Synthesis and SAR of novel GPR39 agonists and positive allosteric modulators.

We report a significant decrease in transcription of the G protein-coupled receptor GPR39 in striatal neurons of Parkinson's disease patients compared to healthy controls, suggesting that a positive modulator of GPR39 may beneficially impact neuroprotection. To test this notion, we developed various structurally diverse tool molecules. While we elaborated on previously reported starting points, we also performed an in silico screen which led to completely novel pharmacophores. In vitro studies indicated that GPR39 agonism does not have a profound effect on neuroprotection.

A dual druggable genome-wide siRNA and compound library screening approach identifies modulators of parkin recruitment to mitochondria.

Genetic and biochemical evidence points to an association between mitochondrial dysfunction and Parkinson's disease (PD). PD-associated mutations in several genes have been identified and include those encoding PTEN-induced putative kinase 1 (PINK1) and parkin. To identify genes, pathways, and pharmacological targets that modulate the clearance of damaged or old mitochondria (mitophagy), here we developed a high-content imaging-based assay of parkin recruitment to mitochondria and screened both a druggable genome-wide siRNA library and a small neuroactive compound library. We used a multiparameter principal component analysis and an unbiased parameter-agnostic machine-learning approach to analyze the siRNA-based screening data. The hits identified in this analysis included specific genes of the ubiquitin proteasome system, and inhibition of ubiquitin-conjugating enzyme 2 N (UBE2N) with a specific antagonist, Bay 11-7082, indicated that UBE2N modulates parkin recruitment and downstream events in the mitophagy pathway. Screening of the compound library identified kenpaullone, an inhibitor of cyclin-dependent kinases and glycogen synthase kinase 3, as a modulator of parkin recruitment. Validation studies revealed that kenpaullone augments the mitochondrial network and protects against the complex I inhibitor MPP+. Finally, we used a microfluidics platform to assess the timing of parkin recruitment to depolarized mitochondria and its modulation by kenpaullone in real time and with single-cell resolution. We demonstrate that the high-content imaging-based assay presented here is suitable for both genetic and pharmacological screening approaches, and we also provide evidence that pharmacological compounds modulate PINK1-dependent parkin recruitment.

Development of CVN424: A Selective and Novel GPR6 Inverse Agonist Effective in Models of Parkinson Disease.

GPR6 is an orphan G-protein-coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT: CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.

Insulin-like peptide 5 is an orexigenic gastrointestinal hormone.

The gut endocrine system is emerging as a central player in the control of appetite and glucose homeostasis, and as a rich source of peptides with therapeutic potential in the field of diabetes and obesity. In this study we have explored the physiology of insulin-like peptide 5 (Insl5), which we identified as a product of colonic enteroendocrine L-cells, better known for their secretion of glucagon-like peptide-1 and peptideYY. i.p. Insl5 increased food intake in wild-type mice but not mice lacking the cognate receptor Rxfp4. Plasma Insl5 levels were elevated by fasting or prolonged calorie restriction, and declined with feeding. We conclude that Insl5 is an orexigenic hormone released from colonic L-cells, which promotes appetite during conditions of energy deprivation.

GO2MSIG, an automated GO based multi-species gene set generator for gene set enrichment analysis.

BACKGROUND: Despite the widespread use of high throughput expression platforms and the availability of a desktop implementation of Gene Set Enrichment Analysis (GSEA) that enables non-experts to perform gene set based analyses, the availability of the necessary precompiled gene sets is rare for species other than human. RESULTS: A software tool (GO2MSIG) was implemented that combines data from various publicly available sources and uses the Gene Ontology (GO) project term relationships to produce GSEA compatible hierarchical GO based gene sets for all species for which association data is available. Annotation sources include the GO association database (which contains data for over 200000 species), the Entrez gene2go table, and various manufacturers' array annotation files. This enables the creation of gene sets from the most up-to-date annotation data available. Additional features include the ability to restrict by evidence code, to remap gene descriptors, to filter by set size and to speed up repeat queries by caching the GO term hierarchy. Synonymous GO terms are remapped to the version preferred by the GO ontology supplied. The tool can be used in standalone form, or via a web interface. Prebuilt gene set collections constructed from the September 2013 GO release are also available for common species including human. In contrast human GO based sets available from the Broad Institute itself date from 2008. CONCLUSIONS: GO2MSIG enables the bioinformatician and non-bioinformatician alike to generate gene sets required for GSEA analysis for almost any organism for which GO term association data exists. The output gene sets may be used directly within GSEA and do not require knowledge of programming languages such as Perl, R or Python. The output sets can also be used with other analysis software such as ErmineJ that accept gene sets in the same format. Source code can be downloaded and installed locally from http://www.bioinformatics.org/go2msig/releases/ or used via the web interface at http://www.go2msig.org/cgi-bin/go2msig.cgi.

