Evidence that human histidine triad nucleotide binding protein 3 (Hint3) is a distinct branch of the histidine triad (HIT) superfamily

Human Hint3 (hHint3) has been classified as a member of the histidine triad nucleotide (Hint) binding protein subfamily. While Hint1 is ubiquitously expressed by both eukaryotes and prokaryotes, Hint3 is found only in eukaryotes. Previously, our laboratory has characterized and compared the aminoacyl-adenylate and nucleoside phosphoramidate hydrolase activity of hHint1 and Escherichia coli hinT. In this study, hHint3-1(Ala36) and its single nucleotide polymorphism, hHint3-2 (A36G variant), were cloned, overexpressed, and purified. Steady-state kinetic studies with a synthetic fluorogenic indolepropinoic acyl-adenylate (AIPA) and with a series of fluorogenic tryptamine nucleoside phosphoramidates revealed that hHint3-1 and hHint3-2 are adenylate and phosphoramidate hydrolases with apparent second-order rate constants (kcat/Km) ranging from 10(2) to 10(6) s(-1) M(-1). Unlike hHint1, hHint3-1 and hHint3-2 prefer AIPA over tryptamine adenosine phosphoramidate by factors of 33- and 16-fold, respectively. In general, hHint3s hydrolyze phosphoramidate 370- to 2000-fold less efficiently than hHint1. Substitution of the potential active-site nucleophile, His145, by Ala was shown to abolish the adenylate and phosphoramidate hydrolase activity for hHint3-1. However, 0.2-0.4% residual activity was observed for the H145A mutant of hHint3-2. Both hHint3-1 and hHint3-2 were found to hydrolyze lysyl-adenylate generated by human lysyl-tRNA synthetase (hLysRS) by proceeding through an adenylated protein intermediate. hLysRS-dependent labeling of hHint3-1 and hHint3-2 was found to depend on His145, which aligns with the His112 of the Hint1 active site. The extent of active-site His145-AMP labeling was shown to be similar to His112-AMP labeling of hHint1. In contrast to all previously characterized members of the histidine triad superfamily, which have been shown to exist exclusively as homodimers, wild type and the H145A of hHint3-1 were found to exist across a range of multimeric states, from dimers to octamers and even larger oligomers, while wild type and the H145A of hHint3-2 exist predominantly in a monomeric state. The differences in oligomeric state may be important in vivo, because unlike tetracysteine-tagged Hint1, which was found along linear arrays exclusively in the cytoplasm in transfected HeLa cells, tagged Hint3-1 and Hint3-2 were found as aggregates both in the cytosol and in the nucleus. Taken together, these results imply that while Hint3 and Hint1 prefer aminoacyl-adenylates as substrates and catalytically interact with aminoacyl-tRNA synthetases, the significant differences in phosphoramidase activity, oligomeric state, and cellular localization suggest that Hint3s should be placed in a distinct branch of the histidine triad superfamily.     

High Rate of Subclinical Chikungunya Virus Infection and Association of Neutralizing Antibody with Protection in a Prospective Cohort in The Philippines

Background

Chikungunya virus (CHIKV) is a globally re-emerging arbovirus for which previous studies have indicated the majority of infections result in symptomatic febrile illness. We sought to characterize the proportion of subclinical and symptomatic CHIKV infections in a prospective cohort study in a country with known CHIKV circulation.

Methods/Findings

A prospective longitudinal cohort of subjects ≥6 months old underwent community-based active surveillance for acute febrile illness in Cebu City, Philippines from 2012-13. Subjects with fever history were clinically evaluated at acute, 2, 5, and 8 day visits, and at a 3-week convalescent visit. Blood was collected at the acute and 3-week convalescent visits. Symptomatic CHIKV infections were identified by positive CHIKV PCR in acute blood samples and/or CHIKV IgM/IgG ELISA seroconversion in paired acute/convalescent samples. Enrollment and 12-month blood samples underwent plaque reduction neutralization test (PRNT) using CHIKV attenuated strain 181/clone25. Subclinical CHIKV infections were identified by ≥8-fold rise from a baseline enrollment PRNT titer <10 without symptomatic infection detected during the intervening surveillance period. Selected CHIKV PCR-positive samples underwent viral isolation and envelope protein-1 gene sequencing. Of 853 subjects who completed all study procedures at 12 months, 19 symptomatic infections (2.19 per 100 person-years) and 87 subclinical infections (10.03 per 100 person-years) occurred. The ratio of subclinical-to-symptomatic infections was 4.6:1 varying with age from 2:1 in 6 month-5 year olds to 12:1 in those >50 years old. Baseline CHIKV PRNT titer ≥10 was associated with 100% (95%CI: 46.1, 100.0) protection from symptomatic CHIKV infection. Phylogenetic analysis demonstrated Asian genotype closely related to strains from Asia and the Caribbean.

Conclusions

Subclinical infections accounted for a majority of total CHIKV infections. A positive baseline CHIKV PRNT titer was associated with protection from symptomatic CHIKV infection. These findings have implications for assessing disease burden, understanding virus transmission, and supporting vaccine development.     

