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Whole virion inactivated vaccine CoronaVac (C) and Spike (S) mRNA BNT162b2 (B) vaccines differ greatly in their ability to elicit neutralizing antibodies but have somewhat comparable effectiveness in protecting from severe COVID-19. We conducted further analyses for a randomized trial (Cobovax study, NCT05057169) of third dose homologous and heterologous booster vaccination, i.e. four interventions CC-C, CC-B, BB-C and BB-B. Here, we assess vaccine immunogenicity beyond neutralizing function, including S and non-S antibodies with Fc receptor (FcR) binding, antibody avidity and T cell specificity to 6 months post-vaccination. Ancestral and Omicron S-specific IgG and FcR binding are significantly higher by BNT162b2 booster than CoronaVac, regardless of first doses. Nucleocapsid (N) antibodies are only increased in homologous boosted CoronaVac participants (CC-C). CoronaVac primed participants have lower baseline S-specific CD4 + IFNγ + cells, but are significantly increased by either CoronaVac or BNT162b2 boosters. Priming vaccine content defined T cell peptide specificity preference, with S-specific T cells dominating B primed groups and non-S structural peptides contributing more in C primed groups, regardless of booster type. S-specific CD4 + T cell responses, N-specific antibodies, and antibody effector functions via Fc receptor binding may contribute to protection and compensate for less potent neutralizing responses in CoronaVac recipients., (© 2024. The Author(s).)

Background: The Oka varicella vaccine strain remains neurovirulent and can establish lifelong latent infection, raising safety concerns about vaccine-related herpes zoster. In this study, we aimed to evaluate the immunogenicity and safety of a skin-attenuated and neuro-attenuated varicella vaccine candidate (v7D vaccine)., Methods: We did this randomised, double-blind, controlled, phase 2a clinical trial in Jiangsu, China. Healthy children aged 3-12 years with no history of varicella infection or vaccination were enrolled and randomly assigned (1:1:1:1) to receive a single subcutaneous injection of the v7D vaccine at 3·3 log 10 plaque forming units (PFU; low-dose v7D group), 3·9 log 10 PFU (medium-dose v7D group), and 4·2 log 10 PFU (high-dose v7D group), or the positive control varicella vaccine (vOka vaccine group). All the participants, laboratory personnel, and investigators other than the vaccine preparation and management staff were masked to the vaccine allocation. The primary outcome was assessment of the geometric mean titres (GMTs) and seroconversion rates of anti-varicella zoster virus immunoglobulin G (IgG) induced by different dose groups of v7D vaccine at 0, 42, 60, and 90 days after vaccination in the per-protocol set for humoral immune response analysis. Safety was a secondary outcome, focusing on adverse events within 42 days post-vaccination, and serious adverse events within 6 months after vaccination. This study was registered on Chinese Clinical Trial Registry, ChiCTR2000034434., Findings: On Aug 18-21, 2020, 842 eligible volunteers were enrolled and randomly assigned treatment. After three participants withdrew, 839 received a low dose (n=211), middle dose (n=210), or high dose (n=210) of v7D vaccine, or the vOka vaccine (n=208). In the per-protocol set for humoral immune response analysis, the anti-varicella zoster virus IgG antibody response was highest at day 90. At day 90, the seroconversion rates of the low-dose, medium-dose, and high-dose groups of v7D vaccine and the positive control vOka vaccine group were 100·0% (95% CI 95·8-100·0; 87 of 87 participants), 98·9% (93·8-100·0; 87 of 88 participants), 97·8% (92·4-99·7; 91 of 93 participants), and 96·4% (89·8-99·2; 80 of 83 participants), respectively; the GMTs corresponded to values of 30·8 (95% CI 26·2-36·0), 31·3 (26·7-36·6), 28·2 (23·9-33·2), and 38·5 (31·7-46·7). The v7D vaccine, at low dose and medium dose, elicited a humoral immune response similar to that of the vOka vaccine. However, the high-dose v7D vaccine induced a marginally lower GMT compared with the vOka vaccine at day 90 (p=0·027). In the per-protocol set, the three dose groups of the v7D vaccine induced a similar humoral immune response at each timepoint, with no statistically significant differences. The incidence of adverse reactions in the low-dose, medium-dose, and high-dose groups of v7D vaccine was significantly lower than that in the vOka vaccine group (17% [35 of 211 participants], 20% [41 of 210 participants], and 13% [27 of 210 participants] vs 24% [50 of 208 participants], respectively; p=0·025), especially local adverse reactions (10% [22 of 211 participants], 14% [30 of 210 participants] and 9% [18 of 210 participants] vs 18% [38 of 208 participants], respectively; p=0·016). None of the serious adverse events were vaccine related., Interpretation: The three dose groups of the candidate v7D vaccine exhibit similar humoral immunogenicity to the vOka vaccine and are well tolerated. These findings encourage further investigations on two-dose vaccination schedules, efficacy, and the potential safety benefit of v7D vaccine in the future., Funding: The National Natural Science Foundation of China, CAMS Innovation Fund for Medical Sciences, the Fundamental Research Funds for the Central Universities, and Beijing Wantai., Translation: For the Chinese translation of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests J-ZJ, J-LH, and X-ZY are employees of and have stock options in Beijing Wantai. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Cross-reactive antibodies with Fc receptor (FcR) effector functions may mitigate pandemic virus impact in the absence of neutralizing antibodies. In this exploratory study, we use serum from a randomized placebo-controlled trial of seasonal trivalent influenza vaccination in children (NCT00792051) conducted at the onset of the 2009 H1N1 pandemic (pH1N1) and monitored for infection. We found that seasonal vaccination increases pH1N1 specific antibodies and FcR effector functions. Furthermore, prospective baseline antibody profiles after seasonal vaccination, prior to pH1N1 infection, show that unvaccinated uninfected children have elevated ADCC effector function, FcγR3a and FcγR2a binding antibodies to multiple pH1N1 proteins, past seasonal and avian (H5, H7 and H9) strains. Whereas, children that became pH1N1 infected after seasonal vaccination have antibodies focussed to seasonal strains without FcR functions, and greater aggregated HA-specific profiles for IgM and IgG3. Modeling to predict infection susceptibility, ranked baseline hemagglutination antibody inhibition as the highest contributor to lack of pH1N1 infection, in combination with features that include pH1-IgG1, H1-stem responses and FcR binding to seasonal vaccine and pH1 proteins. Thus, seasonal vaccination can have benefits against pandemic influenza viruses, and some children already have broadly reactive antibodies with Fc potential without vaccination and may be considered 'elite influenza controllers'., (© 2024. The Author(s).)

Background: Few trials have compared homologous and heterologous third doses of COVID-19 vaccination with inactivated vaccines and mRNA vaccines. The aim of this study was to assess immune responses, safety, and efficacy against SARS-CoV-2 infection following homologous or heterologous third-dose COVID-19 vaccination with either one dose of CoronaVac (Sinovac Biotech; inactivated vaccine) or BNT162b2 (Fosun Pharma-BioNTech; mRNA vaccine)., Methods: This is an ongoing, randomised, allocation-concealed, open-label, comparator-controlled trial in adults aged 18 years or older enrolled from the community in Hong Kong, who had received two doses of CoronaVac or BNT162b2 at least 6 months earlier. Participants were randomly assigned, using a computer-generated sequence, in a 1:1 ratio with allocation concealment to receive a (third) dose of CoronaVac or BNT162b2 (ancestral virus strain), stratified by types of previous COVID-19 vaccination (homologous two doses of CoronaVac or BNT162b2). Participants were unmasked to group allocation after vaccination. The primary endpoint was serum neutralising antibodies against the ancestral virus at day 28 after vaccination in each group, measured as plaque reduction neutralisation test (PRNT 50 ) geometric mean titre (GMT). Surrogate virus neutralisation test (sVNT) mean inhibition percentage and PRNT 50 titres against omicron BA.1 and BA.2 subvariants were also measured. Secondary endpoints included geometric mean fold rise (GMFR) in antibody titres; incidence of solicited local and systemic adverse events; IFNγ + CD4 + and IFNγ + CD8 + T-cell responses at days 7 and 28; and incidence of COVID-19. Within-group comparisons of boost in immunogenicity from baseline and between-group comparisons were done according to intervention received (ie, per protocol) by paired and unpaired t test, respectively, and cumulative incidence of infection was compared using Kaplan-Meier curves and a proportional hazards model to estimate hazard ratio. The trial is registered with ClinicalTrials.gov, NCT05057169., Findings: We enrolled participants from Nov 12, 2021, to Jan 27, 2022. We vaccinated 219 participants who previously received two doses of CoronaVac, including 101 randomly assigned to receive CoronaVac (CC-C) and 118 randomly assigned to receive BNT162b2 (CC-B) as their third dose; and 232 participants who previously received two doses of BNT162b2, including 118 randomly assigned to receive CoronaVac (BB-C) and 114 randomly assigned to receive BNT162b2 (BB-B) as their third dose. The PRNT 50 GMTs on day 28 against ancestral virus were 109, 905, 92, and 816; against omicron BA.1 were 9, 75, 8, and 86; and against omicron BA.2 were 6, 80, 6, and 67 in the CC-C, CC-B, BB-C, and BB-B groups, respectively. Mean sVNT inhibition percentages on day 28 against ancestral virus were 83%, 96%, 87%, and 96%; against omicron BA.1 were 15%, 58%, 19%, and 69%; and against omicron BA.2 were 43%, 85%, 50%, and 90%, in the CC-C, CC-B, BB-C, and BB-B groups, respectively. Participants who had previously received two doses of CoronaVac and a BNT162b2 third dose had a GMFR of 12 (p<0·0001) compared with those who received a CoronaVac third dose; similarly, those who had received two doses of BNT162b2 and a BNT162b2 third dose had a GMFR of 8 (p<0·0001). No differences in CD4 + and CD8 + T-cell responses were observed between groups. We did not identify any vaccination-related hospitalisation within 1 month after vaccination. We identified 58 infections when omicron BA.2 was predominantly circulating, with cumulative incidence of 15·3% and 15·4% in the CC-C and CC-B groups, respectively (p=0·93), and 16·7% and 14·0% in the BB-C and BB-B groups, respectively (p=0·56)., Interpretation: Similar levels of incidence of, presumably, omicron BA.2 infections were observed in each group despite very weak antibody responses to BA.2 in the recipients of a CoronaVac third dose. Further research is warranted to identify appropriate correlates of protection for inactivated COVID-19 vaccines., Funding: Health and Medical Research Fund, Hong Kong., Translation: For the Chinese translation of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests BJC consults for AstraZeneca, Fosun Pharma, GlaxoSmithKline, Haleon, Moderna, Pfizer, Roche and Sanofi Pasteur. BJC has received research funding from Fosun Pharma. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Despite the success in decreasing varicella-related disease burden, live-attenuated Oka vaccine strain of varicella-zoster virus (vOka) remains neuro-virulence and may establish latency and reactivate, raising safety concerns. Here we aimed to evaluate the safety and immunogenicity of a skin- and neuro-attenuated varicella vaccine candidate (v7D)., Methods: This is a randomized, double-blind, placebo-controlled, dose-escalation and age de-escalation phase 1 clinical trial conducted in Liuzhou, China (ChiCTR1900022284). Eligible healthy participants aged 1-49 years, with no history of varicella vaccination and had no history of varicella or herpes zoster were sequentially enrolled and allocated to subcutaneously receive one of the three doses (3.3, 3.9, and 4.2 lg PFU) of v7D, vOka or placebo in a dose-escalation and age de-escalation manner. The primary outcome was safety, assessed by adverse events/reactions within 42 days after vaccination and serious adverse events (SAEs) throughout six months after vaccination. The secondary outcome was immunogenicity, assessed by the VZV IgG antibodies measured with fluorescent antibody to membrane antigen (FAMA) assay., Findings: Between April 2019 and March 2020, totally 224 participants were enrolled. Within 42 days post-vaccination, the incidences of adverse reactions were 37.5%-38.7% in the three doses of v7D groups which were similar to that of the vOka (37.5%) and placebo (34.4%) groups. No SAE has been judged as causally related to vaccination. At 42 days post-vaccination, 100% of children aged 1-12 years in the per-protocol set of immunogenicity cohort of the v7D groups became seropositive. Meanwhile, in the intent-to-treat set of immunogenicity cohort of subjects aged 1-49 years, the geometric mean increases of the three groups of v7D vaccine were 3.8, 5.8 and 3.2, respectively, which were similar to that of the vOka vaccine group (4.4) and significantly higher than that of the placebo group (1.3)., Interpretation: The candidate v7D vaccine has been preliminarily shown to be well-tolerated and immunogenic in humans. The data warrant further evaluation of the safety advantage and efficacy of v7D as a varicella vaccine., Funding: The National Natural Science Foundation of China, CAMS Innovation Fund for Medical Sciences, and Beijing Wantai CO., LTD., Competing Interests: The authors declare the following financial interests/personal relationships that may be considered potential competing interests: The study funder, Beijing Wantai Biological Pharmacy Enterprise, prepared the vaccines in the phase 1 trial. Q-Q S, J-Z J, J-L H, Y-M L and X-Z Y are employees of Beijing Wantai Biological Pharmacy Enterprise. X-Z Y and Y-M L have stock options in Beijing Wantai Biological Pharmacy Enterprise CO. All other authors declare no competing interests., (© 2023 The Author(s).)

