Propitious catalytic response of immobilized α-amylase from G. thermoleovorans in modified APTES-Fe3O4 NPs for industrial bio-processing.

Challenges in enzyme and product recovery are currently intriguing in modern biotechnology. Coping enzyme stability, shelf life and efficiency, nanomaterials-based immobilization were epitomized of industrial practice. Herein, a α-amylase from Geobacillus thermoleovorans was purified and bound effectively on to a modified 3-Aminopropyltriethoxysilane (APTES)-Fe3O4 nanoparticle. It was revealed that the carrier-bound enzyme catalysis (pH 8 and 60 °C) was significant in contrast to the free enzyme (pH 7.5 and 55 °C). Furthermore, Zn2+ and Cu2+ were shown to cause inhibitory effects in both enzyme states. Unlike chloroform, toluene, benzene, and butanol, minimal effects were observed with ethanol, acetone, and hexane. The bound enzyme retained 27.4 % of its initial activity after being stored for 36 days. In addition, the reusability of the bound enzyme showed a gradual decline in activity after the first cycle; however, after 13 cycles, its residual activity at 53 % was observed. These data proved significant enough to use this enzyme for industrial starch and analogous substrate bio-processing.

Green Synthesised TiO2 Nanoparticles-Mediated Terenna asiatica: Evaluation of Their Role in Reducing Oxidative Stress, Inflammation and Human Breast Cancer Proliferation.

Oxidative stress and chronic inflammation interplay with the pathogenesis of cancer. Breast cancer in women is the burning issue of this century, despite chemotherapy and magnetic therapy. The management of secondary complications triggered by post-chemotherapy poses a great challenge. Thus, identifying target-specific drugs with anticancer potential without secondary complications is a challenging task for the scientific community. It is possible that green technology has been employed in a greater way in order to fabricate nanoparticles by amalgamating plants with medicinal potential with metal oxide nanoparticles that impart high therapeutic properties with the least toxicity. Thus, the present study describes the synthesis of Titanium dioxide nanoparticles (TiO2 NPs) using aqueous Terenna asiatica fruit extract, with its antioxidant, anti-inflammatory and anticancer properties. The characterisation of TiO2 NPs was carried out using a powdered X-ray diffractometer (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray diffraction (EDX), high-resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), and zeta-potential. TiO2 NPs showed their antioxidant property by scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals in a dose-dependent manner with an IC50 value of 80.21 µg/µL. To ascertain the observed antioxidant potential of TiO2 NPs, red blood cells (RBC) were used as an in vitro model system. Interestingly, TiO2 NPs significantly ameliorated all the stress parameters, such as lipid peroxidation (LPO), protein carbonyl content (PCC), total thiol (TT), superoxide dismutase (SOD), and catalase (CAT) in sodium nitrite (NaNO2)-induced oxidative stress, in RBC. Furthermore, TiO2 NPs inhibited RBC membrane lysis and the denaturation of both egg and bovine serum albumin, significantly in a dose-dependent manner, suggesting its anti-inflammatory property. Interestingly, TiO2 NPs were found to kill the MCF-7 cells as a significant decrease in cell viability of the MCF-7 cell lines was observed. The percentage of growth inhibition of the MCF-7 cells was compared to that of untreated cells at various doses (12.5, 25, 50, 100, and 200 µg/mL). The IC50 value of TiO2 NPs was found to be (120 µg/mL). Furthermore, the Annexin V/PI staining test was carried out to confirm apoptosis. The assay indicated apoptosis in cancer cells after 24 h of exposure to TiO2 NPs (120 µg/mL). The untreated cells showed no significant apoptosis in comparison with the standard drug doxorubicin. In conclusion, TiO2 NPs potentially ameliorate NaNO2-induced oxidative stress in RBC, inflammation and MCF-7 cells proliferation.

Synthesis of Palladium-Catalysed C-C Bond Forming 5-Chloro Quinolines via Suzuki-Miyaura Coupling; Anti-Pancreatic Cancer Screening on PANC-1 Cell Lines.

