A Paranigral VTA Nociceptin Circuit that Constrains Motivation for Reward.

Nociceptin and its receptor are widely distributed throughout the brain in regions associated with reward behavior, yet how and when they act is unknown. Here, we dissected the role of a nociceptin peptide circuit in reward seeking. We generated a prepronociceptin (Pnoc)-Cre mouse line that revealed a unique subpopulation of paranigral ventral tegmental area (pnVTA) neurons enriched in prepronociceptin. Fiber photometry recordings during progressive ratio operant behavior revealed pnVTAPnoc neurons become most active when mice stop seeking natural rewards. Selective pnVTAPnoc neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTAPnoc nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTAPnoc nucleus caused avoidance and decreased motivation for rewards. These findings provide insight into neuromodulatory circuits that regulate motivated behaviors through identification of a previously unknown neuropeptide-containing pnVTA nucleus that limits motivation for rewards.

TLR2 on CD4+ and CD8+ T cells promotes control of Mycobacterium tuberculosis infection.

Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout mice is due to its role in macrophages, T cells, or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-γ, TNF-α) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells affected control of Mtb in the lungs and spleens of infected mice. This suggests that T-cell co-stimulation by mycobacterial TLR2 ligands in vivo contributes to the control of Mtb infection in the lung and spleen.

Chronic myeloid neoplasms harboring concomitant mutations in myeloproliferative neoplasm driver genes (JAK2/MPL/CALR) and SF3B1.

JAK2, CALR, and MPL are myeloproliferative neoplasm (MPN)-driver mutations, whereas SF3B1 is strongly associated with ring sideroblasts (RS) in myelodysplastic syndrome (MDS). Concomitant mutations of SF3B1 and MPN-driver mutations out of the context of MDS/MPN with RS and thrombocytosis (MDS/MPN-RS-T) are not well-studied. From the cases (<5% blasts) tested by NGS panels interrogating at least 42 myeloid neoplasm-related genes, we identified 18 MDS/MPN-RS-T, 42 MPN, 10 MDS, and 6 MDS/MPN-U cases with an SF3B1 and an MPN-driver mutation. Using a 10% VAF difference to define "SF3B1-dominant," "MPN-mutation dominant," and "no dominance," the majority of MDS/MPN-RS-T clustered in "SF3B1-dominant" and "no dominance" regions. Aside from parameters as thrombocytosis and ≥15% RS required for RS-T, MDS also differed in frequent neutropenia, multilineage dysplasia, and notably more cases with <10% VAF of MPN-driver mutations (60%, p = 0.0346); MPN differed in more frequent splenomegaly, myelofibrosis, and higher VAF of "MPN-driver mutations." "Gray zone" cases with features overlapping MDS/MPN-RS-T were observed in over one-thirds of non-RS-T cases. This study shows that concomitant SF3B1 and MPN-driver mutations can be observed in MDS, MPN, and MDS/MPN-U, each showing overlapping but also distinctively different clinicopathological features. Clonal hierarchy, cytogenetic abnormalities, and additional somatic mutations may in part contribute to different disease phenotypes, which may help in the classification of "gray zone" cases.

Pain, Motivation, Migraine, and the Microbiome: New Frontiers for Opioid Systems and Disease.

For decades the broad role of opioids in addiction, neuropsychiatric disorders, and pain states has been somewhat well established. However, in recent years, with the rise of technological advances, not only is the existing dogma being challenged, but we are identifying new disease areas in which opioids play a critical role. This review highlights four new areas of exploration in the opioid field. The most recent addition to the opioid family, the nociceptin receptor system, shows promise as the missing link in understanding the neurocircuitry of motivation. It is well known that activation of the kappa opioid receptor system modulates negative affect and dysphoria, but recent studies now implicate the kappa opioid system in the modulation of negative affect associated with pain. Opioids are critical in pain management; however, the often-forgotten delta opioid receptor system has been identified as a novel therapeutic target for headache disorders and migraine. Lastly, changes to the gut microbiome have been shown to directly contribute to many of the symptoms of chronic opioid use and opioid related behaviors. This review summarizes the findings from each of these areas with an emphasis on identifying new therapeutic targets. SIGNIFICANCE STATEMENT: The focus of this minireview is to highlight new disease areas or new aspects of disease in which opioids have been implicated; this includes pain, motivation, migraine, and the microbiome. In some cases, this has resulted in the pursuit of a novel therapeutic target and resultant clinical trial. We believe this is very timely and will be a refreshing take on reading about opioids and disease.