Objective: To study the quantity, structure and allocation equity of stomatologists, in order to provide bases and advices for improving the allocation of stomatologists in China. Methods: On the basis of data from China Health Statistics Yearbooks and Brief Book of Administrative Divisions of the People's Republic of China , the general situation of stomatologists was analyzed by descriptive analysis. Gini coefficient was used to evaluate the allocation equity in 2010 and 2020. Results: The total number of stomatologists reached 2 780 hundred in 2020, which increased by 150.5% compared with that in 2010. The overall quality structure of stomatologists had improved. The gender proportion was balanced and the age distribution was reasonable. The team was mainly composed by the young people, in which the numbers under 44 years old accounted for 71.6% (1 991 hundred/2 780 hundred). The proportion of personnel with senior professional titles decreased to 7.9% (220 hundred/2 780 hundred) while the total number increased to 220 thousand. The distribution of stomatologists by population was fair. Gini coefficients of the whole country as well as the eastern, central and western regions were less than 0.3. Conclusions: The quantity, quality and allocation equity of stomatologists were still insufficient in China. It is necessary to optimize the human resources allocation for stomatologists. It is suggested to increase the talents supply through supply-side reform, medicine-education collaboration and multi-agent participation. And it is suggested to optimize criterions to improve the quality of stomatologists.

Vaccines that are broadly cross-protective against current and future SARS-CoV-2 variants of concern (VoC) or across the sarbecoviruses subgenus remain a priority for public health. Virus neutralization is the best available correlate of protection. To define the magnitude and breadth of cross-neutralization in individuals with different exposure to SARS-CoV-2 infection and vaccination, we here use a multiplex surrogate neutralization assay based on virus spike receptor binding domains of multiple SARS-CoV-2 VoC, as well as related bat and pangolin viruses. We include sera from cohorts of individuals vaccinated with two or three doses of mRNA (BNT162b2) or inactivated SARS-CoV-2 (Coronavac or Sinopharm) vaccines with or without a history of previous SARS-CoV-2 or SARS-CoV-1 infection. SARS-CoV-2 or SARS-CoV-1 infection followed by BNT162b2 vaccine, Omicron BA.2 breakthrough infection following BNT162b2 vaccine or a third dose of BNT162b2 following two doses of BNT162b2 or Coronavac elicit the highest and broadest neutralization across VoCs. For both breadth and magnitude of neutralization across all sarbecoviruses, those infected with SARS-CoV-1 immunized with BNT162b2 outperform all other combinations of infection and/or vaccination. These data may inform vaccine design strategies for generating broadly neutralizing antibodies to SARS-CoV-2 variants or across the sarbecovirus subgenus., (© 2022. The Author(s).)

Objective: To explore the effect of P311 microspheres-loaded thermosensitive chitosan hydrogel on the wound healing of full-thickness skin defects in rats. Methods: The method of experimental study was adopted. The polyvinyl alcohol/sodium alginate microspheres (simple microspheres), P311 microspheres, and bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA) microspheres were prepared by water-in-oil emulsification, and then their morphology was observed under a light microscope/inverted fluorescence microscope. Chitosan solution was prepared, chitosan solution and β-glycerol phosphate disodium hydrate were mixed to prepare simple thermosensitive hydrogels, and thermosensitive hydrogels loaded with simple microspheres or P311 microspheres were prepared by adding corresponding substances in simple thermosensitive hydrogels. The morphological changes of the prepared four liquids in the state of tilt was observed at 37 ℃. After being freeze-dried, the micromorphology of the prepared four liquids was observed under a scanning electron microscope. Eighteen 3-4-week-old male Sprague-Dawley rats were divided into normal group without any treatment, dressing group, chitosan group, hydrogel alone group, simple microspheres-loaded hydrogel group, and P311 microspheres-loaded hydrogel group, which were inflicted with one full-thickness skin defect wound on both sides of the back spine and were dealt correspondingly, with 3 rats in each group. Rats with full-thickness skin defects in the five groups were collected, the wound healing was observed on post injury day (PID) 0 (immediately), 5, 10, and 15, and the wound healing rates on PID 5, 10, and 15 were calculated. The wound and wound margin tissue of rats with full-thickness skin defects in the five groups on PID 15 and normal skin tissue in the same site of rats in normal group were collected, hematoxylin and eosin staining was conducted to observe the histological changes, immunohistochemical staining was performed to observe the expressions of CD31 and vascular endothelial growth factor (VEGF), and Western blotting was conducted to detect the protein expressions of CD31 and VEGF. The number of samples was all three. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, and Bonferroni correction. Results: Simple microspheres were spherical, with loose and porous surface. The surfaces of P311 microspheres and FITC-BSA microspheres were smooth without pores, and the FITC-BSA microspheres emitted uniform green fluorescence. The diameters of the three microspheres were basically consistent, being 33.1 to 37.7 μm. Compared with chitosan solution and simple thermosensitive hydrogel, the structures of the two microspheres-loaded hydrogels were more stable in the state of tilt at 37 ℃. The two microspheres-loaded hydrogels had denser network structures than those of chitosan solution and simple thermosensitive hydrogel, and in the cross section of which microspheres with a diameter of about 30 μm could be seen. Within PID 15, the wounds of rats in the five groups were healed to different degrees, and the wound healing of rats in P311 microspheres-loaded hydrogel group was the best. On PID 5, 10, and 15, the wound healing rates of rats in dressing group and chitosan group were (26.6±2.4)%, (38.5±3.1)%, (50.9±1.5)%, (47.6±2.0)%, (58.5±3.6)%, and (66.7±4.1)%, respectively, which were significantly lower than (59.3±4.8)%, (87.6±3.2)%, (97.2±1.0)% in P311 microspheres-loaded hydrogel group ( P <0.05 or P <0.01). The wound healing rates of rats in hydrogel alone group on PID 10 and 15, and in simple microspheres-loaded hydrogel group on PID 15 were (76.0±3.3)%, (84.5±3.6)%, and (88.0±2.6)%, respectively, which were significantly lower than those in P311 microspheres-loaded hydrogel group ( P <0.05). The epidermis, hair follicles, and sebaceous glands could be seen in the normal skin of rats in normal group, without positive expressions of CD31 or VEGF. The wounds of rats in P311 microspheres-loaded hydrogel group on PID 15 were almost completely epithelialized, with more blood vessels, hair follicles, sebaceous glands, and positive expressions of CD31 and VEGF in the wounds than those of rats with full-thickness skin defects in the other four groups, and more protein expressions of CD31 and VEGF than those of rats in the other five groups. Conclusions: The P311 microspheres-loaded thermosensitive chitosan hydrogel can release the encapsulated drug slowly, prolong the drug action time, and promote wound healing in rats with full-thickness skin defects by promoting wound angiogenesis and re-epithelialization.

