Topological analyses of the L-lysine exporter LysO reveal a critical role for a conserved pair of intramembrane solvent-exposed acidic residues.

LysO, a prototypical member of the LysO family, mediates export of L-lysine (Lys) and resistance to the toxic Lys antimetabolite, L-thialysine (Thl) in Escherichia coli. Here, we have addressed unknown aspects of LysO function pertaining to its membrane topology and the mechanism by which it mediates Lys/Thl export. Using substituted cysteine (Cys) accessibility, here we delineated the membrane topology of LysO. Our studies support a model in which both the N- and C-termini of LysO are present at the periplasmic face of the membrane with a transmembrane (TM) domain comprising eight TM segments (TMSs) between them. In addition, a feature of intramembrane solvent exposure in LysO is inferred with the identification of membrane-located solvent-exposed Cys residues. Isosteric substitutions of a pair of conserved acidic residues, one E233, located in the solvent-exposed TMS7 and the other D261, in a solvent-exposed intramembrane segment located between TMS7 and TMS8, abolished LysO function in vivo. Thl, but not Lys, elicited proton release in inside-out membrane vesicles, a process requiring the presence of both E233 and D261. We postulate that Thl may be exported in antiport with H+ and that Lys may be a low-affinity export substrate. Our findings are compatible with a physiological scenario wherein in vivo LysO exports the naturally occurring antimetabolite Thl with higher affinity over the essential cellular metabolite Lys, thus affording protection from Thl toxicity and limiting wasteful export of Lys.

The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-Cout Configuration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function.

ArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export of l-arginine (Arg) in Escherichia coli and l-lysine (Lys) and Arg in Corynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane of E. coli, we have employed a combination of cysteine accessibility in situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Cout configuration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO function in vivo and that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO function in vivo and their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomer in vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit. IMPORTANCE: The orthologous proteins LysE of C. glutamicum and ArgO of E. coli function as exporters of the basic amino acids l-arginine and l-lysine and the basic amino acid l-arginine, respectively, and LysE can functionally substitute for ArgO when expressed in E. coli Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic and in silico studies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

Aspergillus niger PA2: a novel strain for extracellular biotransformation of L-tyrosine into L-DOPA.

L-DOPA (3,4-dihydroxyphenyl-L-alanine), an amino acid derivative is the most widely used drug of choice for the treatment of Parkinson's disease and other neurologic injuries. The present study deals with the elevated biochemical transformation of L-tyrosine to L-DOPA by Aspergillus niger PA2, a potent tyrosinase producer, isolated from decomposed food wastes. This appears to be the first report on A. niger as a notable extracellular tyrosinase producer. The extracellular tyrosinase activity produced remarkably higher levels of L-DOPA, i.e. 2.44 mg mL(-1) when the media was supplemented with 5 mg mL(-1) L-tyrosine. The optimum pH for tyrosinase production was 6.0, with the maximal L-DOPA production at the same pH. The product thus produced was analyzed by thin-layer chromatography, UV spectroscopy, high-performance liquid chromatography and Fourier transform infrared spectroscopy, that had denoted this to be L-DOPA. Kinetic parameters viz. Y p/s, Q s and Q p had further indicated the notable levels of production. Thus, Aspergillus niger PA2 could be a promising resource and may be further exploited for large-scale production of L-DOPA.

Broad-spectrum antiviral inhibitors targeting pandemic potential RNA viruses.

