied to assess its influence on the adhesive properties of the resulting glues. These catechol-free, redox-responsive TPE-adhesives were formulated in aquo and exhibited high optical transparency and strong adhesion even on submerged or moist surfaces of diverse polar substrates such as glass, aluminium, wood, and bone pieces. The adhesives could be cured under water through oxidative disulphide crosslinking of the constituent TPEs. The polyamide backbone provided multi-site H-bonding interactions with the substrates while the disulphide crosslinking provided the cohesive strength to the glue. Strong adhesion of mammalian bones (load bearing capacity upto 7 kg/cm2 ) was achieved using the adhesive containing 30 mol % thiol residues. Higher pH and use of oxidants such as povidone-iodine solution enhanced the curing rate of the adhesives, and so did the use of Tris buffer instead of Phosphate buffer. The porous architecture of the adhesive and its progressive degradation in aqueous medium over the course of three weeks bode well for diverse biomedical applications where temporary adhesion of tissues is required.

Pharmaco-immunomodulatory interventions for averting cytokine storm-linked disease severity in SARS-CoV-2 infection.

The year 2020 is characterised by the COVID-19 pandemic that has quelled more than half a million lives in recent months. We are still coping with the negative repercussions of COVID-19 pandemic in 2021, in which the 2nd wave in India resulted in a high fatality rate. Regardless of emergency vaccine approvals and subsequent meteoric global vaccination drives in some countries, hospitalisations for COVID-19 will continue to occur due to the propensity of mutation in SARS-CoV-2 virus. The immune response plays a vital role in the control and resolution of infectious diseases. However, an impaired immune response is responsible for the severity of the respiratory distress in many diseases. The severe COVID-19 infection persuaded cytokine storm that has been linked with acute respiratory distress syndrome (ARDS), culminates into vital organ failures and eventual death. Thus, safe and effective therapeutics to treat hospitalised patients remains a significant unmet clinical need. In that state, any clue of possible treatments, which save patients life, can be treasured for this time point. Many cohorts and clinical trial studies demonstrated that timely administration of immunomodulatory drugs on severe COVID-19 patients may mitigate the disease severity, hospital stay and mortality. This article addresses the severity and risk factors of hypercytokinemia in COVID-19 patients, with special emphasis on prospective immunomodulatory therapies.

Persistent decrease of renal functional reserve in patients after cardiac surgery-associated acute kidney injury despite clinical recovery.

Background: Cardiac surgery is a leading cause of acute kidney injury (AKI). Such AKI patients may develop progressive chronic kidney disease (CKD). Others, who appear to have sustained no permanent loss of function (normal serum creatinine), may still lose renal functional reserve (RFR). Methods: We extended the follow-up in the observational 'Preoperative RFR Predicts Risk of AKI after Cardiac Surgery' study from hospital discharge to 3 months after surgery for 86 (78.2%) patients with normal baseline estimated glomerular filtration rate (eGFR), and re-measured RFR with a high oral protein load. The primary study endpoint was change in RFR. Study registration at clinicaltrials.gov Identifier: NCT03092947, ISRCTN Registry: ISRCTN16109759. Results: At 3 months, three patients developed new CKD. All remaining patients continued to have a normal eGFR (93.3 ± 15.1 mL/min/1.73 m2). However, when stratified by post-operative AKI and cell cycle arrest (CCA) biomarkers, AKI patients displayed a significant decrease in RFR {from 14.4 [interquartile range (IQR) 9.5 - 24.3] to 9.1 (IQR 7.1 - 12.5) mL/min/1.73 m2; P < 0.001} and patients without AKI but with positive post-operative CCA biomarkers also experienced a similar decrease of RFR [from 26.7 (IQR 22.9 - 31.5) to 19.7 (IQR 15.8 - 22.8) mL/min/1.73 m2; P < 0.001]. In contrast, patients with neither clinical AKI nor positive biomarkers had no such decrease of RFR. Finally, of the three patients who developed new CKD, two sustained AKI and one had positive CCA biomarkers but without AKI. Conclusions: Among elective cardiac surgery patients, AKI or elevated post-operative CCA biomarkers were associated with decreased RFR at 3 months despite normalization of serum creatinine. Larger prospective studies to validate the use of RFR to assess renal recovery in combination with biochemical biomarkers are warranted.

