Highly pathogenic avian influenza H5N1 virus delays apoptotic responses via activation of STAT3

Highly pathogenic avian influenza (HPAI) H5N1 virus continues to pose pandemic threat, but there is a lack of understanding of its pathogenesis. We compared the apoptotic responses triggered by HPAI H5N1 and low pathogenic H1N1 viruses using physiologically relevant respiratory epithelial cells. We demonstrated that H5N1 viruses delayed apoptosis in primary human bronchial and alveolar epithelial cells (AECs) compared to H1N1 virus. Both caspase-8 and -9 were activated by H5N1 and H1N1 viruses in AECs, while H5N1 differentially up-regulated TRAIL. H5N1-induced apoptosis was reduced by TRAIL receptor silencing. More importantly, STAT3 knock-down increased apoptosis by H5N1 infection suggesting that H5N1 virus delays apoptosis through activation of STAT3. Taken together, we demonstrate that STAT3 is involved in H5N1-delayed apoptosis compared to H1N1. Since delay in apoptosis prolongs the duration of virus replication and production of pro-inflammatory cytokines and TRAIL from H5N1-infected cells, which contribute to orchestrate cytokine storm and tissue damage, our results suggest that STAT3 may play a previously unsuspected role in H5N1 pathogenesis.published_or_final_versio

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

<p>Abstract</p> <p>Background</p> <p><it>Laribacter hongkongensis </it>is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements.</p> <p>Results</p> <p>For acid and bile resistance, <it>L. hongkongensis </it>possessed a urease gene cassette, two <it>arc </it>gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent <it>Escherichia coli </it>(<it>E. coli</it>) and enterotoxigenic <it>E. coli</it>. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as <it>E. coli</it>, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases.</p> <p>Conclusions</p> <p>The <it>L. hongkongensis </it>genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified.</p

Transport genes and chemotaxis in Laribacter hongkongensis: a genome-wide analysis

<p>Abstract</p> <p>Background</p> <p><it>Laribacter hongkongensis </it>is a Gram-negative, sea gull-shaped rod associated with community-acquired gastroenteritis. The bacterium has been found in diverse freshwater environments including fish, frogs and drinking water reservoirs. Using the complete genome sequence data of <it>L. hongkongensis</it>, we performed a comprehensive analysis of putative transport-related genes and genes related to chemotaxis, motility and quorum sensing, which may help the bacterium adapt to the changing environments and combat harmful substances.</p> <p>Results</p> <p>A genome-wide analysis using Transport Classification Database TCDB, similarity and keyword searches revealed the presence of a large diversity of transporters (n = 457) and genes related to chemotaxis (n = 52) and flagellar biosynthesis (n = 40) in the <it>L. hongkongensis </it>genome. The transporters included those from all seven major transporter categories, which may allow the uptake of essential nutrients or ions, and extrusion of metabolic end products and hazardous substances. <it>L. hongkongensis </it>is unique among closely related members of <it>Neisseriaceae </it>family in possessing higher number of proteins related to transport of ammonium, urea and dicarboxylate, which may reflect the importance of nitrogen and dicarboxylate metabolism in this assacharolytic bacterium. Structural modeling of two C^₄-dicarboxylate transporters showed that they possessed similar structures to the determined structures of other DctP-TRAP transporters, with one having an unusual disulfide bond. Diverse mechanisms for iron transport, including hemin transporters for iron acquisition from host proteins, were also identified. In addition to the chemotaxis and flagella-related genes, the <it>L. hongkongensis </it>genome also contained two copies of <it>qseB/qseC </it>homologues of the AI-3 quorum sensing system.</p> <p>Conclusions</p> <p>The large number of diverse transporters and genes involved in chemotaxis, motility and quorum sensing suggested that the bacterium may utilize a complex system to adapt to different environments. Structural modeling will provide useful insights on the transporters in <it>L. hongkongensis</it>.</p

Environmental adaptability and stress tolerance of Laribacter hongkongensis: a genome-wide analysis

