Natural Underwater Bioadhesive Offering Cohesion Modulation via Hydrogen Bond Disruptor: A Highly Injectable and in Vivo Stable Remedy for Gastric Ulcer Resolution.

Injectable bioadhesives are attractive for managing gastric ulcers through minimally invasive procedures. However, the formidable challenge is to develop bioadhesives that exhibit high injectability, rapidly adhere to lesion tissues with fast gelation, provide reliable protection in the harsh gastric environment, and simultaneously ensure stringent standards of biocompatibility. Here, a natural bioadhesive with tunable cohesion is developed based on the facile and controllable gelation between silk fibroin and tannic acid. By incorporating a hydrogen bond disruptor (urea or guanidine hydrochloride), the inherent network within the bioadhesive is disturbed, inducing a transition to a fluidic state for smooth injection (injection force <5 N). Upon injection, the fluidic bioadhesive thoroughly wets tissues, while the rapid diffusion of the disruptor triggers instantaneous in situ gelation. This orchestrated process fosters the formed bioadhesive with durable wet tissue affinity and mechanical properties that harmonize with gastric tissues, thereby bestowing long-lasting protection for ulcer healing, as evidenced through in vitro and in vivo verification. Moreover, it can be conveniently stored (≥3 m) postdehydration. This work presents a promising strategy for designing highly injectable bioadhesives utilizing natural feedstocks, avoiding any safety risks associated with synthetic materials or nonphysiological gelation conditions, and offering the potential for minimally invasive application.

Development of growth selection system and pocket engineering of d-amino acid oxidase to enhance selective deamination activity toward d-phosphinothricin.

D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin ( l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d-phosphinothricin ( d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.

Isolation and antioxidant activity of peptides from Chinese hairy tofu.

Hairy tofu is a famous Chinese snack that is made from soybeans and rich in various nutrients. In order to further explore the antioxidant peptides of hairy tofu hydrolysates, seven proteases were used to hydrolyze hairy tofu. The results of in vitro radical scavenging activity showed that hairy tofu hydrolysates obtained by pancreatin exhibited the highest antioxidant activity. After Sephadex G-25 gel filtration and reversed-phase high-performance liquid chromatography (RP-HPLC), 97 peptides were identified in the most antioxidant fraction using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Among them, nine peptides were synthesized and their antioxidant activities were assessed using a H2O2-induced oxidative 293T cell model. Finally, four peptides (QCESHK, LAWNEGR, NLQGENEWDQK, and FTEMWR) at concentrations of < 50 µg/ml significantly decreased the malondialdehyde content compared with the model group, displaying in vivo antioxidant activity and low cytotoxicity. Overall, this research provided the choice of using hairy tofu peptides as antioxidant products in the pharmaceutical and food industries.

An inducible CRISPRi circuit for tunable dynamic regulation of gene expression in Saccharopolyspora erythraea.

Actinomyces are gram-positive bacteria known for their valuable secondary metabolites. Redirecting metabolic flux towards desired products in actinomycetes requires precise and dynamic regulation of gene expression. In this study, we integrated the CRISPR interference (CRISPRi) system with a cumate-inducible promoter to develop an inducible gene downregulation method in Saccharopolyspora erythraea, a prominent erythromycin-producing actinobacterium. The functionality of the cumate-inducible promoter was validated using the gusA gene as a reporter, and the successful inducible expression of the dCas9 gene was confirmed. The developed inducible CRISPRi strategy was then employed to downregulate the expression of target genes rppA in the wild-type strain NRRL2338 and sucC in the high erythromycin-producing strain E3. Through dynamic control of sucC expression, a significant enhancement in erythromycin production was achieved in strain E3. This study demonstrated the effectiveness of an inducible gene downregulation approach using CRISPRi and a cumate-inducible promoter, providing valuable insights for optimizing natural product production in actinomyces.

Recent advances in deformation-assisted microfluidic cell sorting technologies.

