Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs.

The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Short-chain fatty acids propionate and butyrate control growth and differentiation linked to cellular metabolism.

The short-chain fatty acids (SCFA) propionate and butyrate are produced in large amounts by microbial metabolism and have been identified as unique acyl lysine histone marks. In order to better understand the function of these modifications we used ChIP-seq to map the genome-wide location of four short-chain acyl histone marks H3K18pr/bu and H4K12pr/bu in treated and untreated colorectal cancer (CRC) and normal cells, as well as in mouse intestines in vivo. We correlate these marks with open chromatin regions along with gene expression to access the function of the target regions. Our data demonstrate that propionate and butyrate act as promoters of growth, differentiation as well as ion transport. We propose a mechanism involving direct modification of specific genomic regions, resulting in increased chromatin accessibility, and in case of butyrate, opposing effects on the proliferation of normal versus CRC cells.

Short-chain fatty acids propionate and butyrate control growth and differentiation linked to cellular metabolism.

The short-chain fatty acids (SCFA) propionate and butyrate are produced in large amounts by microbial metabolism and have been identified as unique acyl lysine histone marks. In order to better understand the function of these modifications we used ChIP-seq to map the genome-wide location of four short-chain acyl histone marks H3K18pr/bu and H4K12pr/bu in treated and untreated colorectal cancer (CRC) and normal cells, as well as in mouse intestines in vivo . We correlate these marks with open chromatin regions along with gene expression to access the function of the target regions. Our data demonstrate that propionate and butyrate act as promoters of growth, differentiation as well as ion transport. We propose a mechanism involving direct modification of specific genomic regions, resulting in increased chromatin accessibility, and in case of butyrate, opposing effects on the proliferation of normal versus CRC cells.

A Review of Federal and Statewide Guidelines and Their Effects on Orthopedics.

In the past three decades, the use of opioids has risen tremendously. Pain was named the "fifth patient vital sign" in the 1990s, and from that point, opioid usage has continued to grow throughout the 2010s leading to its recognition as a crisis. The United States is responsible for 80% of the global opioid usage while only accounting for less than 5% of the global population. Previously opioids were mostly used to treat acute pain, however, opioids have been most recently used to manage chronic pain as well. The opioid crisis has presented new challenges in treating pain while preventing the abuse of these medications in a system that lacks standardization of treatment guidelines across the United States. Therefore, the authors of this review examine the current national recommendations to help manage the ongoing opioid crisis and explore how they may impact orthopedic patient care.

Serine starvation silences estrogen receptor signaling through histone hypoacetylation.

Loss of estrogen receptor (ER) pathway activity promotes breast cancer progression, yet how this occurs remains poorly understood. Here, we show that serine starvation, a metabolic stress often found in breast cancer, represses estrogen receptor alpha (ERα) signaling by reprogramming glucose metabolism and epigenetics. Using isotope tracing and time-resolved metabolomic analyses, we demonstrate that serine is required to maintain glucose flux through glycolysis and the TCA cycle to support acetyl-CoA generation for histone acetylation. Consequently, limiting serine depletes histone H3 lysine 27 acetylation (H3K27ac), particularly at the promoter region of ER pathway genes including the gene encoding ERα, ESR1. Mechanistically, serine starvation impairs acetyl-CoA-dependent gene expression by inhibiting the entry of glycolytic carbon into the TCA cycle and down-regulating the mitochondrial citrate exporter SLC25A1, a critical enzyme in the production of nucleocytosolic acetyl-CoA from glucose. Consistent with this model, total H3K27ac and ERα expression are suppressed by SLC25A1 inhibition and restored by acetate, an alternate source of acetyl-CoA, in serine-free conditions. We thus uncover an unexpected role for serine in sustaining ER signaling through the regulation of acetyl-CoA metabolism.

Can a private sector engagement intervention that prioritizes pro-poor strategies improve healthcare access and quality? A randomized field experiment in Kenya.

