Forced expression of microRNA-221-3p exerts protective effects against manganese-induced cytotoxicity in human lung epithelial cells.

Manganese (Mn)-induced pulmonary toxicity and the underlying molecular mechanisms remain largely enigmatic. Further, in recent years, microRNAs (miRNAs) have emerged as regulators of several pollutants-mediated toxicity. In this context, our study aimed at elucidating whether miRNAs are involved in manganese (II) chloride (MnCl2) (Mn2+)-induced cytotoxicity in lung epithelial cells. Growth inhibition of Mn2+ towards normal human bronchial epithelial (BEAS-2B) and adenocarcinomic human alveolar basal epithelial (A549) cells was analyzed by MTT assay following 24 or 48 h treatment. Reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm), cell cycle arrest, and apoptosis were evaluated by flow cytometry. RT-qPCR and Western blot were performed to analyze the expression of cyclins, anti-oxidant genes, and miRNAs. We used small RNA sequencing to investigate Mn2+-induced changes in miRNA expression patterns. In both cell lines, Mn2+ treatment inhibited growth in a dose-dependent manner. Further, compared with vehicle-treated cells, Mn2+ (250 µM) treatment induced ROS generation, cell cycle arrest, apoptosis, and decreased ΔΨm as well as altered the expression of cyclins and anti-oxidant genes. Sequencing data revealed that totally 296 miRNAs were differentially expressed in Mn2+-treated cells. Among them, miR-221-3p was one of the topmost down-regulated miRNAs in Mn2+-treated cells. We further confirmed this association in A549 cells. In addition, transient transfection was performed to study gain-of-function experiments. Forced expression of miR-221-3p significantly improved cell viability and reduced Mn2+-induced cell cycle arrest and apoptosis in BEAS-2B cells. In conclusion, miR-221-3p may be the most likely target that accounts for the cytotoxicity of Mn2+-exposed lung epithelial cells.

Assessment of Plasmodium falciparum drug resistance associated molecular markers in Mandla, Madhya Pradesh, India.

BACKGROUND: Resistance against artemisinin-based combination therapy is one of the challenges to malaria control and elimination globally. Mutations in different genes (Pfdhfr, Pfdhps, Pfk-13 and Pfmdr1) confer resistance to artesunate and sulfadoxine-pyrimethamine (AS + SP) were analysed from Mandla district, Madhya Pradesh, to assess the effectiveness of the current treatment regimen against uncomplicated Plasmodium falciparum. METHODS: Dried blood spots were collected during the active fever survey and mass screening and treatment activities as part of the Malaria Elimination Demonstration Project (MEDP) from 2019 to 2020. Isolated DNA samples were used to amplify the Pfdhfr, Pfdhps, Pfk13 and Pfmdr1 genes using nested PCR and sequenced for mutation analysis using the Sanger sequencing method. RESULTS: A total of 393 samples were subjected to PCR amplification, sequencing and sequence analysis; 199, 215, 235, and 141 samples were successfully sequenced for Pfdhfr, Pfdhps, Pfk13, Pfmdr1, respectively. Analysis revealed that the 53.3% double mutation (C59R, S108N) in Pfdhfr, 89.3% single mutation (G437A) in Pfdhps, 13.5% single mutants (N86Y), and 51.1% synonymous mutations in Pfmdr1 in the study area. Five different non-synonymous and two synonymous point mutations found in Pfk13, which were not associated to artemisinin resistance. CONCLUSION: The study has found that mutations linked to SP resistance are increasing in frequency, which may reduce the effectiveness of this drug as a future partner in artemisinin-based combinations. No evidence of mutations linked to artemisinin resistance in Pfk13 was found, suggesting that parasites are sensitive to artemisinin derivatives in the study area. These findings are a baseline for routine molecular surveillance to proactively identify the emergence and spread of artemisinin-resistant parasites.

Significance of nested PCR testing for the detection of low-density malaria infection amongst febrile patients from the Malaria Elimination Demonstration Project in Mandla, Madhya Pradesh, India.