<p>Abstract</p> <p>Background</p> <p><it>Laribacter hongkongensis </it>is associated with community-acquired gastroenteritis and traveler's diarrhea and it can reside in human, fish, frogs and water. In this study, we performed an in-depth annotation of the genes in its genome related to adaptation to the various environmental niches.</p> <p>Results</p> <p><it>L. hongkongensis </it>possessed genes for DNA repair and recombination, basal transcription, alternative σ-factors and 109 putative transcription factors, allowing DNA repair and global changes in gene expression in response to different environmental stresses. For acid stress, it possessed a urease gene cassette and two <it>arc </it>gene clusters. For alkaline stress, it possessed six CDSs for transporters of the monovalent cation/proton antiporter-2 and NhaC Na⁺:H⁺antiporter families. For heavy metals acquisition and tolerance, it possessed CDSs for iron and nickel transport and efflux pumps for other metals. For temperature stress, it possessed genes related to chaperones and chaperonins, heat shock proteins and cold shock proteins. For osmotic stress, 25 CDSs were observed, mostly related to regulators for potassium ion, proline and glutamate transport. For oxidative and UV light stress, genes for oxidant-resistant dehydratase, superoxide scavenging, hydrogen peroxide scavenging, exclusion and export of redox-cycling antibiotics, redox balancing, DNA repair, reduction of disulfide bonds, limitation of iron availability and reduction of iron-sulfur clusters are present. For starvation, it possessed phosphorus and, despite being asaccharolytic, carbon starvation-related CDSs.</p> <p>Conclusions</p> <p>The <it>L. hongkongensis </it>genome possessed a high variety of genes for adaptation to acid, alkaline, temperature, osmotic, oxidative, UV light and starvation stresses and acquisition of and tolerance to heavy metals.</p

Environmental adaptability and stress tolerance of Laribacter hongkongensis: a genome-wide analysis

<p>Abstract</p> <p>Background</p> <p><it>Laribacter hongkongensis </it>is associated with community-acquired gastroenteritis and traveler's diarrhea and it can reside in human, fish, frogs and water. In this study, we performed an in-depth annotation of the genes in its genome related to adaptation to the various environmental niches.</p> <p>Results</p> <p><it>L. hongkongensis </it>possessed genes for DNA repair and recombination, basal transcription, alternative σ-factors and 109 putative transcription factors, allowing DNA repair and global changes in gene expression in response to different environmental stresses. For acid stress, it possessed a urease gene cassette and two <it>arc </it>gene clusters. For alkaline stress, it possessed six CDSs for transporters of the monovalent cation/proton antiporter-2 and NhaC Na⁺:H⁺antiporter families. For heavy metals acquisition and tolerance, it possessed CDSs for iron and nickel transport and efflux pumps for other metals. For temperature stress, it possessed genes related to chaperones and chaperonins, heat shock proteins and cold shock proteins. For osmotic stress, 25 CDSs were observed, mostly related to regulators for potassium ion, proline and glutamate transport. For oxidative and UV light stress, genes for oxidant-resistant dehydratase, superoxide scavenging, hydrogen peroxide scavenging, exclusion and export of redox-cycling antibiotics, redox balancing, DNA repair, reduction of disulfide bonds, limitation of iron availability and reduction of iron-sulfur clusters are present. For starvation, it possessed phosphorus and, despite being asaccharolytic, carbon starvation-related CDSs.</p> <p>Conclusions</p> <p>The <it>L. hongkongensis </it>genome possessed a high variety of genes for adaptation to acid, alkaline, temperature, osmotic, oxidative, UV light and starvation stresses and acquisition of and tolerance to heavy metals.</p

Development of a multi-locus sequence typing scheme for Laribacter hongkongensis, a novel bacterium associated with freshwater fish-borne gastroenteritis and traveler's diarrhea

