Synthesis and antiviral evaluation of thieno[3,4-d]pyrimidine C-nucleoside analogues of 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-adenosine and -inosine

Several thieno[3,4-d]pyrimidine derivatives, including four hitherto unknown 2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydro-C-nucleoside analogues of adenosine and inosine have been synthesized. When evaluated in cell culture experiments against human immunodeficiency virus, none of the tested compounds exhibited any significant antiviral effect, while two of them showed some cytotoxicity.     

A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family

The BAR (Bin/amphiphysin/Rvs) domain defines an emerging superfamily of proteins implicated in fundamental biological processes by sensing and inducing membrane curvature. We identified a novel autoregulatory function for the BAR domain of two related GAPs' (GTPase-activating proteins) of the GRAF (GTPase regulator associated with focal adhesion kinase) subfamily. We demonstrate that the N-terminal fragment of these GAPs including the BAR domain interacts directly with the GAP domain and inhibits its activity. Analysis of various BAR and GAP domains revealed that the BAR domain-mediated inhibition of these GAPs' function is highly specific. These GAPs, in their autoinhibited state, are able to bind and tubulate liposomes in vitro, and to generate lipid tubules in cells. Taken together, we identified BAR domains as cis-acting inhibitory elements that very likely mask the active sites of the GAP domains and thus prevent down-regulation of Rho proteins. Most remarkably, these BAR proteins represent a dual-site system with separate membrane-tubulation and GAP-inhibitory functions that operate simultaneously.     

Impact of cytokine and cytokine receptor gene polymorphisms on cellular immunity after smallpox vaccination

We explored associations between SNPs in cytokine/cytokine receptor genes and cellular immunity in subjects following primary smallpox vaccination. We also analyzed the genotype–phenotype associations discovered in the Caucasian subjects among a cohort of African-Americans. In Caucasians we found 277 associations (p < 0.05) between gene SNPs and inter-individual variations in IFN-α, IL-12p40, IL-1β, IL-2, and TNF-α secretion levels. A collection of SNPs in the IL1RN, IL2RB, IL4R, IL6, IL10RB, IL12A, and IL12RB2 genes had consistent associations among both Caucasians and African-Americans. A regulatory SNP (rs452204) in the IL1RN gene was significantly associated with higher levels of IL-2 secretion in an allele dose-dependent manner in both race groups (p = 0.05 for Caucasians and p = 0.002 for African-Americans). IL12RB2 polymorphism rs3790567 was associated with a dose-related decrease in IL-1β secretion (p = 0.009 for Caucasians and p = 0.01 for African-Americans). Our results demonstrate that variations in smallpox vaccine-induced cytokine responses are modulated by genetic polymorphisms in cytokine and cytokine receptor genes.     

Estimating the Hidden Burden of Bovine Tuberculosis in Great Britain

The number of cattle herds placed under movement restrictions in Great Britain (GB) due to the suspected presence of bovine tuberculosis (bTB) has progressively increased over the past 25 years despite an intensive and costly test-and-slaughter control program. Around 38% of herds that clear movement restrictions experience a recurrent incident (breakdown) within 24 months, suggesting that infection may be persisting within herds. Reactivity to tuberculin, the basis of diagnostic testing, is dependent on the time from infection. Thus, testing efficiency varies between outbreaks, depending on weight of transmission and cannot be directly estimated. In this paper, we use Approximate Bayesian Computation (ABC) to parameterize two within-herd transmission models within a rigorous inferential framework. Previous within-herd models of bTB have relied on ad-hoc methods of parameterization and used a single model structure (SORI) where animals are assumed to become detectable by testing before they become infectious. We study such a conventional within-herd model of bTB and an alternative model, motivated by recent animal challenge studies, where there is no period of epidemiological latency before animals become infectious (SOR). Under both models we estimate that cattle-to-cattle transmission rates are non-linearly density dependent. The basic reproductive ratio for our conventional within-herd model, estimated for scenarios with no statutory controls, increases from 1.5 (0.26–4.9; 95% CI) in a herd of 30 cattle up to 4.9 (0.99–14.0) in a herd of 400. Under this model we estimate that 50% (33–67) of recurrent breakdowns in Britain can be attributed to infection missed by tuberculin testing. However this figure falls to 24% (11–42) of recurrent breakdowns under our alternative model. Under both models the estimated extrinsic force of infection increases with the burden of missed infection. Hence, improved herd-level testing is unlikely to reduce recurrence unless this extrinsic infectious pressure is simultaneously addressed.     

