Verification and Validation of SARS-CoV-2 Assay Performance on the Abbott m2000 and Alinity m Systems.

We verified the analytical performance of the Abbott RealTime SARS-CoV-2 assay on the m2000 system and compared its clinical performance to the CDC 2019-nCoV real-time PCR diagnostic panel and the Thermo Fisher TaqPath RT-PCR COVID-19 kit. We also performed a bridging study comparing the RealTime SARS-CoV-2 assay with the new Abbott Alinity m SARS-CoV-2 assay. A number of standards, reference materials, and commercially available controls were used for the analytical verification to confirm the limit of detection, linearity, and reproducibility. We used nasopharyngeal (NP) swab specimens collected in saline for the clinical verification and bridging studies. Overall, we found 91.2% positive percent agreement (PPA; 95% confidence interval [CI] = 76.2 to 98.14%) and a 100% negative percent agreement (NPA; 95% CI = 97.97 to 100%) between the results of the RealTime SARS-CoV-2 and CDC tests with 217 NP specimens (P = 0.13). We found a PPA of 100% (95% CI = 90.26 to 100%) and an NPA of 95.15% (95% CI = 83.47 to 99.4%) between the results of the RealTime and TaqPath tests with 77 NP specimens (P = 0.24). Finally, we tested 203 NP swab specimens for SARS-CoV-2 on the m2000 on the Alinity m systems. The PPA and NPA were 92.2% (95% CI = 85.3 to 96.59%) and 92% (95% CI = 84.8 to 96.5%), respectively (P = 0.4). Although cycle number (Cn) values obtained for the concordant positive samples were highly correlated (R2 = 0.95), the Cn values were on average 14.14 higher on the Alinity m system due to the unread cycles with the RealTime SARS-CoV-2 assay.

Glycogen Storage Disease Type IV Diagnosed at Fetal Autopsy.

Glycogen storage disease type IV (GSD IV; Andersen's disease) is a rare autosomal recessive disorder that results from defects in the GBE1 gene (3p12.2) and subsequent deficiencies of glycogen branching. We report a case of GSD IV diagnosed at autopsy in a 35 4/7 weeks gestational age female neonate that died shortly after birth. Multisystem blue, ground glass inclusions initially presumed artefactual were periodic acid-Schiff positive, diastase resistant. Chromosomal microarray analysis identified a deletion of exons 2 through 16 of the GBE1 gene and whole exome sequencing identified a nonsense mutation within exon 14, confirming the diagnosis of GSD IV. A strong index of suspicion was required determine GSD IV as the ultimate cause of death, illustrating the need for critical evaluation of postmortem artifact in the setting of fetal demise of unknown etiology and highlighting the role of postmortem molecular diagnostics in a subset of cases.

Optogenetic stimulation of MCH neurons increases sleep.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide present in the hypothalamus of all vertebrates. MCH is implicated in a number of behaviors but direct evidence is lacking. To selectively stimulate the MCH neurons the gene for the light-sensitive cation channel, channelrhodopsin-2, was inserted into the MCH neurons of wild-type mice. Three weeks later MCH neurons were stimulated for 1 min every 5 min for 24 h. A 10 Hz stimulation at the start of the night hastened sleep onset, reduced length of wake bouts by 50%, increased total time in non-REM and REM sleep at night, and increased sleep intensity during the day cycle. Sleep induction at a circadian time when all of the arousal neurons are active indicates that MCH stimulation can powerfully counteract the combined wake-promoting signal of the arousal neurons. This could be potentially useful in treatment of insomnia.

Dysbindin-1 loss compromises NMDAR-dependent synaptic plasticity and contextual fear conditioning.

