Characterisation of Anopheles species composition and genetic diversity in Meghalaya, northeast India, using molecular identification tools.

Malaria in India is declining, in part due to the use of long-lasting insecticide-treated nets (LLINs) and vector control. Historically, the north-eastern region of India has contributed ~10%-12% of the nation's malaria burden. The important mosquito vectors in northeast India have long been considered to be Anopheles baimaii and An. minimus, both associated with forest habitats. Local deforestation and increased rice cultivation, along with widespread LLIN use, may be changing vector species composition. Understanding if and how vector species composition is changing is critical to successful malaria control. In Meghalaya state, malaria is now at a low level of endemicity with occasional seasonal outbreaks. In a biodiverse setting like Meghalaya, where >24 Anopheles mosquito species have been recorded, accurate morphological identification of all species is logistically challenging. To accurately determine Anopheles species richness in the West Khasi Hills (WKH) and West Jaintia Hills (WJH) districts, adult and larval mosquitoes were collected and identified using molecular methods of allele-specific PCR and cytochrome oxidase I DNA barcoding. In 14 villages across both districts, we identified high species richness, 19 species in total. Molecular findings indicated that An. minimus and An. baimaii were rare, while four other species (An. maculatus, An. pseudowillmori, An. jeyporiensis and An. nitidus) were abundant. Anopheles maculatus was highly prevalent in WKH (39% of light trap collections) and An. pseudowillmori in WJH (45%). Larvae of these four species were found in rice fields, suggesting that land cover change is influencing species composition change. Our results suggest that rice fields might be contributing to the observed abundance of An. maculatus and An. pseudowillmori, which could be playing a role in malaria transmission, either independently due to their high abundance, or in combination with An. baimaii and/or An. minimus.

How can the complex epidemiology of malaria in India impact its elimination?

Malaria is a human health hazard in the tropical and subtropical zones of the globe and is poised to be eliminated by the year 2030. Despite a decrease in incidence in the past two decades, many endemic countries, including India, report cases regularly. The epidemiology of malaria in India is unique owing to several features of the Plasmodium parasites, Anopheles vectors, ecoepidemiological situations conducive to disease transmission, and susceptible humans living in rural and forested areas. Limitations in public health reach, and poor health-seeking behaviour of vulnerable populations living in hard-to-reach areas, add to the problem. We bring all of these factors together in a comprehensive framework and opine that, in spite of complexities, targeted elimination of malaria in India is achievable with planned programmatic approaches.

Molecular surveillance of anti-malarial drug resistance genes in Plasmodium falciparum isolates in Odisha, India.

BACKGROUND: Despite significant progress in eliminating malaria from the state of Odisha, India, the disease is still considered endemic. Artesunate plus sulfadoxine-pyrimethamine (AS + SP) has been introduced since 2010 as first-line treatment for uncomplicated Plasmodium falciparum malaria. This study aimed to investigate the prevalence of mutations associated with resistance to chloroquine (CQ), sulfadoxine-pyrimethamine (SP), and artesunate (ART) in P. falciparum parasites circulating in the state. METHODS: A total of 239 isolates of P. falciparum mono infection were collected during July 2018-November 2020 from the four different geographical regions of the state. Genomic DNA was extracted from 200 µL of venous blood and amplified using nested polymerase chain reaction. Mutations on gene associated with CQ (Pfcrt and Pfmdr1) were assessed by PCR amplification and restriction fragment length polymorphism, artemisinin (Pfk13) gene by DNA sequencing and SP (Pfdhfr and Pfdhps) genes by allele-specific polymerase chain reaction (AsPCR). RESULTS: The point mutation in Pfcrt (K76T) was detected 2.1%, in Pfmdr1 (N86Y) 3.4%, and no mutations were found in Pfkelch13 propeller domain. Prevalence of Pfdhfr, Pfdhps and Pfhdfr-Pfdhps (two locus) gene mutations were 50.43%, 47.05% and 49.79% respectively. The single, double, triple and quadruple point mutations in Pfdhfr gene was 11.2%, 8.2%, 17.2% and 3.4% while, in Pfdhps gene was 10.9%,19.5%, 9.5% and 2.7% respectively. Of the total 13 haplotypes found in Pfdhfr, 8 were detected for the first time in the state and of the total 26 haplotypes found in Pfdhps, 7 were detected for the fisrt time in the state. The linked quintuple mutation Pfdhfr (N51I-C59R-S108N)-Pfdhps (A437G-K540E) responsible for clinical failure (RIII level of resistance) of SP resistance and A16V-S108T mutation in Pfdhfr responsible for cycloguanil was absent. CONCLUSION: The study has demonstrated a low prevalence of CQ resistance alleles in the study area. Despite the absence of the Pfkelch13 mutations, high prevalence of Pfdhfr and Pfdhps point mutations undermine the efficacy of SP partner drug, thereby threatening the P. falciparum malaria treatment policy. Therefore, continuous molecular and in vivo monitoring of ACT efficacy is warranted in Odisha.