Discovery and Characterization of Novel CNS-Penetrant GPR55 Agonists.

From our NETSseq-derived human brain transcriptomics data, we identified GPR55 as a potential molecular target for the treatment of motor symptoms in patients with Parkinson's disease. From a high-throughput screen, we identified and optimized agonists with nanomolar potency against both human and rat GPR55. We discovered compounds with either strong or limited ß-arrestin signaling and receptor desensitization, indicating biased signaling. A compound that showed minimal GPR55 desensitization demonstrated a reduction in firing frequency of medium spiny neurons cultured from rat striatum but did not reverse motor deficits in a rat hypolocomotion model. Further profiling of several desensitizing and non-desensitizing lead compounds showed that they are selective over related cannabinoid receptors CB1 and CB2 and that unbound brain concentrations well above the respective GPR55 EC50 can be readily achieved following oral administration. The novel brain-penetrant GPR55 agonists disclosed can be used to probe the role of this receptor in the brain.

Characterisation of C101248: A novel selective THIK-1 channel inhibitor for the modulation of microglial NLRP3-inflammasome.

Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K+. Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K+ channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors. Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: ∼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K+ currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1ß, an effect not seen in THIK-1-depleted microglia. In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1ß from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD.

Pharmacists' impact on quality and financial metrics utilizing virtual care platforms during the coronavirus pandemic.

PURPOSE: Because of rising concern about coronavirus disease 2019 transmission, there has been a large shift from face-to-face, in-office visits to a virtual care model. The purpose of this article is to explain how Ascension Florida Gulf Coast was able to maximize the utility of ambulatory care pharmacists (ACPs) in the primary care setting during the pandemic and to provide guidance for creating a sustainable billing and practice model in the event of another global health crisis. SUMMARY: By employing telehealth services, our ACPs were able to continue to co-manage chronic disease states for documented diagnoses while still maintaining health-system revenue in the midst of a global health crisis. Utilizing various virtual platforms, ACPs were able to accommodate the needs of our patients while addressing concerns related to the compatibility and user friendliness of the platforms for our diverse patient population. ACPs used traditional incident-to evaluation and management services current procedural terminology billing codes with a virtual visit modifier for billing and tracking purposes. CONCLUSION: Utilizing various virtual care platforms, our ACPs completed 447 patient encounters within the first 7 months of the pandemic. During this time, our ACPs addressed various population health metrics, specifically for patients diagnosed with diabetes without an active claim for a cholesterol-lowering medication, in addition to performing chronic disease management. Our ACPs had a 23% success rate for statin initiation in patients reviewed and contacted. By adopting virtual care options, our ACPs were able to effectively co-manage and educate patients while improving quality metrics and generating $50,662.24 in billable encounters for the health system during a global health crisis. The addition of ACP virtual encounters to primary care clinics both increases access to healthcare and improves patient care quality and outcomes while limiting revenue losses in our local health system.

Travel-Related Behaviors and Health Outcomes of Adolescents Compared with Adults on Short-Term International Service Missions.

With an increasing number of adolescents participating in international travel, little is known about travel-related behaviors and health risks in this age group. In the years 2015-2016, we conducted an anonymous, posttravel, questionnaire-based survey with the aim to compare self-reported practices and travel-related symptoms between adolescents (< 18 years old, N = 87) and adults (≥ 18 years old, N = 149) who came to our travel clinic before their humanitarian missions. They had the same pretravel health education, and traveled together to perform similar activities. In univariate analysis, compared with adults, we found that adolescents reported less prior international travel (P < 0.001), more often wore long-sleeved clothing for malaria prevention (P < 0.001) but less often for sun protection (P = 0.009), more often used insect repellents (P = 0.011), and less often had diarrhea (P = 0.024). All other practices and health outcomes were similar between the groups. Multivariate analyses using Bayesian network show strong associations between adults and prior travel experience, and not wearing long-sleeve clothing for malaria prevention. We also found strong associations between prior international travel and sustaining an injury, and having jet lag, as well as between taking malaria prophylaxis and not having diarrhea. Overall, most practices and health outcomes were similar between age groups. Adolescent age and lack of prior international travel experience did not have significant impacts on practices and health outcomes. Our findings highlight the need for more effective strategies to improve the behaviors and health outcomes in both adolescents and adults.