Pancreatic cancer is the most severe among other cancers due to its late detection and less chance of survivability. Heterocycles are proven ring systems in the treatment of various cancers and this is due to the presence of two biodynamic molecules combined, which have a greater synergistic efficacy in many anticancer drugs. Quinoline and pyridine ring systems are brought together to obtain greater potency and this is achieved by coupling both using Pd-catalyst, and in the present investigation, Suzuki-Miyaura coupling (SMC) reactions are adopted to generate potent molecular entities. Pancreatic cancer is difficult to treat due to overexpression of the VEGFR2 protein. VEGFR2 is targeted to design the molecules of quinoline-coupled pyridine moieties and is docked to evaluate the protein-ligand interaction at the binding site. The binding affinity of conjugates revealed the potency and capability of ligands to inhibit the VEGFR2 pathway. The in-silico ADMET properties determined their inherent pharmacokinetic feasibility. The synthesized conjugates have been evaluated by MTT assay against the human pancreatic cancer cell lines (PANC-1). Among the series, compounds 5d, 5e, and 5h exhibited a greater inhibitory activity against the cell lines with an IC50 value of 82.32±1.38, 54.74±1.18 and 80.35±1.68 µM. In the present exploration, 5e exhibited greater inhibitory activity and it could be a promising lead for the development of new chemotherapeutics against pancreatic cancer.

Computational prediction of B and T-cell epitopes of Kyasanur Forest Disease virus marker proteins towards the development of precise diagnosis and potent subunit vaccine.

Kyasanur Forest Disease (KFD), also known as 'monkey fever', caused by KFD Virus (KFDV), is a highly neglected tropical disease endemic to Western Ghat region of Karnataka, India. Recently, KFD, which is fatal for both monkeys and humans with a mortality rate of 2-10% has been found to spread from its epicenter to neighboring districts and states also. The current ELISA based KFD detection method is very non-specific due to cross-reactivity with other flaviviruses. Further, presently available formalin-inactivated vaccine has been found to be less effective leading to disease susceptibility and severity. To address these, the present study was aimed at predicting the potent specific B and T-cell epitopes of KFDV immunogenic marker proteins using diverse computational tools aiming at developing precise diagnostic method and an effective subunit vaccine. Here, we have chosen E, NS1 and NS5 proteins as markers of KFDV by taking into account of their differential and non-overlapping sequences with selected arboviruses. Based on the linear and nonlinear epitope prediction tools and important biophysical parameters, we identified three potential linear and ten nonlinear B-cell epitopes. We also predicted T-cell epitope peptides which binds to MHC class-I and class-II receptors for the effective T-cell activation. Thus, our molecular docking and molecular dynamics simulation analysis has identified six different TH-cell epitopes based on the distribution frequency of MHC-II haplotypes in the human population and one TC-cell epitope from NS5 protein that has maximum interaction with class-I MHC. Overall, we have successfully identified potential B and T-cell epitope marker peptides present in the envelope and two non-structural proteins.Communicated by Ramaswamy H. Sarma.

Effect of Biofunctional Green Synthesized MgO-Nanoparticles on Oxidative-Stress-Induced Tissue Damage and Thrombosis.

The present study describes the green biofunctional synthesis of magnesium oxide (MgO) nanoparticles using the aqueous Tarenna asiatica fruit extract. The characterization of Tarenna asiatica fruit extract MgO nanoparticles (TAFEMgO NPs) was achieved by X-ray powder diffraction, UV-Vis spectroscopy, FTIR, TEM, SEM, and energy-dispersive X-ray diffraction. TAFEMgO NPs scavenged the DPPH free radicals with an IC50 value of 55.95 µg/µL, and it was highly significant compared to the standard. To authenticate the observed antioxidant potential of TAFEMgO NPs, oxidative stress was induced in red blood cells (RBC) using sodium nitrite (NaNO2). Interestingly, TAFEMgO NPs ameliorated the RBC damage from oxidative stress by significantly restoring the stress parameters, such as the protein carbonyl content (PCC), lipid peroxidation (LPO), total thiol (TT), super-oxide dismutase (SOD), and catalase (CAT). Furthermore, oxidative stress was induced in-vivo in Sprague Dawley female rats using diclofenac (DFC). TAFEMgO NPs normalized the stress parameters in-vivo and minimized the oxidative damage in tissues. Most importantly, TAFEMgO NPs restored the function and architecture of the damaged livers, kidneys, and small intestines by regulating biochemical parameters. TAFEMgO NPs exhibited an anticoagulant effect by increasing the clotting time from 193 s in the control to 885 s in the platelet rich plasma. TAFEMgO NPs prolonged the formation of the clot process in the activated partial thromboplastin time and the prothrombin time, suggest the effective involvement in both intrinsic and extrinsic clotting pathways of the blood coagulation cascade. TAFEMgO NPs inhibited adenosine di-phosphate (ADP)-induced platelet aggregation. TAFEMgO NPs did not show hemolytic, hemorrhagic, and edema-inducing properties at the tested concentration of 100 mg/kgbody weight, suggesting its non-toxic property. In conclusion, TAFEMgO NPs mitigates the sodium nitrite (NaNO2)- and diclofenac (DFC)-induced stress due to oxidative damage in both in vitro and in vivo experimental models.