Clinical and Etiological Profile of Fever with Thrombocytopenia - A Tertiary Care Hospital Based Study.

Objectives: Febrile thrombocytopenia is a condition commonly caused by infections. The present study is intended to know the underlying etiology of fever with thrombocytopenia, the various presentations and complications in our community. Material and Methods: A cross-sectional epidemiological study was conducted including 1217 patients aged more than 14 years with fever and thrombocytopenia admitted in the medical wards from October 2013 to September 2014. Detailed clinical examination and routine investigations were done; specific investigations like blood culture, widal test, antigen test for malaria, IgM ELISA leptospira, IgM ELISA dengue, bone marrow aspiration/biopsy etc. were done as and when indicated. The data are presented as percentage and numbers. Rates and ratios are computed. Results: Infection was the commonest cause of thrombocytopenia and dengue was the commonest of the infections followed by malaria. Bleeding manifestations were seen in 42.7% of patients. 91.40% of patients with bleeding tendencies had petechiae/purpura as the commonest bleeding manifestation, followed by spontaneous bleeding in 57%. Spontaneous bleeding was noted when platelet counts were less than 20,000. Petechiae/Purpura were seen more commonly when platelet count was in the range of less than or equal to 50,000. Good recovery was noted in 95%, while 5% had mortality. Septicemia accounted for 85.24% of deaths followed by malaria (6.55%) and dengue (5%). Conclusion: Fever with thrombocytopenia is an important clinical condition commonly caused by infections, particularly dengue and malaria. In majority of patients thrombocytopenia was transient and asymptomatic, but in significant number of cases there were bleeding manifestations. On treating the specific cause drastic improvement in platelet count was noted. Mortality in febrile thrombocytopenia is not directly associated with degree of thrombocytopenia but with concomitant involvement of other organs leading to multiorgan dysfunction.

Delayed Nephrology Consultation and High Mortality on Acute Kidney Injury: A Meta-Analysis.

BACKGROUND: Acute kidney injury (AKI) is a complex syndrome associated with substantial morbidity, mortality and costs. Despite advancements in diagnosis and care practice, AKI remains a disorder usually under/late-recognized with high mortality. One of the hidden reasons for poor outcome might be delayed nephrology consultation, with the involvement of the specialist only in severe stages of AKI when renal replacement therapy (RRT) is required. METHODS: We searched PubMed, EMBASE and Cochrane central register for related work on the subject. Six studies were identified for the meta-analysis, correlating time of nephrology consultation and mortality in AKI. RESULTS: We found that delayed nephrology consultation is associated with higher mortality in AKI, with an OR 0.79 (95% CI 0.48-1.10, p < 0.05). CONCLUSION: Delayed nephrology consultation contributes to higher mortality in AKI. The early involvement of nephrologist may present an advantage in terms of early recognition, prevention and effective treatment of AKI. An early involvement of multidisciplinary task force may contribute to better treatment, before the preventable complications of AKI occur or an emergency RRT is required.

Optimizing a kidney stress test to evaluate renal functional reserve.

BACKGROUND: Renal function reserve (RFR) describes the capacity of the kidney to increase glomerular filtration rate (GFR) in response to physiological or pathological stimuli. The scope of our study was to evaluate the optimal level of stimulation using different doses of protein load (PL) for a standard renal stress test (RST). METHODS: 18 young healthy individuals were given sessions of PL with 1 and 2 g/kg body weight. Endogenous creatinine clearance was calculated. Baseline GFR (bGFR) and stress GFR (sGFR) (post-PL) were obtained; RFR is the difference between stress and baseline GFR. A p-value < 0.05 was considered statistically significant. RESULTS: Mean bGFR was 107.97 ± 12.33 mL/min/1.73m2. sGFR with 1 and 2 g PL were significantly higher than bGFR in all subjects. The sGFR after 2 g PL (141.75 ± 19.90 mL/min/1.73m2) was not statistically different from the sGFR after 1 g PL (142.37 ± 22.35 mL/min/1.73m2). sGFR and therefore RFR were independent from the value of bGFR. CONCLUSIONS: We found no difference between 1 and 2 g/kg body weight PL to elicit sGFR. RST may be useful to predict susceptibility and risk of developing acute kidney injury and/or progression to chronic kidney disease. RST uncovers the possible loss of renal functional mass when this condition is not manifested clinically. Further studies are needed to set this hypothesis.

