Expression Analysis of Nitrogen Metabolism Genes in Lelliottia amnigena PTJIIT1005, Comparison with Escherichia coli K12 and Validation of Nitrogen Metabolism Genes.

Escherichia coli K12 and Lelliottia amnigena PTJIIT1005 bacteria were isolated from the polluted Yamuna River (Delhi, India) site, which can remediate nitrate from groundwater media under anaerobic conditions. BV-BRC (Bacterial and Viral Bioinformatics Resource Center) information system, RAST, and PGAP servers were used to annotate the nitrogen metabolism genes from the genome sequence of these microbes. Here we compared the strains L. amnigena PTJIIT1005 with E. coli K12 in the context of nitrogen metabolism genes. Sequence alignment, similarity percentage, and phylogenetic analysis were done to find similarities between the genes. Common nitrogen genes of these strains, like respiratory nitrate reductase, nitrite reductase, nitric oxide reductase, glutamine synthetase, and hydroxylamine reductase, have found good sequence similarity (83-94%) with each other. The PATRIC tool identified N-operons, and the nitrate reductase gene clusters were also determined as per literature survey. Protein-protein interaction network was constructed using STRING 12.0 database and Cytoscape v 3.10.0 software plug-in Network analyzer. On the basis of network topological parameters NarG, NarZ, NarY, NarH, NarI, NarV, NirB, NirD, NapA, and NapB are the key genes in network of E. coli K12 strain. Nar, NirB, NirD, NasA, NasB, NasC, NasD, NasE, and GlnA are the key genes in network of L. amnigena PTJIIT1005. Among these, NarG and NirB are the superhub genes because of having highest Betweenness centrality (BC) and node degree. The functional enrichment analysis was determined using PANTHER GENE ONTOLOGY and DAVID software exhibited their role in nitrogen metabolism pathway and nitrate assimilation. Further, SWISS-MODEL was used to predict the 3D protein structure of these enzymes, and after, these structures were validated by Ramachandran plot using the PROCHECK tool. The Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) method was used to determine the N-genes expression level in both strains. This study showed that E. coli K12 and L. amnigena PTJIIT1005 have common nitrogen metabolism genes involved in the same functional role, like the denitrification pathway. Additionally, operon arrangement study and PPI network revealed that E. coli K12 has only a denitrification pathway, while L. amnigena PTJIIT1005 has both an assimilation and denitrification pathway. PCR successfully amplified selected N-metabolizing genes, and the expression level of N-genes was high in strain L. amnigena PTJIIT1005. Our previous experimental study exhibited a better nitrate remediation rate in L. amnigena PTJIIT1005 over E. coli K12. This study confirmed the presence of assimilation and denitrification process through amplified N-metabolizing genes and showed high expression of N-genes in L. amnigena PTJIIT1005, which favor the evidence of better nitrate remediation in L. amnigena PTJIIT1005 over E. coli K12.

MDA5 RNA-sensing pathway activation by Mycobacterium tuberculosis promotes innate immune subversion and pathogen survival.

Host cytosolic sensing of Mycobacterium tuberculosis (M. tuberculosis) RNA by the RIG-I-like receptor (RLR) family perturbs innate immune control within macrophages; however, a distinct role of MDA5, a member of the RLR family, in M. tuberculosis pathogenesis has yet to be fully elucidated. To further define the role of MDA5 in M. tuberculosis pathogenesis, we evaluated M. tuberculosis intracellular growth and innate immune responses in WT and Mda5-/- macrophages. Transfection of M. tuberculosis RNA strongly induced proinflammatory cytokine production in WT macrophages, which was abrogated in Mda5-/- macrophages. M. tuberculosis infection in macrophages induced MDA5 protein expression, accompanied by an increase in MDA5 activation as assessed by multimer formation. IFN-γ-primed Mda5-/- macrophages effectively contained intracellular M. tuberculosis proliferation to a markedly greater degree than WT macrophages. Further comparisons of WT versus Mda5-/- macrophages revealed that during M. tuberculosis infection MDA5 contributed to IL-1ß production and inflammasome activation and that loss of MDA5 led to a substantial increase in autophagy. In the mouse TB model, loss of MDA5 conferred host survival benefits with a concomitant reduction in M. tuberculosis bacillary burden. These data reveal that loss of MDA5 is host protective during M. tuberculosis infection in vitro and in vivo, suggesting that M. tuberculosis exploits MDA5 to subvert immune containment.