Impact of oxidative stress in response to malarial infection during pregnancy: Complications, histological changes, and pregnancy outcomes.

Background and Objectives: Pregnancy malaria is a major underestimated global public health problem. To understand the involvement of oxidative stress (OS) in the pathophysiology of placental malaria, OS biomarkers, malondialdehyde (MDA), uric acid (UA), and superoxide dismutase (SOD) levels were analyzed and correlated to placental histopathological changes and pregnancy outcomes. Methods: A hospital-based study was conducted in Mangaluru, Karnataka, India, to analyze the changes in hematological parameters and the serum OS biomarker levels. Histological analysis of placenta, associated complications, and pregnancy outcomes were compared using Kruskal-Wallis test, and pairwise comparison between two groups was made by Mann-Whitney U-test. Correlations were calculated by Pearson's and Spearman's rank correlations. Results: Among 105 pregnant women, 34 were healthy controls and the infected group comprised of Plasmodium Vivax (Pv) (n = 48), Plasmodium falciparum (Pf) (n = 13), and mixed (n = 10) malaria infections. Of 71 infected cases, 67.6% had mild malaria, whereas 32.4% had severe malaria. The white blood cell and C-reactive protein levels were found to increase, whereas hemoglobin, red blood cell, and platelet levels decreased during both types of malarial infections. The MDA and UA values increased and SOD levels decreased particularly during severe Pf infections. Histological changes such as syncytial knots, syncytial ruptures, and fibrinoid necrosis were observed particularly during Pf infections and leukocyte infiltration was observed in Pv malaria. Conclusion: Evaluation of MDA, UA, and SOD levels can serve as an indicator of OS during pregnancy malaria. The OS during pregnancy may lead to complications such as severe anemia, pulmonary edema, intra uterine growth retardation, premature delivery, and low birth weight, not only during Pf but also in Pv malaria. It is important to create awareness among rural and immigrant population residing in Mangaluru and its surroundings about required preventive measures and free government-supported antenatal care services.

Molecular mechanism of Escherichia coli H10407 induced diarrhoea and its control through immunomodulatory action of bioactives from Simarouba amara (Aubl.).

Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of death in children under the age of five in developing countries. ETEC (O78:H11:CFA/I:LT+:ST+) mechanism has been studied in detail with either heat labile (LT) or heat stable (ST) toxins using in vitro and in vivo models. However, there is no adequate information on ETEC pathogenesis producing both the toxins (LT, ST) in BALB/c mice model. In this study, female mice have been employed to understand ETEC H10407 infection induced changes in physiology, biochemical and immunological patterns up to seven days post-infection and the antidiarrhoeal effect of Simarouba amara (Aubl.) bark aqueous extract (SAAE) has also been looked into. The results indicate that BALB/c is sensitive to ETEC infection resulting in altered jejunum and ileum histomorphology. Withal, ETEC influenced cAMP, PGE2, and NO production resulting in fluid accumulation with varied Na+, K+, Cl-, and Ca2+ levels. Meanwhile, ETEC subverted expression of IL-1ß, intestine alkaline phosphatase (IAP), and myeloperoxidase (MPO) in jejunum and ileum. Our data also indicate the severity of pathogenesis reduction which might be due to attainment of equilibrium after reaching optimum rate of infection. Nevertheless, degree of pathogenesis was highly significant (p < 0.01) in all the studied parameters. Besides that, SAAE was successful in reducing the infectious diarrhoea by inhibiting ETEC H10407 in intestine (jejunum and ileum), and shedding in feces. SAAE decreased cAMP, PGE2, and fluid accumulation effectively and boosted the functional activity of immune system in jejunum and ileum IAP, MPO, IL-1ß, and nitric oxide.

