Heterologous expression of nattokinase in E. coli: Biochemical characterization and functional analysis of fibrin binding residues.

Heterologous expression of nattokinase, a potent fibrinolytic enzyme, has been successfully carried out in various microorganisms. However, the successful expression of this enzyme as a soluble protein was not achieved in E. coli. This study delves into the expression of nattokinase in E. coli as a soluble protein followed by its biochemical characterization and functional analysis for fibrinolytic activity. E. coli BL21C41 and pET32a vector host strain with pGro7 protein chaperone induced with IPTG at 16 °C 180 rpm for 16 h enabled the production of recombinant nattokinase in soluble fraction. Enzymatic assays demonstrated its protease activity, while characterization revealed optimal catalytic conditions at 37 °C and pH 8.0, with remarkable stability over a broad pH range (6.0-10.0) and up to 50 °C. The kinetic constants were determined as follows: Km = 25.83 ± 3.43 µM, Vmax = 62.91 ± 1.68 µM/s, kcat = 38.45 ± 1.06 s-1, and kcat/Km = 1.49 × 106 M-1 s-1. In addition, the fibrinolytic activity of NK, quantified by the fibrin plate hydrolysis assay was 1038 ± 156 U/ml, with a corresponding specific activity of 1730 ± 260 U/mg and the assessment of clot lysis time on an artificial clot (1 mg) was found to be 51.5 ± 2.5 min unveiling nattokinase's fibrinolytic potential. Through molecular docking, a substantial binding energy of -6.46 kcal/mol was observed between nattokinase and fibrin, indicative of a high binding affinity. Key fibrin binding residues, including Ser300, Leu302, and Asp303, were identified and confirmed. These mutants affected specifically the fibrin binding and not the proteolytic activity of NK. This comprehensive study provides crucial conditions for the expression of protein in soluble form in E. coli and biochemical properties paving the way for future research and potential applications in medicine and biotechnology.

Systematic Review of Retinal Blood Vessels Segmentation Based on AI-driven Technique.

Image segmentation is a crucial task in computer vision and image processing, with numerous segmentation algorithms being found in the literature. It has important applications in scene understanding, medical image analysis, robotic perception, video surveillance, augmented reality, image compression, among others. In light of this, the widespread popularity of deep learning (DL) and machine learning has inspired the creation of fresh methods for segmenting images using DL and ML models respectively. We offer a thorough analysis of this recent literature, encompassing the range of ground-breaking initiatives in semantic and instance segmentation, including convolutional pixel-labeling networks, encoder-decoder architectures, multi-scale and pyramid-based methods, recurrent networks, visual attention models, and generative models in adversarial settings. We study the connections, benefits, and importance of various DL- and ML-based segmentation models; look at the most popular datasets; and evaluate results in this Literature.

P19 a Parthenin Analog Induces Cell Lineage Dependent Apoptotic and Immunomodulatory Signaling in Acute Lymphoid Leukemia Cells.

Leukemia is a type of cancer that affects the blood and bone marrow. Acute lymphoid leukaemia, also known as ALL, is regarded as one of the deadliest forms of cancer. Due to the rapid increase in various cancer cases and the development of resistance in cancer cells, it is necessary to identify novel lead molecules with more potent anticancer properties. There is a growing interest in using herbal products/analogs as multi-component agents (as anticancer agents and immunomodulators) for cancer treatment. In the present investigation, an attempt has been made to explore the anticancer and immunomodulatory activity of P19, an analog of parthenin in ALL. P19 was reported to exhibit anticancer efficacy by triggering apoptotic signaling events in human leukaemia HL-60 cells by significant NO production. In contrast to this finding, ROS and NO were not required for P19-mediated apoptosis in Raji cells. The mechanism of action of P19 was observed to be cancer cell lineage dependent. P19 demonstrated very effective anticancer properties against ALL (IC50 3µM). Molecular investigations revealed that P19 induced mitochondrion mediated apoptosis by Bax localization to mitochondria and enhanced cytosolic calcium in the cytoplasm. Further activation of the caspase 3, caspase 8 and PARP cleavage suggested the involvement of the caspase-mediated apoptosis. Anti-proliferative activity revealed the telomerase inhibition and cell cycle arrest in G0/G1 phase after P19 treatment. Immunomodulatory effects of the P19 revealed the enhanced INFÉ£ and NO production in Jurkat and THP cells. Owing to its antiproliferative and immunomodulatory potential against leukemia cells P19 can further be explored as effective therapeutics against leukemia.