Identification and examination of nitrogen metabolic genes in Lelliottia amnigena PTJIIT1005 for their ability to perform nitrate remediation.

Lelliottia amnigena PTJIIT1005 is a bacterium that utilizes nitrate as the sole nitrogen source and can remediate nitrate from media. The annotation was done related to nitrogen metabolic genes using the PATRIC, RAST tools, and PGAP from the genome sequence of this bacterium. Multiple sequence alignments and phylogenetic analysis of respiratory nitrate reductase, assimilatory nitrate reductase, nitrite reductase, glutamine synthetase, hydroxylamine reductase, nitric oxide reductase genes from PTJIIT1005 were done to find out sequence identities with the most similar species. The identification of operon arrangement in bacteria was also identified. The PATRIC KEGG feature mapped the N-metabolic pathway to identify the chemical process, and the 3D structure of representative enzymes was also elucidated. The putative protein 3D structure was analyzed using I-TASSER software. It gave good quality protein models of all nitrogen metabolism genes and showed good sequence identity with reference templates, approximately 81-99%, except for two genes; assimilatory nitrate reductase and nitrite reductase. This study suggested that PTJIIT1005 can remove N-nitrate from water because of having N-assimilation and denitrification genes.

Use of the Human Granulysin Transgenic Mice To Evaluate the Role of Granulysin Expression by CD8 T Cells in Immunity To Mycobacterium tuberculosis.

The cytotoxic granules of human NK and CD8 T cells contain the effector molecule granulysin. Although in vitro studies indicate that granulysin is bactericidal to Mycobacterium tuberculosis and human CD8 T cells restrict intracellular M. tuberculosis by granule exocytosis, the role of granulysin in cell-mediated immunity against infection is incompletely understood, in part because a granulysin gene ortholog is absent in mice. Transgenic mice that express human granulysin (GNLY-Tg) under the control of human regulatory DNA sequences permit the study of granulysin in vivo. We assessed whether granulysin expression by murine CD8 T cells enhances their control of M. tuberculosis infection. GNLY-Tg mice did not control pulmonary M. tuberculosis infection better than non-Tg control mice, and purified GNLY-Tg and non-Tg CD8 T cells had a similar ability to transfer protection to T cell deficient mice. Lung CD8 T cells from infected control and GNLY-transgenic mice similarly controlled intracellular M. tuberculosis growth in macrophages in vitro. Importantly, after M. tuberculosis infection of GNLY-Tg mice, granulysin was detected in NK cells but not in CD8 T cells. Only after prolonged in vitro stimulation could granulysin expression be detected in antigen-specific CD8 T cells. GNLY-Tg mice are an imperfect model to determine whether granulysin expression by CD8 T cells enhances immunity against M. tuberculosis. Better models expressing granulysin are needed to explore the role of this antimicrobial effector molecule in vivo. IMPORTANCE Human CD8 T cells express the antimicrobial peptide granulysin in their cytotoxic granules, and in vitro analysis suggest that it restricts growth of Mycobacterium tuberculosis and other intracellular pathogens. The murine model of tuberculosis cannot assess granulysin's role in vivo, as rodents lack the granulysin gene. A long-held hypothesis is that murine CD8 T cells inefficiently control M. tuberculosis infection because they lack granulysin. We used human granulysin transgenic (GNLY-Tg) mice to test this hypothesis. GNLY-Tg mice did not differ in their susceptibility to tuberculosis. However, granulysin expression by pulmonary CD8 T cells could not be detected after M. tuberculosis infection. As the pattern of granulysin expression in human CD8 T cells and GNLY-Tg mice seem to differ, GNLY-Tg mice are an imperfect model to study the role of granulysin. An improved model is needed to answer the importance of granulysin expression by CD8 T cells in different diseases.