Acquired antibody responses against merozoite surface protein-119 antigen during Plasmodium falciparum and P.vivax infections in South Indian city of Mangaluru.

Merozoite surface protein-1 (MSP-1) of malaria parasites has been extensively studied as a malaria vaccine candidate and the antibody response to this protein is an important indicator of protective immunity to malaria. Mangaluru city and its surrounding areas in southwestern India are endemic to malaria with Plasmodium vivax being the most widespread and prevalent species although P. falciparum also frequently infects. However, no information is available on the level of protective immunity in this population. In this regard, a prospective hospital-based study was performed in malarial patients to assess antibody responses against the 19-kDa C-terminal portion of P. vivax and P. falciparum MSP-1 (MSP-119). Serum samples from 51 healthy endemic controls and 267 infected individuals were collected and anti-MSP-119 antibody levels were analyzed by ELISA. The possible association between the antibody responses and morbidity parameters such as malarial anemia and thrombocytopenia was investigated. Among the 267 infected cases, 144 had P. vivax and 123 had P. falciparum infections. Significant levels of anti-MSP-119 antibody were observed both in P. vivax (123/144; 85.4%) and P. falciparum (108/123; 87.9%) infected individuals. In both type of infections, the major antibody isotypes were IgG1 and IgG3. The IgG levels were found to be increased in patients with severe anemia and thrombocytopenia. The antibody levels were also higher in infected individuals who had several previous infections, although antibodies produced during previous infections were short lived. The predominance of cytophilic anti-MSP-119 IgG1 and IgG3 antibodies suggests the possibility of a dual role of Pv MSP-119 and Pf MSP-119 during malarial immunity and pathogenesis.

Optimized Solid-State Fermentation Medium Enhances the Multienzymes Production from Penicillium citrinum and Aspergillus clavatus.

Filamentous fungi play an important role in the production of a range of useful extracellular hydrolytic enzymes for wide industrial applications. The Western Ghats region is known for its rich microbial biodiversity and could be a potential source of several useful fungi that could be exploited for the production of industrially important enzymes. From this soil, we aimed at the isolation of multienzyme producing fungi, optimization of the culture conditions using solid-state fermentation (SSF), partial purification of enzymes and characterization by zymography. Out of seven fungal strains, two isolates, namely Penicillium citrinum and Aspergillus clavatus, were found to produce amylase and cellulase enzymes maximally. The effect of different physicochemical parameters on the production of amylase and cellulase was investigated and the maximum production of multienzymes was achieved in wheat bran substrate. The newly formulated and optimized medium increased the multienzyme production in P. citrinum and A. clavatus as compared to medium with individually optimized parameters. Further, for the first time, different isoforms of amylase and cellulase have been identified from P. citrinum and A. clavatus by zymography. In summary, the present study showed that the filamentous fungi can utilize the industrial waste product such as wheat bran as the substrate for multienzymes production by SSF and could be a promising source of enzymes for biotechnological applications.

Clinical features and haematological parameters among malaria patients in Mangaluru city area in the southwestern coastal region of India.

The aim of this study was to assess the clinical profile, severity and complications of patients suffering from malaria in Mangaluru, a southwestern coastal city in India. A total of 579 patients, who were treated at the District Wenlock Hospital, Mangaluru, and 168 healthy controls were recruited in this study. The clinical profile, haematological and biochemical parameters, and disease complications were assessed. The majority of patients were treated as outpatients and patients who had severe clinical conditions were admitted to the hospital for treatment and supportive care. Among the total 579 patients recruited in this study, the distribution of P. vivax, P. falciparum and mixed infections were 364 (62.9%), 150 (25.9%) and 65 (11.2%), respectively. Among these, 506 (87.4%) had mild malaria, whereas 73 (12.6%) had severe malaria. Overall, the clinical features and severity of malaria in P. vivax and mixed infection patients were comparable to P. falciparum patients, albeit with some significant differences. The clinical complications in severe malaria cases included thrombocytopenia (50.7%), metabolic acidosis (30.1%), severe anaemia (26.0%), jaundice (21.9%), hepatic dysfunction (15.1%), acute renal failure (6.8%), haematuria (8.2%), hypotension (9.6%), cerebral malaria (1.4%) and acute respiratory distress syndrome (1.4%). All the patients with severe malaria recruited in our study were successfully treated and discharged. Majority of patients had mild malaria, likely due to seeking treatment soon after experiencing symptoms and/or having preexisting immune protection. However, a significant number of patients had severe malaria and required hospital admission indicating that there is a substantial need for creating awareness among vulnerable immigrant population. Implementing effective surveillance and vector control measures in malaria hotspot locations in the city and educating people about preventive measures are likely to reduce the malaria burden in this endemic region.

