Evaluation of High-dose versus Standard-dose of Dexamethasone on Mortality among the Mechanically Ventilated COVID-19 Patients.

INTRODUCTION: Anti-inflammatory agents like dexamethasone (DEX) are a mainstay of treatment for COVID-19. Despite randomized trials demonstrating that a 6 mg daily dose of DEX improved patient outcomes in hospitalized COVID-19 patients receiving oxygen, clinicians often prescribe higher doses of corticosteroids without evidence to support this practice. The purpose of this study was to compare outcomes of ventilated COVID-19 patients who received standard dose (SD) versus high dose (HD) DEX. METHOD: This was a multi-site, retrospective, observational study on ventilated COVID-19-positive patients who received DEX for at least three days between June 1, 2020, and January 31, 2022. The primary outcome of this study was the association between mortality and SD (<6mg daily) versus HD (>10mg daily) DEX in ventilated COVID-19 patients. Secondary outcomes included average blood glucose (BG), number of BG readings above 200, incidence of bacterial nosocomial infection, ventilator-free days, length of stay (LOS), and ICU LOS. RESULTS: Of the 212 included patients, 53 (25%) received SD DEX, and 159 (75%) received HD DEX. There was no significant effect of DEX dose on mortality, number of BG readings >200, incidence of nosocomial infections, LOS, or ventilator-free days (p >0.05). After controlling for confounding factors, no difference in mortality persisted (OR 1.34 95% CI 0.62- 2.90). Average daily BG and ICU LOS were significantly greater in the HD group compared to the SD group (p = 0.003, p = 0.019, respectively). CONCLUSION: There was no association between HD DEX and mortality among ventilated COVID-19 patients compared to SD DEX. Moreover, HD DEX is associated with detrimental effects such as prolonged ICU LOS and higher average daily BG. This study supports the use of SD DEX in ventilated COVID-19 patients.

Cannabinoid modulation of brain activation during volitional regulation of negative affect in trauma-exposed adults.

Emotion dysregulation is considered a core component of posttraumatic stress disorder (PTSD). Cognitive reappraisal is one therapeutic emotion regulation strategy that has been widely studied among individuals with mood and anxiety disorders, and numerous differences in brain activation patterns have been shown between individuals with and without PTSD during tasks of cognitive reappraisal. Prior research among healthy subjects suggests that an acute, low dose of Δ9-tetrahydrocannabinol (THC) could attenuate the neurophysiological discrepancies that exist between individuals with and without PTSD during tasks of emotional processing; however, the effect of an acute, low dose of THC on corticolimbic activity during emotion regulation among individuals with PTSD has not yet been studied. The present study aimed to investigate the effect of THC on negative affect and brain activation in a priori regions of interest during cognitive reappraisal among trauma-exposed individuals with and without PTSD. Using a double-blind design, 51 individuals were randomized to receive THC or placebo (PBO) before participating in a well-established emotion regulation task during functional magnetic resonance imaging (fMRI). THC but not PBO reduced negative affect during reappraisal, and THC increased dorsomedial prefrontal cortex (dmPFC) activation in response to neutral images. Individuals with PTSD displayed less activation in the angular gyrus, overall, compared to the trauma-exposed control (TEC) group, however THC increased angular gyrus activation in the PTSD group so that there was no significant difference in angular gyrus activation between the TEC and PTSD groups that received THC. Compared to PBO, THC also increased cerebellar activation during exposure to neutral images in individuals with PTSD. Lastly, in participants that received THC, greater posterior cingulate cortex (PCC)/precuneus activation during reappraisal was associated with less self-reported negative affect following reappraisal blocks. Together these findings suggest that THC may prove to be a beneficial pharmacological adjunct to cognitive reappraisal therapy in the treatment of PTSD.

RTL8 promotes nuclear localization of UBQLN2 to subnuclear compartments associated with protein quality control.