Acidic environment could modulate the interferon-γ expression: Implication on modulation of cancer and immune cells' interactions.

Background: In rapidly growing solid tumors, insufficient vascularization and poor oxygen supply result in an acidic tumor microenvironment, which can alter immune response. Objective: To investigate the role of the acidic microenvironment in immune response modulation along with cancer and immune cells' interactions. Method: To mimic the tumor microenvironment conditions, T cells (Jurkat), macrophages (THP-1), and HeLa (cervical) cells were cultured under acidic conditions (pH 6.9, pH 6.5) and physiological pH (7.4). The HeLa cell culture medium was exploited as a tumor cell conditioned medium. Real-time PCR was carried out to quantify the mRNA levels, while flow cytometry and western blot hybridization was carried out to ascertain the levels of different proteins. Results: The acidic microenvironment around the T cells (Jurkat) and macrophage cells (THP-1) could lead to the downregulation of the interferon gamma (IFN-γ). An increase in IFN-γ expression was observed when Jurkat and macrophage cells were cultured in HeLa cells conditioned medium (HCM) at low pH (pH 6.9, pH 6.5). The HeLa cells under acidic environment (pH 6.9, pH 6.5) upregulated interleukin 18 levels and secreted it as exosome anchored. Additionally, enhanced nuclear localization of NF-κB was observed in Jurkat and THP-1 cells cultured in HCM (pH 6.9, pH 6.5). Jurkat and THP-1 cultured in HCM revealed enhanced cytotoxicity against the HeLa cells upon reverting the pH of the medium from acidic to physiological pH (pH 7.4). Conclusion: Collectively, these results suggest that the acidic microenvironment acted as a key barrier to cancer and immune cells' interactions.

Comparison of Maternal and Neonatal Outcomes among COVID-Positive Pregnant Women Affected in Second and Third Waves in a Dedicated COVID Hospital in Amritsar.

Background: With the emergence of new mutated variants of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), we have witnessed three waves of coronavirus disease (COVID) with varying severity, complication, and outcome in Punjab. The physiological changes of pregnancy make mother more vulnerable for severe infection. Current study is aimed at comparison of maternal and neonatal outcomes of COVID-positive pregnant women in second and third waves. Materials and Methods: This was a retrospective observational single-center study conducted at a dedicated COVID hospital in Punjab, India. Records of all COVID-positive pregnant women admitted from January to June 2021 and from January to February 2022 were reviewed. The demographic details, severity of symptoms, maternal and fetal complications, outcomes, and mortality were noted. Results: There were 220 COVID-positive pregnant patients in the second wave and 65 in third wave. The majority of patients belonged to the age group of 20-34 years (57.73% in the second wave and 70.77% in the third wave). Maternal deaths and severity of disease increased with increasing age of the mother, that is, 6.7% in <35 years and 13.95% in >35 years age group (RR = 2.058, P value = 0.1248). Also, the maternal deaths increased with increasing parity (RR = 2.00, P value = 0.2380). The majority of the study subjects were in the third trimester, with 77.73% in the second wave and 90.77% in the third wave. The majority of the patients in both the waves were asymptomatic or had mild symptoms. In the COVID second wave, 10.91% pregnant patients had moderate COVID symptoms and 8.18% had severe COVID symptoms, whereas none presented with moderate or severe symptoms in the third wave. Eighteen maternal deaths (8.18%) were seen during the COVID second wave, whereas no maternal death occurred in the third COVID wave. 100% of these deaths were because of COVID. Need of oxygen supplementation and intensive care unit admission had statistically significant association with maternal mortality. Conclusion: In the third COVID wave, the morbidity and mortality were significantly reduced. This could be a result of wide-spread vaccination, new strain of COVID, or both. In spite of this, the pregnancy complications such as pre-term birth, IUGR, and IUD were significant. Hence, pregnancies complicated by COVID should be considered as a high risk and closely monitored.

