Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion.

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Glycomic Analysis Reveals That Sialyltransferase Inhibition Is Involved in the Antiviral Effects of Arbidol.

Due to the high mutation rate of influenza virus and the rapid increase of drug resistance, it is imperative to discover host-targeting antiviral agents with broad-spectrum antiviral activity. Considering the discrepancy between the urgent demand of antiviral drugs during an influenza pandemic and the long-term process of drug discovery and development, it is feasible to explore host-based antiviral agents and strategies from antiviral drugs on the market. In the current study, the antiviral mechanism of arbidol (ARB), a broad-spectrum antiviral drug with potent activity at early stages of viral replication, was investigated from the aspect of hemagglutinin (HA) receptors of host cells. N-glycans that act as the potential binding receptors of HA on 16-human bronchial epithelial (16-HBE) cells were comprehensively profiled for the first time by using an in-depth glycomic approach based on TiO2-PGC chip-Q-TOF MS. Their relative levels upon the treatment of ARB and virus were carefully examined by employing an ultra-high sensitive qualitative method based on Chip LC-QQQ MS, showing that ARB treatment led to significant and extensive decrease of sialic acid (SA)-linked N-glycans (SA receptors), and thereby impaired the virus utilization on SA receptors for rolling and entry. The SA-decreasing effect of ARB was demonstrated to result from its inhibitory effect on sialyltransferases (ST), ST3GAL4 and ST6GAL1 of 16-HBE cells. Silence of STs, natural ST inhibitors, as well as sialidase treatment of 16-HBE cells, resulted in similar potent antiviral activity, whereas ST-inducing agent led to the diminished antiviral effect of ARB. These observations collectively suggesting the involvement of ST inhibition in the antiviral effect of ARB. IMPORTANCE This study revealed, for the first time, that ST inhibition and the resulted destruction of SA receptors of host cells may be an underlying mechanism for the antiviral activity of ARB. ST inhibition has been proposed as a novel host-targeting antiviral approach recently and several compounds are currently under exploration. ARB is the first antiviral drug on the market that was found to possess ST inhibiting function. This will provide crucial evidence for the clinical usages of ARB, such as in combination with neuraminidase (NA) inhibitors to exert optimized antiviral effect, etc. More importantly, as an agent that can inhibit the expression of STs, ARB can serve as a novel lead compound for the discovery and development of host-targeting antiviral drugs.

Comprehensive Glycomic Profiling of Respiratory Tract Tissues of Tree Shrews by TiO2-PGC Chip Mass Spectrometry.

Due to its relatively small size, homology to humans, and susceptibility to human viruses, the tree shrew becomes an ideal alternative animal model for the study of human viral infectious diseases. However, there is still no report for the comprehensive glycan profile of the respiratory tract tissues in tree shrews. In this study, we characterized the structural diversity of N-glycans in the respiratory tract of tree shrews using our well-established TiO2-PGC chip-Q-TOF-MS method. As a result, a total of 219 N-glycans were identified. Moreover, each identified N-glycan was quantitated by a high sensitivity and accurate MRM method, in which 13C-labeled internal standards were used to correct the inherent run-to-run variation in MS detection. Our results showed that the N-glycan composition in the turbinate and lung was significantly different from the soft palate, trachea, and bronchus. Meanwhile, 28 high-level N-glycans in turbinate were speculated to be correlated with the infection of H1N1 virus A/California/04/2009. This study is the first to reveal the comprehensive glycomic profile of the respiratory tract of tree shrews. Our results also help to better understand the role of glycan receptors in human influenza infection and pathogenesis.

Colonic self-expanding metal stent (SEMS) as a bridge to surgery in left-sided malignant colonic obstruction: an 8-year review.

