Genomic analysis of Indian isolates of Plasmodium falciparum: Implications for drug resistance and virulence factors.

The emergence of drug resistance to frontline treatments such as Artemisinin-based combination therapy (ACT) is a major obstacle to the control and eradication of malaria. This problem is compounded by the inherent genetic variability of the parasites, as many established markers of resistance do not accurately predict the drug-resistant status. There have been reports of declining effectiveness of ACT in the West Bengal and Northeast regions of India, which have traditionally been areas of drug resistance emergence in the country. Monitoring the genetic makeup of a population can help to identify the potential for drug resistance markers associated with it and evaluate the effectiveness of interventions aimed at reducing the spread of malaria. In this study, we performed whole genome sequencing of 53 isolates of Plasmodium falciparum from West Bengal and compared their genetic makeup to isolates from Southeast Asia (SEA) and Africa. We found that the Indian isolates had a distinct genetic makeup compared to those from SEA and Africa, and were more similar to African isolates, with a high prevalence of mutations associated with antigenic variation genes. The Indian isolates also showed a high prevalence of markers of chloroquine resistance (mutations in Pfcrt) and multidrug resistance (mutations in Pfmdr1), but no known mutations associated with artemisinin resistance in the PfKelch13 gene. Interestingly, we observed a novel L152V mutation in PfKelch13 gene and other novel mutations in genes involved in ubiquitination and vesicular transport that have been reported to support artemisinin resistance in the early stages of ACT resistance in the absence of PfKelch13 polymorphisms. Thus, our study highlights the importance of region-specific genomic surveillance for artemisinin resistance and the need for continued monitoring of resistance to artemisinin and its partner drugs.

Genomic characterization of Plasmodium falciparum genes associated with anti-folate drug resistance and treatment outcomes in eastern India: A molecular surveillance study from 2008 to 2017.

Introduction: After being used vigorously for the previous two decades to treat P. falciparum, chloroquine and sulfadoxine-pyrimethamine were replaced in 2009 with an artemisinin-based combination therapy (artesunate-sulfadoxine-pyrimethamine) in an effort to combat multidrug-resistant parasites. Methods: We set out to assess the genetic variants of sulfadoxine-pyrimethamine resistance and the effectiveness of its treatment in eastern India prior to, during, and 6 to 8 years following the introduction of the new pharmacological regime. In 2008-2009, 318 P. falciparum-positive patients got the recommended doses of sulfadoxine-pyrimethamine. We used 379 additional isolates from 2015 to 2017 in addition to the 106 isolates from 2010. All 803 isolates from two study sites underwent in vitro sulfadoxine-pyrimethamine sensitivity testing and genomic characterisation of sulfadoxine-pyrimethamine resistance (pfdhfr and pfdhps). Results: In Kolkata and Purulia, we observed early treatment failure in 30.7 and 14.4% of patients, respectively, whereas recrudescence was found in 8.1 and 13.4% of patients, respectively, in 2008-2009. In 2017, the proportion of in vitro pyrimethamine and sulfadoxine resistance steadily grew in Kolkata and Purulia despite a single use of sulfadoxine-pyrimethamine. Treatment failures with sulfadoxine-pyrimethamine were linked to quintuple or quadruple pfdhfr- pfdhps mutations (AICII-AGKAT, AICII-AGKAA, AICII-SGKGT, AICII-AGKAA, AICNI-AGKAA) in 2008-2009 (p < 0.001). The subsequent spread of mutant-haplotypes with higher in vitro sulfadoxine-pyrimethamine resistance (p < 0.001), such as the sextuple (dhfr-AIRNI+dhps-AGEAA, dhfr-ANRNL+dhps-AGEAA) and septuple (dhfr-AIRNI+dhps-AGEAT), mutations were observed in 2015-2017. Discussion: This successive spread of mutations with high in vitro sulfadoxine-pyrimethamine resistance confirmed the progressive increase in antifolate resistance even after an 8-year withdrawal of sulfadoxine-pyrimethamine.

A novel nano-anti-malarial induces redox damage and elicits cytokine response to the parasite.

