Discovery of antagonists of tick dopamine receptors via chemical library screening and comparative pharmacological analyses

Ticks transmit a wide variety of disease causing pathogens to humans and animals. Considering the global health impact of tick-borne diseases, there is a pressing need to develop new methods for vector control. We are exploring arthropod dopamine receptors as novel targets for insecticide/acaricide development because of their integral roles in neurobiology. Herein, we developed a screening assay for dopamine receptor antagonists to further characterize the pharmacological properties of the two D₁-like dopamine receptors (Isdop1 and Isdop2) identified in the Lyme disease vector, Ixodes scapularis, and develop a screening assay for receptor antagonists. A cell-based, cyclic AMP luciferase reporter assay platform was implemented to screen the LOPAC¹²⁸⁰ small molecule library for Isdop2 receptor antagonists, representing the first reported chemical library screen for any tick G protein-coupled receptor. Screening resulted in the identification of 85 “hit” compounds with antagonist activity at the Isdop2 receptor. Eight of these chemistries were selected for confirmation assays using a direct measurement of cAMP, and the effects on both Isdop1 and Isdop2 were studied for comparison. Each of these eight compounds showed antagonistic activity at both Isdop1 and Isdop2, although differences were observed regarding their relative potencies. Furthermore, comparison of the pharmacological properties of the tick dopamine receptors with that of the AaDOP2 receptor from the yellow fever mosquito and the human dopamine D₁ receptor (hD₁) revealed species-specific pharmacological profiles of these receptors. Compounds influencing dopaminergic functioning, such as the dopamine receptor antagonists discovered here, may provide lead chemistries for discovery of novel acaricides useful for vector control.     

Biochemical comparison of Anopheles gambiae and human NADPH P450 reductases reveals different 2'-5'-ADP and FMN binding traits

NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical detoxification and insecticide resistance in Anopheles gambiae, the major vector for malaria. Anopheles gambiae CPR (AgCPR) was initially expressed in Eschericia coli but failed to bind 2′, 5′-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidine-tagged version for side-by-side comparisons with human CPR. Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (k _cat) of 105 s⁻¹ and 88 s⁻¹, respectively, for mosquito and human CPR. However, the inhibitory effects of 2′,5′-ADP on activity were different; the IC₅₀ value of AgCPR for 2′, 5′ –ADP was significantly higher (6–10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2′, 5′- ADP. This was confirmed by isothermal titration calorimetry where binding of 2′,5′-ADP to AgCPR (K _d = 410±18 nM) was ∼10 fold weaker than human CPR (K _d = 38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (K_d = 83±2.0 nM) than the equivalent human domain (K_d = 23±0.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC₅₀ = 28 µM±2 and 361±31 µM respectively). Taken together, these results reveal unusual biochemical differences between mosquito CPR and the human form in the binding of small molecules that may aid the development of ‘smart’ insecticides and synergists that selectively target mosquito CPR.     

Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship

Background

Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined.

Methodology/Principal Findings

Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-β activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z)-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1.

Conclusions/Significance

Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-β activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor function. Taken together, these results strongly support phylogenetic analyses indicating that the AmDOP2 and AmOA1 receptor genes are immediate paralogs.     

Protective action of resveratrol in human skin: possible involvement of specific receptor binding sites

Background

Resveratrol is a plant-derived polyphenol with purported protecting action on various disorders associated with aging. It has been suggested that resveratrol could exert its protective action by acting on specific plasma membrane polyphenol binding sites (Han Y.S., et al. (2006) J Pharmacol Exp Ther 318:238–245). The purpose of this study was to investigate, in human skin, the possible existence of specific binding sites that mediate the protective action of resveratrol.

Methods and Findings

Using human skin tissue, we report here the presence of specific [³H]-resveratrol binding sites (K_D  = 180 nM) that are mainly located in the epidermis. Exposure of HaCaT cells to the nitric oxide free radical donor sodium nitroprusside (SNP; 0.3–3 mM) resulted in cell death which was reduced by resveratrol (EC₅₀  = 14.7 µM), and to a much lesser extent by the resveratrol analogue piceatannol (EC₅₀  = 95 µM) and epigallocatechin gallate (EC₅₀  = 200 µM), a green-tea derived polyphenol. The protective action of resveratrol likely relates to its anti-apoptotic effect since at the same range of concentration it was able to reduce both the number of apoptotic cells as well as mitochondrial apoptotic events triggered by SNP.