Private sector engagement in health reform has been suggested to help reduce healthcare inequities in sub-Saharan Africa, where populations with the most need seek the least care. We study the effects of African Health Markets for Equity (AHME), a cluster randomized controlled trial carried out in Kenya from 2012 to 2020 at 199 private health clinics. AHME included four clinic-level interventions: social health insurance, social franchising, SafeCare quality-of-care certification programme and business support. This paper evaluates whether AHME increased the capacity of private health clinics to serve poor clients while maintaining or enhancing the quality of care provided. At endline, clinics that received AHME were 14.5 percentage points (pp) more likely to be empanelled with the National Health Insurance Fund (NHIF), served 51% more NHIF clients and served more clients from the middle three quintiles of the wealth distribution compared to control clinics. Comparing individuals living in households near AHME treatment and control clinics (N = 8241), AHME led to a 6.7-pp increase in the probability of holding any health insurance on average. We did not find any additional effect of AHME on insurance holding among poor households. We measured quality of care using a standardized patient (SP) experiment (N = 596 SP-provider interactions) where recruited and trained SPs were randomized to present as either 'not poor', and able to afford all services provided, or 'poor' by telling the provider they could only afford ∼300 Kenyan Shillings (US$3) in fees. We found that poor SPs received lower levels of both correct and unnecessary services, and AHME did not affect this. More work must be done to ensure that clients of all wealth levels receive high-quality care.

A review of chronic pectoralis major tears: what options are available?

Rupture of the pectoralis major muscle typically occurs in the young, active male. Acute management of these injuries is recommended; however, what if the patient presents with a chronic tear of the pectoralis major? Physical exams and magnetic resonance imaging can help identify the injury and guide the physician with a plan for management. Nonoperative management is feasible, but is recommended for elderly, low-demand patients whose functional goals are minimal. Repair of chronic tears should be reserved for younger, healthier patients with high functional demands. Although operative management provides better functional outcomes, operative treatment of chronic pectoralis tears can be challenging. Tendon retraction, poor tendinous substance and quality of tissue, muscle atrophy, scar formation, and altered anatomy make direct repairs complicated, often necessitating auto- or allograft use. We review the various graft options and fixation methods that can be used when treating patients with chronic pectoralis major tears.

Acute Traumatic Spondyloptosis: A Case Report.

Acute traumatic spondyloptosis (ATS) is a rare condition in the orthopedic literature, with few cases reported. We present a case of ATS in a 35-year-old Hispanic male with multilevel injury, without neurological deficits at the time of injury. The patient was treated in a two-stage method consisting of combined anterior and posterior spinal decompression and fusion. At the six-month follow-up, the patient had no motor/sensory deficits, he remained stable during the one-year period. Conclusion: ATS is rarely seen in patients without neurological deficits on presentation. Although surgical intervention presents significant risks of iatrogenic neurologic compromise, surgical fixation is warranted.

Acetyl-Click Screening Platform Identifies Small-Molecule Inhibitors of Histone Acetyltransferase 1 (HAT1).

HAT1 is a central regulator of chromatin synthesis that acetylates nascent histone H4. To ascertain whether targeting HAT1 is a viable anticancer treatment strategy, we sought to identify small-molecule inhibitors of HAT1 by developing a high-throughput HAT1 acetyl-click assay. Screening of small-molecule libraries led to the discovery of multiple riboflavin analogs that inhibited HAT1 enzymatic activity. Compounds were refined by synthesis and testing of over 70 analogs, which yielded structure-activity relationships. The isoalloxazine core was required for enzymatic inhibition, whereas modifications of the ribityl side chain improved enzymatic potency and cellular growth suppression. One compound (JG-2016 [24a]) showed relative specificity toward HAT1 compared to other acetyltransferases, suppressed the growth of human cancer cell lines, impaired enzymatic activity in cellulo, and interfered with tumor growth. This is the first report of a small-molecule inhibitor of the HAT1 enzyme complex and represents a step toward targeting this pathway for cancer therapy.