Objective: To study the effects of reactive oxygen species (ROS)-responsive antibacterial microneedles (MNs) on the full-thickness skin defect wounds with bacterial colonization in diabetic mice. Methods: Experimental research methods were adopted. The ROS-responsive crosslinker N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-tetramethylpropane-1,3-diaminium (TSPBA) was first synthesized, and then the polyvinyl alcohol (PVA)-TSPBA MNs, PVA-ε-polylysine (ε-PL)-TSPBA MNs, PVA-TSPBA-sodium hyaluronate (SH) MNs, and PVA-ε-PL-TSPBA-SH MNs were prepared by mixing corresponding ingredients, respectively. The PVA-TSPBA MNs were placed in pure phosphate buffer solution (PBS) and PBS containing hydrogen peroxide, respectively. The degradation of MNs immersed for 0 (immediately), 3, 7, and 10 days was observed to indicate their ROS responsiveness. The standard strains of Staphylococcus aureus ( S. aureus ) and Escherichia coli (E. coli) cultured in Luria-Bertani medium containing hydrogen peroxide were divided according to the random number table (the same grouping method below) into blank control group (without any treatment, the same below) and 0 g/L ε-PL group, 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group with which PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL were co-cultured, respectively. Bacterial growth was observed after 24 h of culture, and the relative survival rate of bacteria was calculated ( n =3). The mouse fibroblast cell line 3T3 cells at logarithmic growth stage (the same growth stage below) were divided into blank control group and 0 g/L ε-PL group, 1.0 g /L ε-PL group, 5.0 g /L ε-PL group, and 10.0 g /L ε-PL group in which cells were cultured in medium with the extract from PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL, respectively. Cell growth was observed after 24 h of culture by optical microscopy, and the relative survival rate of cells was detected and calculated by cell counting kit 8 (CCK-8) assay to indicate the cytotoxicity ( n =6). Both PVA-TSPBA MNs and PVA-TSPBA-SH MNs were taken, the morphology of the two kinds of MNs was observed by optical microscopy, and the mechanical properties of the two kinds of MNs were tested by microcomputer controlled electronic universal testing machine (denoted as critical force, n =6). Six male BALB/c mice aged 6-8 weeks (the same gender and age below) were divided into PVA-TSPBA group and PVA-TSPBA-SH group, with 3 mice in each group. After pressing the skin on the back of mice vertically with the corresponding MNs for 1 minute, the skin condition was observed at 0, 10, and 20 min after pressing. Another batch of 3T3 cells were divided into blank control group, 0 g/L ε-PL group and simple 5.0 g/L ε-PL group which were cultured with the extract of PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL, and 5.0 g/L ε-PL+SH group which were cultured with the extract of PVA-ε-PL-TSPBA-SH MNs with 5.0 g/L ε-PL. The CCK-8 assay was performed to detect and calculate the relative survival rate of cells cultured for 24, 48, and 72 h to indicate the cell proliferation activity ( n =6). Eighteen BALB/c mice were induced into diabetic mice model by high-sugar and high-fat diet combined with streptozotocin injection and then divided into sterile dressing group, 0 g/L ε-PL+SH group, and 5.0 g/L ε-PL+SH group, with 6 mice in each group. A full-thickness skin defect wound was made on the back of each mouse, and S. aureus solution was added to make a full-thickness skin defect wound with bacterial colonization model for diabetic mouse. The wounds of mice in 0 g/L ε-PL+SH group and 5.0 g/L ε-PL+SH group were covered with PVA-ε-PL-TSPBA-SH MNs with the corresponding concentration of ε-PL, and the wounds of mice in the 3 groups were all covered with sterile surgical dressings. The wound healing was observed on post injury day (PID) 0, 3, 7, and 12, and the wound healing rate on PID 3, 7, and 12 was calculated. On PID 12, the skin tissue of the wound and the wound margin were stained with hematoxylin and eosin to observe the growth of new epithelium and the infiltration of inflammatory cells. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, Mann-Whitney U test, and Bonferroni test. Results: With the extension of the immersion time, the PVA-TSPBA MNs in PBS containing hydrogen peroxide gradually dissolved and completely degraded after 10 days of immersion. The PVA-TSPBA MNs in pure PBS only swelled but did not dissolve. After 24 h of culture, there was no growth of S. aureus in 5.0 g/L ε-PL group or 10.0 g/L ε-PL group, and there was no growth of E. coli in 10.0 g/L ε-PL group. The relative survival rate of S. aureus was significantly lower in 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group than in blank control group ( P <0.05 or P <0.01). The relative survival rate of E. coli was significantly lower in 5.0 g/L ε-PL group and 10.0 g/L ε-PL group than in blank control group ( P <0.01). After 24 h of culture, the cells in blank control group, 0 g/L ε-PL group, 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group all grew well, and the relative survival rate of cells was similar among the groups ( P >0.05). The needle bodies of PVA-TSPBA MNs and PVA-TSPBA-SH MNs were both quadrangular pyramid-shaped and neatly arranged, and the needle bodies of PVA-TSPBA-SH MNs was more three-dimensional and more angular. The critical force of PVA-TSPBA-SH MNs was significantly higher than that of PVA-TSPBA MNs ( Z =3.317, P <0.01). The MNs in PVA-TSPBA+SH group penetrated the skin of mice at 0 min after pressing, and the pinholes partially disappeared after 10 min and completely disappeared after 20 min, while the MNs in PVA-TSPBA group failed to penetrate the skin of mice. After 24, 48, and 72 h of culture, the proliferation activity of the cells in 5.0 g/L ε-PL+SH group was significantly higher than that of blank control group ( P <0.05 or P <0.01). In sterile dressing group, the wounds of mice healed slowly and exuded more. The wound healing speed of mice in 0 g/L ε-PL+SH group was similar to that of sterile dressing group in the early stage but was faster than that of sterile dressing group in the later stage, with moderate exudation. The wound healing of mice in 5.0 g/L ε-PL+SH group was faster than that in the other two groups, with less exudation. The wound healing rates of mice in 5.0 g/L ε-PL+SH group were (40.6±4.2)%, (64.3±4.1)%, and (95.8±2.4)% on PID 3, 7, and 12, which were significantly higher than (20.4±2.7)%, (38.9±2.2)%, and (59.1±6.2)% in sterile dressing group and (21.6±2.6)%, (44.0±1.7)%, and (82.2±5.3)% in 0 g/L ε-PL+SH group ( P <0.01). The wound healing rates of mice in 0 g/L ε-PL+SH group on PID 7 and 12 were significantly higher than those in sterile dressing group ( P <0.05 or P <0.01). On PID 12, the wounds of mice in 5.0 g/L ε-PL+SH group were almost completely epithelialized with less inflammatory cell infiltration, the wounds of mice in 0 g/L ε-PL+SH group were partially epithelialized with a large number of inflammatory cell infiltration, and no obvious epithelialization but a large number of inflammatory cell infiltration was found in the wounds of mice in sterile dressing group. Conclusions: The composite MNs prepared by TSPBA, PVA, ε-PL, and SH can successfully penetrate mouse skin and slowly respond to ROS in the wound to resolve and release antibacterial substances, inhibit bacterial colonization, and promote the repair of full-thickness skin defect wounds with bacterial colonization in diabetic mice.

Phosphorylation of MAP4 (p-MAP4) causes cardiac remodeling, with the cardiac microvascular endothelium being considered a vital mediator of this process. In the current study, we investigated the mechanism underlying p-MAP4 influences on cardiac microvascular density. We firstly confirmed elevated MAP4 phosphorylation in the myocardium of MAP4 knock-in (KI) mice. When compared with the corresponding control group, we detected the decreased expression of CD31, CD34, VEGFA, VEGFR2, ANG2, and TIE2 in the myocardium of MAP4 KI mice, accompanied by a reduced plasma concentration of VEGF. Moreover, we observed apoptosis and mitochondrial disruption in the cardiac microvascular endothelium of MAP4 KI animals. Consistently, we noted a decreased cardiac microvascular density, measured by CD31 and lectin staining, in MAP4 KI mice. To explore the underlying mechanism, we targeted the NLRP3-related pyroptosis and found increased expression of the corresponding proteins, including NLRP3, ASC, mature IL-1β, IL-18, and GSDMD-N in the myocardium of MAP4 KI mice. Furthermore, we utilized a MAP4 (Glu) adenovirus to mimic cellular p-MAP4. After incubating HUVECs with MAP4 (Glu) adenovirus, the angiogenic ability was inhibited, and NLRP3-related pyroptosis were significantly activated. Moreover, both cytotoxicity and PI signal were upregulated by the MAP4 (Glu) adenovirus. Finally, NLRP3 inflammasome blockage alleviated the inhibited angiogenic ability induced by MAP4 (Glu) adenovirus. These results demonstrated that p-MAP4 reduced cardiac microvascular density by activating NLRP3-related pyroptosis in both young and aged mice. We thus managed to provide clues explaining MAP4 phosphorylation-induced cardiac remodeling and enriched current knowledge regarding the role of MAP4., (© 2021. The Author(s).)

The exact relationships and detailed mechanisms between autophagy and necroptosis remain obscure. Here, we demonstrated the link between accumulated autophagosome and necroptosis by intervening with autophagic flux. We first confirmed that the LC3 interacting region (LIR) domain is present in the protein sequences of RIPK1 and RIPK3. Mutual effects among LC3, RIPK1, and RIPK3 have been identified in myocardium and cardiomyocytes. Direct LC3-RIPK1 and LC3-RIPK3 interactions were confirmed by pull-down assays, and their interactions were deleted after LIR domain mutation. Moreover, after disrupting autophagic flux under normoxia with bafilomycin A1 treatment, or with LC3 or ATG5 overexpression adenovirus, RIPK1, RIPK3, p-RIPK3, and p-MLKL levels increased, suggesting necroptosis activation. Severe disruptions in autophagic flux were observed under hypoxia and bafilomycin A1 co-treated cardiomyocytes and myocardium and led to more significant activation of necroptosis. Conversely, after alleviating hypoxia-induced autophagic flux impairment with LC3 or ATG5 knockdown adenovirus, the effects of hypoxia on RIPK1 and RIPK3 levels were reduced, which resulted in decreased p-RIPK3 and p-MLKL. Furthermore, necroptosis was inhibited by siRNAs against RIPK1 and RIPK3 under hypoxia or normoxia. Based on our results, LIR domain mediated LC3-RIPK1 and LC3-RIPK3 interaction. Besides, autophagosome accumulation under hypoxia lead to necrosome formation and, in turn, necroptosis, while when autophagic flux was uninterrupted, RIPK1 and RIPK3 were cleared through an autophagy-related pathway which inhibited necroptosis. These findings provide novel insights for the role of LC3 in regulating cardiomyocyte necroptosis, indicating its therapeutic potential in the prevention and treatment of hypoxic myocardial injury and other hypoxia-related diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Huang, Feng, Cui, Yang, Zhang, Zhang, Jiang, Zhang, Lv, Jia, Zhang and Huang.)

Objective: To explore the effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 (BNIP3) on the migration and motility of human dermal microvascular endothelial cells (HDMECs) under hypoxia and the mechanism. Methods: The experimental research method was applied. (1) HDMECs were divided into normoxia group received routine culture and hypoxia 6, 12, 24 h groups treated under hypoxia with oxygen volume fraction of 2% for corresponding time according to the random number table (the same grouping method below). Western blotting was used to detect the protein expressions of BNIP3 and microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) in HDMECs. (2) HDMECs were divided into normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group which were transfected with unloaded virus or BNIP3 knockdown virus and were subjected to normoxic or hypoxic treatment. The BNIP3 protein expression was detected by Western blotting and immunofluorescence staining. The scratch area at 24 h post scratching was detected by scratch test, and the healing rate of scratch was calculated. The curve distance of cell movement was measured with the living cell workstation, and the speed of movement was calculated within 3 hours. (3) HDMECs were grouped and treated as experiment (2). Western blotting and immunofluorescence staining were performed to detect the protein expression of LC3Ⅱ. The number of sample was 3 in the above-mentioned experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: (1) Compared with those of normoxia group, the protein expressions of BNIP3 and LC3Ⅱ of cells in hypoxia 6, 12, 24 h groups were significantly increased ( P <0.01). (2) After 6 hours of culture, compared with that of hypoxia+ unloaded group, the BNIP3 protein expressions of cells in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were significantly decreased ( P <0.05 or P <0.01). The red fluorescence denoting BNIP3 protein expression of cells in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group was weak, the red fluorescence of cells in hypoxia+ unloaded group was strong, and the red fluorescence of cells in hypoxia+ BNIP3 knockdown group was significantly decreased compared with that in hypoxia+ unloaded group. After scratching for 24 hours, the scratch of cells in hypoxia+ unloaded group basically healed, while the remaining scratch area in the other three groups were large. The healing rates of scratch of cells in normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group were (61±4)%, (58±4)%, (88±4)%, and (57±4)%, respectively. The healing rate of scratch of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group ( P <0.01) and hypoxia+ BNIP3 knockdown group ( P <0.05). Within 3 hours of observation, the range of cell movement in hypoxia+ unloaded group was significantly larger than that in normoxia+ unloaded group, the range of cell movement in hypoxia+ BNIP3 knockdown group was significantly smaller than that in hypoxia+ unloaded group, and the curve movement velocity of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group ( P <0.01). (3) After 6 hours of culture, compared with hypoxia+ unloaded group, the LC3Ⅱ protein expressions of cells in hypoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were decreased significantly ( P <0.05 or P <0.01). After 6 hours of culture, the red fluorescence denoting LC3 protein expressions of cells was weak in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group, the red fluorescence of cells was significantly enhanced in hypoxia+ unloaded group, and the red fluorescence of cells was significantly inhibited in hypoxia+ BNIP3 knockdown group. Conclusions: BNIP3 can promote the migration and motility of HDMECs under hypoxia, and autophagy may be involved in the regulation migration of HDMECs by BNIP3.

