Genome-wide screening reveals metabolic regulation of stop-codon readthrough by cyclic AMP.

Translational fidelity is critical for microbial fitness, survival and stress responses. Much remains unknown about the genetic and environmental control of translational fidelity and its single-cell heterogeneity. In this study, we used a high-throughput fluorescence-based assay to screen a knock-out library of Escherichia coli and identified over 20 genes critical for stop-codon readthrough. Most of these identified genes were not previously known to affect translational fidelity. Intriguingly, we show that several genes controlling metabolism, including cyaA and crp, enhance stop-codon readthrough. CyaA catalyzes the synthesis of cyclic adenosine monophosphate (cAMP). Combining RNA sequencing, metabolomics and biochemical analyses, we show that deleting cyaA impairs amino acid catabolism and production of ATP, thus repressing the transcription of rRNAs and tRNAs to decrease readthrough. Single-cell analyses further show that cAMP is a major driver of heterogeneity in stop-codon readthrough and rRNA expression. Our results highlight that carbon metabolism is tightly coupled with stop-codon readthrough.

Coordination between aminoacylation and editing to protect against proteotoxicity.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that the ths1-C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, experimental evolution of the mutant results in intragenic mutations that restore heat resistance but not editing. ths1-C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, while the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in either ThrRS aminoacylation or editing is insufficient to cause heat sensitivity, and that ths1-C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.

COVID-19 Impact on Teaching Substance Use Disorders: A Nursing Curricular Thread.

ABSTRACT: Vulnerable populations such as those with substance use disorders (SUDs) are at a higher risk for early morbidities and mortalities yet are less likely to receive primary care and other necessary psychosocial services essential for comprehensive care of these clients. This need has been magnified by the COVID-19 pandemic. Evidence supports an increase in alcohol sales in 2020, and overdoses from illicit drugs have been reported to have more than doubled by May 2020 from the 2018 and 2019 baseline rates, and one reason for these increases is because of COVID-19. The healthcare system is overwhelmed with the cost of treating and addressing the impact of SUDs. Individuals with SUDs often meet providers who are not sufficiently prepared to address their complex issues that include co-occurring mental and physical health disorders. In addition to changes in practice, nursing education must change their curricular approach to meet the challenges in health services across the life span, and nursing education should include lessons being learned during the COVID-19 pandemic. Nurses must be prepared to recognize and screen individuals for SUDs at the undergraduate level as well as assess and treat individuals with SUDs at the advanced practice level in all areas of healthcare services. SUDs should not continue to be siloed and separated into the psychiatric-mental health nursing course within the nursing curriculum but should be addressed in multiple specialties across the curricula and include health responses in regard to the impact that the COVID-19 pandemic is having on SUDs.

p38 MAPK and MKP-1 control the glycolytic program via the bifunctional glycolysis regulator PFKFB3 during sepsis.

Hyperlactatemia often occurs in critically ill patients during severe sepsis/septic shock and is a powerful predictor of mortality. Lactate is the end product of glycolysis. While hypoxia due to inadequate oxygen delivery may result in anaerobic glycolysis, sepsis also enhances glycolysis under hyperdynamic circulation with adequate oxygen delivery. However, the molecular mechanisms involved are not fully understood. Mitogen-activated protein kinase (MAPK) families regulate many aspects of the immune response during microbial infections. MAPK phosphatase (MKP)-1 serves as a feedback control mechanism for p38 and JNK MAPK activities via dephosphorylation. Here, we found that mice deficient in Mkp-1 exhibited substantially enhanced expression and phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) 3, a key enzyme that regulates glycolysis following systemic Escherichia coli infection. Enhanced PFKFB3 expression was observed in a variety of tissues and cell types, including hepatocytes, macrophages, and epithelial cells. In bone marrow-derived macrophages, Pfkfb3 was robustly induced by both E. coli and lipopolysaccharide, and Mkp-1 deficiency enhanced PFKFB3 expression with no effect on Pfkfb3 mRNA stability. PFKFB3 induction was correlated with lactate production in both WT and Mkp-1-/- bone marrow-derived macrophage following lipopolysaccharide stimulation. Furthermore, we determined that a PFKFB3 inhibitor markedly attenuated lactate production, highlighting the critical role of PFKFB3 in the glycolysis program. Finally, pharmacological inhibition of p38 MAPK, but not JNK, substantially attenuated PFKFB3 expression and lactate production. Taken together, our studies suggest a critical role of p38 MAPK and MKP-1 in the regulation of glycolysis during sepsis.

Mitogen-activated protein kinase phosphatase-1 controls PD-L1 expression by regulating type I interferon during systemic Escherichia coli infection.

Mitogen-activated protein kinase phosphatase 1 (Mkp-1) KO mice produce elevated cytokines and exhibit increased mortality and bacterial burden following systemic Escherichia coli infection. To understand how Mkp-1 affects immune defense, we analyzed the RNA-Seq datasets previously generated from control and E. coli-infected Mkp-1+/+ and Mkp-1-/- mice. We found that E. coli infection markedly induced programmed death-ligand 1 (PD-L1) expression and that Mkp-1 deficiency further amplified PD-L1 expression. Administration of a PD-L1-neutralizing monoclonal antibody (mAb) to Mkp-1-/- mice increased the mortality of the animals following E. coli infection, although bacterial burden was decreased. In addition, the PD-L1-neutralizing mAb increased serum interferon (IFN)-γ and tumor necrosis factor alpha, as well as lung- and liver-inducible nitric oxide synthase levels, suggesting an enhanced inflammatory response. Interestingly, neutralization of IFN-α/ß receptor 1 blocked PD-L1 induction in Mkp-1-/- mice following E. coli infection. PD-L1 was potently induced in macrophages by E. coli and lipopolysaccharide in vitro, and Mkp-1 deficiency exacerbated PD-L1 induction with little effect on the half-life of PD-L1 mRNA. In contrast, inhibitors of Janus kinase 1/2 and tyrosine kinase 2, as well as the IFN-α/ß receptor 1-neutralizing mAb, markedly attenuated PD-L1 induction. These results suggest that the beneficial effect of type I IFNs in E. coli-infected Mkp-1-/- mice is, at least in part, mediated by Janus kinase/signal transducer and activator of transcription-driven PD-L1 induction. Our studies also support the notion that enhanced PD-L1 expression contributes to the bactericidal defect of Mkp-1-/- mice.

