MSA: scalable DNA methylation screening BeadChip for high-throughput trait association studies.

The Infinium DNA Methylation BeadChips have significantly contributed to population-scale epigenetics research by enabling epigenome-wide trait association discoveries. Here, we design, describe, and experimentally verify a new iteration of this technology, the Methylation Screening Array (MSA), to focus on human trait screening and discovery. This array utilizes extensive data from previous Infinium platform-based epigenome-wide association studies (EWAS). It incorporates knowledge from the latest single-cell and cell type-resolution whole genome methylome profiles. The MSA is engineered to achieve scalable screening of epigenetics-trait association in an ultra-high sample throughput. Our design encompassed diverse human trait associations, including those with genetic, cellular, environmental, and demographical variables and human diseases such as genetic, neurodegenerative, cardiovascular, infectious, and immune diseases. We comprehensively evaluated this array's reproducibility, accuracy, and capacity for cell-type deconvolution and supporting 5-hydroxymethylation profiling in diverse human tissues. Our first atlas data using this platform uncovered the complex chromatin and tissue contexts of DNA modification variations and genetic variants linked to human phenotypes.

Multi-center clinical evaluation of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab specimens from symptomatic individuals.

The symptomology is overlapping for respiratory infections due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A/B viruses, and respiratory syncytial virus (RSV). Accurate detection is essential for proper medical management decisions. This study evaluated the clinical performance of the Panther Fusion SARS-CoV-2/Flu A/B/RSV assay in nasopharyngeal swab (NPS) specimens from individuals of all ages with signs and symptoms of respiratory infection consistent with COVID-19, influenza, or RSV. Retrospective known-positive and prospectively obtained residual NPS specimens were collected during two respiratory seasons in the USA. Clinical performance was established by comparing Panther Fusion SARS-CoV-2/Flu assay results to a three-molecular assay composite comparator interpretation for SARS-CoV-2 and to the FDA-cleared Panther Fusion Flu A/B/RSV assay results for all non-SARS-CoV-2 targets. A total of 1,900 prospective and 95 retrospective NPS specimens were included in the analyses. The overall prevalence in prospectively obtained specimens was 20.7% for SARS-CoV-2, 6.7% for influenza A, and 0.7% for RSV; all influenza B-positive specimens were retrospective specimens. The positive percent agreement of the Panther Fusion assay was 96.9% (378/390) for SARS-CoV-2, 98.0% (121/123) for influenza A virus, 95.2% (20/21) for influenza B virus, and 96.6% (57/59) for RSV. The negative percent agreement was ≥98.5% for all target viruses. Specimens with discordant Panther Fusion SARS/Flu/RSV assay results all had cycle threshold values of ≥32.4 (by comparator or by Panther Fusion SARS/Flu/RSV assay). Only five co-infections were detected in the study specimens. The Panther Fusion SARS-CoV-2/Flu/RSV assay provides highly sensitive and specific detection of SARS-CoV-2, influenza A virus, influenza B virus, and RSV in NPS specimens.

An anchor-tether 'hindered' HCN1 inhibitor is antihyperalgesic in a rat spared nerve injury neuropathic pain model.

BACKGROUND: Neuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood-brain barrier. METHODS: In silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether 'hindered' variants of 26DTB-P (wherein a hydrophilic 'anchor' is attached in the para-position of 26DTB-P via an acyl chain 'tether') had the desired properties. RESULTS: Molecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing. CONCLUSIONS: These findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets.

Nicotinamide Riboside Improves Enteric Neuropathy in Streptozocin-Induced Diabetic Rats Through Myenteric Plexus Neuroprotection.