INTRODUCTION: Colonic stenting has evolved to be an alternative to emergency laparotomy in the management of acute left-sided malignant colonic obstruction. This retrospective comparative study aimed to review the outcomes of colonic stent as bridge to surgery with emergency operation in a regional hospital in Hong Kong. METHOD: Consecutive patients who were admitted from January 2006 to July 2014 with diagnosis of malignant left-sided colonic obstruction (from splenic flexure to rectosigmoid colon) were included. Patients with peritonitis or disseminated disease were excluded. Colonic stenting was attempted in all eligible patients when fluoroscopy was available in the endoscopy suite during office hour. Otherwise, emergency operation was performed. For patients with clinical success in colonic stenting, interval colectomies were performed. The postoperative outcomes, including the 30-days mortality, the stoma creation rate, the complication rate as well as the survival data were analyzed on an intention-to-treat (ITT) basis. RESULTS: From January 2006 to July 2014, 62 patients underwent colonic stenting and 40 patients underwent emergency operations. The technical success rate and the clinical success rate of stenting were 95.2 and 83.9 %, respectively. Laparoscopic resection was achieved in 74.2 % in the stenting group. More primary anastomoses were performed in the stenting group (71.0 vs. 27.5 %, p = 0.000). The stenting group had a significantly lower permanent stoma rate (16.1 vs. 52.5 %, p < 0.000), fewer Dindo grade III to IV postoperative morbidity (16.1 vs. 40 %, p = 0.007), and the 30-day mortality rate was lower (3.2 vs. 17.5 %, p = 0.018), translating into a better overall 5-year survival rate. The disease-free survival was comparable between the two groups. CONCLUSIONS: Colonic self-expanding metal stent is effective in the management of acute left-sided colonic obstruction. It is associated with reduced stoma creation rate and postoperative morbidity. The oncological safety is not jeopardized by stenting and the interval operation.

Techniques of Laparoscopic Right Posterior Sectionectomy: Glissonian Approach and a Parenchymal Transection Technique.

Laparoscopic liver resections (LLR) have been widely accepted as a treatment option for liver tumors. They offer several advantages over open liver resections, including less blood loss, reduced wound pain, and shorter hospital stays with a comparable oncological outcome. However, laparoscopic resection of lesions in the right posterior section of the liver is challenging due to difficulties in bleeding control and visualizing the surgical field. In the past, laparoscopic right posterior sectionectomy (LRPS) was still in the exploration phase, with undefined risks in the Second International Consensus Conference on LLR in 2014. However, recent technological advancements and increased surgical experience have shown that LRPS can be safe and feasible. It has been found to reduce hospital stay and blood loss compared to open surgery. This manuscript aims to provide a detailed description of the steps involved in LRPS. The key factors contributing to our success in this challenging procedure include proper liver retraction and exposure, the use of an intrahepatic Glissonian approach for inflow control, a technique called the 'ultrasonic scalpel mimic Cavitron ultrasonic surgical aspirator (CUSA)' for parenchymal transection, early identification of the right hepatic vein, and meticulous bleeding control using bipolar diathermy.

Robust quantitation of gangliosides and sulfatides in human brain using UHPLC-MRM-MS: Method development and application in Alzheimer's disease.

Gangliosides (GAs) and sulfatides (STs) are acidic glycosphingolipids that are particularly abundant in the nervous system and are closely related to aging and neurodegenerative disorders. To explore their roles in brain diseases, in-depth molecular profiling, including structural variations of sphingoid backbone, fatty acyl group, and sugar chain of GAs and STs was performed. A total of 210 GAs and 38 STs were characterized in the inferior frontal gyrus (IFG) of human brain, with 90 GAs discovered in brain tissues for the first time. Influential MS parameters for detecting GAs and STs in multiple reaction monitoring (MRM) mode were systematically examined and optimized to minimize in-source fragmentation, resulting in remarkable signal intensity enhancement for GAs and STs, especially for polysialylated species. To eliminate analytical variations, isotopic interference-free internal standards were prepared by simple and fast reduction reaction. The final established method facilitated the simultaneous quantitation of 184 GAs and 30 STs from 25 subtypes, which represents the highest number of GAs quantitated among all quantitation methods recorded in literature so far. The method was further validated and applied to reveal the aberrant change of GAs and STs in the IFG of 12 Alzheimer's disease (AD) patients. Four GAs exhibited high classification capacity for AD (AUC ≥0.80) and were thereby considered the most promising signatures for AD. These findings suggested the close correlation between GAs and the pathogenesis of AD, highlighting the achievements of our robust method for investigating the roles of GAs and STs in various physiological states and diseases.

Emamectin Benzoate Treatment of Hybrid Grouper Infected With Sea Lice in Hong Kong.