The brain-expressed ubiquilins (UBQLNs) 1, 2 and 4 are a family of ubiquitin adaptor proteins that participate broadly in protein quality control (PQC) pathways, including the ubiquitin proteasome system (UPS). One family member, UBQLN2, has been implicated in numerous neurodegenerative diseases including ALS/FTD. UBQLN2 typically resides in the cytoplasm but in disease can translocate to the nucleus, as in Huntington's disease where it promotes the clearance of mutant Huntingtin. How UBQLN2 translocates to the nucleus and clears aberrant nuclear proteins, however, is not well understood. In a mass spectrometry screen to discover UBQLN2 interactors, we identified a family of small (13 kDa), highly homologous uncharacterized proteins, RTL8, and confirmed the interaction between UBQLN2 and RTL8 both in vitro using recombinant proteins and in vivo using mouse brain tissue. Under endogenous and overexpressed conditions, RTL8 localizes to nucleoli. When co-expressed with UBQLN2, RTL8 promotes nuclear translocation of UBQLN2. RTL8 also facilitates UBQLN2's nuclear translocation during heat shock. UBQLN2 and RTL8 colocalize within ubiquitin-enriched subnuclear structures containing PQC components. The robust effect of RTL8 on the nuclear translocation and subnuclear localization of UBQLN2 does not extend to the other brain-expressed ubiquilins, UBQLN1 and UBQLN4. Moreover, compared to UBQLN1 and UBQLN4, UBQLN2 preferentially stabilizes RTL8 levels in human cell lines and in mouse brain, supporting functional heterogeneity among UBQLNs. As a novel UBQLN2 interactor that recruits UBQLN2 to specific nuclear compartments, RTL8 may regulate UBQLN2 function in nuclear protein quality control.

Dorsal ruffles enhance activation of Akt by growth factors.

In fibroblasts, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) stimulate the formation of actin-rich, circular dorsal ruffles (CDRs) and phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of Akt. To test the hypothesis that CDRs increase synthesis of phosphorylated Akt1 (pAkt), we analyzed the contributions of CDRs to Akt phosphorylation in response to PDGF and EGF. CDRs appeared within several minutes of growth factor addition, coincident with a peak of pAkt. Microtubule depolymerization with nocodazole blocked CDR formation and inhibited phosphorylation of Akt in response to EGF but not PDGF. Quantitative immunofluorescence showed increased concentrations of Akt, pAkt and phosphatidylinositol (3,4,5)-trisphosphate (PIP3), the phosphoinositide product of PI3K that activates Akt, concentrated in CDRs and ruffles. EGF stimulated lower maximal levels of pAkt than did PDGF, which suggests that Akt phosphorylation requires amplification in CDRs only when PI3K activities are low. Accordingly, stimulation with low concentrations of PDGF elicited lower levels of Akt phosphorylation, which, like responses to EGF, were inhibited by nocodazole. These results indicate that when receptor signaling generates low levels of PI3K activity, CDRs facilitate local amplification of PI3K and phosphorylation of Akt.This article has an associated First Person interview with the first author of the paper.

Macropinocytosis, mTORC1 and cellular growth control.

The growth and proliferation of metazoan cells are driven by cellular nutrient status and by extracellular growth factors. Growth factor receptors on cell surfaces initiate biochemical signals that increase anabolic metabolism and macropinocytosis, an actin-dependent endocytic process in which relatively large volumes of extracellular solutes and nutrients are internalized and delivered efficiently into lysosomes. Macropinocytosis is prominent in many kinds of cancer cells, and supports the growth of cells transformed by oncogenic K-Ras. Growth factor receptor signaling and the overall metabolic status of the cell are coordinated in the cytoplasm by the mechanistic target-of-rapamycin complex-1 (mTORC1), which positively regulates protein synthesis and negatively regulates molecular salvage pathways such as autophagy. mTORC1 is activated by two distinct Ras-related small GTPases, Rag and Rheb, which associate with lysosomal membranes inside the cell. Rag recruits mTORC1 to the lysosomal surface where Rheb directly binds to and activates mTORC1. Rag is activated by both lysosomal luminal and cytosolic amino acids; Rheb activation requires phosphoinositide 3-kinase, Akt, and the tuberous sclerosis complex-1/2. Signals for activation of Rag and Rheb converge at the lysosomal membrane, and several lines of evidence support the idea that growth factor-dependent endocytosis facilitates amino acid transfer into the lysosome leading to the activation of Rag. This review summarizes evidence that growth factor-stimulated macropinocytosis is essential for amino acid-dependent activation of mTORC1, and that increased solute accumulation by macropinocytosis in transformed cells supports unchecked cell growth.

CXCL12-induced macropinocytosis modulates two distinct pathways to activate mTORC1 in macrophages.

