Extraction and Identification of Three New Urechis unicinctus Visceral Peptides and Their Antioxidant Activity.

The viscera of Urechis unicinctus with polypeptides, fatty acids, and amino acids are usually discarded during processing to food. In order to improve the utilization value of the viscera of Urechis unicinctus and avoid resource waste, antioxidant polypeptides were isolated from the viscera of Urechis unicinctus. First, a protein hydrolysate of Urechis unicinctus (UUPH) was prepared by ultrasonic-assisted enzymatic hydrolysis, and the degree of hydrolysis was as high as 79.32%. Subsequently, three new antioxidant peptides (P1, P2, and P3) were purified from UUPH using ultrafiltration and chromatography, and their amino acid sequences were identified as VTSALVGPR, IGLGDEGLRR, TKIRNEISDLNER, respectively. Then, the antioxidant activity of the polypeptide was predicted by the structure-activity relationship and finally verified by experiments on eukaryotic cells. The P1 peptide exhibited the strongest antioxidant activity among these three antioxidant peptides. Furthermore, P1, P2, and P3 have no toxic effect on RAW264.7 cells at the concentration of 0.01~2 mg/mL and can protect RAW264.7 cells from H2O2-induced oxidative damage in a concentration-dependent manner. These results suggested that these three new antioxidant peptides were isolated from the viscera of Urechis unicinctus, especially the P1 peptide, which might serve as potential antioxidants applied in health-derived food or beverages. This study further developed a new use of the by-product of Urechis unicinctus, which improved the comprehensive utilization of marine biological resources.

Nano-gold-enhanced LAMP method for qualitative visual detection of Salmonella in milk.

Since Salmonella can cause foodborne disease and public health safety issues and requires a robust, rapid, on-site detection method, a novel visual qualitative method with nano-gold-enhanced loop-mediated isothermal amplification (LAMP) reaction was established for detecting Salmonella in an integrated tube. During the experiment, nano-gold were used to enhance LAMP amplification, improving amplification efficiency and shortening the reaction time to within 30 min. Visual qualitative detection is achieved via negative staining, involving the addition of CuSO4 to the final products of the LAMP reaction. Ring-like white accumulation occurs in the absence of Salmonella targets but not when they are present. After completing the LAMP reaction, the integration tube was shaken gently for 1 min to observe the liquid phase system changes, realizing the closed tube detection of Salmonella. The process resolved the challenge presented by cross-contamination, false positives, and nonspecific amplification during the LAMP reaction. This method was used to detect Salmonella in milk, further highlighting its prospects in the field of rapid food safety detection.

The Characteristics of Blood Supply and Tissue Hypoxia in Pathological Scars.

Blood supply is believed to be an important aspect in the development of pathological scars. However, there are controversies about vascular distribution, vascular structure and blood flow in pathological scars. Additionally, hypoxic microenvironment plays an important role in the vascularization of pathological scar tissues, and hypoxic conditions can be reflected by metabolic indexes and some cytokines. Furthermore, the correlation between blood supply and tissue hypoxia is controversial. The aim of this article is to review the literature on the characteristics of blood supply and tissue hypoxia in pathological scars, from which we can see pathological scars have unique characteristics of blood supply that are closely associated with tissue hypoxia. Moreover, development in the treatment of pathological scars is herein reviewed.

Increased blood flow in keloids and adjacent skin revealed by laser speckle contrast imaging.

BACKGROUND AND OBJECTIVES: Keloids are fibroproliferative lesions of unknown origin that are characterized by increased collagen deposition. Vascularization may play a role in the pathogenesis of keloids, but existing reports are contradictory. Thus, we assessed perfusion within keloids and surrounding skin using laser speckle contrast imaging (LSCI). STUDY DESIGN/MATERIALS AND METHODS: Twenty-one patients with 61 untreated keloids were enrolled into this study. LSCI was used to evaluate blood flow in the keloids and surrounding skin. Three regions of interest were manually defined: keloids (K), skin adjacent to keloids (A), and nonadjacent skin separated by at least 0.3 cm from the edge of the keloids (N). Mean perfusion in each of these regions was determined and ratios (K/N, A/N) were calculated. RESULTS: Significantly higher perfusion was noted in keloids and adjacent skin compared with nonadjacent skin (P < 0.05). The mean values (95% confidence intervals) of the ratios were: K/N = 2.41 (2.28-2.54) and A/N = 1.33 (1.28-1.37). A heterogeneous perfusion map was frequently observed. Mean perfusion in keloids and nonadjacent skin in the chest region was significantly higher than that on the back (P < 0.05). There was no statistical signficant difference in K/N at different locations (P > 0.05). CONCLUSIONS: Perfusion values in keloids and adjacent skin were significantly higher than those in nonadjacent skin. LSCI may be a suitable and useful way of assessing perfusion in keloids.