Time for Isoniazid Pharmacogenomic-Guided Therapy of Tuberculosis Based on NAT2 Acetylation Profiles in India.

Tuberculosis (TB), caused by a bacterial pathogen Mycobacterium tuberculosis, is a highly contagious infectious disease. India ranks top in TB burden among other countries in the world. The current treatment of TB in India includes isoniazid (INH) as the first-line drug, based on the "one dose fits all'' concept. It is widely known that INH is not equally tolerated by all patients, as the metabolization process of INH is highly dependent on the acetylation profile of an individual based on the pharmacogenomic profile of the N-acetyltransferase (NAT2) gene. Also, several experimental studies in several TB endemic countries have established that redosing of INH based on genetic profiles of TB patients of the NAT2 can help in effective TB treatment and minimize adverse drug reactions (ADRs). Moreover, acetylation phenotype-based INH dosing has been shown to be cost-effective as well as successful in terms of treatment outcomes and the increase in the quality of life of the patients. Considering a genetically heterogenous population with high vulnerability to TB, we here argue that India could lead in establishing a pharmacogenomic-guided therapy (PGT) under the INH-NAT2 model. With such an approach, not only could an innovative step towards 'End-TB' by the year 2025 be taken but a personalized medicine approach for TB treatment be initiated in India, particularly in tribal communities.

High cumulative risk of colorectal cancers and desmoid tumours and fibromatosis in South Asian APC mutation carriers.

Management of familial adenomatous polyposis (FAP) is guided by the cumulative risk of colorectal cancer (CRC) and aggressive fibromatosis/desmoid (AF/D). The first non-Caucasian FAP cohort with cumulative risk estimates for CRC and AF/D shows distinct differences with the Caucasian and other Asian cohorts. The strong correlation between the adenomatous polyposis coli (APC) mutation location with the FAP phenotype and the geoethnic differences in APC mutation spectrum, genetic constitution, lifestyle and sporadic CRC rates, mandates the use of population-specific cumulative risk estimates for CRC and desmoid for counselling and risk management. On genotype-phenotype correlation in 83 individuals with classical FAP and a confirmed pathogenic/likely Pathogenic (P/LP) APC variant (n=76) or obligate carrier of the family variant (n=7), we observed a high cumulative CRC risk of 40% and 85% by 40 and 60 years, respectively. The observed 30% cumulative risk by 50 years for desmoids was higher than previous European and Asian cohorts and was significantly associated with prophylactic surgery (OR: 4.58, 95% CI 1.06 to 19.78) and APC mutation 3' of codon 1309 (OR: 13.07, 95% CI 3.58 to 47.56) and also 3' of codon 1444 (OR: 8.0, 95% CI 1.83 to 34.94). Global cooperation is required to establish FAP genotype-phenotype associations and population-specific risk estimates to guide genetic counselling and risk management.

Curcumin restores the engraftment capacity of aged hematopoietic stem cells and also reduces PD-1 expression on cytotoxic T cells.