Vitamin D: A Critical Micronutrient for Vascular Health in Type 2 Diabetes Mellitus.

OBJECTIVE: Vitamin D deficiency may be responsible for endothelial dysfunction which in turn affects the onset and progression of vascular disease including coronary artery disease and its risk factor, directly or indirectly through various mechanisms. This study was undertaken to find out association between vitamin D and endothelial dilatation of brachial artery, which may help to suggest possible underlying mechanisms and may be of clinical importance in planning preventive and therapeutic strategies. MATERIAL AND METHODS: 50 cases of type 2 diabetes mellitus aged 40-60 years were taken. 50 cases without type 2 diabetes mellitus matched for confounding factors were taken as controls. Venous blood samples were collected for the investigations including vitamin D levels. Then Participants were subjected to ultrasound examination for measurement of flow mediated dilatation (FMD) and endothelial independent dilatation after ingestion of glyceryl tri-nitrate (GTN). Unpaired student T test and correlation coefficient analysis were used to find out association between different variables. RESULTS: The mean values of FMD were 18.85 ± 5.39% and 10.29 ± 4.91% in controls and cases respectively (p<0.001). The dilatation after GTN was observed to be 26.16 ± 4.25% and 18.74 ± 5.72% in controls and cases respectively (p<0.001). The mean levels of vitamin D among controls and cases were 25.41 ± 12.18 and 14.52 ± 8.28 ng/ml respectively. The correlation between endothelial dependent dilatation (FMD), endothelial independent dilatation (after GTN) and vitamin D was found to be more positive in cases (r=0.870, r=0.798) than controls (r=0.079, r=0.158). CONCLUSIONS: Vitamin D deficiency state is higher among cases of type 2 DM. Endothelial dependent dilatation (FMD) was found to be lower among the patients of type 2 DM. The study gives us an insight to identify the diabetics with vitamin D deficiency which may be at higher risk of vascular complications including coronary artery disease.

Fabry's disease: an example of cardiorenal syndrome type 5.

Cardiorenal syndrome type 5 (CRS-5) includes conditions where there is a simultaneous involvement of the heart and kidney from a systemic disorder. This is a bilateral organ cross talk. Fabry's disease (FD) is a devastating progressive inborn error of metabolism with lysosomal glycosphingolipid deposition in variety of cell types, capillary endothelial cells, renal, cardiac and nerve cells. Basic effect is absent or deficient activity of lysosomal exoglycohydrolase a-galactosidase A. Renal involvement consists of proteinuria, isosthenuria, altered tubular function, presenting in second or third decade leading to azotemia and end-stage renal disease in third to fifth decade mainly due to irreversible changes to glomerular, tubular and vascular structures, especially highlighted by podocytes foot process effacement. Cardiac involvement consists of left ventricular hypertrophy, right ventricular hypertrophy, arrhythmias (sinus node and conduction system impairment), diastolic dysfunction, myocardial ischemia, infarction, transmural replacement fibrosis, congestive heart failure and cardiac death. Management of FD is based on enzymatic replacement therapy and control of renal (with anti-proteinuric agents such as angiotensin-converting enzyme inhibitors-and/or angiotensin II receptor blockers), brain (coated aspirin, clopidogrel and statin to prevent strokes) and heart complications (calcium channel blockers for ischemic cardiomyopathy, warfarin and amiodarone or cardioverter device for arrhythmias).

Cell penetrating synthetic antimicrobial peptides (SAMPs) exhibiting potent and selective killing of mycobacterium by targeting its DNA.