[Relationship between epidermal growth factor receptor gene expression and radiosensitivity of non-small-cell lung cancer cells].

OBJECTIVE: To explore the relationship between epidermal growth factor receptor (EGFR) gene expression and radiosensitivity of non-small-cell lung cancer (NSCLC) cells. METHODS: EGFR sequence-specific double-stranded RNA (dsRNA-EGFR) was chemically synthesized. NSCLC cell line SPC-A1 was transfected with dsRNA-EGFR formulated with Lipofectamine 2000. Western blot and real-time PCR were used to determine the EGFR mRNA and protein expression, respectively. Colony inhibition test was adopted to observe the radiosensitizing effect. To establish the nude mouse tumor models, calculate the tumor growth inhibition rate and make the tumor growth curve by measuring its size and weight. RESULTS: EGFR mRNA levels were 1.51 ± 0.22, 1.38 ± 0.15 and 0.45 ± 0.11 in the control group, dsRNA-unrelated group and dsRNA-EGFR group, respectively (F = 482.7, P < 0.01). The contents of EGFR protein were 2340.87 ± 10.99, 2231.85 ± 35.66 and 832.03 ± 39.13 in the control group, dsRNA-unrelated group and dsRNA-EGFR group, respectively (F = 263.3, P < 0.05). Compared with the control group, dsRNA-EGFR sequence specifically decreased the expressions of EGFR mRNA by 70.2% and EGFR protein by 64.5%. The colony inhibition rates of the control group, dsRNA-unrelated combined with radiotherapy group and dsRNA-EGFR combined with radiotherapy group were 9.3%, 12.5% and 65.5%, and the tumor growth inhibition rates were 21.3%, 24.4% and 64.2%, respectively. The combination of dsRNA-EGFR and radiotherapy significantly inhibited the tumor growth in vitro and in vivo. CONCLUSIONS: DsRNA-EGFR shows an apparent inhibitory effect on the expression of EGFR mRNA and protein of NSCLC cells, effectively inhibit the tumor growth in vivo, and enhance the radiosensitivity of NSCLC.

[Genetic evolution analysis of matrix protein 2 gene of avian influenza H5N1 viruses from boundary of Yunnan province].

OBJECTIVE: To elucidate the variation in characterizations and genetic evolution of the matrix protein 2 or ion channel protein(M2) genes of avian influenza subtype H5N1 viruses in the boundary region of Yunnan province from 2008 to 2012. METHODS: A total of swab samples were collected from foreign poultry such as the junction between Yunnan and Vietnam, Laos,myanmar and wild birds in boundary region of Yunnan province from 2008 to 2012 and screened by H5N1 subtype-specific multiplex RT-PCR. The M genes of H5N1 virus from the positive samples were amplified by RT-PCR and cloned into pMD18-T vectors for sequencing. The alignment and phylogenetic analysis of M2 genes were performed with sequences of the known reference strains. RESULTS: A total of 71 positive samples were found out of 1240 samples and the positive rate was 5.72%. A total of 14 different M2 sequences were obtained from 30 positive samples and were divided into 3 distinct clades or sub-clades(1.2.1, 1.2.2 and 2) by phylogenetic analysis, 5, 7 and 2, respectively. The M2 genes and Hemagglutinin(HA) genes of H5N1 viruses from the boundary region of Yunnan province had showed different relationship of genetic evolution. The substitution or mutation of key amino acids sites had been found among the domains of epitope, adamantane-resistance, and poultry or human original viral strains. CONCLUSION: The M2 genes of H5N1 subtype viruses in boundary region of Yunnan province from 2008 to 2012 showed genetic divergence and the virus of clade 1.2.2 had become dominant epidemic strain in this region.

Interpreting the degradation mechanism of triclosan in microbial fuel cell by combining analysis microbiome community and degradation pathway.