RNA viruses continue to remain a clear and present threat for potential pandemics due to their rapid evolution. To mitigate their impact, we urgently require antiviral agents that can inhibit multiple families of disease-causing viruses, such as arthropod-borne and respiratory pathogens. Potentiating host antiviral pathways can prevent or limit viral infections before escalating into a major outbreak. Therefore, it is critical to identify broad-spectrum antiviral agents. We have tested a small library of innate immune agonists targeting pathogen recognition receptors, including TLRs, STING, NOD, Dectin and cytosolic DNA or RNA sensors. We observed that TLR3, STING, TLR8 and Dectin-1 ligands inhibited arboviruses, Chikungunya virus (CHIKV), West Nile virus (WNV) and Zika virus, to varying degrees. Cyclic dinucleotide (CDN) STING agonists, such as cAIMP, diABZI, and 2',3'-cGAMP, and Dectin-1 agonist scleroglucan, demonstrated the most potent, broad-spectrum antiviral function. Comparative transcriptome analysis revealed that CHIKV-infected cells had larger number of differentially expressed genes than of WNV and ZIKV. Furthermore, gene expression analysis showed that cAIMP treatment rescued cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provided protection against CHIKV in a CHIKV-arthritis mouse model. Cardioprotective effects of synthetic STING ligands against CHIKV, WNV, SARS-CoV-2 and enterovirus D68 (EV-D68) infections were demonstrated using human cardiomyocytes. Interestingly, the direct-acting antiviral drug remdesivir, a nucleoside analogue, was not effective against CHIKV and WNV, but exhibited potent antiviral effects against SARS-CoV-2, RSV (respiratory syncytial virus), and EV-D68. Our study identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses, which can be rapidly deployed to prevent or mitigate future pandemics.

Innate immune pathway modulator screen identifies STING pathway activation as a strategy to inhibit multiple families of arbo and respiratory viruses.

RNA viruses continue to remain a threat for potential pandemics due to their rapid evolution. Potentiating host antiviral pathways to prevent or limit viral infections is a promising strategy. Thus, by testing a library of innate immune agonists targeting pathogen recognition receptors, we observe that Toll-like receptor 3 (TLR3), stimulator of interferon genes (STING), TLR8, and Dectin-1 ligands inhibit arboviruses, Chikungunya virus (CHIKV), West Nile virus, and Zika virus to varying degrees. STING agonists (cAIMP, diABZI, and 2',3'-cGAMP) and Dectin-1 agonist scleroglucan demonstrate the most potent, broad-spectrum antiviral function. Furthermore, STING agonists inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and enterovirus-D68 (EV-D68) infection in cardiomyocytes. Transcriptome analysis reveals that cAIMP treatment rescue cells from CHIKV-induced dysregulation of cell repair, immune, and metabolic pathways. In addition, cAIMP provides protection against CHIKV in a chronic CHIKV-arthritis mouse model. Our study describes innate immune signaling circuits crucial for RNA virus replication and identifies broad-spectrum antivirals effective against multiple families of pandemic potential RNA viruses.

Comparative Analysis of Molecular Pathogenic Mechanisms and Antiviral Development Targeting Old and New World Hantaviruses.

Background: Hantaviruses - dichotomized into New World (i.e. Andes virus, ANDV; Sin Nombre virus, SNV) and Old-World viruses (i.e. Hantaan virus, HTNV) - are zoonotic viruses transmitted from rodents to humans. Currently, no FDA-approved vaccines against hantaviruses exist. Given the recent breakthrough to human-human transmission by the ANDV, an essential step is to establish an effective pandemic preparedness infrastructure to rapidly identify cell tropism, infective potential, and effective therapeutic agents through systematic investigation. Methods: We established human cell model systems in lung (airway and distal lung epithelial cells), heart (pluripotent stem cell-derived (PSC-) cardiomyocytes), and brain (PSC-astrocytes) cell types and subsequently evaluated ANDV, HTNV and SNV tropisms. Transcriptomic, lipidomic and bioinformatic data analyses were performed to identify the molecular pathogenic mechanisms of viruses in different cell types. This cell-based infection system was utilized to establish a drug testing platform and pharmacogenomic comparisons. Results: ANDV showed broad tropism for all cell types assessed. HTNV replication was predominantly observed in heart and brain cells. ANDV efficiently replicated in human and mouse 3D distal lung organoids. Transcriptomic analysis showed that ANDV infection resulted in pronounced inflammatory response and downregulation of cholesterol biosynthesis pathway in lung cells. Lipidomic profiling revealed that ANDV-infected cells showed reduced level of cholesterol esters and triglycerides. Further analysis of pathway-based molecular signatures showed that, compared to SNV and HTNV, ANDV infection caused drastic lung cell injury responses. A selective drug screening identified STING agonists, nucleoside analogues and plant-derived compounds that inhibited ANDV viral infection and rescued cellular metabolism. In line with experimental results, transcriptome data shows that the least number of total and unique differentially expressed genes were identified in urolithin B- and favipiravir-treated cells, confirming the higher efficiency of these two drugs in inhibiting ANDV, resulting in host cell ability to balance gene expression to establish proper cell functioning. Conclusions: Overall, our study describes advanced human PSC-derived model systems and systems-level transcriptomics and lipidomic data to better understand Old and New World hantaviral tropism, as well as drug candidates that can be further assessed for potential rapid deployment in the event of a pandemic.

