Large-scale pathway specific polygenic risk and transcriptomic community network analysis identifies novel functional pathways in Parkinson disease.

Polygenic inheritance plays a central role in Parkinson disease (PD). A priority in elucidating PD etiology lies in defining the biological basis of genetic risk. Unraveling how risk leads to disruption will yield disease-modifying therapeutic targets that may be effective. Here, we utilized a high-throughput and hypothesis-free approach to determine biological processes underlying PD using the largest currently available cohorts of genetic and gene expression data from International Parkinson's Disease Genetics Consortium (IPDGC) and the Accelerating Medicines Partnership-Parkinson's disease initiative (AMP-PD), among other sources. We applied large-scale gene-set specific polygenic risk score (PRS) analyses to assess the role of common variation on PD risk focusing on publicly annotated gene sets representative of curated pathways. We nominated specific molecular sub-processes underlying protein misfolding and aggregation, post-translational protein modification, immune response, membrane and intracellular trafficking, lipid and vitamin metabolism, synaptic transmission, endosomal-lysosomal dysfunction, chromatin remodeling and apoptosis mediated by caspases among the main contributors to PD etiology. We assessed the impact of rare variation on PD risk in an independent cohort of whole-genome sequencing data and found evidence for a burden of rare damaging alleles in a range of processes, including neuronal transmission-related pathways and immune response. We explored enrichment linked to expression cell specificity patterns using single-cell gene expression data and demonstrated a significant risk pattern for dopaminergic neurons, serotonergic neurons, hypothalamic GABAergic neurons, and neural progenitors. Subsequently, we created a novel way of building de novo pathways by constructing a network expression community map using transcriptomic data derived from the blood of PD patients, which revealed functional enrichment in inflammatory signaling pathways, cell death machinery related processes, and dysregulation of mitochondrial homeostasis. Our analyses highlight several specific promising pathways and genes for functional prioritization and provide a cellular context in which such work should be done.

Gene therapy-emulating small molecule treatments in cystic fibrosis airway epithelial cells and patients.

BACKGROUND: Several small molecule corrector and potentiator drugs have recently been licensed for Cystic Fibrosis (CF) therapy. However, other aspects of the disease, especially inflammation, are less effectively treated by these drugs. We hypothesized that small molecule drugs could function either alone or as an adjuvant to licensed therapies to treat these aspects of the disease, perhaps emulating the effects of gene therapy in CF cells. The cardiac glycoside digitoxin, which has been shown to inhibit TNFα/NFκB signaling in CF lung epithelial cells, may serve as such a therapy. METHODS: IB3-1 CF lung epithelial cells were treated with different Vertex (VX) drugs, digitoxin, and various drug mixtures, and ELISA assays were used to assess suppression of baseline and TNFα-activated secretion of cytokines and chemokines. Transcriptional responses to these drugs were assessed by RNA-seq and compared with gene expression in AAV-[wildtype]CFTR-treated IB3-1 (S9) cells. We also compared in vitro gene expression signatures with in vivo data from biopsied nasal epithelial cells from digitoxin-treated CF patients. RESULTS: CF cells exposed to digitoxin exhibited significant suppression of both TNFα/NFκB signaling and downstream secretion of IL-8, IL-6 and GM-CSF, with or without co-treatment with VX drugs. No evidence of drug-drug interference was observed. RNA-seq analysis showed that gene therapy-treated CF lung cells induced changes in 3134 genes. Among these, 32.6% were altered by digitoxin treatment in the same direction. Shared functional gene ontology themes for genes suppressed by both digitoxin and gene therapy included inflammation (84 gene signature), and cell-cell interactions and fibrosis (49 gene signature), while genes elevated by both were enriched for epithelial differentiation (82 gene signature). A new analysis of mRNA data from digitoxin-treated CF patients showed consistent trends in expression for genes in these signatures. CONCLUSIONS: Adjuvant gene therapy-emulating activities of digitoxin may contribute to enhancing the efficacy of currently licensed correctors and potentiators in CF patients.

Parity and tanned white skin as novel predictors of vitamin D status in early pregnancy: a population-based cohort study.