Conclusion

Taken together, these findings suggest that resveratrol, by acting on specific polyphenol binding sites in epidermis, may be useful to prevent skin disorders associated with aging.     

Simukunin from the salivary glands of the black fly Simulium vittatum inhibits enzymes that regulate clotting and inflammatory responses

Background

Black flies (Diptera: Simuliidae) feed on blood, and are important vectors of Onchocerca volvulus, the etiolytic agent of River Blindness. Blood feeding depends on pharmacological properties of saliva, including anticoagulation, but the molecules responsible for this activity have not been well characterized.

Methodology/Principal Findings

Two Kunitz family proteins, SV-66 and SV-170, were identified in the sialome of the black fly Simulium vittatum. As Kunitz proteins are inhibitors of serine proteases, we hypothesized that SV-66 and/or −170 were involved in the anticoagulant activity of black fly saliva. Our results indicated that recombinant (r) SV-66 but not rSV-170 inhibited plasma coagulation. Mutational analysis suggested that SV-66 is a canonical BPTI-like inhibitor. Functional assays indicated that rSV66 reduced the activity of ten serine proteases, including several involved in mammalian coagulation. rSV-66 most strongly inhibited the activity of Factor Xa, elastase, and cathepsin G, exhibited lesser inhibitory activity against Factor IXa, Factor XIa, and plasmin, and exhibited no activity against Factor XIIa and thrombin. Surface plasmon resonance studies indicated that rSV-66 bound with highest affinity to elastase (K_D = 0.4 nM) and to the active site of FXa (K_D = 3.07 nM). We propose the name “Simukunin” for this novel protein.

Conclusions

We conclude that Simukunin preferentially inhibits Factor Xa. The inhibition of elastase and cathepsin G further suggests this protein may modulate inflammation, which could potentially affect pathogen transmission.     

Hypoxia changes the expression of the epidermal growth factor (EGF) system in human hearts and cultured cardiomyocytes

Background

The epidermal growth factor (EGF) receptors HER2 and HER4 and the ligands HB-EGF and NRG1 are crucial for heart development. The purpose of our study was to investigate the role of the complete EGF system in relation to hypoxia of the heart.

Methodology/Principal Findings

We examined the mRNA expression by real time PCR of the 4 receptors and 12 ligands from the EGF-system in paired normoxic and hypoxic biopsies isolated from human hearts during coronary artery bypass operation. Compared to normoxic biopsies, hypoxic samples showed down-regulation of HER2 (P = 0.0005) and NRG1 (both α (P = 0.02) and β (P = 0.03) isoforms). In contrast, HB-EGF (P = 0.0008), NRG2β (P = 0.01) and EGFR (P = 0.02) were up-regulated. As HER2 is essential for heart development and we find its expression reduced under hypoxia we investigated the effect of HER2 inhibition in hypoxic HL-1 cardiomyocytes by treatment with trastuzumab (20 nM). This resulted in inhibition of cardiomyocyte proliferation, but interestingly only in hypoxic cells. Co-treatment of HL-1 cells with HB-EGF (10 nM) but not with NRG-1 (5 ng/ml) rescued the cardiomyocytes from HER2 inhibition. HL-1 cardiomyocytes exposed to hypoxia revealed nuclear translocation of activated MAPK and the activity of this downstream signaling molecule was decreased by HER2 inhibition (20 nM trastuzumab), and re-established by HB-EGF (10 nM).

Conclusions/Significance

Hypoxia in the human heart alters the expression of the EGF system. Mimicking the HER2 down-regulation seen in the human heart in cultured cardiomyocytes inhibited their proliferation under hypoxic conditions. Interestingly, HB-EGF is induced in the hypoxic human hearts, and rescues hypoxic cardiomyocytes from the effect of HER2 inhibition in the in vitro model. The results have implications for future treatment strategies of patients with ischemic heart disease.     

Molecular and Pharmacological Characterization of Serotonin 5-HT2α and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina

Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP₃)/Ca²⁺ and cyclic adenosine 3′,5′-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT₂ and 5-HT₇ receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2α-transfected mammalian cells with 5-HT elevates cytosolic [Ca²⁺] in a dose-dependent manner (EC₅₀ = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP]_i (EC₅₀ = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT_2α or Cv5-HT₇ in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT_2α receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT₇ receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv5-HT₇ in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca²⁺- and cAMP-signalling cascades.     

