Characterization of Intracellular Zn2+ Dynamics and Zn2+- Dependent Gene Expression In Primary Dissociated Hippocampal Neurons

Ionic zinc (Zn2+) is an essential cofactor in many proteins, but labile (non-protein bound) Zn2+ also functions as a signaling ion in many cell types. In specific neurons of the brain and nervous system, Zn2+ is highly enriched and operates as a neurotransmitter that modulates neuronal signaling. Zn2+ has crucial physiological roles in memory consolidation and sensory processing, yet abnormal levels of Zn2+ have been observed to be pathological in neurological disorders ranging from Alzheimer&rsquo;s Disease to depression. Both extracellular and intracellular Zn2+ are necessary for neuronal signaling, but little is known about the dynamics of intracellular Zn2+ during neuronal stimulation and how intracellular Zn2+ influences signaling in physiological or pathological processes. We employed fluorescence imaging with genetically encoded and small molecule Zn2+ sensors to quantify intracellular Zn2+ dynamics during different stimulations in primary dissociated hippocampal neurons. We found that Zn2+ rose transiently during both chemical and electrical stimulation, although there were differences in peak Zn2+ concentrations depending on the stimulus used. Furthermore, we perturbed intracellular Zn2+ signaling to explore Zn2+-dependent changes in gene expression through global mRNA sequencing of hippocampal neurons. Our RNA-Seq results indicated that mild stimulation with potassium chloride induced Zn2+-dependent expression of genes related to synaptic growth and signaling, while more intensive stimulation with glutamate promoted Zn2+-dependent expression of genes involved in endoplasmic reticulum stress. Our results therefore show distinct differences in the role of Zn2+ signaling during potassium chloride and glutamate stimulation of neurons, which may reflect differences between physiological and pathological Zn2+ signaling. In future studies we aim to elucidate the underlying signaling mechanisms behind these differences.</p

Effects of Hypoxia on Swimming and Sensing in a Weakly Electric Fish

Low dissolved oxygen (hypoxia) can severely limit fish performance, especially aerobically expensive behaviours including swimming and acquisition of sensory information. Fishes can reduce oxygen requirements by altering these behaviours under hypoxia, but the underlying mechanisms can be difficult to quantify. We used a weakly electric fish as a model system to explore potential effects of hypoxia on swim performance and sensory information acquisition, which enabled us to non-invasively record electric signalling activity used for active acquisition of sensory information during swimming. To quantify potential effects of hypoxia, we measured critical swim speed (Ucrit) and concurrent electric signalling activity under highand low-dissolved oxygen concentrations in a hypoxia-tolerant African mormyrid fish, Marcusenius victoriae. Fish were maintained under normoxia for 6 months prior to experimental treatments, and then acclimated for 8 weeks to normoxia or hypoxia and tested under both conditions (acute: 4 h exposure). Acute hypoxia exposure resulted in a significant reduction in both Ucrit and electric signalling activity in fish not acclimated to hypoxia. However, individuals acclimated to chronic hypoxia were characterized by a higher Ucrit under both hypoxia and normoxia than fish acclimated to normoxia. Following a 6 month re-introduction to normoxia, hypoxia-acclimated individuals still showed increased performance under acute hypoxic test conditions, but not under normoxia. Our results highlight the detrimental effects of hypoxia on aerobic swim performance and sensory information acquisition, and the ability of fish to heighten aerobic performance through acclimation processes that can still influence performance even months after initial exposure

Intracellular Zn2+ transients modulate global gene expression in dissociated rat hippocampal neurons

Zinc (Zn2+) is an integral component of many proteins and has been shown to act in a regulatory capacity in different mammalian systems, including as a neurotransmitter in neurons throughout the brain. While Zn2+ plays an important role in modulating neuronal potentiation and synaptic plasticity, little is known about the signaling mechanisms of this regulation. In dissociated rat hippocampal neuron cultures, we used fluorescent Zn2+ sensors to rigorously define resting Zn2+ levels and stimulation-dependent intracellular Zn2+ dynamics, and we performed RNA-Seq to characterize Zn2+-dependent transcriptional effects upon stimulation. We found that relatively small changes in cytosolic Zn2+ during stimulation altered expression levels of 931 genes, and these Zn2+ dynamics induced transcription of many genes implicated in neurite expansion and synaptic growth. Additionally, while we were unable to verify the presence of synaptic Zn2+ in these cultures, we did detect the synaptic vesicle Zn2+ transporter ZnT3 and found it to be substantially upregulated by cytosolic Zn2+ increases. These results provide the first global sequencing-based examination of Zn2+-dependent changes in transcription and identify genes that may mediate Zn2+-dependent processes and functions. </div

Test of a Workforce Development Intervention to Expand Opioid Use Disorder Treatment Pharmacotherapy Prescribers: Protocol for a Cluster Randomized Trial