Cell sorting is an essential prerequisite for cell research and has great value in life science and clinical studies. Among the many microfluidic cell sorting technologies, label-free methods based on the size of different cell types have been widely studied. However, the heterogeneity in size for cells of the same type and the inevitable size overlap between different types of cells would result in performance degradation in size-based sorting. To tackle such challenges, deformation-assisted technologies are receiving more attention recently. Cell deformability is an inherent biophysical marker of cells that reflects the changes in their internal structures and physiological states. It provides additional dimensional information for cell sorting besides size. Therefore, in this review, we summarize the recent advances in deformation-assisted microfluidic cell sorting technologies. According to how the deformability is characterized and the form in which the force acts, the technologies can be divided into two categories: (1) the indirect category including transit-time-based and image-based methods, and (2) the direct category including microstructure-based and hydrodynamics-based methods. Finally, the separation performance and the application scenarios of each method, the existing challenges and future outlook are discussed. Deformation-assisted microfluidic cell sorting technologies are expected to realize greater potential in the label-free analysis of cells.

Electrochemical hydrogen evolution on Pt-based catalysts from a theoretical perspective.

Hydrogen evolution reaction (HER) by splitting water is a key technology toward a clean energy society, where Pt-based catalysts were long known to have the highest activity under acidic electrochemical conditions but suffer from high cost and poor stability. Here, we overview the current status of Pt-catalyzed HER from a theoretical perspective, focusing on the methodology development of electrochemistry simulation, catalytic mechanism, and catalyst stability. Recent developments in theoretical methods for studying electrochemistry are introduced, elaborating on how they describe solid-liquid interface reactions under electrochemical potentials. The HER mechanism, the reaction kinetics, and the reaction sites on Pt are then summarized, which provides an atomic-level picture of Pt catalyst surface dynamics under reaction conditions. Finally, state-of-the-art experimental solutions to improve catalyst stability are also introduced, which illustrates the significance of fundamental understandings in the new catalyst design.

Ammonium sulfate supplementation enhances erythromycin biosynthesis by augmenting intracellular metabolism and precursor supply in Saccharopolyspora erythraea.

In this study, the cellular metabolic mechanisms regarding ammonium sulfate supplementation on erythromycin production were investigated by employing targeted metabolomics and metabolic flux analysis. The results suggested that the addition of ammonium sulfate stimulates erythromycin biosynthesis. Targeted metabolomics analysis uncovered that the addition of ammonium sulfate during the late stage of fermentation resulted in an augmented intracellular amino acid metabolism pool, guaranteeing an ample supply of precursors for organic acids and coenzyme A-related compounds. Therefore, adequate precursors facilitated cellular maintenance and erythromycin biosynthesis. Subsequently, an optimal supplementation rate of 0.02 g/L/h was determined. The results exhibited that erythromycin titer (1311.1 µg/mL) and specific production rate (0.008 mmol/gDCW/h) were 101.3% and 41.0% higher than those of the process without ammonium sulfate supplementation, respectively. Moreover, the erythromycin A component proportion increased from 83.2% to 99.5%. Metabolic flux analysis revealed increased metabolic fluxes with the supplementation of three ammonium sulfate rates.

Comparative genomic and transcriptomic analysis guides to further enhance the biosynthesis of erythromycin by an overproducer.

Omics approaches have been applied to understand the boosted productivity of natural products by industrial high-producing microorganisms. Here, with the updated genome sequence and transcriptomic profiles derived from high-throughput sequencing, we exploited comparative omics analysis to further enhance the biosynthesis of erythromycin in an industrial overproducer, Saccharopolyspora erythraea HL3168 E3. By comparing the genome of E3 with the wild type NRRL23338, we identified fragment deletions inside 56 coding sequences and 255 single-nucleotide polymorphisms over the genome of E3. A substantial number of genomic variations were observed in genes responsible for pathways which were interconnected to the biosynthesis of erythromycin by supplying precursors/cofactors or by signal transduction. Furthermore, the transcriptomic data suggested that genes involved in the biosynthesis of erythromycin were significantly upregulated constantly, whereas some genes in biosynthesis clusters of other secondary metabolites contained nonsense mutations and were expressed at extremely low levels. Through comparative transcriptomic analysis, l-glutamine/l-glutamate and 2-oxoglutarate were identified as reporter metabolites. Around the node of 2-oxoglutarate, genomic mutations were also observed. Based on the omics association analysis, readily available strategies were proposed to engineer E3 by simultaneously overexpressing sucB (coding for 2-oxoglutarate dehydrogenase E2 component) and sucA (coding for 2-oxoglutarate dehydrogenase E1 component), which increased the erythromycin titer by 71% compared to E3 in batch culture. This study provides more promising molecular targets to engineer for enhanced production of erythromycin by the overproducer.