Colistin-induced Bartter-like Syndrome: Ponder before Treatment!

Bartter-like syndrome (BLS) is a constellation of biochemical abnormalities which include metabolic alkalosis, hypokalemia, hypocalcemia, hypomagnesemia with normal kidney function. BLS is a very rare syndrome and can be induced by certain diseases, antibiotics, diuretics, and antineoplastic drugs. Colistin is a polymicrobial bactericidal drug and currently re-emerged as the only salvation therapy against multidrug resistant bacilli especially in critically ill patients at intensive care units. Only an anecdotal case report of colistin-induced Bartter-like syndrome has been reported. We here report a case series of four critically ill patients who were on treatment with colistin and presented with serious metabolic disturbances. How to cite this article: Kumari A, Gupta P, Verma H, Kumar A, Thakur P, Sharma K. Colistin-induced Bartter-like Syndrome: Ponder before Treatment! Indian J Crit Care Med 2022;26(2):239-243.

Genomic characterization of Lelliottia amnigena PTJIIT1005, a nitrate tolerant strain isolated from water sample of Yamuna River, Delhi, India.

Here, we report the genome sequence of PTJIIT1005, isolated from a polluted site on the Yamuna River, Delhi. The genome is complete and consists of ~4.5 Mbp with a GC content of 52.62%, 4,259 protein-coding genes, 76 tRNAs, and 4 rRNAs. Strain PTJIIT1005 shows 98.89% average nucleotide identity (ANI) with Lelliottia amnigena.

Doping Independent Work Function and Stable Band Gap of Spinel Ferrites with Tunable Plasmonic and Magnetic Properties.

Tuning optical or magnetic properties of nanoparticles, by addition of impurities, for specific applications is usually achieved at the cost of band gap and work function reduction. Additionally, conventional strategies to develop nanoparticles with a large band gap also encounter problems of phase separation and poor crystallinity at high alloying degree. Addressing the aforementioned trade-offs, here we report Ni-Zn nanoferrites with energy band gap (Eg) of ≈3.20 eV and a work function of ≈5.88 eV. While changes in the magnetoplasmonic properties of the Ni-Zn ferrite were successfully achieved with the incorporation of bismuth ions at different concentrations, there was no alteration of the band gap and work function in the developed Ni-Zn ferrite. This suggests that with the addition of minute impurities to ferrites, independent of their changes in the band gap and work function, one can tune their magnetic and optical properties, which is desired in a wide range of applications such as nanobiosensing, nanoparticle based catalysis, and renewable energy generation using nanotechnology.

Nanoferrites heterogeneous catalysts for biodiesel production from soybean and canola oil: a review.

Fossil fuel depletion and pollution are calling for alternative, renewable energies such as biofuels. Actual challenges include the design of efficient processes and catalysts to convert various feedstocks into biofuels. Here, we review nanoferrites heterogeneous catalysts to produce biodiesel from soybean and canola oil. For that, transesterification is the main synthesis route and offers simplicity, cost-effectiveness, better process control, and high conversion yield. Catalysis with nanoferrites and composites allow to obtain yields higher than 95% conversion with less than 5.0 wt.% of catalyst loading at 80 °C in 1-2 h. More than 90% conversion yields can be achieved with a moderate alcohol/oil molar ratio, i.e., between 12:1 to 16:1. Catalyst recovery is easy due to the magnetic properties of nanoferrite, which can be effectively reused up to 4 times with less than 10% loss of catalytic efficiency.