Rapidly-customizable, scalable 3D-printed wireless optogenetic probes for versatile applications in neuroscience.

Optogenetics is an advanced neuroscience technique that enables the dissection of neural circuitry with high spatiotemporal precision. Recent advances in materials and microfabrication techniques have enabled minimally invasive and biocompatible optical neural probes, thereby facilitating in vivo optogenetic research. However, conventional fabrication techniques rely on cleanroom facilities, which are not easily accessible and are expensive to use, making the overall manufacturing process inconvenient and costly. Moreover, the inherent time-consuming nature of current fabrication procedures impede the rapid customization of neural probes in between in vivo studies. Here, we introduce a new technique stemming from 3D printing technology for the low-cost, mass production of rapidly customizable optogenetic neural probes. We detail the 3D printing production process, on-the-fly design versatility, and biocompatibility of 3D printed optogenetic probes as well as their functional capabilities for wireless in vivo optogenetics. Successful in vivo studies with 3D printed devices highlight the reliability of this easily accessible and flexible manufacturing approach that, with advances in printing technology, can foreshadow its widespread applications in low-cost bioelectronics in the future.

Magnetic Resonance Imaging Findings of Crystalloid-Containing Parotid Cysts.

Crystalloids are occasionally encountered on fine needle aspiration of cystic parotid lesions. This goal of this study was to retrospectively characterize the MRI features of a series benign crystalloid-containing parotid cysts. A total of 4 patients with fine needle aspiration findings of crystalloids and available parotid MRI scans were identified. Review of the imaging revealed that the cystic lesions contain layering material that corresponds to crystals.

NOP Receptor Signaling Cascades.

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is a G protein-coupled receptor with wide distribution throughout the peripheral and central nervous system. Similar to other opioid receptors, NOP receptors couple to intracellular second messengers and regulatory proteins to affect biological systems. In this chapter, we review the current literature for NOP signaling cascades including their role as classic GPCRs, the investigation of their kinase and arrestin signaling pathways, and the importance of examining biased signaling to critically evaluate the therapeutic potential of novel NOP agonists.

Research: Continuous Surveillance of Sleep Apnea Patients in a Medical-Surgical Unit.

This report consists of two separate studies on the use of continuous capnography monitoring conducted in an effort to improve patient safety at Virtua Health System. The desire for improved patient safety is motivating continuous monitoring and improved surveillance in clinical areas not traditionally equipped for such monitoring. We explored the use of remote monitoring of capnography, using enterprise middleware, in patients recovering from surgery in a medical-surgical unit. Continuous monitoring traditionally has been used in higher-acuity settings, such as intensive care units. Patients diagnosed or suspected to have obstructive or central sleep apnea may benefit from the increased surveillance afforded by continuous monitoring. Pain management in this cohort of patients, recovering from bariatric, joint replacement, or other major surgery, often involves administration of opioids (e.g., hydromorphone, morphine sulfate), which are known to increase risk of respiratory depression. Continuous monitoring of these patients increases the likelihood of detecting adverse clinical events. Our goal was to implement continuous monitoring in order to identify alarm conditions caused by adverse clinical events requiring intervention (e.g., opioid-induced respiratory depression) and artifacts related to patient movement, suspect measurements, or other medical device-generated alarm signals.

