Alginate Composition Effects on a Neural Stem Cell-Seeded Scaffold

The purpose of this study was to evaluate the effects of alginate composition on the neurotrophic factor release, viability, and proliferation of encapsulated neural stem cells (NSCs), as well as on the mechanical stability of the scaffold itself. Four compositions were tested: a high guluronic acid (68%) and a high mannuronic acid (54%) content alginate, with or without a poly-L-lysine (PLL) coating layer. Enzyme-linked immunosorbent assay was used to quantify the release of brain-derived neurotrophic factor, glial-derived neurotrophic factor, and nerve growth factor from the encapsulated cells. All three factors were detected from encapsulated cells only when a high L-guluronic acid alginate without PLL was used. Additionally, capsules with this composition remained intact more frequently when exposed to solutions of low osmolarity, potentially indicating superior mechanical stability. Alginate beads with a PLL-coated, high D-mannuronic acid composition were the most prone to breakage in the osmotic pressure test, and were too fragile for histology and proliferation assays after 1 week in vitro. NSCs survived and proliferated in the three remaining alginate compositions similarly over the 21-day study course irrespective of scaffold condition. NSC-seeded alginate beads with a high L-guluronic acid, non-PLL-coated composition may be useful in the repair of injured nervous tissue, where the mechanism is the secretion of neuroprotective factors. We verify the neuroprotective effects of medium conditioned by NSC-seeded alginate beads on the serum withdrawal-mediated death of PC-12 cells here.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78133/1/ten.tec.2008.0302.pd

Netrin-1-Mediated Axon Guidance in Mouse Embryonic Stem Cells Overexpressing Neurogenin-1

Stem cell therapy holds great promise for treating neurodegenerative disease, but major barriers to effective therapeutic strategies remain. A complete understanding of the derived phenotype is required for predicting cell response once introduced into the host tissue. We sought to identify major axonal guidance cues present in neurons derived from the transient overexpression of neurogenin-1 (Neurog1) in mouse embryonic stem cells (ESCs). Neurog1 upregulated the netrin-1 axon guidance receptors DCC (deleted in colorectal cancer) and neogenin (NEO1). Quantitative polymerase chain reaction results showed a 2-fold increase in NEO1 mRNA and a 36-fold increase in DCC mRNA in Neurog1-induced compared with control ESCs. Immunohistochemistry indicated that DCC was primarily expressed on cells positive for the neuronal marker TUJ1. DCC was preferentially localized to the cell soma and growth-cones of induced neurons. In contrast, NEO1 expression showed less specificity, labeling both TUJ1-positive and TUJ1-negative cells as well as uninduced control cells. Axonal outgrowth was directed preferentially toward aggregates of HEK293 cells secreting a recombinant active fragment of netrin-1. These data indicate that DCC and NEO1 are downstream products of Neurog1 and may guide the integration of Neurog1-induced ESCs with target cells secreting netrin-1. Differential expression profiles for netrin receptors could indicate different roles for this guidance cue on neuronal and non-neuronal cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98473/1/scd%2E2011%2E0437.pd

Unique Features of Alarmone Metabolism in \u3ci\u3eClostridioides difficile\u3c/i\u3e

The “magic spot” alarmones (pp)pGpp, previously implicated in Clostridioides difficile antibiotic survival, are synthesized by the RelA-SpoT homolog (RSH) of C. difficile (RSHCd) and RelQCd. These enzymes are transcriptionally activated by diverse environmental stresses. RSHCd has previously been reported to synthesize ppGpp, but in this study, we found that both clostridial enzymes exclusively synthesize pGpp. While direct synthesis of pGpp from a GMP substrate, and (p)ppGpp hydrolysis into pGpp by NUDIX hydrolases, have previously been reported, there is no precedent for a bacterium synthesizing pGpp exclusively. Hydrolysis of the 5′ phosphate or pyrophosphate from GDP or GTP substrates is necessary for activity by the clostridial enzymes, neither of which can utilize GMP as a substrate. Both enzymes are remarkably insensitive to the size of their metal ion cofactor, tolerating a broad array of metals that do not allow activity in (pp)pGpp synthetases from other organisms. It is clear that while C. difficile utilizes alarmone signaling, its mechanisms of alarmone synthesis are not directly homologous to those in more completely characterized organisms

Spatial Transcriptomics as a Novel Approach to Redefine Electrical Stimulation Safety