Naturally occurring antimicrobial peptides (AMPs) are powerful defence tools to tackle pathogenic microbes. However, limited natural production and high synthetic costs in addition to poor selectivity limit large-scale use of AMPs in clinical settings. Here, we present a series of synthetic AMPs (SAMPs) that exhibit highly selective and potent killing of Mycobacterium (minimum inhibitory concentration <20 µg mL(-1)) over E. coli or mammalian cells. These SAMPs are active against rapidly multiplying as well as growth saturated Mycobacterium cultures. These SAMPs are not membrane-lytic in nature, and are readily internalized by Mycobacterium and mammalian cells; whereas in E. coli, the lipopolysaccharide layer inhibits their cellular uptake, and hence, their antibacterial action. Upon internalization, these SAMPs interact with the unprotected genomic DNA of mycobacteria, and impede DNA-dependent processes, leading to bacterial cell death.

Initiation of renal replacement therapy in the intensive care unit in Vicenza (IRRIV) score.

INTRODUCTION: One of the top research priorities in acute kidney injury is related to the timing of renal replacement therapy (RRT) initiation. The purpose was to develop an index that might serve as a standardized concept of timing of initiation of RRT. METHODS: A previously described database was used. We applied a multivariable Cox regression model with backward selection to characterize parameters present in those patients who received RRT compared with those who did not receive RRT. RESULTS: We studied 590 patients. We identified independent risk factors for RRT and a risk score was devised. The Area Under the Curve of the receiver operating characteristic curve was 0.81 (95% CI 0.74-0.86) for predicting the need for RRT. CONCLUSIONS: We have developed a simple Score (IRRIV Score) to identify patients at high risk of requiring RRT. This score may serve as a standardized definition of the timing of initiation of RRT.

Renal functional reserve and renal recovery after acute kidney injury.

Renal functional reserve (RFR) represents the capacity of the kidney to increase glomerular filtration rate (GFR) in response to certain physiological or pathological stimuli or conditions. Once baseline GFR is determined, RFR can be assessed clinically after an oral protein load or intravenous amino acid infusion. In clinical practice, baseline GFR displays variable levels due to diet or other factors. RFR is the difference between peak 'stress' GFR induced by the test (p.o. or i.v.) and the baseline GFR. In clinical scenarios where hyperfiltration is present (high baseline GFR due to pregnancy, hypertension or diabetic nephropathy, in solitary kidney or kidney donors), RFR may be fully or partially used to achieve normal or supranormal renal function. Since commonly used renal function markers, such as GFR, may remain within normal ranges until 50% of nephrons are lost or in patients with a single remnant kidney, the RFR test may represent a sensitive and early way to assess the functional decline in the kidney. RFR assessment may become an important tool to evaluate the ability of the kidney to recover completely or partially after a kidney attack. In case of healing with a defect and progressive fibrosis, recovery may appear complete clinically, but a reduced RFR may be a sign of a maladaptive repair or subclinical loss of renal mass. Thus, a reduction in RFR may represent the equivalent of renal frailty or susceptibility to insults. The main aim of this article is to review the concept of RFR, its utility in different clinical scenarios, and future perspective for its use.

High cutoff membrane to reduce systemic inflammation due to differentiation syndrome: a case report.

BACKGROUND: Differentiation syndrome is a life-threatening complication of therapy that is carried out with agents used for acute promyelocytic leukemia. Its physiopathology comprehends the production of inflammatory mediators by differentiating granulocytes, endothelial and alveolar cells due to stimulation by all-trans retinoic acid and leading to sustained systemic inflammation. METHODS: Treatment with high cut-off continuous veno-venous hemodialysis (HCO-CVVHD) was performed to reduce the circulating mediators of systemic inflammation. RESULTS: After 52 h of treatment, an important reduction was observed in inflammatory mediators (IL-1ß: from 10 to 2 pg/ml; IL-8: from 57 to 40 pg/ml; TNF-α: from 200 to 105 pg/ml; IL-6: from 263 to 91 pg/ml), as well as in anti-inflammatory mediators (IL-10: from 349 to 216 pg/ml). CONCLUSIONS: HCO-CVVHD should be explored as a part of treatment in systemic inflammation states other than sepsis (e.g., differentiation syndrome). Furthermore, its immunomodulatory effects could be particularly useful in immunocompromised patient treated with corticosteroids.