BACKGROUND: Diabetes Mellitus causes a systemic oxidative stress due in part to the hyperglycemia and the reactive oxygen species generated. Up to 75% of diabetic patients present with an autonomic neuropathy affecting the Enteric Nervous System. Deficits in the human population are chronic dysmotilities with either increased (i.e., constipation) or decreased (i.e., diarrhea) total gastrointestinal transit times. These are recapitulated in the streptozocin-induced diabetic rat, which is a model of Type I Diabetes Mellitus. AIMS: Examine the effects that a precursor of nicotinamide adenosine dinucleotide (NAD), nicotinamide riboside (NR), had on the development of dysmotility in induced diabetic rats and if fecal microbiota transplant (FMT) could produce the same results. MATERIALS AND METHODS: Utilizing a 6-week treatment paradigm, NR was administered intraperitoneally every 48 h. Total gastrointestinal transit time was assessed weekly utilizing the carmine red method. Three weeks following hyperglycemic induction, FMT was performed between NR-treated animals and untreated animals. SIGNIFICANT RESULTS: There is improvement in overall gastrointestinal transit time with the use of NR. 16S microbiome sequencing demonstrated decreased alpha and beta diversity in induced diabetic rats without change in animals receiving FMT. Improvements in myenteric plexus ganglia density in small and large intestines in diabetic animals treated with NR were seen. CONCLUSIONS: NR treatment led to functional improvement in total gastrointestinal transit time in induced diabetic animals. This was associated with neuroprotection in the myenteric plexuses of both small and large intestines of induced diabetic rats. This represents an important first step in showing NR's benefit as a treatment for diabetic enteric neuropathy. Streptozocin-induced diabetic rats have improved transit times and increased myenteric plexus ganglia density when treated with intraperitoneal nicotinamide riboside.

Failure to Upregulate the RNA Binding Protein ZBP After Injury Leads to Impaired Regeneration in a Rodent Model of Diabetic Peripheral Neuropathy.

Most diabetes patients eventually suffer from peripheral nerve degeneration. Unfortunately, there is no treatment for the condition and its mechanisms are not well understood. There is, however, an emerging consensus that the inability of peripheral nerves to regenerate normally after injury contributes to the pathophysiology. We have previously shown that regeneration of peripheral axons requires local axonal translation of a pool of axonal mRNAs and that the levels and members of this axonal mRNA pool are altered in response to injury. Here, we show that following sciatic nerve injury in a streptozotocin rodent model of type I diabetes, this mobilization of RNAs into the injured axons is attenuated and correlates with decreased axonal regeneration. This failure of axonal RNA localization results from decreased levels of the RNA binding protein ZBP1. Over-expression of ZBP1 rescues the in vitro growth defect in injured dorsal root ganglion neurons from diabetic rodents. These results provide evidence that decreased neuronal responsiveness to injury in diabetes is due to a decreased ability to alter the pool of axonal mRNAs available for local translation, and may open new therapeutic opportunities for diabetic peripheral neuropathy.

Zeb2 Is a Regulator of Astrogliosis and Functional Recovery after CNS Injury.

The astrocytic response to injury is characterized on the cellular level, but our understanding of the molecular mechanisms controlling the cellular processes is incomplete. The astrocytic response to injury is similar to wound-healing responses in non-neural tissues that involve epithelial-to-mesenchymal transitions (EMTs) and upregulation in ZEB transcription factors. Here we show that injury-induced astrogliosis increases EMT-related genes expression, including Zeb2, and long non-coding RNAs, including Zeb2os, which facilitates ZEB2 protein translation. In mouse models of either contusive spinal cord injury or transient ischemic stroke, the conditional knockout of Zeb2 in astrocytes attenuates astrogliosis, generates larger lesions, and delays the recovery of motor function. These findings reveal ZEB2 as an important regulator of the astrocytic response to injury and suggest that astrogliosis is an EMT-like process, which provides a conceptual connection for the molecular and cellular similarities between astrogliosis and wound-healing responses in non-neural tissue.

Manipulating Adult Neural Stem and Progenitor Cells with G-Quadruplex Ligands.