CONTEXT: In pregnancy, vitamin D insufficiency and deficiency, defined as serum 25-hydroxyvitamin D (25(OH)D) <50 nM, and <25 nM, respectively, may have adverse effects for both mother and child. Prevalence estimates, and identification of subgroups at special risk, may be useful for the planning of preventive strategies. OBJECTIVE: To study the prevalence and risk factors of hypovitaminosis D in early pregnancy. DESIGN AND METHODS: In a cross-sectional study of 1348 women in early pregnancy from the Odense Child Cohort, Denmark, 25(OH)D was determined and correlated to demographic and lifestyle variables (age, nationality, skin tone, parity, prepregnancy body mass index (BMI), smoking and sun exposure), using multiple linear and logistic regression analyses for all year, or stratified for summer and winter. The risk of vitamin D insufficiency was expressed as odds ratios (OR) with 95% confidence intervals in brackets. RESULTS: The prevalence of vitamin D insufficiency and deficiency was estimated to 27·8% and 3·5% respectively. In adjusted analyses, vitamin D insufficiency was directly associated with winter season, OR = 1·89 (1·35-2·63); increasing prepregnancy BMI, OR = 1·06 (1·03-1·10); and smoking, OR = 2·7 (1·34-5·41); but was less frequent in nulliparous, OR = 0·47 (0·33-0·68) and tanned Caucasians, OR = 0·63 (0·41-0·97). Season-specific associations having parental origin from outside Europe in summer, OR = 4·13 (1·41-12·13); in winter smoking, OR = 3·15 (1·19-8·36); and prepregnancy BMI, OR = 1·12 (1·06-1·18). CONCLUSIONS: Vitamin D insufficiency was widespread in early pregnancy. Associations to smoking, prepregnancy BMI and origin outside Europe varied with season. Multiparity and not being tanned in Caucasians represent new risk factors of vitamin D insufficiency.

Sami dietary habits and the risk of cardiometabolic disease: a systematic review.

This systematic literary review investigates if an association between Sami dietary habits and cardiometabolic outcomes exists, and examines the dietary characteristics and cardiometabolic status of the Sami population. Included were all articles assessing Sami dietary habits and cardiometabolic disease or risk factors. Embase, Medline and SweMed were searched on 26 September 2019 and articles were screened for eligibility in October 2019. Data were extracted according to Moose Guidelines and the Newcastle Ottawa Scale (NOS) was used to assess risk of bias. The initial search generated 4,195 articles in total. Nine articles met all inclusion criteria. Two were cohort studies and seven were cross-sectional. Rating by NOS ranked from 2/7 to 8/9 stars. The studies were largely descriptive and only few had results regarding a direct association between Sami dietary habits and cardiometabolic outcomes. The findings demonstrated no association between consumption of certain Sami food items and blood-lipids or mortality from CVD/CHD. A higher intake of fat, protein, reindeer-meat and coffee and a slightly lower blood pressure and mortality from CVD/CHD was seen among Sami compared with non-Sami. The limited amount and descriptive nature of the eligible articles indicate that resaerch within the fielt is limited. Thus, additional longitudinal studies are suggested.

The MCP-4/MCP-1 ratio in plasma is a candidate circadian biomarker for chronic post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is psychiatric disease, which can occur following exposure to traumatic events. PTSD may be acute or chronic, and can have a waxing and waning course of symptoms. It has been hypothesized that proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) or plasma might be mediators of the psychophysiological mechanisms relating a history of trauma exposure to changes in behavior and mental health disorders, and medical morbidity. Here we test the cytokine/chemokine hypothesis for PTSD by examining levels of 17 classical cytokines and chemokines in CSF, sampled at 0900 hours, and in plasma sampled hourly for 24 h. The PTSD and healthy control patients are from the NIMH Chronic PTSD and healthy control cohort, initially described by Bonne et al. (2011), in which the PTSD patients have relatively low comorbidity for major depressive disorder (MDD), drug or alcohol use. We find that in plasma, but not CSF, the bivariate MCP4 (CCL13)/ MCP1(CCL2) ratio is ca. twofold elevated in PTSD patients compared with healthy controls. The MCP-4/MCP-1 ratio is invariant over circadian time, and is independent of gender, body mass index or the age at which the trauma was suffered. By contrast, MIP-1ß is a candidate biomarker for PTSD only in females, whereas TARC is a candidate biomarker for PTSD only in males. It remains to be discovered whether these disease-specific differences in circadian expression for these specific immune signaling molecules are biomarkers, surrogates, or drivers for PTSD, or whether any of these analytes could contribute to therapy.

