Fear of Contagion Among Nursing Students in the Era of COVID-19.

BACKGROUND: COVID-19 quickly spread to pandemic proportions, resulting in anxiety and fear for many students. The Fear of Contagion model was explored in first-semester nursing students. METHOD: This study used a qualitative design with content analysis of narrative responses to questions derived from the Fear of Contagion model and an a priori template based on the model. RESULTS: The results included five themes: (a) doing their part to prevent the spread but wanting to do more; (b) making specific behavior changes to prevent the spread and effects of the virus; (c) experiencing fear, anxiety, and stress; (d) uncertainty but hopefulness; and (e) positive regard and concern for others. CONCLUSION: Fear of contagion in this study reflected many of the elements of the original model. Interventions to not only reduce fear but also facilitate education are recommended for nursing educators. [J Nurs Educ. 2021;60(7):404-407.].

Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance.

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.

Regulation of GSK3ß by Ser389 Phosphorylation During Neural Development.

GSK3ß is a constitutively active kinase that promotes cell death, which requires strict regulatory mechanisms. Although Akt-mediated phosphorylation at Ser9 is the default mechanism to inactivate GSK3ß, phosphorylation of GSK3ß at Ser389 by p38 MAPK has emerged as an alternative inhibitory pathway that provides cell protection and repair in response to DNA damage. Phosphorylation of Ser389 GSK3ß has been detected in adult brain, where it has been related to neuronal survival and behavior. However, the use of this pathway to regulate GSK3ß in the neonatal developing brain is unknown. In this study, we show that phosphorylation of GSK3ß at Ser389 in the brain is developmentally regulated, with the highest levels corresponding to the first 2 weeks of age. Moreover, we found that the phosphorylation of GSK3ß at Ser389 is the preferential mechanism for inactivating brain GSK3ß in 2-week-old mice. Importantly, we show that phospho-Ser389 GSK3ß expression is predominant in neuronal cell cultures from neonatal brain relative to other cell populations. However, phospho-Ser389 GSK3ß is triggered by DNA double-strand breaks in all developing neural cell types examined. Thus, the phosphorylation of GSK3ß on Ser389 could be a central regulatory mechanism to restrain GSK3ß during neurogenesis early in life.

Two Weeks of Variable Stress Increases Gamma-H2AX Levels in the Mouse Bed Nucleus of the Stria Terminalis.

Recent reports demonstrate that DNA damage is induced, and rapidly repaired, in circuits activated by experience. Moreover, stress hormones are known to slow DNA repair, suggesting that prolonged stress may result in persistent DNA damage. Prolonged stress is known to negatively impact physical and mental health; however, DNA damage as a factor in stress pathology has only begun to be explored. Histone H2A-X phosphorylated at serine 139 (γH2AX) is a marker of DNA double-strand breaks (DSB), a type of damage that may lead to cell death if unrepaired. We hypothesized that a 14-day period of variable stress exposure sufficient to alter anxiety-like behavior in male C57BL/6J mice would produce an increase in γH2AX levels in the bed nucleus of the stria terminalis (BNST), a region implicated in anxiety and stress regulation. We observed that 14 days of variable stress, but not a single stress exposure, was associated with increased levels of γH2AX 24 h after termination of the stress paradigm. Further investigation found that phosphorylation levels of a pair of kinases associated with the DNA damage response, glycogen synthase kinase 3 ß (GSK3ß) and p38 mitogen-activated protein kinase (MAPK) were also elevated following variable stress. Our results suggest that unrepaired DNA DSBs and/or repetitive attempted repair may represent an important component of the allostatic load that stress places on the brain.

Failure to Inactivate Nuclear GSK3ß by Ser389-Phosphorylation Leads to Focal Neuronal Death and Prolonged Fear Response.

GSK3ß plays an essential role in promoting cell death and is emerging as a potential target for neurological diseases. Understanding the mechanisms that control neuronal GSK3ß is critical. A ubiquitous mechanism to repress GSK3ß involves Akt-mediated phosphorylation of Ser9. Here we show that phosphorylation of GSK3ß on Ser389 mediated by p38 MAPK specifically inactivates nuclear GSK3ß in the cortex and hippocampus. Using GSK3ß Ser389 to Ala mutant mice, we show that failure to inactivate nuclear GSK3ß by Ser389 phosphorylation causes neuronal cell death in subregions of the hippocampus and cortex. Although this focal neuronal death does not impact anxiety/depression-like behavior or hippocampal-dependent spatial learning, it leads to an amplified and prolonged fear response. This phenotype is consistent with some aspects of post-traumatic stress disorder (PTSD). Our studies indicate that inactivation of nuclear GSK3ß by Ser389 phosphorylation plays a key role in fear response, revealing new potential therapeutic approaches to target PTSD.