Cytokine removal with high cut-off membrane: review of literature.

During the last decade, blood purification therapies have been proposed as an effective therapy to control the cytokines dysregulation in systemic inflammatory syndromes. Among them, the treatment with high cut-off membranes is characterized by larger pore size and more effective clearance for middle molecular weight molecules (cytokines). In this paper, we performed a thoughtful review of the literature on HCO being used for blood purification indications in all systemic inflammation syndromes. Clinical and experimental studies show that the use of high effluent flows in a pure diffusive treatment effectively removes serum cytokines with a safe profile in albumin clearance. In clinical studies, the removal of these inflammatory mediators is associated with a significant improvement in hemodynamic condition, oxygenation indices, and organ dysfunction.

Functional Outcome Analysis of Autologous Stromal Vascular Fraction (SVF) (Sahaj Therapy®) Using Direct Sonication in Osteonecrosis of the Femoral Head (ONFH): A 6-Year Follow-Up Study.

Introduction: We investigated the safety, efficacy, functional, and clinical outcomes of intra-osseous implantation of mechanically isolated, autologous stromal vascular fraction (SVF), an Australian patented direct ultrasonication technology (Sahaj Therapy®) in osteonecrosis of the femoral head (ONFH). Materials and Methods: A total of 32 cases of ONFH were enrolled in the study after confirming with an MRI of the affected hip. All cases were treated with an intra-osseous autologous SVF implantation [4-5 cc with the cellular dosage of 8.0 × 107 cells with a viability of > 85% SVF cells] on the same surgical sitting. All the cases were followed up clinically, functionally, and radiologically at regular intervals. A comparison of mean HOOS scores at different follow-ups was done using Paired 't'-test. A P value of < 0.05 was considered significant. Results: In our study, male preponderance was seen (53.1%). According to the modified Ficat and Arlet classification, the most common grade of ONFH was grade 2 [right: 25 hips and left: 25 hips]. There was a statistically significant improvement in the mean HOOS score of the right hip (n = 10) and left hip (n = 9) from preoperative time till 72 months (P < 0.05). The follow-up MRI of the affected hips shows improved osteogenesis without any further worsening of the contour of the femoral head. No adverse effects were seen in any of the study participants. Conclusion: For individuals with ONFH, treated with intra-osseous autologous SVF implantation in the same surgical procedure is an innovative and promising treatment modality. Even after 6 years of follow-up, the study participants with ONFH have shown good clinical and functional outcomes with autologous SVF.

Prevalence of drug resistant Enterobacteriaceae in a Nepalese tertiary care hospital.

Antimicrobial resistance in Enterobacteriaceae is an emerging global public health problem. Numerous studies have reported community-acquired AmpC beta-lactamase and extended spectrum beta-lactamase (ESBL) producing Enterobacteriaceae in Nepal. However, there are limited data on community-acquired Metallo-beta-lactamase (MBL) producing Enterobacteriaceae. A hospital-based descriptive cross-sectional study was conducted using 294 Enterobacteriaceae isolates from a total of 2,345 different clinical specimens collected from patients attending a tertiary care hospital in Nepal. Bacteria were isolated using standard microbiological growth media and identified using biochemical tests. For antimicrobial susceptibility testing, Kirby-Bauer disc diffusion technique was used. AmpC, ESBL, and MBL productions were detected by using combined disc method. AmpC, ESBL, and MBL productions were detected in 19.4%, 29.6%, and 8.5% of total Enterobacteriaceae isolates respectively. Higher rates of beta-lactamases production were seen among the isolates from in-patients in comparison with those from out-patients. However, 11.6%, 25%, and 3.7% of the total isolates from out-patients were AmpC, ESBL, and MBL producers respectively. The co-production of the beta-lactamases was also detected, with two Klebsiella pneumoniae isolates producing all three beta-lactamases. One MBL producing Proteus vulgaris isolate that was pan-resistant with no remaining treatment options was also isolated. Prevalence of drug resistant Enterobacteriaceae in our study was very high. Detection of AmpC, ESBL, and MBL positive isolates from out-patients, who did not have recent history of hospital visit, indicated the community dissemination of the drug resistant bacteria. This is a matter of great concern and an immediate attention to formulate strategies to prevent further development and spread of antibiotic resistance is required.