<p>Abstract</p> <p>Background</p> <p>Laribacter hongkongensis is a newly discovered, facultative anaerobic, Gram-negative, motile, sea gull-shaped rod associated with freshwater fish borne gastroenteritis and traveler's diarrhea. A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of <it>L. hongkongensis</it>. In this study, a multilocus sequence typing (MLST) system was developed for <it>L. hongkongensis</it>. The system was used to characterize 146 <it>L. hongkongensis </it>isolates, including 39 from humans and 107 from fish.</p> <p>Results</p> <p>Fragments (362 to 504 bp) of seven housekeeping genes were amplified and sequenced. Among the 3068 bp of the seven loci, 332 polymorphic sites were observed. The median number of alleles at each locus was 34 [range 22 (<it>ilvC</it>) to 45 (<it>thiC</it>)]. All seven genes showed very low <it>d</it>_<it>n</it>/<it>d</it>_{<it>s </it>}ratios of < 0.04, indicating that no strong positive selective pressure is present. A total of 97 different sequence types (STs) were assigned to the 146 isolates, with 80 STs identified only once. The overall discriminatory power was 0.9861. eBURST grouped the isolates into 12 lineages, with six groups containing only isolates from fish and three groups only isolates from humans. Standardized index of association (<it>I</it>^<it>S</it>_<it>A</it>) measurement showed significant linkage disequilibrium in isolates from both humans and fish. The <it>I</it>^<it>S</it>_{<it>A </it>}for the isolates from humans and fish were 0.270 and 0.636, indicating the isolates from fish were more clonal than the isolates from humans. Only one interconnected network (<it>acnB</it>) was detected in the split graphs. The P-value (P = 0) of sum of the squares of condensed fragments in Sawyer's test showed evidence of intragenic recombination in the <it>rho, acnB </it>and <it>thiC </it>loci, but the P-value (P = 1) of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. Congruence analysis showed that all the pairwise comparisons of the 7 MLST loci were incongruent, indicating that recombination played a substantial role in the evolution of <it>L. hongkongensis</it>. A website for <it>L. hongkongensis </it>MLST was set up and can be accessed at <url>http://mlstdb.hku.hk:14206/MLST_index.html</url>.</p> <p>Conclusion</p> <p>A highly reproducible and discriminative MLST system was developed for <it>L. hongkongensis</it>.</p

Pan-RAF and MEK vertical inhibition enhances therapeutic response in non-V600 BRAF mutant cells

BACKGROUND: Currently, there are no available targeted therapy options for non-V600 BRAF mutated tumors. The aim of this study was to investigate the effects of RAF and MEK concurrent inhibition on tumor growth, migration, signaling and apoptosis induction in preclinical models of non-V600 BRAF mutant tumor cell lines. METHODS: Six BRAF mutated human tumor cell lines CRL5885 (G466 V), WM3629 (D594G), WM3670 (G469E), MDAMB231 (G464 V), CRL5922 (L597 V) and A375 (V600E as control) were investigated. Pan-RAF inhibitor (sorafenib or AZ628) and MEK inhibitor (selumetinib) or their combination were used in in vitro viability, video microscopy, immunoblot, cell cycle and TUNEL assays. The in vivo effects of the drugs were assessed in an orthotopic NSG mouse breast cancer model. RESULTS: All cell lines showed a significant growth inhibition with synergism in the sorafenib/AZ628 and selumetinib combination. Combination treatment resulted in higher Erk1/2 inhibition and in increased induction of apoptosis when compared to single agent treatments. However, single selumetinib treatment could cause adverse therapeutic effects, like increased cell migration in certain cells, selumetinib and sorafenib combination treatment lowered migratory capacity in all the cell lines. Importantly, combination resulted in significantly increased tumor growth inhibition in orthotropic xenografts of MDAMB231 cells when compared to sorafenib - but not to selumetinib - treatment. CONCLUSIONS: Our data suggests that combined blocking of RAF and MEK may achieve increased therapeutic response in non-V600 BRAF mutant tumors

Clinical Characteristics and Transmission of COVID-19 in Children and Youths During 3 Waves of Outbreaks in Hong Kong

IMPORTANCE: Schools were closed intermittently across Hong Kong to control the COVID-19 outbreak, which led to significant physical and psychosocial problems among children and youths. OBJECTIVE: To compare the clinical characteristics and sources of infection among children and youths with COVID-19 during the 3 waves of outbreaks in Hong Kong in 2020. DESIGN, SETTING AND PARTICIPANTS: This cross-sectional study involved children and youths aged 18 years or younger with COVID-19 in the 3 waves of outbreaks from January 23 through December 2, 2020. Data were analyzed from December 2020 through January 2021. MAIN OUTCOMES AND MEASURES: Demographic characteristics, travel and contact histories, lengths of hospital stay, and symptoms were captured through the central electronic database. Individuals who were infected without recent international travel were defined as having domestic infections. RESULTS: Among 397 children and youths confirmed with COVID-19 infections, the mean (SD) age was 9.95 (5.34) years, 220 individuals (55.4%) were male, and 154 individuals (38.8%) were asymptomatic. There were significantly more individuals who were infected without symptoms in the second wave (59 of 118 individuals [50.0%]) and third wave (94 of 265 individuals [35.5%]) than in the first wave (1 of 14 individuals [7.1%]) (P = .001). Significantly fewer individuals who were infected in the second and third waves, compared with the first wave, had fever (first wave: 10 individuals [71.4%]; second wave: 22 individuals [18.5%]; third wave: 98 individuals [37.0%]; P < .001) or cough (first wave: 6 individuals [42.9%]; second wave: 15 individuals [12.7%]; third wave: 52 individuals [19.6%]; P = .02). Among all individuals, 394 individuals (99.2%) had mild illness. One patient developed chilblains (ie, COVID toes), 1 patient developed multisystem inflammatory syndrome in children, and 1 patient developed post–COVID-19 autoimmune hemolytic anemia. In all 3 waves, 204 patients with COVID-19 (51.4%) had domestic infections. Among these individuals, 186 (91.2%) reported having a contact history with another individual with COVID-19, of which most (183 individuals [90.0%]) were family members. In the third wave, 18 individuals with domestic infections had unknown contact histories. Three schoolmates were confirmed with COVID-19 on the same day and were reported to be close contacts. CONCLUSIONS AND RELEVANCE: his cross-sectional study found that nearly all children and youths with COVID-19 in Hong Kong had mild illness. These findings suggest that household transmission was the main source of infection for children and youths with domestic infections and that the risk of being infected at school was small