Proteomic signatures of in vivo muscle oxidative capacity in healthy adults

Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23–87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by ³¹P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τ_PCr) in the vastus lateralis muscle, with smaller τ_PCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC‐MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.     

Stability and structural constraints of random brain networks with excitatory and inhibitory neural populations

The stability of brain networks with randomly connected excitatory and inhibitory neural populations is investigated using a simplified physiological model of brain electrical activity. Neural populations are randomly assigned to be excitatory or inhibitory and the stability of a brain network is determined by the spectrum of the network’s matrix of connection strengths. The probability that a network is stable is determined from its spectral density which is numerically determined and is approximated by a spectral distribution recently derived by Rajan and Abbott. The probability that a brain network is stable is maximum when the total connection strength into a population is approximately zero and is shown to depend on the arrangement of the excitatory and inhibitory connections and the parameters of the network. The maximum excitatory and inhibitory input into a structure allowed by stability occurs when the net input equals zero and, in contrast to networks with randomly distributed excitatory and inhibitory connections, substantially increases as the number of connections increases. Networks with the largest excitatory and inhibitory input allowed by stability have multiple marginally stable modes, are highly responsive and adaptable to external stimuli, have the same total input into each structure with minimal variance in the excitatory and inhibitory connection strengths, and have a wide range of flexible, adaptable, and complex behavior.     

French Experience of 2009 A/H1N1v Influenza in Pregnant Women

Background

The first reports on the pandemic influenza 2009 A/H1N1v from the USA, Mexico, and Australia indicated that this disease was associated with a high mortality in pregnant women. The aim of this study was to describe and compare the characteristics of severe critically ill and non-severe pregnant women with 2009 A/H1N1v-related illness in France.

Methodology/Principal Findings

A national registry was created to screen pregnant women with laboratory-confirmed 2009 A/H1N1v influenza. Three hundred and fifteen patients from 46 French hospitals were included: 40 patients were admitted to intensive care units (severe outcomes), 111 were hospitalized in obstetric or medical wards (moderate outcomes), and 164 were outpatients (mild outcomes). The 2009 A/H1N1v influenza illness occurred during all pregnancy trimesters, but most women (54%), notably the severe patients (70%), were in the third trimester. Among the severe patients, twenty (50%) underwent mechanical ventilation, and eleven (28%) were treated with extracorporeal membrane oxygenation. Three women died from A/H1N1v influenza. We found a strong association between the development of a severe outcome and both co-existing illnesses (adjusted odds ratio [OR], 5.1; 95% confidence interval [CI], 2.2–11.8) and a delay in oseltamivir treatment after the onset of symptoms (>3 or 5 days) (adjusted OR, 4.8; 95% CI, 1.9–12.1 and 61.2, 95% CI; 14.4–261.3, respectively). Among the 140 deliveries after 22 weeks of gestation known to date, 19 neonates (14%) were admitted to a neonatal intensive care unit, mainly for preterm delivery, and two neonates died. None of these neonates developed 2009 A/H1N1v infection.

Conclusions

This series confirms the high incidence of complications in pregnant women infected with pandemic A/H1N1v observed in other countries but depicts a lower overall maternal and neonatal mortality and morbidity than indicated in the USA or Australia. Moreover, our data demonstrate the benefit of early oseltamivir treatment in this specific population.     

2,4-Diaminopyrimidine MK2 inhibitors. Part I: Observation of an unexpected inhibitor binding mode

MK2 is a Ser/Thr kinase of significant interest as an anti-inflammatory drug discovery target. Here we describe the development of in vitro tools for the identification and characterization of MK2 inhibitors, including validation of inhibitor interactions with the crystallography construct and determination of the unique binding mode of 2,4-diaminopyrimidine inhibitors in the MK2 active site. Use of these tools in the optimization of a potent and selective inhibitor lead series is described in the accompanying Letter.