An Observational Study Comparing the Effects of Chloroquine and Artemisinin-Based Combination Therapy on Hematological Recovery in Patients With Plasmodium vivax Malaria.

Introduction Malaria, a common parasitic disease in tropical regions, produces hematological changes in patients. In the present study, we compare hematologic recovery between the chloroquine and artemether-lumefantrine treatment groups of patients with Plasmodium vivax malaria. Methodology This was a cross-sectional observational study comparing hematological parameters, including total and differential white blood cell counts, and platelet counts between two patient groups: one group with 48 patients receiving chloroquine, and the other with 47 patients receiving the artemether-lumefantrine combination. Both groups received primaquine to combat the hypnozoite stage. Results The rate of platelet count recovery was significantly faster in patients treated with the artemether-lumefantrine combination (p-value 0.002). Rates of recovery of the total white blood cell count and neutrophil count were faster with the artemether-lumefantrine combination, while the recovery of the lymphocyte count was faster in patients treated with chloroquine. However, these changes were statistically insignificant (p-values = 0.69, 0.42, and 0.65, respectively). Conclusion Based on hematological recovery, artemisinin combination therapy may be preferred over treatment with chloroquine in cases of P. vivax malaria. However, factors such as the adverse effect profile, cost-effectiveness, and chloroquine resistance need to be considered for the practical applicability of the same.

Combating the Progression of Novel Coronavirus SARS-CoV-2 Infectious Disease: Current State and Future Prospects in Molecular Diagnostics and Drug Discovery.

A highly infectious and life-threatening virus was first reported in Wuhan, China, in late 2019, and it rapidly spread all over the world. This novel virus belongs to the coronavirus family and is associated with severe acute respiratory syndrome (SARS), causing respiratory disease known as COVID-19. In March 2020, WHO has declared the COVID-19 outbreak a global pandemic. Its morbidity and mortality rates are swiftly rising day by day, with the situation becoming more severe and fatal for the comorbid population. Many COVID-19 patients are asymptomatic, but they silently spread the infection. There is a need for proper screening of infected patients to prevent the epidemic transmission of disease and for early curative interventions to reduce the risk of developing severe complications from COVID-19. To date, the diagnostic assays are of two categories, molecular detection of viral genetic material by real-time RTpolymerase chain reaction and serological test, which relies on detecting antiviral antibodies. Unfortunately, there are no effective prophylactics and therapeutics available against COVID-19. However, a few drugs have shown promising antiviral activity against it, and these presently are being referred for clinical trials, albeit FDA has issued an Emergency Use Authorization (EUA) for the emergency use of a few drugs for SARSCoV- 2 infection. This review provides an insight into current progress, challenges and future prospects of laboratory detection methods of COVID-19, and highlights the clinical stage of the major evidence-based drugs/vaccines recommended against the novel SARS-CoV-2 pandemic virus.

Optically transparent hydrogels from an auxin-amino-acid conjugate super hydrogelator and its interactions with an entrapped dye.

