Spatio-temporal variability of public water supply characteristics and associated health hazards for children and adults in selected locations of Ambala, India.

The contamination of public water supply and groundwater resources is a major concern in many parts of developing nations. Polluted water poses serious health risks to humans and the environment. This research was conducted to investigate the seasonal variations of the water quality parameters in the public water supply. To assess the supply water quality in different blocks of Ambala District, hydro-chemical analysis was conducted after a series of systematic sampling in various locations. The statistical tools for water quality indexing including water quality indexing (WQI), heavy metal pollution indexing (HMPI), pollution indexing (PI), overall pollution indexing (OPI), metal indexing (MI), and hazard indexing (HI) were used for data as well as the health hazard analysis through water pathway. Overall, 40 water samples were taken from the public water supply systems covering winter and summer seasons, and the levels of pH, total dissolved solids (TDS), EC, F- , Cl- , NO3 - , SO4 2- , HCO3 - , As, B, Cd, Co, Pb, Zn, Cr, Fe, and Mn were investigated. The weight arithmetic index method was used for WQI, and water pollution indices such as HMPI, PI, OPI, and MI were calculated using different models to check the severity of contamination. The mean hazard quotient and hazard index values calculated using the concentration levels of As, B, Cd, Co, Pb, Cr, Fe, Mn, Zn, F- , and NO3 - reveal that supply water may pose a significant health risk to both adults and children that further varies with temporal and spatial changes. During both seasons, a high carcinogenic risk for both adults and children was observed in the studied area because of high levels of As, Pb, Cd, and NO3 - . PRACTITIONER POINTS: The quality of public supply water was assessed at the selected sites of Ambala, India. High levels of NO3 - , As, Cd, and Pb were observed posing a health risk to adults and children via water pathway. 95% of the samples qualified for the excellent water quality category with respect to the levels of F- , Cl- , NO3 - , SO4 2- , HCO3 - , pH, EC, and TDS. Statistical analysis (HMPI, PI, MI, OPI, HI) using different models revealed water contamination with reference to the levels of NO3 - , As, Pb, Cr, Ni, and Cd. Immediate measures are needed to uphold the safety and health of the natives.

The M. tuberculosis Rv1523 Methyltransferase Promotes Drug Resistance Through Methylation-Mediated Cell Wall Remodeling and Modulates Macrophages Immune Responses.

The acquisition of antibiotics resistance is a major clinical challenge limiting the effective prevention and treatment of the deadliest human infectious disease tuberculosis. The molecular mechanisms by which initially Mycobacterium tuberculosis (M.tb) develop drug resistance remain poorly understood. In this study, we report the novel role of M.tb Rv1523 MTase in the methylation of mycobacterial cell envelope lipids and possible mechanism of its contribution in the virulence and drug resistance. Initial interactome analyses predicted association of Rv1523 with proteins related to fatty acid biosynthetic pathways. This promoted us to investigate methylation activity of Rv1523 using cell wall fatty acids or lipids as a substrate. Rv1523 catalyzed the transfer of methyl group from SAM to the cell wall components of mycobacterium. To investigate further the in vivo methylating role of Rv1523, we generated a recombinant Mycobacterium smegmatis strain that expressed the Rv1523 gene. The M. smegmatis strain expressing Rv1523 exhibited altered cell wall lipid composition, leading to an increased survival under surface stress, acidic condition and resistance to antibiotics. Macrophages infected with recombinant M. smegmatis induced necrotic cell death and modulated the host immune responses. In summary, these findings reveal a hitherto unknown role of Rv1523 encoded MTase in cell wall remodeling and modulation of immune responses. Functional gain of mycolic acid Rv1523 methyltransferase induced virulence and resistance to antibiotics in M. smegmatis. Thus, mycolic acid methyltransferase may serve as an excellent target for the discovery and development of novel anti-TB agents.

PGRS Domain of Rv0297 of Mycobacterium tuberculosis Is Involved in Modulation of Macrophage Functions to Favor Bacterial Persistence.