Objective: To investigate the effects and mechanism of mitochondrial transcription factor A (TFAM) and cytochrome c oxidase (COX) pathway in the energy production of hypoxic cardiomyocytes of rats regulated by tumor necrosis factor receptor associated protein 1 (TRAP1). Methods: The cardiomyocytes were isolated from 135 neonatal Sprague-Dawley rats (aged 1-3 d) and cultured for the following experiments. (1) Cells were collected and divided into normoxia blank control (NBC) group, hypoxia blank control (HBC) group, hypoxia+ TRAP1 over-expression control (HTOC) group, and hypoxia+ TRAP1 over-expression (HTO) group according to the random number table (the same grouping method below), with 1 bottle in each group. Cells in NBC group were cultured routinely, cells in HBC group were cultured in hypoxic condition for 6 hours after routine culture, cells in HTOC and HTO groups were respectively added with TRAP1 over-expression empty virus vector and TRAP1 over-expression adenovirus vector virus suspension for transfection for 48 hours after routine culture and then cultured in hypoxic condition for 6 hours. The protein expression of TFAM of cells in each group was detected by Western blotting. (2) Cells were collected and divided into NBC, HBC, HTOC, HTO, HTO+ TFAM interference control (HTOTIC), and HTO+ TFAM interference (HTOTI) groups, with 1 well in each group. Cells in the former 4 groups were dealt with the same methods as the corresponding groups in experiment (1). Cells in HTOTIC and HTOTI groups were respectively added with TFAM interference empty virus vector and TFAM interference adenovirus vector virus suspension for transfection for 48 hours, and the other processing methods were the same as those in HTO group. The content of ATP of cells in each group was determined by ATP determination kit and microplate reader, and the COX activity of cells in each group was determined by COX activity assay kit and microplate reader. (3) Cells were collected and divided into NBC group, normoxia+ sodium azide (NSA) group, HBC group, and hypoxia+ sodium azide (HSA) group, with 1 well in each group. Cells in NBC and HBC groups were respectively dealt with the same methods as the corresponding groups in experiment (1). Cells in NSA and HSA groups were respectively added with 32 nmol sodium azide at 30 min before experiment or hypoxia, and then cells in HSA group were cultured in hypoxic condition for 6 hours. The content of ATP was determined by the same method as above. The above three experiments were repeated for three times. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: (1) Compared with that in NBC group, the protein expression of TFAM of cells in HBC group was significantly decreased ( P <0.01). Compared with that in HBC group or HTOC group, the protein expression of TFAM of cells in HTO group was significantly increased ( P <0.01). (2) Compared with 0.552±0.041 and 1.99±0.15 in NBC group, the COX activity (0.270±0.044) and ATP content (1.09±0.11) of cells in HBC group were significantly decreased ( P <0.01). Compared with 0.269±0.042 and 1.17±0.12 in HBC group and those in HTOC group, the COX activity (0.412±0.032 and 0.404±0.016) and ATP content (1.75±0.06 and 1.69±0.07) of cells in HTO and HTOTIC groups were significantly increased ( P <0.01). Compared with those in HTO and HTOTIC groups, the COX activity (0.261±0.036) and ATP content (1.23±0.07) of cells in HTOTI group were significantly decreased ( P <0.01). (3) Compared with that in NBC group, the ATP content of cells in NSA and NBC groups was significantly decreased ( P <0.01). Compared with that in HBC group, the ATP content of cells in HSA group was significantly decreased ( P <0.01). Conclusions: TRAP1 can increase the COX activity of cardiomyocytes by raising the expression of TFAM, and finally alleviate the impairment in energy production of cardiomyocytes caused by hypoxia.

Objective: To explore the mechanism of CD38-mediated cardiac damage under hypoxic-ischemic (H/I) conditions. Methods: Twenty CD38(-/-) male mice (8-week-old) and 20 wild-type (WT) male C57BL/6J mice (8-week-old) were randomly selected to construct the model of approximately 25% of the total body surface area (TBSA) burn injury. The cardiomyocytes (CMs) were separated from neonatal mice (1day) to construct the H/I injury model. Ad-CD38 adenovirus was transfected into CD38(-)/- primary CMs to callback CD38 expression. Animal experiments were grouped into WT-control group, CD38(-/-)-control group, WT-burn group, and CD38(-/-)-burn group (10 mice in each group). Primary CMs were divided into 6 groups: WT-normoxia group, CD38(-/-)-normoxia group, CD38(-/-)+Ad-CD38-normoxia group, WT-H/I group, CD38(-/-)-H/I group, CD38(-/-)+Ad-CD38-H/I group. The release of lactic dehydrogenase (LDH) from CMs and the cell viability were measured to estimate the level of myocardial injury. Ultrastructure of cardiomyocytes was examined by electron microscope. CD38 protein level and mitochondrial apoptosis-related proteins were detected by Western blot. Flow cytometry was used to detect mitochondrial reactive oxygen species (MitoSOX) of CMs under H/I condition. Cardiac function of mice was detected by ultrasonic apparatus. Results: (1) Animal experiments: The expression level of cardiac CD38 in WT-burn group was significantly higher than that in sham group ( P< 0.001). The heart function of CD38(-/-)-burn group was obviously better than WT-burn group [ejection fraction (EF)%: (84.70±2.31)% vs (76.10±2.96)%, shortening fraction (FS)%: (48.90±5.00)% vs (38.10±2.80)%] (both P< 0.001). (2) Cell experiments: The expression level of cardiac CD38 in WT CMs under H/I condition was significantly higher than that in WT CMs under normoxia condition ( P< 0.05). The level of LDH, apoptotic cell and MitoSOX in CD38(-/-)-H/I group were fewer than WT-H/I group and CD38(-/-)+Ad-CD38(-)H/I group [(11.2±3.0)% vs (18.2±3.4)% and (17.6±4.0)%, (13.0±2.8)% vs (23.1±4.9)% and (23.3±6.0)%, (162±11)% vs (228±18)% and (220±18)%] (all P< 0.001). The levels of cleaved-caspase3, Cytochrome-C in CD38(-/-)-H/I group were significantly lower than those in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group ( P< 0.001). The cell viability in CD38(-/-)-H/I group was higher than that in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group (0.355±0.043 vs 0.280±0.051 and 0.291±0.024) (all P< 0.05). Electron microscopy results showed that structure of mitochondria in CD38(-/-)-H/I group was better than in WT-H/I group and CD38(-/-)+Ad-CD38-H/I group. Conclusion: Overexpression of CD38 contributes to cardiac damage by stimulating mitochondrial apoptotic pathway.

Objective: To explore the effect and potential mechanism of cardiac adipose triglyceride lipase (ATGL) overexpression on burn-induced cardiac injury. Methods: Eight-week-old C57BL/6J mice with cardiac ATGL overexpression driven by the myosin heavy chain (MHC) promoter (MHC-ATGL burn group) and wild-type (wild-type burn group) mice were randomly chose to the following experiments with burn injury after 24 h ( n= 8/group), MHC-ATGL mice and wild-type mice with corresponded age and sex were included as control. Cardiac ATGL protein expression, serum levels of cardiac troponin T and cardiac kinase-MB (CK-MB), cardiac free fatty acid and reactive oxygen species were detected. The wild-type and MHC-ATGL burn groups were not only compared with their corresponded control groups, but also compared between each other. Results: The hair color and development were shown little difference between each group. ATGL protein expression is elevated in wild-type burn group (1.00±0.68 vs 3.09±0.93, P= 0.023) and decreased in MHC-ATGL burn group (17.84±2.41 vs 10.36±2.22, P< 0.001), while ATGL protein expression is still increased in MHC-ATGL burn group compared with wild-type burn group ( P< 0.001). Serum levels of cardiac troponin T and CK-MB were both elevated in wild-type burn group and MHC-ATGL burn group [(0.456±0.131) vs (0.076±0.019) μg/L and (0.219±0.089) vs (0.060±0.019) μg/L, (1 421±162) vs (221±67) U/L and (761±142) vs (221±41) U/L] (all P< 0.001), while serum levels of cardiac troponin T and CK-MB was still decreased in MHC-ATGL burn group compared with wild-type burn group ( P< 0.001). In addition, cardiac free fatty acid was increased in wild-type burn group and little difference was found in MHC-ATGL burn group [(2.54±0.51) vs (0.46±0.27) mmol/L, P< 0.001, and (0.81±0.38) vs (0.59±0.25) mmol/L, P= 0.251], while cardiac free fatty acid was significant reduction in MHC-ATGL burn group compared with wild-type burn group ( P< 0.001). Levels of cardiac reactive oxygen species was both elevated in wild-type burn group and MHC-ATGL burn group [(1.89±0.23) vs (1.00±0.18) and (1.38±0.17) vs (0.95±0.13)] (both P< 0.001), while levels of cardiac reactive oxygen was reduction in MHC-ATGL burn group compared with wild-type burn group ( P< 0.001). Conclusion: Cardiac ATGL overexpression may protect against burn-induced cardiac injury through reducing free fatty acid and reactive oxygen species production.

Lysosomal membrane permeabilization (LMP) has recently been recognized as an important cell death pathway in various cell types. However, studies regarding the correlation between LMP and cardiomyocyte death are scarce. Lysosomal membrane-associated protein 2 (Lamp2) is an important component of lysosomal membranes and is involved in both autophagy and LMP. In the present study, we found that the protein content of Lamp2 gradually decreased in response to oxygen, glucose and serum deprivation (OGD) treatment in vitro . To further elucidate its role in ischemic cardiomyocytes, particularly with respect to autophagy and LMP, we infected cardiomyocytes with adenovirus carrying full-length Lamp2 to restore its protein level in cells. We found that OGD treatment resulted in the occurrence of LMP and a decline in the viability of cardiomyocytes, which were remarkably reversed by Lamp2 restoration. Exogenous expression of Lamp2 also significantly alleviated the autophagic flux blockade induced by OGD treatment by promoting the trafficking of cathepsin B (Cat B) and cathepsin D (Cat D). Through drug intervention and gene regulation to alleviate and exacerbate autophagic flux blockade respectively, we found that impaired autophagic flux in response to ischemic injury contributed to the occurrence of LMP in cardiomyocytes. In conclusion, our present data suggest that Lamp2 overexpression can improve autophagic flux blockade probably by promoting the trafficking of cathepsins and consequently conferring cardiomyocyte resistance against lysosomal cell death (LCD) that is induced by ischemic injury. These results may indicate a new therapeutic target for ischemic heart damage., (Copyright © 2020 Cui, Zhao, Ye, Yang, Huang, Jiang, Zhang, Jia, Zhang and Huang.)