Impact of aquatic microplastics and nanoplastics pollution on ecological systems and sustainable remediation strategies of biodegradation and photodegradation.

The extreme degree of microplastics contamination and its negative impact on ecosystems has become a serious and emerging global concern. Microplastics are mainly generated from products that are used primarily in our everyday lives and are also generated from the fragmentation of larger plastic wastes. It easily penetrates the food chain and, when ingested by aquatic animals or humans, can pose serious health problems. Recently, several technologies have been developed to control the unrestricted spread of microplastics and possibly eradicate them; however, still under investigation. In this review, we have illustrated the types of microplastics, their harmful effect on living things, and the progress to degrade them to protect the environment and life on earth. Several promising and eco-friendly technologies including microbial and enzymatic approaches are enticing to eliminate the microplastics. Also, the photodegradation of microplastics contaminations appeals as a more fascinating approach. The metal oxide-assisted photodegradation of microplastics has also been taken into account. This work presented an impact on the comprehensive research for the effective degradation of different microplastic compositions as well as emerging green approaches for a sustainable environment and a healthier life.

Determination of disulfiram by micellar liquid chromatography in illicit preparations.

Presently, disulfiram is used in aversion therapy for recovering alcoholics. It acts by inhibiting aldehyde dehydrogenase, leading to high blood levels of acetaldehyde. A simple direct injection micellar liquid chromatographic procedure was developed to determine disulfiram in illicit preparations (ayurvedic, herbal, divine ash, and traditional medicine), as well as in pharmaceuticals and biological samples (urine). After application of a predictive optimization strategy, the proposed method was developed using a 0.1 M sodium dodecyl sulfate-butanol 4% (v/v) buffered to pH 7 as the mobile phase at a flow rate of 1 mL/min, an octyl silyl (C8) 150 mm column, and diode array detection at 248 nm. Under the above conditions, the analysis time was below 8 min. Validation studies were based on U.S. Food and Drug Administration guidelines. The LOD (3 x SD criterion) was 15 ng/mL and LOQ (10 x SD criterion) was 70 ng/mL for disulfiram. The intraday and interday precisions were below 3.5%, recoveries were in the range of 97-102%, and robustness was below 3%. The optimized and validated micellar liquid chromatographic method was successfully applied to the determination of disulfiram in ayurvedic, herbal, divine ash, and other samples. The procedure developed could also be used in the fields of QC, routine analysis, and pharmacokinetic studies.

Validation of absolute point dosimetry by the analytical anisotropic algorithm and Acuros XB algorithm employing intensity-modulated radiotherapy technique on an in-house develop cost-effective heterogeneous thorax phantom.

INTRODUCTION: Dose validation inside the human body needs a medium which can simulate the actual heterogeneities of a specific body site. The aim of the present work is to study the properties of a cost-effective heterogeneous thorax phantom (HTP) developed in-house by the author and its application for the evaluation of patient-specific absolute point dosimetry by employing analytic anisotropic algorithm (AAA) and Acuros XB (AXB) algorithm. MATERIALS AND METHODS: HTP was made from the dust of porous pinewood, rib cage, and honeybee's wax. Density and central axis isodose depth distribution was measured on computed tomography images of actual patient and on HTP. Absolute point dose verification of 35 patients was done using AAA and AXB algorithm. The difference in the calculated dose by AAA and AXB was compared using the Wilcoxon signed-rank test. RESULTS: Density distribution and central axis depth dose inside the HTP compare well with that of an actual patient. The mean percentage variation between the planned and the measured doses inside the HTP was found to be 4.85 (standard deviation [SD] = 3.38) and 1.3 (SD = 2.93), respectively, using AAA and AXB algorithm. The difference in the measured dose and the planned dose was found to be significant for AAA with the significance level of 0.01 (p-value < 0.00001), whereas it was found to be insignificant (p-value < 0.00001) for AXB. CONCLUSION: The results of this study showed that the HTP is resembled with the human thorax in terms of its heterogeneities and radiological properties and can be used for pretreatment plan verification.