Patients with body mass index ≥25 kg/m2 as a target population for improvement of rate of follow-up duplex venous ultrasound examinations following initial incomplete examinations.

OBJECTIVE: Obesity is highly prevalent and a major risk factor for deep vein thrombosis (DVT) and chronic venous disease. It can also technically limit duplex ultrasound evaluations for lower extremity DVT. We compared the rates and results of repeat lower extremity venous duplex ultrasound (LEVDUS) after an initial incomplete and negative (IIN) LEVDUS in overweight (body mass index [BMI] ≤25-30 kg/m2) and obese (BMI ≥30 kg/m2) patients with those of patients with a BMI <25 kg/m2 to evaluate whether increasing the rate of follow-up examinations in overweight and obese patients might facilitate improved patient care. METHODS: We performed a retrospective review of 617 patients with an IIN LEVDUS study from December 31, 2017 to December 31, 2020. Demographic and imaging data of the patients with an IIN LEVDUS and the frequency of repeat studies performed within 2 weeks were abstracted from the electronic medical records. The patients were divided into three BMI-based groups: normal (BMI <25 kg/m2), overweight (BMI 25-30 kg/m2), and obese (BMI ≥30 kg/m2). RESULTS: Of the 617 patients with an IIN LEVDUS, 213 (34.5%) were normal weight, 177 (29%) were overweight, and 227 (37%) were obese. The repeat LEVDUS rates were significantly different across the three weight groups (P < .001). After an IIN LEVDUS, the rate of repeat LEVDUS for the normal weight, overweight, and obese groups was 46% (98 of 213), 28% (50 of 227), and 32% (73 of 227), respectively. The overall rates of thrombosis (both DVT and superficial vein thrombosis) in the repeat LEVDUS examinations were not significantly different among the normal weight (14%), overweight (11%), and obese (18%) patients (P = .431). CONCLUSIONS: Overweight and obese patients (BMI ≥25 kg/m2) received fewer follow-up examinations after an IIN LEVDUS. Follow-up LEVDUS examinations of overweight and obese patients after an IIN LEVDUS study have similar rates of venous thrombosis compared with normal weight patients. Targeting improving usage of follow-up LEVDUS studies for all patients, but especially for those who are overweight and obese, with an IIN LEVDUS through quality improvement efforts could help minimize missed diagnoses of venous thrombosis and improve the quality of patient care.

Prevalence of renal and bone risk factors among individuals prescribed oral pre-exposure prophylaxis for HIV.

Objectives: The only available oral pre-exposure prophylaxis (PrEP) regimens approved in the United States to prevent HIV infection during the period covered by this study were emtricitabine/tenofovir alafenamide (F/TAF) and emtricitabine/tenofovir disoproxil fumarate (F/TDF). Both agents have similar efficacy, however F/TAF exhibits improved bone and renal health safety endpoints over F/TDF. In 2021, the United States Preventive Services Task Force recommended individuals have access to the most medically appropriate PrEP regimen. To understand the impact of these guidelines, the prevalence of risk factors to renal and bone health was evaluated among individuals prescribed oral PrEP. Methods: This prevalence study utilized the electronic health records of people prescribed oral PrEP between January 1, 2015 and February 29, 2020. Renal and bone risk factors (age, comorbidities, medication, renal function, and body mass index) were identified using International Classification of Diseases (ICD) and National Drug Code (NDC) codes. Results: Among 40 621 individuals prescribed oral PrEP, 62% had ≥1 renal risk factor and 68% had ≥1 bone risk factor. Comorbidities were the most frequent (37%) class of renal risk factors. Concomitant medications were the most prominent (46%) class of bone-related risk factors. Conclusions: The high prevalence of risk factors suggests the importance of their consideration when choosing the most appropriate regimen for individuals who may benefit from PrEP.

Mitochondrial Uncoupling Induces Epigenome Remodeling and Promotes Differentiation in Neuroblastoma.

