NS1643 Interacts around L529 of hERG to Alter Voltage Sensor Movement on the Path to Activation

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V_1/2 of activation of L529I (−34 ± 4 mV) is similar to wild-type (WT) (−37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V_1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (−131 ± 4 mV for K525C and −120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.     

Epistasis among Deleterious Mutations in the HIV-1 Protease

A central goal in molecular evolution is to understand how genetic interactions between protein mutations shape protein function and fitness. While intergenic epistasis has been extensively explored in eukaryotes, bacteria, and viruses, intragenic epistatic interactions have been insufficiently studied. Here, we employ a model system in which lambda phage fitness correlates with the enzymatic activity of human immunodeficiency virus type 1 (HIV-1) protease to systematically determine the epistatic interactions between intragenic pairs of deleterious protein substitutions. We generated 114 genotypes of the HIV-1 protease, each carrying pairs of nucleotide substitution mutations whose separated and combined deleterious effects on fitness were then determined. A high proportion (39%) of pairs displayed lethality. Several pairs exhibited significant interactions for fitness, including positive and negative epistasis. Significant negative epistatic interactions predominated (15%) over positive interactions (2%). However, the average ± SD epistatic effect, ē = 0.0025 ± 0.1334, was not significantly different from zero (p = 0.8368). Notably, epistatic interactions, regardless of epistatic direction, tend to be more frequent in the context of less deleterious mutations. In the present study, the high frequencies of lethality and negative epistasis indicate that the HIV-1 protease is highly sensitive to the effects of deleterious mutations. Therefore, proteins may not be as robust to mutational change as is usually expected.     

Substitution of NS5 N-terminal Domain of Dengue Virus Type 2 RNA with Type 4 Domain Caused Impaired Replication and Emergence of Adaptive Mutants with Enhanced Fitness

Flavivirus NS3 and NS5 are required in viral replication and 5′-capping. NS3 has NS2B-dependent protease, RNA helicase, and 5′-RNA triphosphatase activities. NS5 has 5′-RNA methyltransferase (MT)/guanylyltransferase (GT) activities within the N-terminal 270 amino acids and the RNA-dependent RNA polymerase (POL) activity within amino acids 271–900. A chimeric NS5 containing the D4MT/D4GT and the D2POL domains in the context of wild-type (WT) D2 RNA was constructed. RNAs synthesized in vitro were transfected into baby hamster kidney cells. The viral replication was analyzed by an indirect immunofluorescence assay to monitor NS1 expression and by quantitative real-time PCR. WT D2 RNA-transfected cells were NS1- positive by day 5, whereas the chimeric RNA-transfected cells became NS1-positive ∼30 days post-transfection in three independent experiments. Sequence analysis covering the entire genome revealed the appearance of a single K74I mutation within the D4MT domain ∼16 days post-transfection in two experiments. In the third, D290N mutation in the conserved NS3 Walker B motif appeared ≥16 days post-transfection. A time course study of serial passages revealed that the 30-day supernatant had gradually evolved to gain replication fitness. Trans-complementation by co-expression of WT D2 NS5 accelerated viral replication of chimeric RNA without changing the K74I mutation. However, the MT and POL activities of NS5 WT D2 and the chimeric NS5 proteins with or without the K74I mutation are similar. Taken together, our results suggest that evolution of the functional interactions involving the chimeric NS5 protein encoded by the viral genome species is essential for gain of viral replication fitness.     

Near-Neighbor Interactions in the NS3-4A Protease of HCV Impact Replicative Fitness of Drug-Resistant Viral Variants