Theoretical study of the electron affinities of the alkaline-earth tetramers possessing T d symmetry: Be4 and Mg4

The electron affinities of beryllium and magnesium tetramers are calculated at the ROMP2 level employing the Dunning-type aug-cc-pVQZ basis set. The vertical electron detachment energy (VEDE) amounts to 1.685 eV for Be₄^– and 0.943 eV for Mg₄ . The decomposition of the VEDE into physical components and an atomic orbital population analysis are used to elucidate the nature of the outer electron binding in these anions.Figure The lowest unoccupied molecular orbitals in the ground state of Mg4 : a LUMO, symmetry A₁, b LUMO + 1, symmetry T₂; c the highest occupied molecular orbital (HOMO), symmetry A1 in the ground state of Mg₄^–.      

Environmental factors function as constraints on soil nitrous oxide fluxes in bioenergy feedstock cropping systems

Nitrous oxide (N₂O) is a potent greenhouse gas and major component of the net global warming potential of bioenergy feedstock cropping systems. Numerous environmental factors influence soil N₂O production, making direct correlation difficult to any one factor of N₂O fluxes under field conditions. We instead employed quantile regression to evaluate whether soil temperature, water‐filled pore space (WFPS), and concentrations of soil nitrate () and ammonium () determined upper bounds for soil N₂O flux magnitudes. We collected data over 6 years from a range of bioenergy feedstock cropping systems including no‐till grain crops, perennial warm‐season grasses, hybrid poplar, and polycultures of tallgrass prairie species each with and without nitrogen (N) addition grown at two sites. The upper bounds for soil N₂O fluxes had a significant and positive correlation with all four environmental factors, although relatively large fluxes were still possible at minimal values for nearly all factors. The correlation with was generally weaker, suggesting it is less important than in driving large fluxes. Quantile regression slopes were generally lower for unfertilized perennials than for other systems, but this may have resulted from a perpetual state of nitrogen limitation, which prevented other factors from being clear constraints. This framework suggests efforts to reduce concentrations of in the soil may be effective at reducing high‐intensity periods—”hot moments”—of N₂O production.     

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.     

Accuracy of Protein-Protein Binding Sites in High-Throughput Template-Based Modeling

The accuracy of protein structures, particularly their binding sites, is essential for the success of modeling protein complexes. Computationally inexpensive methodology is required for genome-wide modeling of such structures. For systematic evaluation of potential accuracy in high-throughput modeling of binding sites, a statistical analysis of target-template sequence alignments was performed for a representative set of protein complexes. For most of the complexes, alignments containing all residues of the interface were found. The full interface alignments were obtained even in the case of poor alignments where a relatively small part of the target sequence (as low as 40%) aligned to the template sequence, with a low overall alignment identity (<30%). Although such poor overall alignments might be considered inadequate for modeling of whole proteins, the alignment of the interfaces was strong enough for docking. In the set of homology models built on these alignments, one third of those ranked 1 by a simple sequence identity criteria had RMSD<5 Å, the accuracy suitable for low-resolution template free docking. Such models corresponded to multi-domain target proteins, whereas for single-domain proteins the best models had 5 Å<RMSD<10 Å, the accuracy suitable for less sensitive structure-alignment methods. Overall, ∼50% of complexes with the interfaces modeled by high-throughput techniques had accuracy suitable for meaningful docking experiments. This percentage will grow with the increasing availability of co-crystallized protein-protein complexes.     

Exercise reverses high-fat diet-induced impairments on compartmentalization and activation of components of the insulin-signaling cascade in skeletal muscle

The aims of this investigation were 1) to determine whether endurance exercise training could reverse impairments in insulin-stimulated compartmentalization and/or activation of aPKCzeta/lambda and Akt2 in skeletal muscle from high-fat-fed rodents and 2) to assess whether the PPARgamma agonist rosiglitazone could reverse impairments in skeletal muscle insulin signaling typically observed after high-fat feeding. Sprague-Dawley rats were placed on chow (NORCON, n = 16) or high-fat (n = 64) diets for 4 wk. During a subsequent 4-wk experimental period, high-fat-fed rats were allocated (n = 16/group) to either sedentary control (HFC), exercise training (HFX), rosiglitazone treatment (HFRSG), or a combination of both exercise training and rosiglitazone (HFRX). Following the 4-wk experimental period, animals underwent hindlimb perfusions. Insulin-stimulated plasma membrane-associated aPKCzeta and -lambda protein concentration, aPKCzeta/lambda activity, GLUT4 protein concentration, cytosolic Akt2, and aPKCzeta/lambda activities were reduced (P < 0.05) in HFC compared with NORCON. Cytosolic Akt2, aPKCzeta, and aPKClambda protein concentrations were not affected in HFC compared with NORCON. Exercise training reversed the deleterious effects of the high-fat diet such that insulin-stimulated compartmentalization and activation of components of the insulin-signaling cascade in HFX were normalized to NORCON. High-fat diet-induced impairments to skeletal muscle glucose metabolism were not reversed by rosiglitazone administration, nor did rosiglitazone augment the effect of exercise. Our findings indicate that chronic exercise training, but not rosiglitazone, reverses high-fat diet induced impairments in compartmentalization and activation of components of the insulin-signaling cascade in skeletal muscle.