Regulation of Pseudomonas putida genes involved in the metabolism of acidic amino acids

Pseudomonas putida KT2440 has the ability to utilize a wide range of amino acids as carbon and nitrogen sources. Rapid growth of this organism is supported by the acidic amino acids (Asp and Glu) and their amides (Asn and Gln) when supplied as sole source of carbon and nitrogen, or in combination with other carbon and nitrogen sources such as glucose and NH4+. A proteomics study based on two-dimensional gel electrophoresis revealed that during growth of P. putida KT2440 in Glu containing medium a set of at least 9 major proteins were up-regulated in a coordinate fashion, whereas 4 other proteins were specifically induced during growth in NH4+/Glucose. Most of the identified proteins have some role in the uptake and utilization of amino acids. Based on their assigned functions genetic organization, we propose that they form a regulon involved in the metabolism of amino acids. By transposon mutagenesis it was found that the expression of that regulon depends on a functional gltB gene which encodes the major subunit of glutamate synthase (GOGAT). Finally, a novel two-component system (aau) was identified which seems to be involved in the utilization of acidic amino acids. Disruption mutants defective in the response regulator (AauR) and the sensor kinase component (AauS), respectively, were constructed and the resulting phenotype analyzed. Growth of both mutants was severely impaired in glutamate and glutamine-containing media. By contrast, both strains grew at normal rates when succinate was supplied in addition to amino acids. This finding indicate that the aau system is related to, but not identical with the dct two-component system which is involved in the utilization of succinate by rhizobia

Regulation of Pseudomonas putida genes involved in the metabolism of acidic amino acids

Pseudomonas putida KT2440 has the ability to utilize a wide range of amino acids as carbon and nitrogen sources. Rapid growth of this organism is supported by the acidic amino acids (Asp and Glu) and their amides (Asn and Gln) when supplied as sole source of carbon and nitrogen, or in combination with other carbon and nitrogen sources such as glucose and NH4+. A proteomics study based on two-dimensional gel electrophoresis revealed that during growth of P. putida KT2440 in Glu containing medium a set of at least 9 major proteins were up-regulated in a coordinate fashion, whereas 4 other proteins were specifically induced during growth in NH4+/Glucose. Most of the identified proteins have some role in the uptake and utilization of amino acids. Based on their assigned functions genetic organization, we propose that they form a regulon involved in the metabolism of amino acids. By transposon mutagenesis it was found that the expression of that regulon depends on a functional gltB gene which encodes the major subunit of glutamate synthase (GOGAT). Finally, a novel two-component system (aau) was identified which seems to be involved in the utilization of acidic amino acids. Disruption mutants defective in the response regulator (AauR) and the sensor kinase component (AauS), respectively, were constructed and the resulting phenotype analyzed. Growth of both mutants was severely impaired in glutamate and glutamine-containing media. By contrast, both strains grew at normal rates when succinate was supplied in addition to amino acids. This finding indicate that the aau system is related to, but not identical with the dct two-component system which is involved in the utilization of succinate by rhizobia

Nanoparticle Vaccines Against Infectious Diseases

Due to emergence of new variants of pathogenic micro-organisms the treatment and immunization of infectious diseases have become a great challenge in the past few years. In the context of vaccine development remarkable efforts have been made to develop new vaccines and also to improve the efficacy of existing vaccines against specific diseases. To date, some vaccines are developed from protein subunits or killed pathogens, whilst several vaccines are based on live-attenuated organisms, which carry the risk of regaining their pathogenicity under certain immunocompromised conditions. To avoid this, the development of risk-free effective vaccines in conjunction with adequate delivery systems are considered as an imperative need to obtain desired humoral and cell-mediated immunity against infectious diseases. In the last several years, the use of nanoparticle-based vaccines has received a great attention to improve vaccine efficacy, immunization strategies, and targeted delivery to achieve desired immune responses at the cellular level. To improve vaccine efficacy, these nanocarriers should protect the antigens from premature proteolytic degradation, facilitate antigen uptake and processing by antigen presenting cells, control release, and should be safe for human use. Nanocarriers composed of lipids, proteins, metals or polymers have already been used to attain some of these attributes. In this context, several physico-chemical properties of nanoparticles play an important role in the determination of vaccine efficacy. This review article focuses on the applications of nanocarrier-based vaccine formulations and the strategies used for the functionalization of nanoparticles to accomplish efficient delivery of vaccines in order to induce desired host immunity against infectious diseases

Toxicity and antibacterial assessment of chitosancoated silver nanoparticles on human pathogens and macrophage cells