3-D macro/microporous-nanofibrous bacterial cellulose scaffolds seeded with BMP-2 preconditioned mesenchymal stem cells exhibit remarkable potential for bone tissue engineering.

Bone repair using BMP-2 is a promising therapeutic approach in clinical practices, however, high dosages required to be effective pose issues of cost and safety. The present study explores the potential of low dose BMP-2 treatment via tissue engineering approach, which amalgamates 3-D macro/microporous-nanofibrous bacterial cellulose (mNBC) scaffolds and low dose BMP-2 primed murine mesenchymal stem cells (C3H10T1/2 cells). Initial studies on cell-scaffold interaction using unprimed C3H10T1/2 cells confirmed that scaffolds provided a propitious environment for cell adhesion, growth, and infiltration, owing to its ECM-mimicking nano-micro-macro architecture. Osteogenic studies were conducted by preconditioning the cells with 50 ng/mL BMP-2 for 15 min, followed by culturing on mNBC scaffolds for up to three weeks. The results showed an early onset and significantly enhanced bone matrix secretion and maturation in the scaffolds seeded with BMP-2 primed cells compared to the unprimed ones. Moreover, mNBC scaffolds alone were able to facilitate the mineralization of cells to some extent. These findings suggest that, with the aid of 'osteoinduction' from low dose BMP-2 priming of stem cells and 'osteoconduction' from nano-macro/micro topography of mNBC scaffolds, a cost-effective bone tissue engineering strategy can be designed for quick and excellent in vivo osseointegration.

Production and characterization of Komagataeibacter xylinus SGP8 nanocellulose and its calcite based composite for removal of Cd ions.

In the present study, fermentative production of bacterial nanocellulose (BNC) by using Komagataeibacter xylinus strain SGP8 and characterization of nanocellulose is presented. The bacterium was able to produce 1.82 g L-1 of cellulose in the form of pellicle in standard Hestrin-Schramn (HS) medium. The morpho-structural characterization of the BNC using scanning electron microscopy (SEM) and X-ray diffraction (XRD) studies, respectively revealed nanofibrillar structure and high crystallinity index (~86%). The thermogravimetric analysis (TGA) showed the stability of BNC up to 280 °C, further rise in temperature to 350 °C results in depolymerization of the sample. In order to show the applicability of produced BNC, it was modified first using calcite (CaCO3) and thereafter characterized using SEM, XRD, FTIR, and TGA studies. The BNC-CaCO3 composites as a sorbent resulted in >99% removal of initial 10 mg L-1 of Cd (II) at pH 5, 7 and 9 after 12 h of treatment. Moreover, the composite was also found to be competent in removing high concentrations of Cd (25 and 50 mg L-1) from the solution (69-70%). Overall, the above results suggest that cellulose produced by K. xylinus strain SGP8 showed excellent material properties, and modified BNC (BNC-CaCO3 composite) could effectively be used for remediation of toxic levels of Cd from the contaminated system.

Biotransformation of sweet lime pulp waste into high-quality nanocellulose with an excellent productivity using Komagataeibacter europaeus SGP37 under static intermittent fed-batch cultivation.

Herein, sweet lime pulp waste (SLPW) was utilized as a low- or no-cost feedstock for the production of bacterial nanocellulose (BNC) alone and in amalgamation with other nutritional supplements by the isolate K. europaeus SGP37 under static batch and static intermittent fed-batch cultivation. The highest yield (26.2±1.50gL-1) was obtained in the hot water extract of SLPW supplemented with the components of HS medium, which got further boosted to 38±0.85gL-1 as the cultivation strategy was shifted from static batch to static intermittent fed-batch. BNC obtained from various SLPW medium was similar or even superior to that obtained with standard HS medium in terms of its physicochemical properties. The production yields of BNC thus obtained are significantly higher and fit well in terms of industrial scale production.

Plasmodium Hsp40 and human Hsp70: A potential cochaperone-chaperone complex.