Cephalosporin C biosynthesis and fermentation in Acremonium chrysogenum.

Cephalosporins are currently the most widely used antibiotics in clinical practice. The main strain used for the industrial production cephalosporin C (CPC) is Acremonium chrysogenum. CPC has the advantages of possessing a broad antibacterial spectrum and strong antibacterial activity. However, the yield and titer of cephalosporins obtained from A. chrysogenum are much lower than penicillin, which is also a ß-lactam antibiotic produced by Penicillium chrysogenum. Molecular biology research into A. chrysogenum has focused on gene editing technologies, multi-omics research which has provided information on the differences between high- and low-yield strains, and metabolic engineering involving different functional genetic modifications and hierarchical network regulation to understand strain characteristics. Furthermore, optimization of the fermentation process is also reviewed as it provides the optimal environment to realize the full potential of strains. Combining rational design to control the metabolic network, high-throughput screening to improve the efficiency of obtaining high-performance strains, and real-time detection and controlling in the fermentation process will become the focus of future research in A. chrysogenum. This minireview provides a holistic and in-depth analysis of high-yield mechanisms and improves our understanding of the industrial value of A. chrysogenum. KEY POINTS: • Review of the advances in A. chrysogenum characteristics improvement and process optimization • Elucidate the molecular bases of the mechanisms that control cephalosporin C biosynthesis and gene expression in A. chrysogenum • The future development trend of A. chrysogenum to meet industrial needs.

Engineering of succinyl-CoA metabolism in view of succinylation regulation to improve the erythromycin production.

As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metabolism regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3ΔsucC was identified by using a high-resolution mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metabolism, and antibiotics biosynthesis in S. erythraea. Enzymatic experiments in vivo and in vitro were further conducted to determine the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3ΔsucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermentation. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metabolism changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sg1 was increased by 54.7% compared with E3, which was 1200.5 mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea. KEY POINTS: • We reported the first systematic profiling of the S. erythraea succinylome. • We found that the succinylation regulation on the activity of aconitase. • We enhanced the production of erythromycin by using CRISPRi to regulate the transcription of gene sucC.

Engineering the methyltransferase through inactivation of the genK and genL leads to a significant increase of gentamicin C1a production in an industrial strain of Micromonospora echinospora 49-92S.

In this study, a single-component high-yielding Micromonospora echinospora strain 49-92S-KL01 was constructed by deleting methyltransferase-encoding genes genK and genL. In 5-L fermentation trials, gentamicin C1a titers in the mutant strain were 3.22-fold higher than that in the parental strain (211 U/mL vs. 50 U/mL). The glycolysis pathway and tricarboxylic acid cycle fluxes were reduced by 26.8% and 26.6%, respectively, compared to the parental strain according to the metabolic flux analysis during the stationary phase, resulting in lower levels of energy supplements required for the cellular maintenance. Meanwhile, a significant enhancement in precursor (paromamine) accumulation and availability was observed in 49-92S-KL01 compared to parental strain. These results indicate that genK and genL significantly affect the synthesis of gentamicin C1a. In addition, this study provides a more rational strategy for gentamicin C1a production.

Ex Vivo Hydrostatic Pressure Loading of Atrial Tissues Activates Profibrotic Transcription via TGF-ß Signal Pathway.

