Meta-analysis on Plasmodium falciparum sulfadoxine-pyrimethamine resistance-conferring mutations in India identifies hot spots for genetic surveillance.

BACKGROUND: India is on track to eliminate malaria by 2030 but emerging resistance to first-line antimalarials is a recognised threat. Two instances of rapid development, spread, and natural selection of drug-resistant mutant parasites in India (chloroquine across the country and artesunate + sulfadoxine-pyrimethamine [AS+SP] in the northeastern states) translated into drug policy changes for Plasmodium falciparum malaria in 2010 and 2013, respectively. Considering these rapid changes in the SP drug resistance-conferring mutation profile of P. falciparum, there is a need to systematically monitor the validated mutations in Pfdhfr and Pfdhps genes across India alongside AS+SP therapeutic efficacy studies. There has been no robust, systematic countrywide surveillance reported for these parameters in India, hence the current study was undertaken. METHODS: Studies that reported data on WHO-validated SP resistance markers in P. falciparum across India from 2008 to January 2023 were included. Five major databases, PubMedⓇ, Web of ScienceTM, ScopusⓇ, EmbaseⓇ, and Google Scholar, were exhaustively searched. Individual and pooled prevalence estimates of mutations were obtained through random- and fixed-effect models. Data were depicted using forest plots created with a 95% confidence interval. The study is registered with PROSPERO (CRD42021236012). RESULTS: A total of 37 publications, and 533 Pfdhfr and 134 Pfdhps National Centre of Biotechnology Information (NCBI) DNA sequences were included from >4000 samples. The study included information from 80 districts, 21 states and 3 union territories (UTs) from India. The two PfDHFR mutations, C59R (62%) and S108N (74%), were the most prevalent mutations (pooled estimates 61% and 71%, respectively) and appeared to be stabilised/fixed. Although rarest overall, the prevalence of I164L was observed to be as high as 32%. The PfDHFR double mutants were the most prevalent overall (51%; pooled 42%). The prevalence of triple and quadruple mutations was 6% and 5%, respectively, and is an immediate concern for some states. The most prevalent PfDHPS mutation was A437G (39%), followed by K540E (25%) and A581G (12%). There was a low overall prevalence of PfDHFR/PfDHPS quintuple and sextuple mutations but surveillance for these mutations is critical for some areas. CONCLUSION: The analyses span the two critical policy changes, highlight the areas of concern, and guide policymakers in strategising and refining the anti-malaria drug policy for malaria elimination. The results of the analyses also highlight the SP-resistance hot spots, critical gaps and challenges, and indicate that focal and local malaria genetic surveillance (including drug-resistance markers) is needed until malaria is successfully eliminated.

Exploring natural products as multi-target-directed drugs for Parkinson's disease: an in-silico approach integrating QSAR, pharmacophore modeling, and molecular dynamics simulations.

Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the midbrain. Current treatments provide limited symptomatic relief without halting disease progression. A multi-targeting approach has shown potential benefits in treating neurodegenerative diseases. In this study, we employed in silico approaches to explore the COCONUT natural products database and identify novel drug candidates with multi-target potential against relevant Parkinson's disease targets. QSAR models were developed to screen for potential bioactive molecules, followed by a hybrid virtual screening approach involving pharmacophore modeling and molecular docking against MAO-B, AA2AR, and NMDAR. ADME evaluation was performed to assess drug-like properties. Our findings revealed 22 candidates that exhibited the desired pharmacophoric features. Particularly, two compounds: CNP0121426 and CNP0242698 exhibited remarkable binding affinities, with energies lower than -10 kcal/mol and promising interaction profiles with the chosen targets. Furthermore, all the ligands displayed desirable pharmacokinetic properties for brain-targeted drugs. Lastly, molecular dynamics simulations were conducted on the lead candidates, belonging to the dihydrochalcone and curcuminoid class, to evaluate their stability over a 100 ns timeframe and compare their dynamics with reference complexes. Our findings revealed the curcuminoid CNP0242698 to have an overall better stability with the three targets compared to the dihydrochalcone, despite the high ligand RMSD, the curcuminoid CNP0242698 showed better protein stability, implying ligand exploration of different orientations. Similarly, AA2AR exhibited higher stability with CNP0242698 compared to the reference complex, despite the high initial ligand RMSD due to the bulkier active site. In NMDAR, CNP0242698 displayed good stability and less fluctuations implying a more restricted conformation within the smaller active site of NMDAR. These results may serve as lead compounds for the development and optimization of natural products as multi-target disease-modifying natural remedies for Parkinson's disease patients. However, experimental assays remain necessary to validate these findings.Communicated by Ramaswamy H. Sarma.