Association between inflammatory cytokine levels and anemia during Plasmodium falciparum and Plasmodium vivax infections in Mangaluru: A Southwestern Coastal Region of India.

BACKGROUND AND OBJECTIVES: Dysregulated production of inflammatory cytokines might play important role in anemia during malaria infection. The objective of this study was to assess the extent of anemia due to malaria, associated complications, and inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin [IL]-6, and IL-10) across varying anemic intensity during malaria infections. MATERIALS AND METHODS: A hospital-based cross-sectional study was conducted at District Wenlock hospital in Mangaluru city. Samples from 627 patients and 168 healthy controls (HC) were analyzed for level of hemoglobin (Hb), red blood cells (RBCs), and inflammatory cytokines. The blood cell parameters and inflammatory cytokines levels across varying intensity of anemia were analyzed using Kruskal-Wallis test and pair-wise comparison between two groups were by Mann-Whitney U-test. Correlations were calculated by Pearson's and Spearman rank correlations. RESULTS: Compared to HC, Hb, and RBC levels were significantly lower in infected patients. On comparison with mild anemia patients (Hb 8-10.9 g/dL), the levels of TNF-α and IL-6 were significantly elevated, whereas IL-10 levels were lower during severe anemia (SA) (Hb <5 g/dL). In this endemic setting, we found a strong negative association between Hb levels and parasitemia, Hb and TNF-α, and positive relationship with IL-10; anemic patients also had significantly high TNF-α/IL-10 ratios. SA was associated with complications such as acute renal failure (16.0%), jaundice (16.0%), metabolic acidosis (24.0%), hypoglycemia (12.0%), hyperparasitemia (4.0%), and hepatic dysfunction (16.0%). CONCLUSIONS: Contrary to its benign reputation, Plasmodium vivax (Pv) infections can also result in severe malarial anemia (SMA) and its associated severe complications similar to Plasmodium falciparum infections. Dysregulated inflammatory cytokine responses play an important role in the pathogenesis of SMA, especially during Pv infections.

Association between Inflammatory Cytokine Levels and Thrombocytopenia during Plasmodium falciparum and P. vivax Infections in South-Western Coastal Region of India.

BACKGROUND: Thrombocytopenia is a most commonly observed complication during malaria infections. Inflammatory cytokines such as IL-1, IL-6, and IL-10 have been documented in malaria induced thrombocytopaenia. This study was aimed to understand the possible relationship between inflammatory cytokines across varying degrees of thrombocytopenia during P. vivax, P. falciparum, and mixed infections. METHODS: A hospital-based cross sectional study was conducted at District Wenlock Hospital in Mangaluru, a city situated along the south-western coastal region of Arabian Sea in India. In this study, blood samples from 627 malaria patients were analyzed for infected parasite species, clinical conditions, platelet levels, and key cytokines that are produced in response to infection; samples from 176 uninfected healthy individuals were used as controls. RESULTS: The results of our study showed a high prevalence of malarial thrombocytopenia (platelets <150 ×103/µl) in this endemic settings. About 62.7% patients had mild-to-moderate levels of thrombocytopenia and 16% patients had severe thrombocytopenia (platelets <50 × 103/µl). Upon comparison of cytokines across varying degrees of thrombocytopenia, irrespective of infecting species, the levels of TNF-α and IL-10 were significantly higher during thrombocytopenia, whereas IL-6 levels were considerably lower in severe thrombocytopenia patients suffering from P. vivax or P. falciparum infections. The severe clinical complications observed in patients with malarial thrombocytopenia included severe anemia (17.5%), acute renal failure (12.7%), jaundice (27.0%), metabolic acidosis (36.5%), spontaneous bleeding (3.2%), hypoglycemia (25.4%), hyperparasitemia (4.8%), acute respiratory distress syndrome (1.6%), pulmonary edema (19.0%), and cerebral malaria (1.6%) in various combinations. CONCLUSION: Overall, the results of our study suggest that inflammatory cytokines influence the transformation of mild forms of thrombocytopenia into severe forms during malarial infections. Further studies are needed to understand the association of inflammatory cytokine responses with severe malaria complications and thrombocytopenia.