Genetic variants in DTNBP1 encoding the protein dysbindin-1 have often been associated with schizophrenia and with the cognitive deficits prominent in that disorder. Because impaired function of the hippocampus is thought to play a role in these memory deficits and because NMDAR-dependent synaptic plasticity in this region is a proposed biological substrate for some hippocampal-dependent memory functions in schizophrenia, we hypothesized that reduced dysbindin-1 expression would lead to impairments in NMDAR-dependent synaptic plasticity and in contextual fear conditioning. Acute slices from male mice carrying 0, 1, or 2 null mutant alleles of the Dtnbp1 gene were prepared, and field recordings from the CA1 striatum radiatum were obtained before and after tetanization of Schaffer collaterals of CA3 pyramidal cells. Mice homozygous for the null mutation in Dtnbp1 exhibited significantly reduced NMDAR-dependent synaptic potentiation compared to wild type mice, an effect that could be rescued by bath application of the NMDA receptor coagonist glycine (10 µM). Behavioral testing in adult mice revealed deficits in hippocampal memory processes. Homozygous null mice exhibited lower conditional freezing, without a change in the response to shock itself, indicative of a learning and memory deficit. Taken together, these results indicate that a loss of dysbindin-1 impairs hippocampal plasticity which may, in part, explain the role dysbindin-1 plays in the cognitive impairments of schizophrenia.

Next-Generation Sequencing Informatic Architecture Considerations.

Clinically relevant sequencing methodologies continue to expand in number, diversity, complexity, and scale. This evolving and varied landscape requires unique implementations in all aspects of the assay, including the wet bench, bioinformatics, and reporting. Following implementation, the informatics of many of these tests continue to change over time, from software and annotation source updates, guidelines, and knowledgebase changes to changes in underlying information technology (IT) infrastructure. Key principles can be applied when implementing the informatics of a new clinical test which can greatly improve the lab's ability to deal with these updates rapidly and reliably. In this chapter, we discuss a variety of informatics issues which span all NGS applications. In particular, there is the need for implementing a reliable, repeatable, redundant, and version-controlled bioinformatics pipeline and architecture and a discussion of common methodologies to address these needs.

Pipeline for Integrated Microarray Expression Normalization Tool Kit (PIMENTo) for Tumor Microarray Profiling Experiments.

We have developed a Pipeline for Integrated Microarray Expression & Normalization Tool kit (PIMENTo) with the aim of streamlining the processes necessary for gene expression analysis in tumor tissue using DNA microarrays. Built with the R programming language and leveraging several open-source packages available through CRAN and Bioconductor, PIMENTo enables researchers to perform complex tasks with a minimal number of operations. Here, we describe the pipeline, review necessary data inputs, examine data outputs and quality control assessments and explore the commands to perform such analysis.

A high prevalence of biallelic RPE65 mutations in Costa Rican children with Leber congenital amaurosis and early-onset retinal dystrophy.

BACKGROUND: Leber congenital amaurosis (LCA) and early-onset retinal dystrophy (EORD), are primary causes of inherited childhood blindness. Both are autosomal recessive diseases, with mutations in more than 25 genes explaining approximately ~70% of cases. However, the genetic cause for many cases remains unclear. Sequencing studies from genetically isolated populations with increased prevalence of a disorder has proven useful for rare variant studies, making Costa Rica an ideal place to study LCA/EORD genetics. MATERIALS AND METHODS: Twenty-eight affected children (25 LCA, three EORD) and their immediate family members, totaling 52 individuals (30 affected) from 22 families, were sequenced. Whole exome sequencing was performed on all affected individuals. Available parents were analyzed either by whole exome sequencing (WES) or Sanger sequencing to determine transmission. RESULTS: All affected individuals demonstrated compound heterozygous or homozygous mutations in known Inherited Retinal Disease (IRD) associated genes. Twelve variants were identified in at least one individual in three genes, RDH12, RPE65, and USH2A. Four recurrent RPE65 mutations were observed in 97% of individuals and 95% of families. All patients with LCA and two of the three individuals with EORD had biallelic mutations in RPE65; one child with EORD had a homozygous RDH12 mutation. CONCLUSIONS: These data suggest that the majority of LCA/EORD in Costa Rica is due to four founder mutations in RPE65 which have been maintained in this genetically isolated population. This finding is of great clinical significance due to the availability of gene therapy recently approved in the US and European Union for patients with biallelic RPE65 defects.

