OBJECTIVE: To identify the epidemiological characteristics and transmission patterns of pediatric patients with the 2019 novel coronavirus disease in China.METHODS: Nationwide case series of 2135 pediatric patients with COVID-19 reported to the Chinese Center for Disease Control and Prevention from January 16, 2020, to February 8, 2020, were included. The epidemic curves were constructed by key dates of disease onset and case diagnosis. Onset-to-diagnosis curves were constructed by fitting a log-normal distribution to data on both onset and diagnosis dates.RESULTS: There were 728 (34.1%) laboratory-confirmed cases and 1407 (65.9%) suspected cases. The median age of all patients was 7 years (interquartile range: 2-13 years), and 1208 case patients (56.6%) were boys. More than 90% of all patients had asymptomatic, mild, or moderate cases. The median time from illness onset to diagnoses was 2 days (range: 0-42 days). There was a rapid increase of disease at the early stage of the epidemic, and then there was a gradual and steady decrease. The disease rapidly spread from Hubei province to surrounding provinces over time. More children were infected in Hubei province than any other province.CONCLUSIONS: Children of all ages appeared susceptible to COVID-19, and there was no significant sex difference. Although clinical manifestations of children's COVID-19 cases were generally less severe than those of adult patients, young children, particularly infants, were vulnerable to infection. The distribution of children's COVID-19 cases varied with time and space, and most of the cases were concentrated in Hubei province and surrounding areas. Furthermore, this study provides strong evidence of human-to-human transmission.

IntroductionPlatelet glycoprotein (GP) Ib-IX-V complex binds to von Willebrand factor (VWF) deposited at the injured blood vessel wall, mediating initial platelet rolling and adhesion to the injury site. Ligation of VWF to the GP Ib␣ subunit in the complex also transmits a signal to the platelet that leads to platelet activation and aggregation. 1,2 In addition to mediating outside-in signals, the GP Ib-IX-V complex is also capable of transmitting intracellular signals to the outside. [3][4][5][6][7] How this receptor complex mediates signaling in both directions is not clear, but recent work has suggested the likely interaction between the Ib␣ and Ib␤ subunits as a potential key element of the mechanism. [8][9][10] Thus, understanding the interaction between GP Ib␣ and GP Ib␤, and in general the organizing principle of the GP Ib-IX-V complex, will help to unveil the mechanism underlying transmembrane signaling mediated by the complex.It is widely accepted that the GP Ib-IX-V complex contains 7 subunits composed of 4 different polypeptides: Ib␣, Ib␤, IX, and V with a respective stoichiometry of 2:2:2:1. [11][12][13][14][15] Each subunit is a type I transmembrane protein. A disulfide bond links GP Ib␣ and GP Ib␤, forming a complex known as GP Ib, that in turn interacts noncovalently with GP IX and GP V to generate the Ib-IX-V complex. The Ib␣ extracellular domain contains 9 Cys residues, 7 of which reside in the N-terminal domain that contains the binding site for VWF. The N-terminal domain contains 3 disulfides, C4-C17, C209-C248, and C211-C264. 16 C65 is buried in the hydrophobic core of the N-terminal domain and is therefore unpaired. 17,18 The other 2 Cys residues in GP Ib␣, C484 and C485, are located near the transmembrane (TM) domain and are conserved across species. The Ib␣/Ib␤ ratio of 1:1 in the receptor complex predicts that only 1 of the 2 Cys residues forms a disulfide bond with the membrane-proximal C122 in GP Ib␤ (Figure 1). However, it is not clear which one is paired with C122, and, more intriguingly, what role the free thiol group of the other Cys residue plays. It seems paradoxic that 2 neighboring Cys residues are exposed to presumably similar, if not the same, oxidizing environments, yet they have entirely different fates.In the present study, we sought to assign the disulfide between GP Ib␣ and GP Ib␤. To our surprise, we found that both C484 and C485 are linked to GP Ib␤ by disulfide bonds. In other words, contrary to the currently prevailing model, 1 Ib␣ subunit is linked through disulfide bonds to 2 Ib␤ subunits. Materials and methods Vectors and antibodiesThe vector pDX 19,20 was used in the expression of GP Ib-IX complex in Chinese hamster ovary (CHO) cells. The CHO K1 cell line was obtained from ATCC (Manassas, VA). The expression vector pGEX-4T-3 was purchased from Amersham Biosciences (Piscataway, NJ). WM23, an anti-Ib␣ monoclonal antibody, was kindly provided by Dr M. Berndt. Other antibodies against individual subunits of GP Ib-IX complex, including FMC25, Gi27, SZ2, and AK2, were p...