The Warburg effect is the major metabolic hallmark of cancer. According to Warburg himself, the consequence of the Warburg effect is cell dedifferentiation. Therefore, reversing the Warburg effect might be an approach to restore cell differentiation in cancer. In this study, we used a mitochondrial uncoupler, niclosamide ethanolamine (NEN), to activate mitochondrial respiration, which induced neural differentiation in neuroblastoma cells. NEN treatment increased the NAD+/NADH and pyruvate/lactate ratios and also the α-ketoglutarate/2-hydroxyglutarate (2-HG) ratio. Consequently, NEN treatment induced promoter CpG island demethylation and epigenetic landscape remodeling, activating the neural differentiation program. In addition, NEN treatment upregulated p53 but downregulated N-Myc and ß-catenin signaling in neuroblastoma cells. Importantly, even under hypoxia, NEN treatment remained effective in inhibiting 2-HG generation, promoting DNA demethylation, and suppressing hypoxia-inducible factor signaling. Dietary NEN intervention reduced tumor growth rate, 2-HG levels, and expression of N-Myc and ß-catenin in tumors in an orthotopic neuroblastoma mouse model. Integrative analysis indicated that NEN treatment upregulated favorable prognosis genes and downregulated unfavorable prognosis genes, which were defined using multiple neuroblastoma patient datasets. Altogether, these results suggest that mitochondrial uncoupling is an effective metabolic and epigenetic therapy for reversing the Warburg effect and inducing differentiation in neuroblastoma. SIGNIFICANCE: Targeting cancer metabolism using the mitochondrial uncoupler niclosamide ethanolamine leads to methylome reprogramming and differentiation in neuroblastoma, providing a therapeutic opportunity to reverse the Warburg effect and suppress tumor growth. See related commentary by Byrne and Bell, p.167.

A phase II study of talazoparib monotherapy in patients with wild-type BRCA1 and BRCA2 with a mutation in other homologous recombination genes.

Talazoparib, a PARP inhibitor, is active in germline BRCA1 and BRCA2 (gBRCA1/2)-mutant advanced breast cancer, but its activity beyond gBRCA1/2 is poorly understood. We conducted Talazoparib Beyond BRCA ( NCT02401347 ), an open-label phase II trial, to evaluate talazoparib in patients with pretreated advanced HER2-negative breast cancer (n = 13) or other solid tumors (n = 7) with mutations in homologous recombination (HR) pathway genes other than BRCA1 and BRCA2. In patients with breast cancer, four patients had a Response Evaluation Criteria in Solid Tumors (RECIST) partial response (overall response rate, 31%), and three additional patients had stable disease of ≥6 months (clinical benefit rate, 54%). All patients with germline mutations in PALB2 (gPALB2; encoding partner and localizer of BRCA2) had treatment-associated tumor regression. Tumor or plasma circulating tumor DNA (ctDNA) HR deficiency (HRD) scores were correlated with treatment outcomes and were increased in all gPALB2 tumors. In addition, a gPALB2-associated mutational signature was associated with tumor response. Thus, talazoparib has been demonstrated to have efficacy in patients with advanced breast cancer who have gPALB2 mutations, showing activity in the context of HR pathway gene mutations beyond gBRCA1/2.

Hydroxychloroquine/chloroquine for the treatment of hospitalized patients with COVID-19: An individual participant data meta-analysis.