Utility of Antigen-Based Rapid Diagnostic Test for Detection of SARS-CoV-2 Virus in Routine Hospital Settings.

OBJECTIVE: This study aims to evaluate the performance of an antigen-based rapid diagnostic test (RDT) for the detection of the SARS-CoV-2 virus. METHODS: A cross-sectional study was conducted on 677 patients. Two nasopharyngeal swabs and 1 oropharyngeal swab were collected from patients. The RDT was performed onsite by a commercially available immune-chromatographic assay on the nasopharyngeal swab. The nasopharyngeal and oropharyngeal swabs were examined for SARS-CoV-2 RNA by real-time reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay. RESULTS: The overall sensitivity of the SARS-CoV-2 RDT was 34.5% and the specificity was 99.8%. The positive predictive value and negative predictive value of the test were 96.6% and 91.5%, respectively. The detection rate of RDT in RT-qPCR positive results was high (45%) for cycle threshold values <25. CONCLUSION: The utility of RDT is in diagnosing symptomatic patients and may not be particularly suited as a screening tool for patients with low viral load. The low sensitivity of RDT does not qualify its use as a single test in patients who test negative; RT-qPCR continues to be the gold standard test.

Relative and Quantitative Phosphoproteome Analysis of Macrophages in Response to Infection by Virulent and Avirulent Mycobacteria Reveals a Distinct Role of the Cytosolic RNA Sensor RIG-I in Mycobacterium tuberculosis Pathogenesis.

Comparative phosphoproteomics of Mycobacterium tuberculosis (Mtb)- and Mycobacterium bovis BCG (BCG)-infected macrophages could be instrumental in understanding the characteristic post-translational modifications of host proteins and their subsequent involvement in determining Mtb pathogenesis. To identify proteins acquiring a distinct phosphorylation status, herein, we compared the phosphorylation profile of macrophages upon exposure to Mtb and BCG. We observed a significant dephosphorylation of proteins following Mtb infection relative to those with uninfected or BCG-infected cells. A comprehensive tandem mass tag mass spectrometry (MS) approach detected ∼10% phosphosites on a variety of host proteins that are modulated in response to infection. Interestingly, the innate immune-enhancing interferon (IFN)-stimulated genes were identified as a class of proteins differentially phosphorylated during infection, including the cytosolic RNA sensor RIG-I, which has been implicated in the immune response to bacterial infection. We show that Mtb infection results in the activation of RIG-I in primary human macrophages. Studies using RIG-I knockout macrophages reveal that the Mtb-mediated activation of RIG-I promotes IFN-ß, IL-1α, and IL-1ß levels, dampens autophagy, and facilitates intracellular Mtb survival. To our knowledge, this is the first study providing exhaustive information on relative and quantitative changes in the global phosphoproteome profile of host macrophages that can be further explored in designing novel anti-TB drug targets. The peptide identification and MS/MS spectra have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013171.

A review on MnZn ferrites: Synthesis, characterization and applications.

Researchers are taking great interest in the synthesis and characterization of MnZn ferrites due to their wide range of applications in many areas. MnZn ferrites are a class of soft magnetic materials that have very good electrical, magnetic and optical properties. The properties of MnZn ferrites include high value of resistivity, permeability, permittivity, saturation magnetization, low power losses and coercivity. The above mentioned advantageous features of MnZn ferrites make them suitable for the use in various applications. In biomedical field these ferrites are used for cancer treatment and MRI. MnZn ferrites are also used in electronic applications for making transformers, transducers and inductors. These ferrites are also used in magnetic fluids, sensors and biosensors. MnZn ferrite is highly useful material for several electrical and electronic applications. It finds applications in almost every household appliances like mobile charger, LED bulb, TV, refrigerator, juicer mixer, washing machine, iron, microwave oven, mobile, laptop, desktop, printer and so on. Therefore, the present review focuses on different techniques for synthesis of MnZn ferrites in literature, their characterization tools, effect of doping on the properties of MnZn ferrite and finally we will discuss about their applications.