Microbes play a dominant role for the transformation of organic contaminants in the environment, while a significant gap exists in understanding the degradation mechanism and the function of different species. Herein, the possible bio-degradation of triclosan in microbial fuel cell was explored, with the investigation of degradation kinetics, microbial community, and possible degradation products. 5 mg/L of triclosan could be degraded within 3 days, and an intermediate degradation product (2,4-dichlorophen) could be further degraded in system. 32 kinds of dominant bacteria (relative intensity >0.5%) were identified in the biofilm, and 10 possible degradation products were identified. By analyzing the possible involved bioreactions (including decarboxylation, dehalogenation, dioxygenation, hydrolysis, hydroxylation, and ring-cleavage) of the dominant bacteria and possible degradation pathway of triclosan based on the identified products, biodegradation mechanism and function of the bacteria involved in the degradation of triclosan was clarified simultaneously. This study provides useful information for further interpreting the degradation mechanism of organic pollutants in mixed flora by combining analysis microbiome community and degradation pathway.

Intelligent biosensing strategies for rapid detection in food safety: A review.

With the development of the economy and progress in science and technology, people are paying increasing attention to food safety, and food safety testing technology has also developed rapidly. Biosensors, one type of detection technology, stand out due to their high sensitivity and specificity. In combination with emerging technologies, such as smartphones, 3D printing, artificial sensing and the Internet of Things (IOT), biosensor technology has been increasingly developed. The future development of food detection requires intelligence, portability and sensitivity. Based on these factors, we reviewed the research progress regarding the intelligence of biosensors in recent years, expound the research situation based on various biosensor intelligent technologies and equipment, and forecast the future applications of intelligent biosensors.

The Fundus Structural and Functional Predictions of DME Patients After Anti-VEGF Treatments.

Diabetic retinopathy (DR) is an important complication with a high incidence of 34.6% in the diabetic populations. DR could finally lead to vision impairment without effective interventions, during which, diabetic macular edema (DME) is a key phase causing visual loss. Up to date, antivascular endothelial growth factor (anti-VEGF) therapy is the first-line treatment for DME which has achieved relatively better clinical outcomes than traditional treatments. However, there are several kinds of anti-VEGF medicines, and patients are sensitive to different anti-VEGF treatments. In addition, its effectiveness is unstable. Considering the patients' need to accept continual anti-VEGF treatments and its price is comparatively high, it is clinically important to predict the prognosis after different anti-VEGF treatments. In our research, we used the demographic and clinical data of 254 DME patients and 2,763 optical coherence tomography (OCT) images from three countries to predict the fundus structural and functional parameters and treatment plan in 6 months after different anti-VEGF treatments. Eight baseline features combined with 11 models were applied to conduct seven prediction tasks. Accuracy (ACC), the area under curve (AUC), mean absolute error (MAE), and mean square error (MSE) were respectively used to evaluate the classification and regression tasks. The ACC and AUC of structural predictions of retinal pigment epithelial detachment were close to 1.000. The MAE and MSE of visual acuity predictions were nearly 0.3 to 0.4 logMAR. The ACC of treatment plan regarding continuous injection was approaching 70%. Our research has achieved great performance in the predictions of fundus structural and functional parameters as well as treatment plan, which can help ophthalmologists improve the treatment compliance of DME patients.

Novel VUV/g-C3N4 system with high adaptability to varied environmental conditions and outstanding degradation capacity for chlorophenols.

Given the limitations of conventional vacuum ultraviolet (VUV) systems, a novel vacuum ultraviolet/graphite carbon nitride (VUV/g-C3N4) system with high adaptability to varying environmental conditions was developed. Compared with conventional VUV and UV/g-C3N4 systems, the VUV/g-C3N4 system demonstrates a much higher ability for the efficient degradation of chlorophenols (CPs). In particular, the VUV/g-C3N4 system exhibits outstanding performance even at low pH and high concentrations of humic acid and SO42-. Alkaline conditions and the presence of HCO3- can further promote CP removal. In addition, the feasibility of the VUV/g-C3N4 system was verified by its stable operation in both river water and tap water. Unlike conventional photochemical systems relying on •OH, the dominant reactive species for CP degradation by the VUV/g-C3N4 system was identified to be •O2-. This study conclusively provided a novel system for the efficient photocatalytic treatment of pollutants.