Excessive mechanical stress causes fibrosis-related atrial arrhythmia. Herein, we tried to investigate the mechanism of atrial fibrogenesis in response to mechanical stress by ex vivo approach. We collected atrial tissues from mice and then cultured them as "explants" under atmospheric pressure (AP group) or 50 mmHg hydrostatic pressure loading (HP group) conditions. Pathway-specific PCR array analysis on the expression of fibrosis-related genes indicated that the loading of atrial tissues to 50 mmHg for 24 hours extensively upregulated a series of profibrotic genes. qRT-PCR data also showed that loading atrial tissues to 50 mmHg enhanced Rhoa, Rock2, and Thbs1 expression at different time points. Interestingly, the enhanced expression of Thbs1 at 1 hour declined at 6-24 hours and then increased again at 72 hours. In contrast, an enhanced expression of Tgfb1 was observed at 72 hours. In contrast, daily loading to 50 mmHg for 3 hours significantly accelerated the outgrowth of mesenchymal stem-like stromal cells from atrial tissues; however, we did not observe significant phenotypic changes in these outgrowing cells. Our ex vivo experimental data clearly show the induction of profibrotic transcription of atrial tissues by HP loading, which confirms the common pathological feature of atrial fibrosis following pressure overload.

Exploring the metabolic fate of propanol in industrial erythromycin-producing strain via 13C labeling experiments and enhancement of erythromycin production by rational metabolic engineering of Saccharopolyspora erythraea.

Propanol had been widely used as a precursor for erythromycin synthesis in industrial production. However, the knowledge on the exact metabolic fate of propanol was still unclear. In the present study, the metabolic fate of propanol in industrial erythromycin-producing strain Saccharopolyspora erythraea E3 was explored via 13C labeling experiments. An unexpected pathway in which propanol was channeled into tricarboxylic acid cycle was uncovered, resulting in uneconomic catabolism of propanol. By deleting the sucC gene, which encodes succinyl-CoA synthetase that catalyse a reaction in the unexpected propanol utilization pathway, a novel strain E3-ΔsucC was constructed. The strain E3-ΔsucC showed a significant enhancement in erythromycin production in the chemically defined medium compared to E3 (786.61 vs 392.94 mg/L). Isotopically nonstationary 13C metabolic flux analysis were employed to characterize the metabolic differences between Saccharopolyspora erythraea E3 and E3-ΔsucC. The results showed that compared with the starting strain E3, the fluxes of pentose phosphate pathway in E3-△sucC increased by almost 200%. The flux of the metabolic reaction catalyzed by succinyl-CoA synthetase in E3-ΔsucC was almost zero, while the glyoxylate bypass flux significantly increased. These new insights into the precursor utilization of antibiotic biosynthesis by rational metabolic engineering in Saccharopolyspora erythraea provided the new vision in increasing industrial production of secondary metabolites.

Effect of Helicobacter Pylori on Plasma Metabolic Phenotype in Patients With Gastric Cancer.

BACKGROUND: Although Helicobacter pylori (Hp) as high risk factor for gastric cancer have been investigated from human trial, present data is inadequate to explain the effect of Hp on the changes of metabolic phenotype of gastric cancer in different stages. PURPOSE: Herein, plasma of human superficial gastritis (Hp negative and positive), early gastric cancer and advanced gastric cancer analyzed by UPLC-HDMS metabolomics can not only reveal metabolic phenotype changes in patients with gastric cancer of different degrees (30 Hp negative, 30 Hp positive, 20 early gastric cancer patients, and 10 advanced gastric cancer patients), but also auxiliarily diagnose gastric cancer. RESULTS: Combined with multivariate statistical analysis, the results represented biomarkers different from Hp negative, Hp positive, and the alterations of metabolic phenotype of gastric cancer patients. Forty-three metabolites are involved in amino acid metabolism, and lipid and fatty acid metabolism pathways in the process of cancer occurrence, especially 2 biomarkers glycerophosphocholine and neopterin, were screened in this study. Neopterin was consistently increased with gastric cancer progression and glycerophosphocholine tended to consistently decrease from Hp negative to advanced gastric cancer. CONCLUSION: This method could be used for the development of rapid targeted methods for biomarker identification and a potential diagnosis of gastric cancer.