Background: Overdoses due to non-medical use of prescription opioids and other opiates have become the leading cause of accidental deaths in the USA. Buprenorphine and extended-release naltrexone are key evidence-based pharmacotherapies available to addiction treatment providers to address opioid use disorder (OUD) and prevent overdose deaths. Treatment organizations’ efforts to provide these pharmacotherapies have, however, been stymied by limited success in recruiting providers (physicians, nurse practitioners, and physician assistants) to prescribe these medications. Historically, the addiction treatment field has not attracted physicians, and many barriers to implementing OUD pharmacotherapy exist, ranging from lack of confidence in treating OUD patients to concerns regarding reimbursement. Throughout the USA, the prevalence of OUD far exceeds the capacity of the OUD pharmacotherapy treatment system. Poor access to OUD pharmacotherapy prescribers has become a workforce development need for the addiction treatment field and a significant health issue. Methods: This cluster randomized controlled trial (RCT) is designed to increase buprenorphine and extended-release naltrexone treatment capacity for OUD. The implementation intervention to be tested is a bundle of OUD pharmacotherapy capacity building practices called the Prescriber Recruitment Bundle (PRB), which was developed and piloted in a previous statewide buprenorphine implementation study. For this cluster RCT, organizational sites will be recruited and then randomized into one of two arms: (1) control, with treatment as usual and access to a website with PRB resources, or (2) intervention, with organizations implementing the PRB using the Network for the Improvement of Addiction Treatment organizational change model over a 24-month intervention period and a 10-month sustainability period. The primary treatment outcomes for each organizational site are self-reported monthly counts of buprenorphine slots, extended-release naltrexone capacity, number of buprenorphine patients, and number of extended-release naltrexone patients. This trial will be conducted in Florida, Ohio, and Wisconsin, resulting in 35 sites in each arm, for a total sample size of 70 organizations. Discussion: This study addresses three issues of substantial public health significance: (1) the pressing opioid misuse epidemic, (2) the low uptake of OUD treatment pharmacotherapies, and (3) the need to increase prescriber participation in the addiction treatment workforce. Trial Registration: ClinicalTrials.gov NCT02926482

Screening Surgeons for HIV Infection: Assessment of a Potential Public Health Program

Objective: To develop a model to assess the impact of a program of testing surgeons for human immunodeficiency virus (HIV) on the risk of HIV acquisition by their patients. Design: A Monte Carlo simulation model of physician-to-patient transmission of human immunodeficiency virus (HIV) infection using three different rates of physician-to-patient transmission per percutaneous exposure event (0.15%, 0.3%, 0.6%). Data from the odel were developed from a review of the medical literature and from subjective probability estimates when data were not available. We used this model to estimate on a national basis the annual number of cases of HIV transmission from surgeons to patients with and without surgeon testing and practice limitations. Results: The annual number of transmitted cases would range from 0.5 (+ or - 0.3), assuming a surgeon HIV prevalence of 0.1% and a surgeon-to-patient transmission rate of 0.15%, to 36.9 (+ or - 11.6), assuming a surgeon HIV prevalence of 2% and a surgeon-to-patient transmission rate of 0.6%. After one screening cycle, a mandatory screening program would be expected to reduce the annual transmissions to 0.05 (+ or - 0.03) and 3.1 (+ or - 1.1), respectively. Conclusion: Patients are at a low risk of acquiring HIV infection from an infected physician during an invasive procedure. The potential costs of such a program extended beyond the costs of testing and counseling. In communities with high HIV prevalence, screening surgeons and limiting their practices may decrease patient access to care. A disability insurance program also would be required to protect surgeons and trainees performing invasive procedures. Screening surgeons for HIV infection would be a costly undertaking that would reduce but not completely eliminate this risk.Sociolog

Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration

Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are transferred to lysine residues of proteins, in a reaction regulated by acetyltransferases. The tight balance between acetyltransferases and their antagonistic counterparts histone deacetylases is a well-known determining factor for the acetylation status of proteins. However, the influence of Coenzyme A levels on protein acetylation is unknown. Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models. We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival. Decreased protein acetylation and the concurrent phenotypes are partly rescued by pantethine and HDAC inhibitors, suggesting possible directions for future PKAN therapy development

Summarizing performance for genome scale measurement of miRNA: reference samples and metrics

Background: The potential utility of microRNA as biomarkers for early detection of cancer and other diseases is being investigated with genome-scale profiling of differentially expressed microRNA. Processes for measurement assurance are critical components of genome-scale measurements. Here, we evaluated the utility of a set of total RNA samples, designed with between-sample differences in the relative abundance of miRNAs, as process controls. Results: Three pure total human RNA samples (brain, liver, and placenta) and two different mixtures of these components were evaluated as measurement assurance control samples on multiple measurement systems at multiple sites and over multiple rounds. In silico modeling of mixtures provided benchmark values for comparison with physical mixtures. Biomarker development laboratories using next-generation sequencing (NGS) or genome-scale hybridization assays participated in the study and returned data from the samples using their routine workflows. Multiplexed and single assay reverse-transcription PCR (RT-PCR) was used to confirm in silico predicted sample differences. Data visualizations and summary metrics for genome-scale miRNA profiling assessment were developed using this dataset, and a range of performance was observed. These metrics have been incorporated into an online data analysis pipeline and provide a convenient dashboard view of results from experiments following the described design. The website also serves as a repository for the accumulation of performance values providing new participants in the project an opportunity to learn what may be achievable with similar measurement processes. Conclusions: The set of reference samples used in this study provides benchmark values suitable for assessing genome-scale miRNA profiling processes. Incorporation of these metrics into an online resource allows laboratories to periodically evaluate their performance and assess any changes introduced into their measurement process

Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

Although manganese is an essential trace metal, little is known about its transport and homeostatic regulation. Here we have identified a cohort of patients with a novel autosomal recessive manganese transporter defect caused by mutations in SLC39A14. Excessive accumulation of manganese in these patients results in rapidly progressive childhood-onset parkinsonism-dystonia with distinctive brain magnetic resonance imaging appearances and neurodegenerative features on post-mortem examination. We show that mutations in SLC39A14 impair manganese transport in vitro and lead to manganese dyshomeostasis and altered locomotor activity in zebrafish with CRISPR-induced slc39a14 null mutations. Chelation with disodium calcium edetate lowers blood manganese levels in patients and can lead to striking clinical improvement. Our results demonstrate that SLC39A14 functions as a pivotal manganese transporter in vertebrates.Action Medical ResearchThis is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1160