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The many layers of the neuroretina contain a complex, interconnected network of specialized neurons that both process visual stimuli and conduct processed information to higher brain areas. Neural networks rely on proteostatic control mechanisms to maintain proper protein homeostasis both in cell bodies as well as within synapses; protein chaperones play an important role in regulating and supporting this process. ProSAAS is a small neuronal chaperone that functions as an anti-aggregant in in vitro assays and is released upon depolarization in neuronal primary cultures. We here report a potential role for proSAAS in the retina. A review of human and mouse retinal RNAseq studies reveals that proSAAS expression is abundant within the retina. Single cell sequencing data from mouse and human studies show that proSAAS levels are highest in retinal ganglion cells (RGCs) and horizontal cells. Using proSAAS antibodies in combination with antisera to known retinal cell markers in mouse retinal sections, we confirm RNAseq data showing that proSAAS expression is highest in RGCs and horizontal cells. The proSAAS signal is concentrated within the ganglion cell layer and the inner plexiform layer, a dense synaptic layer connecting retinal neurons. Western blotting of mouse retinal extracts indicates the presence of two processed proSAAS forms, a 21 kDa C-terminally processed form, and a small 13 kDa species which, based on antibody specificity, likely represents an internal fragment. This fragment is also found in extracts prepared from human retinas. Taken together, our data provide support for the hypothesis that retinal synapses utilize the proSAAS chaperone to support visual signaling.

Age-related macular degeneration (AMD) is a multifactorial neurodegenerative disorder. Although molecular mechanisms remain elusive, deficits in autophagy have been associated with AMD. Here we show that deficiency of calcium and integrin binding protein 2 (CIB2) in mice, leads to age-related pathologies, including sub-retinal pigment epithelium (RPE) deposits, marked accumulation of drusen markers APOE, C3, Aβ, and esterified cholesterol, and impaired visual function, which can be rescued using exogenous retinoids. Cib2 mutant mice exhibit reduced lysosomal capacity and autophagic clearance, and increased mTORC1 signaling—a negative regulator of autophagy. We observe concordant molecular deficits in dry-AMD RPE/choroid post-mortem human tissues. Mechanistically, CIB2 negatively regulates mTORC1 by preferentially binding to ‘nucleotide empty’ or inactive GDP-loaded Rheb. Upregulated mTORC1 signaling has been implicated in lymphangioleiomyomatosis (LAM) cancer. Over-expressing CIB2 in LAM patient-derived fibroblasts downregulates hyperactive mTORC1 signaling. Thus, our findings have significant implications for treatment of AMD and other mTORC1 hyperactivity-associated disorders. Age-related macular degeneration (AMD) has been connected to deficits in autophagy. Here, the authors demonstrate, in mice and dry-AMD patient samples, that calcium and integrin binding protein 2 (CIB2) regulates Rheb-mTORC1 signaling axis, and subsequently autophagy.

The visual cycle is an important pathway in the retinal pigment epithelium (RPE) which regenerates 11- cis retinal chromophore for the retinal photoreceptors. The central enzyme in the visual cycle is RPE65 retinol isomerase. Expression of RPE65 mRNA and protein levels are significantly lower in RPE cell culture models when compared to native RPE. This limits the use of these models to study the visual cycle. To determine the main drivers of RPE65 regulation we compared the transcriptional profiles of native and cell culture models of RPE with various levels of RPE65 expression. We also compared the levels of RPE65 expression between ARPE-19 cells grown in media supplemented with 1 mM pyruvate (PYR) or 10 mM nicotinamide (NAM). In addition, we performed experiments directed at transcriptional and translational regulation of RPE65. We show that RPE65 mRNA and protein expression is significantly higher in NAM media grown cells than PYR cells. Transfection of cells with a variety of different vectors containing RPE65 ORFs with different promoters, codon optimization, IRES, 3’ UTRs, suggest that translational effects are less important than transcriptional status. Importantly, we found that feeding with rod outer segments (ROS) decreases RPE65 expression in NAM grown cells, suggesting that certain primary functions of the RPE (here, visual cycle and phagocytosis) are not positively linked. Analysis of differentially regulated microRNAs (miRs) provides a basis for this downregulation. It appears that the regulation of RPE65 expression in ARPE-19 cells, in particular, is multifactorial, involving primarily metabolic and transcriptional status of the cells, with translation of RPE65 mRNA playing a smaller role.