Objective: To investigate the effect of human antigen R on lysosomal acidification during autophagy in mouse cardiomyocytes cultured in vitro. Methods: The hearts of 20 C57BL/6 mice aged 1-2 days no matter male or female were isolated to culture primary cardiomyocytes which were used in the following experiments. (1) The cells were divided into 5 groups according to the random number table (the same grouping method below), i. e., normal control group and sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups. The cells in normal control group were routinely cultured for 54.0 h with Dulbecco's modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), and the cells in sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups were firstly regularly cultured for 53.5, 53.0, 51.0, 48.0 h and then cultured with replaced sugar-free serum-free medium for 0.5, 1.0, 3.0, and 6.0 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 Ⅱ (LC3Ⅱ), autophagy-related protein 5, and adenosine triphosphatase V1 region E1 subunit (ATP6V1E1) were detected by Western blotting. (2) The cells were divided into normal control group and sugar-free serum-free 3.0 h group. The cells in corresponding groups were treated the same as those in experiment (1), and the cell lysosomal acidification level was observed and detected under a laser scanning confocal microscope. (3) Two batches of cells were grouped and treated the same as those in experiment (1). The protein expression of human antigen R in the whole protein of cells of one batch and its protein expression in the cytoplasm and nucleus protein of cells of the other batch were detected by Western blotting. (4) The cells were divided into normal control group, simple control small interfering RNA (siRNA) group, simple human antigen R-siRNA1 (HuR-siRNA1) group, simple HuR-siRNA2 group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group. After 48 hours of regular culture, the cells in simple control siRNA group and sugar-free serum-free+ control siRNA group were transfected with negative control siRNA for 6 h, the cells in simple HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA1 group were transfected with HuR-siRNA1 for 6 h, and the cells in simple HuR-siRNA2 group and sugar-free serum-free+ HuR-siRNA2 group were transfected with HuR-siRNA2 for 6 h. Hereafter, the cells in these 8 groups were continuously cultured for 48 h with regular conditon, and then the cells in normal control group and each simple siRNA-treated group were replaced with DMEM/F12 medium, the cells in the other groups were replaced with sugar-free serum-free medium, and they were cultured for 3 h. The protein expression of human antigen R in the whole protein of cells was detected by Western blotting. (5) Two batches of cells were divided into sugar-free serum-free+ control siRNA group and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The distribution and expression of human antigen R in the cells of one batch were observed and detected by immunofluorescence method, and the lysosomal acidification level in the cells of the other batch was observed and detected under a laser scanning confocal microscope. (6) Three batches of cells were divided into sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, sugar-free serum-free+ HuR-siRNA1 group, and sugar-free serum-free+ HuR-siRNA2 group, and the cells in corresponding groups were treated the same as those in experiment (4). The protein expressions of cathepsin D in the whole protein of cells of one batch, human antigen R in the cytoplasm protein of cells of one batch, and ATP6V1E1 in the whole protein of cells of the other batch were detected by Western blotting. (7) The cells were divided into normal control group, sugar-free serum-free 3.0 h group, sugar-free serum-free+ control siRNA group, and sugar-free serum-free+ HuR-siRNA1 group, and the cells in corresponding groups were treated the same as those in experiment (4). The mRNA expression of ATP6V1E1 in cells was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The sample number of each experiment was 3. Data were processed with independent data t test, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normal control group, the protein expressions of LC3Ⅱ and ATP6V1E1 in the whole protein of cells of sugar-free serum-free 1.0, 3.0, and 6.0 h groups were significantly increased ( t =12.16, 4.05, 4.82, 11.64, 3.29, 8.37, P <0.05 or P <0.01). Compared with that of normal control group, the protein expression of autophagy-related protein 5 in the whole protein of cells of sugar-free serum-free 0.5, 1.0, 3.0, and 6.0 h groups was significantly increased ( t =6.88, 10.56, 5.76, 9.91, P <0.05 or P <0.01). (2) Compared with 1.03±0.08 of normal control group, the lysosomal acidification level in the cells of sugar-free serum-free 3.0 group (2.92±0.30) was significantly increased ( t =6.01, P <0.01). (3) There was no statistically significant difference in the overall comparison of protein expression of human antigen R in the whole protein of cells among the 5 groups ( F =1.09, P >0.05). Compared with that of normal control group, the protein expression of human antigen R in the cytoplasm protein of cells was significantly increased in sugar-free serum-free 1.0, 3.0, and 6.0 h groups ( t =43.05, 11.07, 5.39, P <0.05 or P <0.01), while the protein expression of human antigen R in the nucleus protein of cells was significantly decreased in sugar-free serum-free 3.0 and 6.0 h groups ( t =11.18, 12.71, P <0.01). (4) Compared with that of simple control siRNA group, the protein expression of human antigen R in the whole protein of cells of simple HuR-siRNA1 group and simple HuR-siRNA2 group was significantly decreased ( t =4.82, 4.44, P <0.05). Compared with that of sugar-free serum-free+ control siRNA group, the protein expression of human antigen R in the whole protein of cells of sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group was significantly decreased ( t =4.39, 6.27, P <0.05). (5) Compared with those of sugar-free serum-free+ control siRNA group, the distribution of human antigen R in the cytoplasm of cells and its expression level were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group ( t =10.13, P <0.01). Compared with 1.00±0.06 of sugar-free serum-free+ control siRNA group, the lysosomal acidification level (0.73±0.06) in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased ( t =3.28, P <0.01). (6) Compared with those of sugar-free serum-free+ control siRNA group, the protein expressions of cathepsin D in the whole protein of cells, human antigen R in the cytoplasm protein of cells, and ATP6V1E1 in the whole protein of cells were significantly decreased in sugar-free serum-free+ HuR-siRNA1 group and sugar-free serum-free+ HuR-siRNA2 group ( t =4.16, 3.99, 4.81, 5.07, 11.68, 12.97, P <0.05 or P <0.01). (7) Compared with that of normal control group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free 3.0 h group was significantly increased ( t =5.51, P <0.05). Compared with that of sugar-free serum-free+ control siRNA group, the mRNA expression of ATP6V1E1 in the cells of sugar-free serum-free+ HuR-siRNA1 group was significantly decreased ( t =5.97, P <0.05). Conclusions: After sugar-free serum-free treatment in vitro, the autophagy in mouse primary cardiomyocytes is activated, the lysosomal acidification is enhanced, and the expression of human antigen R in cytoplasm is increased. Human antigen R function is activated and involved in maintaining lysosomal acidification during autophagy in mouse cardiomyocytes.

Objective: To explore the effects of transient receptor potential vanilloid 1 (TRPV1) on autophagy in early hypoxic mouse cardiomyocytes and the mechanism in vitro. Methods: The hearts of 120 C57BL/6 mice aged 1-2 days, no matter male or female, were isolated, and then primary cardiomyocytes were cultured and used for the following experiments, the random number table was used for grouping. (1) The cells were divided into normoxia group and hypoxia 3, 6, and 9 h groups, with one well in each group. The cells in normoxia group were routinely cultured (the same below), the cells in hypoxia 3, 6, and 9 h groups were treated with fetal bovine serum-free and glucose-free Dulbecco' s modified Eagle medium under low oxygen condition in a volume fraction of 1% oxygen, 5% carbon dioxide, and 94% nitrogen for 3, 6, and 9 h, respectively. The protein expressions of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, TRPV1 were determined with Western botting. (2) The cells were divided into normoxia group and hypoxia group, with two coverslips in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above. The positive expression of TRPV1 was detected by immunofluorescence assay. (3) The cells were divided into 4 groups, with one well in each group. The cells in simple hypoxia group were treated with hypoxia for 6 h as above, and the cells in hypoxia+ 0.1 μmol/L capsaicin group, hypoxia+ 1.0 μmol/L capsaicin group, and hypoxia+ 10.0 μmol/L capsaicin group were respectively treated with 0.1, 1.0, 10.0 μmol/L capsaicin for 30 min before hypoxia for 6 h. The protein expressions of LC3, Beclin-1, and TRPV1 were detected by Western blotting. (4) The cells were divided into 5 groups, with 5 wells in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ chloroquine group, hypoxia+ capsaicin group, and hypoxia+ capsaicin+ chloroquine group were treated with hypoxia for 6 h after being cultured with 50 μmol/L chloroquine, 10.0 μmol/L capsaicin, and 50 μmol/L chloroquine+ 10.0 μmol/L capsaicin for 30 min, respectively. Viability of cells was detected by cell counting kit 8 assay. (5) The cells were divided into simple hypoxia group and hypoxia+ 10.0 μmol/L capsaicin group, with one well in each group. The cells in hypoxia group were treated with hypoxia for 6 h as above, the cells in hypoxia+ 10.0 μmol/L capsaicin group were treated with 10.0 μmol/L capsaicin for 30 minutes and then with hypoxia for 6 h. The protein expressions of lysosomal associated membrane protein 1 (LAMP-1) and LAMP-2 were detected by Western blotting. Each experiment was repeated for 3 or 5 times. Data were processed with one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: (1) Compared with those of normoxia group, the protein expressions of LC3, Beclin-1, and TRPV1 were significantly increased in cardiomyocytes of hypoxia 3, 6, and 9 h groups ( t (3 h)=4.891, 5.890, 4.928; t (6 h)=9.790, 6.750, 10.590; t (9 h)=6.948, 6.764, 5.049, P <0.05 or P <0.01), which of hypoxia 6 h group were the highest (1.08±0.05, 1.12±0.10, 0.953±0.071, respectively). (2) The density of TRPV1 in cell membrane and inside the cardiomyocytes in hypoxia group was significantly increased with lump-like distribution, and the expression of TRPV1 was higher than that in normoxia group. (3) Compared with those of simple hypoxia group, the protein expression of Beclin-1 in cardiomyocytes of hypoxia+ 0.1 μmol/L capsaicin group was increased ( t =10.488, P <0.01), while the protein expressions of LC3 and TRPV1 were increased without statistically significant differences ( t =4.372, 3.026, P >0.05); the protein expressions of LC3, TRPV1, and Beclin-1 in cardiomyocytes of hypoxia+ 1.0 μmol/L capsaicin group and hypoxia+ 10.0 μmol/L capsaicin group were significantly increased ( t =15.505, 5.773, 13.430; 20.915, 8.054, 16.384; P <0.05 or P <0.01), which of hypoxia+ 10.0 μmol/L capsaicin group were the highest (2.33±0.09, 1.34±0.07, 1.246±0.053, respectively). (4) Compared with 0.585±0.045 in normoxia group, the cardiomyocyte viability in hypoxia group was significantly decreased (0.471±0.037, t =4.365, P <0.05). Compared with that in hypoxia group, the cardiomyocyte viability in hypoxia+ chloroquine group was further decreased (0.350±0.023, t =6.216, P <0.01), while 0.564±0.047 in hypoxia+ capsaicin group was significantly increased ( t =3.489, P <0.05). Compared with that in hypoxia+ chloroquine group, the cardiomyocyte viability in hypoxia+ capsaicin+ chloroquine group did not significantly change (0.364±0.050, t =0.545, P >0.05). (5) Compared with 0.99±0.04 and 0.54±0.04 in simple hypoxia group, the protein expressions of LAMP-1 and LAMP-2 in hypoxia+ 10.0 μmol/L capsaicin group were significantly increased (1.49±0.06, 0.81±0.05, t =12.550, 7.442, P <0.01). Conclusions: TRPV1 can further promote the expression of autophagy-related proteins in hypoxic cardiomyocytes through autophagy-lysosomal pathway, enhance autophagy activity, and improve autophagic flow for alleviating early hypoxic cardiomyocyte injury.

