Establishment of a Mouse Model of Mycoplasma pneumoniae-Induced Plastic Bronchitis.

Plastic bronchitis (PB) constitutes a life-threatening pulmonary disorder, predominantly attributed to Mycoplasma pneumoniae (MP) infection. The pathogenic mechanisms involved remain largely unexplored, leading to the absence of reliable approaches for early diagnosis and clear treatment. Thus, the present investigation aimed to develop an MP-induced mouse model of PB, thereby enhancing our understanding of this complex condition. In the first stage, healthy BALB/c mice were utilized to investigate the optimal methods for establishing PB. This involved the application of nebulization (15-20 min) and intratracheal administration (6-50 µL) with 2-chloroethyl ethyl sulfide (CEES) concentrations ranging from 4.5% to 7.5%. Subsequently, the MP model was induced by administering an MP solution (2 mL/kg/day, 108 CFU/50 µL) via the intranasal route for a duration of five consecutive days. Ultimately, suitable techniques were employed to induce plastic bronchitis in the MP model. Pathological changes in lung tissue were analyzed, and immunohistochemistry was employed to ascertain the expression levels of vascular endothelial growth factor receptor 3 (VEGFR-3) and the PI3K/AKT/mTOR signaling pathway. The administration of 4.5% CEES via a 6 µL trachea was the optimal approach to establishing a PB model. This method primarily induced neutrophilic inflammation and fibrinous exudate. The MP-infected group manifested symptoms indicative of respiratory infection, including erect hair, oral and nasal secretions, and a decrease in body weight. Furthermore, the pathological score of the MP+CEES group surpassed that of the groups treated with MP or CEES independently. Notably, the MP+CEES group demonstrated significant activation of the VEGFR-3 and PI3K/AKT/mTOR signaling pathways, implying a substantial involvement of lymphatic vessel impairment in this pathology. This study successfully established a mouse model of PB induced by MP using a two-step method. Lymphatic vessel impairment is a pivotal element in the pathogenetic mechanisms underlying this disease entity. This accomplishment will aid in further research into treatment methods for patients with PB caused by MP.

Antigenic Characterization of Circulating and Emerging SARS-CoV-2 Variants in the U.S. throughout the Delta to Omicron Waves.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages with unique spike mutations and caused multiple epidemics domestically and globally. Although COVID-19 vaccines are available, new variants with the capacity for immune evasion continue to emerge. To understand and characterize the evolution of circulating SARS-CoV-2 variants in the U.S., the Centers for Disease Control and Prevention (CDC) initiated the National SARS-CoV-2 Strain Surveillance (NS3) program and has received thousands of SARS-CoV-2 clinical specimens from across the nation as part of a genotype to phenotype characterization process. Focus reduction neutralization with various antisera was used to antigenically characterize 143 SARS-CoV-2 Delta, Mu and Omicron subvariants from selected clinical specimens received between May 2021 and February 2023, representing a total of 59 unique spike protein sequences. BA.4/5 subvariants BU.1, BQ.1.1, CR.1.1, CQ.2 and BA.4/5 + D420N + K444T; BA.2.75 subvariants BM.4.1.1, BA.2.75.2, CV.1; and recombinant Omicron variants XBF, XBB.1, XBB.1.5 showed the greatest escape from neutralizing antibodies when analyzed against post third-dose original monovalent vaccinee sera. Post fourth-dose bivalent vaccinee sera provided better protection against those subvariants, but substantial reductions in neutralization titers were still observed, especially among BA.4/5 subvariants with both an N-terminal domain (NTD) deletion and receptor binding domain (RBD) substitutions K444M + N460K and recombinant Omicron variants. This analysis demonstrated a framework for long-term systematic genotype to antigenic characterization of circulating and emerging SARS-CoV-2 variants in the U.S., which is critical to assessing their potential impact on the effectiveness of current vaccines and antigen recommendations for future updates.