Emergence and spread of resistant parasites to the newest chemotherapeutic anti-malarial agents are the biggest challenges against malaria control programs. Therefore, developing a novel effective treatment to reduce the overgrowing burden of multidrug resistant malaria is a pressing need. Herein, we have developed a biocompatible and biodegradable, non-toxic chitosan-tripolyphosphate-chloroquine (CS-TPP CQ) nanoparticle. CS-TPP CQ nanoparticles effectively kill the parasite through redox generation and induction of the pro- and anti-inflammatory cytokines in both sensitive and resistant parasite in vitro. The in vitro observations showed a strong inhibitory effect (p < 0.01) on pro-inflammatory cytokines more specifically on TNF-α and IFN-γ whereas CS-TPP CQ nanoparticles significantly elevated the anti-inflammatory cytokines- IL-10 and TGF-ß. In addition, CS-TPP CQ nanoparticle significantly increased NO generation (p < 0.01) and altered the GSH/GSSG ratio 72 h after parasite co-culture with peripheral blood mononuclear cells culminating in the free radical induced parasite killing. CS-TPP CQ nanoparticle had an effective dose of 100 ng/ml against CQ-sensitive parasite lines (p < 0.001) whereas effective dose against CQ-resistant parasite line was 200 ng/ml CS-TPP CQ with an effective duration of 72 h (p < 0.001). Our studies suggest that CS-TPP CQ nanoparticle has a potential to modulate the pro- and anti-inflammatory responses, and to trigger the redox-mediated parasite killing. It can be a novel nano-based futuristic approach towards malaria control.

LmjF.36.3850, a novel hypothetical Leishmania major protein, contributes to the infection.

Leishmania is a protozoan parasite that resides in mammalian macrophages and inflicts the disease known as leishmaniasis. Although prevalent in 88 countries, an anti-leishmanial vaccine remains elusive. While comparing the virulent and avirulent L. major transcriptomes by microarray, PCR and functional analyses for identifying a novel virulence-associated gene, we identified LmjF.36.3850, a hypothetical protein significantly less expressed in the avirulent parasite and without any known function. Motif search revealed that LmjF.36.3850 protein shared phosphorylation sites and other structural features with sucrose non-fermenting protein (Snf7) that shuttles virulence factors. LmjF.36.3850 was predicted to bind diacylglycerol (DAG) with energy value similar to PKCα and PKCß, to which DAG is a cofactor. Indeed, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue, enhanced the phosphorylation of PKCα and PKCßI. We cloned LmjF.36.3850 gene in a mammalian expression vector and primed susceptible BALB/c mice followed by challenge infection. We observed a higher parasite load, comparable antibody response and higher anti-inflammatory cytokines such as IL-4 and IL-10, while expression of major anti-leishmanial cytokine, IFN-γ, remained unchanged in LmjF.36.3850-vaccinated mice. CSA restimulated LN cells from vaccinated mice after challenge infection secreted comparable IL-4 and IL-10 but reduced IFN-γ, as compared to controls. These observations suggest a skewed Th2 response, diminished IFN-γ secreting Th1-TEM cells and increased central and effector memory subtype of Th2, Th17 and Treg cells in the vaccinated mice. These data indicate that LmjF.36.3850 is a plausible virulence factor that enhances disease-promoting response, possibly by interfering with PKC activation and by eliciting disease-promoting T cells.

Pro-Oxidant Therapeutic Activities of Cerium Oxide Nanoparticles in Colorectal Carcinoma Cells.

Given that basal levels of reactive oxygen species (ROS) are higher in cancer cells, there is a growing school of thought that endorses pro-oxidants as potential chemotherapeutic agents. Intriguingly, cerium oxide (CeO2) nanoparticles can manifest either anti- or pro-oxidant activity as a function of differential pH of various subcellular localizations. In an acidic pH environment, for example, in extracellular milieu of cancer cells, CeO2 would function as a pro-oxidant. Based on this concept, the present study is designed to investigate the pro-oxidant activities of CeO2 in human colorectal carcinoma cell line (HCT 116). For comparison, we have also studied the effect of ceria nanoparticles on human embryonic kidney (HEK 293) cells. Dose-dependent viability of cancerous as well as normal cells has been assessed by treating them independently with CeO2 nanoparticles of different concentrations (5-100 µg/mL) in the culture media. The half maximal inhibitory concentration (IC50) of nanoceria for HCT 116 is found to be 50.48 µg/mL while that for the HEK 293 cell line is 92.03 µg/mL. To understand the intricate molecular mechanisms of CeO2-induced cellular apoptosis, a series of experiments have been conducted. The apoptosis-inducing ability of nanoceria has been investigated by Annexin V-FITC staining, caspase 3/9 analysis, cytochrome c release, intracellular ROS analysis, and mitochondrial membrane potential analysis using flow cytometry. Experimental data suggest that CeO2 treatment causes DNA fragmentation through enhanced generation of ROS, which ultimately leads to cellular apoptosis through the p53-dependent mitochondrial signaling pathway.