Current standards for safe delivery of electrical stimulation to the central nervous system are based on foundational studies which examined post-mortem tissue for histological signs of damage. This set of observations and the subsequently proposed limits to safe stimulation, termed the “Shannon limits,” allow for a simple calculation (using charge per phase and charge density) to determine the intensity of electrical stimulation that can be delivered safely to brain tissue. In the three decades since the Shannon limits were reported, advances in molecular biology have allowed for more nuanced and detailed approaches to be used to expand current understanding of the physiological effects of stimulation. Here, we demonstrate the use of spatial transcriptomics (ST) in an exploratory investigation to assess the biological response to electrical stimulation in the brain. Electrical stimulation was delivered to the rat visual cortex with either acute or chronic electrode implantation procedures. To explore the influence of device type and stimulation parameters, we used carbon fiber ultramicroelectrode arrays (7 μm diameter) and microwire electrode arrays (50 μm diameter) delivering charge and charge density levels selected above and below reported tissue damage thresholds (range: 2–20 nC, 0.1–1 mC/cm2). Spatial transcriptomics was performed using Visium Spatial Gene Expression Slides (10x Genomics, Pleasanton, CA, United States), which enabled simultaneous immunohistochemistry and ST to directly compare traditional histological metrics to transcriptional profiles within each tissue sample. Our data give a first look at unique spatial patterns of gene expression that are related to cellular processes including inflammation, cell cycle progression, and neuronal plasticity. At the acute timepoint, an increase in inflammatory and plasticity related genes was observed surrounding a stimulating electrode compared to a craniotomy control. At the chronic timepoint, an increase in inflammatory and cell cycle progression related genes was observed both in the stimulating vs. non-stimulating microwire electrode comparison and in the stimulating microwire vs. carbon fiber comparison. Using the spatial aspect of this method as well as the within-sample link to traditional metrics of tissue damage, we demonstrate how these data may be analyzed and used to generate new hypotheses and inform safety standards for stimulation in cortex

Next-Generation Diamond Electrodes for Neurochemical Sensing: Challenges and Opportunities

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Carbon-based electrodes combined with fast-scan cyclic voltammetry (FSCV) enable neurochemical sensing with high spatiotemporal resolution and sensitivity. While their attractive electrochemical and conductive properties have established a long history of use in the detection of neurotransmitters both in vitro and in vivo, carbon fiber microelectrodes (CFMEs) also have limitations in their fabrication, flexibility, and chronic stability. Diamond is a form of carbon with a more rigid bonding structure (sp3-hybridized) which can become conductive when boron-doped. Boron-doped diamond (BDD) is characterized by an extremely wide potential window, low background current, and good biocompatibility. Additionally, methods for processing and patterning diamond allow for high-throughput batch fabrication and customization of electrode arrays with unique architectures. While tradeoffs in sensitivity can undermine the advantages of BDD as a neurochemical sensor, there are numerous untapped opportunities to further improve performance, including anodic pretreatment, or optimization of the FSCV waveform, instrumentation, sp2 /sp3 character, doping, surface characteristics, and signal processing. Here, we review the state-of-the-art in diamond electrodes for neurochemical sensing and discuss potential opportunities for future advancements of the technology. We highlight our team’s progress with the development of an all-diamond fiber ultramicroelectrode as a novel approach to advance the performance and applications of diamond-based neurochemical sensors

Genomewide Association Study for Determinants of HIV-1 Acquisition and Viral Set Point in HIV-1 Serodiscordant Couples with Quantified Virus Exposure

Host genetic factors may be important determinants of HIV-1 sexual acquisition. We performed a genome-wide association study (GWAS) for host genetic variants modifying HIV-1 acquisition and viral control in the context of a cohort of African HIV-1 serodiscordant heterosexual couples. To minimize misclassification of HIV-1 risk, we quantified HIV-1 exposure, using data including plasma HIV-1 concentrations, gender, and condom use.We matched couples without HIV-1 seroconversion to those with seroconversion by quantified HIV-1 exposure risk. Logistic regression of single nucleotide polymorphisms (SNPs) for 798 samples from 496 HIV-1 infected and 302 HIV-1 exposed, uninfected individuals was performed to identify factors associated with HIV-1 acquisition. In addition, a linear regression analysis was performed using SNP data from a subset (n = 403) of HIV-1 infected individuals to identify factors predicting plasma HIV-1 concentrations.After correcting for multiple comparisons, no SNPs were significantly associated with HIV-1 infection status or plasma HIV-1 concentrations.This GWAS controlling for HIV-1 exposure did not identify common host genotypes influencing HIV-1 acquisition. Alternative strategies, such as large-scale sequencing to identify low frequency variation, should be considered for identifying novel host genetic predictors of HIV-1 acquisition

Cholesterol Influences Voltage-Gated Calcium Channels and BK-Type Potassium Channels in Auditory Hair Cells

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitabilityare unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassiumcurrent by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (∼30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology

Association Analysis of the FTO Gene with Obesity in Children of Caucasian and African Ancestry Reveals a Common Tagging SNP