BACKGROUND: Low-density malaria infections (LDMI) are defined as infections that are missed by the rapid diagnostic test (RDT) and/or microscopy which can lead to continued transmission and poses a challenge in malaria elimination efforts. This study was conducted to investigate the prevalence of LDMI in febrile cases using species-specific nested Polymerase Chain Reaction (PCR) tests in the Malaria Elimination Demonstration Project, where routine diagnosis was conducted using RDT. METHODS: Every 10th fever case from a cross-sectional community based fever surveillance was tested with RDT, microscopy and nested PCR. Parasite DNA was isolated from the filter paper using Chelex based method. Molecular diagnosis by nested PCR was performed targeting 18SrRNA gene for Plasmodium species. RESULTS: The prevalence of malaria was 2.50% (436/17405) diagnosed by PCR, 1.13% (196/17405) by RDT, and 0.68% (118/ 17,405) by microscopy. Amongst 17,405 febrile samples, the prevalence of LDMI was 1.51% (263/17405) (95% CI 1.33-1.70), which were missed by conventional methods. Logistic regression analysis revealed that illness during summer season [OR = 1.90 (p < 0.05)] and cases screened within three days of febrile illness [OR = 5.27 (p < 0.001)] were the statistically significant predictors of LDMI. CONCLUSION: The prevalence of malaria among febrile cases using PCR was 2.50% (436/17405) as compared to 1.13% (196/17405) by RDT. Higher number of the LDMI cases were found in subjects with ≤ 3 days mean duration of reported fever, which was statistically significant (p < 0.001). This observation suggests that an early detection of malaria with a more sensitive diagnostic method or repeat testing of the all negative cases may be useful for curtailing malaria transmission. Therefore, malaria elimination programme would benefit from using more sensitive and specific diagnostic methods, such as PCR.

Mass screening and treatment (MSaT) for identifying and treating asymptomatic cases of malaria-malaria elimination demonstration project (MEDP), Mandla, Madhya Pradesh.

BACKGROUND: Mass screening and treatment (MSaT) aims at reducing the spread of malaria in communities by identifying and treating infected persons regardless of the symptoms. This study was conducted to identify and treat asymptomatic cases using MSaT approaches in the community. METHODS: Three rounds of MSaT using cluster combination approaches were carried out during September 2018 to December 2019 to identify and treat asymptomatic malaria cases in the community. All individuals who were present in the household were screened using RDT irrespective of malaria related symptoms. Simultaneously thick and thin blood smear and blood spot were collected for further analysis using microscopy and diagnostic PCR done in a subset of the samples. RESULTS: Logistic regression analysis revealed that asymptomatic malaria cases significantly less among the older age groups compared with < 5 years children (OR ranged between 0.52 and 0.61; p < 0.05), lowest in cluster 4 (OR = 0.01; p < 0.0001); during third round of MSaT survey (OR = 0.11; p < 0.0001) and significantly higher in moderate to high endemic areas (OR = 88.30; p < 0.0001). CONCLUSION: Over the three rounds of MSaT, the number of asymptomatic cases were significantly less in the older age groups, and during third round. Similarly, the asymptomatic cases were significantly less in the low endemic area with API < 1 (cluster four). Therefore, the malaria elimination programme may consider the MSaT strategy to identify asymptomatic cases that would be otherwise missed by routine fever based surveillance. This MSaT strategy would help accomplish the malaria elimination goal in an expedited manner.

Activation of ERK/NF-kB Pathways Contributes to the Inflammatory Response in Epithelial Cells and Macrophages Following Manganese Exposure.

Different types of metals, including manganese (Mn), are constantly encountered in various environmental matrices due to natural and anthropogenic activities. They induce a sustained inflammatory response in various organs, which is considered to be an important priming event in the pathogenesis of several diseases. Mn-induced neuroinflammation and subsequent neurodegeneration are well recognized. However, emerging data suggest that occupationally and environmentally relevant levels may affect various organs, including the lungs. Therefore, the present study was carried out to investigate the effects of Mn (as Mn2+) exposure on the inflammatory response in human normal bronchial (BEAS-2B) and adenocarcinoma alveolar basal (A549) epithelial cells, as well as in murine macrophages (J774). Mn2+ exposure significantly induced mRNA and protein expression of various pro-inflammatory mediators (cytokines and chemokines) in all cells compared to corresponding vehicle controls. Furthermore, Mn2+ treatment also led to increased phosphorylation of extracellular-signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-kB) p65 in both epithelial cells and macrophages. As expected, cells treated with inhibitors of ERK1/2 (PD98059) and NF-kB p65 (IMD0354) effectively mitigated the expression of various pro-inflammatory mediators induced by Mn2+, suggesting that ERK/NF-kB pathways have a critical role in the Mn2+-induced inflammatory response. Further, in vivo studies are required to confirm these in vitro findings to support clinical translation.

Non-malignant respiratory illness associated with exposure to arsenic compounds in the environment.

Arsenic (As), a toxic metalloid, primarily originates from both natural and anthropogenic activities. Reports suggested that millions of people globally exposed to high levels of naturally occurring As compounds via inhalation and ingestion. There is evidence that As is a well-known lung carcinogen. However, there has been relatively little evidence suggesting its non-malignant lung effects. This review comprehensively summarises current experimental and clinical studies implicating the association of As exposure and the development of several non-malignant lung diseases. Experimental studies provided evidence that As exposure induces redox imbalance, apoptosis, inflammatory response, epithelial-to-mesenchymal transition (EMT), and affected normal lung development through alteration of the components of intracellular signaling cascades. In addition, we also discuss the sources and possible mechanisms of As influx and efflux in the lung. Finally, current experimental studies on treatment strategies using phytochemicals and our perspective on future research with As are also discussed.