BACKGROUND: Results from observational studies and randomized clinical trials (RCTs) have led to the consensus that hydroxychloroquine (HCQ) and chloroquine (CQ) are not effective for COVID-19 prevention or treatment. Pooling individual participant data, including unanalyzed data from trials terminated early, enables more detailed investigation of the efficacy and safety of HCQ/CQ among subgroups of hospitalized patients. METHODS: We searched ClinicalTrials.gov in May and June 2020 for US-based RCTs evaluating HCQ/CQ in hospitalized COVID-19 patients in which the outcomes defined in this study were recorded or could be extrapolated. The primary outcome was a 7-point ordinal scale measured between day 28 and 35 post enrollment; comparisons used proportional odds ratios. Harmonized de-identified data were collected via a common template spreadsheet sent to each principal investigator. The data were analyzed by fitting a prespecified Bayesian ordinal regression model and standardizing the resulting predictions. RESULTS: Eight of 19 trials met eligibility criteria and agreed to participate. Patient-level data were available from 770 participants (412 HCQ/CQ vs 358 control). Baseline characteristics were similar between groups. We did not find evidence of a difference in COVID-19 ordinal scores between days 28 and 35 post-enrollment in the pooled patient population (odds ratio, 0.97; 95% credible interval, 0.76-1.24; higher favors HCQ/CQ), and found no convincing evidence of meaningful treatment effect heterogeneity among prespecified subgroups. Adverse event and serious adverse event rates were numerically higher with HCQ/CQ vs control (0.39 vs 0.29 and 0.13 vs 0.09 per patient, respectively). CONCLUSIONS: The findings of this individual participant data meta-analysis reinforce those of individual RCTs that HCQ/CQ is not efficacious for treatment of COVID-19 in hospitalized patients.

A Case-Control Study Evaluating Risk Factors and Outcomes of Hospitalized Children With ESBL-UTI.

Urinary tract infections (UTIs) are among the most common causes of hospitalization in children, with a rising prevalence of extended-spectrum beta-lactamase-producing organisms (ESBL). The purpose of this study was to identify risk factors and treatment outcomes of children with ESBL-UTI. A retrospective case-control study of hospitalized children was performed from July 2014 till December 2017. Medical records from patients with a positive urine culture were reviewed and included in the study if they met criteria for UTI. Cases were defined as ESBL-UTI, while controls were defined as non-ESBL-UTI patients. This study confirmed that there are certain risk factors, such as previous UTI, recent antibiotic use, urinary tract abnormalities, recent hospital admission, and nonrenal comorbidities, that are associated with ESBL-UTI. Most of the patients with ESBL-UTI responded to discordant antibiotics. Other significant outcomes in patients with ESBL-UTI included a longer length of stay and longer intravenous antibiotic therapy.

Using gamification to enhance clinical trial start-up activities.

Background: The Trial Innovation Network (TIN) is a collaborative initiative within the National Center for Advancing Translational Science (NCATS) Clinical and Translational Science Awards (CTSA) Program. To improve and innovate the conduct of clinical trials, it is exploring the uses of gamification to better engage the trial workforce and improve the efficiencies of trial activities. The gamification structures described in this article are part of a TIN website gamification toolkit, available online to the clinical trial scientific community. Methods: The game designers used existing electronic trial platforms to gamify the tasks required to meet trial start-up timelines to create friendly competitions. Key indicators and familiar metrics were mapped to scoreboards. Webinars were organized to share and applaud trial and game performance. Results: Game scores were significantly associated with an increase in achieving start-up milestones in activation, institutional review board (IRB) submission, and IRB approval times, indicating the probability of completing site activation faster by using games. Overall game enjoyment and feelings that the game did not apply too much pressure appeared to be an important moderator of performance in one trial but had little effect on performance in a second. Conclusion: This retrospective examination of available data from gaming experiences may be a first-of-kind use in clinical trials. There are signals that gaming may accelerate performance and increase enjoyment during the start-up phase of a trial. Isolating the effect of gamification on trial outcomes will depend on a larger sampling from future trials, using well-defined, hypothesis-driven statistical analysis plans.

Rationally targeted anti-VISTA antibody that blockades the C-C' loop region can reverse VISTA immune suppression and remodel the immune microenvironment to potently inhibit tumor growth in an Fc independent manner.