Substance use remains a leading cause of preventable death globally. A model of intervention known as screening, brief intervention, and referral to treatment (SBIRT) was developed decades ago to facilitate time- and resource-sensitive interventions in acute care and outpatient settings. SBIRT, which includes a psychosocial intervention incorporating the principles of motivational interviewing, has been shown to be effective in reducing alcohol consumption and consequences in unhealthy drinkers both in primary care and emergency department settings. Subsequently, SBIRT for unhealthy alcohol use has been endorsed by governmental agencies and professional societies in multiple countries. Although most trials support the efficacy of SBIRT for unhealthy alcohol use (McQueen et al. in Cochrane Database Syst Rev 8, 2011 ; Kaner et al. in Cochrane Database Syst Rev 2, 2007 ; O’Donnell et al. in Alcohol Alcohol 49(1):66–78, 2014 ), results are heterogenous; negative studies exist. A newer approach to screening and intervention for substance use can incorporate initiation of medication management at the index visit, for individuals willing to do so, and for providers and healthcare systems that are appropriately trained and resourced. Our group has conducted two successful trials of an approach we call screening, treatment initiation, and referral (STIR). In one trial, initiation of nicotine pharmacotherapy coupled with screening and brief counseling in adult smokers resulted in sustained biochemically confirmed abstinence. In a second trial, initiation of buprenorphine for opioid dependent individuals resulted in greater engagement in treatment at 30 days and greater self-reported abstinence. STIR may offer a new, clinically effective approach to the treatment of substance use in clinical care settings.

The rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION) is similar in many of its pathophysiological responses to clinical NAION. Like human NAION, there is significant variability in the severity of the lesion produced, and little is known of the parameters associated with rNAION induction severity or if pre- or early post-induction biomarkers can be identified that enable prediction of lesion severity and ultimate loss of retinal ganglion cells (RGCs). Adult male Sprague-Dawley outbred rats were evaluated for various parameters including physiological characteristics (heart rate, respiratory rate, temperature, hematocrit [Hct]), optic nerve head (ONH) appearance, pre- and post-induction mean diameter, and intravenous fluorescein and indocyanine green angiographic patterns of vascular leakage at 5 hours post-induction, performed using a spectral domain-optical coherence tomography (SD-OCT) instrument. Early changes were correlated with ultimate RGC loss by Brn3a (+) immunohistology. RGC loss also was correlated with the relative level of laser exposure. The severity of ONH edema 2d, but not 5hr, post induction was most closely associated with the degree of RGC loss, revealing a threshold effect, and consistent with a compartment syndrome where a minimum level of capillary compression within a tight space is responsible for damage. RGC loss increased dramatically as the degree of laser exposure increased. Neither physiological parameters nor the degree of capillary leakage 5hr post induction were informative as to the ultimate degree of RGC loss. Similar to human NAION, the rNAION model exhibits marked variability in lesion severity. Unlike clinical NAION, pre-induction ONH diameter likely does not contribute to ultimate lesion severity; however, cross-sectional ONH edema can be used as a biomarker 2d post-induction to determine randomization of subjects prior to inclusion in specific neuroprotection or neuroregeneration studies.

Background Ciliary neurotrophic factor (CNTF) has been shown to protect retinal ganglion cells (RGCs) in traumatic optic nerve injury. We sought to evaluate this neuroprotective effect of CNTF after an ischaemic event using rodent anterior ischaemic optic neuropathy (rAION), a mouse model of non-arteritic anterior ischaemic optic neuropathy (NAION). Methods We induced rAION in Thy1-cyan fluorescent protein (CFP) transgenic mice by exposing the optic nerve to frequency doubled neodymium yttrium aluminium garnet laser pulses following intravenous rose bengal injection. One day after rAION induction, an intravitreal injection of 0.75 μg CNTF or vehicle (sham injection) was given. Animals were euthanised on day 15 after induction, tissues isolated and CFP cells in the RGC layer were counted using stereology in flat-mounted retina. The average number of CFP-positive (CFP+) cells was determined for each study group and the percentages of RGC loss were compared between the different groups. Results Two weeks after rAION induction, significantly more (CFP+) cells were preserved in CNTF-treated eyes than in sham-injected controls. Sham-treated animals showed a 58% loss of CFP+ cells. In contrast, CFP+ cell density in CNTF-treated eyes decreased by only 10%, when compared with untreated control eyes. This increased survival was statistically significant (p<0.05). Conclusions CNTF exerts a neuroprotective effect in ischaemic optic nerve injury and promotes RGC survival, suggesting that CNTF may be effective in the clinical treatment of human NAION.

