GAS CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS OF BIOACTIVE COMPONENTS FROM THE RHIZOME EXTRACT OF NARDOSTACHYS JATAMANSI DC

ABSTRACTIn this study, the bioactive compounds of Nardostachys jatamansi have been evaluated using gas chromatography-mass spectrometry (GC-MS). Thechemical composition of the rhizome of N. jatamansi, petroleum ether, chloroform, and ethanol extract was investigated using agilent 7890 GC-MSinstrument. N. jatamansi extract contains 61 compounds, i.e., actinidine (11.2%), indane (28.7%), aristolene (7.2%), gurjuenen (5.5%), valencene(8.9%), globulol (8.2%), betapatachoulene (8.4%), etc. Actinidine and indane are the major compounds in the ethanol extract along with few minorcompounds.Keywords: Chloroform extract, Gas chromatography-mass spectrometry analysis, Nardostachys jatamansi

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

Developmental Stage- and Cell Cycle Number-Dependent Changes in Characteristics of Plasmodium falciparum-Infected Erythrocyte Adherence to Placental Chondroitin-4-Sulfate Proteoglycan▿

The adherence of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin-4-sulfate (C4S). Although IRBC binding to C4S has been unequivocally established, the adherence characteristics of IRBCs at different stages of parasite development and through successive parasite generations after selection for C4S adherence are not known. Here we show that IRBCs acquire a significant capacity to bind to C4S at as early as 14 h and exhibit maximum binding at 22 to 26 h postinvasion. Surprisingly, the IRBC binding ability decreases by ∼50% at the late trophozoite and schizont stages. The binding strength of the IRBCs also gradually decreases during successive generations after selection for C4S binding, and at the 32nd generation, the binding capacity was only ∼31% of that of IRBCs at the 2nd generation, suggesting that IRBCs eventually lose their C4S-adherent capacity. We also tested the susceptibility of the adhesive protein(s) on the IRBC surface to trypsin treatment at different stages of parasite development. The data show that IRBCs with late trophozoites are more resistant to trypsin treatment than those containing early trophozoites, indicating that parasite proteins expressed on the IRBC surface during trophozoite maturation partially mask accessibility of adhesive protein for binding to C4S. These data provide important insights into the expression pattern of the C4S-adhesive protein(s) on the IRBC surface, emphasizing the need for understanding the regulation of genes involved in IRBC binding to C4S. Our data also define the parasite stage at which IRBCs are suitable for studying structural interactions with C4S

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

Abstract 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

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</p

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

Abstract 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

Chondroitin Sulfate Proteoglycan Expression and Binding of Plasmodium falciparum-Infected Erythrocytes in the Human Placenta during Pregnancy

A characteristic feature of malaria during pregnancy is the sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the intervillous spaces of the placenta. We have recently shown that unusually low-sulfated chondroitin sulfate proteoglycans (CSPGs) present in the intervillous spaces mediate the adherence of IRBCs in the placenta. In areas of endemicity, the prevalence of P. falciparum infection in pregnant women peaks during weeks 13 to 20 and then gradually declines, implying that the placental CSPGs are available for IRBC adhesion early during the pregnancy. However, there is no information on the expression and composition of CSPGs during pregnancy. In this study, the expression pattern of CSPGs during the course of pregnancy was investigated. The CSPGs were purified from placentas of various gestational ages, characterized, and tested for the ability to bind IRBCs. The data demonstrate that the CSPGs are present in the intervillous spaces throughout the second and third trimesters. The levels of CSPGs expressed per unit tissue weight were similar in placentas of various gestational ages. However, the structures of the intervillous-space CSPGs changed considerably during the course of pregnancy. In particular, the molecular weight was decreased, with an accompanying gradual increase in the CSPG size polydispersity, from 16 weeks until 38 weeks. The sulfate content was increased considerably after 24 weeks. Despite these structural changes, the CSPGs of placentas of various gestational ages efficiently supported the binding of IRBCs. These results demonstrate that CSPGs can mediate the sequestration of IRBCs in the intervillous spaces of the placenta during the entire second and third trimesters and possibly during the later part of the first trimester as well

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

Abstract 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