Aging reduces motivation through decreased Bdnf expression in the ventral tegmental area.

Age-associated reduced motivation is a hallmark of neuropsychiatric disorders in the elderly. In our rapidly aging societies, it is critical to keep motivation levels high enough to promote healthspan and lifespan. However, how motivation is reduced during aging remains unknown. Here, we used multiple mouse models to evaluate motivation and related affective states in young and old mice. We also compared the effect of social isolation, a common stressor, to those of aging. We found that both social isolation and aging decreased motivation in mice, but that Bdnf expression in the ventral tegmental area (VTA) was selectively decreased during aging. Furthermore, VTA-specific Bdnf knockdown in young mice recapitulated reduced motivation observed in old mice. These results demonstrate that maintaining Bdnf expression in the VTA could promote motivation to engage in effortful activities and potentially prevent age-associated neuropsychiatric disorders.

Development and Optimization of an Unbiased, Metagenomics-Based Pathogen Detection Workflow for Infectious Disease and Biosurveillance Applications.

Rapid, specific, and sensitive identification of microbial pathogens is critical to infectious disease diagnosis and surveillance. Classical culture-based methods can be applied to a broad range of pathogens but have long turnaround times. Molecular methods, such as PCR, are time-effective but are not comprehensive and may not detect novel strains. Metagenomic shotgun next-generation sequencing (NGS) promises specific identification and characterization of any pathogen (viruses, bacteria, fungi, and protozoa) in a less biased way. Despite its great potential, NGS has yet to be widely adopted by clinical microbiology laboratories due in part to the absence of standardized workflows. Here, we describe a sample-to-answer workflow called PanGIA (Pan-Genomics for Infectious Agents) that includes simplified, standardized wet-lab procedures and data analysis with an easy-to-use bioinformatics tool. PanGIA is an end-to-end, multi-use workflow that can be used for pathogen detection and related applications, such as biosurveillance and biothreat detection. We performed a comprehensive survey and assessment of current, commercially available wet-lab technologies and open-source bioinformatics tools for each workflow component. The workflow includes total nucleic acid extraction from clinical human whole blood and environmental microbial forensic swabs as sample inputs, host nucleic acid depletion, dual DNA and RNA library preparation, shotgun sequencing on an Illumina MiSeq, and sequencing data analysis. The PanGIA workflow can be completed within 24 h and is currently compatible with bacteria and viruses. Here, we present data from the development and application of the clinical and environmental workflows, enabling the specific detection of pathogens associated with bloodstream infections and environmental biosurveillance, without the need for targeted assay development.

Use of a Vanadate Oxidation Conjugated Bilirubin Assay to Reduce Test Cancellations Resulting from Hemolyzed Specimens in Pediatric Patients.

OBJECTIVES: We sought to replace the highly hemolysis-susceptible diazo conjugated bilirubin (Bc) assay with the more robust vanadate oxidation method and determine its impact on test cancellation in the pediatric population. METHODS: Analytical validation of the Randox vanadate assay and comparison with the Roche diazo method were performed. The frequency of pediatric sample cancellation because of hemolysis was compared between the diazo and vanadate methods by retrospective analysis of clinical test data. RESULTS: The vanadate assay demonstrated no clinically significant interference from hemolysis up to a hemolysis index of 1,300 (approximately 13 g/L hemoglobin). There was a strong correlation with the diazo method (r2 = 0.97) but with a positive slope bias of 1.27. Implementing the vanadate method resulted in a significantly lower proportion of pediatric samples cancelled because of hemolysis compared with the diazo method (0.6% of 688 patients vs 30.6% of 10,464 patients, respectively; P < .001), with a 0.6% (n = 513) vs 43.2% (n = 6,464) reduction in test cancellations (P < .001) for children younger than 6 months of age. CONCLUSIONS: The vanadate method showed robust performance against hemolysis. Its implementation resulted in a significant decrease in pediatric tests cancelled because of hemolysis.