A variety of amino acid substitutions in the NS3-4A protease of the hepatitis C virus lead to protease inhibitor (PI) resistance. Many of these significantly impair the replication fitness of the resistant variants in a genotype- and subtype-dependent manner, a critical factor in determining the probability with which resistant variants will persist. However, the underlying molecular mechanisms are unknown. Here, we present a novel residue-interaction network approach to determine how near-neighbor interactions of PI resistance mutations in NS3-4A can impact protease functional sites dependent on their genomic background. We constructed subtype-specific consensus residue networks for subtypes 1a and 1b from protease structure ensembles combined with biological properties of protein residues and evolutionary amino acid conservation. By applying local and global network topology analysis and visual exploration, we characterize PI resistance-associated sites and outline differences in near-neighbor interactions. We find local residue-interaction patterns and features at protease functional sites that are subtype specific. The noncovalent bonding patterns indicate higher fitness costs conferred by PI resistance mutations in a subtype 1b genomic background and explain the prevalence of Q80K and R155K in subtype 1a. Based on local residue interactions, we predict a subtype-specific role for the protease residue NS3–Q80 in molecular mechanisms related to the assembly of infectious virus particles that is supported by experimental data on the capacity of Q80K variants to replicate and produce infectious virus in subtype 1a and 1b cell culture.     

Deep mutational scanning and massively parallel kinetics of plasminogen activator inhibitor-1 functional stability to probe its latency transition

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor superfamily of proteins, is unique among serine protease inhibitors for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ∼1 to 2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 °C. Of the 7201 possible single amino acid substitutions in PAI-1, we identified 439 that increased the functional stability of PAI-1 beyond that of the WT protein. We also found 1549 single amino acid substitutions that retained inhibitory activity toward the canonical target protease of PAI-1 (urokinase-like plasminogen activator), whereas exhibiting functional stability less than or equal to that of WT PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying the latency transition of PAI-1 and provide a database for interpreting human PAI-1 genetic variants.     

Stress response of biomolecules (carbohydrate,protein and lipid profiles) in fish Channa punctatus inhabiting river polluted by Thermal Power Plant effluent

Qualitative and quantitative assessment of heavy metals in the Thermal Power Plant effluent was performed to study the impact of their toxic effects on various biomarkers (carbohydrate, protein and lipid profiles). Heavy metals present in the water were in the order Fe > Cu > Zn > Mn > Ni > Co > Cr. Fe and Ni exceeded and Cr was equal to the USA standards set by UNEPGEMS. Glycogen in liver (p < 0.001) and muscle (p < 0.01) depleted significantly. Insignificant (p < 0.05) decline in blood glucose (−21.0%) and significant (p < 0.05) elevation in both total protein and globulin in serum, liver and muscle was noted. Albumin decreased significantly (p < 0.01) in serum but showed significant (p < 0.05) increase in liver and muscle. Thus A:G ratio fell in serum and rose in liver and muscle. Similarly lipid profile also gets altered where significant elevation in serum total lipid (p < 0.01), total cholesterol (p < 0.01), phospholipid (p < 0.05), triglycerides (p < 0.001), LDL (p < 0.01) was observed but significant (p < 0.05) decline in VLDL was recorded. These biomarkers suggested that fish become hypoglycemic, hyperlipidemic and hypercholesterolemic. Heavy metals also provoked immune response as evident from the rise in globulin. In conclusion the Thermal Power Plant wastewater containing heavy metals induced stress, making fish weak and vulnerable to diseases.     

Pleiotropic Effects of a Mitochondrial–Nuclear Incompatibility Depend upon the Accelerating Effect of Temperature in Drosophila

Interactions between mitochondrial and nuclear gene products that underlie eukaryotic energy metabolism can cause the fitness effects of mutations in one genome to be conditional on variation in the other genome. In ectotherms, the effects of these interactions are likely to depend upon the thermal environment, because increasing temperature accelerates molecular rates. We find that temperature strongly modifies the pleiotropic phenotypic effects of an incompatible interaction between a Drosophila melanogaster polymorphism in the nuclear-encoded, mitochondrial tyrosyl-transfer (t)RNA synthetase and a D. simulans polymorphism in the mitochondrially encoded tRNA^Tyr. The incompatible mitochondrial–nuclear genotype extends development time, decreases larval survivorship, and reduces pupation height, indicative of decreased energetic performance. These deleterious effects are ameliorated when larvae develop at 16° and exacerbated at warmer temperatures, leading to complete sterility in both sexes at 28°. The incompatible genotype has a normal metabolic rate at 16° but a significantly elevated rate at 25°, consistent with the hypothesis that inefficient energy metabolism extends development in this genotype at warmer temperatures. Furthermore, the incompatibility decreases metabolic plasticity of larvae developed at 16°, indicating that cooler development temperatures do not completely mitigate the deleterious effects of this genetic interaction. Our results suggest that the epistatic fitness effects of metabolic mutations may generally be conditional on the thermal environment. The expression of epistatic interactions in some environments, but not others, weakens the efficacy of selection in removing deleterious epistatic variants from populations and may promote the accumulation of incompatibilities whose fitness effects will depend upon the environment in which hybrids occur.     