Fine-Tuning of CD8(+) T Cell Mitochondrial Metabolism by the Respiratory Chain Repressor MCJ Dictates Protection to Influenza Virus.

Mitochondrial respiration is regulated in CD8(+) T cells during the transition from naive to effector and memory cells, but mechanisms controlling this process have not been defined. Here we show that MCJ (methylation-controlled J protein) acted as an endogenous break for mitochondrial respiration in CD8(+) T cells by interfering with the formation of electron transport chain respiratory supercomplexes. Metabolic profiling revealed enhanced mitochondrial metabolism in MCJ-deficient CD8(+) T cells. Increased oxidative phosphorylation and subcellular ATP accumulation caused by MCJ deficiency selectively increased the secretion, but not expression, of interferon-γ. MCJ also adapted effector CD8(+) T cell metabolism during the contraction phase. Consequently, memory CD8(+) T cells lacking MCJ provided superior protection against influenza virus infection. Thus, MCJ offers a mechanism for fine-tuning CD8(+) T cell mitochondrial metabolism as an alternative to modulating mitochondrial mass, an energetically expensive process. MCJ could be a therapeutic target to enhance CD8(+) T cell responses.

Inactivation of nuclear GSK3ß by Ser(389) phosphorylation promotes lymphocyte fitness during DNA double-strand break response.

Variable, diversity and joining (V(D)J) recombination and immunoglobulin class switch recombination (CSR) are key processes in adaptive immune responses that naturally generate DNA double-strand breaks (DSBs) and trigger a DNA repair response. It is unclear whether this response is associated with distinct survival signals that protect T and B cells. Glycogen synthase kinase 3ß (GSK3ß) is a constitutively active kinase known to promote cell death. Here we show that phosphorylation of GSK3ß on Ser(389) by p38 MAPK (mitogen-activated protein kinase) is induced selectively by DSBs through ATM (ataxia telangiectasia mutated) as a unique mechanism to attenuate the activity of nuclear GSK3ß and promote survival of cells undergoing DSBs. Inability to inactivate GSK3ß through Ser(389) phosphorylation in Ser(389)Ala knockin mice causes a decrease in the fitness of cells undergoing V(D)J recombination and CSR. Preselection-Tcrß repertoire is impaired and antigen-specific IgG antibody responses following immunization are blunted in Ser(389)GSK3ß knockin mice. Thus, GSK3ß emerges as an important modulator of the adaptive immune response.

Mitochondrial Ca²âº and membrane potential, an alternative pathway for Interleukin 6 to regulate CD4 cell effector function.

IL-6 plays an important role in determining the fate of effector CD4 cells and the cytokines that these cells produce. Here we identify a novel molecular mechanism by which IL-6 regulates CD4 cell effector function. We show that IL-6-dependent signal facilitates the formation of mitochondrial respiratory chain supercomplexes to sustain high mitochondrial membrane potential late during activation of CD4 cells. Mitochondrial hyperpolarization caused by IL-6 is uncoupled from the production of ATP by oxidative phosphorylation. However, it is a mechanism to raise the levels of mitochondrial Ca(2+) late during activation of CD4 cells. Increased levels of mitochondrial Ca(2+) in the presence of IL-6 are used to prolong Il4 and Il21 expression in effector CD4 cells. Thus, the effect of IL-6 on mitochondrial membrane potential and mitochondrial Ca(2+) is an alternative pathway by which IL-6 regulates effector function of CD4 cells and it could contribute to the pathogenesis of inflammatory diseases.

The emerging role of p38 mitogen-activated protein kinase in multiple sclerosis and its models.

Multiple sclerosis (MS), the most common disabling neurologic disease of young adults, is considered a classical T cell-mediated disease and is characterized by demyelination, axonal damage, and progressive neurological dysfunction. The currently available disease-modifying therapies are limited in their efficacy, and improved understanding of new pathways contributing to disease pathogenesis could reveal additional novel therapeutic targets. The p38 mitogen-activated protein kinase (MAPK) signaling pathway is known to be triggered by stress stimuli and to contribute to inflammatory responses. Importantly, a number of recent studies have identified this signaling pathway as a central player in MS and its principal animal model, experimental allergic encephalomyelitis. Here, we review the evidence from mouse and human studies supporting the role of p38 MAPK in regulating key immunopathogenic mechanisms underlying autoimmune inflammatory disease of the central nervous system and the potential of targeting this pathway as a disease-modifying therapy in MS.

