Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256.

In the renal collecting duct, binding of AVP to the V2 receptor triggers signaling changes that regulate osmotic water transport. Short-term regulation of water transport is dependent on vasopressin-induced phosphorylation of aquaporin-2 (AQP2) at Ser256. The protein kinase that phosphorylates this site is not known. We use Bayes' theorem to rank all 521 rat protein kinases with regard to the likelihood of a role in Ser256 phosphorylation on the basis of prior data and new experimental data. First, prior probabilities were estimated from previous transcriptomic and proteomic profiling data, kinase substrate specificity data, and evidence for kinase regulation by vasopressin. This ranking was updated using new experimental data describing the effects of several small-molecule kinase inhibitors with known inhibitory spectra (H-89, KN-62, KN-93, and GSK-650394) on AQP2 phosphorylation at Ser256 in inner medullary collecting duct suspensions. The top-ranked kinase was Ca2+/calmodulin-dependent protein kinase II (CAMK2), followed by protein kinase A (PKA) and protein kinase B (AKT). Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based in vitro phosphorylation studies compared the ability of three highly ranked kinases to phosphorylate AQP2 and other inner medullary collecting duct proteins, PKA, CAMK2, and serum/glucocorticoid-regulated kinase (SGK). All three proved capable of phosphorylating AQP2 at Ser256, although CAMK2 and PKA were more potent than SGK. The in vitro phosphorylation experiments also identified candidate protein kinases for several additional phosphoproteins with likely roles in collecting duct regulation, including Nedd4-2, Map4k4, and 3-phosphoinositide-dependent protein kinase 1. We conclude that Bayes' theorem is an effective means of integrating data from multiple data sets in physiology.

Methamphetamines and Serious Injection-Related Infections: Opioid Use Care Continuum and Opportunities to End Alabama's Drug Crisis.

Background: Increasingly, injection opioid use and opioid use disorder (OUD) are complicated by methamphetamine use, but the impact of stimulant use on the care of people who inject drugs (PWID) with serious injection-related infections (SIRIs) is unknown. The objective of this study was to explore hospital outcomes and postdischarge trends for a cohort of hospitalized PWID to identify opportunities for intervention. Methods: We queried the electronic medical record for patients hospitalized at the University of Alabama at Birmingham with injection drug use-related infections between 1/11/2016 and 4/24/2021. Patients were categorized as having OUD only (OUD), OUD plus methamphetamine use (OUD/meth), or injection of other substance(s) (other). We utilized statistical analyses to assess group differences across hospital outcomes and postdischarge trends. We determined the OUD continuum of care for those with OUD, with and without methamphetamine use. Results: A total of 370 patients met inclusion criteria-many with readmissions (98%) and high mortality (8%). The majority were White, male, and uninsured, with a median age of 38. One in 4 resided outside of a metropolitan area. There were significant differences according to substance use in terms of sociodemographics and hospital outcomes: patients with OUD/meth were more likely to leave via patient-directed discharge, but those with OUD only had the greatest mortality. Comorbid methamphetamine use did not significantly impact the OUD care continuum. Conclusions: The current drug crisis in AL will require targeted interventions to engage a young, uninsured population with SIRI in evidence-based addiction and infection services.

A Study Protocol to Increase Engagement in Evidence Based Hospital and Community Based Care Using a Serious Injection Related Infections (SIRI) Checklist and Enhanced Peer for Hospitalized PWID (ShaPe).

Background: With the opioid crisis, surging methamphetamine use, and healthcare disruptions due to SARS-CoV-2, serious injection related infections (SIRIs), like endocarditis, have increased significantly. Hospitalizations for SIRI provide a unique opportunity for persons who inject drugs (PWID) to engage in addiction treatment and infection prevention, yet many providers miss opportunities for evidence-based care due to busy inpatient services and lack of awareness. To improve hospital care, we developed a 5-item SIRI Checklist for providers as a standardized reminder to offer medication for opioid use disorder (MOUD), HIV and HCV screening, harm reduction counseling, and referral to community-based care. We also formalized an Intensive Peer Recovery Coach protocol to support PWID on discharge. We hypothesized that the SIRI Checklist and Intensive Peer Intervention would increase use of hospital-based services (HIV, HCV screening, MOUD) and linkage to community-based care: PrEP prescription, MOUD prescription, and related outpatient visit(s). Methods: This is a feasibility study and randomized control trial of a checklist and intensive peer intervention for hospitalized PWID with SIRI admitted to UAB Hospital. We will recruit 60 PWID who will be randomized to one of 4 groups (SIRI Checklist, SIRI Checklist + Enhanced Peer, Enhanced Peer, and Standard of Care). Results will be analyzed using a 2x2 factorial design. We will use surveys to collect data on drug use behaviors, stigma, HIV risk, and PrEP interest and awareness. Our primary outcome of feasibility will include the ability to recruit hospitalized PWID and retain them in the study to determine post-discharge clinical outcomes. Additionally, we will explore clinical outcomes using a combination of patient surveys and electronic medical record data (HIV, HCV testing, MOUD and PrEP prescriptions).This study is approved by UAB IRB #300009134. Discussion: This feasibility study is a necessary step in designing and testing patient-centered interventions to improve public health for rural and Southern PWID. By testing low barrier interventions that are accessible and reproducible in states without access to Medicaid expansion and robust public health infrastructure, we aim to identify models of care that promote linkage and engagement in community care. Trial Registration: NCT05480956.

HIV and Addiction Services for People Who Inject Drugs: Healthcare Provider Perceptions on Integrated Care in the U.S. South.

This qualitative study evaluates physician training and experience with treatment and prevention services for people who inject drugs (PWID) including medications for opioid use disorder (MOUD) and HIV pre-exposure prophylaxis (PrEP). The Behavioral Model of Healthcare Utilization for Vulnerable Populations was applied as a framework for data analysis and interpretation. Two focus groups were conducted, one with early career physicians (n = 6) and one with mid- to late career physicians (n = 3). Focus group transcripts were coded and analyzed using thematic analysis to identify factors affecting implementation of treatment and prevention services for PWID. Respondents identified that increasing the availability of providers prescribing MOUD was a critical enabling factor for PWID seeking and receiving care. Integrated, interdisciplinary services were identified as an additional resource although these remain fragmented in the current healthcare system. Barriers to care included provider awareness, stigma associated with substance use, and access limitations. Providers identified the interwoven risk factors associated with injection drug use that must be addressed, including the risk of HIV acquisition, notably more at the forefront in the minds of early career physicians. Additional research is needed addressing the medical education curriculum, health system, and healthcare policy to address the addiction and HIV crises in the U.S. South.

Identifying protein kinase target preferences using mass spectrometry.

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called "PhosphoLogo," uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions -2 and -3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3ß, Wnk1, and Wnk4.

Revamping Residency Education during a Pandemic with Twitter-Based Learning.

Amidst the increasing clinical demands and social distancing constraints of COVID-19, Twitter-based, resident-driven education offers adaptability for the current predicament faced by residency programs and sparks curiosity that will outlast this pandemic.