Association between HLA class I and class II alleles and the outcome of West Nile virus infection: an exploratory study

Background

West Nile virus (WNV) infection is asymptomatic in most individuals, with a minority developing symptoms ranging from WNV fever to serious neuroinvasive disease. This study investigated the impact of host HLA on the outcome of WNV disease.

Methods

A cohort of 210 non-Hispanic mostly white WNV⁺ subjects from Canada and the U.S. were typed for HLA-A, B, C, DP, DQ, and DR. The study subjects were divided into three WNV infection outcome groups: asymptomatic (AS), symptomatic (S), and neuroinvasive disease (ND). Allele frequency distribution was compared pair-wise between the AS, S, and ND groups using χ2 and Fisher''s exact tests and P values were corrected for multiple comparisons (Pc). Allele frequencies were compared between the groups and the North American population (NA) used as a control group. Logistic regression analysis was used to evaluate the potential synergistic effect of age and HLA allele phenotype on disease outcome.

Results

The alleles HLA-A*68, C*08 and DQB*05 were more frequently associated with severe outcomes (ND vs. AS, P _A*68 = 0.013/Pc = 0.26, P _C*08 = 0.0075/Pc = 0.064, and P _DQB1*05 = 0.029/Pc = 0.68), However the apparent DQB1*05 association was driven by age. The alleles HLA-B*40 and C*03 were more frequently associated with asymptomatic outcome (AS vs. S, P _B*40 = 0.021/Pc = 0.58 and AS vs. ND P _C*03 = 0.039/Pc = 0.64) and their frequencies were lower within WNV⁺ subjects with neuroinvasive disease than within the North American population (NA vs. S, P _B*40 = 0.029 and NA vs. ND, P _C*03 = 0.032).

Conclusions

Host HLA may be associated with the outcome of WNV disease; HLA-A*68 and C*08 might function as “susceptible” alleles, whereas HLA-B*40 and C*03 might function as “protective” alleles.     

Selective κ Opioid Antagonists nor-BNI,GNTI and JDTic Have Low Affinities for Non-Opioid Receptors and Transporters

Background

Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets.

Results

In binding assays, the three antagonists showed no detectable affinity (K _i≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α_2C-adrenoceptor (K _i = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α_1A-adrenoceptor (EC₅₀ = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M₁ receptor antagonist (K _B = 3.7 µM). JDTic bound to the noradrenaline transporter (K _i = 54 nM), but only weakly inhibited transport (IC₅₀ = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K _i = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers.

Conclusions

Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α_1A-adrenoceptors). This may contribute to GNTI''s severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.     

Estimation of individual muscle force using elastography

Background

Estimation of an individual muscle force still remains one of the main challenges in biomechanics. In this way, the present study aimed: (1) to determine whether an elastography technique called Supersonic Shear Imaging (SSI) could be used to estimate muscle force, (2) to compare this estimation to that one provided by surface electromyography (EMG), and (3) to determine the effect of the pennation of muscle fibers on the accuracy of the estimation.

Methods and Results

Eleven subjects participated in two experimental sessions; one was devoted to the shear elastic modulus measurements and the other was devoted to the EMG recordings. Each session consisted in: (1) two smooth linear torque ramps from 0 to 60% and from 0 to 30% of maximal voluntary contraction, for the first dorsal interosseous and the abductor digiti minimi, respectively (referred to as “ramp contraction”); (2) two contractions done with the instruction to freely change the torque (referred to as “random changes contraction”). Multi-channel surface EMG recordings were obtained from a linear array of eight electrodes and the shear elastic modulus was measured using SSI. For ramp contractions, significant linear relationships were reported between EMG activity level and torque (R² = 0.949±0.036), and between shear elastic modulus and torque (R² = 0.982±0.013). SSI provided significant lower RMS_deviation between measured torque and estimated torque than EMG activity level for both types of contraction (1.4±0.7 vs. 2.8±1.4% of maximal voluntary contraction for “ramp contractions”, p<0.01; 4.5±2.3 vs. 7.9±5.9% of MVC for “random changes contractions”, p<0.05). No significant difference was reported between muscles.

Conclusion

The shear elastic modulus measured using SSI can provide a more accurate estimation of individual muscle force than surface EMG. In addition, pennation of muscle fibers does not influence the accuracy of the estimation.     

