Investigating the functional implications of the Clock 3111T/C SNP

Introduction: Bipolar disorder (BD) is a debilitating mental illness proven to be very difficult to treat. By studying the molecular mechanisms that underlie BD, we can learn how to improve treatments. The CLOCK protein is a key transcription factor involved in the persistence of circadian rhythms. Studies have found that manipulations of the Clock gene are sufficient to produce manic-like behavior in mice. The Clock 3111T/C single-nucleotide polymorphism (SNP) is a variation of the human Clock gene that is associated with increased frequency of mania in BD patients. In this study, we sought to examine the functional implications of the Clock 3111T/C SNP on Clock and Per2 expression over 24 hours. Methods and Materials: Plasmid construction: The human Clock gene was cloned and site-directed mutagenesis was performed to produce the 3111T and 3111C versions of the gene. An Npas2 shRNA was also constructed in order to knockdown Npas2 expression, which has been shown to be significantly upregulated in Clock KO MEFs. MEFs: Mouse embryonic fibroblasts (MEFs) were collected from Clock Knockout (KO) mice. Clock KO MEFs were used to avoid confounds of endogenous Clock expression. Transfections: MEFs were transfected with either the Clock T or C constructs, in addition with an Npas2 shRNA. Quantitative PCR: RNA from transfected MEFs was isolated, and then reverse-transcribed to cDNA. Quantitative polymerase chain reaction (qPCR) was performed over a 24 hour period. Results: Quantitative PCR revealed significantly increased levels of Clock and Per2 mRNA over a 24-hour time course in MEFs expressing the 3111C SNP, as compared with 3111T. Conclusions: The CLOCK protein controls expression of genes beyond the circadian clock. These include dopamine regulators, which are known to be involved in decreased depression-like behaviors and increased impulsivity/drug intake. From this, it is clear that altered Clock expression may have tremendous implications for a variety of diseases, including BD

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

NPAS2 Regulation of Anxiety-Like Behavior and GABAA Receptors

Abnormal circadian rhythms and circadian genes are strongly associated with several psychiatric disorders. Neuronal PAS Domain Protein 2 (NPAS2) is a core component of the molecular clock that acts as a transcription factor and is highly expressed in reward- and stress-related brain regions such as the striatum. However, the mechanism by which NPAS2 is involved in mood-related behaviors is still unclear. We measured anxiety-like behaviors in mice with a global null mutation in Npas2 (Npas2 null mutant mice) and found that Npas2 null mutant mice exhibit less anxiety-like behavior than their wild-type (WT) littermates (in elevated plus maze, light/dark box and open field assay). We assessed the effects of acute or chronic stress on striatal Npas2 expression, and found that both stressors increased levels of Npas2. Moreover, knockdown of Npas2 in the ventral striatum resulted in a similar reduction of anxiety-like behaviors as seen in the Npas2 null mutant mouse. Additionally, we identified Gabra genes as transcriptional targets of NPAS2, found that Npas2 null mutant mice exhibit reduced sensitivity to the GABAa positive allosteric modulator, diazepam and that knockdown of Npas2 reduced Gabra1 expression and response to diazepam in the ventral striatum. These results: (1) implicate Npas2 in the response to stress and the development of anxiety; and (2) provide functional evidence for the regulation of GABAergic neurotransmission by NPAS2 in the ventral striatum

Effect of Antiplatelet Therapy on Survival and Organ Support–Free Days in Critically Ill Patients With COVID-19

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