Customizable, wireless and implantable neural probe design and fabrication via 3D printing.

This Protocol Extension describes the low-cost production of rapidly customizable optical neural probes for in vivo optogenetics. We detail the use of a 3D printer to fabricate minimally invasive microscale inorganic light-emitting-diode-based neural probes that can control neural circuit activity in freely behaving animals, thus extending the scope of two previously published protocols describing the fabrication and implementation of optoelectronic devices for studying intact neural systems. The 3D-printing fabrication process does not require extensive training and eliminates the need for expensive materials, specialized cleanroom facilities and time-consuming microfabrication techniques typical of conventional manufacturing processes. As a result, the design of the probes can be quickly optimized, on the basis of experimental need, reducing the cost and turnaround for customization. For example, 3D-printed probes can be customized to target multiple brain regions or scaled up for use in large animal models. This protocol comprises three procedures: (1) probe fabrication, (2) wireless module preparation and (3) implantation for in vivo assays. For experienced researchers, neural probe and wireless module fabrication requires ~2 d, while implantation should take 30-60 min per animal. Time required for behavioral assays will vary depending on the experimental design and should include at least 5 d of animal handling before implantation of the probe, to familiarize each animal to their handler, thus reducing handling stress that may influence the result of the behavioral assays. The implementation of customized probes improves the flexibility in optogenetic experimental design and increases access to wireless probes for in vivo optogenetic research.

Petri-plate, bacteria, and laser optical scattering sensor.

Classical microbiology has paved the path forward for the development of modern biotechnology and microbial biosensing platforms. Microbial culturing and isolation using the Petri plate revolutionized the field of microbiology. In 1887, Julius Richard Petri invented possibly the most important tool in microbiology, the Petri plate, which continues to have a profound impact not only on reliably isolating, identifying, and studying microorganisms but also manipulating a microbe to study gene expression, virulence properties, antibiotic resistance, and production of drugs, enzymes, and foods. Before the recent advances in gene sequencing, microbial identification for diagnosis relied upon the hierarchal testing of a pure culture isolate. Direct detection and identification of isolated bacterial colonies on a Petri plate with a sensing device has the potential for revolutionizing further development in microbiology including gene sequencing, pathogenicity study, antibiotic susceptibility testing , and for characterizing industrially beneficial traits. An optical scattering sensor designated BARDOT (bacterial rapid detection using optical scattering technology) that uses a red-diode laser, developed at the beginning of the 21st century at Purdue University, some 220 years after the Petri-plate discovery can identify and study bacteria directly on the plate as a diagnostic tool akin to Raman scattering and hyperspectral imaging systems for application in clinical and food microbiology laboratories.

End-User Perspectives on Using Quantitative Real-Time PCR and Genomic Sequencing in the Field.

Quantitative real-time PCR and genomic sequencing have become mainstays for performing molecular detection of biological threat agents in the field. There are notional assessments of the benefits, disadvantages, and challenges that each of these technologies offers according to findings in the literature. However, direct comparison between these two technologies in the context of field-forward operations is lacking. Most market surveys, whether published in print form or provided online, are directed to product manufacturers who can address their respective specifications and operations. One method for comparing these technologies is surveying end-users who are best suited for discussing operational capabilities, as they have hands-on experience with state-of-the-art molecular detection platforms and protocols. These end-users include operators in military defense and first response, as well as various research scientists in the public sector such as government and service laboratories, private sector, and civil society such as academia and nonprofit organizations performing method development and executing these protocols in the field. Our objective was to initiate a survey specific to end-users and their feedback. We developed a questionnaire that asked respondents to (1) determine what technologies they currently use, (2) identify the settings where the technologies are used, whether lab-based or field-forward, and (3) rate the technologies according to a set list of criteria. Of particular interest are assessments of sensitivity, specificity, reproducibility, scalability, portability, and discovery power. This article summarizes the findings from the end-user perspective, highlighting technical and operational challenges.