Antibody Responses Against Plasmodium falciparum MSP3 Protein During Natural Malaria Infection in Individuals Living in Malaria-Endemic Regions of India.

The humoral immune responses to blood-stage malaria proteins are requisite for the inhibition of parasite invasion. Plasmodium falciparum merozoite surface protein 3 (MSP3) is a secretory, expressed abundantly, merozoite surface protein that is important for the parasite invasion process. It has been shown to induce antibody responses during natural infections and is, therefore, considered to be the potential vaccine candidates against Plasmodium. Elucidating the immunogenicity and prevalence of anti-parasite antibodies is important in identifying potential targets as candidates for malarial diagnosis and anti-malarial vaccine. The present study concerns the presence of antibodies against the MSP3 proteins of human malaria parasite- P. falciparum in infected individuals from endemic regions of India. Seventy-one anonymized P. falciparum infected serum samples were procured from the malaria fever clinic of ICMR-National Institute of Malaria Research (NIMR), New Delhi to detect the presence of antibodies against MSP3 protein by ELISA. The IgM antibody response against recombinant MSP3 was detected at significantly higher levels during acute malaria. The protein was found to be immunogenic and did not demonstrate any cross-reactivity with the serum of uninfected individuals or individuals infected with other Plasmodium species. The protein has hydrophilic regions in its N- and C-terminus which may contain immunogenic linear and conformational B-cell epitopes. The results from this study suggest that the MSP3 is immunogenic and likely a potential candidate for antibody-based diagnosis or vaccine development against the blood-stage of P. falciparum.

Glutamate dehydrogenase: a novel candidate to diagnose Plasmodium falciparum through rapid diagnostic test in blood specimen from fever patients.

In recent years, Plasmodium falciparum histidine-rich protein 2 gene deletion has been reported in India. Such isolates are prone to selective transmission and thus form a challenge to case management. As most of the rapid malaria diagnostic tests are based on the detection of HRP2 protein in the blood, we attempted to use Glutamate Dehydrogenase (GDH) as a biomarker for the diagnosis of P. falciparum. Recombinant PfGDH was successfully cloned, expressed and purified using the Ni-NTA approach. Polyclonal antibodies were raised against full-length rPfGDH and its peptides. Antibodies for rPfGDH showed a strong immune response against the recombinant protein. However, antibody showed no affinity towards the peptides, which suggests they failed as antigen. Antibodies for rPfGDH significantly detected the GDH in human blood specimens. This is the first report where P. falciparum GDH was detected in malaria cases from various parts of India. The raised polyclonal antibodies had shown an affinity for PfGDH in quantitative ELISA and are capable to be exploited for RDTs. This research needs further statistical validation on a large number and different sample types from candidates infected with P. falciparum and other species.

Insights into the early liver stage biology of Plasmodium.

Even though malaria is preventable and curable, it has become a serious threat to mankind. In 2016, there were an estimated 216 million cases of malaria across the world. The biology of its causative agent, i.e. Plasmodium parasite is full of complex mechanisms. There are five Plasmodium species responsible for malaria in humans, viz. Plasmodium falciparum, P. vivax, P. malariae, P. ovale and recently identified P. knowlesi that normally infect apes. In humans, malaria is spread by the injection of Plasmodium sporozoites through the bite of infectious Anopheles' female mosquito during their blood meal. From the time of entry into human skin till the development into the asexual forms, the parasite undergoes several transformations. This review attempts to understand the science behind the pre-erythrocytic liver stage of Plasmodium. Research articles explaining parasite biology, cell-traversal, transformation stages, cell-egress process, etc. were retrieved from PubMed and google scholar database. Various known and unknown mechanisms and strategies used by the malaria parasite P. berghei in rodent models have been discussed in this review. Limited or no information was available for humans, due to technical feasibility and complexity of parasite's life cycle. Hence, it was concluded that there is an urgent need to investigate the hepatic invasion, traversal and egress mechanism of P. falciparum and P. vivax for developing novel therapeutics to fight against malaria.

