Development of a Covalent Inhibitor of c-Jun N-Terminal Protein Kinase (JNK) 2/3 with Selectivity over JNK1.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1-JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher Kinact/KI compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.

Factors Associated with COVID-19 Vaccine Hesitancy in South Central Appalachia.

Introduction: The newly emergent COVID-19 virus reached pandemic levels in March 2020. By the middle of August 2020, there were over 1 million deaths attributed to COVID-19 in the U.S., with those in rural areas outpacing urban counterparts. Prior to emergency approval of the Pfizer, Moderna and Johnson & Johnson vaccine formulations, mitigation efforts addressing individual behavior were challenging. However, even with the entrance of these three new vaccines, herd immunity was not achieved in rural areas, as vaccine uptake remained low there. Although there has since been an abundance of COVID-19-related research addressing health literacy, vaccine hesitancy and overall medical mistrust, few of these studies focus on Appalachia. Purpose: This study identifies barriers and facilitators to adherence with COVID-19 mitigation, focusing specifically on vaccine hesitancy in South Central Appalachia. Methods: A secondary data study was conducted with a subset of Appalachian residents from the COVID-19 Public Health survey. Participants were grouped by county using ARC economic county designations for analysis. The dependent variable, vaccine hesitancy, was explored in relation to five categories of independent variable: (1) demographics (with four conceptual areas); (2) belief; (3) action; (4) medical mistrust; and (5) health literacy. Results: Findings indicate vaccine hesitancy attributes include beliefs addressing COVID-19 threat, overstatement of severity of illness, risk of vaccines, vaccine safety information not present from manufacturer, and independent decision to vaccinate. Findings from this study are comparable to HPV vaccine studies in Appalachia. Implications: As interventions are developed for Appalachia, it is paramount to focus vaccine administration at the individual and population level.

LIMS-Kinase provides sensitive and generalizable label-free in vitro measurement of kinase activity using mass spectrometry.

Measurements of kinase activity are important for kinase-directed drug development, analysis of inhibitor structure and function, and understanding mechanisms of drug resistance. Sensitive, accurate, and miniaturized assay methods are crucial for these investigations. Here, we describe a label-free, high-throughput mass spectrometry-based assay for studying individual kinase enzymology and drug discovery in a purified system, with a focus on validated drug targets as benchmarks. We demonstrate that this approach can be adapted to many known kinase substrates and highlight the benefits of using mass spectrometry to measure kinase activity in vitro, including increased sensitivity. We speculate that this approach to measuring kinase activity will be generally applicable across most of the kinome, enabling research on understudied kinases and kinase drug discovery.

Therapeutic Targeting the Allosteric Cysteinome of RAS and Kinase Families.

Allosteric mechanisms are pervasive in nature, but human-designed allosteric perturbagens are rare. The history of KRASG12C inhibitor development suggests that covalent chemistry may be a key to expanding the armamentarium of allosteric inhibitors. In that effort, irreversible targeting of a cysteine converted a non-deal allosteric binding pocket and low affinity ligands into a tractable drugging strategy. Here we examine the feasibility of expanding this approach to other allosteric pockets of RAS and kinase family members, given that both protein families are regulators of vital cellular processes that are often dysregulated in cancer and other human diseases. Moreover, these heavily studied families are the subject of numerous drug development campaigns that have resulted, sometimes serendipitously, in the discovery of allosteric inhibitors. We consequently conducted a comprehensive search for cysteines, a commonly targeted amino acid for covalent drugs, using AlphaFold-generated structures of those families. This new analysis presents potential opportunities for allosteric targeting of validated and understudied drug targets, with an emphasis on cancer therapy.

Presence and Therapeutic Listening.

Presence and therapeutic listening are complementary therapies that are incorporated into daily nursing practice. This article presents the nurse's role in facilitating complementary therapies of presence and therapeutic listening, barriers encountered, and the behaviors and techniques to be used. Also discussed is the impact that using presence and therapeutic listening has on patient outcomes. These interventions have the potential to significantly alter patients' perceptions of care and improve patient outcomes. Presence and therapeutic listening are vital to developing a therapeutic nurse-patient relationship. This relationship is what drives positive patient outcomes and increases overall satisfaction and quality of care.

Workplace Financial Wellness Programs Help Employees Manage Health Care Changes.

Employers and employees are navigating major changes in health insurance benefits, including the move to high-deductible health plans in conjunction with health savings accounts (HSAs). The HSA offers unique benefits that could prove instrumental in helping workers both navigate current health care expenses and build a nest egg for much larger health care costs in retirement. Yet employees often don't understand the HSA and how to best use it. How can employers help employees make wise benefits choices that work for their personal financial circumstances?