The effects of different thawing methods (air thawing, water soak thawing, refrigeration thawing, low frequency ultrasound thawing at 160, 240, 320 and 400 W) on thawing time, thawing loss, cooking loss, water-holding capacity and texture of frozen squid were investigated. The results showed that thawing loss and thawing time were reduced significantly ( p < 0.05) by ultrasound thawing compared with the water soak thawing and air thawing, but the cooking loss had no significant difference ( p > 0.05). Results of the ultrasound thawing especially at 160 and 240 W on microstructure showed less destructive effect on muscle. The microstructure of the muscle was destroyed significantly after air thawing and water soak thawing compared with the ultrasound thawing, which showed that more fibre structure was broken and the gap between the muscle fibres was increased significantly. Low-field NMR results showed that the ability of immobile water shifting to free water after ultrasound thawing was lower than air thawing and water soak thawing, which was consistent with the results of thawing loss and cooking loss. Ultrasound thawing might be chosen as an alternative method to enhance the quality during thawing process.

Objective: To investigate the role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro. Methods: The hearts of totally six male and female C57BL/6 mice aged from 1 to 2 days were isolated to culture primary cardiomyocytes which were used for the following experiments. (1) The cells were divided into 6 groups according to the random number table (the same grouping method below), i. e., normal control 3, 6, and 9 h groups and ischemia-hypoxia 3, 6, and 9 h groups, with 4 wells in each group. After being regularly cultured for 48 h with Dulbecco's modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), the cells in normal control 3, 6, and 9 h groups were cultured with replaced fresh DMEM/F12 medium for 3, 6, and 9 h, respectively, and the cells in ischemia-hypoxia 3, 6, and 9 h groups were cultured with replaced sugar-free serum-free medium in the low-oxygen incubator with a volume fraction of 1% oxygen and a volume fraction of 5% carbon dioxide at 37 ℃ (the same hypoxic culture condition below) for 3, 6, and 9 h, respectively. Cell viability was measured by the cell counting kit 8 (CCK-8) method. (2) The cells were grouped and treated the same as those in experiment (1), with 1 well in each group. Western blotting was used to detect the protein expressions of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ), LC3Ⅱ, p62, and hexokinase Ⅱ. (3) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, and ischemia-hypoxia 9 h+ 2-deoxyglucose (2-DG) group, with 4 wells in each group. After a regular culture for 48 h, the cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h; the cells in simple ischemia-hypoxia 9 h group were replaced with sugar-free serum-free medium, and the cells in ischemia-hypoxia 9 h+ 2-DG group were replaced with sugar-free serum-free medium in which 2-DG was dissolved in a concentration of 10 mmol/L (20 μmol), and then they were cultured with hypoxia for 9 h. Cell viability was measured by CCK-8 method. (4) The cells were grouped and treated the same as those in experiment (3), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, and p62. (5) The cells were grouped and treated the same as those in experiment (3), with 2 wells in each group. Transmission electron microscope was used to observe autophagosomes/autolysosomes in cardiomyocytes. (6) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ hexosinase Ⅱ small interfering RNA1 (HK-ⅡsiRNA1) group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group, with 4 wells in each group. The cells in normal control group and simple ischemia-hypoxia 9 h group were regularly cultured for 48 h, and the cells in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were respectively transfected with 200 nmol/L HK-ⅡsiRNA1 and HK-ⅡsiRNA2 and then also cultured for 48 h. The cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h, and the cells in simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were cultured with replaced sugar-free serum-free medium and hypoxia for 9 h. Cell viability was measured by CCK-8 method. (7) The cells were grouped and treated the same as those in experiment (6), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, p62, and hexokinase Ⅱ. Except for experiment (5), each experiment was repeated 3 times. Data were processed with one-way analysis of variance and lest significant difference t test, and Bonferroni correction. Results: (1) The viabilities of cardiomyocytes in ischemia-hypoxia 3, 6, and 9 h groups were 0.450±0.022, 0.385±0.010, and 0.335±0.015, respectively, which were significantly lower than 0.662±0.026, 0.656±0.028, and 0.661±0.021 of the corresponding normal control 3, 6, and 9 h groups, respectively ( t =6.21, 9.12, 12.48, P <0.01). (2) Compared with those of corresponding normal control 3, 6, and 9 h groups, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 3, 6, and 9 h groups were significantly increased ( t (3 h)=16.15, 10.99, 5.30, t (6 h)=6.79, 10.42, 9.42, t (9 h)=15.76, 16.51, 7.20, P <0.05 or P <0.01). (3) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.353±0.022, which was significantly lower than 0.673±0.027 of normal control group ( t =9.29, P <0.01). The viability of cardiomyocytes in ischemia-hypoxia 9 h+ 2-DG group was 0.472±0.025, which was significantly higher than that of simple ischemia-hypoxia 9 h group ( t =3.60, P <0.05). (4) Compared with those of normal control group, the LC3Ⅱ/Ⅰ ratio and protein expression of p62 in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased ( t =9.45, 8.40, P <0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰratio and protein expression of p62 in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group were significantly decreased ( t =4.39, 4.74, P <0.05). (5) In cardiomyocytes of normal control group, only single autophagosome/autolysosome with bilayer membrane structure was observed. Compared with that of normal control group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of simple ischemia-hypoxia 9 h group was increased significantly. Compared with that of simple ischemia-hypoxia 9 h group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group was significantly decreased. (6) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.358±0.023, which was significantly lower than 0.673±0.026 in normal control group ( t =9.12, P <0.01). The viabilities of cardiomyocytes in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were 0.487±0.027 and 0.493±0.022, respectively, which were significantly higher than the viability in simple ischemia-hypoxia 9 h group ( t =3.63, 4.28, P <0.05). (7) Compared with those of normal control group, the LC3Ⅱ/Ⅰratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased ( t =6.08, 6.31, 4.83, P <0.05 or P <0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were significantly decreased ( t =5.10, 7.76, 15.33, 4.17, 8.42, 12.11, P <0.05 or P <0.01). Conclusions: Ischemia-hypoxia upregulates the expression level of hexokinase Ⅱ protein in mouse cardiomyocytes cultured in vitro, which decreases the viability of cardiomyocytes by impairing autophagic flow. To inhibit the activity of hexokinase Ⅱ or its expression can alleviate the ischemia-hypoxia damage of cardiomyocytes.

Several lines of evidence suggested that abnormal levels of certain chemical elements may contribute to the development of autism spectrum disorders (ASD). The present work aimed to investigate the multiple chemical elements profile in the erythrocytes of autistic versus typically developing children (TDC) of China. Analyses were carried out to explore the possible association between levels of elements and the risk as well as the severity of ASD. Erythrocyte levels of 11 elements (32%) among 34 detected elements in autistic group were significantly different from those in the TDC group. To our knowledge, this is the first study which compared the levels of rare earth elements in erythrocytes between children with or without ASD. Five elements including Pb, Na, Ca, Sb, and La are associated with the Childhood Autism Rating Scale (CARS) total score. Also, a series of tendencies were found in this research which was believed to affect auditory response, taste, smell, and touch, as well as fear or nervousness. It can be concluded that Chinese autistic children suffer from multi-chemical element imbalances which involves a complex combination of genetic and environmental factors. The results showed a significant correlation between abnormal levels of several chemical elements and the severity of the autistic syndrome., Lay Summary: It is suggested that abnormal levels of some chemical elements may contribute to the development of autism spectrum disorders (ASD). In this work, the impact of element imbalances on the risk and severity of ASD was investigated, focusing on the analysis of abnormal levels of the multi-chemical elements profile in erythrocytes compared with typically developing children. Furthermore, the results showed a significant correlation between abnormal levels of several chemical elements and the severity of the autistic syndrome. Autism Res 2018, 11: 834-845. © 2018 International Society for Autism Research, Wiley Periodicals, Inc., (© 2018 International Society for Autism Research, Wiley Periodicals, Inc.)

In crop plants, a high-density genetic linkage map is essential for both genetic and genomic researches. The complexity and the large size of wheat genome have hampered the acquisition of a high-resolution genetic map. In this study, we report a high-density genetic map based on an individual mapping population using the Affymetrix Wheat660K single-nucleotide polymorphism (SNP) array as a probe in hexaploid wheat. The resultant genetic map consisted of 119 566 loci spanning 4424.4 cM, and 119 001 of those loci were SNP markers. This genetic map showed good collinearity with the 90 K and 820 K consensus genetic maps and was also in accordance with the recently released wheat whole genome assembly. The high-density wheat genetic map will provide a major resource for future genetic and genomic research in wheat. Moreover, a comparative genomics analysis among gramineous plant genomes was conducted based on the high-density wheat genetic map, providing an overview of the structural relationships among theses gramineous plant genomes. A major stable quantitative trait locus (QTL) for kernel number per spike was characterized, providing a solid foundation for the future high-resolution mapping and map-based cloning of the targeted QTL.

Objective: To explore the effects of change of activity of vacuolar adenosine triphosphatase (V-ATPase) of myocardial lysosome on myocardial damage in rats after severe burn and its mechanism. Methods: The myocardial lysosomes were extracted from the hearts of 12 SD rats with ultra-high speed gradient density centrifugation, then Western blotting and transmission electron microscope observation were conducted for identification. One hundred and twenty rats were divided into pure burn group, ATP group, normal control group, and bafilomycin group according to the random number table, with 30 rats in each group. Rats in pure burn group and ATP group were inflicted with 40% TBSA full-thickness scald on the back. Immediately after injury, rats in pure burn group were intraperitoneally injected with lactated Ringer's solution in 4 mL·%TBSA(-1)·kg(-1,) and rats in ATP group were intraperitoneally injected with ATP in 0.4 mg/kg at 12 h before burn, immediately after burn, and 12 h after burn. Rats in normal control group did not receive any treatment, and rats in bafilomycin group were intraperitoneally injected with bafilomycin A1 in 0.3 mg/kg at the same time points as those of ATP group. At 24 h after burn, 30 rats from each group were collected for determining activity of V-ATPase of myocardial lysosome with coupled-enzyme assay and the expression of myocardium autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3) and P62 by Western blotting. Left ventricular arterial blood was collected to detect the content of 5 items of myocardial enzyme spectrum and cardiac troponin T (cTnT). Data were processed with one-way analysis of variance and t test. Results: (1) After identification, both the expression level of lysosome-related membrane protein 1 and purity of lysosome in the sample were high, and the structure of lysosome was intact. (2) At 24 h after burn, the activity values of V-ATPase of myocardial lysosome in rats of pure burn group, ATP group, normal control group, and bafilomycin group were (2.03±0.67), (3.01±0.58), (4.29±0.26), and (1.83±0.52) μmol·mg(-1)·h(-1,) respectively. The activity value of V-ATPase of myocardial lysosome in rats of pure burn group was significantly lower than the values in ATP group and normal control group (with t values respectively 3.14 and 8.87, P values below 0.01). The activity values of V-ATPase of rats in normal control group were significantly higher than those in bafilomycin group ( t =11.87, P <0.01). At 24 h after burn, the expressions of myocardial LC3 and P62 in pure burn group were significantly higher than those in ATP group and normal control group (with t values from 3.73 to 5.88, P values below 0.01). The expressions of myocardial LC3 and P62 in normal control group were significantly lower than those in bafilomycin group (with t values respectively 2.64 and 3.07, P <0.05 or P <0.01). At 24 h after burn, the content of 5 items of myocardial enzyme spectrum and cTnT in pure burn group was significantly higher than that in ATP group and normal control group (with t values from 3.24 to 16.72, P values below 0.01). The content of 5 items of myocardial enzyme spectrum and cTnT in normal control group was significantly lower than that in bafilomycin group (with t values from 2.39 to 10. 70, P values below 0.01). Conclusions: The activity of V-ATPase of myocardial lysosome decreased in rats after severe burn, which can result in myocardial damage by inhibiting myocardial autophagy flux.