Lack of evidence for changing virulence of HIV-1 in North America

Background

Several long-term cohort studies and in-vitro fitness assays have resulted in inconsistent reports on changes in HIV-1 virulence, including reports of decreasing, stable, and increasing virulence over the course of the AIDS pandemic. We tested the hypothesis of changing HIV-1 virulence by examining trends in prognostic clinical markers of disease progression from 1984 to 2005 among nearly 400 antiretroviral-naïve participants in the United States Multicenter AIDS Cohort Study (MACS), a longitudinal study of HIV infection in men who have sex with men (MSM).

Methodology/Principal Findings

Because clinical AIDS endpoints could not be used (due to antiretroviral therapies and prophylaxis), three prognostic markers of disease progression were used as proxies for HIV-1 virulence: plasma viral RNA load and CD4+ T cell count at “set point” (between ∼9 and ∼15 months after seroconversion), and rate of CD4 cell decline within three years after seroconversion. We performed multivariate analyses of the association between these markers and seroconversion year, with covariates including MACS site, race/ethnic group, seroconversion age, and CCR5Δ32 status. No statistically significant association was found between year of seroconversion and “set point” plasma viral load (at ∼9 months after seroconversion: slope = −0.004 log₁₀ copies/mL/year, p = 0.76; at ∼15 months: slope = −0.005 log₁₀ copies/mL/year, p = 0.71), CD4 cell count after seroconversion (at ∼9 months: slope = −0.112 cells/µL/year, p = 0.22; at ∼15 months: slope = −0.047 cells/µL/year, p = 0.64), or rate of CD4 cell decline over the first three years after seroconversion (slope = −0.010 cells/ul/yr², p = 0.88).

Conclusions/Significance

The lack of significant trends from 1984 to 2005 in these prognostic markers of HIV disease progression suggests no major change in HIV-1 virulence over the AIDS pandemic in MSM in the US.     

Association between IgM anti-herpes simplex virus and plasma amyloid-beta levels

Objective

Herpes simplex virus (HSV) reactivation has been identified as a possible risk factor for Alzheimer''s disease (AD) and plasma amyloid-beta (Aβ) levels might be considered as possible biomarkers of the risk of AD. The aim of our study was to investigate the association between anti-HSV antibodies and plasma Aβ levels.

Methods

The study sample consisted of 1222 subjects (73.9 y in mean) from the Three-City cohort. IgM and IgG anti-HSV antibodies were quantified using an ELISA kit, and plasma levels of Aβ_1–40 and Aβ_1–42 were measured using an xMAP-based assay technology. Cross-sectional analyses of the associations between anti-HSV antibodies and plasma Aβ levels were performed by multi-linear regression.

Results

After adjustment for study center, age, sex, education, and apolipoprotein E-e4 polymorphism, plasma Aβ_1–42 and Aβ_1–40 levels were specifically inversely associated with anti-HSV IgM levels (β = −20.7, P = 0.001 and β = −92.4, P = 0.007, respectively). In a sub-sample with information on CLU- and CR1-linked SNPs genotyping (n = 754), additional adjustment for CR1 or CLU markers did not modify these associations (adjustment for CR1 rs6656401, β = −25.6, P = 0.002 for Aβ_1–42 and β = −132.7, P = 0.002 for Aβ_1–40; adjustment for CLU rs2279590, β = −25.6, P = 0.002 for Aβ_1–42 and β = −134.8, P = 0.002 for Aβ_1–40). No association between the plasma Aβ_1–42-to-Aβ_1–40 ratio and anti-HSV IgM or IgG were evidenced.

Conclusion

High anti-HSV IgM levels, markers of HSV reactivation, are associated with lower plasma Aβ_1–40 and Aβ_1–42 levels, which suggest a possible involvement of the virus in the alterations of the APP processing and potentially in the pathogenesis of AD in human.     

Grifonin-1: a small HIV-1 entry inhibitor derived from the algal lectin, Griffithsin

Background

Griffithsin, a 121-residue protein isolated from a red algal Griffithsia sp., binds high mannose N-linked glycans of virus surface glycoproteins with extremely high affinity, a property that allows it to prevent the entry of primary isolates and laboratory strains of T- and M-tropic HIV-1. We used the sequence of a portion of griffithsin''s sequence as a design template to create smaller peptides with antiviral and carbohydrate-binding properties.