G-quadruplexes are pervasive nucleic acid secondary structures in mammalian genomes and transcriptomes that regulate gene expression and genome duplication. Small molecule ligands that modify the stability of G-quadruplexes are widely studied in cancer, but whether G-quadruplex ligands can also be used to manipulate cell function under normal development and homeostatic conditions is largely unexplored. Here we show that two related G-quadruplex ligands (pyridostatin and carboxypyridostatin) can reduce proliferation of adult neural stem cell and progenitor cells derived from the adult mouse subventricular zone both in vitro and in vivo. Studies with neurosphere cultures show that pyridostatin reduces proliferation by a mechanism associated with DNA damage and cell death. By contrast, selectively targeting RNA G-quadruplex stability with carboxypyridostatin diminishes proliferation through a mechanism that promotes cell cycle exit and the production of oligodendrocyte progenitors. The ability to generate oligodendrocyte progenitors by targeting RNA G-quadruplex stability, however, is dependent on the cellular environment. Together, these findings show that ligands that can selectively stabilize RNA G-quadruplexes are an important, new class of molecular tool for neural stem and progenitor cell engineering, whereas ligands that target DNA G-quadruplexes have limited utility due to their toxicity.

HDAC6 inhibition promotes α-tubulin acetylation and ameliorates CMT2A peripheral neuropathy in mice.

Charcot-Marie-Tooth type 2A (CMT2A) peripheral neuropathy, the most common axonal form of CMT, is caused by dominantly inherited point mutations in the Mitofusin 2 (Mfn2) gene. It is characterized by progressive length-dependent degeneration of motor and sensory nerves with corresponding clinical features of motor and sensory impairment. There is no cure for CMT, and therapeutic approaches are limited to physical therapy, orthopedic devices, surgery, and analgesics. In this study we focus on histone deacetylase 6 (HDAC6) as a therapeutic target in a mouse model of mutant MFN2 (MFN2R94Q)-induced CMT2A. We report that these mice display progressive motor and sensory dysfunction as well as a significant decrease in α-tubulin acetylation in distal segments of long peripheral nerves. Treatment with a new, highly selective HDAC6 inhibitor, SW-100, was able to restore α-tubulin acetylation and ameliorate motor and sensory dysfunction when given either prior to or after the onset of symptoms. To confirm HDAC6 is the target for ameliorating the CMT2A phenotype, we show that genetic deletion of Hdac6 in CMT2A mice prevents the development of motor and sensory dysfunction. Our findings suggest α-tubulin acetylation defects in distal parts of nerves as a pathogenic mechanism and HDAC6 as a therapeutic target for CMT2A.

Hypoxia-Inducible Factor 1α (HIF-1α) Counteracts the Acute Death of Cells Transplanted into the Injured Spinal Cord.

Cellular transplantation is in clinical testing for a number of central nervous system disorders, including spinal cord injury (SCI). One challenge is acute transplanted cell death. To prevent this death, there is a need to both establish when the death occurs and develop approaches to mitigate its effects. Here, using luciferase (luc) and green fluorescent protein (GFP) expressing Schwann cell (SC) transplants in the contused thoracic rat spinal cord 7 d postinjury, we establish via in vivo bioluminescent (IVIS) imaging and stereology that cell death occurs prior to 2-3 d postimplantation. We then test an alternative approach to the current paradigm of enhancing transplant survival by including multiple factors along with the cells. To stimulate multiple cellular adaptive pathways concurrently, we activate the hypoxia-inducible factor 1α (HIF-1α) transcriptional pathway. Retroviral expression of VP16-HIF-1α in SCs increased HIF-α by 5.9-fold and its target genes implicated in oxygen transport and delivery (VEGF, 2.2-fold) and cellular metabolism (enolase, 1.7-fold). In cell death assays in vitro, HIF-1α protected cells from H2O2-induced oxidative damage. It also provided some protection against camptothecin-induced DNA damage, but not thapsigargin-induced endoplasmic reticulum stress or tunicamycin-induced unfolded protein response. Following transplantation, VP16-HIF-1α increased SC survival by 34.3%. The increase in cell survival was detectable by stereology, but not by in vivo luciferase or ex vivo GFP IVIS imaging. The results support the hypothesis that activating adaptive cellular pathways enhances transplant survival and identifies an alternative pro-survival approach that, with optimization, could be amenable to clinical translation.