Objective: To explore the effects of rapamycin on the migration of human epidermal cell line HaCaT, and to analyze its molecular mechanism., Methods: HaCaT cells were conventionally cultured with RPMI 1640 culture medium containing 10% fetal calf serum (hereinafter referred to as culture medium). (1) According to the random number table, HaCaT cells in logarithmic phase were divided into control group and 1, 5, 50, 100, 200 nmol/L rapamycin groups, with 6 wells in each group. The cells in rapamycin groups were cultured with culture medium containing rapamycin in corresponding mass concentration, and the cells in control group were cultured with culture medium containing dimethyl sulfoxide (DMSO) instead. After being conventionally cultured for 4 hours, proliferative activity of cells was determined with microplate reader (denoted as absorbance value). (2) HaCaT cells in logarithmic phase were grouped and cultured as that in experiment (1), with 1 well in each group. After being conventionally cultured for 4 hours, range of movement of cells in 3 hours was observed under live cell imaging workstation, and their curvilinear movement speeds were calculated. Then the suitable concentration of rapamycin was selected for experiments (3) and (4). (3) HaCaT cells in logarithmic phase were divided into control group and rapamycin group according to the random number table, with 1 well in each group. The cells in rapamycin group were cultured with culture medium containing 50 nmol/L rapamycin, and the cells in control group were cultured with culture medium containing DMSO. After being conventionally cultured for 4 hours, cells were collected for scratch assay. Wound area was observed at post scratching hour (PSH) 0, 5, 10, and 15, and the migration rates of cells at PSH 5, 10, and 15 were calculated respectively. (4) HaCaT cells in logarithmic phase were grouped and cultured as that in experiment (3), with 1 well in each group. Activity of focal adhesion kinase (FAK) was determined with Western blotting (denoted as the ratio of gray value of phosphorylated FAK to that of FAK). Above-mentioned experiments were independently repeated for three or five times. Data were processed with one-way analysis of variance, LSD test, and t test., Results: (1) Proliferative activity of cells in control group and 1, 5, 50, 100, 200 nmol/L rapamycin groups was respectively 1.22±0.28, 1.29±0.38, 1.12±0.27, 1.20±0.29, 1.15±0.30, 1.39±0.40, without statistically significant differences among these groups (F=2.112, P=0.068). (2) The ranges of movement of cells in 1, 5 nmol/L rapamycin groups were similar to the range of movement of cells in control group, while those of cells in 50, 100, 200 nmol/L rapamycin groups were obviously smaller than the range of movement of cells in control group. There were statistically significant differences in cell curvilinear movement speeds among the 6 groups (F=3.525, P=0.004). The curvilinear movement speeds of cells in 1, 5 nmol/L rapamycin groups were respectively (0.8±0.4) and (0.8±0.8) μm/min, and they were similar to the curvilinear movement speed of cells in control group [(0.9±0.5) μm/min, with P values above 0.05]. The curvilinear movement speeds of cells in 50, 100, 200 nmol/L rapamycin groups were respectively (0.7±0.5), (0.7±0.4), (0.7±0.4) μm/min, and they were significantly lower than the curvilinear movement speed of cells in control group (with P values below 0.01). Thus, 50 nmol/L rapamycin was selected for experiments (3) and (4). (3) Compared with those of control group, wound areas of rapamycin group showed no obvious change at PSH 0 and 5, while they were obviously increased at PSH 10 and 15. At PSH 5, migration rate of cells in control group [(17.5±2.6)%] was similar to that in rapamycin group [(15.8±3.5)%, t=1.951, P>0.05]. Migration rates of cells of rapamycin group at PSH 10 and 15 [(42.5±4.0)% and (71.3±9.2)%, respectively] were obviously decreased as compared with those of control group [(46.9±6.7)% and (88.0±7.7)%, with t values respectively 2.732 and 6.746, P values below 0.01]. (4) Compared with that in control group (0.46±0.14), FAK activity of cells in rapamycin group (0.16±0.08) was significantly down-regulated (t=4.967, P<0.01)., Conclusions: FAK signal pathway is sensitive to rapamycin in HaCaT cells. Inhibition effects of rapamycin on migration of HaCaT cells may be mediated by down-regulated activity of FAK.

Acute obstruction of coronary arteries leads to acute myocardial infarction (AMI), which causes unexpected death in humans. However, AMI cannot be easily detected in forensic examinations with traditional hematoxylin and eosin (H&E) staining. We analyzed whether cardiac troponin inhibitor (CTnI) could serve as a sensitive and specific early marker for diagnosing AMI in forensic medicine. We established an AMI model in rabbits by ligating the left ventricular branch and observed CTnI expression with immunohistochemistry after different ligation times. We found increased CTnI staining at the 0.5-h time point and depletion of CTnI staining with a 1-h ligation. The areas in which CTnI staining was depleted as seen with immunohistochemical analysis were consistent with the results of H&E staining. Next, human myocardium tissues from 30 persons who died from AMI and were subsequently examined in our forensic center were studied using immunohistochemistry with an antibody to human CTnI. Areas of infarction also showed depletion of CTnI staining. These findings suggested that immunohistochemical detection of CTnI is earlier, more sensitive, and myocardial tissue - specific as compared with H&E staining. CTnI may serve as an ideal marker for diagnosing AMI in forensic investigations., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Objective: To estimate the application of prognosis evaluation of ulnar nerve injury by 1H-magnetic resonance spectroscopy (1H-MRS)., Methods: The metabolites of first dorsal interossei (FDI) of two hands from 12 healthy volunteers and 1 volunteer with complete ulnar nerve injury were detected by 1H-MRS and the data were statistically analyzed., Results: For the FDI of healthy adults, the female peaks area of extra-myocellular lipids (EMCL) was higher than the male (P < 0.05); There was no significant difference in Cho, Cr and intra-myocellular lipids (IMCL) between male and female (P > 0.05); There was no significant difference in all the peaks area between the left and right hand (P > 0.05). The EMCL peak of the injury side was higher than that of the healthy side, and the area of FDI was reduced in the volunteer with ulnar nerve injury., Conclusion: Noninvasive and quantitative detection of 1H-MRS may be valuable for prognosis evaluation of peripheral nerve injury.

Hypoxia-induced microtubule disruption and mitochondrial permeability transition (mPT) are crucial events leading to fatal cell damage and recent studies showed that microtubules (MTs) are involved in the modulation of mitochondrial function. Dynein light chain Tctex-type 1 (DYNLT1) is thought to be associated with MTs and mitochondria. Previously we demonstrated that DYNLT1 knockdown aggravates hypoxia-induced mitochondrial permeabilization, which indicates a role of DYNLT1 in hypoxic cytoprotection. But the underlying regulatory mechanism of DYNLT1 remains illusive. Here we aimed to investigate the phosphorylation alteration of DYNLT1 at serine 82 (S82) in hypoxia (1% O2). We therefore constructed recombinant adenoviruses to generate S82E and S82A mutants, used to transfect H9c2 and HeLa cell lines. Development of hypoxia-induced mPT (MMP examining, Cyt c release and mPT pore opening assay), hypoxic energy metabolism (cellular viability and ATP quantification), and stability of MTs were examined. Our results showed that phosph-S82 (S82-P) expression was increased in early hypoxia; S82E mutation (phosphomimic) aggravated mitochondrial damage, elevated the free tubulin in cytoplasm and decreased the cellular viability; S82A mutation (dephosphomimic) seemed to diminish the hypoxia-induced injury. These data suggest that DYNLT1 phosphorylation at S82 is involved in MTs and mitochondria regulation, and their interaction and cooperation contribute to the cellular hypoxic tolerance. Thus, we provide new insights into a DYNLT1 mechanism in stabilizing MTs and mitochondria, and propose a potential therapeutic target for hypoxia cytoprotective studies.

Leaf senescence can impact crop production by either changing photosynthesis duration, or by modifying the nutrient remobilization efficiency and harvest index. The doubling of the grain yield in major cereals in the last 50 years was primarily achieved through the extension of photosynthesis duration and the increase in crop biomass partitioning, two things that are intrinsically coupled with leaf senescence. In this review, we consider the functionality of a leaf as a function of leaf age, and divide a leaf's life into three phases: the functionality increasing phase at the early growth stage, the full functionality phase, and the senescence and functionality decreasing phase. A genetic framework is proposed to describe gene actions at various checkpoints to regulate leaf development and senescence. Four categories of genes contribute to crop production: those which regulate (I) the speed and transition of early leaf growth, (II) photosynthesis rate, (III) the onset and (IV) the progression of leaf senescence. Current advances in isolating and characterizing senescence regulatory genes are discussed in the leaf aging and crop production context. We argue that the breeding of crops with leaf senescence ideotypes should be an essential part of further crop genetic improvement., (© 2012 Institute of Botany, Chinese Academy of Sciences.)

The cytokinin oxidase/dehydrogenase (CKX) gene plays a principal role in controlling cytokinin levels and has been shown to be a major quantitative trait locus (QTL) affecting grain number in rice. However, the function and evaluation of the haplotypes of the wheat CKX gene have yet to be illustrated. In this study, TaCKX6-D1, a wheat ortholog of rice OsCKX2, was cloned and its haplotype variants were determined to be significantly associated with the 1000-grain weight on the basis of linkage mapping, association analysis and gene expression analysis. Five TaCKX6-D1 haplotypes, designated a-e, were identified. An indel marker was developed to identify haplotype a, which was associated with higher grain weight. Haplotype a showed decreased expression relative to haplotype b in seeds at 8 d after pollination. Sequence variations among modern cultivars, landraces and wild species suggest a significant domestication signature at the TaCKX6-D1 locus in Chinese wheat germplasm. TaCKX6-D1 may serve as a useful gene for the breeding of high-yielding wheat. A strategy for allele mining and utilization of TaCKX6-D1 was proposed. Our study also sheds light on the mechanisms of grain development and domestication of wheat, as well as the functional divergence of orthologs in comparative genomics., (© 2012 Institute of Crop Sciences. CAAS New Phytologist © 2012 New Phytologist Trust.)