Mycobacterium tuberculosis (M. tb) Rv0297-encoded PE_PGRS5 has been known to be expressed at the later stages of infection and in acidified phagosomes during transcriptome and proteomic studies. The possible role of Rv0297 in the modulation of phagosomal maturation and in providing protection against a microbicidal environment has been hypothesized. We show that Rv0297PGRS is involved in modulating the calcium homeostasis of macrophages followed by impedance of the phagolysosomal acidification process. This is evident from the downregulation of the late endosomal markers (Rab7 and cathepsin D) in the macrophages infected with recombinant Mycobacterium smegmatis (rM.smeg)-M.smeg_Rv0297 and M.smeg_Rv0297PGRS-or treated with recombinant Rv0297PGRS protein. Macrophages infected with rM.smeg expressing Rv0297 produce nitric oxide and undergo apoptosis, which may aid in the dissemination of pathogen in the later stages of infection. Rv0297 was also found to be involved in rescuing the bacterium from oxidative and hypoxic stress employed by macrophages and augmented the survivability of the recombinant bacterium. These results attribute to the functional significance of this protein in M.tb virulence mechanism. The fact that this protein gets expressed at the later stages of lung granulomas during M.tb infection suggests that the bacterium possibly employs Rv0297 as its dissemination and survival strategy.

Interaction of Mycobacterium tuberculosis Virulence Factor RipA with Chaperone MoxR1 Is Required for Transport through the TAT Secretion System.

UNLABELLED: Mycobacterium tuberculosis is a leading cause of death worldwide. The M. tuberculosis TAT (twin-arginine translocation) protein secretion system is present at the cytoplasmic membrane of mycobacteria and is known to transport folded proteins. The TAT secretion system is reported to be essential for many important bacterial processes that include cell wall biosynthesis. The M. tuberculosis secretion and invasion protein RipA has endopeptidase activity and interacts with one of the resuscitation antigens (RpfB) that are expressed during pathogen reactivation. MoxR1, a member of the ATPase family that is associated with various cellular activities, was predicted to interact with RipA based on in silico analyses. A bimolecular fluorescence complementation (BiFC) assay confirmed the interaction of these two proteins in HEK293T cells. The overexpression of RipA in Mycobacterium smegmatis and copurification with MoxR1 further validated their interaction in vivo. Recombinant MoxR1 protein, expressed in Escherichia coli, displays ATP-enhanced chaperone activity. Secretion of recombinant RipA (rRipA) protein into the E. coli culture filtrate was not observed in the absence of RipA-MoxR interaction. Inhibition of this export system in M. tuberculosis, including the key players, will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing ß-lactam antibiotics, opening up new candidates for drug repurposing. IMPORTANCE: The virulence mechanism of mycobacteria is very complex. Broadly, the virulence factors can be classified as secretion factors, cell surface components, enzymes involved in cellular metabolism, and transcriptional regulators. The mycobacteria have evolved several mechanisms to secrete its proteins. Here, we have identified one of the virulence proteins of Mycobacterium tuberculosis, RipA, possessing peptidoglycan hydrolase activities secreted by the TAT secretion pathway. We also identified MoxR1 as a protein-protein interaction partner of RipA and demonstrated chaperone activity of this protein. We show that MoxR1-mediated folding is critical for the secretion of RipA within the TAT system. Inhibition of this export system in M. tuberculosis will prevent localization of peptidoglycan hydrolase and result in sensitivity to existing ß-lactam antibiotics, opening up new candidates for drug repurposing.

Comparison of extramedullary and intramedullary devices for treatment of subtrochanteric femoral fractures at tertiary level center.