Ultrasound-guided stellate ganglion block benefits the postoperative recovery of patients undergoing laparoscopic colorectal surgery: a single-center, double-blinded, randomized controlled clinical trial.

BACKGROUND: With the increasing prevalence of colorectal cancer (CRC), optimizing perioperative management is of paramount importance. This study investigates the potential of stellate ganglion block (SGB), known for its stress response-mediating effects, in improving postoperative recovery. We postulate that preoperative SGB may enhance the postoperative recovery of patients undergoing laparoscopic CRC surgery. METHODS: We conducted a randomized controlled trial of 57 patients undergoing laparoscopic colorectal cancer surgery at a single center. Patients, aged 18-70 years, were randomly assigned to receive either preoperative SGB or standard care. SGB group patients received 10 mL of 0.2% ropivacaine under ultrasound guidance prior to surgery. Primary outcome was time to flatus, with secondary outcomes encompassing time to defecation, lying in bed time, visual analog scale (VAS) pain score, hospital stays, patient costs, intraoperative and postoperative complications, and 3-year mortality. A per-protocol analysis was used. RESULTS: Twenty-nine patients in the SGB group and 28 patients in the control group were analyzed. The SGB group exhibited a significantly shorter time to flatus (mean [SD] hour, 20.52 [9.18] vs. 27.93 [11.69]; p = 0.012), accompanied by decreased plasma cortisol levels (mean [SD], postoperatively, 4.01 [3.42] vs 7.75 [3.13], p = 0.02). Notably, postoperative pain was effectively managed, evident by lower VAS scores at 6 h post-surgery in SGB-treated patients (mean [SD], 4.70 [0.91] vs 5.35 [1.32]; p = 0.040). Furthermore, patients in the SGB group experienced reduced hospital stay length (mean [SD], day, 6.61 [1.57] vs 8.72 [5.13], p = 0.042). CONCLUSIONS: Preoperative SGB emerges as a promising approach to enhance the postoperative recovery of patients undergoing laparoscopic CRC surgery. CLINICAL TRIAL REGISTRATION: ChiCTR1900028404, Principal investigator: Xia Feng, Date of registration: 12/20/2019.

Remodeling of the Intra-Conduit Inflammatory Microenvironment to Improve Peripheral Nerve Regeneration with a Neuromechanical Matching Protein-Based Conduit.

Peripheral nerve injury (PNI) remains a challenging area in regenerative medicine. Nerve guide conduit (NGC) transplantation is a common treatment for PNI, but the prognosis of NGC treatment is unsatisfactory due to 1) neuromechanical unmatching and 2) the intra-conduit inflammatory microenvironment (IME) resulting from Schwann cell pyroptosis and inflammatory-polarized macrophages. A neuromechanically matched NGC composed of regenerated silk fibroin (RSF) loaded with poly(3,4-ethylenedioxythiophene): poly(styrene sulfonate) (P:P) and dimethyl fumarate (DMF) are designed, which exhibits a matched elastic modulus (25.1 ± 3.5 MPa) for the peripheral nerve and the highest 80% elongation at break, better than most protein-based conduits. Moreover, the NGC can gradually regulate the intra-conduit IME by releasing DMF and monitoring sciatic nerve movements via piezoresistive sensing. The combination of NGC and electrical stimulation modulates the IME to support PNI regeneration by synergistically inhibiting Schwann cell pyroptosis and reducing inflammatory factor release, shifting macrophage polarization from the inflammatory M1 phenotype to the tissue regenerative M2 phenotype and resulting in functional recovery of neurons. In a rat sciatic nerve crush model, NGC promoted remyelination and functional and structural regeneration. Generally, the DMF/RSF/P:P conduit provides a new potential therapeutic approach to promote nerve repair in future clinical treatments.

Biomimetic Structural Protein Based Magnetic Responsive Scaffold for Enhancing Bone Regeneration by Physical Stimulation on Intracellular Calcium Homeostasis.