Malaria Severity in Mangaluru City in the Southwestern Coastal Region of India.

Dakshina Kannada district in the Southwestern region of Karnataka state, India, including Mangaluru city is endemic to malaria. About 80% of malaria infections in Mangaluru and its surrounding areas are caused by Plasmodium vivax and the remainder is due to Plasmodium falciparum. Malaria-associated clinical complications significantly occur in this region. Here, we report the pathological conditions of 41 cases of fatal severe malaria, admitted to the district government hospital in Mangaluru city during January 2013 through December 2016. The results of clinical, hematological, and biochemical analyses showed that most of these severe malaria cases were associated with thrombocytopenia, anemia, metabolic acidosis, acute respiratory distress, and single or multi-organ dysfunction involving liver, kidney, and brain. Of the 41 fatal malaria cases, 24, 10, and seven patients had P. vivax, P. falciparum, and P. vivax and P. falciparum mixed infections, respectively. These data suggest that besides P. falciparum that is known to extensively cause severe and fatal malaria illnesses, P. vivax causes fatal illnesses substantially in this region, an observation that is consistent with recent findings in other regions.

Epidemiology, drug resistance, and pathophysiology of Plasmodium vivax malaria.

Malaria, caused by the protozoan parasites of the genus Plasmodium, is a major health problem in many countries of the world. Five parasite species namely, Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi, cause malaria in humans. Of these, P. falciparum and P. vivax are the most prevalent and account for the majority of the global malaria cases. In most areas of Africa, P. vivax infection is essentially absent because of the inherited lack of Duffy antigen receptor for chemokines on the surface of red blood cells that is involved in the parasite invasion of erythrocytes. Therefore, in Africa, most malaria infections are by P. falciparum and the highest burden of P. vivax infection is in Southeast Asia and South America. Plasmodium falciparum is the most virulent and as such, it is responsible for the majority of malarial mortality, particularly in Africa. Although, P. vivax infection has long been considered to be benign, recent studies have reported life-threatening consequences, including acute respiratory distress syndrome, cerebral malaria, multi-organ failure, dyserythropoiesis and anaemia. Despite exhibiting low parasite biomass in infected people due to parasite's specificity to infect only reticulocytes, P. vivax infection triggers higher inflammatory responses and exacerbated clinical symptoms than P. falciparum, such as fever and chills. Another characteristic feature of P. vivax infection, compared to P. falciparum infection, is persistence of the parasite as dormant liver-stage hypnozoites, causing recurrent episodes of malaria. This review article summarizes the published information on P. vivax epidemiology, drug resistance and pathophysiology.

Presence of novel triple mutations in the pvdhfr from Plasmodium vivax in Mangaluru city area in the southwestern coastal region of India.

BACKGROUND: Genes encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) are the targets of sulfadoxine-pyrimethamine (SP) present in artemisinin based combination therapy (ACT; artesunate + sulfadoxine pyrimethamine) for Plasmodium falciparum. Although SP is generally not used to treat vivax infection, mutations in dhfr and dhps that confer antifolate resistance in Plasmodium vivax are common; which may be attributed to its sympatric existence with P. falciparum. Current study was aimed to determine the pattern of mutations in dhfr and dhps in P. vivax isolates from Mangaluru region. METHODS: A total of 140 blood samples were collected from P. vivax-infected people attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of 140 isolates, 25 (18%) and 50 (36%) isolates were selected randomly for sequence analysis of pvdhfr and pvdhps genes respectively. Fragment of pvdhps and full length pvdhfr were amplified, sequenced and analysed for single nucleotide polymorphisms. dhps was analysed by PCR-RFLP also, to detect the two specific mutations (A383G and A553G). RESULTS: Analysis of pvdhps sequences from 50 isolates revealed single and double mutants at 38 and 46% respectively. Three non-synonymous mutations (K55R, S58R and S117N) were identified for pvdhfr. Among these, K55R was detected for the first time. CONCLUSIONS: The current study indicates that P. vivax dhps and dhfr mutant alleles are prevalent in this area, suggesting significant SP pressure.