Rv3539 (PPE63) of Mycobacterium Tuberculosis Promotes Survival of Mycobacterium Smegmatis in Human Macrophages Cell Line via Cell Wall Modulation of Bacteria and Altering Host's Immune Response.

The modulation of host's immune response plays an important role in the intracellular survival of Mycobacterium tuberculosis. The intracellular pathogen counteracts environmental stresses with help of the expression of several genes. The M. tuberculosis genome encodes several immune-modulatory proteins including PE (proline-glutamic acid)/PPE (proline-proline-glutamic acid) superfamily proteins. It is unclear how the unique PE/PPE proteins superfamily contributes to survival under different stress and pathophysiology conditions. Previously, we showed that PPE63 (Rv3539) has C-terminal esterase extension and was localized as a membrane attached and in extracellular compartment. Therefore, the probability of these proteins interacting with the host to modulate the host immune response cannot be ruled out. The physiological role of PPE63 was characterized by expressing the PPE63 in the M. smegmatis, a non-pathogenic strain intrinsically deficient of PPE63. The recombinant M. smegmatis expressing PPE63 altered the colony morphology, lipid composition, and integrity of the cell wall. It provided resistance to multiple hostile environmental stress conditions and several antibiotics. MS_Rv3539 demonstrated higher infection and intracellular survival in comparison to the MS_Vec in the PMA-differentiated THP-1 cells. The decreased intracellular level of ROS, NO, and expression of iNOS was observed in THP-1 cells upon infection with MS_Rv3539 in comparison to MS_Vec. Further, the decrease in expression of pro-inflammatory cytokines like IL-6, TNF-α, and IL-1ß and enhanced anti-inflammatory cytokines like IL-10, pointed toward its role in immune modulation. Overall this study suggested the role of Rv3539 in enhanced intracellular survival of M. smegmatis via cell wall modulation and altered immune response of host.

Cell wall and immune modulation by Rv1800 (PPE28) helps M. smegmatis to evade intracellular killing.

Rv1800 is predicted as PPE family protein found in pathogenic mycobacteria only. Under acidic stress, the rv1800 gene was expressed in M. tuberculosis H37Ra. In-silico study showed lipase/esterase activity in C-terminus PE-PPE domain having pentapeptide motif with catalytic Ser-Asp-His residue. Full-length Rv1800 and C-terminus PE-PPE domain proteins showed esterase activity with pNP-C4 at the optimum temperature of 40 °C and pH 8.0. However, the N-terminus PPE domain showed no esterase activity, but involved in thermostability of Rv1800 full-length protein. M. smegmatis expressing rv1800 (MS_Rv1800) showed altered colony morphology and a significant resistance to numerous environmental stresses, antibiotics and higher lipid content. In extracellular and membrane fraction, Rv1800 protein was detected, while C terminus PE-PPE was present in cytoplasm, suggesting the role of N-terminus PPE domain in transportation of protein. MS_Rv1800 infected macrophage showed higher intracellular survival and low production of ROS, NO and expression levels of iNOS and pro-inflammatory cytokines, while induced expression of the anti-inflammatory cytokines. The Rv1800, PPE and PE-PPE showed antibody-mediated immunity in MDR-TB and PTB patients. Overall, these results confirmed the esterase activity in the C-terminus and function of N-terminus in thermostabilization and transportation; predicting the role of Rv1800 in immune/lipid modulation to support intracellular mycobacterium survival.

MSMEG_5850, a stress-induced TetR protein, involved in global transcription regulation in Mycobacterium smegmatis.

Aim: To decipher the role of MSMEG_5850 in the physiology of mycobacteria. Methods: MSMEG_5850 was knocked out and RNA sequencing was performed. MSMEG_5850 protein was purified from the Escherichia coli pET28a system. Electrophoretic mobility shift assay and size exclusion chromatography were used to determine the binding of MSMEG_5850 to its motif and binding stoichiometry. The effect of nutritional stress was monitored. Results: Transcriptome analysis revealed the differential expression of 148 genes in an MSMEG_5850 knockout strain. MSMEG_5850 had control over 50 genes because those genes had a binding motif upstream of their sequence. The electrophoretic mobility shift assay showed MSMEG_5850 bound to its motif as a monomer. MSMEG_5850 was upregulated under nutritional stress and promoted the survival of mycobacteria. Conclusion: The study confirms the role of MSMEG_5850 in global transcriptional regulation.