Lack of expression of glycoprotein (GP)Ib Platelet glycoprotein (GP)2 Ib-IX-V complex mediates the initial tethering and rolling of platelets on von Willebrand factor (vWF) localized at the vascular injury site (1, 2). Upon ligation with the A1 domain of vWF, the GP Ib-IX-V complex transmits inward a signal that leads to activation of integrin ␣IIb␤3 and eventual platelet activation and aggregation (3, 4).Although the GP Ib-IX-V complex is widely considered to comprise GP Ib␣, GP Ib␤, GP IX, and GP V with a 2:2:2:1 stoichiometry (5-7), it is not clear how these subunits assemble into a functional receptor complex in the membrane, partly because intersubunit interactions are largely unknown.Abnormally low expression of the GP Ib-IX-V complex in human platelets can result from mutations in either GP Ib␣, GP Ib␤, or GP IX, but not GP V (8). Such phenomena can be reproduced in transfected Chinese hamster ovary (CHO) cells; missing any of the three subunits leads to no or significantly lowered surface expression of the GP Ib-IX complex (9, 10). In contrast, GP V is not required for efficient expression of the GP Ib-IX complex, although there are mixed reports on whether it enhances the complex expression (11, 12). Furthermore, it has been shown in transfected cells that the GP Ib-IX complex assembles in the endoplasmic reticulum before being transported as a single entity to the plasma membrane. Failure of proper complex assembly results in prompt degradation of GP Ib␣ in the lysosome and accumulation of nonnative GP IX inside the cell (13,14).Although the interdependence among the subunits in the GP Ib-IX complex is well documented, the underlying reasons are not clear. The requirement of all three subunits to efficient surface expression of the complex posits the importance of intersubunit interactions to the proper assembly and therefore stability of the receptor complex. Based on the effects of mutations in GP Ib␤ on the expression level of GP IX, the N-terminal cysteine knot region in GP Ib␤ was suggested to mediate the interaction with GP IX (15). It helps to explain the dependence of GP IX expression on GP Ib␤. Why the expression of GP Ib␣ is dependent on GP Ib␤ and/or IX nonetheless remains unclear.The transmembrane (TM) domains of GP Ib␣ and IX may be important to efficient surface expression of the GP Ib-IX complex. Deletion of either the extracellular or cytoplasmic domain of GP Ib␣ does not significantly affect complex expression in transfected cells (16,17). Consistently, the Ib␣ extracellular domain, but not the combined extracellular and TM domains, can be replaced with counterparts from the interleukin-4 receptor while maintaining reasonable expression level of the receptor complex (18). In addition, a single-site mutation in the IX TM domain was recently identified to cause decreased expression of the GP Ib-IX