BACKGROUND: Despite significant progress in cancer immunotherapy in recent years, resistance to existing immune checkpoint therapies (ICT) is common. V-domain Ig suppressor of T cell activation (VISTA), a predominantly myeloid immune checkpoint regulator, represents a promising therapeutic target due to its role in suppressing proinflammatory antitumor responses in myeloid-enriched tumor microenvironments. However, uncertainty around the cognate VISTA ligand has made the development of effective anti-VISTA antibodies challenging. The expression of VISTA on normal immune cell subtypes argues for a neutralizing non-depleting antibody, however, previous reported anti-VISTA antibodies use IgG1 Fc isotypes that deplete VISTA+ cells by antibody dependent cellular cytotoxicity/complement dependent cytotoxicity and these antibodies have shown fast serum clearance and immune toxicities. METHOD: Here we used a rational antibody discovery approach to develop the first Fc-independent anti-VISTA antibody, HMBD-002, that binds a computationally predicted functional epitope within the C-C-loop, distinct from other known anti-VISTA antibodies. This epitope is species-conserved allowing robust in vitro and in vivo testing of HMBD-002 in human and murine models of immune activation and cancer including humanized mouse models. RESULTS: We demonstrate here that blockade by HMBD-002 inhibits VISTA binding to potential partners, including V-Set and Immunoglobulin domain containing 3, to reduce myeloid-derived suppression of T cell activity and prevent neutrophil migration. Analysis of immune cell milieu suggests that HMBD-002 treatment stimulates a proinflammatory phenotype characterized by a Th1/Th17 response, recapitulating a phenotype previously noted in VISTA knockout models. This mechanism of action is further supported by immune-competent syngenic and humanized mouse models of colorectal, breast and lung cancer where neutralizing VISTA, without depleting VISTA expressing cells, significantly inhibited tumor growth while decreasing infiltration of suppressive myeloid cells and increasing T cell activity. Finally, we did not observe either the fast serum clearance or immune toxicities that have been reported for IgG1 antibodies. CONCLUSION: In conclusion, we have shown that VISTA-induced immune suppression can be reversed by blockade of the functional C-C' loop region of VISTA with a first-in-class rationally targeted and non-depleting IgG4 isotype anti-VISTA antibody, HMBD-002. This antibody represents a highly promising novel therapy in the VISTA-suppressed ICT non-responder population.

Hydroxychloroquine/chloroquine for the treatment of hospitalized patients with COVID-19: An individual participant data meta-analysis.

Background: Results from observational studies and randomized clinical trials (RCTs) have led to the consensus that hydroxychloroquine (HCQ) and chloroquine (CQ) are not effective for COVID-19 prevention or treatment. Pooling individual participant data, including unanalyzed data from trials terminated early, enables more detailed investigation of the efficacy and safety of HCQ/CQ among subgroups of hospitalized patients. Methods: We searched ClinicalTrials.gov in May and June 2020 for US-based RCTs evaluating HCQ/CQ in hospitalized COVID-19 patients in which the outcomes defined in this study were recorded or could be extrapolated. The primary outcome was a 7-point ordinal scale measured between day 28 and 35 post enrollment; comparisons used proportional odds ratios. Harmonized de-identified data were collected via a common template spreadsheet sent to each principal investigator. The data were analyzed by fitting a prespecified Bayesian ordinal regression model and standardizing the resulting predictions. Results: Eight of 19 trials met eligibility criteria and agreed to participate. Patient-level data were available from 770 participants (412 HCQ/CQ vs 358 control). Baseline characteristics were similar between groups. We did not find evidence of a difference in COVID-19 ordinal scores between days 28 and 35 post-enrollment in the pooled patient population (odds ratio, 0.97; 95% credible interval, 0.76-1.24; higher favors HCQ/CQ), and found no convincing evidence of meaningful treatment effect heterogeneity among prespecified subgroups. Adverse event and serious adverse event rates were numerically higher with HCQ/CQ vs control (0.39 vs 0.29 and 0.13 vs 0.09 per patient, respectively). Conclusions: The findings of this individual participant data meta-analysis reinforce those of individual RCTs that HCQ/CQ is not efficacious for treatment of COVID-19 in hospitalized patients.