C-terminal lysine residues enhance plasminogen activation by inducing conformational flexibility and stabilization of activator complex of staphylokinase with plasmin.

Staphylokinase (SAK), a potent fibrin-specific plasminogen activator secreted by Staphylococcus aureus, carries a pair of lysine at the carboxy-terminus that play a key role in plasminogen activation. The underlaying mechanism by which C-terminal lysins of SAK modulate its function remains unknown. This study has been undertaken to unravel role of C-terminal lysins of SAK in plasminogen activation. While deletion of C-terminal lysins (Lys135, Lys136) drastically impaired plasminogen activation by SAK, addition of lysins enhanced its catalytic activity 2-2.5-fold. Circular dichroism analysis revealed that C-terminally modified mutants of SAK carry significant changes in their beta sheets and secondary structure. Structure models and RING (residue interaction network generation) studies indicated that the deletion of lysins has conferred extensive topological alterations in SAK, disrupting vital interactions at the interface of SAK.plasmin complex, thereby leading significant impairment in its functional activity. In contrast, addition of lysins at the C-terminus enhanced its conformational flexibility, creating a stronger coupling at the interface of SAK.plasmin complex and making it more efficient for plasminogen activation. Taken together, these studies provided new insights on the role of C-terminal lysins in establishment of precise intermolecular interactions of SAK with the plasmin for the optimal function of activator complex.

N-terminal PPE domain plays an integral role in extracellular transportation and stability of the immunomodulatory Rv3539 protein of the Mycobacterium tuberculosis.

Multigene PE/PPE family is exclusively present in mycobacterium species. Only few selected genes of this family have been characterized till date. Rv3539 was annotated as PPE63 with conserved PPE domain at N-terminal and PE-PPE at C-terminal. An α/ß hydrolase structural fold, characteristic of lipase/esterase, was present in the PE-PPE domain. To assign the biochemical function to Rv3539, the corresponding gene was cloned in pET-32a (+) as full-length, PPE, and PE-PPE domains individually, followed by expression in E. Coli C41 (DE3). All three proteins demonstrated esterase activity. However, the enzyme activity in the N-terminal PPE domain was very low. The enzyme activity of Rv3539 and PE-PPE proteins was approximately same with the pNP-C4 as optimum substrate at 40 °C and pH 8.0. The loss of enzyme activity after mutating the predicted catalytic triad (Ser296Ala, Asp369Ala, and His395Ala) found only in the PE-PPE domain, confirmed the candidature of the bioinformatically predicted active site residue. The optimal activity and thermostability of the Rv3539 protein was altered by removing the PPE domain. CD-spectroscopy analysis confirmed the role of PPE domain to the thermostability of Rv3539 by maintaining the structural integrity at higher temperatures. The presence of the N-terminal PPE domain directed the Rv3539 protein to the cell membrane/wall and the extracellular compartment. The Rv3539 protein could generate humoral response in TB patients. Therefore, results demonstrated that Rv3539 demonstrated esterase activity. PE-PPE domain of Rv3539 is functionally automated, however, N-terminus domain played a role in protein stabilization and its transportation. Both domains participated in immunomodulation.

Integrated analysis of smRNAome, transcriptome, and degradome data to decipher microRNAs regulating costunolide biosynthesis in Saussurea lappa.