The bone matrix has distinct architecture and biochemistry which present a barrier to synthesizing bone-mimetic regenerative scaffolds. To mimic the natural structures and components of bone, biomimetic structural decellularized extracellular matrix (ECM)/regenerated silk fibroin (RSF) scaffolds incorporated with magnetic nanoparticles (MNP) are prepared using a facile synthetic methodology. The ECM/RSF/MNP scaffold is a hierarchically organized and interconnected porous structure with silk fibroin twined on the collagen nanofibers. The scaffold demonstrates saturation magnetization due to the presence of MNP, along with good cytocompatibility. Moreover, the ß-sheet crystalline domain of RSF and the chelated MNP could mimic the deposition of hydroxyapatite and enhance compressive modulus of the scaffold by ≈20%. The results indicate that an external static magnetic field (SMF) with a magnetic responsive scaffold effectively promotes cell migration, osteogenic differentiation, neogenesis of endotheliocytes in vitro, and new bone formation in a critical-size femur defect rat model. RNA sequencing reveals that the molecular mechanisms underlying this osteogenic effect involve calsequestrin-2-mediated Ca2+ release from the endoplasmic reticulum to activate Ca2+ /calmodulin/calmodulin-dependent kinase II signaling axis. Collectively, bionic magnetic scaffolds with SMF stimulation provide a potent strategy for bone regeneration through internal structural cues, biochemical composition, and external physical stimulation on intracellular Ca2+ homeostasis.

Antiviral susceptibility of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses isolated from birds and mammals in the United States, 2022.

Clade 2.3.4.4b highly pathogenic avian influenza (HPAI) A(H5N1) viruses that are responsible for devastating outbreaks in birds and mammals pose a potential threat to public health. Here, we evaluated their susceptibility to influenza antivirals. Of 1,015 sequences of HPAI A(H5N1) viruses collected in the United States during 2022, eight viruses (∼0.8%) had a molecular marker of drug resistance to an FDA-approved antiviral: three adamantane-resistant (M2-V27A), four oseltamivir-resistant (NA-H275Y), and one baloxavir-resistant (PA-I38T). Additionally, 31 viruses contained mutations that may reduce susceptibility to inhibitors of neuraminidase (NA) (n = 20) or cap-dependent endonuclease (CEN) (n = 11). A panel of 22 representative viruses was tested phenotypically. Overall, clade 2.3.4.4b A(H5N1) viruses lacking recognized resistance mutations were susceptible to FDA-approved antivirals. Oseltamivir was least potent at inhibiting NA activity, while the investigational NA inhibitor AV5080 was most potent, including against NA mutants. A novel NA substitution T438N conferred 12-fold reduced inhibition by zanamivir, and in combination with the known marker N295S, synergistically affected susceptibility to all five NA inhibitors. In cell culture-based assays HINT and IRINA, the PA-I38T virus displayed 75- to 108-fold and 37- to 78-fold reduced susceptibility to CEN inhibitors, baloxavir and the investigational AV5116, respectively. Viruses with PA-I38M or PA-A37T showed 5- to 10-fold reduced susceptibilities. As HPAI A(H5N1) viruses continue to circulate and evolve, close monitoring of drug susceptibility is needed for risk assessment and to inform decisions regarding antiviral stockpiling.

Piezoresistive MXene/Silk fibroin nanocomposite hydrogel for accelerating bone regeneration by Re-establishing electrical microenvironment.

