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.

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.

Natural sources and encapsulating materials for probiotics delivery systems: Recent applications and challenges in functional food development.

Probiotics are known as the live microorganisms which upon adequate administration elicit a health beneficial response inside the host by decreasing the luminal pH, eliminating the pathogenic bacteria in the gut as well as producing short chain fatty acids (SCFA). With advancements in research; probiotics have been explored as potential ingredients in foods. However, their use and applications in food industry have been limited due to restrictions of maintaining the viability of probiotic cells and targeting the successful delivery to gut. Encapsulation techniques have significant influence on increasing the viability rates of probiotic cells with the successful delivery of cells to the target site. Moreover, encapsulating techniques also prevent the live cells from harsh physiological conditions of gut. This review discusses several encapsulating techniques as well as materials derived from natural sources and nutraceutical compounds. In addition to this, this paper also comprehensively discusses the factors affecting the probiotics viability and evaluation of successful release and survival of probiotics under simulated gastric, intestinal conditions as well as bile, acid tolerant conditions. Lastly applications and challenges of using encapsulated bacteria in food industry for the development of novel functional foods have also been discussed in detail too. Future studies must include investigating the use of encapsulated bacterial formulations in in-vivo models for effective health beneficial properties as well as exploring the mechanisms behind the successful release of these formulations in gut, hence helping us to understand the encapsulation of probiotic cells in a meticulous manner.

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.

In vitro & in vivo evaluations of PLGA nanoparticle based combinatorial drug therapy for baclofen and lamotrigine for neuropathic pain management.

AIM: Baclofen and Lamotrigine via PLGA nanoparticles were developed for nose-to-brain delivery for the treatment of Neuropathic pain. METHODS: Nanoparticles were prepared using the modified nano-precipitation method. The prepared NPs were characterised and further in vitro and in vivo studies were performed. RESULTS: The Bcf-Ltg-PLGA-NPs were ∼177.7 nm with >75%(w/w) drugs encapsulated. In vitro dissolution studies suggested zero-order release profiles following the Korsmeyer-Peppas model. In vitro cytotoxicity and staining studies on mammalian cells showed dose dependant cytotoxicity where nanoparticles were significantly less toxic (>95% cell-viability). ELISA studies on RAW-macrophages showed Bcf-Ltg-PLGA-NPs as a potential pro-inflammatory-cytokines inhibitor. In vivo gamma-scintigraphy studies on rats showed intra-nasal administration of 99mTc-Bcf-Ltg-PLGA-NPs showed Cmax 3.6%/g at Tmax = 1.5h with DTE% as 191.23% and DTP% = 38.61% in brain. Pharmacodynamics evaluations on C57BL/6J mice showed a significant reduction in licks/bites during inflammation-induced phase II pain. CONCLUSION: The findings concluded that the combination of these drugs into a single nanoparticle-based formulation has potential for pain management.

Baclofen and Lamotrigine loaded PLGA nanoparticles were prepared with a size of 177.7nm, PDI 0.057 and Zeta Potential −15.8 mVIn vitro cell lines based studies showed dose dependant cytotoxicity and Bcf-Ltg-PLGA-NPs were found to be pro-inflammatory cytokines inhibitorsIn vivo Pharmacokinetic studies showed C_max 3.6%/g at T_max = 1.5 h with Drug Targeting Efficiency 191.23% and Drug Target Organ Transport 38.61% in the brain for prepared nanoparticlesIn vivo pharmacodynamics studies showed a significant reduction in licks/bites during inflammation-induced phase II pain.

Phytoremediation of aspirin and tetracycline by Brassica juncea.

With the increasing release of pharmaceutical drugs in the environment, research is in progress for investigating alternative methods for their remediation. Various studies have shown the phytoremediation potential of Brassica juncea for metals. The current study was aimed at evaluating the phytoremediation potential of B. juncea for two different pharmaceutical drugs i.e. aspirin and tetracycline in in-vitro conditions. The seeds of B. juncea were germinated and grown for a period of 28 and 24 days for aspirin and tetracycline, respectively. The study analyzed the remediation rate of B. juncea for the selected drugs in three different sets of varying concentration along with any phytotoxic effects exerted by the drugs on the seeds. Preliminary results showed that the average remediation rate of aspirin and tetracycline at the end of experiment was approximately 90% and 71%, respectively. As initial drug concentrations were increased in the media, the remediation rate also improved. However, at higher concentrations, the plants showed phytotoxicity as depicted by the decrease in shoot length of the germinated seeds. These preliminary results indicated that B. juncea could tolerate and remediate pharmaceutical drugs such as analgesics and antibiotics.