OBJECTIVE: The treatment of subtrochanteric fractures is challenging and treatment modalities and implants are constantly evolving. This study attempts to revisit and compare extramedullary vs. intramedullary devices in relatively young population. METHODS: Thirty patients with subtrochanteric fractures were enrolled and treated with extramedullary or intramedullary devices and follow-up continued one year for clinico-radiological assessment. RESULTS: The mean age of patients was 37.53 years. Most were males between 21-40 years. The dominant mode of injury was traffic accidents (66%). Fractures were classified according to Russell-Taylor classification. Forty percent were Russell-Taylor type IA, 37% type IB and 23% type IIA. Average time to surgery was 3.6 days from the time of admission to hospital. Mean duration of surgery was 45 minutes for intramedullary device (group A) and 105 minutes for extramedullary device (group B). Average blood loss was 100 ml in group A and 200 ml in group B. Mean duration of radiation exposure was 130 seconds and 140 seconds for groups A and B, while average duration of hospital stay was 12 days and 16 days respectively. Excellent results were seen in 47% of cases in group A and 33% of cases in group B. CONCLUSION: Intramedullary device is a reliable implant for subtrochanteric fractures. It has high rates of union with minimal soft-tissue damage. Intramedullary fixation has biological and biomechanical advantages, but surgery is technically demanding. Gradual learning and patience is needed to make this method truly rewarding.

Expression, purification, and characterization of pro-phenoloxidase-activating serine protease from Spodoptera litura.

One of the important trigger molecules for innate immunity is a serine protease that activates zymogen phenol oxidase (PPO). Central to wound healing response is the activation of phenol oxidase zymogen. Molecular characterization of phenol oxidase has been recently reported by us. Here, we report isolation, cloning, expression, and purification of prophenol oxidase activating enzyme 1 (slppae1) from polyphagous pest, Spodoptera litura. SLPPAE1 is induced within 6 h of physical injury. The structural features of the mature polypeptide are reminiscent of other lepidopteran PPAE in having a signal peptide, propeptide, and catalytically active polypeptide. The cDNA has been expressed in Sf21 cells using baculovirus expression vector. Fractionation of expressing Sf21 cells revealed its expression in the membranes. The recombinant protein was solubilized from membranes and purified by Ni-NTA affinity chromatography. The purified enzyme is catalytically active on chromogenic substrate, activates recombinantly expressed prophenol oxidase (PPO) of S. litura, and is sensitive to inhibition by aprotenin. N-terminal sequencing of processed phenol oxidase revealed 11 kDa propeptide instead of in-silico predicted 6 kDa polypeptide.

Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin.

The bacterium Bacillus thuringiensis produces ICPs (insecticidal crystal proteins) that are deposited in their spore mother cells. When susceptible lepidopteran larvae ingest these spore mother cells, the ICPs get solubilized in the alkaline gut environment. Of approx. 140 insecticidal proteins described thus far, insecticidal protein Cry1Ac has been applied extensively as the main ingredient of spray formulation as well as the principal ICP introduced into crops as transgene for agricultural crop protection. The 135 kDa Cry1Ac protein, upon ingestion by the insect, is processed successively at the N- and C-terminus by the insect midgut proteases to generate a 65 kDa bioactive core protein. The activated core protein interacts with specific receptors located at the midgut epithilium resulting in the lysis of cells and eventual death of the larvae. A laboratory-reared population of Helicoverpa armigera displayed 72-fold resistance to the B. thuringiensis insecticidal protein Cry1Ac. A careful zymogram analysis of Cry1Ac-resistant insects revealed an altered proteolytic profile. The altered protease profile resulted in improper processing of the insecticidal protein and as a consequence increased the LC50 concentrations of Cry1Ac. The 135 kDa protoxin-susceptible insect larval population processed the protein to the biologically active 65 kDa core protein, while the resistant insect larval population yielded a mixture of 95 kDa and 68 kDa Cry1Ac polypeptides. N-terminal sequencing of these 95 and 68 kDa polypeptides produced by gut juices of resistant insects revealed an intact N-terminus. Protease gene transcription profiling by semi-quantitative RT (reverse transcription)-PCR led to the identification of a down-regulated HaSP2 (H. armigera serine protease 2) in the Cry1Ac-resistant population. Protease HaSP2 was cloned, expressed and demonstrated to be responsible for proper processing of insecticidal protoxin. The larval population displaying resistance to Cry1Ac do not show an altered sensitivity against another insecticidal protein, Cry2Ab. The implications of these observations in the context of the possibility of development of resistance and its management in H. armigera to Cry1Ac through transgenic crop cultivation are discussed.

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite.

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.