Engineering trypsin for inhibitor resistance

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [k_cat/K_M = (1.2 ± 0.3) × 10⁷ M⁻¹ s⁻¹. Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.     

Comparative study of high sensitivity troponin T and heart-type fatty acid-binding protein in STEMI patients

Aim and background

Heart-type fatty acid-binding proteins (H-FABP) which are detected within 2–3 h of acute myocardial infarction are involved in uptake of free fatty acids in the myocardium. Our aim in the present study is to compare window periods of H-FABP to high sensitivity troponin T (hs-Trop T) in acute ST elevation myocardial infarction (STEMI).

Methods

160 STEMI diagnosed patient’s serum samples are analyzed for hs-Trop T and H-FABP. Different window periods of chest pain onset (<3 h, 3–6 h and >6 h) are compared with complications, in-hospital mortality and statistically analyzed.

Results

From 160 patients, 53 (33%) cases are presented in <3 h, 75 (47%) in 3–6, and 32 (20%) after >6 h respectively. Accordingly sensitivity of hs-Trop T was 92%, 94% and 97% while H-FABP was 75%, 88% and 84%, respectively. Overall sensitivity was 94% and 82% respectively. Statistically significant difference between mean hs-Trop T values with respect to window period <3, 3–6 and >6 h was 0.21, 0.35 and 0.80 ng/ml respectively, p value < 0.0001. No significant difference in H-FABP values was observed.Hs-Trop T positively correlated with age (r = 0.153, P = 0.05), window period (r = 0.363, P < 0.0001), TIMI score (r = 0.208, P = 0.008), ejection fraction (r = 0.191, P = 0.008), serum H-FABP (r = 0.229, P = 0.004), and serum hs-CRP (r = 0.326, p < 0.001). There was a statistically significant difference of mean hs-Trop T values with or without in hospital mortality (0.35 vs. 0.85 ng/ml, respectively, p = 0.008).No significant correlation to age, TIMI score, ejection fraction and hs-CRP values for H-FABP was observed.

Conclusion

It appears that hs-Trop T is a more sensitive marker than H-FABP in early hours of AMI and higher hs-Trop T predicts increase in-hospital mortality.     

A Multi-Variant,Viral Dynamic Model of Genotype 1 HCV to Assess the in vivo Evolution of Protease-Inhibitor Resistant Variants

Variants resistant to compounds specifically targeting HCV are observed in clinical trials. A multi-variant viral dynamic model was developed to quantify the evolution and in vivo fitness of variants in subjects dosed with monotherapy of an HCV protease inhibitor, telaprevir. Variant fitness was estimated using a model in which variants were selected by competition for shared limited replication space. Fitness was represented in the absence of telaprevir by different variant production rate constants and in the presence of telaprevir by additional antiviral blockage by telaprevir. Model parameters, including rate constants for viral production, clearance, and effective telaprevir concentration, were estimated from 1) plasma HCV RNA levels of subjects before, during, and after dosing, 2) post-dosing prevalence of plasma variants from subjects, and 3) sensitivity of variants to telaprevir in the HCV replicon. The model provided a good fit to plasma HCV RNA levels observed both during and after telaprevir dosing, as well as to variant prevalence observed after telaprevir dosing. After an initial sharp decline in HCV RNA levels during dosing with telaprevir, HCV RNA levels increased in some subjects. The model predicted this increase to be caused by pre-existing variants with sufficient fitness to expand once available replication space increased due to rapid clearance of wild-type (WT) virus. The average replicative fitness estimates in the absence of telaprevir ranged from 1% to 68% of WT fitness. Compared to the relative fitness method, the in vivo estimates from the viral dynamic model corresponded more closely to in vitro replicon data, as well as to qualitative behaviors observed in both on-dosing and long-term post-dosing clinical data. The modeling fitness estimates were robust in sensitivity analyses in which the restoration dynamics of replication space and assumptions of HCV mutation rates were varied.     