Saussurea lappa (S. lappa) has been known to synthesize medicinally important, costunolide. Due to its immense therapeutic importance, understanding of regulatory mechanism associated with its biosynthesis is crucial. The identification of genes and transcription factors (TFs) in S. lappa, created a clear picture of costunolide biosynthesis pathways. Further to understand the regulation of costunolide biosynthesis by miRNAs, an integrated study of transcriptome, miRNAs, and degradome was performed. Identified candidate miRNAs and associated feed-forward loops (FFLs) illustrates their regulatory role in secondary metabolite biosynthesis. Small RNA and degradome sequencing were performed for leaf and root tissues to determine miRNAs-targets pairs. A total of 711 and 525 such targets were obtained for novel and known miRNAs respectively. This data was used to generate costunolide-specific miRNA-TF-gene interactome to perform systematic analyses through graph theoretical approach. Interestingly, miR171c.1 and sla-miR121 were identified as key regulators to connect and co-regulate both mevalonate and sesquiterpenoid pathways to bio-synthesize costunolide. Tissue-specific FFLs were identified to be involved in costunolide biosynthesis which further suggests the evolutionary co-relation of root-specific networks in synthesis of secondary metabolites in addition to leaf-specific networks. This integrative approach allowed us to determine candidate miRNAs and associated tissue-specific motifs involved in the diversification of secondary metabolites. MiRNAs identified in present study can provide alternatives for bioengineering tool to enhance the synthesis of costunolide and other secondary metabolites in S. lappa.

Protein Engineering Strategies for Tailoring the Physical and Catalytic Properties of Enzymes for Defined Industrial Applications.

BACKGROUND: Highly evolved biocatalysts that can endure harsh environmental conditions during industrial processes are highly desirable. The availability of suitable biocatalysts with high enzyme activity, substrate selectivity, and stability could lower the production costs in the pharmaceutical, chemical, and food industries, resulting in more economical products. OBJECTIVES: Naturally evolved enzymes could not be exploited in industrial applications because of their compromised properties. Till date, protein engineering strategies have helped us to improve the desired physical and catalytic properties of enzymes to meet their performance needs in industrial and medical applications. RESULTS: Protein engineering technologies such as directed evolution and rational designing are wellsuited for improving biocatalytic properties. Each approach has its own set of limitations, and the implementation of techniques is contingent on the availability of prerequisite information about the biocatalyst. Protein structure information is essential for rational design, but no prior structural knowledge is required for directed evolution. Furthermore, semi-rational approaches and enzyme designing are also being used. Considering these facts, this study outlines the various molecular techniques used to improve the physical and catalytic properties of enzymes. It also emphasises the magnitude of strategies used to improve the properties of biocatalysts to meet the needs of industrial processes. CONCLUSION: Protein engineering frequently employs for improving crucial enzyme characteristics. A semi-rational approach has now emerged as the preferred technology for protein engineering. However, adopting an engineering strategy to achieve the desired characteristic depends on the availability of resources and subject-matter knowledge.

Use of artificial intelligence for cancer clinical trial enrollment: a systematic review and meta-analysis.

BACKGROUND: The aim of this study is to provide a comprehensive understanding of the current landscape of artificial intelligence (AI) for cancer clinical trial enrollment and its predictive accuracy in identifying eligible patients for inclusion in such trials. METHODS: Databases of PubMed, Embase, and Cochrane CENTRAL were searched until June 2022. Articles were included if they reported on AI actively being used in the clinical trial enrollment process. Narrative synthesis was conducted among all extracted data: accuracy, sensitivity, specificity, positive predictive value, and negative predictive value. For studies where the 2x2 contingency table could be calculated or supplied by authors, a meta-analysis to calculate summary statistics was conducted using the hierarchical summary receiver operating characteristics curve model. RESULTS: Ten articles reporting on more than 50 000 patients in 19 datasets were included. Accuracy, sensitivity, and specificity exceeded 80% in all but 1 dataset. Positive predictive value exceeded 80% in 5 of 17 datasets. Negative predictive value exceeded 80% in all datasets. Summary sensitivity was 90.5% (95% confidence interval [CI] = 70.9% to 97.4%); summary specificity was 99.3% (95% CI = 81.8% to 99.9%). CONCLUSIONS: AI demonstrated comparable, if not superior, performance to manual screening for patient enrollment into cancer clinical trials. As well, AI is highly efficient, requiring less time and human resources to screen patients. AI should be further investigated and implemented for patient recruitment into cancer clinical trials. Future research should validate the use of AI for clinical trials enrollment in less resource-rich regions and ensure broad inclusion for generalizability to all sexes, ages, and ethnicities.