Parent B2 N2 -Perylenes with Different BN Orientations.

Introducing BN units into polycyclic aromatic hydrocarbons expands the chemical space of conjugated materials with novel properties. However, it is challenging to achieve accurate synthesis of BN-PAHs with specific BN positions and orientations. Here, three new parent B2 N2 -perylenes with different BN orientations are synthesized with BN-naphthalene as the building block, providing systematic insight into the effects of BN incorporation with different orientations on the structure, (anti)aromaticity, crystal packing and photophysical properties. The intermolecular dipole-dipole interaction shortens the π-π stacking distance. The crystal structure, (anti)aromaticity, and photophysical properties vary with the change of BN orientation. The revealed BN doping effects may provide a guideline for the synthesis of BN-PAHs with specific stacking structures, and the synthetic strategy employed here can be extended toward the synthesis of larger BN-embedded PAHs with adjustable BN patterns.

Nicaraven Attenuates Postoperative Systemic Inflammatory Responses-Induced Tumor Metastasis.

BACKGROUND: Inflammation has been demonstrated to promote cancer metastasis. Due to the well-known systemic inflammatory responses (SIR) after major surgery, it is critical to investigate and attenuate SIR-induced tumor metastasis of cancer patients suffering surgical procedures. METHODS: C57BL/6 mice were intravenously injected with Lewis lung cancer cells at 6, 24, and 72 h after the induction of intestinal ischemia/reperfusion (I/R) injury. We found that the number of tumor nodules significantly increased in lungs of mice injected with cancer cells at 6 h but not at 24 and 72 h after I/R injury. The administration of nicaraven 30 min before and 24 h after I/R injury effectively attenuated the enhanced tumor metastasis to lungs. Protein array showed the increase of various cytokines in plasma of mice at 6 h after I/R injury, but many of them were attenuated by the administration of nicaraven. Immunostaining indicated the increase of Ly6g-, CD206-, and CD11c-positive inflammatory cells in the lungs, but it was also attenuated by nicaraven administration. CONCLUSIONS: Postoperative SIR-induced tumor metastasis have been clearly evidenced in our experimental model, and the administration of nicaraven may ameliorate the SIR-induced tumor metastasis by suppressing inflammatory responses.

Aerosol Synthesis of High Entropy Alloy Nanoparticles.

Homogeneously mixing multiple metal elements within a single particle may offer new material property functionalities. High entropy alloys (HEAs), nominally defined as structures containing five or more well-mixed metal elements, are being explored at the nanoscale, but the scale-up to enable their industrial application is an extremely challenging problem. Here, we report an aerosol droplet-mediated technique toward scalable synthesis of HEA nanoparticles with atomic-level mixing of immiscible metal elements. An aqueous solution of metal salts is nebulized to generate ∼1 µm aerosol droplets, which when subjected to fast heating/quenching result in decomposition of the precursors and freezing-in of the zero-valent metal atoms. Atomic-level resolution scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy analysis reveals that all metal elements in the nanoparticles are homogeneously mixed at the atomic level. We believe that this approach offers a facile and flexible aerosol droplet-mediated synthesis technique that will ultimately enable bulk processing starting from a particulate HEA.

Stability and anion diffusion kinetics of Yttria-stabilized zirconia resolved from machine learning global potential energy surface exploration.

