Taiwan experienced a series of outbreaks of nosocomial severe acute respiratory syndrome (SARS) infections in 2003. Two months after the final outbreak, we recruited 658 employees from the hospital that suffered the first and most severe SARS infections to help us investigate epidemiological and genetic factors associated with the SARS coronavirus (SARS-CoV). SARS-CoV infections were detected by using enzyme immunoassays and confirmed by a combination of Western blot assays, neutralizing antibody tests, and commercial SARS tests. Risk factors were analyzed via questionnaire responses and sequence-specific oligonucleotide probes of human leukocyte antigen (HLA) alleles. Our results indicate that 3% (20/658) of the study participants were seropositive, with one female nurse identified as a subclinical case. Identified SARS-CoV infection risk factors include working in the same building as the hospital's emergency room and infection ward, providing direct care to SARS patients, and carrying a Cw*0801 HLA allele. The odds ratio for contracting a SARS-CoV infection among persons with either a homozygous or a heterozygous Cw*0801 genotype was 4.4 (95% confidence interval, 1.5 to 12.9; P ‫؍‬ 0.007).

Cell tracing dyes are very frequently utilized in cellular biology research because they provide highly sensitive fluorescent tags that do not compromise cellular functions such as growth and proliferation. In many investigations concerning cellular adhesion and mechanics, fluorescent dyes have been employed with the assumption of little impact on the results. Using the single-cell compression technique developed by our team, the single-cell mechanics of MDA-MB-468 and MLC-SV40 cells were investigated as a function of dye uptake. Cell tracing dyes increase living cell stiffness 3-6 times and cell-to-probe adhesion up to 7 times. These results suggest a more significant effect than toxins, such as Thrombin. A simple analytical model was derived to enable the extraction of the Young's moduli of the cell membrane and cytoskeleton from the force-deformation profiles measured for individual cells. The increase in Young's modulus of the membrane is 3-7 times, which is more significant than that of the cytoskeleton (1.1-3.4 times). We propose that changes in cell mechanics upon the addition of fluorescent tracing dye are primarily due to incorporation of amphiphilic dye molecules into the cellular plasma membrane, which increases the lateral interaction among phospholipid chains and thus enhances their rigidity and adhesion.

C-terminal tensin-like (cten) is a focal adhesion molecule belonging to the tensin family. Previous studies have suggested that cten may function as a prostate-specific tumor suppressor. Here, we show that although cten is expressed at a very low level in normal colon, its expression is significantly up-regulated in colon cancer. Furthermore, a high population of cten is found in the nucleus, where it interacts with Bcatenin, a critical player in the canonical Wnt pathway. This interaction may contribute to the role of cten in enhancing the colony formation, anchorage-independent growth, and invasiveness of colon cancer cells. Our studies have identified cten as a novel nuclear partner of B-catenin, showed an oncogenic activity of cten in colon cancers, and revealed cten as a potential biomarker and target for colon cancers.

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) uses dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) to facilitate cell entry via cellular receptor-angiotensin-converting enzyme 2. For this project, we used recombinant baculoviruses expressing different lengths of SARS-CoV spike (S) protein in a capture assay to deduce the minimal DC-SIGN binding region. Our results identified the region location between amino acid (aa) residues 324 to 386 of the S protein. We then generated nine monoclonal antibodies (MAbs) against the S protein to map the DC-SIGN-binding domain using capture assays with pseudotyped viruses and observed that MAb SIa5 significantly blocked S protein-DC-SIGN interaction. An enhancement assay using the HKU39849 SARS-CoV strain and human immature dendritic cells confirmed our observation. Data from a pepscan analysis and M13 phage peptide display library system mapped the reactive MAb SIa5 epitope to aa residues 363 to 368 of the S protein. Results from a capture assay testing three pseudotyped viruses with mutated N-linked glycosylation sites of the S protein indicate that only two pseudotyped viruses (N330Q and N357Q, both of which lost glycosylation sites near the SIa5 epitope) had diminished DC-SIGN-binding capacity. We also noted that MAb SIb4 exerted a neutralizing effect against HKU39849; its reactive epitope was mapped to aa residues 435 to 439 of the S protein. We offer the data to facilitate the development of therapeutic agents and preventive vaccines against SARS-CoV infection.Severe acute respiratory syndrome (SARS) causes progressive respiratory failure and death in approximately 10% of infected individuals (14,35). A SARS-associated coronavirus (SARS-CoV) has been identified as the causal agent (15,17,26,35), and angiotensin-converting enzyme 2 (ACE2) and dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) have been identified as SARS-CoV cellular receptors (30,33,44).The SARS-CoV spike (S) protein is 1,255 amino acids (aa) in length; its 43 strains share 97.7% sequence identity (28). It contains two domains-SI (aa residues 17 to 680) and SII (aa residues 681 to 1,255)-which are, respectively, responsible for receptor binding and membrane fusion (38,40). The receptor binding domain (aa residues 318 to 510) of the S protein contains a major neutralization determinant capable of inducing potent neutralizing antibodies in mice (22). A recombinant protein (RP) containing aa residues 310 to 510 of the S protein absorbs and removes most neutralizing antibodies in various animals inoculated with a modified vaccinia virus Ankara that expresses a full-length S protein (8). According to these findings, SARS-CoV S protein receptor binding domain is a critical target for vaccine and therapeutic pharmaceutical development.DC-SIGN, a C-type lectin receptor expressed on dendritic cells (DCs), was initially identified as a human immunodeficiency virus (HIV) attachment factor (13, 18) but has since been found to be a receptor for hepatitis C virus (36),...

DLC1 is a RhoGAP-containing tumor suppressor and many of DLC1’s functions are absolutely dependent on its RhoGAP activity. Through its RhoGAP domain, DLC1 inhibits the activity of RhoA GTPase, which regulates actin cytoskeleton networks and dis/assembly of focal adhesions. Tensin1 (TNS1) is a focal adhesion molecule that links the actin cytoskeleton to integrins and forms signaling complexes through its multiple binding domains. Here, we report that TNS1 enhances RhoA activity in a DLC1-dependent manner. This is accomplished by binding to DLC1 through TNS1’s C2, SH2, and PTB domains. Point mutations at these three sites disrupt TNS1’s interaction with DLC1 as well as its effect on RhoA activity. The biological relevance of this TNS1-DLC1-RhoA signaling axis is investigated in TNS1 knockout (KO) cells and mice. Endothelial cells isolated from TNS1 KO mice or those silenced with TNS1 siRNA show significant reduction in proliferation, migration, and tube formation activities. Concomitantly, the RhoA activity is down-regulated in TNS1 KO cells and this reduction is restored by further silencing of DLC1. Furthermore, the angiogenic process is compromised in TNS1 KO mice. These studies demonstrate that TNS1 binds to DLC1 and fine-tunes its RhoGAP activity toward RhoA and that the TNS1-DLC1-RhoA signaling axis is critical in regulating cellular functions that lead to angiogenesis.