Knockout of MAPK Phosphatase-1 Exaggerates Type I IFN Response during Systemic Escherichia coli Infection.

We have previously shown that Mkp-1-deficient mice produce elevated TNF-α, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coli-infected wild-type and Mkp-1 -/- mice. A large number of IFN-stimulated genes were more robustly expressed in E. coli-infected Mkp-1 -/- mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-ß, IFN-γ, TNF-α, IL-1α and ß, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coli-infected Mkp-1 -/- mice relative to wild-type mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1 -/- mice prior to E. coli infection augmented mortality and disease severity. Mkp-1 -/- bone marrow-derived macrophages (BMDM) produced higher levels of IFN-ß mRNA and protein than did wild-type BMDM upon treatment with LPS, E. coli, polyinosinic:polycytidylic acid, and herring sperm DNA. Augmented IFN-ß induction in Mkp-1 -/- BMDM was blocked by a p38 inhibitor but not by an JNK inhibitor. Enhanced Mkp-1 expression abolished IFN-ß induction by both LPS and E. coli but had little effect on the IFN-ß promoter activity in LPS-stimulated RAW264.7 cells. Mkp-1 deficiency did not have an overt effect on IRF3/7 phosphorylation or IKK activation but modestly enhanced IFN-ß mRNA stability in LPS-stimulated BMDM. Our results suggest that Mkp-1 regulates IFN-ß production primarily through a p38-mediated mechanism and that IFN-ß plays a beneficial role in E. coli-induced sepsis.

Simulation Clinical Experience of Veteran Care Competence for Psychiatric Mental Health Nurse Practitioner Students with Standardized Patients.

OBJECTIVE: The purpose of this simulation educational activity was to assist psychiatric mental health nurse practitioner students (PMHNPs) with identifying military veterans as a vulnerable group with health care disparities and provide competent military veteran care in the private sector. Mindful of all the varied terms for military service, this article will use the term military veteran. METHODS: The simulation educational activity included applying the conceptual frameworks of the healthcare disparities framework (HDF) and nursing education simulation framework (NESF). The psychiatric nurse practitioner students participated in a mandatory clinical standardized patient (SP) simulation for veteran care competence. RESULTS: The learning was assessed in the debriefing following the student's performance in the SP simulation scenario. Anecdotally, the students reported that the activity was well received and a valuable learning experience for their practice. CONCLUSIONS: SP simulation may increase PMHNPs' quality of assessment of military veterans and treatment to decrease healthcare disparities. Additionally, the education of PMHNPs with military veteran care competence will increase the availability of health care providers in the private sector, where many military veterans are seeking care.

Screening, brief intervention, and referral to treatment: a need for educational reform in nursing.

With the prevalence of addiction-related health consequences, all nurses must maintain a basic level of knowledge and skills regarding addictions. Nurses are ideally positioned to screen, assess, refer; and, at the advanced practice level, treat clients for addiction disorders, provided the knowledge and willingness exists to intervene. A vision for nursing education is to achieve minimal competencies for all generalist nurses, facilitated by incorporation of substance-related disorder concepts into nursing education. An urgent need exists to disseminate the most recent knowledge and skills in nursing school curricula throughout the United States and internationally.

Nursing students' personal experiences involving alcohol problems.

This article discusses the views and beliefs of nursing students toward people who abuse alcohol. An original study published in a separate article [Archives of Psychiatric Nursing , (2003); (4) 17 : 156-164.] examined this relationship with both a quantitative and a qualitative design. Three open-ended questions allowed for further qualitative exploration about relationships with others who have alcohol problems and beliefs about recovery. The chronic nature of alcoholism was clearly identified by students who described it as a lifelong process. Most students (79%) expressed belief that recovery was possible whether they had personal experience with people who have alcohol problems or not. The level of optimism was surprisingly high in this sample of nursing students, especially because many had had a personal experience with someone who abused alcohol. Students come to the educational setting with a clear and accurate view of the lifelong commitment that may be needed to recover from alcohol addiction, but they also come with an overly optimistic view of recovery. How this optimism impacts future care is unknown. If nursing students hold an unrealistically positive view of recovery, they may be ill prepared to handle the disappointments associated with treatment such as relapse, interpersonal conflict, health deterioration, or other related sequelae.

Examining the relationship of addiction education and beliefs of nursing students toward persons with alcohol problems.

This study examined the effectiveness of two methods of teaching nursing students about alcohol addiction. Each student who agreed to participate was given pretests, posttests, and 3-month follow-up tests that measured knowledge about and beliefs held toward people who abuse alcohol. Group 1 received lecture only, whereas group 2 received lecture and discussion with a person who had been sober for many years. Both groups showed improved scores in knowledge and certain aspects of beliefs, however, group 2 showed greater knowledge and more accurate beliefs overall toward this population than group 1. The introduction of a person successfully remaining sober was shown to be an even more effective teaching strategy than lecture alone.