Diversity and expression of Plasmodium falciparum var gene in severe and mild malaria cases from Central India.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein is encoded by a highly variable multicopy var gene family known to play a key role in malaria pathogenicity. Therefore, we investigated sequence variation, expression profile and immune response of the Duffy binding-like domain (DBLα) region of the var gene. METHODS: Blood samples were collected from patients with cerebral, severe and mild malaria in Chhattisgarh, India, a region with endemic malaria. Polymerase chain reaction amplicons were cloned and sequenced to determine sequence variation. The expression level was analyzed targeting the upstream region of var gene using the Delta-Delta-Ct method. Immunoglobulin G (IgG) level was determined against the 6 synthetic peptides of the DBLα region. RESULTS: The study identified that group 1 and group 5 sequences (cysteine/position of limited variability (cys/PoLV) classification) along with cys2/cys4 and MFK*/REY motifs and short amino acid length were significantly associated with malaria severity. The specific PoLV (MFKS, LREA, PTNL) were restricted to cerebral malaria. The expression level of var group A was higher than var groups B and C, demonstrating its prognostic characteristic. All peptides showed high-quality IgG response, while VAR P5 appeared to be a good marker for severity. CONCLUSIONS: The present study illustrates the presence of specific sequences of DBLα tags involved in severe malaria that could be targeted in future interventions for malaria control and elimination.

Genetic diversity and expression profile of Plasmodium falciparum Pf34 gene supports its immunogenicity.

PURPOSE OF THE STUDY: Genetic variation is one of the major obstacles in the development of effective vaccines. A multivalent malaria vaccine is required to increase efficacy and confer long term protection. In this context, we analysed the genetic diversity, expression profile, and immune response against Pf34. METHODS: Phylogenetic analysis was carried out using Pf34 orthologues sequences of various Plasmodium species. Genetic diversity was analysed by PCR amplification and Sanger dideoxy sequencing of Pf34 gene from Plasmodium falciparum positive human blood samples. The expression level of Pf34 gene was studied during erythrocytic stage by real time qPCR at four-hour interval, and immune response against synthetic peptides of Pf34 (P1 and P2) was analysed using ELISA. RESULTS: Phylogenetic analysis revealed the conserved nature of Pf34 gene. Genetic diversity analysis showed that majority (92%) of Plasmodium falciparum isolates in available database bore wild type Pf34 gene (Hd = 0.160 ± 0.030, π = 0.00021), including the present study (89.3%). The P. falciparum specific amino acid repeats (NNDK, NNDLK, and NNNNNN) in the B cell epitope regions were conserved. Furthermore, Pf34 gene is expressed throughout the erythrocytic cycle and comparatively high expression was observed in early ring and schizont stage. High IgG response was observed against both the peptides P1 and P2 of Pf34 containing asparagine NNNNNN and NNDLK repeat respectively. CONCLUSION: The limited genetic diversity, presence of conserved amino acid repeats within B cell epitope and high IgG response suggests that Pf34 may be a potential vaccine candidate for malaria. However, further validation studies are required.

Association of Angiopoietin Dysregulation in Placental Malaria with Adverse Birth Outcomes.

Malaria in pregnancy causes adverse birth outcomes due to sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Angiopoietins are critical regulators of vascular development and formation of placental villous vasculature. Angiopoietin-1 and Angiopoietin-2 concentrations were measured in peripheral and placental plasma samples from 70 malaria-infected and 216 control women using commercially available DuoSet ELISA development kit. Angiopoietins increased in placental plasma (ANG1-5833.5 pg/ml and ANG2-9580.6 pg/ml) as compared to peripheral plasma (ANG1-2293.1 pg/ml and ANG2-1198.9 pg/ml, p < 0.0001). The concentration of placental and peripheral ANG1 (6099.23 pg/ml and 2320.5 pg/ml) was significantly lower (5013.5 pg/ml, 2208.5 pg/ml), and ANG2 (9553.3 pg/ml, 1180.92 pg/ml) was significantly higher (9664.6 pg/ml, 1254.4 pg/ml) in malaria-positive cases as compared to malaria-negative (p < 0.0001). The association of dysregulated angiopoietins in malaria with adverse birth outcomes showed that the peripheral and placental ANG1 concentration was lower and ANG2 concentration was higher in low-birth-weight baby and stillbirth birth outcome as compared to normal deliveries among malaria-positive group. Therefore, ANG1 and ANG2 could be considered a biomarker for adverse outcome during malaria in pregnancy.

Prevalence of malaria in two highly endemic Community Health Centers in the Bastar district, Chhattisgarh showing mixed infections with Plasmodium species.