Habitual Behavior Is Mediated by a Shift in Response-Outcome Encoding by Infralimbic Cortex.

The ability to flexibly switch between goal-directed actions and habits is critical for adaptive behavior. The infralimbic prefrontal cortex (IfL-C) has been consistently identified as a crucial structure for the regulation of response strategies. To investigate the role of the IfL-C, the present study employed two validated reinforcement schedules that either promote habits or goal-directed actions in mice. The results reveal that information about action-outcome relationships is differentially encoded in the IfL-C during actions and habits as evidenced by encoding of behavioral outcomes during goal-directed actions that is lost during habits. Optogenetic inhibition of the IfL-C selectively at press during habitual behavior (when firing rates are reduced during unreinforced goal-directed actions) resulted in restoration of sensitivity to change of action-outcome contingency. These results reveal a novel functional mechanism by which IfL-C promotes habitual behavior, and provide insight into strategies for the treatment and prevention of pathological, inflexible behavior common in neuropsychiatric illness.

Genomics pipelines and data integration: challenges and opportunities in the research setting.

INTRODUCTION: The emergence and mass utilization of high-throughput (HT) technologies, including sequencing technologies (genomics) and mass spectrometry (proteomics, metabolomics, lipids), has allowed geneticists, biologists, and biostatisticians to bridge the gap between genotype and phenotype on a massive scale. These new technologies have brought rapid advances in our understanding of cell biology, evolutionary history, microbial environments, and are increasingly providing new insights and applications towards clinical care and personalized medicine. Areas covered: The very success of this industry also translates into daunting big data challenges for researchers and institutions that extend beyond the traditional academic focus of algorithms and tools. The main obstacles revolve around analysis provenance, data management of massive datasets, ease of use of software, interpretability and reproducibility of results. Expert commentary: The authors review the challenges associated with implementing bioinformatics best practices in a large-scale setting, and highlight the opportunity for establishing bioinformatics pipelines that incorporate data tracking and auditing, enabling greater consistency and reproducibility for basic research, translational or clinical settings.

Methamphetamine self-administration produces attentional set-shifting deficits and alters prefrontal cortical neurophysiology in rats.

BACKGROUND: Chronic methamphetamine abusers exhibit deficits in tasks requiring intact prefrontal cortex function, and prefrontal cortex dysfunction has been implicated in the loss of control over drug use. This study used a combination of behavioral and electrophysiologic assessments in rats with a history of long access methamphetamine self-administration to determine methamphetamine-induced changes in prefrontal cortex-dependent attentional set-shifting performance, drug-seeking, and prefrontal cortex neuronal activity. METHODS: Male Long-Evans rats self-administered methamphetamine (.02 mg/infusion, intravenous) or received yoked saline infusions for 6 hours a day for 14 days. Cognitive flexibility was assessed using an attentional set-shifting task before 2 weeks of self-administration and 1 day after self-administration. Animals then underwent 11 days of abstinence, followed by three subsequent tests for context-induced drug seeking. Finally, animals were anesthetized, and single-unit in vivo extracellular recordings were performed in the dorsomedial prefrontal cortex. RESULTS: Methamphetamine-experienced rats showed escalated drug intake and context-induced drug-seeking following abstinence. During the extradimensional set-shift component, meth-experienced rats showed selective impairments that were identical to deficits produced by excitotoxic lesions of the prefrontal cortex. Rats with a history of chronic methamphetamine intake also exhibited higher basal firing frequency and a significantly greater proportion of burst-firing cells in the prefrontal cortex compared with yoked-saline controls. CONCLUSIONS: Prefrontal cortex-specific alterations in neuronal function may play a key role in methamphetamine-induced attentional deficits and drug-seeking. These data support the possibility that targeting prefrontal cortex pathology may improve treatment outcome in methamphetamine addiction.