Mammalian expression cloning of nucleic acid binding proteins by agarose thin-layer gelshift clone selection.

Here we report a functional screening technique to identify cDNAs encoding mammalian nucleic acid binding proteins. We have combined cDNA expression cloning with the agarose thin-layer gelshift assay technique to detect specific nucleic acid binding proteins from a mammalian expression library. We divided this cDNA expression library into multiple pools and transfected mammalian cells with the individual pools. Following transfection, we tested the expressed proteins for DNA-binding activity by agarose thin-layer electrophoretic gelshift assay. After we identified a single expression poolfor the presence of a DNA-binding protein, the corresponding cDNA pool was further divided into smaller aliquots. Then, the cDNA expression and gelshift clone selection was repeated until a single clone was isolated In contrast to traditional polyacrylamide gels, the agarose thin-layer is significantly faster and resolves larger DNA-protein complexes. This method can be widely used for the cDNA cloning of DNA- and RNA-binding proteins from various mammalian host cells.

Saturated fat intake is reduced in patients with ischemic heart disease 1 year after comprehensive counseling but not after brief counseling.

OBJECTIVE: To examine the dietary habits of patients with ischemic heart disease 1 year after they received either dietary advice on using the Plate Model and how to increase intakes of fruits and vegetables in a 10-minute session (brief counseling group, BCG) or dietary advice primarily based on the National Cholesterol Education Program Step I diet provided in 2 individually tailored 50-minute sessions held 3 months apart (comprehensive counseling group, CCG). DESIGN: A randomized study that included dietary intake evaluation on basis of 7-day weighed food records completed at 3 occasions: immediately before counseling (week zero), 12 weeks after counseling, and 52 weeks after counseling. SUBJECTS: BCG was composed of 15 men and 2 women and CCG was composed of 16 men and 3 women with ischemic heart disease age 70 years or younger recruited from the Department of Cardiology, Odense University Hospital, Odense, Denmark. STATISTICAL ANALYSES PERFORMED: ANOVA, unpaired t tests, and multiple regression analysis, as well as nonparametric statistical analyses were carried out. RESULTS: The comprehensive counseling resulted in significant improvements from week 0 to 52 in the percent of energy from fat (33% to 28%), saturated fat (12% to 9%) and carbohydrate (51% to 54%) consumed by the subjects. The corresponding values in BCG did not differ significantly (31% to 32%, 11% to 12%, 53% to 52% respectively). Differences from week 0 to 52 between groups were significant for fat, saturated fat, and carbohydrate intake. In CCG, median intakes of fish, fruits, and vegetables were 44 g/day, 172 g/day, and 315 g/day, respectively, at week 52. The corresponding values in BCG were 44 g/day, 129 g/day, and 224 g/day. There was no significant difference either within or between the groups. CONCLUSION: This study suggests that sustained improvements in dietary behavior require individualized and reinforced counseling in patients with ischemic heart disease. Changes in intakes of fish, fruits, and vegetables need to be specifically targeted.

Valproic acid inhibits neurosphere formation by adult subventricular cells by a lithium-sensitive mechanism.

The mood stabilizer valproic acid (VPA) decreases neural progenitor proliferation and promotes neurogenesis in the adult hippocampus. However, the effects of VPA on progenitor cells in the adult subventricular zone (SVZ) are not as well characterized. Here we report VPA blocks neurosphere formation and inhibits DNA synthesis in cultured NSCs from the SVZ of adult mice. Inhibition of DNA synthesis is associated with the up-regulation of the differentiation transcription factors Egr1 and Neurod1 and down-regulation of transcription factors associated with "stemness". Co-treatment of VPA with the mood stabilizer lithium antagonizes the anti-proliferative effects of VPA on adult NSCs and abolishes VPA activation of Egr1. Co-treatment of VPA with the MEK1/2 inhibitor PD980589 similarly abolishes Egr1 activation consistent with VPA activation and lithium antagonism of MEK-ERK signaling in adult NSCs. However, Western blot reveals VPA significantly suppresses ERK2 phosphorylation in adult NSCs grown in proliferating culture conditions and that lithium co-treatment does not attenuate this effect. Combined the data indicate VPA inhibition of adult NSC proliferation and activation of Egr1 by VPA, along with the antagonism of these effects by lithium, are the effects of cumulative changes in multiple signaling pathways and are not attributable to a common kinase target.