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

The incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The half-life of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (T_m) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of K_m and k_cat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications.     

Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis

The aggregation of α-synuclein (α-Syn) is linked to Parkinson’s disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of α-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free α-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) α-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of α-Syn dimers could lead to different aggregation pathways.     

Deconvoluting the Composition of Low-Frequency Hepatitis C Viral Quasispecies: Comparison of Genotypes and NS3 Resistance-Associated Variants between HCV/HIV Coinfected Hemophiliacs and HCV Monoinfected Patients in Japan

Pre-existing low-frequency resistance-associated variants (RAVs) may jeopardize successful sustained virological responses (SVR) to HCV treatment with direct-acting antivirals (DAAs). However, the potential impact of low-frequency (∼0.1%) mutations, concatenated mutations (haplotypes), and their association with genotypes (Gts) on the treatment outcome has not yet been elucidated, most probably owing to the difficulty in detecting pre-existing minor haplotypes with sufficient length and accuracy. Herein, we characterize a methodological framework based on Illumina MiSeq next-generation sequencing (NGS) coupled with bioinformatics of quasispecies reconstruction (QSR) to realize highly accurate variant calling and genotype-haplotype detection. The core-to-NS3 protease coding sequences in 10 HCV monoinfected patients, 5 of whom had a history of blood transfusion, and 11 HCV/HIV coinfected patients with hemophilia, were studied. Simulation experiments showed that, for minor variants constituting more than 1%, our framework achieved a positive predictive value (PPV) of 100% and sensitivities of 91.7–100% for genotyping and 80.6% for RAV screening. Genotyping analysis indicated the prevalence of dominant Gt1a infection in coinfected patients (6/11 vs 0/10, p = 0.01). For clinical samples, minor genotype overlapping infection was prevalent in HCV/HIV coinfected hemophiliacs (10/11) and patients who experienced whole-blood transfusion (4/5) but none in patients without exposure to blood (0/5). As for RAV screening, the Q80K/R and S122K/R variants were particularly prevalent among minor RAVs observed, detected in 12/21 and 6/21 cases, respectively. Q80K was detected only in coinfected patients, whereas Q80R was predominantly detected in monoinfected patients (1/11 vs 7/10, p < 0.01). Multivariate interdependence analysis revealed the previously unrecognized prevalence of Gt1b-Q80K, in HCV/HIV coinfected hemophiliacs [Odds ratio = 13.4 (3.48–51.9), p < 0.01]. Our study revealed the distinct characteristics of viral quasispecies between the subgroups specified above and the feasibility of NGS and QSR-based genetic deconvolution of pre-existing minor Gts, RAVs, and their interrelationships.     

Robust prognostic model based on immune infiltration‐related genes and clinical information in ovarian cancer

Immune infiltration of ovarian cancer (OV) is a critical factor in determining patient''s prognosis. Using data from TCGA and GTEx database combined with WGCNA and ESTIMATE methods, 46 genes related to OV occurrence and immune infiltration were identified. Lasso and multivariate Cox regression were applied to define a prognostic score (IGCI score) based on 3 immune genes and 3 types of clinical information. The IGCI score has been verified by K‐M curves, ROC curves and C‐index on test set. In test set, IGCI score (C‐index = 0.630) is significantly better than AJCC stage (C‐index = 0.541, p < 0.05) and CIN25 (C‐index = 0.571, p < 0.05). In addition, we identified key mutations to analyse prognosis of patients and the process related to immunity. Chi‐squared tests revealed that 6 mutations are significantly (p < 0.05) related to immune infiltration: BRCA1, ZNF462, VWF, RBAK, RB1 and ADGRV1. According to mutation survival analysis, we found 5 key mutations significantly related to patient prognosis (p < 0.05): CSMD3, FLG2, HMCN1, TOP2A and TRRAP. RB1 and CSMD3 mutations had small p‐value (p < 0.1) in both chi‐squared tests and survival analysis. The drug sensitivity analysis of key mutation showed when RB1 mutation occurs, the efficacy of six anti‐tumour drugs has changed significantly (p < 0.05).     