Aging affects the functionality of hematopoietic stem cells (HSCs), and therefore, aged individuals are not preferred as donors in HSC transplantation. Such elimination leads to the restriction of donor cohort. Several efforts are being done to rejuvenate aged HSCs. Here, we show that treatment of aged mice with curcumin rejuvenates their HSCs by restoring the expression of autophagy-inducing messenger RNAs in them, and improves their engraftment capacity. Importantly, we show that curcumin is effective in rejuvenation of HSCs when administered via both, intraperitoneal as well as oral routes. Aging also affects the immune system. While elderly individuals are not immuno-deficient, they do not respond optimally to immunizations, and hence, a strategy needs to be developed to make them immunologically responsive. Programmed cell death 1 (PD-1), one of the inhibitory coreceptors, plays an important role in the regulation of autoimmunity, infectious immunity, and cancer immunity. Its expression on T cells is indicative of their exhaustion. Here, we show that curcumin reduces the frequency of PD1+ cytotoxic T cells in the spleens of aged mice. Curcumin has a proven safety profile, and hence, can be used to treat aged donors to boost the functionality of their HSCs and also to improve the immunological profile of aged individuals. These data could have implications in various other regenerative medicine protocols as well.

Establishment of human iPSC line from patient of Indian ethnicity carrying homozygous CD8/9 (+G) beta thalassemia mutation.

This study shows generation of iPSCs from peripheral blood mononuclear cells (PBMNCs) of a male patient having homozygous CD 8/9 (+G) beta thalassemia (major) mutation. Cells were nucleofected with episomal vectors containing Oct4, Sox2, L-Myc, Lin28, Klf4 and p53DD (dominant negative p53 mutation). Cell line exhibited presence of pluripotency markers by immunofluorescence, flow-cytometry and PCR. The plasmids were lost from cells by subsequent passages, observed by PCR. Karyotype analysis demonstrated a stable genome. The cells had capability to differentiate into three-germ lineages in vitro. This iPSC line can be used as a tool for drug design and gene therapy studies.

Transforming growth factor ß1 accelerates and enhances in vitro red blood cell formation from hematopoietic stem cells by stimulating mitophagy.

BACKGROUND: Generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) in vitro takes about 21 days, making it unaffordable for clinical applications. Acceleration of the in vitro erythropoiesis process by using small molecules could eventually make the large-scale production of these cells commercially viable. Transforming Growth Factor ß1 (TGF-ß1) has been shown to have a dose-dependent activity on the HSCs: at high concentration it inhibits, whereas at low concentration it stimulates the HSCs growth. At high concentration, it also inhibits erythropoiesis but accelerates terminal erythroid differentiation of cell lines and erythroid progenitors. Here we examined whether the use of low concentration of TGF-ß1 would be beneficial for increasing RBC production by stimulating HSC growth and also supporting erythroid differentiation. Such a strategy could make RBC production in vitro more efficient and cost-effective for clinical applications. METHODS: HSCs isolated from Apheresis samples were differentiated into mature RBCs by the sequential addition of specific combinations of growth factors for 21 days. In the control set, only EPO (3 IU/ml) was added whereas, in the test set, TGF-ß1 at a concentration of 10 pg/ml was added along with EPO (3 IU/ml) from day 0. RESULTS: We found that a low concentration of TGF-ß1 has no inhibitory effect on the proliferation of the early stages of erythropoiesis. Additionally, it significantly accelerates terminal stages of erythroid differentiation by promoting BNIP3L/NIX-mediated mitophagy. CONCLUSIONS: Incorporation of TGF-ß1 at 10 pg/ml concentration in the differentiation medium accelerates the in vitro erythropoiesis process by 3 days. This finding could have potential applications in transfusion medicine.

Neutrophils generated in vitro from hematopoietic stem cells isolated from apheresis samples and umbilical cord blood form neutrophil extracellular traps.

Neutrophils release neutrophil extracellular traps (NET) comprising of decondensed chromatin that immobilizes and kills pathogens. In vitro generation of neutrophils on a large scale from hematopoietic stem cells (HSCs) may be a useful strategy for treating neutropenic patients in future, though it is not in clinical practice yet. Microbial infections lead to major cause of morbidity and mortality in these patients. Despite the importance of NET in preventing infection, efficacy of in vitro-generated neutrophils from HSCs to form NET is not tested. We show that functional neutrophils could be generated in vitro from HSCs/MNCs isolated from umbilical cord blood (UCB) and apheresis-derived peripheral blood (APBL). Neutrophils generated from UCB showed properties comparable to those isolated from peripheral blood. We also show that isolation of HSCs is not absolutely essential for in vitro neutrophil generation. Further, we show that neutrophils generated from HSCs express PADI4 enzyme and their NET-forming ability is comparable to peripheral blood neutrophils. Taken together, our data show that fully functional neutrophils can be generated in vitro from HSCs. NET-forming ability of in vitro-generated neutrophils is an important parameter to determine their functionality and thus, should be studied along with other standard functional assays.