Opposite impacts of K+ and Ca2+ on membrane fouling by humic acid and cleaning process: Evaluation and mechanism investigation.

Understanding the influences of cations on membrane fouling was important to improve the performance of membrane filtration system, however, opposite conclusions were made in different studies. Meanwhile, although the influences of cation concentration have been studied extensively, few attentions have been paid to the cation valence. To clarify it, the effects of typical cations on membrane fouling and cleaning, as well as the related mechanisms were investigated systemically in this study. K+ and Ca2+ were chosen as the representative cations, and humic acid (HA) was chosen as the membrane foulants. The results demonstrated Ca2+ promoted the formation of reversible fouling, meanwhile higher removal efficiency of HA could also be achieved with the assistance of filtration cake containing HA + Ca2+. However, K+ led to the formation of more recalcitrant irreversible fouling. By comparing the concentration of cations in feed and permeate, analyzing the influence of cations on size of HA flocs, and the detailed SEM, AFM and TEM observation, it could be found that different mechanisms dominated the interaction between cations and HA. The bridging effect induced by Ca2+ attributed to the extension of HA molecules, while the electrostatic shielding effect induced by K+ led to the compression of them. Moreover, the different characteristics of hydrated Ca2+ and K+ also contributed to the different structures of foulant layers formed by HA + Ca2+ and HA + K+. Given the abundance of K+ and Ca2+ in natural water, results of this study can provide valuable advice for practical membrane filtration process.

Size effect of elemental selenium nanoparticles (Nano-Se) at supranutritional levels on selenium accumulation and glutathione S-transferase activity.

It has been shown that 36 nm Nano-Se has lower toxicity than selenite or selenomethionine, but these forms of selenium (Se) all possess similar ability to increase selenoenzyme levels. The size of nanoparticles plays an important role in their biological activity: as expected, 5-200 nm Nano-Se can directly scavenge free radicals in vitro in a size-dependent fashion. However, in Se-deficient cells and Se-deficient mice, the size effect of Nano-Se on increasing selenoenzymes and liver Se disappears unexpectedly. We hypothesize that under conditions of Se deficiency, the avidity of Se uptake mechanisms may be increased to maintain the biosynthesis of selenoenzymes, which are fundamental for redox homeostasis. This increased avidity may override the potential advantage of small size Nano-Se seen under Se-replete conditions, thereby eliminating the size effect. Once selenoenzymes have been saturated, Se uptake mechanisms may downregulate; accordingly, the size effect of Nano-Se can then reappear. To test this hypothesis, Se-deficient mice were administered either 36 or 90 nm Nano-Se at supranutritional doses, in both a short-term model and a single-dose model. Under these conditions, Nano-Se showed a size effect on Se accumulation and glutathione S-transferase (GST) activity. A size effect of Nano-Se was found in 15 out of 18 total comparisons between sizes at the same dose and time in the two models. Furthermore, the magnitude of the size effect was more prominent on Se accumulation than on GST activity. GST is strictly regulated by transcriptional and translational mechanisms, so its increase in activity normally does not exceed 3-fold. In contrast, the homeostasis of Se accumulation is not as tightly controlled. In the present experiments, GST activity had reached or was approaching saturation, but liver Se was far below saturation. Therefore, our results strongly suggest that the saturation profile of the tested biomarker has an impact on the size effect of Nano-Se. Since both GST and small molecular weight selenocompounds accumulated in vivo are important intermediates for chemoprevention by Se, our results also suggest that Nano-Se should be most effective as a chemopreventive agent at smaller particle size.

Improved humoral immunity against tuberculosis ESAT-6 antigen by chimeric DNA prime and protein boost strategy.