The rs340874 PROX1 type 2 diabetes mellitus risk variant is associated with visceral fat accumulation and alterations in postprandial glucose and lipid metabolism

Large-scale meta-analyses of genome-wide association studies have recently confirmed that the rs340874 single-nucleotide polymorphism in PROX1 gene is associated with fasting glycemia and type 2 diabetes mellitus; however, the mechanism of this link was not well established. The aim of our study was to evaluate the functional/phenotypic differences related to rs340874 PROX1 variants. The study group comprised 945 subjects of Polish origin (including 634 with BMI > 25) without previously known dysglycemia. We analyzed behavioral patterns (diet, physical activity), body fat distribution and glucose/fat metabolism after standardized meals and during the oral glucose tolerance test. We found that the carriers of the rs340874 PROX1 CC genotype had higher nonesterified fatty acids levels after high-fat meal (p = 0.035) and lower glucose oxidation (p = 0.014) after high-carbohydrate meal in comparison with subjects with other PROX1 genotypes. Moreover, in subjects with CC variant, we found higher accumulation of visceral fat (p < 0.02), but surprisingly lower daily food consumption (p < 0.001). We hypothesize that lipid metabolism alterations in subjects with the PROX1 CC genotype may be a primary cause of higher glucose levels after glucose load, since the fatty acids can inhibit insulin-stimulated glucose uptake by decreasing carbohydrate oxidation. Our observations suggest that the PROX1 variants have pleiotropic effect on disease pathways and it seem to be a very interesting goal of research on prevention of obesity and type 2 diabetes mellitus. The study may help to understand the mechanisms of visceral obesity and type 2 diabetes mellitus risk development.     

Real-life use of vitamin D3-fortified bread and milk during a winter season: the effects of CYP2R1 and GC genes on 25-hydroxyvitamin D concentrations in Danish families,the VitmaD study

Common genetic variants rs10741657 and rs10766197 in CYP2R1 and rs4588 and rs842999 in GC and a combined genetic risk score (GRS) of these four variants influence late summer 25-hydroxyvitamin D (25(OH)D) concentrations. The objectives were to identify those who are most at risk of developing low vitamin D status during winter and to assess whether vitamin D₃-fortified bread and milk will increase 25(OH)D concentrations in those with genetically determined low 25(OH)D concentrations at late summer. We used data from the VitmaD study. Participants were allocated to either vitamin D₃-fortified bread and milk or non-fortified bread and milk during winter. In the fortification group, CYP2R1 (rs10741657) and GC (rs4588 and rs842999) were statistically significantly associated with winter 25(OH)D concentrations and CYP2R1 (rs10766197) was borderline significant. There was a negative linear trend between 25(OH)D concentrations and carriage of 0–8 risk alleles (p < 0.0001). No association was found for the control group (p = 0.1428). There was a significant positive linear relationship between different quintiles of total vitamin D intake and the increase in 25(OH)D concentrations among carriers of 0–2 (p = 0.0012), 3 (p = 0.0001), 4 (p = 0.0118) or 5 (p = 0.0029) risk alleles, but not among carriers of 6–8 risk alleles (p = 0.1051). Carriers of a high GRS were more prone to be vitamin D deficient compared to carriers of a low GRS. Furthermore, rs4588-AA carriers have a low but very stable 25(OH)D concentration, and interestingly, also low PTH level.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0413-7) contains supplementary material, which is available to authorized users.     