Can the personalized medicine approach contribute in controlling tuberculosis in general and India in particular?

Poor drug compliance and drug-resistant Mycobacterium tuberculosis are the two principal obstacles in controlling tuberculosis (TB) in endemic regions including India, which has contributed the most to global TB burden. We argue here that a personalized medicine approach, to start with the N-acetyl transferase-2-isoniazid (NAT2-INH) model, could be a step forward in dealing with both these limitations in controlling TB in India.

Catalase incorporation in freezing mixture leads to improved recovery of cryopreserved iPSC lines.

Among the various types of stem cells, induced pluripotent stem cells (iPSCs) have gained much attention due to their pluripotent nature. iPSCs help us to understand the processes that regulate pluripotency and specialization. However, in order to use them in various applications in regenerative medicine, their efficient cryopreservation and recovery after the freezing injury is critical. Here we have used an antioxidant catalase, as an additive to the conventional freezing mixture containing 50% FBS and 10% DMSO. The hiPSCs were frozen as aggregates by using a programmable freezer and then stored in liquid nitrogen at -196 °C. It was seen that catalase improved the revival efficiency by reducing the late apoptotic populations and increasing the live cell fraction. Catalase also retained the pluripotent nature of iPSCs in a better way post revival. This improvement could be attributed to reduction of total ROS and apoptosis, which are the two main factors that cause damage during freezing. Our data suggest that catalase could be a useful additive while freezing hiPSCs.

Freezing of dendritic cells with trehalose as an additive in the conventional freezing medium results in improved recovery after cryopreservation.

BACKGROUND: Dendritic cell (DC) vaccination involves administration of multiple doses. Cryopreservation of tumor antigen-pulsed DCs can provide a ready to use vaccine source and eliminate the need of frequent withdrawal of the patient's blood for vaccine preparation. The aim of this study was to assess the effect of addition of trehalose in the freezing medium on the recovery of DCs after cryopreservation. STUDY DESIGN AND METHODS: DCs were generated from mononuclear cells from apheresis samples of healthy donors. For long-term storage of 6 months, cells were frozen with a rate-controlled programmable freezer and stored in liquid nitrogen. For short-term storage of 1 month, cells were frozen and stored at -80°C. DCs frozen with Iscove's Modified Dulbecco's Medium + 10% dimethyl sulfoxide + 20% fetal bovine serum served as the control group, while the test group was additionally supplemented with 50 µg/mL of trehalose. After revival of control and test DCs, they were assessed for viability, morphology, phenotype, and functions. RESULTS: The addition of trehalose to the conventional freezing medium helped to preserve the viability and functionality of DCs better than dimethyl sulfoxide alone in both long- and short-term cryopreservation. Trehalose also protected the mitochondrial membrane potential and cytoskeleton integrity of DCs, which are necessary for their functionality. Mediators of the intrinsic apoptotic pathway like Caspase-9 and Bim-1 were found to be low in the test. CONCLUSION: Supplementation of conventional freezing medium with trehalose results in better quality of DCs revived after cryopreservation. This finding could help improve DC vaccine preparation for cancer immunotherapy.

Derivation of human iPSC line NCCSi002-A from umbilical cord blood (UCB) CD34+cells of donor from Indian ethnicity.

We discuss the reprogramming of CD34+ cells isolated from UCB of a healthy female child of Indian ethnicity. The CD34+cells were nucleofected using episomal vectors expressing Oct4, Sox2, L-Myc, Klf4, Lin28 and p53DD (negative mutation in p53). The colonies were stained for alkaline phosphatase and evaluated for pluripotency marker expression by PCR, immunofluorescence and flow-cytometry. The safety of cells was confirmed by absence of plasmid in subsequent passages by PCR. G-banded karyotype demonstrated a stable genome. The ability of tri-lineage differentiation was confirmed by specific marker expression by immunofluorescence invitro and teratoma formation invivo.