Science Production in Germany, France, Belgium, and Luxembourg: Comparing the Contributions of Research Universities and Institutes to Science, Technology, Engineering, Mathematics, and Health.

Charting significant growth in science production over the 20th century in four European Union member states, this neo-institutional analysis describes the development and current state of universities and research institutes that bolster Europe's position as a key region in global science. On-going internationalization and Europeanization of higher education and science has been accompanied by increasing competition as well as collaboration. Despite the policy goals to foster innovation and further expand research capacity, in cross-national and historical comparison neither the level of R&D investments nor country size accounts completely for the differential growth of scientific productivity. Based on a comprehensive historical database from 1900 to 2010, this analysis uncovers both stable and dynamic patterns of production and productivity in Germany, France, Belgium, and Luxembourg. Measured in peer-reviewed research articles collected in Thomson Reuters' Science Citation Index Expanded, which includes journals in the fields of Science, Technology, Engineering, Mathematics, and Health, we show the varying contributions of different organizational forms, especially research universities and research institutes. Comparing the institutionalization pathways that created the conditions necessary for continuous and strong growth in scientific productivity in the European center of global science emphasizes that the research university is the key organizational form across countries.

Genetic approaches to unraveling G protein-coupled receptor biology.

Genetic approaches to validating G protein-coupled receptors (GPCRs) have proven to be a powerful research tool, especially knockout studies in rodents. To date, data related to in vivo function have been published on approximately half of the human rhodopsin-like family-1 GPCRs, which can be attributed to the use of mouse knockouts. It is likely that many currently unknown yet important therapeutic mechanisms will be uncovered through knockout screens in mice. One such recent discovery is the elucidation of the in vivo function of the GPCR GPR54 through mouse genetics, and its subsequent validation in human populations. Although previously unsuspected, GPR54 has been found to be a master-regulator of the hypothalamic-pituitary-gonadal axis.

Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes.

The ability to generate tagged mutants of Rhodococcus spp. will facilitate a deeper understanding of this medically and commercially important genus. The absence of efficient transposon systems in these organisms has here been overcome by the use of Tn5-based DNA-protein transposition complexes which can transpose at high efficiency. To achieve this, electroporation efficiencies and antibiotic selection were optimized. A Rhodococcus rhodochrous CW25 Tn5 insertion library of 1500 mutants was created. Southern blotting of 23 representative mutants demonstrated random insertion. A number of auxotrophic mutants were isolated and the disrupted regions involved were identified by inverse PCR and subsequent sequencing. Transposition of Tn5 was confirmed by the presence of 9 bp direct repeats of Rhodococcus DNA flanking the transposon insertion site. To further test this system, a Tn5 insertion library was constructed in a wild-type soil isolate of Rhodococcus spp. This is the first viable transposon knockout system reported for Rhodococcus.

Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes.

The compact genome of Fugu rubripes has been sequenced to over 95% coverage, and more than 80% of the assembly is in multigene-sized scaffolds. In this 365-megabase vertebrate genome, repetitive DNA accounts for less than one-sixth of the sequence, and gene loci occupy about one-third of the genome. As with the human genome, gene loci are not evenly distributed, but are clustered into sparse and dense regions. Some "giant" genes were observed that had average coding sequence sizes but were spread over genomic lengths significantly larger than those of their human orthologs. Although three-quarters of predicted human proteins have a strong match to Fugu, approximately a quarter of the human proteins had highly diverged from or had no pufferfish homologs, highlighting the extent of protein evolution in the 450 million years since teleosts and mammals diverged. Conserved linkages between Fugu and human genes indicate the preservation of chromosomal segments from the common vertebrate ancestor, but with considerable scrambling of gene order.