A review on possible modes of action of chloroquine/hydroxychloroquine: repurposing against SAR-CoV-2 (COVID-19) pandemic.

Chloroquine (CQ) and its analogue hydroxychloroquine (HCQ) have long been used worldwide as frontline drugs for the treatment and prophylaxis of human malaria. Since the first reported cases in Wuhan, China, in late December 2019, humans have been under threat from coronavirus disease 2019 (COVID-19) caused by the novel coronavirus SARS-CoV-2 (previously known as 2019-nCoV), subsequently declared a pandemic. While the world is searching for expedited approval for a vaccine, which may be only preventative and not a cure, physicians and country leaders are considering several concerted clinical trials suggesting that the age-old antimalarial drugs CQ/HCQ could be a potent therapeutic against COVID-19. Based on accumulating scientific reports, here we highlight the possible modes of action of CQ/HCQ that could justify its use against viral infections. Considering the global health crisis of the COVID-19 pandemic, the option of repurposing old drugs, e.g. CQ/HCQ, particularly HCQ, for the treatment of SARS-CoV-2 infection could be a good choice. CQ/HCQ has diverse modes of action, including alteration of the acidic environment inside lysosomes and late endosomes, preventing endocytosis, exosome release and phagolysosomal fusion, and inhibition of the host cytokine storm. One or more diverse mechanisms might work against viral infections and reduce mortality. As there is no cure for COVID-19, clinical testing of HCQ is urgently required to determine its potency against SARS-CoV-2, as this is the currently available treatment option. There remains a need to find other innovative drug candidates as possible candidates to enter clinical evaluation and testing.

Self assembled arjunolic acid acts as a smart weapon against cancer through TNF- α mediated ROS generation.

Arjunolic acid (AA) a plant derived pentacyclic triterpenoid which showed effective anticancer activity against MCF-7 and HeLa cells as well as no significant toxic effect was observed against normal lymphocytes. In the current study the self assemble property of arjunolic acid gives an extra emphasis on anticancer activity which was proved by several fluorescence studies like ROS generation, EtBr/AO and DAPI staining. At a selected dose of 50µg/ml AA disrupt the redox balance inside the cancer cells by producing reactive oxygen species. The apoptotic event was mediated by two key regulator proteins TNF-α and NF-κß which was proved here. The increment of the pro-inflammatory cytokines indicates the ROS mediated pathway of cancer cell apoptosis.

Immunostimulatory effect of chitosan conjugated green copper oxide nanoparticles in tumor immunotherapy.

Current study demonstrates the immunogenic role of biopolymer coated green synthesized copper oxide nanoparticles by the induction of cellular immunity through the activation immune cells. Alongside humoral immunity response was triggered by the surface coated NPs through IgG response which indicate the adjuvanic role of the nano conjugate. Th1 (Type 1 and Type 2 helper T cells) and Th2 cells were activated after the treatment with nano conjugate and act as an immunostimulant which would inhibit the proliferation of breast cancer (MCF-7) and cervical cancer (HeLa) cells in in vitro. Solid tumor induced by 4 T1 cells were also inhibited in in vivo Balb/C mice model. Secretion of pro-inflammatory cytokines and the increase in CD + 4 populations indicate the activation of immune cells in the current study. Immunotherapy by the help of metal nano conjugate can be an effective tool to eradicate the cancer cells from the system.

Leishmania major adenylate kinase immunization offers partial protection to a susceptible host.

Leishmania major causes mild-to-severe cutaneous lesions resulting in significant disfigurations, if untreated. The drugs are toxic, and drug-resistance parasites are emerging. Therefore, a prophylactic vaccination is an urgent need. As no vaccine is available, we compared the genes expressed by virulent and avirulent parasites. We identify L major adenylate kinase (AdeK) as a probable vaccine candidate after a series of experimentations. We cloned the gene in mammalian pcDNA6/HisA and pet28a+ vector for in vivo expression following immunization and in vitro protein expression for booster, respectively. We observed that immunization of susceptible BALB/c mice with AdeK resulted in significant protection against L major challenge infection. The protection was accompanied by increased IFN-γ producing lymphocytes and reduced IL-4, IL-17 and IL-10 secreting central and effector Th2, Th17 and Treg memory cells, respectively. These observations indicate L major AdeK as a potential vaccine candidate.

LmjMAPK10 offers protection against Leishmania donovani infection.