Novel mutations and phenotypic associations identified through APC, MUTYH, NTHL1, POLD1, POLE gene analysis in Indian Familial Adenomatous Polyposis cohort.

Colo-Rectal Cancer is a common cancer worldwide with 5-10% cases being hereditary. Familial Adenomatous Polyposis (FAP) syndrome is due to germline mutations in the APC or rarely MUTYH gene. NTHL1, POLD1, POLE have been recently reported in previously unexplained FAP cases. Unlike the Caucasian population, FAP phenotype and its genotypic associations have not been widely studied in several geoethnic groups. We report the first FAP cohort from South Asia and the only non-Caucasian cohort with comprehensive analysis of APC, MUTYH, NTHL1, POLD1, POLE genes. In this cohort of 112 individuals from 53 FAP families, we detected germline APC mutations in 60 individuals (45 families) and biallelic MUTYH mutations in 4 individuals (2 families). No NTHL1, POLD1, POLE mutations were identified. Fifteen novel APC mutations and a new Indian APC mutational hotspot at codon 935 were identified. Eight very rare FAP phenotype or phenotypes rarely associated with mutations outside specific APC regions were observed. APC genotype-phenotype association studies in different geo-ethnic groups can enrich the existing knowledge about phenotypic consequences of distinct APC mutations and guide counseling and risk management in different populations. A stepwise cost-effective mutation screening approach is proposed for genetic testing of south Asian FAP patients.

Implication of nanofibers in oral drug delivery.

Nanofibers has gained significant prominence in recent years due to its wide applications in medicinal pharmacy, textile, tissue engineering and in various drug delivery system. In oral drug delivery system (DDS), nanofibers can be delivered as Nanofiber scaffolds, electrosponge nanofibers as oral fast delivery system, multilayered nanofiber loaded mashes, surface modified cross-linked electrospun nanofibers. Nanofibers are of 50- 1000 nm size fibres having large surface area, high porosity, small pore size, low density. Various approaches for formulation of nanofibers are molecular assembly, thermally induced phase separation, electrospining. Most commonly used by using electrospining polymer nanofibres with different range can be produced collective usage of electro spinning with pharmaceutical polymers offers novel tactics for developing drug delivery system (DDS). Different polymers used in preparation of nanofibers include biodegradable hydrophilic polymers, hydrophobic polymers and amphiphilic polymers. Electrospun nanofibers are often used to load insoluble drugs for enhancing their dissolution properties due to their high surface area per unit mass. Besides the water insoluble drugs freely water soluble sodium can also spun into the fibers. The most commonly polymers used for nanofibers are gelatin, dextran, nylon, polystyrene, polyacrylonitrile, polycarbonate, polyimides, poly vinyl alchol, polybenzimidazole. Delivery systems reviewed rely on temporal control, changes in pH along the GIT, the action of local enzymes to trigger drug release, and changes in intraluminal pressure. Dissolution of enteric polymer coatings due to a change in local pH and reduction of azo-bonds to release an active agent are both used in commercially marketed products. In vitro and in vivo studies have demonstrated that the release rates of drugs from these nanofiber formulations are enhanced compared to those from original drug substance. This review is focused on the different type of polymers used, different used in the preparation of nanofibers, cytotoxicity studies and application of nanofiber by using oral drug delivery.

Role of polymer-drug conjugates in organ-specific delivery systems.

Polymers have been utilized to deliver the drug to targeted site in controlled manner, achieving the high-therapeutic efficacy. Polymeric drug conjugates having variable ligands as attachments have been proved to be biodegradable, stimuli sensitive and targeted systems. Numerous polymeric drug conjugates having linkers degraded by acidity or intracellular enzymes or sensitive to over expressed groups of diseased organ/tissue have been synthesized during last decade to develop targeted delivery systems. Most of these organs have number of receptors attached with different cells such as Kupffer cells of liver have mannose-binding receptors while hepatocytes have asialoglycoprotein receptors on their surface which mainly bind with the galactose derivatives. Such ligands can be used for achieving high targeting and intracellular delivery of the drug. This review presents detailed aspects of receptors found in different cells of specific organ and ligands with binding efficiency to these specific receptors. This review highlights the need of further studies on organ-specific polymer-drug conjugates by providing detailed account of polymeric conjugates synthesized till date having organ-specific targeting.