Translational control of NKT cell cytokine production by p38 MAPK.

NKT cells are known to rapidly produce a large amount of cytokines upon activation. Although a number of signaling pathways that regulate the development of NKT cells have been identified, the signaling pathways involved in the regulation of NKT cell cytokine production remain unclear. In this study, we show that the p38 MAPK pathway is dispensable for the development of NKT cells. However, NKT cell cytokine production and NKT-mediated liver damage are highly dependent on activation of this pathway. p38 MAPK does not substantially affect cytokine gene expression in NKT cells, but it regulates the synthesis of cytokines through the Mnk-eIF4E pathway. Thus, in addition to gene expression, translational regulation by p38 MAPK could be a novel mechanism that contributes to the overall production of cytokine by NKT cells.

Nuclear localization of p38 MAPK in response to DNA damage.

p38 MAP kinase (MAPK) is activated in response to environmental stress, cytokines and DNA damage, and mediates death, cell differentiation and cell cycle checkpoints. The intracellular localization of p38 MAPK upon activation remains unclear, and may depend on the stimulus. We show here that activation of p38 MAPK by stimuli that induce DNA double strand breaks (DSBs), but not other stimuli, leads to its nuclear translocation. In addition, naturally occurring DSBs generated through V(D)J recombination in immature thymocytes also promote nuclear accumulation of p38 MAPK. Nuclear translocation of p38 MAPK does not require its catalytic activity, but is induced by a conformational change of p38 MAPK triggered by phosphorylation within the active site. The selective nuclear accumulation of p38 MAPK in response to DNA damage could be a mechanism to facilitate the phosphorylation of p38 MAPK nuclear targets for the induction of a G2/M cell cycle checkpoint and DNA repair.

Non-classical p38 map kinase functions: cell cycle checkpoints and survival.

The p38 MAPK kinase pathway is activated in response to a wide range of cellular stress stimuli and cytokines. Our understanding of the important functions of p38 MAPK in the process of differentiation and cell death has grown considerably in the recent years and is now relatively established. Here we discuss the role of p38 MAPK in the mediation of cell cycle checkpoints and cell survival, processes that have received less attention. We describe how p38 MAPK regulates both the G2/M as well as a G1/S cell cycle checkpoint in response to cellular stress such as DNA damage. While p38 MAPK has classically been associated with the induction of apoptosis, we discuss that p38 MAPK can also mediate cell survival in specific situations, such as in response to DNA damage. It is important to recognize these less appreciated functions of p38 MAPK when considering the potential use of pharmacological inhibitors of p38 MAPK in therapeutic treatments for disease.

Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation.

Glycogen synthase kinase 3beta (GSK3beta) is involved in metabolism, neurodegeneration, and cancer. Inhibition of GSK3beta activity is the primary mechanism that regulates this widely expressed active kinase. Although the protein kinase Akt inhibits GSK3beta by phosphorylation at the N terminus, preventing Akt-mediated phosphorylation does not affect the cell-survival pathway activated through the GSK3beta substrate beta-catenin. Here, we show that p38 mitogen-activated protein kinase (MAPK) also inactivates GSK3beta by direct phosphorylation at its C terminus, and this inactivation can lead to an accumulation of beta-catenin. p38 MAPK-mediated phosphorylation of GSK3beta occurs primarily in the brain and thymocytes. Activation of beta-catenin-mediated signaling through GSK3beta inhibition provides a potential mechanism for p38 MAPK-mediated survival in specific tissues.

Direct manipulation of activator protein-1 controls thymocyte proliferation in vitro.