Prajna Jena1, Soumitra Mohanty1, Rojee Mallick1, Biju Jacob2, Avinash Sonawane11School of Biotechnology, KIIT University, Bhubaneswar, Orissa, India; 2Center for Innovation, Technopark Technology Business Incubator, Bangalore, Karnataka, IndiaBackground: Pathogenic bacteria are able to develop various strategies to counteract the bactericidal action of antibiotics. Silver nanoparticles (AgNPs) have emerged as a potential alternative to conventional antibiotics because of their potent antimicrobial properties. The purpose of this study was to synthesize chitosan-stabilized AgNPs (CS-AgNPs) and test for their cytotoxic, genotoxic, macrophage cell uptake, antibacterial, and antibiofilm activities.Methods: AgNPs were synthesized using chitosan as both a stabilizing and a reducing agent. Antibacterial activity was determined by colony-forming unit assay and scanning electron microscopy. Genotoxic and cytotoxic activity were determined by DNA fragmentation, comet, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Cellular uptake and intracellular antibacterial activity were tested on macrophages.Results: CS-AgNPs exhibited potent antibacterial activity against different human pathogens and also impeded bacterial biofilm formation. Scanning electron microscopy analysis indicated that CS-AgNPs kill bacteria by disrupting the cell membrane. CS-AgNPs showed no significant cytotoxic or DNA damage effect on macrophages at the bactericidal dose. Propidium iodide staining indicated active endocytosis of CS-AgNPs resulting in reduced intracellular bacterial survival in macrophages.Conclusion: The present study concludes that at a specific dose, chitosan-based AgNPs kill bacteria without harming the host cells, thus representing a potential template for the design of antibacterial agents to decrease bacterial colonization and to overcome the problem of drug resistance.Keywords: chitosan-silver nanoparticles, antibiofilm, cytotoxicity, genotoxicit

Cationic Antimicrobial Peptides and Biogenic Silver Nanoparticles Kill Mycobacteria without Eliciting DNA Damage and Cytotoxicity in Mouse Macrophages

ABSTRACT With the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal, and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria. Mycobacterium smegmatis was found to be more susceptible to AgNPs compared to M. marinum . We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at a 0.5-ppm concentration, whereas LLKKK-18 showed antibacterial activity only with NP-2 at 0.5-ppm dose against M. smegmatis . In case of M. marinum NK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing of M. smegmatis in combination with the antituberculosis drug rifampin. The sizes and shapes of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, whereas treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed fluorescein isothiocyanate-labeled AgNPs resulting in nitric oxide independent intracellular killing of M. smegmatis . Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G 1 -phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template. </jats:p

Mycobacterium tuberculosis EsxL inhibits MHC-II expression by promoting hypermethylation in class-II transactivator loci in macrophages

Mycobacterium tuberculosis (Mtb) is known to modulate the host immune responses to facilitate its persistence inside the host cells. One of the key mechanisms includes repression of class-II transactivator (CIITA) and MHC-II expression in infected macrophages. However, the precise mechanism of CIITA and MHC-II down-regulation is not well studied. Mtb 6-kDa early secretory antigenic target (ESAT-6) is a known potent virulence and antigenic determinant. Mtb genome encodes 23 such ESAT-6 family proteins. We herein report that Mtb and M. bovis-BCG infection down-regulated the expression of CIITA/MHC-II by inducing hypermethylation in histone H3 Lysine 9 (H3K9me2/3). Further, we show that Mtb ESAT-6 family protein EsxL, encoded by Rv1198, is responsible for the down-regulation of CIITA/MHC-II by inducing H3K9me2/3. We further report that Mtb esxL induced the expression of nitric oxide synthetase (iNOS), NO production and p38-MAPK pathway, which in turn was responsible for the increased H3K9me2/3 in CIITA via up-regulation of euchromatic histone-lysine N-methyltransferase 2 (G9a). In contrast, inhibition of iNOS, p38-MAPK and G9a abrogated H3K9me2/3 resulting in increased CIITA expression. Chromatin immune precipitation assay confirmed that hypermethylation at the promoter IV (pIV) region of CIITA is mainly responsible for the CIITA down regulation and subsequently antigen presentation. We found that co-culture of macrophages infected with esxL expressing M. smegmatis and mouse spleenocytes led to down-regulation of IL-2, a key cytokine involved in T-cell proliferation. In summary, we show that Mtb esxL inhibits antigen presentation by enhancing H3K9me2/3 on CIITA promoter thereby repressing its expression through NO and p38-MAPK activation

Search for long-lived particles decaying to a pair of muons in proton-proton collisions at s=13\sqrt s = 13 TeV.