Yttria-stabilized zirconia (YSZ) is an important material with wide industrial applications particularly for its good conductivity in oxygen anion transportation. The conductivity is known to be sensitive to Y concentration: 8 mol. % YSZ (8YSZ) achieves the best performance, which, however, degrades remarkably under ∼1000 °C working conditions. Here, using the recently developed SSW-NN method, stochastic surface walking global optimization based on global neural network potential (G-NN), we establish the first ternary Y-Zr-O G-NN potential by fitting 28 803 first principles dataset screened from more than 107 global potential energy surface (PES) data and explore exhaustively the global PES of YSZ at different Y concentrations. Rich information on the thermodynamics and the anion diffusion kinetics of YSZ is, thus, gleaned, which helps resolve the long-standing puzzles on the stability and conductivity of the 8YSZ. We demonstrate that (i) 8YSZ is the cubic phase YSZ with the lowest possible Y concentrations. It is thermodynamically unstable, tending to segregate into the monoclinic phase of 6.7YSZ and the cubic phase of 20YSZ. (ii) The O anion diffusion in YSZ is mediated by O vacancy sites and moves along the ⟨100⟩ direction. In 8YSZ and 10YSZ, despite different Y concentrations, their anion diffusion barriers are similar, ∼ 1 eV, but in 8YSZ, the O diffusion distance is much longer due to the lack of O vacancy aggregation along the ⟨112⟩ direction. Our results illustrate the power of G-NN potential in solving challenging problems in material science, especially those requiring a deep knowledge on the complex PES.

The study of methylation and single nucleotide polymorphisms of cancer-related genes in patients with early-stage ulcerative colitis.

Aim: To investigate the methylation status and single nucleotide polymorphisms (SNPs) of cancer-associated genes in ulcerative colitis (UC) patients and explore the potential mechanism for high cancer risk of UC. Methods: A total of 103 patients were enrolled in our study, which included 30 healthy subjects, 41 patients with early-stage UC, and 32 patients with colorectal cancer (CRC). Methylation status of cyclooxygenase 2 (COX2) and human RUNT-related transcription factor 3 (RUNX3) genes in colonic mucosa from 3 groups of subjects were detected by methylation-specific polymerase chain reaction (PCR). The SNPs TNF-α rs1800629 and IL-1 rs1143627 were genotyped by PCR and direct sequencing. Results: The methylation rate of RUNX3 gene within CRC group was 35.7%, which was significantly higher than the other two groups (Healthy control 5.9%, UC 15.4%, p = .040). There was no significant difference in the methylation rate of RUNX3 between early-stage UC group and healthy control group (p = .633). The methylation rate of COX2 gene, the genotypes (GG, AG) and alleles (A, G) of rs1800629, and the genotypes (CC,CT,TT) and alleles (C,T) of rs1143627 were not statistically different among three groups. Conclusion: In the early stage of UC, the methylation rate of cancer-related genes RUNX3 and COX2 and SNPs TNF-α rs1800629 and IL-1 rs1143627 were not significantly different compared with healthy subjects. The methylation rate of RUNX3 in CRC increased, while the methylation rate of COX2 and SNPs TNF-α rs1800629 and IL-1 rs1143627 did not change significantly compared with the other two groups.

Isolation and genetic characterization of avian influenza virus H4N6 from ducks in China.

An avian influenza virus (AIV) strain belonging to the H4 subtype and provisionally designated as A/duck/China/J1/2012(H4N6) was isolated from diseased ducks with respiratory disease at a commercial poultry farm in Shandong, China, in 2012. The genomic coding sequences of all eight segments of this J1 isolate were determined and used for subsequent analysis. Phylogenetic analysis of all eight segments showed that this duck H4N6 virus was of Eurasian lineage and not American lineage. The results show that the virus probably emerged because of a reassortment event involving other avian H4N6 and H6N1 viruses. Interestingly, this H4N6 virus had all the conserved features common to low-pathogenic AIVs, including the HA cleavage sequence, receptor-binding sequences for the 2,3-linked sialic acid receptor in avian species, and the PB2 627E motif. These results suggest that the duck H4N6 isolate could not cross the species barrier to infect and replicate in mammals, including humans. In addition, screening of the duck serum samples showed that only 0.57 % (2/352) of the individuals had weak but measurable hemagglutination inhibition (HI) antibody titers. The low antibody prevalence data were also supported by the failure to detect H4N6 virus (0/56) in clinical nasal swabs of the ducks. These data indicate an alternate reservoir for the H4N6 virus.