Malaria is a major public health problem in India and in the Chhattisgarh state. The diagnosis of malaria presents a major challenge in remote areas The prevalence of malaria in Darbha and Kilepal Community Health Centers (CHCs) of the Jagdalpur district, Chhattisgarh affected by conflict was determined using microscopy and polymerase chain reaction (PCR). In the year 2015, 29.4% and 21.5% cases were found to be positive for malaria at the Darbha and Kilepal CHCs, respectively, by microscopy, and 7.4% and 1.6% of cases had mixed infections, respectively. Among the suspected cases of mixed infections and doubtful diagnoses, 21% had mixed infections with two or more species at the Darbha CHC, and 17% from the Kilepal CHC, as determined by PCR. Both the P. vivax subspecies Pv210 (56%) and Pv247 (44%) and the P. ovale curtisi subspecies were found in this area. The high proportion of mixed malaria parasitic infections detected in this study indicate the need to adequately train health staff involved in diagnosing malaria. This study showed that there is a need for site-specific data to understand the epidemiological picture and to develop appropriate intervention strategies and management guidelines for controlling and eliminating malaria in India.

Sympatric distribution of Plasmodium ovale curtisi and P. ovale wallikeri in India: implication for the diagnosis of malaria and its control.

BACKGROUND: We report the first evidence of sympatric distribution of Plasmodium ovale curtisi and P. ovale wallikeri from India. METHODS: Fingerprick blood samples were collected from fever cases in district Bastar, Chhattisgarh State for malaria screening by microscopy and PCR. RESULTS: Two cases of mono infection of P. ovale, and a fatal case of cerebral malaria with a mixed infection of P. vivax, P. falciparum and P. ovale were confirmed by PCR. Sequencing analysis revealed the presence of P. ovale curtisi and P. ovale wallikeri. CONCLUSIONS: This study highlights the need of molecular diagnosis of malaria cases in forested areas for treatment and control.

Burden of complicated malaria in a densely forested Bastar region of Chhattisgarh State (Central India).

BACKGROUND: A prospective study on severe and complicated malaria was undertaken in the tribal dominated area of Bastar division, Chhattisgarh (CG), Central India, with an objective to understand the clinical epidemiology of complicated malaria in patients attending at a referral hospital. METHODS: Blood smears, collected from the general medicine and pediatric wards of a government tertiary health care facility located in Jagdalpur, CG, were microscopically examined for malaria parasite from July 2010 to December 2013. The Plasmodium falciparum positive malaria cases who met enrollment criteria and provided written informed consent were enrolled under different malaria categories following WHO guidelines. PCR was performed to reconfirm the presence of P.falciparum mono infection among enrolled cases. Univariate and multivariate logistic regression analysis was done to identify different risk factors using STATA 11.0. RESULTS: A total of 40,924 cases were screened for malaria. The prevalence of malaria and P.falciparum associated complicated malaria (severe and cerebral both) in the hospital was 6% and 0.81%, respectively. P.falciparum malaria prevalence, severity and associated mortality in this region peaked at the age of > 4-5 years and declined with increasing age. P.falciparum malaria was significantly more prevalent in children than adults (P < 0.00001). Among adults, males had significantly more P.falciparum malaria than females (P < 0.00001). Case fatality rate due to cerebral malaria and severe malaria was, respectively, 32% and 9% among PCR confirmed mono P.falciparum cases. Coma was the only independent predictor of mortality in multivariate regression analysis. Mortality was significantly associated with multi-organ complication score (P = 0.0003). CONCLUSION: This study has revealed that the pattern of morbidity and mortality in this part of India is very different from earlier reported studies from India. We find that the peak morbidity and mortality in younger children regardless of seasonality. This suggests that this age group needs special care for control and clinical management.

In silico 3D structure prediction and hydrogen peroxide binding study of wheat catalase.

Catalase (CAT) is one of the most active enzyme catalysts found in plants, animals and in all aerobic microorganisms. The major function of the enzyme is to decompose H2O2, produced by cellular metabolic activities under normal and stressful conditions to water and oxygen. The present study involves 3D structure modeling of wheat catalase from Triticum aestivum by MODELLER9v7 and its binding study with H2O2. The Evaluation of the model was based on Discrete Optimized Protein Energy Score (DOPE). The structure was also validated using PROCHECK comprising of 95.0% amino acid residues in favored regions of Ramachandran plot, Verify3D and ProsA which confirm that the model is reliable. The 3D model of the molecule was found to consist of ten strands and seventeen helices having a common fold characterised by ß-pleated sheet flanked either side by helices. The docking study with H2O2 indicates that Gln352 and Arg353 are two important determinant residues in binding H2O2 as these residues have strong hydrogen bonding contacts with the substrate. These hydrogen-bonding interactions play a significant role in the stability of the complex.