The electrical microenvironment plays an important role in bone repair. However, the underlying mechanism by which electrical stimulation (ES) promotes bone regeneration remains unclear, limiting the design of bone microenvironment-specific electroactive materials. Herein, by simple co-incubation in aqueous suspensions at physiological temperatures, biocompatible regenerated silk fibroin (RSF) is found to assemble into nanofibrils with a ß-sheet structure on MXene nanosheets, which has been reported to inhibit the restacking and oxidation of MXene. An electroactive hydrogel based on RSF and bioencapsulated MXene is thus prepared to promote efficient bone regeneration. This MXene/RSF hydrogel also acts as a piezoresistive pressure transducer, which can potentially be utilized to monitor the electrophysiological microenvironment. RNA sequencing is performed to explore the underlying mechanisms, which can activate Ca2+/CALM signaling in favor of the direct osteogenesis process. ES is found to facilitate indirect osteogenesis by promoting the polarization of M2 macrophages, as well as stimulating the neogenesis and migration of endotheliocytes. Consistent improvements in bone regeneration and angiogenesis are observed with MXene/RSF hydrogels under ES in vivo. Collectively, the MXene/RSF hydrogel provides a distinctive and promising strategy for promoting direct osteogenesis, regulating immune microenvironment and neovascularization under ES, leading to re-establish electrical microenvironment for bone regeneration.

Differential neutralization and inhibition of SARS-CoV-2 variants by antibodies elicited by COVID-19 mRNA vaccines.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccine-mediated protection from disease. To ascertain and rank the risk of VOCs and VOIs, we analyze the ability of 14 variants (614G, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, Mu, and Omicron) to escape from mRNA vaccine-induced antibodies. The variants show differential reductions in neutralization and replication by post-vaccination sera. Although the Omicron variant (BA.1, BA.1.1, and BA.2) shows the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retain moderate neutralizing activity against that variant. Therefore, vaccination remains an effective strategy during the COVID-19 pandemic.

Defining the risk of SARS-CoV-2 variants on immune protection.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced after infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases within the National Institutes of Health established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants that could potentially affect the transmission, virulence, and resistance to infection- and vaccine-induced immunity. The SAVE programme is a critical data-generating component of the US Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines and therapeutics, and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models and notable findings facilitated by this collaborative approach and identify future challenges. This programme is a template for the response to rapidly evolving pathogens with pandemic potential by monitoring viral evolution in the human population to identify variants that could reduce the effectiveness of countermeasures.

Injectable Mussel-Inspired highly adhesive hydrogel with exosomes for endogenous cell recruitment and cartilage defect regeneration.

In the early stage of osteoarthritis (OA), cartilage degradation in the surface region leads to superficial cartilage defect. However, enhancing the regeneration of cartilage defect remains a great challenge for existing hydrogel technology because of the weak adhesion to wet tissue. In the present study, an injectable mussel-inspired highly adhesive hydrogel with exosomes was investigated for endogenous cell recruitment and cartilage defect regeneration. The hydrogel with high bonding strength to the wet surface was prepared using a crosslinked network of alginate-dopamine, chondroitin sulfate, and regenerated silk fibroin (AD/CS/RSF). Compared with commercial enbucrilate tissue adhesive, the AD/CS/RSF hydrogel provided a comparative lap shear strength of 120 kPa, with a similar gelation time and a higher capacity for maintaining adhesive strength. The AD/CS/RSF/EXO hydrogel with encapsulated exosomes recruited BMSCs migration and inflation, promoted BMSCs proliferation and differentiation. Most importantly, the AD/CS/RSF/EXO hydrogel accelerated cartilage defect regeneration in situ, and extracellular matrix remodeling after injection in rat patellar grooves. The exosomes released by the hydrogels could recruit BMSCs into the hydrogel and neo-cartilage via the chemokine signaling pathway. Our findings reveal an injectable and adhesive hydrogel for superficial cartilage regeneration, which is a promising approach for minimally treating cartilage defect with arthroscopic assistance.

Cardiac Derived CD51-Positive Mesenchymal Stem Cells Enhance the Cardiac Repair Through SCF-Mediated Angiogenesis in Mice With Myocardial Infarction.

