Structure modeling and antidiabetic activity of a seed protein of Momordica charantia in non-obese diabetic (NOD) mice

Momordica charantia is a well known medicinal plant used in the traditional medicinal system for the treatment of various diseases including diabetes mellitus. Recently, a novel protein termed as ADMc1 from the seed extract of M. charantia has been identified and isolated showing significant antihyperglycemic activity in type 1 diabetic rats in which diabetes was induced. However, the structure of this protein has not yet been analyzed. Homology modeling approach was used to generate a high quality protein 3D structure for the amino acid sequence of the ADMc1 protein in this study. The comparative assessment of secondary structures revealed ADMc1 as an all-alpha helix protein with random coils. Tertiary structure predicted on the template structure of Napin of B. Napus (PDB ID: 1SM7) with which the ADMc1 showed significant sequence similarity, was validated using protein structure validation tools like PROCHECK, WHAT_CHECK, VERIFY3D and ProSA. Arrangement of disulfide bridges formed by cysteine residues were predicted by the Dianna 1.1 server. The presence of multiple disulfide bond confers the stable nature of the ADMc1 protein. Further, the biological activity of the ADMc1 was assessed in non-obese diabetic (NOD) mice which are spontaneous model of type 1 diabetes. Significant reduction in the blood glucose levels of NOD mice was observed up to 8 h post administration of the rADMc1 protein. Overall, the structural characterizations with antihyperglycemic activity of this seed protein of Momordica charantia demonstrate its potential as an antidiabetic agent.     

Comparative Evaluation of Bivalent Malaria Rapid Diagnostic Tests versus Traditional Methods in Field with Special Reference to Heat Stability Testing in Central India

Background

Malaria presents a diagnostic challenge in areas where both Plasmodium falciparum and P.vivax are co-endemic. Bivalent Rapid Diagnostic tests (RDTs) showed promise as diagnostic tools for P.falciparum and P.vivax. To assist national malaria control programme in the selection of RDTs, commercially available seven malaria RDTs were evaluated in terms of their performance with special reference to heat stability.

Methodology/Principal Findings

This study was undertaken in four forested districts of central India (July, 2011– March, 2012). All RDTs were tested simultaneously in field along with microscopy as gold standard. These RDTs were stored in their original packing at 25°C before transport to the field or they were stored at 35°C and 45°C upto 100 days for testing the performance of RDTs at high temperature. In all 2841 patients with fever were screened for malaria of which 26% were positive for P.falciparum, and 17% for P.vivax. The highest sensitivity of any RDT for P.falciparum was 98% (95% CI; 95.9–98.8) and lowest sensitivity was 76% (95% CI; 71.7–79.6). For P.vivax highest and lowest sensitivity for any RDT was 80% (95% CI; 94.9 - 83.9) and 20% (95% CI; 15.6–24.5) respectively. Heat stability experiments showed that most RDTs for P.falciparum showed high sensitivity at 45°C upto 90 days. While for P.vivax only two RDTs maintained good sensitivity upto day 90 when compared with RDTs kept at room temperature. Agreement between observers was excellent for positive and negative readings for both P.falciparum and P.vivax (Kappa >0.6–0.9).

Conclusion

This is first field evaluation of RDTs regarding their temperature stability. Although RDTs are useful as diagnostic tool for P.falciparum and P.vivax even at high temperature, the quality of RDTs should be regulated and monitored more closely.     

Exploring the Chemical Space around 8-Mercaptoguanine as a Route to New Inhibitors of the Folate Biosynthesis Enzyme HPPK

As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg^2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (K_D ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC₅₀ ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N ⁹ substitution, or tautomerization arising from N ⁷ substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N ⁹-H···Val46 hydrogen bond, combined with the limited space available around the N ⁹ position. The water-filled pocket under the N ⁷ position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N ⁷ (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (K_D ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N ⁷ position towards the Mg²⁺-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg²⁺ centres or Mg²⁺ -binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.     

Increased Carotid Intima-Media Thickness and Reduced Distensibility in Human Class III Obesity: Independent and Differential Influences of Adiposity and Blood Pressure on the Vasculature

Carotid intima-media-thickness (cIMT) and carotid distensibility (distensibility), structural and functional properties of carotid arteries respectively, are early markers, as well as strong predictors of cardiovascular disease (CVD). The characteristic of these two parameters in individuals with BMI>40.0 kg/m² (Class III obesity), however, are largely unknown. The present study was designed to document cIMT and distensibility in this population and to relate these to other factors with established association with CVD in obesity. The study included 96 subjects (65 with BMI>40.0 kg/m² and 31, age- and gender-matched, with BMI of 18.5 to 30.0 kg/m²). cIMT and distensibility were measured by non-invasive high resolution ultrasonography, circulatory CD133⁺/KDR⁺ angiogenic cells and endothelial microparticles (EMP) by flow cytometry, and plasma levels of adipokines, growth factors and cytokines by Luminex immunoassay kits. The study results demonstrated increased cIMT (0.62±0.11 mm vs. 0.54±0.08 mm, P = 0.0002) and reduced distensibility (22.52±10.79 10⁻³kpa⁻¹ vs. 29.91±12.37 10⁻³kpa⁻¹, P<0.05) in individuals with BMI>40.0 kg/m². Both cIMT and distensibility were significantly associated with traditional CVD risk factors, adiposity/adipokines and inflammatory markers but had no association with circulating angiogenic cells. We also demonstrated, for the first time, elevated plasma EMP levels in individuals with BMI>40.0 kg/m². In conclusion, cIMT is increased and distensibility reduced in Class III obesity with the changes predominantly related to conventional CVD risk factors present in this condition, demonstrating that both cIMT and distensibility remain as CVD markers in Class III obesity.     