Drug resistance genes: pvcrt-o and pvmdr-1 polymorphism in patients from malaria endemic South Western Coastal Region of India.

BACKGROUND: Malaria is highly prevalent in many parts of India and is mostly caused by the parasite species Plasmodium vivax followed by Plasmodium falciparum. Chloroquine (CQ) is the first-line treatment for blood stage P. vivax parasites, but cases of drug resistance to CQ have been reported from India. One of the surveillance strategies which is used to monitor CQ drug resistance, is the analysis of single nucleotide polymorphisms (SNPs) of the associated gene markers. Susceptibility to CQ can also be determined by copy number assessment of multidrug resistant gene (mdr-1). The current study has examined the prevalence of SNPs in P. vivax orthologs of P. falciparum chloroquine resistant and multi-drug resistant genes (pvcrt-o and pvmdr-1, respectively) and pvmdr-1 copy number variations in isolates from the highly endemic Mangaluru city near the South Western Coastal region of India. METHODS: A total of 140 blood samples were collected from P. vivax infected patients attending Wenlock Hospital Mangaluru during July 2014 to January 2016. Out of these 140 samples, sequencing was carried out for 54 (38.5%) and 85 (60.7%) isolates for pvcrt-o and pvmdr-1, respectively. Single nucleotide polymorphisms (SNPs) in the pvcrt-o and pvmdr-1 genes were analysed by direct sequencing method, while copy number variations of 60 isolates (42. 8%) were determined by real time PCR. RESULTS: Out of 54 clinical isolates analysed for pvcrt-o, three (5.6%) showed K10 insertion and the rest had wild type sequence. This is the first report to show K10 insertion in P. vivax isolates from India. Further, out of 85 clinical isolates of P. vivax analysed for mutations in pvmdr-1 gene, only one isolate had wild type sequence (~ 1%) while the remaining (99%) carried mutant alleles. Seven non-synonymous mutations with two novel mutations (I946V and Y1028C) were observed. Of all the observed mutations in pvmdr-1 gene, T958M was most highly prevalent (present in 90% of samples) followed by F1076L (76%), and Y976F (7%). Amplification of pvmdr-1 gene was observed in 31.6% of the isolates, out of 60 amplified. CONCLUSION: The observed variations both in pvmdr-1 and pvcrt-o genes indicate a trend towards parasite acquiring CQ resistance in this endemic area.

Optimization of novel halophilic lipase production by Fusarium solani strain NFCCL 4084 using palm oil mill effluent.

Among different sources of lipases, fungal lipases have continued to attract a wide range of applications. Further, halophilic lipases are highly desirable for biodiesel production due to the need to mitigate environmental pollution caused as result of extensive use of fossil fuels. However, currently, the high production cost limits the industrial application of lipases. In order to address this issue, we have attempted to optimize lipase production by Fusarium solani NFCCL 4084 and using palm oil mill effluent (POME) based medium. The production was optimized using a combinatory approach of Plackett-Burman (PB) design, one factor at a time (OFAT) design and face centred central composite design (FCCCD). The variables (malt extract, (NH4)2SO4, CaCl2, MgSO4, olive oil, peptone, K2HPO4, NaNO3, Tween-80, POME and pH) were analyzed using PB design and the variables with positive contrast coefficient were found to be K2HPO4, NaNO3, Tween-80, POME and pH. The significant variables selected were further analyzed for possible optimum range by using OFAT approach and the findings revealed that K2HPO4, NaNO3, and Tween-80 as the most significant medium components, and thus were further optimized by using FCCCD. The optimum medium yielded a lipase with an activity of 7.8 U/ml, a significant 3.2-fold increase compared to un-optimized medium. The present findings revealed that POME is an alternative and suitable substrate for halophilic lipase production at low cost. Also, it is clearly evident that the combinatory approach employed here proved to be very effective in producing high activity halophilic lipases, in general.

Malaria prevalence in Mangaluru city area in the southwestern coastal region of India.