Objective: Many tissues contained resident mesenchymal stromal/stem cells (MSCs) that facilitated tissue hemostasis and repair. However, there is no typical marker to identify the resident cardiac MSCs. We aimed to determine if CD51 could be an optimal marker of cardiac MSCs and assess their therapeutic potential for mice with acute myocardial infarction (AMI). Methods: Cardiac-derived CD51+CD31-CD45-Ter119- cells (named CD51+cMSCs) were isolated from C57BL/6 mice(7-day-old) by flow cytometry. The CD51+cMSCs were characterized by proliferation capacity, multi-differentiation potential, and expression of typical MSC-related markers. Adult C57BL/6 mice (12-week-old) were utilized for an AMI model via permanently ligating the left anterior descending coronary artery. The therapeutic efficacy of CD51+cMSCs was estimated by echocardiography and pathological staining. To determine the underlying mechanism, lentiviruses were utilized to knock down gene (stem cell factor [SCF]) expression of CD51+cMSCs. Results: In this study, CD51 was expressed in the entire layers of the cardiac wall in mice, including endocardium, epicardium, and myocardium, and its expression was decreased with age. Importantly, the CD51+cMSCs possessed potent self-renewal potential and multi-lineage differentiation capacity in vitro and also expressed typical MSC-related surface proteins. Furthermore, CD51+cMSC transplantation significantly improved cardiac function and attenuated cardiac fibrosis through pro-angiogenesis activity after myocardial infarction in mice. Moreover, SCF secreted by CD51+cMSCs played an important role in angiogenesis both in vivo and in vitro. Conclusions: Collectively, CD51 is a novel marker of cardiac resident MSCs, and CD51+cMSC therapy enhances cardiac repair at least partly through SCF-mediated angiogenesis.

Pulmonary Metastasectomy in Patients with Lung Metastases from Nasopharyngeal Carcinoma.

BACKGROUND: Approximately 10% of patients with nasopharyngeal carcinoma (NPC) develop lung-only metastases. Data regarding the potential role of lung metastasectomy are limited. OBJECTIVE: The aim of this case-control study was to determine whether lung metastasectomy prolongs survival in patients with NPC with lung-only metastases. METHODS: The resectability of 215 consecutive patients diagnosed with lung-only metastases from 2001 to 2018 was reviewed by doctors blinded to the patient groups. The propensity score matching method was used to balance the potential probability of being assigned to treatment groups based on pretherapeutic information. Postmetastatic survival (PMS) and cumulative incidence of local failure were compared between the surgical and nonsurgical arms. RESULTS: Overall, 120 potentially resectable cases were enrolled, and 45 and 22 patients who underwent and did not undergo metastasectomy, respectively, were included in the propensity-matched cohort. Patients who underwent pulmonary resection had better PMS and a lower cumulative incidence of local failure than those who did not undergo surgery. The 5-year PMS rates were 75.53% and 47.81% in the surgical and nonsurgical arms, respectively (difference 27.72%; 95% confidence interval 3.95-51.49%). Younger patients (≤ 45 years), and those with a lower primary N stage (N0-1), longer disease-free interval (> 2 years), smaller total diameter of the metastatic lesions (≤ 3 cm), unilateral distribution of metastases, no mediastinal/hilar lymph node involvement, and adjuvant chemotherapy showed a significantly longer PMS after metastasectomy by multivariable analysis. CONCLUSIONS: Lung metastasectomy may improve PMS and decrease the chance of local treatment failure in NPC patients with potentially resectable lung-only metastases.

Genetically and Antigenically Divergent Influenza A(H9N2) Viruses Exhibit Differential Replication and Transmission Phenotypes in Mammalian Models.