Monitor alarms are prevalent in the ED. Continuous electronic monitoring of patients' vital signs may alert staff to physiologic decompensation. However, repeated false alarms may lead to desensitization of staff to alarms. Mitigating this could involve prioritizing the most clinically-important alarms. There are, however, little data on which ED monitor alarms are clinical meaningful. We evaluated whether and which ED monitor alarms led to observable changes in patients' ED care. This prospective, observational study was conducted in an urban, academic ED. An ED physician completed 53 h of observation, recording patient characteristics, alarm type, staff response, whether the alarm was likely real or false, and whether it changed clinical management. The primary outcome was whether the alarm led to an observable change in patient management. Secondary outcomes included the type of alarms and staff responses to alarms. There were 1049 alarms associated with 146 patients, for a median of 18 alarms per hour of observation. The median number of alarms per patient was 4 (interquartile range 2–8). Alarms changed clinical management in 8 out of 1049 observed alarms (0.8%, 95% CI, 0.3%, 1.3%) in 5 out of the 146 patients (3%, 95% CI, 0.2%, 5.8%). Staff did not observably respond to most alarms (63%). Most ED monitor alarms did not observably affect patient care. Efforts at improving the clinical significance of alarms could focus on widening alarm thresholds, customizing alarms parameters for patients' clinical status, and on utilizing monitoring more selectively.

It is vital to determine how patient characteristics that precede COVID-19 illness relate to COVID-19 mortality. This is a retrospective cohort study of patients hospitalized with COVID-19 across 21 healthcare systems in the US. All patients (N = 145,944) had COVID-19 diagnoses and/or positive PCR tests and completed their hospital stays from February 1, 2020 through January 31, 2022. Machine learning analyses revealed that age, hypertension, insurance status, and healthcare system (hospital site) were especially predictive of mortality across the full sample. However, multiple variables were especially predictive in subgroups of patients. The nested effects of risk factors such as age, hypertension, vaccination, site, and race accounted for large differences in mortality likelihood with rates ranging from about 2–30%. Subgroups of patients are at heightened risk of COVID-19 mortality due to combinations of preadmission risk factors; a finding of potential relevance to outreach and preventive actions.

Polyamidoamine dendrimer nanoparticles (~ 4 nanometers) are inert polymers that can be linked to biologically active compounds. These dendrimers selectively target and accumulate in inflammatory cells upon systemic administration. Dendrimer-linked compounds enable sustained release of therapeutic compounds directly at the site of damage. The purpose of this study was to determine if dendrimers can be used to target the optic nerve (ON) ischemic lesion in our rodent and nonhuman primate models of nonarteritic anterior ischemic optic neuropathy (NAION), a disease affecting >10,000 individuals in the US annually, and for which there currently is no effective treatment. NAION was induced in male Long-Evans rats (rNAION) and in one adult male rhesus monkey (pNAION) using previously described procedures. Dendrimers were covalently linked to near-infrared cyanine-5 fluorescent dye (D-Cy5) and injected both intravitreally and systemically (in the rats) or just systemically (in the monkey) to evaluate D-Cy5 tissue accumulation in the eye and optic nerve following induction of NAION. Following NAION induction, Cy-5 dendrimers selectively accumulated in astrocytes and circulating macrophages. Systemic dendrimer administration provided the best penetration of the ON lesion site when injected shortly after induction. Systemic administration 1 day post-induction in the pNAION model gave localization similar to that seen in the rats. Dendrimers selectively target the ischemic ON lesion after induction of both rNAION and pNAION. Systemic nanoparticle-linked therapeutics thus may provide a powerful, targeted and safe approach to NAION treatment by providing sustained and focused treatment of the cells directly affected by ischemia.

The COVID-19 pandemic was managed in part by the rapid development of vaccines, diagnostics, and therapeutics including antiviral agents and advances in emergency airway and ventilatory management. The impact of these therapeutic advances on clinically pertinent metrics of emergency care have not been well-studied. We abstracted data from emergency department (ED) visits made to 21 US health systems during the first two years of the pandemic, from February 1, 2020 to January 31, 2022. These health systems were participants in the NIH-supported COVID EHR Cohort, in which the University of Wisconsin served as the coordinating site. Limited patient-level data files were submitted monthly. Data elements included demographic and clinical variables, as well as standard measures of ED outcomes including 72-h returns, 72-h returns leading to readmission, and in-hospital mortality. Multivariable models were fitted to identify correlates of each of the dependent variables. A test for trend was used to detect changes in outcomes over time. During the two-year period, 150,357 individuals aged 18 years or older visited the ED. The median age was 45.4 years (IQR 27), 58.1 % were female, 49 % were White, 18.3 % Hispanic/Latino, and 45 % were publicly insured or uninsured. The prevalence of 72-h ED returns, readmissions, and in-hospital mortality significantly declined across the two-year period. SARS-CoV-2 vaccination was associated with reduced ED returns and mortality. Therapeutic agents were associated with increased mortality risk but were likely confounded by unmeasured covariates. Operational and clinical outcomes of ED-based treatment of individuals with COVID-19 improved in the first two years of the pandemic. This improvement is likely multifactorial and includes the development and deployment of SARS-CoV-2-specific vaccines, therapeutic agents, and improved healthcare delivery in the ED and elsewhere addressing management of airway and ventilatory status, as well as increased innate immunity in the general population.