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.     

Protection of blood retinal barrier and systemic vasculature by insulin-like growth factor binding protein-3

Previously, we showed that insulin growth factor (IGF)-1 binding protein-3 (IGFBP-3), independent of IGF-1, reduces pathological angiogenesis in a mouse model of the oxygen-induced retinopathy (OIR). The current study evaluates novel endothelium-dependent functions of IGFBP-3 including blood retinal barrier (BRB) integrity and vasorelaxation. To evaluate vascular barrier function, either plasmid expressing IGFBP-3 under the regulation of an endothelial-specific promoter or a control plasmid was injected into the vitreous humor of mouse pups (P1) and compared to the non-injected eyes of the same pups undergoing standard OIR protocol. Prior to sacrifice, the mice were given an injection of horseradish peroxidase (HRP). IGFBP-3 plasmid-injected eyes displayed near-normal vessel morphology and enhanced vascular barrier function. Further, in vitro IGFBP-3 protects retinal endothelial cells from VEGF-induced loss of junctional integrity by antagonizing the dissociation of the junctional complexes. To assess the vasodilatory effects of IGFBP-3, rat posterior cerebral arteries were examined in vitro. Intraluminal IGFBP-3 decreased both pressure- and serotonin-induced constrictions by stimulating nitric oxide (NO) release that were blocked by L-NAME or scavenger receptor-B1 neutralizing antibody (SRB1-Ab). Both wild-type and IGF-1-nonbinding mutant IGFBP-3 (IGFBP-3NB) stimulated eNOS activity/NO release to a similar extent in human microvascular endothelial cells (HMVECs). NO release was neither associated with an increase in intracellular calcium nor decreased by Ca(2+)/calmodulin-dependent protein kinase II (CamKII) blockade; however, dephosphorylation of eNOS-Thr(495) was observed. Phosphatidylinositol 3-kinase (PI3K) activity and Akt-Ser(473) phosphorylation were both increased by IGFBP-3 and selectively blocked by the SRB1-Ab or PI3K blocker LY294002. In conclusion, IGFBP-3 mediates protective effects on BRB integrity and mediates robust NO release to stimulate vasorelaxation via activation of SRB1. This response is IGF-1- and calcium-independent, but requires PI3K/Akt activation, suggesting that IGFBP-3 has novel protective effects on retinal and systemic vasculature and may be a therapeutic candidate for ocular complications such as diabetic retinopathy.     

Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma

Allergic asthma rates have increased steadily in developed countries, arguing for an environmental aetiology. To assess the influence of gut microbiota on experimental murine allergic asthma, we treated neonatal mice with clinical doses of two widely used antibiotics--streptomycin and vancomycin--and evaluated resulting shifts in resident flora and subsequent susceptibility to allergic asthma. Streptomycin treatment had little effect on the microbiota and on disease, whereas vancomycin reduced microbial diversity, shifted the composition of the bacterial population and enhanced disease severity. Neither antibiotic had a significant effect when administered to adult mice. Consistent with the 'hygiene hypothesis', our data support a neonatal, microbiota-driven, specific increase in susceptibility to experimental murine allergic asthma.     

The Vitis vinifera C-repeat binding protein 4 (VvCBF4) transcriptional factor enhances freezing tolerance in wine grape

Chilling and freezing can reduce significantly vine survival and fruit set in Vitis vinifera wine grape. To overcome such production losses, a recently identified grapevine C‐repeat binding factor (CBF) gene, VvCBF4, was overexpressed in grape vine cv. ‘Freedom’ and found to improve freezing survival and reduced freezing‐induced electrolyte leakage by up to 2 °C in non‐cold‐acclimated vines. In addition, overexpression of this transgene caused a reduced growth phenotype similar to that observed for CBF overexpression in Arabidopsis and other species. Both freezing tolerance and reduced growth phenotypes were manifested in a transgene dose‐dependent manner. To understand the mechanistic basis of VvCBF4 transgene action, one transgenic line (9–12) was genotyped using microarray‐based mRNA expression profiling. Forty‐seven and 12 genes were identified in unstressed transgenic shoots with either a >1.5‐fold increase or decrease in mRNA abundance, respectively. Comparison of mRNA changes with characterized CBF regulons in woody and herbaceous species revealed partial overlaps, suggesting that CBF‐mediated cold acclimation responses are widely conserved. Putative VvCBF4‐regulon targets included genes with functions in cell wall structure, lipid metabolism, epicuticular wax formation and stress‐responses suggesting that the observed cold tolerance and dwarf phenotypes are the result of a complex network of diverse functional determinants.     