An inclusive search for long-lived exotic particles decaying into a pair of muons is presented. The experimental signature is a pair of oppositely charged muons originating in a common secondary vertex displaced from the proton-proton collision point by distances ranging from several hundred micrometers to several meters. The search is conducted using data collected in 2016 and 2018 by the CMS experiment at the LHC in proton-proton collisions with a center of mass energy $\sqrt{s} = 13$ TeV in 2016 and 2018, corresponding to an integrated luminosity of 97.6 fb$^{-1}$. The results are interpreted in the framework of the Hidden Abelian Higgs Model, and a simplified model in which Long Lived Particles are produced in the decay of an exotic heavy neutral scalar boson

The AauR-AauS Two-Component System Regulates Uptake and Metabolism of Acidic Amino Acids in Pseudomonas putida

Pseudomonas putida KT2440 metabolizes a wide range of carbon and nitrogen sources, including many amino acids. In this study, a σ(54)-dependent two-component system that controls the uptake and metabolism of acidic amino acids was identified. The system (designated aau, for acidic amino acid utilization) involves a sensor histidine kinase, AauS, encoded by PP1067, and a response regulator, AauR, encoded by PP1066. aauR and aauS deletion mutants were unable to efficiently utilize aspartate (Asp), glutamate (Glu), and glutamine (Gln) as sole sources of carbon and nitrogen. Growth of the mutants was partially restored when the above-mentioned amino acids were supplemented with glucose or succinate as an additional carbon source. Uptake of Gln, Asp, and asparagine (Asn) by the aauR mutant was moderately reduced, while Glu uptake was severely impaired. In the absence of glucose, the aauR mutant even secreted Glu into the medium. Furthermore, disruption of aauR affected the activities of several key enzymes of Glu and Asp metabolism, leading to the intracellular accumulation of Glu and greatly reduced survival times under conditions of nitrogen starvation. By a proteomics approach, four major proteins were identified that are downregulated during growth of the aauR mutant on Glu. Two of these were identified as periplasmic glutaminase/asparaginase and the solute-binding protein of a Glu/Asp transporter. Transcriptional analysis of lacZ fusions containing the putative promoter regions of these genes confirmed that their expression is indeed affected by the aau system. Three further periplasmic solute-binding proteins were strongly expressed during growth of the aauR deletion mutant on Glu but downregulated during cultivation on glucose/NH(4)(+). These systems may be involved in amino acid efflux

Computational Investigations of Spray Atomization and Evaporation Under Cold-Start Conditions of a Diesel Engine

Abstract Unburnt hydrocarbon emissions and combustion instability are severe issues in diesel engines during cold starting. This simulation study aims to provide insights for improving the cold-start issues of diesel engines. Computational analysis of the diesel spray and evolution of plumes from a 7-hole injector was done in a constant volume quiescent spray chamber for analyzing the effect of fuel temperature. This study was based on a comprehensive numerical analysis using CONVERGE computational fluid dynamics (CFD) software, where an Eulerian–Lagrangian approach was adopted in a large eddy simulation (LES) framework. Diesel engine-like cold-start conditions were replicated by reducing the fuel temperatures to 250 K to simulate freezing conditions outside. This computational study compares fuel sprays at 250 K with 312 K into a relatively colder ambient temperature of 626 K vis-a-vis high-temperature diesel engine-like ambient conditions with ambient temperature to 961 K to investigate the degree of spray characteristics improvements due to increased fuel temperature. The predicted liquid spray penetration obtained by simulations agreed well with the experimental data for fuel temperatures injected into the ambient at cold (626 K) and hot (961 K) engine-like ambient conditions. The available empirical relations justify the simulation results of this study. Results showed that fuel and ambient temperatures significantly affected the spray atomization and evaporation characteristics. A higher reduction in liquid penetration length was found with increasing fuel temperature at hot ambient conditions. Increasing ambient temperature also improved the evaporation characteristics of the spray droplets. Vapor formation for the same increase in fuel temperature was higher at hot ambient temperature than the cold. Fuel temperature had a major role in the spray atomization process, whereas the ambient temperature affected the spray evaporation process. However, fuel and ambient temperatures had only a minor effect on the distribution of total kinetic energy (TKE). Among all test conditions, a fuel temperature of 312 K sprayed in hot ambient conditions showed superior fuel spray atomization and evaporation. Hence, to tackle the cold-start of diesel engines, measures taken to increase the fuel and ambient temperatures simultaneously proved to be useful.</jats:p