Management, entrepreneurship, continuing professional development, and leadership education in United States doctor of pharmacy curricula: A scoping review.

BACKGROUND: As doctor of pharmacy (PharmD) curricula must fulfill accreditation standards emphasizing managerial skills, entrepreneurship, continuing professional development (CPD), and leadership, there is interest among faculty to incorporate high-quality, evidence-based educational strategies. To date there has not been a comprehensive review of these four topics in one paper; therefore, we aimed to describe the landscape of the published literature and areas for future research. METHODS: A keyword search of Academic Search Complete/Premier and OvidMedline databases identified articles published between 1 January 2000 and 31 December 2020. Inclusion criteria included primary, peer-reviewed literature describing the implementation and evaluation of teaching methodologies on aspects of management, entrepreneurship, CPD, or leadership in United States PharmD programs. Titles and abstracts were screened for inclusion. Key information regarding instructional design and assessment were retrieved to develop narrative summaries of activities and outcomes and to chart the student year of study, sample size, mode of delivery (didactic/experiential/co-curricular), type of experience (required/optional), and learning activity. RESULTS: Thirty-five articles met inclusion criteria and were categorized: management (n = 12), entrepreneurship (n = 2), CPD (n = 7), and leadership (n = 14). The articles provided example activities that faculty across the country can consider implementing. Identified gaps included a focus on episodic and/or optional experiences and a relative lack of objective and longitudinal assessment practices. IMPLICATIONS: This review describes educational methodologies for management, entrepreneurship, CPD, and leadership that can be replicated or adapted. Additional reports of innovative educational practices assessed through valid and reliable methods are needed.

Scalable and modular wireless-network infrastructure for large-scale behavioural neuroscience.

The use of rodents to acquire understanding of the function of neural circuits and of the physiological, genetic and developmental underpinnings of behaviour has been constrained by limitations in the scalability, automation and high-throughput operation of implanted wireless neural devices. Here we report scalable and modular hardware and software infrastructure for setting up and operating remotely programmable miniaturized wireless networks leveraging Bluetooth Low Energy for the study of the long-term behaviour of large groups of rodents. The integrated system allows for automated, scheduled and real-time experimentation via the simultaneous and independent use of multiple neural devices and equipment within and across laboratories. By measuring the locomotion, feeding, arousal and social behaviours of groups of mice or rats, we show that the system allows for bidirectional data transfer from readily available hardware, and that it can be used with programmable pharmacological or optogenetic stimulation. Scalable and modular wireless-network infrastructure should facilitate the remote operation of fully automated large-scale and long-term closed-loop experiments for the study of neural circuits and animal behaviour.

Evaluation of the Truvian Easy Check COVID-19 IgM/IgG Lateral Flow Device for Rapid Anti-SARS-CoV-2 Antibody Detection.

OBJECTIVES: To evaluate the analytical and clinical performance of the Truvian Easy Check coronavirus disease 2019 (COVID-19) IgM/IgG anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody test.Serologic assays have become increasingly available for surveillance through the Food and Drug Administration emergency use authorization in the ongoing COVID-19 global pandemic. However, widespread application of serologic assays has been curbed by reports of faulty or inaccurate tests. Therefore, rapid COVID-19 antibody tests need to be thoroughly validated prior to their implementation. METHODS: The Easy Check device was analytically evaluated and its performance was compared with the Roche Elecsys anti-SARS-CoV-2 antibody assay. The test was further characterized for cross-reactivity using sera obtained from patients infected by other viruses. Clinical performance was analyzed with polymerase chain reaction-confirmed samples and a 2015 prepandemic reference sample set. RESULTS: The Easy Check device showed excellent analytical performance and compares well with the Roche Elecsys antibody assay, with an overall concordance of 98.6%. Clinical performance showed a sensitivity of 96.6%, a specificity of 98.2%, and an overall accuracy of 98.1%. CONCLUSIONS: The Easy Check device is a simple, reliable, and rapid test for detection of SARS-CoV-2 seropositivity, and its performance compares favorably against the automated Roche Elecsys antibody assay.