Although growth factors and chemokines elicit different overall effects on cells-growth and chemotaxis, respectively-and activate distinct classes of cell-surface receptors, nonetheless, they trigger similar cellular activities and signaling pathways. The growth factor M-CSF and the chemokine CXCL12 both stimulate the endocytic process of macropinocytosis, and both activate the mechanistic target of rapamycin complex 1 (mTORC1), a protein complex that regulates cell metabolism. Recent studies of signaling by M-CSF in macrophages identified a role for macropinocytosis in the activation of mTORC1, in which delivery of extracellular amino acids into lysosomes via macropinocytosis was required for activation of mTORC1. Here, we analyzed the regulation of macropinosome (MP) formation in response to CXCL12 and identified 2 roles for macropinocytosis in the activation of mTORC1. Within 5 min of adding CXCL12, murine macrophages increased ruffling, macropinocytosis and amino acid-dependent activation of mTORC1. Inhibitors of macropinocytosis blocked activation of mTORC1, and various isoform-specific inhibitors of type 1 PI3K and protein kinase C (PKC) showed similar patterns of inhibition of macropinocytosis and mTORC1 activity. However, unlike the response to M-CSF, Akt phosphorylation (pAkt) in response to CXCL12 required the actin cytoskeleton and the formation of macropinocytic cups. Quantitative fluorescence microscopy showed that phosphatidylinositol (3,4,5)-trisphosphate (PIP3), a product of PI3K and an upstream activator of Akt, localized to macropinocytic cups and that pAkt occurred primarily in cups. These results indicate that CXCL12 activates mTORC1 via 2 mechanisms: 1) that the macropinocytic cup localizes Akt signaling and 2) that MPs convey extracellular nutrients to lysosomes.

Growth factor signaling to mTORC1 by amino acid-laden macropinosomes.

The rapid activation of the mechanistic target of rapamycin complex-1 (mTORC1) by growth factors is increased by extracellular amino acids through yet-undefined mechanisms of amino acid transfer into endolysosomes. Because the endocytic process of macropinocytosis concentrates extracellular solutes into endolysosomes and is increased in cells stimulated by growth factors or tumor-promoting phorbol esters, we analyzed its role in amino acid-dependent activation of mTORC1. Here, we show that growth factor-dependent activation of mTORC1 by amino acids, but not glucose, requires macropinocytosis. In murine bone marrow-derived macrophages and murine embryonic fibroblasts stimulated with their cognate growth factors or with phorbol myristate acetate, activation of mTORC1 required an Akt-independent vesicular pathway of amino acid delivery into endolysosomes, mediated by the actin cytoskeleton. Macropinocytosis delivered small, fluorescent fluid-phase solutes into endolysosomes sufficiently fast to explain growth factor-mediated signaling by amino acids. Therefore, the amino acid-laden macropinosome is an essential and discrete unit of growth factor receptor signaling to mTORC1.

Differential signaling during macropinocytosis in response to M-CSF and PMA in macrophages.

The cellular movements that construct a macropinosome have a corresponding sequence of chemical transitions in the cup-shaped region of plasma membrane that becomes the macropinosome. To determine the relative positions of type I phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) in this pathway, we analyzed macropinocytosis in macrophages stimulated by the growth factor macrophage-colony-stimulating factor (M-CSF) and by the diacylglycerol (DAG) analog phorbol 12-myristate 13-acetate (PMA). In cells stimulated with M-CSF, microscopic imaging of fluorescent probes for intracellular lipids indicated that the PI3K product phosphatidylinositol (3,4,5)-trisphosphate (PIP3) appeared in cups just prior to DAG. We then tested the hypothesis that PMA and DAG function after PI3K and prior to Ras and protein kinase C (PKC) during macropinosome formation in macrophages. Although the PI3K target Akt was activated by M-CSF, the Akt inhibitor MK-2206 did not inhibit macropinocytosis. The phospholipase C (PLC) inhibitor U73122 blocked macropinocytosis by M-CSF but not PMA. Macropinocytosis in response to M-CSF and PMA was inhibited by the Ras inhibitor farnesyl thiosalicylate (FTS), by the PKC inhibitor Calphostin C and by the broad specificity inhibitor rottlerin. These studies support a model in which M-CSF stimulates PI3K in macropinocytic cups, and the resulting increase in PIP3 activates PLC, which in turn generates DAG necessary for activation of PKC, Ras and the late stages of macropinosome closure.