Evaluation and Recommendations for Routine Genotyping Using Skim Whole Genome Re-sequencing in Canola.

Whole genome sequencing offers genome wide, unbiased markers, and inexpensive library preparation. With the cost of sequencing decreasing rapidly, many plant genomes of modest size are amenable to skim whole genome resequencing (skim WGR). The use of skim WGR in diverse sample sets without the use of imputation was evaluated in silico in 149 canola samples representative of global diversity. Fastq files with an average of 10x coverage of the reference genome were used to generate skim samples representing 0.25x, 0.5x, 1x, 2x, 3x, 4x, and 5x sequencing coverage. Applying a pre-defined list of SNPs versus de novo SNP discovery was evaluated. As skim WGR is expected to result in some degree of insufficient allele sampling, all skim coverage levels were filtered at a range of minimum read depths from a relaxed minimum read depth of 2 to a stringent read depth of 5, resulting in 28 list-based SNP sets. As a broad recommendation, genotyping pre-defined SNPs between 1x and 2x coverage with relatively stringent depth filtering is appropriate for a diverse sample set of canola due to a balance between marker number, sufficient accuracy, and sequencing cost, but depends on the intended application. This was experimentally examined in two sample sets with different genetic backgrounds: 1x coverage of 1,590 individuals from 84 Australian spring type four-parent crosses aimed at maximizing diversity as well as one commercial F1 hybrid, and 2x coverage of 379 doubled haploids (DHs) derived from a subset of the four-parent crosses. To determine optimal coverage in a simpler genetic background, the DH sample sequence coverage was further down sampled in silico. The flexible and cost-effective nature of the protocol makes it highly applicable across a range of species and purposes.

Regulation and overexpression studies of YidC in Mycobacterium tuberculosis.

The preprotein translocase, YidC is an envelope protein which controls respiratory metabolism in Mycobacterium tuberculosis. Previously, we have established that depletion of yidC is deleterious for both extra- and intracellular proliferation of M. tuberculosis; however, it remains unclear how YidC expression is regulated under different growth conditions and whether its altered expression impact mycobacterial physiology. Herein, we show that yidC is expressed as an operon with upstream genes. Interestingly, expression analysis under various stress conditions reveals a distinct paradox in the profile of the yidC mRNA transcripts and the YidC protein. While YidC protein level is moderately elevated upon bacterial exposure to cell surface stresses, the corresponding mRNA transcript levels are significantly repressed under these conditions. In contrast, overexpression of M. tuberculosis yidC under a strong anhydrotetracycline-inducible promoter results in significant induction of YidC protein. Additionally, we also observe that overexpression of M. tuberculosis yidC, and not of its counterpart from fast-growing M. smegmatis, results in altered in vitro growth of bacteria, compromised integrity of bacterial cell envelope and differential expression of a small set of genes including those which are regulated under detergent stress. Overall findings of our study suggest that YidC proteins of slow- and fast-growing mycobacteria are functionally distinct despite exhibiting a great deal of identity.

Exploitation of data from breeding programs supports rapid implementation of genomic selection for key agronomic traits in perennial ryegrass.