A New Miocene-Divergent Lineage of Old World Racer Snake from India

A distinctive early Miocene-divergent lineage of Old world racer snakes is described as a new genus and species based on three specimens collected from the western Indian state of Gujarat. Wallaceophis gen. et. gujaratenesis sp. nov. is a members of a clade of old world racers. The monotypic genus represents a distinct lineage among old world racers is recovered as a sister taxa to Lytorhynchus based on ~3047bp of combined nuclear (cmos) and mitochondrial molecular data (cytb, ND4, 12s, 16s). The snake is distinct morphologically in having a unique dorsal scale reduction formula not reported from any known colubrid snake genus. Uncorrected pairwise sequence divergence for nuclear gene cmos between Wallaceophis gen. et. gujaratenesis sp. nov. other members of the clade containing old world racers and whip snake is 21–36%.     

Cost Effectiveness of Implementing Integrated Management of Neonatal and Childhood Illnesses Program in District Faridabad,India

Introduction

Despite the evidence for preventing childhood morbidity and mortality, financial resources are cited as a constraint for Governments to scale up the key health interventions in some countries. We evaluate the cost effectiveness of implementing IMNCI program in India from a health system and societal perspective.

Methods

We parameterized a decision analytic model to assess incremental cost effectiveness of IMNCI program as against routine child health services for infant population at district level in India. Using a 15-years time horizon from 2007 to 2022, we populated the model using data on costs and effects as found from a cluster-randomized trial to assess effectiveness of IMNCI program in Haryana state. Effectiveness was estimated as reduction in infant illness episodes, deaths and disability adjusted life years (DALY). Incremental cost per DALY averted was used to estimate cost effectiveness of IMNCI. Future costs and effects were discounted at a rate of 3%. Probabilistic sensitivity analysis was undertaken to estimate the probability of IMNCI to be cost effective at varying willingness to pay thresholds.

Results

Implementation of IMNCI results in a cumulative reduction of 57384 illness episodes, 2369 deaths and 76158 DALYs among infants at district level from 2007 to 2022. Overall, from a health system perspective, IMNCI program incurs an incremental cost of USD 34.5 (INR 1554) per DALY averted, USD 34.5 (INR 1554) per life year gained, USD 1110 (INR 49963) per infant death averted. There is 90% probability for ICER to be cost effective at INR 2300 willingness to pay, which is 5.5% of India’s GDP per capita. From a societal perspective, IMNCI program incurs an additional cost of USD 24.1 (INR 1082) per DALY averted, USD 773 (INR 34799) per infant death averted and USD 26.3 (INR 1183) per illness averted in during infancy.

Conclusion

IMNCI program in Indian context is very cost effective and should be scaled-up as a major child survival strategy.     

Is MTHFR 677 C>T Polymorphism Clinically Important in Polycystic Ovarian Syndrome (PCOS)? A Case-Control Study,Meta-Analysis and Trial Sequential Analysis

Background

Optimum efficiency of the folate pathway is considered essential for adequate ovarian function. 677 C>T substitution in the 5, 10-methylene tertrahydrofolatereductase (MTHFR) gene compromises activity of the MTHFR enzyme by about 50%. The significance of correlation between 677C>T substitution and PCOS remains dubious due to the low power of published studies.

Methods and Results

We analyzed MTHFR 677 C>T site in ethnically two different PCOS case-control groups (total 261 cases and 256 controls) from India. The data analysis revealed a lack of association between this polymorphism and PCOS [OR = 1.11 (95%CI = 0.71–1.72), P = 0.66]. Group-wise analysis on the basis of ethnicity also revealed no association in any of the ethnic groups [Indo-Europeans, P = 1; Dravidians, P = 0.70]. Homocysteine levels did not differ significantly between cases (15.51 μmol/L, SD = 2.89) and controls (15.89 μmol/L, SD = 2.23). We also undertook a meta-analysis on 960 cases and 1028 controls, which suggested a significant association of the substitution with PCOS in the dominant model of analysis (OR = 1.47 (95%CI = 1.04–2.09), P = 0.032]. Trial sequential analysis corroborated findings of the traditional meta-analysis. However, we found that the conclusions of meta-analysis were strongly influenced by studies that deviated from the Hardy Weinberg equilibrium. A careful investigation of each study and a trial sequential analysis suggested that 677 C>T substitution holds no clinical significance in PCOS in most of the populations.

Conclusion

In conclusion, MTHFR 677 C>T polymorphism does not affect PCOS risk in India. The association seen in the meta-analysis is due to an outlier study and studies showing deviation from the Hardy Weinberg equilibrium.