Platelet GPIb-IX receptor complex has 3 subunits GPIb␣, GPIb␤, and GPIX, which assemble with a ratio of 1:2:1. Dysfunction in surface expression of the complex leads to Bernard-Soulier syndrome. We have crystallized the GPIb␤ ectodomain (GPIb␤ E ) and determined the structure to show a single leucine-rich repeat with Nand C-terminal disulphide-bonded capping regions. The structure of a chimera of GPIb␤ E and 3 loops (a,b,c) taken from the GPIX ectodomain sequence was also determined. The chimera (GPIb␤ Eabc ), but not GPIb␤ E , forms a tetramer in the crystal, showing a quaternary interface between GPIb␤ and GPIX. Central to this interface is residue Tyr106 from GPIb␤, which inserts into a pocket generated by 2 loops (b,c) from GPIX. Mutagenesis studies confirmed this interface as a valid representation of interactions between GPIb␤ and GPIX in the full-length complex. Eight GPIb␤ missense mutations identified from patients with BernardSoulier syndrome were examined for changes to GPIb-IX complex surface expression. Two mutations, A108P and P74R, were found to maintain normal secretion/folding of GPIb␤ E but were unable to support GPIX surface expression. The close structural proximity of these mutations to Tyr106 and the GPIb␤ E interface with GPIX indicates they disrupt the quaternary organization of the GPIb-IX complex. (Blood. 2011;118(19):5292-5301) IntroductionGPIb-IX-V complex is an abundant membrane receptor complex on the platelet surface that plays a critical role in mediating platelet adhesion to the damaged vessel wall under conditions of high shear stress. 1 Platelets adhere, and integrins are subsequently activated by interactions of GPIb-IX-V with VWF bound to the subendothelium. How GPIb-IX-V transmits the VWF-binding signal across the membrane is not clear, partly because the structure and organization of this complex receptor remain to be elucidated. Because GPV is only weakly associated with the receptor complex and is not essential for complex expression, assembly, VWF binding, or signal transduction, 2,3 we focus on the GPIb-IX complex here.The GPIb-IX complex contains 3 subunits, GPIb␣, GPIb␤, and GPIX, with a 1:2:1 stoichiometry. 4 Each subunit is a type I transmembrane (TM) protein, containing an ectodomain with leucine-rich repeats (LRRs), 5 a single TM helix, and a relatively short cytoplasmic tail. The GPIb␣ ectodomain contains binding sites for a growing list of hemostatically important ligands, including VWF and thrombin. 6-8 Covalent and noncovalent interactions are important to the quaternary stabilization of the receptor. GPIb␣ links to 2 GPIb␤ subunits through membrane-proximal disulfide bonds to constitute the GPIb complex. 4 GPIX tightly associates with GPIb through noncovalent interactions. 9 Assembly of these subunits into a tightly integrated complex is also supported by genetic evidence. Bernard-Soulier syndrome (BSS) is a hereditary bleeding disorder that is characterized in most cases by giant platelets, low platelet counts, and little or no expression of GPIb-IX on the p...

Summary. Background: The glycoprotein (GP) Ib-IX complex is critically involved in platelet adhesion to von Willebrand factor and in the initial step of platelet activation. How this complex is assembled is not clear. We previously showed that the transmembrane (TM) domains of the GPIba and GPIbb subunits interact and participate in complex assembly. Objectives and methods: Here, we have investigated the role of the TM and cytoplasmic domains of GPIX in assembly of the GPIb-IX complex, by analyzing the mutational effects on complex expression and assembly in transiently transfected Chinese hamster ovary cells. Results: Replacing the cytoplasmic domain of GPIX with a poly-alanine sequence had little effect on surface expression and structural integrity of the GPIb-IX complex. In contrast, replacing the GPIX TM domain (residues 132-153) with a poly-leucine-alanine sequence markedly disrupted complex formation of GPIX with GPIba, interfered with GPIb formation, and decreased surface expression of the host complex. We further analyzed the contributions of a number of GPIX TM residues to complex formation by mutagenesis and found significant roles for Asp135 and several Leu residues. Conclusions: The TM domain, rather than the cytoplasmic domain, of GPIX plays an important role in expression and assembly of the GPIb-IX complex by interacting with its counterparts of GPIb. These TM domains may form a parallel four-helical bundle structure in the complex.