Severely Calcified Pericardium Causing Constrictive Pericarditis.

Constrictive pericarditis (CP) is a type of diastolic heart failure caused by an inelastic pericardium that impairs cardiac filling. Diagnosing CP can be challenging, and a variety of imaging techniques may be necessary. We present a unique case of severely calcified pericardium leading to CP.

Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2.

Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cations other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion-pumping characteristics by mutations is therefore of great interest. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence proton pumping is observed. Our data reveal specific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spectroscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.

Microbial remediation and plant-microbe interaction under arsenic pollution.

Arsenic contamination in aquatic and terrestrial ecosystem is a serious environmental issue. Both natural and anthropogenic processes can introduce it into the environment. The speciation of the As determine the level of its toxicity. Among the four oxidation states of As (-3, 0, +3, and + 5), As(III) and As(V) are the common species found in the environment, As(III) being the more toxic with adverse impact on the plants and animals including human health. Therefore, it is very necessary to remediate arsenic from the polluted water and soil. Different physicochemical as well as biological strategies can be used for the amelioration of arsenic polluted soil. Among the microbial approaches, oxidation of arsenite, methylation of arsenic, biosorption, bioprecipitation and bioaccumulation are the promising transformation activities in arsenic remediation. The purpose of this review is to discuss the significance of the microorganisms in As toxicity amelioration in soil, factors affecting the microbial remediation, interaction of the plants with As resistant bacteria, and the effect of microorganisms on plant arsenic tolerance mechanism. In addition, the exploration of genetic engineering of the bacteria has a huge importance in bioremediation strategies, as the engineered microbes are more potent in terms of remediation activity along with quick adaptively in As polluted sites.

Depression in Childhood Asthma vs. Adult-Onset Asthma: A Cross-Sectional Study from the National Health and Nutrition Examination Survey (NHANES).

Background: asthma, a chronic respiratory disease caused by inflammation and narrowing of the small airways in the lungs, is the most common chronic childhood disease. Prevalence of childhood asthma in the United States is 5.8%. In boys, prevalence is 5.7% and it is 6% in girls. Asthma is associated with other comorbidities such as major depressive disorder and anxiety disorder. This study explores the association between asthma and depression. Methods: we conducted a retrospective cross-sectional study using NHANES data from 2013 to 2018. Asthma and childhood onset asthma were assessed using questionnaires MCQ010 and MCQ025, respectively. Sociodemographic variables were summarized, and univariate analysis was performed to determine the association between asthma and major depressive disorder and its individual symptoms. Results: there were 402,167 participants from 2013−2018 in our study: no asthma in 84.70%; asthma in 15.30%. Childhood onset asthma (COA) included 10.51% and adult-onset asthma (AOA) included 4.79%. Median age of COA is 5 years and AOA is 41 years. Among the asthma groups, most AOA were females (67.77%, p < 0.0001), most COA were males (52.16%, p < 0.0001), and ethnicity was predominantly White in AOA (42.39%, p < 0001) and in COA (35.24%, p < 0.0001). AOA mostly had annual household income from $0−24,999 (35.91%, p < 0.0001), while COA mostly had annual household income from $25,000−64,999 (36.66%, p < 0.0001). There was a significantly higher prevalence of MDD in COA (38.90%) and AOA (47.30%) compared to NOA (31.91%). Frequency of symptoms related to MDD were found to have a significantly higher prevalence and severity in the asthma groups compared to no asthma, and slightly greater and more severe in AOA than in COA. Symptoms include having little interest in doing things (COA 18.38% vs. AOA 22.50% vs. NOA 15.44%), feeling down, depressed, or hopeless (COA 20.05% vs. AOA 22.77% vs. NOA 15.85%), having trouble sleeping or sleeping too much (COA 27.38% vs. AOA 23.15% vs. NOA 22.24%), feeling tired or having little energy (COA 39.17% vs. AOA 34.24% vs. NOA 33.97%), having poor appetite or overeating (COA 19.88% vs. AOA 20.02% vs. NOA 15.11%), feeling bad about yourself (COA 13.90% vs. AOA 13.79% vs. NOA 10.78%), having trouble concentrating on things (COA 12.34% vs. AOA 14.41% vs. NOA 10.06%), moving or speaking slowly or too fast (COA 8.59% vs. AOA 9.72% vs. NOA 6.09%), thinking you would be better off dead (COA 3.12% vs. AOA 4.38% vs. NOA 1.95%) and having the difficulties these problems have caused (COA 21.66% vs. AOA 26.73% vs. NOA 19.34%, p < 0.0001). Conclusion: MDD and related symptoms were significantly higher and more severe in participants with asthma compared to no asthma. Between adult-onset asthma compared to childhood onset asthma, adult-onset asthma had slightly greater and more severe MDD and related symptoms compared to childhood onset asthma.

