Rationale: The coronavirus disease (COVID-19) pandemic is now a global health concern. Objectives: We compared the clinical characteristics, laboratory examinations, computed tomography images, and treatments of patients with COVID-19 from three different cities in China. Methods: A total of 476 patients were recruited from January 1, 2020, to February 15, 2020, at three hospitals in Wuhan, Shanghai, and Anhui. The patients were divided into four groups according to age and into three groups (moderate, severe, and critical) according to the fifth edition of the Guidelines on the Diagnosis and Treatment of COVID-19 issued by the National Health Commission of China. Measurements and Main Results: The incidence of comorbidities was higher in the severe (46.3%) and critical (67.1%) groups than in the moderate group (37.8%). More patients were taking angiotensinconverting enzyme inhibitors/angiotensin II receptor blockers in the moderate group than in the severe and critical groups. More patients had multiple lung lobe involvement and pleural effusion in the critical group than in the moderate group. More patients received antiviral agents within the first 4 days in the moderate group than in the severe group, and more patients received antibiotics and corticosteroids in the critical and severe groups. Patients .75 years old had a significantly lower survival rate than younger patients. Conclusions: Multiple organ dysfunction and impaired immune function were the typical characteristics of patients with severe or critical illness. There was a significant difference in the use of angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers among patients with different severities of disease. Involvement of multiple lung lobes and pleural effusion were associated with the severity of COVID-19. Advanced age (>75 yr) was a risk factor for mortality.

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.

Dysfunctional immune response in the COVID-19 patients is a recurrent theme impacting symptoms and mortality, yet the detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes were associated with distinct clinical features including age, sex, severity, and disease stages of COVID-19. SARS-CoV-2 RNAs were found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within viral positive cells. Systemic up-regulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis and developing effective therapeutic strategies for COVID-19.

Notch signaling is a central mechanism for controlling embryogenesis. However, its in vivo function during mesenchymal cell differentiation, and specifically, in bone homeostasis remains largely unknown. Here, we show that osteoblast-specific gain of Notch function causes severe osteosclerosis due to increased proliferation of immature osteoblasts. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by up-regulating Cyclin D, Cyclin E, and Osterix. Notch also regulates terminal osteoblastic differentiation by directly binding Runx2 and repressing its transactivation function. In contrast, loss of all physiologic Notch signaling in osteoblasts, generated by deletion of Presenilin 1 and 2 in bone, is associated with late onset, age-related osteoporosis resulting from increased osteoblast-dependent osteoclastic activity due to decreased production of Osteoprotegerin. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis and may provide direction for novel therapeutic applications.Evolutionarily conserved Notch signaling plays a critical role in cell fate determination, and various developmental processes by translating cell-cell interactions into specific transcriptional programs 1, 2 . Temporal and spatial modulation of this pathway can significantly affect proliferation, differentiation and apoptotic events 3 . Moreover, the timing of Notch signaling can lead to diverse effects within the same cell lineage 4, 5 . In mammals, activation of up to four Notch receptors by membrane-bound ligands initiates a process leading to presenilin-mediated cleavage and release of the Notch intracellular domain (NICD) from the membrane that then traffics to the nucleus. NICD subsequently regulates the expression of genes in cooperation with the transcription factor RBP-Jκ and Mastermind-like proteins.The observation that mutations in the Notch ligand Delta homologue-3 (Dll-3) and γ-secretase Presenilin1 both cause axial skeletal phenotypes originally linked Notch signaling with skeletal development 6, 7 . Recently, several in vitro studies with conflicting results implicated the Notch pathway in the regulation of osteoblast differentiation, but the in vivo role of Notch signaling in bone homeostasis still remains unknown 8-12 .Corresponding Author: Brendan Lee, M.D., Ph.D., One Baylor Plaza, Rm 635E, Houston, Tx 77030,, Email E-mail: blee@bcm.tmc.edu. In this study, we investigate the tissue, cellular, and molecular consequences of both gain and loss of function of Notch signaling in committed osteoblasts. NIH Public Access RESULTS Gain of function of Notch signaling results in severe osteosclerosisTo determine the pathological consequences of in vivo gain of Notch function during bone formation and homeostasis, we generated transgenic mice expressing the Notch1 intracellular domain (N1ICD) under the control of the type I collagen (Col1a1) promoter (Suppl. Fig. 1a,b). Here, gain of Notch function would occur in committed osteoblastic ce...

