Investigating a putative transcriptional regulatory protein encoded by Rv1719 gene of Mycobacterium tuberculosis

The Protein Journal - Mycobacterium tuberculosis, the causative agent of tuberculosis, demonstrates immense plasticity with which it adapts to a highly dynamic and hostile host environment. This is...     

Targeting AMPK signaling pathway by natural products for treatment of diabetes mellitus and its complications

Diabetes affects a large population of the world. Lifestyle, obesity, dietary habits, and genetic factors contribute to this metabolic disease. A target pathway to control diabetes is the 5′-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway. AMPK is a heterotrimeric protein with α, β, and γ subunits. In several studies, AMPK activation enhanced glucose uptake into cells and inhibited intracellular glucose production. Impairment of AMPK activity is present in diabetes, according to some studies. Drugs used in the treatment of diabetes, such as metformin, are also known to act through regulation of AMPK. Thus, drugs that activate and regulate AMPK are potential candidates for the treatment of diabetes. In addition, many patients encounter important adverse effects, like hypoglycemia, while using allopathic drugs. As a result, the investigation of plant-derived natural drugs that lack adverse side effects and treat diabetes is necessary. Natural products like berberine, quercetin, resveratrol, and so forth have shown significant potential in regulating and activating the AMPK pathway which can lead to manage diabetes mellitus and its complications.     

Nod1-mediated lipolysis promotes diacylglycerol accumulation and successive inflammation via PKCδ-IRAK axis in adipocytes

Chronic inflammation contributes to obesity mediated metabolic disturbances, including insulin resistance. Obesity is associated with altered microbial load in metabolic tissues that can contribute to metabolic inflammation. Different bacterial components such as, LPS, peptidoglycans have been shown to underpin metabolic disturbances through interaction with host innate immune receptors. Activation of Nucleotide-binding oligomerization domain-containing protein 1 (Nod1) with specific peptidoglycan moieties promotes insulin resistance, inflammation and lipolysis in adipocytes. However, it was not clear how Nod1-mediated lipolysis and inflammation is linked. Here, we tested if Nod1-mediated lipolysis caused accumulation of lipid intermediates and promoted cell autonomous inflammation in adipocytes. We showed that Nod1-mediated lipolysis caused accumulation of diacylglycerol (DAG) and activation of PKCδ in 3T3-L1 adipocytes, which was prevented with a Nod1 inhibitor. Nod1-activated PKCδ caused downstream stimulation of IRAK1/4 and was associated with increased expression of proinflammatory cytokines such as, IL-1β, IL-18, IL-6, TNFα and MCP-1. Pharmacological inhibition or siRNA mediated knockdown of IRAK1/4 attenuated Nod1-mediated activation of NF-κB, JNK, and the expression of proinflammatory cytokines. These results reveal that Nod1-mediated lipolysis promoted accumulation of DAG, which engaged PKCδ and IRAK1/4 to augment inflammation in 3T3-L1 adipocytes.     

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional ‘single target–single drug’ approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3’-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC₅₀ and EC₅₀ values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.     

Designing an immunoinformatic vaccine for peri-implantitis using a structural biology approach

ObjectivesPeri-implantitis is a destructive inflammatory process that affects the soft and hard tissues around dental implants. porphyromonas gingivalis, an anaerobic gram-negative bacterium, appears to be the main culprit. Since there is no efficient and specific vaccine to treat peri-implantitis, the goal of our research has been to develop a multi-epitope vaccination utilizing an immunoinformatics approach that targeted P. gingivalis type I fim A.Materials and methodsP. gingivalis peptides 6JKZ and 6KMF are suitable for vaccine development. B- and T-cell epitopes from 6KMF and 6JKZ were detected and evaluated based on critical factors to produce a multi-epitope vaccine construct. It was assessed based on allergenicity, antigenicity, stability. The vaccine's dual major histocompatibility complex (MHC-I and MHC-II) binding epitopes allowed it to reach a larger population. P. gingivalis fimbriae induce immune subversion through TLR -CXCR4 receptor complex pathway. The ClusPro 2.0 server was used to do the molecular docking using TLR2 - CXCR4 and vaccine epitopes as receptor and ligand respectively.ResultsThe designed vaccine was non-allergenic and had a high antigenicity, solubility, and stability. The 3D structure of the vaccine revealed strong interaction with CXCR4(TLR2) using molecular docking. The vaccine-CXCR4 interface was more consistent, possibly because the vaccination has a higher affinity for the CXCR4-TLR2 complex.ConclusionThis study details the vaccine's distinct and sustained interaction with the CXCR4(TLR2) immunological receptor and its consistent and effective utterance in the bacterial system. As a result, our vaccine formulation will evoke a significant memory response and induce an adaptive immune response against P. gingivalis.     

An Efficient Large-Area Grating Coupler for Surface Plasmon Polaritons

We report the design, fabrication, and characterization of a periodic grating of shallow rectangular grooves in a metallic film with the goal of maximizing the coupling efficiency of an extended plane wave (PW) of visible or near-infrared light into a single surface plasmon polariton (SPP) mode on a flat metal surface. A PW-to-SPP power conversion factor >45% is demonstrated at a wavelength of 780?nm, which exceeds by an order of magnitude the experimental performance of SPP grating couplers reported to date at any wavelength. Conversion efficiency is maximized by matching the dissipative SPP losses along the grating surface to the local coupling strength. This critical coupling condition is experimentally achieved by tailoring the groove depth and width using a focused ion beam.     

Allethrin-induced genotoxicity and oxidative stress in Swiss albino mice

Allethrin (C(19)H(26)O(3)) is non-cyano-containing pyrethroid insecticide that is used extensively for controlling flies and mosquitoes. Apart from its neurotoxic effects in non-target species, allethrin is reported to be mutagenic in bacterial systems. In this study, we observed oxidative damage-mediated genotoxicity caused by allethrin in Swiss albino mice. The genotoxic potential of allethrin was evaluated using chromosome aberrations (CAs) and a micronuclei (MN) induction assay as genetic end-points. The oral intubation of allethrin (25 and 50mg/kg b.wt.) significantly induces CAs and MN in mouse bone marrow cells. The DNA-damaging potential of allethrin was estimated in mouse liver using the DNA alkaline unwinding assay (DAUA) and by measuring the levels of 8-hydroxy-2'-deoxy-guanosine (8-OH-dG). Furthermore, a dose-dependent increase in reactive oxygen species (ROS) generation and lipid peroxidation (LPO), with a concurrent decrease in superoxide dismutase (SOD) and catalase, confirm its pro-oxidant potential. The DNA-damaging potential of allethrin was found to be mediated through the modulation of p53, p21, GADD45α and MDM-2. These results confirm the genotoxic and the pro-oxidant potential of allethrin in Swiss albino mice.     

Substrate specificity of the deazaflavin-dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.