Aflatoxins in corn and rice sold in Lahore, Pakistan.

A survey was conducted to evaluate aflatoxin contamination of commonly used food in Lahore, Pakistan. In this context, 1125 samples of various food commodities were collected from various areas of Lahore city. In corn-based products AFB1 was detected in 52% (range 2.0-1405.3 µg kg(-1)) and AFB2 in 25% (range 1.0-55.2 µg kg(-1)) of the samples. In super kernel basmati rice, 13.3% of the samples (range 1.1-32.9 µg kg(-1)) showed the presence of AFB1, 1.9% was contaminated with AFB2 (range 1.0-8.1 µg kg(-1)) and only one sample exhibited the presence of AFG1. As far as the status of basmati rice is concerned, 18.3% was contaminated with AFB1 (range 1.0-15.4 µg kg(-1)) and 2% was contaminated with AFG1. In 42.9% of parboiled rice (range 1.1-9.2 µg kg(-1)) and 36.4% of broken rice (range 2.1-25.3 µg kg(-1)), samples were contaminated with AFB1.

Polyunsaturated fatty acids confer cryoresistance on megakaryocytes generated from cord blood and also enhance megakaryocyte production from cryopreserved cord blood cells.

BACKGROUND AIMS: Previous data have shown that the addition of docosahexanoic acid (DHA)/arachidonic acid (AA) has a beneficial effect on cytokine-mediated in vitro generation of megakaryocytes (MK) from umbilical cord blood (UCB).Cryopreservation forms an inherent part of UCB banking and MK progenitors are known to be very sensitive to the stresses of freezing. It is therefore imperative to generate functional cells from cryopreserved cells, and the generated cells need to be cryopreserved until used. In the present study, cryopreservation of ex vivo-expanded MK as well as MK generation from cryopreserved UCB samples was investigated. METHODS: MK generated with or without DHA/AA were cryopreserved in freezing medium containing 10% dimethyl sulfoxide (DMSO). Freezing efficacy was tested by quantitating MK after revival. Cryopreserved CD34(+) cells were cultured with stem cell factor (SCF) and thrombopoietin (TPO), in the presence and absence of DHA/AA for 10 days, and then quantitated for MK. Results. We observed a 1.5-3-fold increase in MK numbers, their progenitor content and their expression of phenotypic markers and MK-related transcription factors. DHA/AA sets showed a 2-5-fold improved engraftment in NOD/SCID mice. These data showed that the beneficial effect of DHA/AA obtained during MK expansion was not altered after freezing stress. The enhancement in MK generation obtained from fresh cord blood (CB) cells was reproduced with comparable efficiency when we used cryopreserved CB samples. CONCLUSIONS: Taken together, our data suggest that in vitro-generated DHA/AA MK survive cryoinjuries in a functionally better state. DHA/AA support a more efficient generation of MK from cryopreserved UCB.

Occurrence of aflatoxins in export-quality Pakistani rice.

A survey was conducted to assess the extent of aflatoxin B1, B2, G1 and G2 contamination of export-quality rice. Five hundred and nineteen batches of rice (including white, brown and sella rice) from various exporters were analysed for aflatoxins using Mycosep column clean-up and quantification with high-performance liquid chromatography, during the year 2010. Mean concentrations for aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) were 0.56 µg kg(-1) and 0.03 µg kg(-1) for brown rice, 0.49 µg kg(-1) and 0.03 µg kg(-1) for white rice and 0.73 µg kg(-1) and 0.02 µg kg(-1) for sella rice, with an overall positive (≥ the limit of detection of 0.1 µg kg(-1) for AFB1 and AFG1; and 0.05 µg kg(-1) for AFB2 and AFG2) incidence of 49%. Out of all analysed samples, only 1.9% was found positive for aflatoxin G1. Highly contaminated samples were found in the month of August, with findings for aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) of 16.65 µg kg(-1) and 2.64 µg kg(-1), respectively. During the whole year, monitoring aflatoxin concentrations exhibited a distinct variation according to the climatic conditions, with relatively high levels during March, July and August, which could be related to favourable environmental conditions.