AIMS: This study aimed at evaluating the DNA vaccination efficacy of Leishmania major-derived MAPK10 against Leishmania donovani infection. METHODS AND RESULTS: MAPK10 is one of the 15 mitogen-activated protein kinases (MAPKs) of Leishmania major. Herein, we expressed the gene through a mammalian vector and tested whether priming with this gene would offer protection against L donovani infection. We report that LmjMAPK10 DNA vaccination using a mammalian expression vector significantly reduces the parasite burden. The protection is accompanied by host-protective T-cell functions, TH 1-type cytokines and elevated leishmanial antigen-specific IgG2a isotype response. T-cell response to the L donovani/challenge infection is associated with increase in IL-12 and IFN-γ, but reduced IL-10 and IL-4 production. CONCLUSIONS: LmjMAPK10 is cross-protective against L donovani infection.

Anti-Leishmanial Vaccines: Assumptions, Approaches, and Annulments.

Leishmaniasis is a neglected protozoan parasitic disease that occurs in 88 countries but a vaccine is unavailable. Vaccination with live, killed, attenuated (physically or genetically) Leishmania have met with limited success, while peptide-, protein-, or DNA-based vaccines showed promise only in animal models. Here, we critically assess several technical issues in vaccination and expectation of a host-protective immune response. Several studies showed that antigen presentation during priming and triggering of the same cells in infected condition are not comparable. Altered proteolytic processing, antigen presentation, protease-susceptible sites, and intracellular expression of pathogenic proteins during Leishmania infection may vary dominant epitope selection, MHC-II/peptide affinity, and may deter the reactivation of desired antigen-specific T cells generated during priming. The robustness of the memory T cells and their functions remains a concern. Presentation of the antigens by Leishmania-infected macrophages to antigen-specific memory T cells may lead to change in the T cells' functional phenotype or anergy or apoptosis. Although cells may be activated, the peptides generated during infection may be different and cross-reactive to the priming peptides. Such altered peptide ligands may lead to suppression of otherwise active antigen-specific T cells. We critically assess these different immunological issues that led to the non-availability of a vaccine for human use.

Facet-Dependent Photodegradation of Methylene Blue Using Pristine CeO2 Nanostructures.

This work comprises the shape- and facet-dependent catalytic efficacies of different morphologies of CeO2, namely, hexagonal, rectangular, and square. The formation of different shapes of CeO2 is controlled using polyvinyl pyrrolidone as a surfactant. The surface reactivity of formation of differently exposed CeO2 facets is thoroughly investigated using UV-visible, photoluminescence, Raman, and X-ray photoelectron spectroscopies. A correlation between the growth of a surface-reactive facet and the corresponding oxygen vacancies is also established. Considering the tremendous contamination, caused by the textile effluents, the present study articulates the facet-dependent photocatalytic activities of pristine CeO2 for complete degradation of methylene blue within 175 min. The observed degradation time deploying pristine CeO2 as a catalyst is the shortest to be reported in the literature to our best knowledge.

Biodistribution and toxickinetic variances of chemical and green Copper oxide nanoparticles in vitro and in vivo.

In this study, chemical (S1) and green (S2) Copper Oxide nanoparticles (NPs) were synthesized to determine their biodistribution and toxicokinetic variances in vitro and in vivo. Both NPs significantly released Copper ions (Cu) in lymphocytes and were primarily deposited in the mononuclear phagocyte system (MPS) such as the liver and spleen in mice. In particular, S2NPs seemed to be prominently stored in the spleen, whereas the S1NPs were widely stored in more organs including the liver, heart, lungs, kidney and intestine. The circulation in the blood and fecal excretions both showed higher S2NPs contents respectively. Measurements of cell viability, Hemolysis assay, Reactive Oxygen Species (ROS) generation, biochemical estimation and apoptotic or necrotic study in lymphocytes after 24 h and measurements of body and organ weight, serum chemistry evaluation, cytokines level, protein expressions and histopathology of Balb/C mice after 15 days indicated significant toxicity difference between the S1NPs and S2NPs. Our observations proved that the NPs physiochemical properties influence toxicity and Biodistribution profiles in vitro and in vivo.

Antigen conjugated nanoparticles reprogrammed the tumor-conditioned macrophages toward pro-immunogenic type through regulation of NADPH oxidase and p38MAPK.

Tumor associated macrophages (TAMs) are pertinent to cancer cell growth in the tumor microenvironment. Indeed, TAMs differentiate from monocytes (MΦ) due to specific growth factors present in the tumor microenvironment. TAMs show mostly an M2-like phenotype is due to the absence of pro-inflammatory signals and supply fuel to tumor growth. Several attempts have been taken to switch TAMs into a pro-immunogenic type. To address context, we used a tumor microenvironment by in vitro coculturing human blood MΦ with cancer cell conditioned media (TC-MΦ). We showed that the antigen cobalt oxide nanoparticles (Ag-NPs) can reprogram TC-MΦ to pro-immunogenic type to build up an antitumor immune response. Our results demonstrate that NPs-Ag induced a marked activation of NADPH oxidase in TC-MΦ, likely through stimulation of ROS linked to activation of p38 MAPK. These activated p38 MAPK up-regulated the IFN-γ, TNF-α and initial IL-12 production, in turn, the activation of IFN-γ prolonged IL-12 production.