B cell activating transcription factor (BATF) belongs to the activator protein-1 (AP-1) superfamily of basic leucine zipper transcription factors and forms heterodimers with Jun that possess minimal transcriptional activity. Mice carrying a p56(lck)HA-BATF transgene were created to observe the effects of constitutive expression of this well-characterized AP-1 inhibitor on T cell proliferation. Consistent with the role of AP-1 in promoting the proliferation of many cell types, BATF-transgenic thymocytes proliferate poorly in vitro when stimulated with anti-CD3epsilon and anti-CD28 antibodies or with Concanavalin A. However, when BATF-transgenic thymocytes were stimulated using a standard treatment of PMA and ionomycin, proliferation is normal. The responsiveness to PMA and ionomycin can be attributed to the dramatic disappearance of the hemagglutinin antigen (HA)-tagged BATF protein which is a PKC-dependent process caused by the down-regulation of the p56(lck) proximal promoter coupled with the rapid turnover of the HA-BATF protein. These studies describe conditions of T cell stimulation that negatively influence transcription of the widely used p56(lck) proximal promoter expression cassette. In addition, the unique circumstances of this regulation were exploited to demonstrate that inhibition of AP-1 activity by BATF exerts a direct, and reversible, effect on T cell proliferation in vitro.

Phosphorylation of BATF regulates DNA binding: a novel mechanism for AP-1 (activator protein-1) regulation.

BATF is a member of the AP-1 (activator protein-1) family of bZIP (basic leucine zipper) transcription factors that form transcriptionally inhibitory, DNA binding heterodimers with Jun proteins. In the present study, we demonstrate that BATF is phosphorylated in vivo on multiple serine and threonine residues and at least one tyrosine residue. Reverse-polarity PAGE revealed that serine-43 and threonine-48 within the DNA binding domain of BATF are phosphorylated. To model phosphorylation of the BATF DNA binding domain, serine-43 was replaced by an aspartate residue. BATF(S43D) retains the ability to dimerize with Jun proteins in vitro and in vivo, and the BATF(S43D):Jun heterodimer localizes properly to the nucleus of cells. Interestingly, BATF(S43D) functions like wild-type BATF to reduce AP-1-mediated gene transcription, despite the observed inability of the BATF(S43D):Jun heterodimer to bind DNA. These data demonstrate that phosphorylation of serine-43 converts BATF from a DNA binding into a non-DNA binding inhibitor of AP-1 activity. Given that 40% of mammalian bZIP transcription factors contain a residue analogous to serine-43 of BATF in their DNA binding domains, the phosphorylation event described here represents a mechanism that is potentially applicable to the regulation of many bZIP proteins.

EBNA2 and activated Notch induce expression of BATF.

The immortalization of human B lymphocytes by Epstein-Barr virus (EBV) requires the virus-encoded transactivator EBNA2 and the products of both viral and cellular genes which serve as EBNA2 targets. In this study, we identified BATF as a cellular gene that is up-regulated dramatically within 24 h following the infection of established and primary human B cells with EBV. The transactivation of BATF is mediated by EBNA2 in a B-cell-specific manner and is duplicated in non-EBV-infected B cells by the expression of mammalian Notch proteins. In contrast to other target genes activated by EBNA2, the BATF gene encodes a member of the AP-1 family of transcription factors that functions as a negative regulator of AP-1 activity and as an antagonist of cell growth. A potential role for BATF in promoting EBV latency is supported by studies in which BATF was shown to negatively impact the expression of a BZLF1 reporter gene and to reduce the frequency of lytic replication in latently infected cells. The identification of BATF as a cellular target of EBV provides important new information on how programs of viral and cellular gene expression may be coordinated to promote viral latency and control lytic-cycle entry.

SPINDLY is a nuclear-localized repressor of gibberellin signal transduction expressed throughout the plant.

SPY (SPINDLY) encodes a putative O-linked N-acetyl-glucosamine transferase that is genetically defined as a negatively acting component of the gibberellin (GA) signal transduction pathway. Analysis of Arabidopsis plants containing a SPY::GUS reporter gene reveals that SPY is expressed throughout the life of the plant and in most plant organs examined. In addition to being expressed in all organs where phenotypes due to spy mutations have been reported, SPY::GUS is expressed in the root. Examination of the roots of wild-type, spy, and gai plants revealed phenotypes indicating that SPY and GAI play a role in root development. A second SPY::GUS reporter gene lacking part of the SPY promoter was inactive, suggesting that sequences in the first exon and/or intron are required for detectable expression. Using both subcellular fractionation and visualization of a SPY-green fluorescent protein fusion protein that is able to rescue the spy mutant phenotype, the majority of SPY protein was shown to be present in the nucleus. This result is consistent with the nuclear localization of other components of the GA response pathway and suggests that SPY's role as a negative regulator of GA signaling involves interaction with other nuclear proteins and/or O-N-acetyl-glucosamine modification of these proteins.