Low-molecular-weight organic hydrogelators (LMHGs) that can rigidify water into soft materials are desirable in various applications. Herein, we report the excellent hydrogelating properties of a simple synthetic auxin-amino-acid conjugate, naphthalene-1-acetamide of L-phenylalanine (1-NapF, M(w)=333.38 Da), which gelated water even at 0.025 wt %, thereby making it the most-efficient LMHG known. Optically transparent gels that exhibited negligible scattering in the range 350-900 nm were obtained. A large shift from the theoretical pK(a) value of the gelator was observed. The dependence of the minimum gelator concentration (MGC) and the gel-melting temperatures on the pH value indicated the importance of H-bonding between the carboxylate groups on adjacent phenylalanine molecules in the gelator assembly. FTIR spectroscopy of the xerogels showed a ß-sheet-like assembly of the gelator. Variable-temperature (1)H NMR spectroscopy demonstrated that π stacking of the aromatic residues was also partly involved in the gelator assembly. TEM of the xerogel showed the presence of a dense network of thin, high-aspect-ratio fibrillar assemblies with diameters of about 5 nm and lengths that exceeded a few microns. Rheology studies showed the formation of stable gels. The entrapment of water-soluble dyes afforded extremely fluorescent gels that involved the formation of J-aggregates by the dye within gel. A strong induced-CD band established that the RhoB molecules were interacting closely with the chiral gelator aggregates. H-bonding and electrostatic interactions, rather than intercalation, seemed to be involved in RhoB binding. The addition of chaotropic reagents, as well as increasing the pH value, disassembled the gel and promoted the release of the entrapped dye with zero-order kinetics.

A Phagosomally Expressed Gene, rv0428c, of Mycobacterium tuberculosis Demonstrates Acetyl Transferase Activity and Plays a Protective Role Under Stress Conditions.

Mycobacterium tuberculosis genome is composed of several hypothetical gene products that need to be characterized for understanding the physiology of bacteria. Rv0428c was one of the 11 proteins exclusively identified within the phagosomal compartment of macrophages infected with mycobacteria and marked as hypothetical. The expression of rv0428c gene was upregulated under acidic and nutritive stress conditions in M. tuberculosis H37Ra, which was supported by potential sigma factor binding sites in the region upstream to the rv0428c gene. The bioinformatics analysis predicted it to be a GCN5- acetyl transferase, belonging to the Histone acetyl transferase (HAT) family. The docking analysis predicted formation of hydrogen bonds and hydrophobic interactions between donor acetyl-co-A and histone H3 tail region. rv0428c gene was cloned and expressed in E. coli. The protein was purified to homogeneity and was fairly stable over a wide range of pH 5.0-9.0 and temperature up to 40 °C. The HAT activity of purified Rv0428c was confirmed by in vitro acetylation assay using recombinant H3 histone expressed in bacteria as substrate, which increased in time dependent manner. The results suggested that it is the second confirmed acetyl transferase in M. tuberculosis H37Rv. Furthermore, rv0428c was over expressed in surrogate host M. smegmatis, which led to enhanced growth rate and altered colony morphology. The expression of rv0428c in M. smegmatis promoted the survival of bacteria under acidic and nutritive stress conditions. In conclusion, Rv0428c, a phagosomal acetyl transferase of M. tuberculosis, might be involved in survival under stress conditions.

Transcript analysis and expression of the glbO gene, encoding truncated hemoglobin,O, of M. Smegmatis implicate its role under hypoxia and oxidative stress.

Although truncated hemoglobin O, (trHbO), is ubiquitous among mycobacteria, its physiological function is not very obvious and may be diverse. In an attempt to understand role of trHbO in cellular metabolism of a non-pathogenic mycobacterium, we analysed expression profile of the glbO gene, encoding trHbO, in M. smegmatis and studied implications of its overexpression on physiology of its host under different environmental conditions. Quantitative RT-PCR indicated that transcript level of the glbO gene remains low at a basal level under aerobic growth cycle of M. smegmatis but its level gets induced significantly during low oxygen, oxidative stress and macrophage infection. Overexpression of the glbO gene enhanced growth of M. smegmatis under hypoxia, promoted pellicle biofilm formation and provided resistance towards oxidative stress. Additionally, glbO gene overexpressing M. smegmatis exhibited enhanced cell survival over isogenic control cells and altered the level of pro- and anti- inflammatory cytokines during intracellular infection. These results suggested important role of trHbO, in supporting the cellular metabolism and survival of M, smegmatis both under low oxygen and oxidative stress.