Methodology/Results

The new peptides derived from a trio of homologous β-sheet repeats that comprise the motifs responsible for its biological activity. Our most active antiviral peptide, grifonin-1 (GRFN-1), had an EC₅₀ of 190.8±11.0 nM in in vitro TZM-bl assays and an EC₅₀ of 546.6±66.1 nM in p24^gag antigen release assays. GRFN-1 showed considerable structural plasticity, assuming different conformations in solvents that differed in polarity and hydrophobicity. Higher concentrations of GRFN-1 formed oligomers, based on intermolecular β-sheet interactions. Like its parent protein, GRFN-1 bound viral glycoproteins gp41 and gp120 via the N-linked glycans on their surface.

Conclusion

Its substantial antiviral activity and low toxicity in vitro suggest that GRFN-1 and/or its derivatives may have therapeutic potential as topical and/or systemic agents directed against HIV-1.     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

Risk factors of Streptococcus suis infection in Vietnam. A case-control study

Background

Streptococcus suis infection, an emerging zoonosis, is an increasing public health problem across South East Asia and the most common cause of acute bacterial meningitis in adults in Vietnam. Little is known of the risk factors underlying the disease.

Methods and Findings

A case-control study with appropriate hospital and matched community controls for each patient was conducted between May 2006 and June 2009. Potential risk factors were assessed using a standardized questionnaire and investigation of throat and rectal S. suis carriage in cases, controls and their pigs, using real-time PCR and culture of swab samples. We recruited 101 cases of S. suis meningitis, 303 hospital controls and 300 community controls. By multivariate analysis, risk factors identified for S. suis infection as compared to either control group included eating “high risk” dishes, including such dishes as undercooked pig blood and pig intestine (OR₁ = 2.22; 95%CI = [1.15–4.28] and OR₂ = 4.44; 95%CI = [2.15–9.15]), occupations related to pigs (OR₁ = 3.84; 95%CI = [1.32–11.11] and OR₂ = 5.52; 95%CI = [1.49–20.39]), and exposures to pigs or pork in the presence of skin injuries (OR₁ = 7.48; 95%CI = [1.97–28.44] and OR₂ = 15.96; 95%CI = [2.97–85.72]). S. suis specific DNA was detected in rectal and throat swabs of 6 patients and was cultured from 2 rectal samples, but was not detected in such samples of 1522 healthy individuals or patients without S. suis infection.

Conclusions

This case control study, the largest prospective epidemiological assessment of this disease, has identified the most important risk factors associated with S. suis bacterial meningitis to be eating ‘high risk’ dishes popular in parts of Asia, occupational exposure to pigs and pig products, and preparation of pork in the presence of skin lesions. These risk factors can be addressed in public health campaigns aimed at preventing S. suis infection.     

A characterization of internet dating network structures among nordic men who have sex with men

Background

The Internet has become an important venue for seeking sexual partners and may facilitate transmission of sexually transmitted infections.

Methods

We examined a 64-day data log of flirt messages expressing sexual interest among MSM within the Qruiser.com community. We used logistic regression to analyze characteristics of MSM sending and receiving flirt messages and negative binomial regression to examine individual activity and popularity. The structural properties, including the core structure of the flirt network, were analyzed.

Results

The MSM population consisted of approximately 40% homosexuals and 37% bisexuals, while the remaining 23% included men who identified as heterosexual but searched for sex with men and “experimental”. MSM were more likely to send flirt messages if they were homosexual and aged 40+ years; young people aged < 30 years were more likely to receive a flirt. Possession of a webcam was strongly associated with both sending flirt messages and being a flirt target. The distributions of flirts sent (max k_out = 2162) and received (max k_in = 84) were highly heterogeneous. Members in central cores were more likely homosexuals, singles, and aged 31–40 years. The probability of a matched flirt (flirt returned from target) increased from 1% in the outer core to 18% in the central core (core size = 4).

Discussion

The flirt network showed high degree heterogeneity similar to the structural properties of real sexual contact networks with a single central core. Further studies are needed to explore use of webcam for Internet dating.     

Associations between XPD Asp312Asn polymorphism and risk of head and neck cancer: a meta-analysis based on 7,122 subjects

Background

To investigate the association between XPD Asp312Asn polymorphism and head and neck cancer risk through this meta-analysis.

Methods

We performed a meta-analysis of 9 published case-control studies including 2,670 patients with head and neck cancer and 4,452 controls. An odds ratio (OR) with a 95% confidence interval (CI) was applied to assess the association between XPD Asp312Asn polymorphism and head and neck cancer risk.