Ag85A and ESAT-6 proteins of Mycobacterium tuberculosis (M.TB) are important protective antigens. The 32-kDa Ag85A is a strong immunogen in both small and large animals. However, the 6-kDa ESAT-6 has relatively low inherent immunogenicity, especially in large animals. To improve the immunogenicity of ESAT-6 in animals, we made chimeric DNA vaccines, HG856K and HG856A, by inserting the esat-6 gene into the Kpn I or Acc I endonuclease restriction site of the ag85a gene, respectively. BALB/c mice were injected intramuscularly three times with the 10-microg singular DNA vaccine (HG85 encoding for Ag85A or HG6 encoding for ESAT-6) or chimeric DNA vaccine (HG856K or HG856A) followed by electroporation (EP). Ten days after the last DNA vaccination, mice received a booster immunization intraperitoneally with 50-microg pure recombinant protein Ag85A or ESAT-6 without adjuvant. Additional groups of mice immunized with chimeric DNA vaccines were boosted with two mixed proteins (Ag85A/ESAT-6) at the same time. The results showed that the immunogenicity of M.TB ESAT-6 antigen was not improved by priming with the HG6 DNA vaccine. However, the humoral immunity against the ESAT-6 antigen was significantly increased in the mice primed with chimeric DNA vaccines, HG856K or HG856A, followed by boosting with ESAT-6 or ESAT-6/Ag85A mixed proteins.

Effects of metal ions on the conformation and activity of acutolysin D from Agkistrodon Acutus venom.

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A (1%) (280)) of acutolyisn D have been determined to be 47,850 +/- 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca(2+)-binding sites and two Zn(2+)-binding sites determined by atomic absorption spectrophotometer. Zn(2+) is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn(2+) significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)(2) precipitate formation. Ca(2+) is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca(2+) markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu(2+), Co(2+), Mn(2+) or Tb(3+) and inhibited completely by Cd(2+), is enhanced by Mg(2+). The fluorescence intensity of the protein decreases in the presence of Cu(2+), Co(2+), Cd(2+) or Mn(2+), but neither for Ca(2+), Mg(2+) nor for Tb(3+). Zn(2+), Ca(2+), Mg(2+), Cu(2+), Mn(2+), Co(2+ )and Tb(3+) have slight effects on its secondary structure contents. In addition, Cd(2+) causes a marked increase of antiparallel beta-sheet content from 45.5% to 60.2%.

The studies of FT-IR and CD spectroscopy on catechol oxidase I from tobacco.

A novel copper-containing enzyme named COI (catechol oxidase I) has been isolated and purified from tobacco by extracting acetone-emerged powder with phosphate buffer, centrifugation at low temperature, ammonium sulfate fractional precipitation, and column chromatography on DEAE-sephadex (A-50), sephadex (G-75), and DEAE-celluse (DE-52). PAGE, SDS-PAGE were used to detect the enzyme purity, and to determine its molecular weight. Then the secondary structures of COI at different pH, different temperatures and different concentrations of guanidine hydrochloride (GdnHCl) were studied by the FT-IR, Fourier self-deconvolution spectra, and circular dichroism (CD). At pH 2.0, the contents of both alpha-helix and anti-parallel beta-sheet decrease, and that of random coil increases, while beta-turn is unchanged compared with the neutral condition (pH 7.0). At pH 11.0, the results indicate that the contents of alpha-helix, anti-parallel beta-sheet and beta-turn decrease, while random coil structure increases. According to the CD measurements, the relative average fractions of alpha-helix, anti-parallel beta-sheet, beta-turn/parallel beta-sheet, aromatic residues and disulfide bond, and random coil/gamma-turn are 41.7%, 16.7%, 23.5%, 11.3%, and 6.8% at pH 7.0, respectively, while 7.2%, 7.7%, 15.2%, 10.7%, 59.2% at pH 2.0, and 20.6%, 9.5%, 15.2%, 10.5%, 44.2% at pH 11.0. Both alpha-helix and random coil decrease with temperature increasing, and anti-parallel beta-sheet increases at the same time. After incubated in 6 mol/L guanidine hydrochloride for 30 min, the fraction of alpha-helix almost disappears (only 1.1% left), while random coil/gamma-turn increases to 81.8%, which coincides well with the results obtained through enzymatic activity experiment.

Effects of metal ions and an inhibitor on the fluorescence and activity of acutolysin A from Agkistrodon acutus venom.