The α1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte–specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive β-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2 +/− background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2-null mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

Osteogenesis Imperfecta (OI) is a heritable disorder of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations1. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms2. Here, we show that excessive transforming growth factor-beta (TGFβ) signaling is a mechanism of OI in both recessive (Crtap−/−) and dominant (Col1a2tm1.1Mcbr) OI mouse models. In the skeleton, we find higher expression of TGFβ target genes, ratio of pSmad2/Smad2 protein, and in vivo Smad2 reporter activity. Anti-TGFβ treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI, and improves the lung abnormalities in Crtap−/− mice. Moreover, type I collagen of Crtap−/− mice shows reduced binding to the small leucine rich proteoglycan decorin, a known regulator of TGFβ activity3–4. Hence, altered TGFβ matrix-cell signaling is a primary mechanism in the pathogenesis of OI, and could be a promising target for the treatment of OI.

Mesenchymal stem cell-derived osteochondroprogenitors express two master transcription factors, SOX9 and RUNX2, during condensation of the skeletal anlagen. They are essential for chondrogenesis and osteogenesis, respectively, and their haploinsufficiency causes human skeletal dysplasias. We show that SOX9 directly interacts with RUNX2 and represses its activity via their evolutionarily conserved high-mobility-group and runt domains. Ectopic expression of full-length SOX9 or its RUNX2-interacting domain in mouse osteoblasts results in an osteodysplasia characterized by severe osteopenia and down-regulation of osteoblast differentiation markers. Thus, SOX9 can inhibit RUNX2 function in vivo even in established osteoblastic lineage. Finally, we demonstrate that this dominant inhibitory function of SOX9 is physiologically relevant in human campomelic dysplasia. In campomelic dysplasia, haploinsufficiency of SOX9 results in up-regulation of the RUNX2 transcriptional target COL10A1 as well as all three members of RUNX gene family. In summary, SOX9 is dominant over RUNX2 function in mesenchymal precursors that are destined for a chondrogenic lineage during endochondral ossification.differentiation ͉ mesenchymal ͉ skeletal dysplasias ͉ osteoblasts ͉ transcriptional repressor D uring embryogenesis, the majority of bones are formed via endochondral ossification; mesenchymal progenitor cells differentiate into chondrocytes that are eventually replaced by osteoblasts (1, 2). It is a well coordinated process regulated by a complex transcriptional network in which the transcription factors Runx2 and Sox9 play essential roles. Runx2 is required for osteoblast differentiation and chondrocyte maturation both in vivo and in vitro (3). We and others have shown that mutations in RUNX2 cause cleidocranial dysplasia, a dominantly inherited skeletal dysplasia characterized by hypoplastic clavicles, large fontanels, dental anomalies, and delayed skeletal development (4, 5). Sox9 is a potent transcriptional activator for chondrocyte-specific genes such as Col2a1 and Col11a1, and mouse genetic studies demonstrate that it is required for the successive steps of chondrocyte differentiation and cartilage formation (6-8). Mutations in human SOX9 result in campomelic dysplasia (CMD1), a disorder characterized by generalized hypoplasia of endochondral bones (9, 10).Although Runx2 is a strong transcriptional activator for osteoblast-specific and hypertrophic chondrocyte-specific genes, its embryonic expression is present in osteochondroprogenitor cells during mesenchymal condensations as early as embryonic day 10 (E10), before overt chondrocyte differentiation or osteoblast differentiation (11, 12). Hence, a strong context-dependent inhibition of Runx2 must occur before cell fate commitment to the chondrogenic lineage. Because Sox9 is also highly expressed in all osteochondroprogenitor cells and in proliferating (prehypertrophic) chondrocytes (6), we hypothesize that, in addition to its well established role as transcriptional activator for ch...

Nitric Oxide (NO) plays a critical role in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (Asl) deficiency exhibits a distinct phenotype manifest by multi-organ dysfunction and NO deficiency. Loss of Asl leads to reduced NO synthesis due to decreased endogenous arginine synthesis as well as reduced utilization of extracellular arginine for NO production in both humans and mice. Hence, ASL as seen in other species through evolution has a structural function in addition to its catalytic activity. Importantly, therapy with nitrite rescued the tissue autonomous NO deficiency in hypomorphic Asl mice, while a NOS independent NO donor restored NO-dependent vascular reactivity in subjects with ASL deficiency. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as treatment of NO-related diseases.