Altered gene expression in the emerging cerebellar primordium of Neurog1-/- mice.

Expression of the basic helix-loop-helix (bHLH) transcription factor Neurogenin1 (Neurog1) coincides with the emergence of the cerebellum and Neurog1-expressing progenitors are fated to become Purkinje cells and later interneurons. However, the gene regulatory functions of Neurog1 in cerebellar development have not been characterized. We performed a genome-wide analysis of gene expression in the cerebellar primordium of E11.5 Neurog1 null (Neurog1-/-) mice to identify the Neurog1 transcriptome in the emerging cerebellum. This screen identified 117 genes differentially enriched in Neurog1-/- versus control sample sets with a high presence of gene sets enriched for functions in nervous system development. Hierarchical clustering revealed complete stratification of differentially expressed genes based on Neurog1 gene deletion status. In silico analysis of promoter regions identifies high probability Neurog1 regulatory (E-box) binding sites in 94 of the 117 differentially expressed genes and Pax6 binding motifs in 25 of these 94 promoters. Our data provide a framework for investigating Neurog1 transcriptional programs in early cerebellar development and suggest functional Neurog1-Pax6 cross-talk in the activation of downstream targets.

Intraocular pressure in the first days after implantation of the semiflexible McGhan/3 M, style 70, anterior chamber lens.

Intracapsular cataract extraction employing a corneal incision and with implantation of the semiflexible McGhan/3 M, style 70, anterior chamber lens was performed in a group of 25 patients with senile cataract. Intraocular pressure (IOP) was measured prior to and daily in the first 4 days after the operation. IOP was slightly, but significantly (P less than or equal to 0.05) lower in the observed post-operative period as compared to the pre-operative measurement. Similar results have previously been found after cataract extraction with an identical technique, but without lens implantation. This probably means that early post-operative intraocular homeostasis with respect to IOP is unaffected by the implantation of this type of lens. The fine regulation of early post-operative IOP is probably related to the fact that the described surgical technique usually is associated with only minor gonioscopically visible changes of the trabecular meshwork.

Functional partitioning of beta1 integrins revealed by activating and inhibitory mAbs.

Integrins exist in different activation states on the surfaces of cells. Addition of the proper signal, ligand, or antibody can alter the activation state of these molecules. We report here the identification of two immunocytochemically distinct populations of beta1 integrins on fixed embryonic chick dermal fibroblasts. One population, recognized by the integrin activating mAb TASC, localizes to discrete regions of the cell, most likely focal contacts. These integrins co-localize with other proteins, such as vinculin and F-actin, and their retention at these sites is dependent on the actin cytoskeleton. The other population, identified with the inhibitory mAb W1B10, is more evenly distributed throughout the cell surface, and its pattern remains unchanged after disruption of the actin cytoskeleton. Double labeling experiments using Fab fragments of TASC alongside whole W1B10 IgG revealed non-overlapping staining patterns. These results show that it is possible to visualize and study discrete populations of integrins on cell surfaces using two different antibodies. We hypothesize that these antibodies report differences in the distribution of receptors in two different states. A model is proposed describing the ligand independent recruitment of integrins based on these findings and results from other labs.

Mercury in the Umbilical Cord: Implications for Risk Assessment for Minamata Disease.

Umbilical cord tissue was obtained from 50 births in the Faroe Islands, where high mercury intake is due to ingestion of pilot whale meat. The mercury concentration correlated significantly with the frequency of maternal whale meat dinners during pregnancy and with mercury concentrations in umbilical cord blood and in maternal hair. The results were compared with published values for mercury in umbilical cord tissue from 12 infants diagnosed with congenital methylmercury poisoning in Minamata, Japan. From the regression coefficients obtained in the Faroese samples, the median umbilical cord mercury concentration of 4.95 nmol/g dry weight in Minamata would correspond to 668 nmol/l cord blood and 114 nmol/g maternal hair. These levels agree well with other evidence of susceptibility of the fetus to increased exposure to methylmercury.