Results

Overall, no significant association between XPD Asp312Asn polymorphism and head and neck cancer risk was found in this meta-analysis (Asn/Asn vs. Asp/Asp: OR = 0.95, 95%CI = 0.80–1.13, P = 0.550, P _{heterogeneity} = 0.126; Asp/Asn vs. Asp/Asp: OR = 1.11, 95%CI = 0.99–1.24, P = 0.065, P _{heterogeneity} = 0.663; Asn/Asn+Asp/Asn vs. Asp/Asp: OR = 1.07, 95%CI = 0.97–1.19, P = 0.189, P _{heterogeneity} = 0.627; Asn/Asn vs. Asp/Asp+Asp/Asn: OR = 0.87, 95%CI = 0.68–1.10, P = 0.243, P _{heterogeneity} = 0.089). In the subgroup analysis by HWE, ethnicity, and study design, there was still no significant association detected in all genetic models.

Conclusions

This meta-analysis demonstrates that XPD Asp312Asn polymorphism may not be a risk factor for developing head and neck cancer.     

Variance of the SGK1 gene is associated with insulin secretion in different European populations: results from the TUEF, EUGENE2, and METSIM studies

Hypothesis

Serum- and Glucocorticoid-inducible Kinase 1 (SGK1) is involved in the regulation of insulin secretion and may represent a candidate gene for the development of type 2 diabetes mellitus in humans.

Methods

Three independent European populations were analyzed for the association of SGK1 gene (SGK) variations and insulin secretion traits. The German TUEF project provided the screening population (N = 725), and four tagging SNPs (rs1763527, rs1743966, rs1057293, rs9402571) were investigated. EUGENE2 (N = 827) served as a replication cohort for the detected associations. Finally, the detected associations were validated in the METSIM study, providing 3798 non-diabetic and 659 diabetic (type 2) individuals.

Results

Carriers of the minor G allele in rs9402571 had significantly higher C-peptide levels in the 2 h OGTT (+10.8%, p = 0.04; dominant model) and higher AUC_C-Peptide/AUC_Glc ratios (+7.5%, p = 0.04) compared to homozygous wild type TT carriers in the screening population. As interaction analysis for BMI×rs9402571 was significant (p = 0.04) for the endpoint insulin secretion, we stratified the TUEF cohort for BMI, using a cut off point of BMI = 25. The effect on insulin secretion only remained significant in lean TUEF participants (BMI≤25). This finding was replicated in lean EUGENE2 rs9402571 minor allele carriers, who had a significantly higher AUC_Ins/AUC_Glc (TT: 226±7, XG: 246±9; p = 0.019). Accordingly, the METSIM trial revealed a lower prevalence of type 2 diabetes (OR: 0.85; 95%CI: 0.71–1.01; p = 0.065, dominant model) in rs9402571 minor allele carriers.

Conclusions

The rs9402571 SGK genotype associates with increased insulin secretion in lean non-diabetic TUEF/EUGENE2 participants and with lower diabetes prevalence in METSIM. Our study in three independent European populations supports the conclusion that SGK variability affects diabetes risk.     

Two Acidic,Anticoagulant PLA2 Isoenzymes Purified from the Venom of Monocled Cobra Naja kaouthia Exhibit Different Potency to Inhibit Thrombin and Factor Xa via Phospholipids Independent,Non-Enzymatic Mechanism

Background

The monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A₂ (PLA₂; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA₂ isoenzymes, Nk-PLA₂α (13463.91 Da) and Nk-PLA₂β (13282.38 Da) purified from the venom of N. kaouthia.

Principal Findings

By LC-MS/MS analysis, these PLA₂s showed highest similarity (23.5% sequence coverage) with PLA₂ III isolated from monocled cobra venom. The catalytic activity of Nk-PLA₂β exceeds that of Nk-PLA₂α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA₂ isoenzymes. The anticoagulant potency of Nk-PLA₂α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA₂β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA₂s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA₂α and Nk-PLA₂β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA₂ isoenzymes inhibit their “pharmacological target(s)” by uncompetitive mechanism without the requirement of phospholipids/Ca²⁺. The anticoagulant potency of Nk-PLA₂β which is higher than that of Nk-PLA₂α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA₂ isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA₂β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.

Conclusion/Significance

In order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA₂α and Nk-PLA₂β for the treatment and/or prevention of cardiovascular disorders are proposed.