Morphometric study of Kalophrynus palmatissimus at two forest reserves in Malaysia

A research study on morphometrics of Kalophrynus palmatissimus (commonly known as Lowland Grainy Frog) at Ayer Hitam Forest Reserve (AHFR), Selangor and Pasoh Forest Reserve (PFR), Negeri Sembilan was carried out from 12 November 2016 to 13 September 2017. The study was to examine data on the morphometric traits of K. palmatissimus at the two forest reserves. 15 morphometric traits of K. palmatissimus that were taken by using vernier calipers. Frog surveys were done by using 15 and 18 nocturnal 400 m transect lines with an interval distance of 20 m at AHFR and PFR, respectively. The GPS coordinates for all frog samples were recorded to ensure the precise geographic location. In addition, five climatic data were recorded. The results showed that most morphometric traits in AHFR (n = 34) and PFR (n = 31) were positively correlated with each other. On the other hand, climatic factor, which was soil pH, had a significant positive influence on most of the morphometric traits (p < .01), except for tympanum diameter and upper eyelid width (p ≥ .05). Meanwhile, the temperature had a significantly negative influence on all morphometric traits (p < .01). General linear model (GLM) analysis showed that snout‐vent length (SVL) influenced most morphometric traits (F ≤ 80.86, p < .01), except for hand length (HAL: F = 0.299, p > .05). Later, it was found that the snout‐vent length of K. palmatissimus at AHFR was slightly larger than at PFR (AHFR: μ = 37.00 mm, SE = 1.16 c.f. PFR: μ = 30.29 mm, SE = 1.07). It showed that there were variations in morphometric traits of K. palmatissimus at AHFR and PFR. From PCA analysis, morphometric traits are grouped into two components for AHFR and PFR, respectively. In AHFR, head length, eye diameter, head width, internarial distance, interorbital distance, forearm length, tibia length, foot length, and thigh length were strongly correlated, while snout length and eye‐nostril distance were strongly correlated. In PFR, eye diameter, head width, internarial distance, interorbital distance, foot length, and thigh length were strongly correlated, though snout length and eye‐nostril distance were strongly correlated, hence, suggested that all morphometric traits grow simultaneously in K. palmatissimus with eye‐nostril distance (EN), and snout length (SL) growing almost simultaneously at AHFR (r = .91) and PFR (r = .97). There is still a lack of available information regarding the distribution and morphometric studies of K. palmatissimus in Malaysia, especially at AHFR and PFR. This study showed 15 different morphometric traits of K. palmatisssimus between AHFR and PFR, with K. palmatissimus at AHFR were found to be slightly larger than at PFR.     

The performance of the SD BIOLINE Dengue DUO? rapid immunochromatographic test kit for the detection of NS1 antigen,IgM and IgG antibodies during a dengue type 1 epidemic in Jamaica

Background

Dengue is an important mosquito-borne viral infection that affects millions of persons worldwide. Early diagnosis is necessary to effect appropriate management and decrease mortality. Immunochromatographic tests are advantageous in producing dengue test results within 30 min but these results should be sensitive and specific. In this study we evaluated the diagnostic performance of the SD BIOLINE Dengue DUO® rapid immunochromatographic test kit. A panel of 309 dengue and 30 non-dengue single serum samples characterized by using reference enzyme-linked immunosorbent assays (ELISAs) was used. These samples were received in the virology laboratory for routine testing during a dengue type 1 outbreak between October to December, 2012.

Results

The overall diagnostic sensitivities of the SD BIOLINE Dengue DUO® rapid testfor IgM, IgG and NSI were 49.3 % (95 % CI: 41.3-57.4), 39.1 % (95 % CI: 33.3-45.2) and 90 % (95 % CI: 82.1-94.7), respectively. The IgM and IgG detection rates were significantly lower than that of the NSI (p < 0.001). However the combination of the IgM detection with NS1 detection or both NS1 and IgG resulted in a significant (p < 0.001) increase in sensitivity to 97.5 % (95 % CI: 92.9-99.2) and 98.9 % (95 % CI: 96.0-99.7), respectively. These higher sensitivities were achieved without any decrease in specificities.

Conclusions

This study revealed that combining two or more parameters of the SD BIOLINE Dengue DUO® rapid kit significantly improved the sensitivity of diagnosis of dengue virus infection and supports its usefulness in the Jamaican setting.