Improper Tagging of the Non-Essential Small Capsid Protein VP26 Impairs Nuclear Capsid Egress of Herpes Simplex Virus

To analyze the subcellular trafficking of herpesvirus capsids, the small capsid protein has been labeled with different fluorescent proteins. Here, we analyzed the infectivity of several HSV1(17(+)) strains in which the N-terminal region of the non-essential small capsid protein VP26 had been tagged at different positions. While some variants replicated with similar kinetics as their parental wild type strain, others were not infectious at all. Improper tagging resulted in the aggregation of VP26 in the nucleus, prevented efficient nuclear egress of viral capsids, and thus virion formation. Correlative fluorescence and electron microscopy showed that these aggregates had sequestered several other viral proteins, but often did not contain viral capsids. The propensity for aggregate formation was influenced by the type of the fluorescent protein domain, the position of the inserted tag, the cell type, and the progression of infection. Among the tags that we have tested, mRFPVP26 had the lowest tendency to induce nuclear aggregates, and showed the least reduction in replication when compared to wild type. Our data suggest that bona fide monomeric fluorescent protein tags have less impact on proper assembly of HSV1 capsids and nuclear capsid egress than tags that tend to dimerize. Small chemical compounds capable of inducing aggregate formation of VP26 may lead to new antiviral drugs against HSV infections.     

α-Synuclein genetic variants predict faster motor symptom progression in idiopathic Parkinson disease

Currently, there are no reported genetic predictors of motor symptom progression in Parkinson's disease (PD). In familial PD, disease severity is associated with higher α-synuclein (SNCA) expression levels, and in postmortem studies expression varies with SNCA genetic variants. Furthermore, SNCA is a well-known risk factor for PD occurrence. We recruited Parkinson's patients from the communities of three central California counties to investigate the influence of SNCA genetic variants on motor symptom progression in idiopathic PD. We repeatedly assessed this cohort of patients over an average of 5.1 years for motor symptom changes employing the Unified Parkinson's Disease Rating Scale (UPDRS). Of 363 population-based incident PD cases diagnosed less than 3 years from baseline assessment, 242 cases were successfully re-contacted and 233 were re-examined at least once. Of subjects lost to follow-up, 69% were due to death. Adjusting for covariates, risk of faster decline of motor function as measured by annual increase in motor UPDRS exam score was increased 4-fold in carriers of the REP1 263bp promoter variant (OR 4.03, 95%CI:1.57-10.4). Our data also suggest a contribution to increased risk by the G-allele for rs356165 (OR 1.66; 95%CI:0.96-2.88), and we observed a strong trend across categories when both genetic variants were considered (p for trend = 0.002). Our population-based study has demonstrated that SNCA variants are strong predictors of faster motor decline in idiopathic PD. SNCA may be a promising target for therapies and may help identify patients who will benefit most from early interventions. This is the first study to link SNCA to motor symptom decline in a longitudinal progression study.     

Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway

The secretory pathway in mammalian cells has evolved to facilitate the transfer of cargo molecules to internal and cell surface membranes. Use of automated microscopy-based genome-wide RNA interference screens in cultured human cells allowed us to identify 554 proteins influencing secretion. Cloning, fluorescent-tagging and subcellular localization analysis of 179 of these proteins revealed that more than two-thirds localize to either the cytoplasm or membranes of the secretory and endocytic pathways. The depletion of 143 of them resulted in perturbations in the organization of the COPII and/or COPI vesicular coat complexes of the early secretory pathway, or the morphology of the Golgi complex. Network analyses revealed a so far unappreciated link between early secretory pathway function, small GTP-binding protein regulation, actin cytoskeleton organization and EGF-receptor-mediated signalling. This work provides an important resource for an integrative understanding of global cellular organization and regulation of the secretory pathway in mammalian cells.     

Morphology, anatomy, and molecular studies of the ectomycorrhiza formed axenically by the fungus Sistotrema sp. (Basidiomycota)

Several species of the corticioid (resupinate) genus Sistotrema of the cantharelloid clade (Basidiomycota) were recently found to be ectomycorrhizal. This changed the traditional assertion that all Sistotrema species are strictly wood rotting and suggests that the genus may be polyphyletic. In the present investigation, a still unknown root tip-associated fungal specimen (EW63) was isolated and found to be associated with an above-ground fruiting body. Sequencing of the ITS and the nucLSU DNA regions and phylogenetic analyses verified that the root-associated fungus and the fruiting body represented the same species, which was found to belong to the genus Sistotrema. To prove the ectomycorrhizal status of this strain, axenic Pinus sylvestris resyntheses in flask cultures were conducted. Growth parameters of the seedlings were determined and the morphology and anatomy of the synthesized mycorrhizas were described. Length and dry mass of the Pinus shoot as well as those of the total root tips were found to be enhanced as a result of the mycorrhizal association. Mycorrhizal frequency was high (51.5%) in these cultures. Mycorrhizal root tips were cottony light ochre with a thin plectenchymatic hyphal mantle. The clamps of the fruiting body hyphae as well as the mycorrhiza were ampullately inflated. This is the first report proving in axenic culture that a fungus belonging to the genus Sistotrema forms true ectomycorrhiza.     