BACKGROUND: Malaria is highly prevalent in many parts of India and the Indian subcontinent. Mangaluru, a city in the southwest coastal region of Karnataka state in India, and surrounding areas are malaria endemic with 10-12 annual parasite index. Despite high endemicity, to-date, very little has been reported on the epidemiology and burden of malaria in this area. METHODS: A cross-sectional surveillance of malaria cases was performed among 900 febrile symptomatic native people (long-time residents) and immigrant labourers (temporary residents) living in Mangaluru city area. During each of dry, rainy, and end of rainy season, blood samples from a group of 300 randomly selected symptomatic people were screened for malaria infection. Data on socio-demographic, literacy, knowledge of malaria, and treatment-seeking behaviour were collected to understand the socio-demographic contributions to malaria menace in this region. RESULTS: Malaria is prevalent in Mangaluru region throughout the year and Plasmodium vivax is predominant species compared to Plasmodium falciparum. The infection frequency was found to be high during rainy season. Infections were markedly higher in males than females, and in adults aged 16-45 years than both younger and older age groups. Also, malaria incidence was high among immigrants compared to native population. In both groups, infection rate was directly correlated with their literacy level, knowledge on malaria, dwelling environment, and protective measures used. There was also a significant difference in treatment-seeking behaviour between these two groups. CONCLUSIONS: Malaria incidences in Mangaluru region are predominantly localized to certain hotspot areas within the city, where socioeconomically underprivileged and immigrant labourers are densely populated. These areas have inadequate sanitation and constant water stagnation, harbouring high vector density and contributing to high infection incidences. Additionally, people in these areas seldom practice preventive measures such as using bed nets. The high incidences of malaria in adults are due to minimal cloth wearing, and long working hours stretching to late evenings in places with high vector density. Instituting heightened preventive public measures by governments and creating awareness on using preventive protective and environmental hygienic measures through educational programmes may substantially reduce the risk of contracting infections in these areas and spreading to other areas.

Malaria Transmission Under an Unusual Circumstance Causing Death in Two Siblings.

Two school-going siblings from a family residing in a presumed malaria non-endemic locality ∼90 km from Mangalore city in southwestern India contracted Plasmodium falciparum infection. In both cases, misunderstanding of initial clinical symptoms as due to viral hepatitis resulted in progression to severe malaria before malaria treatment was initiated. Despite treatment at a tertiary hospital, the children died of cerebral malaria and multi-organ dysfunction. Active case detection in the affected locality suggested that the infection was transmitted from infected individuals who worked in nearby malaria-endemic areas and periodically visited their families. A lesson from this study is that lethal falciparum malaria can be transmitted in regions of India, believed to be non-endemic for the disease, resulting in fatal outcomes if diagnosis is missed or delayed. Implementation of effective surveillance and control measures as well as preparedness for malaria detection and diagnosis are necessary in areas that are potentially disposed to malaria transmission even though they are presumed to be non-endemic.

Synthesis and biological evaluation of new imidazo[2,1-b][1,3,4]thiadiazole-benzimidazole derivatives.

In this report, we describe the synthesis and biological evaluation of a new series of 2-(imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-1H-benzimidazole derivatives (5a-ac). The molecules were analyzed by (1)H NMR, (13)C NMR, mass spectral and elemental data. The structure of one of the pre-final compounds, 6-(4-methoxyphenyl)-2-(4-methylphenyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde (4d) and that of a target compound, 2-[2-methyl-6-(4-methyl phenyl) imidazo[2,1-b][1,3,4]thiadiazol-5-yl]-1H-benzimidazole (5aa) were confirmed by single crystal XRD studies. All the target compounds were screened for in vitro anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv strain. Seven (5c, 5d, 5l, 5p, 5r, 5z and 5aa) out of twenty nine compounds showed potent anti-tubercular activity with a MIC of 3.125 µg/mL. A p-substituted phenyl group (p-tolyl or p-chlorophenyl) in the imidazo[2,1-b][1,3,4]thiadiazole ring and/or a chloro group in the benzimidazole ring enhance anti-tuberculosis activity whereas a nitro group in the benzimidazole ring reduces the activity. In the antibacterial screening, compounds 5i, 5w and 5ac showed promising activity against the tested bacterial strains. Further, antifungal and antioxidant activities of these molecules were also investigated. In the cytotoxicity study, the active antitubercular compounds exhibited very low toxicity against a normal cell line.