Sea lice (Copepoda: Caligidae) are ectoparasites which negatively impact marine aquaculture species around the world. There are a limited number of treatments licensed for use against sea lice in tropical and semi-tropical farmed fish species. Emamectin benzoate (EB) was an effective pharmaceutical drug against sea lice infestations in several salmon industries before resistance to the product developed. This drug has not been extensively tested in marine fish within Asia. The objective of this study was to determine whether this drug could be used to treat oral infections with sea lice in hybrid grouper (Mycteroperca tigris × Epinephelus lanceolatus) cultured in saltwater net-pen sites in Hong Kong. We observed an overall reduction in sea lice infections over time, starting on the last day of the treatment up to the end of our study (i.e., 14 days after the last EB treatment). We also observed a large variation in concentrations of EB in fish on the last day of the treatment, which provides an explanation for the variation in response to the treatment. It also suggests that distribution of the medication to fish in saltwater net-pens is difficult, especially when medication is hand-mixed in the feed and possibly unevenly distributed in the daily rations. Overall, this study provides preliminary evidence that EB could be used to treat sea lice found in Hong Kong and potentially in other regions of SE Asia.

A viral fusogen hijacks the actin cytoskeleton to drive cell-cell fusion.

Cell-cell fusion, which is essential for tissue development and used by some viruses to form pathological syncytia, is typically driven by fusogenic membrane proteins with tall (>10 nm) ectodomains that undergo conformational changes to bring apposing membranes in close contact prior to fusion. Here we report that a viral fusogen with a short (<2 nm) ectodomain, the reptilian orthoreovirus p14, accomplishes the same task by hijacking the actin cytoskeleton. We show that phosphorylation of the cytoplasmic domain of p14 triggers N-WASP-mediated assembly of a branched actin network. Using p14 mutants, we demonstrate that fusion is abrogated when binding of an adaptor protein is prevented and that direct coupling of the fusogenic ectodomain to branched actin assembly is sufficient to drive cell-cell fusion. This work reveals how the actin cytoskeleton can be harnessed to overcome energetic barriers to cell-cell fusion.

Evaluation of the performance of a modified Acute Physiology and Chronic Health Evaluation (APACHE II) scoring system for critically ill patients in emergency departments in Hong Kong.

INTRODUCTION: Numerous prognostic predictive models have been developed for critically ill patients, many of which are primarily designed for use in intensive care units. The objective of this study was to evaluate the accuracy of a modified Acute Physiology and Chronic Health Evaluation (APACHE II) scoring system in predicting the mortality for critically ill patients managed in emergency department (ED) resuscitation rooms in Hong Kong. METHOD: A multi-centre, prospective study was conducted for patients managed in the resuscitation rooms of the EDs of four major hospitals, including one university teaching hospital. The primary outcome measure was 14 day all-cause mortality and the secondary outcome measure was the length of stay in hospital. RESULTS: Of 867 patients recruited between 4 and 30 April 2004, 106 (12.2%) patients died. The modified APACHE II score was found to be significantly higher in non-survivors compared to survivors (mean+/-S.D.: 21.2+/-7.7 versus 14.4+/-7.1, p<0.001). The area under the curve for modified APACHE II in predicting mortality was 0.743 (95% CI, 0.696-0.790). CONCLUSION: The modified APACHE II score is only a moderate predictor of mortality for critically ill patients managed in the resuscitation rooms of EDs in Hong Kong. A more ED specific scoring method is required.

Rh2E2, a novel metabolic suppressor, specifically inhibits energy-based metabolism of tumor cells.

Energy metabolism in cancer cells is often increased to meet their higher proliferative rate and biosynthesis demands. Suppressing cancer cell metabolism using agents like metformin has become an attractive strategy for treating cancer patients. We showed that a novel ginsenoside derivative, Rh2E2, is as effective as aspirin in preventing the development of AOM/DSS-induced colorectal cancer and suppresses tumor growth and metastasis in a LLC-1 xenograft. A sub-chronic and acute toxicity LD50 test of Rh2E2 showed no harmful reactions at the maximum oral dosage of 5000 mg/kg body weight in mice. Proteomic profiling revealed that Rh2E2 specifically inhibited ATP production in cancer cells via down-regulation of metabolic enzymes involving glycolysis, fatty acid ß-oxidation and the tricarboxylic acid cycle, leading to specific cytotoxicity and S-phase cell cycle arrest in cancer cells. Those findings suggest that Rh2E2 possesses a novel and safe anti-metabolic agent for cancer patients by specific reduction of energy-based metabolism in cancer cells.