Seagrass resource assessment using remote sensing methods in St. Joseph Sound and Clearwater Harbor, Florida, USA.

In the event of a natural or anthropogenic disturbance, environmental resource managers require a reliable tool to quickly assess the spatial extent of potential damage to the seagrass resource. The temporal availability of the Landsat 5 Thematic Mapper (TM) imagery provided a suitable option to detect and assess damage of the submerged aquatic vegetation (SAV). This study examined Landsat TM imagery classification techniques to create two-class (SAV presence/absence) and three-class (SAV estimated coverage) SAV maps of the seagrass resource. The Mahalanobis Distance method achieved the highest overall accuracy (86%) and validation accuracy (68%) for delineating the seagrass resource (two-class SAV map). The Maximum Likelihood method achieved the highest overall accuracy (74%) and validation accuracy (70%) for delineating the seagrass resource three-class SAV map. The Landsat 5 TM imagery classification provided a seagrass resource map product with similar accuracy to the aerial photointerpretation maps (validation accuracy 71%). The results support the application of remote sensing methods to analyze the spatial extent of the seagrass resource.

The p-type ATPase superfamily.

P-type ATPases function to provide homeostasis in higher eukaryotes, but they are essentially ubiquitous, being found in all domains of life. Thever and Saier [J Memb Biol 2009;229:115-130] recently reported analyses of eukaryotic P-type ATPases, dividing them into nine functionally characterized and 13 functionally uncharacterized (FUPA) families. In this report, we analyze P-type ATPases in all major prokaryotic phyla for which complete genome sequence data are available, and we compare the results with those for eukaryotic P-type ATPases. Topological type I (heavy metal) P-type ATPases predominate in prokaryotes (approx. tenfold) while type II ATPases (specific for Na(+),K(+), H(+) Ca(2+), Mg(2+) and phospholipids) predominate in eukaryotes (approx. twofold). Many P-type ATPase families are found exclusively in prokaryotes (e.g. Kdp-type K(+) uptake ATPases (type III) and all ten prokaryotic FUPA familes), while others are restricted to eukaryotes (e.g. phospholipid flippases and all 13 eukaryotic FUPA families). Horizontal gene transfer has occurred frequently among bacteria and archaea, which have similar distributions of these enzymes, but rarely between most eukaryotic kingdoms, and even more rarely between eukaryotes and prokaryotes. In some bacterial phyla (e.g. Bacteroidetes, Flavobacteria and Fusobacteria), ATPase gene gain and loss as well as horizontal transfer occurred seldom in contrast to most other bacterial phyla. Some families (i.e. Kdp-type ATPases) underwent far less horizontal gene transfer than other prokaryotic families, possibly due to their multisubunit characteristics. Functional motifs are better conserved across family lines than across organismal lines, and these motifs can be family specific, facilitating functional predictions. In some cases, gene fusion events created P-type ATPases covalently linked to regulatory catalytic enzymes. In one family (FUPA Family 24), a type I ATPase gene (N-terminal) is fused to a type II ATPase gene (C-terminal) with retention of function only for the latter. Several pseudogene-encoded nonfunctional ATPases were identified. Genome minimalization led to preferential loss of P-type ATPase genes. We suggest that in prokaryotes and some unicellular eukaryotes, the primary function of P-type ATPases is protection from extreme environmental stress conditions. The classification of P-type ATPases of unknown function into phylogenetic families provides guides for future molecular biological studies.

Common variants on chromosome 2 and risk of primary open-angle glaucoma in the Afro-Caribbean population of Barbados.

Primary open-angle glaucoma (POAG) is the second leading cause of blindness worldwide. Although a number of genetic loci have shown association or genetic linkage to monogenic forms of POAG, the identified genes and loci do not appear to have a major role in the common POAG phenotype. We seek to identify genetic loci that appear to be major risk factors for POAG in the Afro-Caribbean population of Barbados, West Indies. We performed linkage analyses in 146 multiplex families ascertained through the Barbados Family Study of Glaucoma (BFSG) and identified a strong linkage signal on chromosome 2p (logarithm of odds score = 6.64 at = 0 with marker D2S2156). We subsequently performed case-control analyses using unrelated affected individuals and unaffected controls. A set of SNPs on chromosome 2p was evaluated in two independent groups of BFSG participants, a discovery group (130 POAG cases, 65 controls) and a replication group (122 POAG cases, 65 controls), and a strong association was identified with POAG and rs12994401 in both groups (P < 3.34 E-09 and P < 1.21E-12, respectively). The associated SNPs form a common disease haplotype. In summary, we have identified a locus with a major impact on susceptibility to the common POAG phenotype in an Afro-Caribbean population in Barbados. Our approach illustrates the merit of using an isolated population enriched with common disease variants as an efficient method to identify genetic underpinning of POAG.