Novel pfkelch13 Gene Polymorphism Associates With Artemisinin Resistance in Eastern India.

BACKGROUND: Artesunate-sulfadoxine-pyrimethamine (ASSP) is the frontline artemisinin combination therapy (ACT) in India. Random, irrational, subtherapeutic artemisinin doses and self-medication with ACT along with predominance of sulfadoxine-pyrimethamine resistance parasite invoked a strong possibility of emerging artemisinin-resistant malaria parasites. METHODS: This study involved 226 patients with uncomplicated Plasmodium falciparum infection who had successfully completed the 42 days follow-up after ASSP combination therapy from April 2014 to January 2016. We assessed the ASSP treatment efficacy by evaluating parasite clearance half-life, pfkelch13, and other (pfdhfr, pfdhps, pfmdr1, pfcrt) gene mutations and survival of parasites as detected by an ex vivo ring-stage survival assay (RSA). FINDINGS: Slow-clearing infections with longer parasite clearance half-lives (>5 hours) were observed in 12% isolates. Cure rate after ASSP treatment was declining to about 84.1%. ASSP failure was recorded in 15.9% (early treatment failure, 7.9%; late treatment failure, 7.9%) of isolates. In sum, 24 patients (10.6%) had parasite clearance half-lives greater than 5 hours with pfkelch13 polymorphism after 441 codon; in 15 of those patients (6.6%), parasites had not cleared by 72 hours after initiation of therapy. Median ex vivo ring-stage survival rate of these isolates was very high (12.2%; 95% confidence interval [CI], 10.9-13.8) from that of cured patients (0.9%; 95% CI, 0.09-1.07). Of these 15 patients, 13 patients had pfkelch13 G625R polymorphism, whereas 2 patients contained R539T polymorphism. As per the World Health Organization guideline, these 15 isolates were true artemisinin-resistant isolates. INTERPRETATION: Identification of artemisinin-resistant isolates in India together with new mutations and increasing combination therapy failures blow alarms for urgent malaria control.

Green synthesis of Nerium oleander-conjugated gold nanoparticles and study of its in vitro anticancer activity on MCF-7 cell lines and catalytic activity.

The phytochemicals present in the stem bark extract of Nerium oleander (commonly known as Karabi) have been utilized for the green synthesis of stable gold-conjugated nanoparticles at room temperature under very mild conditions. The green synthesized gold-conjugated nanoparticles were characterized by surface plasmon resonance spectroscopy, High resolution transmission electron microscopy, X-ray diffraction studies and dynamic light scattering. A mechanism for the synthesis and stabilization of gold-conjugated nanoparticles (AuNPs) has been proposed. Anticancer activity of the stabilized AuNPs studied against MCF-7 breast cancer cell line revealed that the stabilized AuNPs were highly effective for the apoptosis of cancer cells selectively. The antioxidant activity of the stem bark extract of Nerium oleander has also been studied against a long lived 2,2-diphenylpicrylhydrazyl radical at room temperature. Moreover, the utilization of the stabilized AuNPs as a catalyst has also been demonstrated.

Development of Low-ppm CO Sensors Using Pristine CeO2 Nanospheres with High Surface Area.

Mesoporous CeO2 nanospheres with appreciably high surface area are prepared using reversed micelles by a water-in-oil microemulsion method. The structural morphology and semiconducting properties of the nanoparticles are thoroughly investigated using X-ray diffraction, field effect scanning electron microscopy, transmission electron microscopy, and UV-visible spectroscopic techniques. Even after high-temperature calcination, the morphological retention of the material is apparent by electron microscopy. The deployment of undoped CeO2 nanospheres for the detection of low-ppm CO yields superior performances in terms of sensitivity, response-recovery times, and selectivity compared to those of other sensors of the same genre. These CO sensors exhibit ∼ 52% sensitivity with a response time of only 13 s. The sensor parameters are analyzed as a function of both temperature and gas concentration. In addition to that on the cost-effective and scalable synthesis of CeO2 nanospheres, this article also reports on the fabrication of packaged CO sensors, which can be potentially utilized for industrial and environmental monitoring purposes.