Functionalizable hydrogel microparticles of tunable size and stiffness for soft-tissue filler applications.

Particle size, stiffness and surface functionality are important in determining the injection site, safety and efficacy of injectable soft-tissue fillers. Methods to produce soft injectable biomaterials with controlled particle characteristics are therefore desirable. Here we report a method based on suspension photopolymerization and semi-interpenetrating network (semi-IPN) to synthesize soft, functionalizable, spherical hydrogel microparticles (MP) of independently tunable size and stiffness. MP were prepared using acrylated forms of polyethylene glycol (PEG), gelatin and hyaluronic acid. Semi-IPN MP of PEG-diacrylate and PEG were used to study the effect of process parameters on particle characteristics. The process parameters were systematically varied to produce MP with size ranging from 115 to 515µm and stiffness ranging from 190 to 1600Pa. In vitro studies showed that the MP thus prepared were cytocompatible. The ratio and identity of the polymers used to make the semi-IPN MP were varied to control their stiffness and to introduce amine groups for potential functionalization. Slow-release polymeric particles loaded with Rhodamine or dexamethasone were incorporated in the MP as a proof-of-principle of drug incorporation and release from the MP. This work has implications in preparing injectable biomaterials of natural or synthetic polymers for applications as soft-tissue fillers.

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum).

A TaqMan quantitative real-time PCR detection system was developed to examine transgene copy number in cotton. GhUBC1, a gene validated to be present as a single copy per haploid Gossypium hirsutum genome, was used as the endogenous reference to estimate copy number of GFP and selection marker NPTII in 28 T0 plants. This system was found to be more accurate than genomic Southern blot hybridization and could effectively tell homozygotes from heterozygotes in a T1 transgenic cotton population. Therefore it is suitable for efficient and cost effective early screening of transgenic seedlings and identifying transgene homozygotes in segregation populations.

Transgene integration and organization in cotton (Gossypium hirsutum L.) genome.

While genetically modified upland cotton (Gossypium hirsutum L.) varieties are ranked among the most successful genetically modified organisms (GMO), there is little knowledge on transgene integration in the cotton genome, partly because of the difficulty in obtaining large numbers of transgenic plants. In this study, we analyzed 139 independently derived T0 transgenic cotton plants transformed by Agrobacterium tumefaciens strain AGL1 carrying a binary plasmid pPZP-GFP. It was found by PCR that as many as 31% of the plants had integration of vector backbone sequences. Of the 110 plants with good genomic Southern blot results, 37% had integration of a single T-DNA, 24% had two T-DNA copies and 39% had three or more copies. Multiple copies of the T-DNA existed either as repeats in complex loci or unlinked loci. Our further analysis of two T1 populations showed that segregants with a single T-DNA and no vector sequence could be obtained from T0 plants having multiple T-DNA copies and vector sequence. Out of the 57 T-DNA/T-DNA junctions cloned from complex loci, 27 had canonical T-DNA tandem repeats, the rest (30) had deletions to T-DNAs or had inclusion of vector sequences. Overlapping micro-homology was present for most of the T-DNA/T-DNA junctions (38/57). Right border (RB) ends of the T-DNA were precise while most left border (LB) ends (64%) had truncations to internal border sequences. Sequencing of collinear vector integration outside LB in 33 plants gave evidence that collinear vector sequence was determined in agrobacterium culture. Among the 130 plants with characterized flanking sequences, 12% had the transgene integrated into coding sequences, 12% into repetitive sequences, 7% into rDNAs. Interestingly, 7% had the transgene integrated into chloroplast derived sequences. Nucleotide sequence comparison of target sites in cotton genome before and after T-DNA integration revealed overlapping microhomology between target sites and the T-DNA (8/8), deletions to cotton genome in most cases studied (7/8) and some also had filler sequences (3/8). This information on T-DNA integration in cotton will facilitate functional genomic studies and further crop improvement.