KEY MESSAGE: Exploitation of data from a ryegrass breeding program has enabled rapid development and implementation of genomic selection for sward-based biomass yield with a twofold-to-threefold increase in genetic gain. Genomic selection, which uses genome-wide sequence polymorphism data and quantitative genetics techniques to predict plant performance, has large potential for the improvement in pasture plants. Major factors influencing the accuracy of genomic selection include the size of reference populations, trait heritability values and the genetic diversity of breeding populations. Global diversity of the important forage species perennial ryegrass is high and so would require a large reference population in order to achieve moderate accuracies of genomic selection. However, diversity of germplasm within a breeding program is likely to be lower. In addition, de novo construction and characterisation of reference populations are a logistically complex process. Consequently, historical phenotypic records for seasonal biomass yield and heading date over a 18-year period within a commercial perennial ryegrass breeding program have been accessed, and target populations have been characterised with a high-density transcriptome-based genotyping-by-sequencing assay. Ability to predict observed phenotypic performance in each successive year was assessed by using all synthetic populations from previous years as a reference population. Moderate and high accuracies were achieved for the two traits, respectively, consistent with broad-sense heritability values. The present study represents the first demonstration and validation of genomic selection for seasonal biomass yield within a diverse commercial breeding program across multiple years. These results, supported by previous simulation studies, demonstrate the ability to predict sward-based phenotypic performance early in the process of individual plant selection, so shortening the breeding cycle, increasing the rate of genetic gain and allowing rapid adoption in ryegrass improvement programs.

Shigella sonnei Sepsis in an Infant: A Case Report.

Shigella bacteraemia is a complication of gastroenteritis caused by Shigella spp. which occurs mainly in immunocompromised children. Other risk factors may include young malnourished children, patients with chronic disease, post transplant patients etc. We present a case of six-month-old infant who developed an episode of gastroenteritis caused by Shigella sonnei which was followed by septicaemia. The patient was managed well and he recovered. Shigella sonnei was isolated in both stool as well as blood culture. S.sonnei bacteraemia is a rare condition but we should always be vigilant. Blood cultures as well as stool cultures should be performed in patients presenting with acute febrile gastroenteritis, whether immunocompromised or not. This simple, early and prompt investigation may help in precise diagnosis, which will guide the clinician about appropriate intervention like antibiotic therapy and supportive care.

Stenotrophomonas maltophilia: An Emerging Pathogen in Paediatric Population.

INTRODUCTION: Stenotrophomonas maltophilia (formerly Pseudomonas maltophilia/Xanthomonas maltophilia), a Gram- negative, non-fermenting bacillus, is being increasingly recognized as a threatening nosocomial pathogen, associated with significant mortality. AIM: To determine the prevalence of infection, antimicrobial susceptibility pattern and clinical outcome of S. maltophilia in a paediatric population. MATERIALS AND METHODS: This was a retrospective study conducted over a period of eight months, i.e., October 2015 to May 2016. All clinical samples received in the microbiology laboratory during the study period were processed using standard microbiological procedures. S. maltophilia isolates were selected. Antibiotic susceptibility was performed for levofloxacin and trimethoprim-sulphamethoxazole by Vitek 2C system (Biomerieux, France). Average length of stay and mortality caused by S. maltophilia infection was compared with age and sex matched controls without S. maltophilia infection. RESULTS: A total of 16,234 clinical specimens were received in the microbiology laboratory in the study period, with 2,734 pathogenic bacteria isolated. A total of 1,339 (1.7% of total isolates) Gram-negative bacteria were isolated, out of which 414 were non-fermenters. Among the non-fermenters, 23 (5.5%) were S. maltophilia. Out of the 23 isolates, 15 (65.2%) were isolated from blood, 4 (17.3%) were isolated from urine and tracheal aspirate each. A total of 91.3% of strains were susceptible and 8.6% were resistant to trimethoprim-sulphamethoxazole. Total 80% of strains were sensitive and 20% had intermediate susceptibility for levofloxacin. None of the strains were resistant to levofloxacin. Average length of stay of patients with S. maltophilia infection was found out to be 23.3 days as compared to 44.8 days in controls. The average mortality of patients with S. maltophilia infection was found to be same as that of controls (35.2%). CONCLUSION: S. maltophilia is becoming an important nosocomial pathogen and its isolation rate is reported to be increasing. Trimethoprim-sulphamethoxazole still remains the drug of choice but resistance has been reported for this drug as well. As its isolation is increasing, it is important to study the epidemiology, antimicrobial susceptibility profile and clinical outcomes of these isolates.