Out of the total forty four members of Plasmodium falciparum Hsp40 protein family, nineteen of them possess a PEXEL motif, and are predicted to be exported into the cytosol of an infected RBC. It is speculated that the human Hsp70 (hHsp70), which resides into the cytosol of the host erythrocyte, along with the exported PfHsp40s assists in the folding of parasitic proteins, thus playing a crucial role in the establishment of virulence. However, till date no experimental evidence supports this hypothesis. Our work establishes that the PEXEL motifs containing Type II PfDNAJ proteins specifically interact with hHsp70 (HSPA1A). It suggests that there exists a specific factor in PfDNAJ that determines the choice of cognate Hsp70. This opens up an interesting avenue of malaria research.

From rotten grapes to industrial exploitation: Komagataeibacter europaeus SGP37, a micro-factory for macroscale production of bacterial nanocellulose.

Bacterial nanocellulose (BNC), being ultrapure and unique in its properties, is a booming and ageless precursor of several breakthrough technologies of materials sciences; however, its low yield and high cost has created a challenge for its usage at industrial level. Herein, we report a novel, high yielding bacterial cell factory Komagataeibacter europaeus SGP37, isolated from rotten grapes, for the production of high quality and value added BNC. The strain was kinetically analyzed to evaluate BNC production under different physiological conditions and had demonstrated the production of 9.98±0.24gL-1 BNC at the expense of 12.08±1.94gL-1 sugar following 2 weeks of cultivation, thus having the conversion yield of 0.82g BNC/g sugar which seems to be the maximum reported yield so far. The analysis of produced pellicle using FTIR, 13C CP MAS NMR, FE-SEM, XRD and TGA had shown similar structural, morphological and chemical characteristics with that of bacterial nanocellulose. Thus, K. europaeus SGP37 appears to be a potential strain and may offer a promising platform for industrial scale production of nanocelluloses.

Aspergillus niger PA2 Tyrosinase Covalently Immobilized on a Novel Eco-Friendly Bio-Composite of Chitosan-Gelatin and Its Evaluation for L-DOPA Production.

Tyrosinase (EC 1.14.18.1) a copper-containing monooxygenase, isolated from a fungal isolate Aspergillus niger PA2 was subjected for immobilization onto a composite consisting of chitosan and gelatin biopolymers. The homogeneity of the chitosan-gelatin biocomposite film was characterized by X-ray diffraction analyses. To evaluate immobilization efficiency, chitosan-gelatin-Tyr bio-composite films were analyzed by field emission scanning electron microscopy, atomic force microscopy and UV-spectroscopy. The rough morphology of the film led to a high loading of enzyme and it could retain its bioactivity for a longer period. The enzyme adsorbed onto the film exhibited 72% of its activity after 10 days and exhibited good repeatability for up to nine times, after intermittent storage. Moreover, the immobilized enzyme exhibited broader pH and temperature profile as compared to free counterpart. Immobilized enzyme was further evaluated for the synthesis of L-DOPA (2,4-dihydroxy phenylalanine) which is a precursor of dopamine and a potent drug for the treatment of Parkinson's disease and for myocardium neurogenic injury.

KAP Study on HIV/AIDS Among Antenatal Women Attending Central Referral Hospital of North East India.

BACKGROUND: This study was conducted to determine whether antenatal mothers in Sikkim have adequate knowledge about awareness, attitude, and preventive practices regarding HIV infection. METHODS: Cross-sectional study using structured questionnaire. 220 Antenatal mothers attending the outpatient department of Central Referral Hospital of Sikkim were taken for the study for a period of 1 year from April 2011 to April 2012. Questionnaire form filled by pregnant women during their first antenatal visit was the source of data for this study. Systematic sampling technique was used where every alternate pregnant women registering for ANC visit were voluntarily recruited into the study. RESULTS: 2.27 % (5) women had not heard about HIV. 84 % (38) women had the knowledge that HIV was related to STI, while 50 % (110) did not. Television was the best method of increasing the knowledge (48 %). 68 % (150) of the women were aware about mother-to-child transmission (MTCT) of HIV during antenatal period. Only 2.66 % (6) women knew that HIV can be transmitted to child through breast milk. 90 % (198) knew that HIV is spread by having unsafe sex, 48 % (106) women knew using condoms would protect against it. 69.4 % (153) women wanted partner testing, and 84 % (185) of women consented that all pregnant women should be tested for HIV. CONCLUSIONS: The current study revealed high levels of knowledge, positive attitude, and preventive practices regarding HIV; however, this population lacked knowledge about MTCT and its prevention.