High-resolution crystal structure of a polyextreme GH43 glycosidase from Halothermothrix orenii with α-L-arabinofuranosidase activity.

A gene from the heterotrophic, halothermophilic marine bacterium Halothermothrix orenii has been cloned and overexpressed in Escherichia coli. This gene encodes the only glycoside hydrolase of family 43 (GH43) produced by H. orenii. The crystal structure of the H. orenii glycosidase was determined by molecular replacement and refined at 1.10 Šresolution. As for other GH43 members, the enzyme folds as a five-bladed ß-propeller. The structure features a metal-binding site on the propeller axis, near the active site. Based on thermal denaturation data, the H. orenii glycosidase depends on divalent cations in combination with high salt for optimal thermal stability against unfolding. A maximum melting temperature of 76°C was observed in the presence of 4 M NaCl and Mn(2+) at pH 6.5. The gene encoding the H. orenii GH43 enzyme has previously been annotated as a putative α-L-arabinofuranosidase. Activity was detected with p-nitrophenyl-α-L-arabinofuranoside as a substrate, and therefore the name HoAraf43 was suggested for the enzyme. In agreement with the conditions for optimal thermal stability against unfolding, the highest arabinofuranosidase activity was obtained in the presence of 4 M NaCl and Mn(2+) at pH 6.5, giving a specific activity of 20-36 µmol min(-1) mg(-1). The active site is structurally distinct from those of other GH43 members, including arabinanases, arabinofuranosidases and xylanases. This probably reflects the special requirements for degrading the unique biomass available in highly saline aqueous ecosystems, such as halophilic algae and halophytes. The amino-acid distribution of HoAraf43 has similarities to those of mesophiles, thermophiles and halophiles, but also has unique features, for example more hydrophobic amino acids on the surface and fewer buried charged residues.

Biochemical and structural characterization of a thermostable ß-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis.

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial ß-glucosidases (BGL) with significant ß-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific ß-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal ß-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70 °C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are ß-D-Galp-(1→6)-D-Lac (6GALA) and ß-D-Galp-(1→3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophile Halothermothrix orenii.

A ribokinase gene (rbk) from the anaerobic halothermophilic bacterium Halothermothrix orenii was cloned and overexpressed in Escherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 45.6, b = 61.1, c = 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

Expression, purification and preliminary crystallographic analysis of the recombinant ß-glucosidase (BglA) from the halothermophile Halothermothrix orenii.

The ß-glucosidase A gene (bglA) has been cloned from the halothermophilic bacterium Halothermothrix orenii and the recombinant enzyme (BglA; EC 3.2.1.21) was bacterially expressed, purified using metal ion-affinity chromatography and subsequently crystallized. Orthorhombic crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystal structure with two molecules in the asymmetric unit was solved by molecular replacement using a library of known glucosidase structures. Attempts to collect higher resolution diffraction data from crystals grown under different conditions and structure refinement are currently in progress.

Molecular epidemiological characteristics of Shigella spp. isolated from river Narmada during 2005-2006.

Shigellosis is an acute gastroenteritis caused by Shigella species. Forty isolates of Shigella spp. were obtained from the river Narmada during 2005-2006. Twenty-three isolates were identified as S. flexneri, 10 as S. sonnei, and seven as S. dysenteriae on the basis of biochemical tests and serotyping. All the isolates harbored at least one plasmid (range: 1-4) and exhibited 12 distinct plasmid profile patterns. Out of 40 isolates, 90% were found to be resistant against more than two antibiotics. All of the isolates were resistant to ciprofloxacin. It is noteworthy that all of the S. dysenteriae strains were resistant to chloramphenicol and trimethoprim, and that all of the S. flexneri and S. sonnei strains were resistant to cephotaxime, amikacin, and norfloxacin, which can be used for the treatment of shigellosis. Forty-two and a half percent of Shigella isolates were found to be Congo red positive. Since the Congo red binding test is cheap and simple, it can be used to determine the virulence properties of Shigella species. We could not find a specific correlation between serotype, antimicrobial resistance, and plasmid profile.