With the ongoing outbreak of COVID-19 around the world, it has become a worldwide health concern. One previous study reported a family cluster with an asymptomatic transmission of COVID-19. Here, we report another series of cases and further demonstrate the repeatability of the transmission of COVID-19 by pre-symptomatic carriers. Methods: A familial cluster of five patients associated with COVID-19 was enrolled in the hospital. We collected epidemiological and clinical characteristics, laboratory outcomes from electronic medical records, and also verified them with the patients and their families. Results: Among them, three family members (Case 3/4/5) had returned from Wuhan. Additionally, two family members, those who had not traveled to Wuhan, also contracted COVID-19 after contacting with the other three family members. Case 1 developed severe pneumonia and was admitted to the ICU. Case 3 and Case 5 presented fever and cough on days two through three of hospitalization and had ground-glass opacity changes in their lungs. Case 4 presented with diarrhea and pharyngalgia after admission without radiographic abnormalities. Case 2 presented no clinical nor radiographic abnormalities. All five cases had an increasing level of C-reactive protein.Conclusions: Our findings indicate that COVID-19 can be transmitted by asymptomatic carriers during the incubation period.

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CD4+CD25+Foxp3+ regulatory T cells (Tregs) can inhibit cytotoxic responses. Though several studies have analyzed Treg frequency in the peripheral blood mononuclear cells (PBMCs) of pancreatic ductal adenocarcinoma (PDA) patients using flow cytometry (FCM), few studies have examined how intratumoral Tregs might contribute to immunosuppression in the tumor microenvironment. Thus, the potential role of intratumoral Tregs in PDA patients remains to be elucidated. In this study, we found that the percentages of Tregs, CD4+ T cells and CD8+ T cells were all increased significantly in tumor tissue compared to control pancreatic tissue, as assessed via FCM, whereas the percentages of these cell types in PBMCs did not differ between PDA patients and healthy volunteers. The percentages of CD8+ T cells in tumors were significantly lower than in PDA patient PBMCs. In addition, the relative numbers of CD4+CD25+Foxp3+ Tregs and CD8+ T cells were negatively correlated in the tissue of PDA patients, and the abundance of Tregs was significantly correlated with tumor differentiation. Additionally, Foxp3+ T cells were observed more frequently in juxtatumoral stroma (immediately adjacent to the tumor epithelial cells). Patients showing an increased prevalence of Foxp3+ T cells had a poorer prognosis, which was an independent factor for patient survival. These results suggest that Tregs may promote PDA progression by inhibiting the antitumor immunity of CD8+ T cells at local intratumoral sites. Moreover, a high proportion of Tregs in tumor tissues may reflect suppressed antitumor immunity.

Signal transducer and activator of transcription (Stat) 3 is an oncogene constitutively activated in many cancer systems where it contributes to carcinogenesis. To develop chemical probes that selectively target Stat3, we virtually screened 920,000 small drug-like compounds by docking each into the peptide-binding pocket of the Stat3 SH2 domain, which consists of three sites—the pY-residue binding site, the +3 residue-binding site and a hydrophobic binding site, which served as a selectivity filter. Three compounds satisfied criteria of interaction analysis, competitively inhibited recombinant Stat3 binding to its immobilized pY-peptide ligand and inhibited IL-6-mediated tyrosine phosphorylation of Stat3. These compounds were used in a similarity screen of 2.47 million compounds, which identified 3 more compounds with similar activities. Examination of the 6 active compounds for the ability to inhibit IFN-γ-mediated Stat1 phosphorylation revealed that 5 of 6 were selective for Stat3. Molecular modeling of the SH2 domains of Stat3 and Stat1 bound to compound revealed that compound interaction with the hydrophobic binding site was the basis for selectivity. All 5 selective compounds inhibited nuclear-to-cytoplasmic translocation of Stat3, while 3 of 5 compounds induced apoptosis preferentially of breast cancer cell lines with constitutive Stat3 activation. Thus, virtual ligand screening of compound libraries that targeted the Stat3 pY-peptide binding pocket identified for the first time 3 lead compounds that competitively inhibited Stat3 binding to its pY-peptide ligand; these compounds were selective for Stat3 vs. Stat1 and induced apoptosis preferentially of breast cancer cells lines with constitutively activated Stat3.

Collagen is an extracellular matrix structural component that can regulate cellular processes through its interaction with the integrins, ␣1␤1, ␣2␤1, ␣10␤1, and ␣11␤1. Collagen-like proteins have been identified in a number of bacterial species. Here, we used Scl2 from Streptococcus pyogenes serotype M28 strain MGAS6274 as a backbone for the introduction of discrete integrin-binding sequences. The introduced sequences GLPGER, GFPGER, or GFPGEN did not affect triple helix stability of the Scl (Streptococcal collagen-like) protein. Using ELISA and surface plasmon resonance, we determined that Scl2 GLPGER and Scl2 GFPGER bound to recombinant human ␣1 and ␣2 I-domains in a metal ion-dependent manner and without a requirement for hydroxyproline. We predicted a novel and selective integrinbinding sequence, GFPGEN, through the use of computer modeling and demonstrated that Scl2 GFPGEN shows specificity toward the ␣1 I-domain and does not bind the ␣2 I-domain. Using C2C12 cells, we determined that intact integrins interact with the modified Scl2 proteins with the same selectivity as recombinant I-domains. These modified Scl2 proteins also acted as cell attachment substrates for fibroblast, endothelial, and smooth muscle cells. However, the modified Scl2 proteins were unable to aggregate platelets. These results indicate that Scl2 is a suitable backbone for the introduction of mammalian integrin-binding sequences, and these sequences may be manipulated to individually target ␣1␤1 and ␣2␤1.Collagen is a major constituent of the extracellular matrix where it functions as a structural component. In addition, collagen can directly or indirectly interact with cellular receptors and regulate a variety of cellular processes (1). There are at least 28 identified mammalian collagens each consisting of three polypeptides that can be genetically identical or distinct (2). A defining feature of collagens is the tightly packed left-handed triple helix made of polypeptide segments with repeating GXY triplets. The small Gly residue fits in the interior of the triple helix, and the X and Y positions are often occupied by proline and hydroxyproline residues. Hydroxylation of proline residues stabilizes the triple helical structure and inhibition of posttranslational hydroxylation decreases the melting temperature of the mammalian collagen triple helix by ϳ15°C (3).Surface proteins with collagen-like domains recently have been found on a number of prokaryotic organisms, including Streptococcus pyogenes, Streptococcus equi, and Bacillus anthracis (4 -6). These collagen-like domains contain conserved GXY repeats but lack the hydroxyproline found in mammalian collagens. S. pyogenes often contain two collagen-like proteins, Scl1 and Scl2 (4, 7-13). The primary sequences and length of different domains, including the GXY repeat segments, vary considerably in Scl1 and Scl2 proteins from different strains (4). In electron micrographs, both proteins form a lollipop-like structure with an N-terminal variable globular domain connected to an extende...