Recently an association was demonstrated between the single nucleotide polymorphism (SNP), rs9939609, within the FTO locus and obesity as a consequence of a genome wide association (GWA) study of type 2 diabetes in adults. We examined the effects of two perfect surrogates for this SNP plus 11 other SNPs at this locus with respect to our childhood obesity cohort, consisting of both Caucasians and African Americans (AA). Utilizing data from our ongoing GWA study in our cohort of 418 Caucasian obese children (BMI≥95th percentile), 2,270 Caucasian controls (BMI<95th percentile), 578 AA obese children and 1,424 AA controls, we investigated the association of the previously reported variation at the FTO locus with the childhood form of this disease in both ethnicities. The minor allele frequencies (MAF) of rs8050136 and rs3751812 (perfect surrogates for rs9939609 i.e. both r2 = 1) in the Caucasian cases were 0.448 and 0.443 respectively while they were 0.391 and 0.386 in Caucasian controls respectively, yielding for both an odds ratio (OR) of 1.27 (95% CI 1.08–1.47; P = 0.0022). Furthermore, the MAFs of rs8050136 and rs3751812 in the AA cases were 0.449 and 0.115 respectively while they were 0.436 and 0.090 in AA controls respectively, yielding an OR of 1.05 (95% CI 0.91–1.21; P = 0.49) and of 1.31 (95% CI 1.050–1.643; P = 0.017) respectively. Investigating all 13 SNPs present on the Illumina HumanHap550 BeadChip in this region of linkage disequilibrium, rs3751812 was the only SNP conferring significant risk in AA. We have therefore replicated and refined the association in an AA cohort and distilled a tag-SNP, rs3751812, which captures the ancestral origin of the actual mutation. As such, variants in the FTO gene confer a similar magnitude of risk of obesity to children as to their adult counterparts and appear to have a global impact

Genome-Wide Gene-Environment Study Identifies Glutamate Receptor Gene GRIN2A as a Parkinson's Disease Modifier Gene via Interaction with Coffee

Our aim was to identify genes that influence the inverse association of coffee with the risk of developing Parkinson's disease (PD). We used genome-wide genotype data and lifetime caffeinated-coffee-consumption data on 1,458 persons with PD and 931 without PD from the NeuroGenetics Research Consortium (NGRC), and we performed a genome-wide association and interaction study (GWAIS), testing each SNP's main-effect plus its interaction with coffee, adjusting for sex, age, and two principal components. We then stratified subjects as heavy or light coffee-drinkers and performed genome-wide association study (GWAS) in each group. We replicated the most significant SNP. Finally, we imputed the NGRC dataset, increasing genomic coverage to examine the region of interest in detail. The primary analyses (GWAIS, GWAS, Replication) were performed using genotyped data. In GWAIS, the most significant signal came from rs4998386 and the neighboring SNPs in GRIN2A. GRIN2A encodes an NMDA-glutamate-receptor subunit and regulates excitatory neurotransmission in the brain. Achieving P2df = 10−6, GRIN2A surpassed all known PD susceptibility genes in significance in the GWAIS. In stratified GWAS, the GRIN2A signal was present in heavy coffee-drinkers (OR = 0.43; P = 6×10−7) but not in light coffee-drinkers. The a priori Replication hypothesis that “Among heavy coffee-drinkers, rs4998386_T carriers have lower PD risk than rs4998386_CC carriers” was confirmed: ORReplication = 0.59, PReplication = 10−3; ORPooled = 0.51, PPooled = 7×10−8. Compared to light coffee-drinkers with rs4998386_CC genotype, heavy coffee-drinkers with rs4998386_CC genotype had 18% lower risk (P = 3×10−3), whereas heavy coffee-drinkers with rs4998386_TC genotype had 59% lower risk (P = 6×10−13). Imputation revealed a block of SNPs that achieved P2df<5×10−8 in GWAIS, and OR = 0.41, P = 3×10−8 in heavy coffee-drinkers. This study is proof of concept that inclusion of environmental factors can help identify genes that are missed in GWAS. Both adenosine antagonists (caffeine-like) and glutamate antagonists (GRIN2A-related) are being tested in clinical trials for treatment of PD. GRIN2A may be a useful pharmacogenetic marker for subdividing individuals in clinical trials to determine which medications might work best for which patients

A Common CNR1 (Cannabinoid Receptor 1) Haplotype Attenuates the Decrease in HDL Cholesterol That Typically Accompanies Weight Gain

We have previously shown that genetic variability in CNR1 is associated with low HDL dyslipidemia in a multigenerational obesity study cohort of Northern European descent (209 families, median  = 10 individuals per pedigree). In order to assess the impact of CNR1 variability on the development of dyslipidemia in the community, we genotyped this locus in all subjects with class III obesity (body mass index >40 kg/m2) participating in a population-based biobank of similar ancestry. Twenty-two haplotype tagging SNPs, capturing the entire CNR1 gene locus plus 15 kb upstream and 5 kb downstream, were genotyped and tested for association with clinical lipid data. This biobank contains data from 645 morbidly obese study subjects. In these subjects, a common CNR1 haplotype (H3, frequency 21.1%) is associated with fasting TG and HDL cholesterol levels (p = 0.031 for logTG; p = 0.038 for HDL-C; p = 0.00376 for log[TG/HDL-C]). The strength of this relationship increases when the data are adjusted for age, gender, body mass index, diet and physical activity. Mean TG levels were 160±70, 155±70, and 120±60 mg/dL for subjects with 0, 1, and 2 copies of the H3 haplotype. Mean HDL-C levels were 45±10, 47±10, and 48±9 mg/dL, respectively. The H3 CNR1 haplotype appears to exert a protective effect against development of obesity-related dyslipidemia