Meta-analysis of genome-wide scans for total body BMD in children and adults reveals allelic heterogeneity and age-specific effects at the WNT16 locus

To identify genetic loci influencing bone accrual, we performed a genome-wide association scan for total-body bone mineral density (TB-BMD) variation in 2,660 children of different ethnicities. We discovered variants in 7q31.31 associated with BMD measurements, with the lowest P = 4.1 × 10(-11) observed for rs917727 with minor allele frequency of 0.37. We sought replication for all SNPs located ± 500 kb from rs917727 in 11,052 additional individuals from five independent studies including children and adults, together with de novo genotyping of rs3801387 (in perfect linkage disequilibrium (LD) with rs917727) in 1,014 mothers of children from the discovery cohort. The top signal mapping in the surroundings of WNT16 was replicated across studies with a meta-analysis P = 2.6 × 10(-31) and an effect size explaining between 0.6%-1.8% of TB-BMD variance. Conditional analyses on this signal revealed a secondary signal for total body BMD (P = 1.42 × 10(-10)) for rs4609139 and mapping to C7orf58. We also examined the genomic region for association with skull BMD to test if the associations were independent of skeletal loading. We identified two signals influencing skull BMD variation, including rs917727 (P = 1.9 × 10(-16)) and rs7801723 (P = 8.9 × 10(-28)), also mapping to C7orf58 (r(2) = 0.50 with rs4609139). Wnt16 knockout (KO) mice with reduced total body BMD and gene expression profiles in human bone biopsies support a role of C7orf58 and WNT16 on the BMD phenotypes observed at the human population level. In summary, we detected two independent signals influencing total body and skull BMD variation in children and adults, thus demonstrating the presence of allelic heterogeneity at the WNT16 locus. One of the skull BMD signals mapping to C7orf58 is mostly driven by children, suggesting temporal determination on peak bone mass acquisition. Our life-course approach postulates that these genetic effects influencing peak bone mass accrual may impact the risk of osteoporosis later in life.     

Codon-optimized bacterial genes improve L-Arabinose fermentation in recombinant Saccharomyces cerevisiae

Bioethanol produced by microbial fermentations of plant biomass hydrolysates consisting of hexose and pentose mixtures is an excellent alternative to fossil transportation fuels. However, the yeast Saccharomyces cerevisiae, commonly used in bioethanol production, can utilize pentose sugars like l-arabinose or d-xylose only after heterologous expression of corresponding metabolic pathways from other organisms. Here we report the improvement of a bacterial l-arabinose utilization pathway consisting of l-arabinose isomerase from Bacillus subtilis and l-ribulokinase and l-ribulose-5-P 4-epimerase from Escherichia coli after expression of the corresponding genes in S. cerevisiae. l-Arabinose isomerase from B. subtilis turned out to be the limiting step for growth on l-arabinose as the sole carbon source. The corresponding enzyme could be effectively replaced by the enzyme from Bacillus licheniformis, leading to a considerably decreased lag phase. Subsequently, the codon usage of all the genes involved in the l-arabinose pathway was adapted to that of the highly expressed genes encoding glycolytic enzymes in S. cerevisiae. Yeast transformants expressing the codon-optimized genes showed strongly improved l-arabinose conversion rates. With this rational approach, the ethanol production rate from l-arabinose could be increased more than 2.5-fold from 0.014 g ethanol h(-1) (g dry weight)(-1) to 0.036 g ethanol h(-1) (g dry weight)(-1) and the ethanol yield could be increased from 0.24 g ethanol (g consumed l-arabinose)(-1) to 0.39 g ethanol (g consumed l-arabinose)(-1). These improvements make up a new starting point for the construction of more-efficient industrial l-arabinose-fermenting yeast strains by evolutionary engineering.     

Energy gain and energy gap in normal-weight children: longitudinal data of the KOPS

Population-based prevention of overweight needs evidence-based goals consistent with our present knowledge about energy gap (i.e., daily imbalance between energy intake and energy expenditure resulting in overweight). Longitudinal data of normal-weight children (1,029 girls and 1,028 boys; Kiel Obesity Prevention Study, KOPS) were used to calculate energy gain (i.e., increase in fat mass (FM) and fat-free mass (FFM)) in normal-weight children staying normal weight (persistent children) or becoming overweight (incident children). Taking into account weight gain in proportion to height gain (normal development) energy gap was calculated from increases in FM and FFM exceeding normal development. Children were divided into two groups and were followed from age 6 to 10 (group A) and 10 to 14 years (group B). FM and FFM were measured. Medians of 4-year BMI- (kg/m(2))/weight changes (kg) were +1.8/+13.2 (A) and +3.0/+18.7 (B) in girls, and +1.6/+12.8 (A) and +2.6/21.7 (B) in boys. Corresponding data for FM/FFM (kg) were +3.1/+10.2 (A) and +5.1/12.7 (B) in girls, and +2.3/10.8 (A) and +3.0/18.6 (B) in boys. The 4-year-incidence of overweight (%) were 9.4 (A) and 5.4 (B) in girls, and 11.0 (A) and 3.8 (B) in boys, respectively. Mean energy gains (kcal/day) were 26.8 (A) and 46.4 (B) in girls, and 22.1 (A) and 32.5 (B) in boys. The 90th percentile of energy gap (kcal/day) in incident children were 58.1 (A) and 72.0 (B) in girls and 46.0 (A) and 53.2 (B) in boys. To prevent overweight in children energy gap should not exceed 46-72 kcal/day.