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.

Genomic Insights into Omega-3 Polyunsaturated Fatty Acid Producing Shewanella sp. N2AIL from Fish Gut.

The genus Shewanella is widely distributed in niches ranging from an aquatic environment to spoiled fish and is loaded with various ecologically and commercially important metabolites. Bacterial species under this genus find application in bioelectricity generation and bioremediation due to their capability to use pollutants as the terminal electron acceptor and could produce health-beneficial omega-3 fatty acids, particularly eicosapentaenoic acid (EPA). Here, the genome sequence of an EPA-producing bacterium, Shewanella sp. N2AIL, isolated from the gastrointestinal tract of Tilapia fish, is reported. The genome size of the strain was 4.8 Mb with a GC content of 46.3% containing 4385 protein-coding genes. Taxonogenomic analysis assigned this strain to the genus Shewanella on the basis of average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH), phylogenetically most closely related with S. baltica NCTC 10735T. The comparative genome analysis with the type strain of S. baltica revealed 693 unique genes in the strain N2AIL, highlighting the variation at the strain level. The genes associated with stress adaptation, secondary metabolite production, antibiotic resistance, and metal reduction were identified in the genome suggesting the potential of the bacterium to be explored as an industrially important strain. PUFA synthase gene cluster of size ~20.5 kb comprising all the essential domains for EPA biosynthesis arranged in five ORFs was also identified in the strain N2AIL. The study provides genomic insights into the diverse genes of Shewanella sp. N2AIL, which is particularly involved in adaptation strategies and prospecting secondary metabolite potential, specifically the biosynthesis of omega-3 polyunsaturated fatty acids.

Mutation in Eth A protein of Mycobacterium tuberculosis conferred drug tolerance against enthinoamide in Mycobacterium smegmatis mc2155.

EthA is an NADPH-specific flavin adenine dinucleotide (FAD) containing monooxygenase that activates the -second-line drug ethionamide (ETH). ETH gets converted to an active form after interaction with the EthA (monooxygenase) protein. Upon activation, ETH interacts with NAD+ to form an ETH-NAD adduct, which hampers the activity of InhA (Enoyl-[(acyl-carrier-protein) reductase (NADH)]. This, in turn, inhibits the cell wall synthesis, thus killing the Mycobacterium tuberculosis (Mtb). Mutations in the EthA gene can modulate ETH activation. The mutation at 202 position (Val202-Leu) of EthA protein has been reported frequently in ETH resistance. In this study, the effect of this mutation on the function of the EthA protein was examined through structural and functional analysis. Molecular docking of wild type and mutated EthA protein with ETH were compared to inspect the effect of mutation on molecular mechanism of drug resistant. Docking results corroborated that the lower docking score of the mutant protein, larger binding cavity, and lower affinity towards ETH resulted in a less compact and energetically less stable structure than the wild type protein. The computational outcome was authenticated by in-vitro experiments. The wild type and mutated genes were cloned and expressed in M. smegmatis, a surrogate host. Antibiotic susceptibility testing demonstrated that the mutant showed high growth and survival in the presence of the ETH drug. Overall, the results indicated that a mutation in the intergenic region of EthA protein could result in the altered conversion of ETH to the active form, resulting in differential ETH sensitivity for M. smegmatis carrying the wild type and mutant gene.