Epidemiology of Acute Myocarditis/Pericarditis in Hong Kong Adolescents Following Comirnaty Vaccination

BACKGROUND: Age-specific incidence of acute myocarditis/pericarditis in adolescents following Comirnaty vaccination in Asia is lacking. This study aimed to study the clinical characteristics and incidence of acute myocarditis/pericarditis among Hong Kong adolescents following Comirnaty vaccination. METHODS: This is a population cohort study in Hong Kong that monitored adverse events following immunization through a pharmacovigilance system for COVID-19 vaccines. All adolescents aged between 12 and 17 years following Comirnaty vaccination were monitored under the COVID-19 vaccine Adverse Event Response and Evaluation Programme. The clinical characteristics and overall incidence of acute myocarditis/pericarditis in adolescents following Comirnaty vaccination were analysed. RESULTS: Between 14 June 2021 and 4 September 2021, 33 Chinese adolescents who developed acute myocarditis/pericarditis following Comirnaty vaccination were identified. 29 (87.88%) were males and 4 (12.12%) were females, with a median age of 15.25 years. 27 (81.82%) and 6 (18.18%) cases developed acute myocarditis/pericarditis after receiving the second and first dose, respectively. All cases are mild and required only conservative management.The overall incidence of acute myocarditis/pericarditis was 18.52 (95% Confidence Interval [CI], 11.67-29.01) per 100,000 persons vaccinated. The incidence after the first and second doses were 3.37 (95%CI 1.12-9.51) and 21.22 (95%CI 13.78-32.28 per 100,000 persons vaccinated, respectively. Among male adolescents, the incidence after the first and second doses were 5.57 (95% CI 2.38-12.53) and 37.32 (95% CI 26.98-51.25) per 100,000 persons vaccinated. CONCLUSIONS: There is a significant increase in the risk of acute myocarditis/pericarditis following Comirnaty vaccination among Chinese male adolescents, especially after the second dose

Plato's Cave Algorithm: Inferring Functional Signaling Networks from Early Gene Expression Shadows

Improving the ability to reverse engineer biochemical networks is a major goal of systems biology. Lesions in signaling networks lead to alterations in gene expression, which in principle should allow network reconstruction. However, the information about the activity levels of signaling proteins conveyed in overall gene expression is limited by the complexity of gene expression dynamics and of regulatory network topology. Two observations provide the basis for overcoming this limitation: a. genes induced without de-novo protein synthesis (early genes) show a linear accumulation of product in the first hour after the change in the cell's state; b. The signaling components in the network largely function in the linear range of their stimulus-response curves. Therefore, unlike most genes or most time points, expression profiles of early genes at an early time point provide direct biochemical assays that represent the activity levels of upstream signaling components. Such expression data provide the basis for an efficient algorithm (Plato's Cave algorithm; PLACA) to reverse engineer functional signaling networks. Unlike conventional reverse engineering algorithms that use steady state values, PLACA uses stimulated early gene expression measurements associated with systematic perturbations of signaling components, without measuring the signaling components themselves. Besides the reverse engineered network, PLACA also identifies the genes detecting the functional interaction, thereby facilitating validation of the predicted functional network. Using simulated datasets, the algorithm is shown to be robust to experimental noise. Using experimental data obtained from gonadotropes, PLACA reverse engineered the interaction network of six perturbed signaling components. The network recapitulated many known interactions and identified novel functional interactions that were validated by further experiment. PLACA uses the results of experiments that are feasible for any signaling network to predict the functional topology of the network and to identify novel relationships