Low-pathogenicity avian influenza A(H9N2) viruses, enzootic in poultry populations in Asia, are associated with fewer confirmed human infections but higher rates of seropositivity compared to A(H5) or A(H7) subtype viruses. Cocirculation of A(H5) and A(H7) viruses leads to the generation of reassortant viruses bearing A(H9N2) internal genes with markers of mammalian adaptation, warranting continued surveillance in both avian and human populations. Here, we describe active surveillance efforts in live poultry markets in Vietnam in 2018 and compare representative viruses to G1 and Y280 lineage viruses that have infected humans. Receptor binding properties, pH thresholds for HA activation, in vitro replication in human respiratory tract cells, and in vivo mammalian pathogenicity and transmissibility were investigated. While A(H9N2) viruses from both poultry and humans exhibited features associated with mammalian adaptation, one human isolate from 2018, A/Anhui-Lujiang/39/2018, exhibited increased capacity for replication and transmission, demonstrating the pandemic potential of A(H9N2) viruses.IMPORTANCE A(H9N2) influenza viruses are widespread in poultry in many parts of the world and for over 20 years have sporadically jumped species barriers to cause human infection. As these viruses continue to diversify genetically and antigenically, it is critical to closely monitor viruses responsible for human infections, to ascertain if A(H9N2) viruses are acquiring properties that make them better suited to infect and spread among humans. In this study, we describe an active poultry surveillance system established in Vietnam to identify the scope of influenza viruses present in live bird markets and the threat they pose to human health. Assessment of a recent A(H9N2) virus isolated from an individual in China in 2018 is also reported, and it was found to exhibit properties of adaptation to humans and, importantly, it shows similarities to strains isolated from the live bird markets of Vietnam.

Characteristics of Human OAS1 Isoform Proteins.

The human OAS1 (hOAS1) gene produces multiple possible isoforms due to alternative splicing events and sequence variation among individuals, some of which affect splicing. The unique C-terminal sequences of the hOAS1 isoforms could differentially affect synthetase activity, protein stability, protein partner interactions and/or cellular localization. Recombinant p41, p42, p44, p46, p48, p49 and p52 hOAS1 isoform proteins expressed in bacteria were each able to synthesize trimer and higher order 2'-5' linked oligoadenylates in vitro in response to poly(I:C). The p42, p44, p46, p48 and p52 isoform proteins were each able to induce RNase-mediated rRNA cleavage in response to poly(I:C) when overexpressed in HEK293 cells. The expressed levels of the p42 and p46 isoform proteins were higher than those of the other isoforms, suggesting increased stability in mammalian cells. In a yeast two-hybrid screen, Fibrillin1 (FBN1) was identified as a binding partner for hOAS1 p42 isoform, and Supervillin (SVIL) as a binding partner for the p44 isoform. The p44-SVIL interaction was supported by co-immunoprecipitation data from mammalian cells. The data suggest that the unique C-terminal regions of hOAS1 isoforms may mediate the recruitment of different partners, alternative functional capacities and/or different cellular localization.

Novel prognostic model for stratifying survival in stage I lung adenocarcinoma patients.

PURPOSE: We combined conventional clinical and pathological characteristics and pathological architectural grading scores to develop a prognostic model to identify a specific group of patients with stage I lung adenocarcinomas with poor survival following surgery. METHODS: This retrospective study included 198 patients with stage I lung adenocarcinomas recruited from 2004 to 2013. Multivariate analyses were used to confirm independent risk factors, which were checked for internal validity using the bootstrapping method. The prognostic scores, derived from ß-coefficients using the Cox regression model, classified patients into high- and low-risk groups. The predictive performance and discriminative ability of the model were assessed by the area under the receiver operating characteristic curve (AUC), concordance index (C-index) and Kaplan-Meier survival analyses. RESULTS: Three risk factors were identified: T2 (rounding of ß-coefficients = 81), necrosis (rounding of ß-coefficients = 67), and pathological architectural score of 5-6 (rounding of ß-coefficients = 58). The final prognostic score was the sum of points. The derived prognostic scores stratified patients into low- (score ≤ 103) and high- (score > 103) risk groups, with significant differences in 5-year overall survival (high vs. low risk: 49.3% vs. 88.0%, respectively; hazard ratio: 4.55; p < 0.001). The AUC for the proposed model was 0.717. The C-index of the model was 0.693. CONCLUSION: An integrated prognostic model was developed to discriminate resected stage I adenocarcinoma patients into low- and high-risk groups, which will help clinicians select individual treatment strategies.