Significance of High-Containment Biological Laboratories Performing Work During the COVID-19 Pandemic: Biosafety Level-3 and -4 Labs.

High containment biological laboratories (HCBL) are required for work on Risk Group 3 and 4 agents across the spectrum of basic, applied, and translational research. These laboratories include biosafety level (BSL)-3, BSL-4, animal BSL (ABSL)-3, BSL-3-Ag (agriculture livestock), and ABSL-4 laboratories. While SARS-CoV-2 is classified as a Risk Group 3 biological agent, routine diagnostic can be handled at BSL-2. Scenarios involving virus culture, potential exposure to aerosols, divergent high transmissible variants, and zoonosis from laboratory animals require higher BSL-3 measures. Establishing HCBLs especially those at BSL-4 is costly and needs continual investments of resources and funding to sustain labor, equipment, infrastructure, certifications, and operational needs. There are now over 50 BSL-4 laboratories and numerous BSL-3 laboratories worldwide. Besides technical and funding challenges, there are biosecurity and dual-use risks, and local community issues to contend with in order to sustain operations. Here, we describe case histories for distinct HCBLs: representative national centers for diagnostic and reference, nonprofit organizations. Case histories describe capabilities and assess activities during COVID-19 and include capacities, gaps, successes, and summary of lessons learned for future practice.

A photoswitchable GPCR-based opsin for presynaptic inhibition.

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has proven more elusive. Here, we leveraged the natural ability of inhibitory presynaptic GPCRs to suppress synaptic transmission and characterize parapinopsin (PPO) as a GPCR-based opsin for terminal inhibition. PPO is a photoswitchable opsin that couples to Gi/o signaling cascades and is rapidly activated by pulsed blue light, switched off with amber light, and effective for repeated, prolonged, and reversible inhibition. PPO rapidly and reversibly inhibits glutamate, GABA, and dopamine release at presynaptic terminals. Furthermore, PPO alters reward behaviors in a time-locked and reversible manner in vivo. These results demonstrate that PPO fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition and has broad utility for spatiotemporal control of inhibitory GPCR signaling cascades.

Comparison of Molecular Methods and BRAF Immunohistochemistry (VE1 Clone) for the Detection of BRAF V600E Mutation in Papillary Thyroid Carcinoma: A Meta-Analysis.

The evaluation of surgically resected papillary thyroid carcinomas (PTC) by immunohistochemistry (IHC) for BRAF mutation has diagnostic, prognostic and therapeutic implications. The goal of this meta-analysis was to perform a systematic review of studies using the VE1 clone (specific for detection of the BRAF V600E mutation) on formalin-fixed paraffin embedded (FFPE) thyroid surgical resection specimens for primary papillary thyroid carcinoma. The authors' molecular techniques, immunohistochemistry protocols, and scoring methods for VE1 immunostaining were also evaluated. This study included 4079 PTCs representing data from 23 studies. The results extracted from each study were split into two different groups, direct sequencing group or PCR group, based on the molecular "gold standard" method used to compare VE1 IHC staining. In the direct sequencing group, the IHC sensitivity was 100% (95% CI 0.97-1.00) and specificity 84% (95% 0.72-0.91). In the PCR group the sensitivity was 98% (95% CI 0.96-0.99) and specificity 89% (95% CI 0.82-0.94). Although immunohistochemical procedures varied by author, the overall performance of the VE1 clone shows that it is highly sensitive and relatively specific for detecting the BRAF V600E mutation in surgical resection specimens. However, standardization of immunohistochemical procedural method and scoring/interpretation criteria may improve the reliability and reproducibility for the use of VE1 clone for future practice.