Neuroblastoma (NB) is the most common pediatric extra-cranial solid tumor with heterogeneous characteristics, and the prognosis of patients with high-risk NB is usually poor. Discovery of novel biomarkers for early detection and investigation of the underlying mechanisms governing invasion and metastasis of NB are urgently needed. Recently, exosomal microRNAs (miRNAs) have been shown to play vital regulatory or communication roles in the process of various types of cancers. However, the roles and mechanisms of exosomal miRNAs in NB remain unknown. Thus, the present study aims to investigate the detailed functions of tumor-derived exosomal miRNAs in progression and migration of NB in vivo and in vitro . By examining different exosomal miRNA expression profiles in the plasma of NB patients, we identified that the expression of hsa-miR199a-3p from exosomes was significantly upregulated, which was correlated with the severity of NB patients. Furthermore, we confirmed that exosomal hsa-miR199a-3p could facilitate proliferation and migration of NB via regulating NEDD4 expression. In summary, our data, for the first time, revealed that exosomal hsa-miR199a-3p could promote tumor proliferation and migration via decreasing NEDD4 expression in NB, suggesting that exosomal hsa-miR199a-3p may be applicated as a fast, easy, and non-invasive detection biomarker and contribute to the development of novel therapeutic strategies for NB in the future.

BackgroundRespiratory tract infections (RTIs) are the most common illness in children, and rapid diagnosis is required for the optimal management of RTIs, especially severe infections.MethodsNasopharyngeal swab or sputum specimens were collected from children aged 19 days to 15 years who were admitted to a hospital in Shanghai and diagnosed with RTIs. The specimens were tested with the FilmArray Respiratory Panel, a multiplex PCR assay that detects 16 viruses, Mycoplasma pneumoniae (M. pneumoniae), Bordetella pertussis (B. pertussis) and Chlamydophila pneumoniae (C. pneumoniae).ResultsAmong the 775 children studied, 626 (80.8%, 626/775) tested positive for at least one organism, and multiple organisms were detected in 198 (25.5%). Rhinoviruses/enteroviruses (25.5%, 198/775) were detected most often, followed by respiratory syncytial virus (19.5%, 151/775), parainfluenza virus 3 (14.8%, 115/775), influenza A or B (10.9%), adenovirus (10.8%), M. pneumoniae (10.6%) and B. pertussis (6.3%). The prevalence of organisms differed by age, and most of the viruses were more common in winter. Of the 140 children suspected of having pertussis, 35.0% (49/140) tested positive for B. pertussis.ConclusionsFilmArray RP allows the rapid simultaneous detection of a wide number of respiratory organisms, with limited hands-on time, in Chinese pediatric patients with RTIs.Electronic supplementary materialThe online version of this article (10.1186/s12879-018-3429-6) contains supplementary material, which is available to authorized users.

Summary. Background: The mechanism of assembly of the platelet glycoprotein (GP) Ib-IX complex from GPIba, GPIbb and GPIX subunits is not entirely clear. In this complex, ectodomains of both GPIbb and GPIX subunits contain two leucine-rich repeats (LRR) and share high sequence similarity. However, they differ noticeably in stability, hampering further analysis of their interaction. Objectives and methods: Guided by analysis of the LRR structure, we report a well-folded Ibb/IX chimera and its usage in dissecting GPIX function. Results: In this chimera, three non-contiguous sequences that may constitute the putative convex surface of the GPIbb ectodomain are replaced by their GPIX counterparts. Like GPIbb but unlike GPIX ectodomain, it can secrete from transfected Chinese hamster ovary cells and fold into a stable conformation. Furthermore, replacing the ectodomain in GPIX with the Ibb/ IX chimera, but not the GPIbb ectodomain, preserved its interaction with GPIbb as demonstrated by its native-like GPIbb-induced increase in surface expression and coimmunoprecipitation. Conclusions: The putative convex surface of the LRR domain in GPIX is sufficient, in the context of full-length subunit, to mediate its association with GPIbb.