Acutolysin A, a protein isolated from the venom of Chinese Five-pace snake (Agkistrodon acutus) has shown marked hemorrhagic and proteolytic activities. In the present study, the effects of metal ions and an inhibitor EDTA on the fluorescence and function of autolysin A have been studied, by following fluorescence and activity measurements. Acutolysin A contains a Ca(2+)-binding site, which provides it with important structural stability, and a Zn(2+)-binding site, which is essential for its enzymatic activities. The removal of metal ions in acutolysin A by incubation with EDTA results in irreversible inhibition and complete denaturation, and a marked decrease in its fluorescence intensity. The fluorescence intensity of acutolysin A is also decreased in the presence of Cu2+, Co2+, Mn2+ or Mg2+, but does not change in the presence of Ca2+, Cd2+, or Tb3+. Caseinolytic activity of acutolysin A is enhanced by Co2+, Ca2+ and Mg2+, but is partly inhibited by Cu2+, Mn2+ and Tb3+, and completely inhibited by Cd2+. Both Zn2+ and Co2+ recover the loss of activity of the protein caused by Cd2+.

[Research on the mechanism of fluorescence quenching of HRP by NaN3].

The fluorescence and synchronous fluorescence spectra were employed to study the binding between sodium azide (NaN3) and horseradish peroxidase (HRP), which was affected by the molecular conformation and the microenvironment of the fluorescence residues. The mechanism of the fluorescence quenching of HRP by NaN3 was discussed and the binding constant and the number of binding sites of non-covalent bond between them were calculated.

Overexpression of short TRPM8 variant α promotes cell migration and invasion, and decreases starvation-induced apoptosis in prostate cancer LNCaP cells.

The aim of the present study was to investigate the function of a transient receptor potential melastatin 8 (TRPM8) splice variant, short TRMP8α (sM8α), in the androgen-dependent prostate cancer LNCaP cell line, and to evaluate the potential involvement of the mitogen-activated protein kinase (MAPK) signaling pathway. The coding DNA for sM8α was cloned and transfected into LNCaP cells to generate cells that overexpress this isoform of TRPM8. Cellular proliferation was determined by performing an MTT assay, and flow cytometry was used to analyze apoptosis and cell cycle distribution. Furthermore, cellular migration and invasion were evaluated using Transwell® migration assays. The subcellular location of recombinant sM8α was detected by quantum dots-based immunofluorescent imaging, western blotting was performed to examine the expression levels of proteins in the MAPK signaling pathway and reverse transcription-polymerase chain reaction was used to determine the expression of sM8α mRNA transcripts. The present study demonstrated that sM8α mRNA was expressed at a low level in the LNCaP, DU145 and PC-3 prostate cancer cell lines. Additionally, the recombinant sM8α protein was located in the cytoplasm of LNCaP cells and its overexpression significantly reduced starvation-induced apoptosis in these cells (P<0.05), possibly by means of reduced activation of phosphorylated-c-Jun N-terminal kinase (p-JNK). The migration and invasion of the LNCaP cells were markedly enhanced by the overexpression of sM8α, possibly via activation of MMP-2. Furthermore, overexpression of sM8α in LNCaP cells did not alter the expression of full-length TRPM8 and had no effect on cellular proliferation. Overall, the results of the present study indicate that sM8α may be important in the regulation of prostate cancer cell migration and invasion through the activation of matrix metalloproteinase-2, as well as in the regulation of apoptosis through the activation of p-JNK in the MAPK signaling pathway.

Emergence of novel clade 2.3.4 influenza A (H5N1) virus subgroups in Yunnan Province, China.

From December 2013 to March 2014, a major wave of highly pathogenic avian influenza outbreak occurred in poultry in Yunnan Province, China. We isolated and characterized eight highly pathogenic avian influenza A (H5N1) viruses from poultry. Full genome influenza sequences and analyses have been performed. Sequence analyses revealed that they belonged to clade 2.3.4 but did not fit within the three defined subclades. The isolated viruses were provisional subclade 2.3.4.4e. The provisional subclade 2.3.4.4e viruses with six internal genes from avian influenza A (H5N2) viruses in 2013 were the novel reassortant influenza A (H5N1) viruses which were associated with the outbreak of H5N1 occurred in egg chicken farms in Yunnan Province. The HA genes were similar to subtype H5 viruses isolated from January to March of 2014 in Asia including H5N6 and H5N8. The NA genes were most closely related to A/chicken/Vietnam/NCVD-KA423/2013 (H5N1) from the subclade 2.3.2. The HI assay demonstrated a lack of antigenic relatedness between clades 2.3.4.4e and 2.3.4.1 (RE-5 vaccine strain) or 2.3.2.2 (RE-6 vaccine strain).