Plasticity of DNA methylation in mouse T cell activation and differentiation

Background

Circulating CD4⁺ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4⁺ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells.

Results

Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status.

Conclusion

We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.     

Structural and Functional Properties of Glycerol-3-Phosphate Dehydrogenase from a Mammalian Hibernator

Glycerol-3-phosphate dehydrogenase (G3PDH; E.C.1.1.1.8) was purified from liver and skeletal muscle of black-tailed prairie dogs (Cynomys ludivicianus), a hibernating species. Native and subunit molecular masses of the dimeric enzyme were 77 and 40 kD, respectively, and both tissues contained a single isozyme with a pI of 6.4. Kinetic parameters of purified G3PDH from prairie dog liver and muscle were characterized at 22 and 5 °C and compared with rabbit muscle G3PDH. Substrate affinities for hibernator muscle G3PDH were stable (NAD) or increased significantly (K_m G3P and DHAP decreased) at low temperature whereas K_m NAD and DHAP of rabbit G3PDH increased. Prairie dog G3PDH showed greater conservation of K_m G3P over a wide temperature range as well as greater thermal stability and resistance to chemical denaturation by guanidine hydrochloride than the rabbit enzyme. In addition, using the protein sequence of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and bioinformatics tools, the deduced protein structure of G3PDH was compared between heterothermic and homeothermic mammals. Structural and functional characteristics of G3PDH from the hibernating species would support enzyme function over a wide range of core body temperatures over cycles of torpor and arousal.     

Human Fidgetin is a microtubule severing the enzyme and minus-end depolymerase that regulates mitosis

Fidgetin is a member of the AAA protein superfamily with important roles in mammalian development. Here we show that human Fidgetin is a potent microtubule severing and depolymerizing the enzyme used to regulate mitotic spindle architecture, dynamics and anaphase A. In vitro, recombinant human Fidgetin severs taxol-stabilized microtubules along their length and promotes depolymerization, primarily from their minus-ends. In cells, human Fidgetin targets to centrosomes, and its depletion with siRNA significantly reduces the velocity of poleward tubulin flux and anaphase A chromatid-to-pole motion. In addition, the loss of Fidgetin induces a microtubule-dependent enlargement of mitotic centrosomes and an increase in the number and length of astral microtubules. Based on these data, we propose that human Fidgetin actively suppresses microtubule growth from and attachment to centrosomes.     

Efficiency and power as a function of sequence coverage, SNP array density, and imputation

High coverage whole genome sequencing provides near complete information about genetic variation. However, other technologies can be more efficient in some settings by (a) reducing redundant coverage within samples and (b) exploiting patterns of genetic variation across samples. To characterize as many samples as possible, many genetic studies therefore employ lower coverage sequencing or SNP array genotyping coupled to statistical imputation. To compare these approaches individually and in conjunction, we developed a statistical framework to estimate genotypes jointly from sequence reads, array intensities, and imputation. In European samples, we find similar sensitivity (89%) and specificity (99.6%) from imputation with either 1× sequencing or 1 M SNP arrays. Sensitivity is increased, particularly for low-frequency polymorphisms (MAF < 5%), when low coverage sequence reads are added to dense genome-wide SNP arrays--the converse, however, is not true. At sites where sequence reads and array intensities produce different sample genotypes, joint analysis reduces genotype errors and identifies novel error modes. Our joint framework informs the use of next-generation sequencing in genome wide association studies and supports development of improved methods for genotype calling.