Survival of stage II nasopharyngeal carcinoma patients with or without concurrent chemotherapy: A propensity score matching study.

BACKGROUND: To ascertain if concurrent chemotherapy (CCT) benefits people with stage II nasopharyngeal carcinoma (NPC) treated with two-dimensional radiotherapy (2DRT) or intensity-modulated radiotherapy (IMRT). METHODS: A total of 4157 patients diagnosed with stage II NPC were evaluated. Patients received radiotherapy (RT) with/without CCT. Patients were divided into 2DRT and IMRT subgroups. After propensity score matching, the role of CCT was explored in these two subgroups. Overall survival (OS) was the primary endpoint and progression-free survival (PFS), locoregional relapse-free survival (LRFS) and distant metastasis-free survival (DMFS) were secondary endpoints. RESULTS: In the 2DRT subgroup, CCT addition to RT benefited cases with T1N1/T2N1 in OS, PFS and LRFS (P < .001, P = .003 and P = .003, respectively) significantly, but no difference was observed in patients with T2N0. DMFS were similar in the two arms. CCT was a significant protective factor for OS, PFS, and LRFS for patients with stage N1. In the IMRT subgroup, RT alone could maintain equivalent OS, PFS, LRFS and DMFS (P = .209, .448, .477 and .602 respectively) and cause less acute toxicity compared with concurrent chemoradiotherapy (CCRT). CONCLUSION: CCRT was better than 2DRT alone among patients with T1-2N1M0 stage. CCT application for NPC patients receiving IMRT led to no survival benefit and greater toxic effects.

Detection of highly pathogenic avian influenza A(H5N6) viruses in waterfowl in Bangladesh.

Bangladesh has reported repeated outbreaks of highly pathogenic avian influenza (HPAI) A(H5) viruses in poultry since 2007. Because of the large number of live poultry markets (LPM) relative to the population density of poultry throughout the country, these markets can serve as sentinel sites for HPAI A(H5) detection. Through active LPM surveillance during June 2016-June 2017, HPAI A(H5N6) viruses along with 14 other subtypes of influenza A viruses were detected. The HPAI A(H5N6) viruses belonged to clade 2.3.4.4 and were likely introduced into Bangladesh around March 2016. Human infections with influenza clade 2.3.4.4 viruses in Bangladesh have not been identified, but the viruses had several molecular markers associated with potential human infection. Vigilant surveillance at the animal-human interface is essential to identify emerging avian influenza viruses with the potential to threaten public and animal health.

Comparison of nucleic acid extraction methods for next-generation sequencing of avian influenza A virus from ferret respiratory samples.

Influenza A virus is a negative-sense RNA virus with a segmented genome consisting of eight RNA segments. Avian influenza A virus (AIV) primarily infects avian hosts and sporadically infects mammals, which can lead to adaptation to new species. Next-generation sequencing (NGS) of emerging AIV genomes extracted from respiratory samples collected on sequential days from animal models and clinical patients enables analysis of the emergence of evolutionary variants within the virus population over time. However, obtaining codon complete AIV genome at a sufficient coverage depth for nucleotide variant calling remains a challenge, especially from post-inoculation respiratory samples collected at late time points that have low viral titers. In this study, nasal wash samples from ferrets inoculated with different subtypes of AIV were collected on various days post-inoculation. Each nasal wash sample was aliquoted and extracted using five commercially available nucleic acid extraction methods. Extracted influenza virus RNA was amplified and NGS conducted using Illumina Mi-Seq. For each nasal wash sample, completeness of AIV genome segments and coverage depth were compared among five extraction methods. Nucleic acids extracted by MagNA pure compact RNA isolation consistently yielded codon complete sequences for all eight genome segments at the required coverage depth at each time point sampled. The study revealed that DNase treatment was critical to the amplification of influenza genome segments and the downstream success of codon complete NGS from nasal wash samples. The findings from this study can be applied to improve NGS of influenza and other RNA viruses that infect the respiratory tract and are collected from respiratory samples.