Two hundred and six F2:3 families from the cross between TD22 and HT-1-1-1-1 were used for dynamic QTL research of tomato soluble solid content and correlative traits, and correlation analysis of soluble solid content (SSC) with fruit weight (FW), fruit shape index (FSI), soluble sugar, vitamin C (VC), and organic acid at three different development stages. The results showed that there were differences in QTL loci for soluble solid content during the three stages of tomato fruit development. Four and eight QTLs were detected in green ripe stage and red ripe stage, respectively. These QTLs showed dynamic changes, and two markers LEaat006 and Tomato|TC162363 were detected in two stages, which might be useful in molecular-marker assisted selection (MAS). The result also showed that there was extremely significant difference in SSC at the three different stages, and its main correlative traits were different at different stages. Soluble solid content was positively correlated with soluble sugar, but negatively correlated with FW at green ripe stage; SSC was positively correlated with soluble sugar and organic acid at yellow ripe stage; SSC was positively correlated with soluble sugar and organic acid, but negatively correlated with fruit weight at red ripe stage. Based on correlation analysis of these traits, linear regression model was constructed. Non-tested varieties were used to test the fitness, and the result showed that it is well fitted, and the fitness is above 95%.

Objective: To find out the economic laws regulating medical service demand in accordance with influencing factors at the township level, thus to provide references for further adjusting the medical service demand reasonably in the future., Methods: The model of medical service demand was established to measure the elasticity of demand in 49 township health clinics in 1995, 1999, 2003 and 2007., Results: The price elasticity of outpatient and inpatient demand was stable during the four periods, and the average value was -0.029 and -0.132 respectively; the average value of income elasticity was 0.973 and 0.977, registering a downward trend in general., Conclusion: The medical service demand at the township level is price inelastic, indicating that it is a necessity for rural residents. The downward trend of income elasticity under the influence of some health policies illustrates a lightening in economic burden for medical service demand among rural residents in township health clinics.

Objective: The exogenous substrate, thymol, was firstly biotransformed by using suspension hairy roots of transgenic Polygonum multflorum, and its biotransformed situation was also investigated., Methods: After five days co-cultivated period, the transformed product was isolated by Thin Layer Chromatograph and Column Chromatograph, with the structure elucidated by physic-chemical methods and spectra data. Meanwhile, the time course of biotransformation (T-C) for thymol was also measured by HPLC to illuminate its bio-transformed situation., Results: The glycosylated product, namely DMP, was isolated and purified, which structure was determined as 5-methyl-2-(1-methylethyl) phenyl- beta-D-glucopyranoside. And the distribution of DMP in the medium or culture was varied in different co-cultivated periods, and for five days co-cultivated period, it mainly existed in the medium., Conclusion: The hairy roots of Polygonum multiflorum were able to convert the aromatic exogenous substrate, thymol, into its glycoside. Furthermore, the time course indicated the relationship between DMP and co-cultivated period.

Objective: To study the clinical efficacy of lymphadenectomy of lung cancer by video-assisted thoracic small incision surgery., Methods: Radical lobectomy or pneumonectomy and systemic lymphadenectomy were performed in 197 patients with NSCLC through video-assisted thoracoscopic surgery. Pathological type and grade, total number of dissected lymph nodes, number of metastatic lymph nodes, survival and life quality were evaluated., Results: The average of dissected lymph nodes was 11.61 and 471 lymph nodes from 382 regions were confirmed to have metastasis. The metastatic rates were 24.1% and 20.6% respectively. There were no significant difference between video-assisted thoracic small incision surgery and conventional thoracotomy. The 1, 3, 5-year survival rates of stage I , II and III a had no significant difference between video-assisted thoracic small incision surgery and conventional thoracotomy., Conclusion: Video-assisted thoracic small incision surgery achieves a similar efficacy as the traditional lymphadenectomy and lung cancer radical resection.

The frequency and distribution of the major vernalization requirement genes and their effects on growth habits were studied. Of the 551 bread wheat genotypes tested, seven allelic combinations of the three Vrn-1 genes were found to be responsible for the spring habit, three for the facultative habit and one for the winter habit. The three Vrn-1 genes behaved additively with the dominant allele of Vrn-A1 exerting the strongest effect. The allele combinations of the facultative genotypes and the discovery of spring genotypes with "winter" allele of Vrn-1 implied the presence of as yet unidentified alleles/genes for vernalization response. The dominant alleles of the three Vrn-1 genes were found in all ten ecological regions where wheat is cultivated in China, with Vrn-D1 as the most common allele in nine and Vrn-A1 in one. The combination of vrn-A1vrn-B1Vrn-D1 was the predominant genotype in seven of the regions. Compared with landraces, improved varieties contain a higher proportion of the spring type. This was attributed by a higher frequency of the dominant Vrn-A1 and Vrn-B1 alleles in the latter. Correlations between Vrn-1 allelic constitutions and heading date, spike length, plant type as well as cold tolerance were established.

A dominant wheat stripe rust resistance gene, temporarily designated as YrY201, was identified in an accession Y201 of Aegilops tauschii. By bulk segregation analysis, three microsatellite markers Xgwm273b, Xgwm37 and Wmc14 were found to be linked to YrY201 with genetic distance of 11.5, 5.8 and 10.9 cM , respectively. According to the locations of the linked markers, the resistance gene was located on chromosome 7DL. Based on the chromosomal location and the resistance pattern of the gene, we proposed that YrY201 was a novel stripe rust resistance gene, and could be selected by marker-assisted selection.

Calcium-dependent protein kinases (CDPKs) are crucial sensors of calcium concentration changes in plant cells under diverse endogenous and environmental stimuli. We identified 20 CDPK genes from bread wheat and performed a comprehensive study on their structural, functional and evolutionary characteristics. Full-length cDNA sequences of 14 CDPKs were obtained using various approaches. Wheat CDPKs were found to be similar to their counterparts in rice in genomic structure, GC content, subcellular localization, and subgroup classification. Divergence time estimation of wheat CDPK gene pairs and wheat-rice orthologs suggested that most duplicated genes already existed in the common ancestor of wheat and rice. The number of CDPKs in diploid wheat genome was estimated to be at least 26, a number close to that in rice, Arabidopsis, and poplar. However, polymorphism among EST sequences uncovered transcripts of all three homoeologous alleles for 13 out of 20 CDPKs. Thus, the hexaploid wheat should have 2-3 fold more CDPK genes expressing in their cells than the diploid species. Wheat CDPK genes were found to respond to various biotic and abiotic stimuli, including cold, hydrogen peroxide (H(2)O(2)), salt, drought, powdery mildew (Blumeria graminis tritici, Bgt), as well as phytohormones abscisic acid (ABA) and gibberellic acid (GA). Each CDPK gene often responded to multiple treatments, suggesting that wheat CDPKs are converging points for multiple signal transduction pathways. The current work represents the first comprehensive study of CDPK genes in bread wheat and provides a foundation for further functional study of this important gene family in Triticeae.

Objective: This study aimed to clarify the morphology of the Martin-Gruber anastomosis (MGA) in Chinese., Methods: One hundred and five Chinese upper limbs (36 males and 20 femalese) were dissected to find the connections between medial nerve and ulnar nerve. The MGA was classified as previously described by Lee., Results: MGA was found in 24 cases (22.9%), in 11 of the 36 male and 5 of the 20 female. There was no obvious difference in the frequency of MGA in both upper limbs. Most MGA ulnar position was located at the medial and distal segment of the forearm., Conclusion: MGA anatomy could play important role in forensic diagnosis of ulnar nerve injury in Chinese population.

The use of full-length cDNA libraries is an effective tool to obtain complete gene information in a high-efficiency, high-throughput manner, especially in organisms with huge genomes that are not amenable to whole genome sequencing. In this review, we outlined several methods of full-length cDNA library construction and compared their advantages and disadvantages based on their respective principles. Drawing on our own experience, we described the Cap-trapper method in detail, with an emphasis on its application in wheat full-length cDNA library construction as well as the determination of the ratio of full-length cDNA in a library.

Objective: To study the relationship between postmortem interval and the myofibril fragmentation index of skeletal muscle., Methods: Rabbit skeletal muscle were left in the room temperature for different postmortem intervals, and the protein concentration of each sample was detected by using biuret method. Furthermore, the myofibril fragmentation index of each sample was measured under the protein concentration level of 0.5 mg/mL., Results: The myofibril fragmentation index increased obviously according to the postmortem interval prolongation., Conclusion: The myofibril fragmentation index may be used on the estimation of early postmortem interval.

A powdery mildew resistance gene from Triticum urartu Tum. accession UR206 was successfully transferred into hexaploid wheat (Triticum aestivum L.) through crossing and backcrossing. The F1 plants, which had 28 chromosomes and an average of 5.32 bivalents and 17.36 univalents in meiotic pollen mother cells (PMC), were obtained through embryos rescued owing to shriveling of endosperm in hybrid seed of cross Chinese Spring (CS) x UR206. Hybrid seeds were produced through backcrossing F1 with common wheat parents. The derivative lines had normal chromosome numbers and powdery mildew resistance similar to the donor UR206, indicating that the powdery mildew resistance gene originating from T. urartu accession UR206 was successfully transferred and expressed in a hexaploid wheat background. Genetic analysis indicated that a single dominant gene controlled the powdery mildew resistance at the seedling stage. To map and tag the powdery mildew resistance gene, 143 F2 individuals derived from a cross UR206 x UR203 were used to construct a linkage map. The resistant gene was mapped on the chromosome 7AL based on the mapped microsatellite makers. The map spanned 52.1 cM and the order of these microsatellite loci agreed well with the established microsatellite map of chromosome arm 7AL. The resistance gene was flanked by the microsatellite loci Xwmc273 and Xpsp3003, with the genetic distances of 2.2 cM and 3.8 cM, respectively. On the basis of the origin and chromosomal location of the gene, it was temporarily designated PmU.

To discover new genes in a throughput manner,the cap-trapper method published previously was optimized for raising the efficiency in the construction of full-length cDNA library. Using the optimized protocol,we successfully constructed a full-length cDNA library of Aegilops speltoides,which contained 3.0 x 10(6) clones and more than 99% of plaques were recombinant phages. Sequence analysis results indicated that more than 89% of the clones were full-length.

Wheat 1BL/1RS translocations are widely planted in China as well as in most of the wheat producing area in the world for their good qualities of disease resistance and high yield. 1BL/1RS translocations are however poor in bread making, partially caused by a family of small monomeric proteins, omega-secalins, which are encoded by genes on 1RS. Based on published sequence of a rye omega-secalin gene we designed a pair of primers to cover the whole mature protein coding sequence. A major band could be amplified from 1BL/1RS translocations but not from euploid wheat. Using this primer set we conducted PCR amplification by using high fidelity Pfu polymerase on the genomic DNAs and cDNAs purified from a 1BL/1RS translocation Lankao 906. Sequencing analysis indicated that this gene family contains several members of 1150 bp, 1076 bp, 1075 bp, 1052 bp and 1004 bp genes, including two pseudogenes and three active genes. The gene transcripts were differentially expressed in developing seeds.

One 134 bp fragment was amplified in anthers of male sterile and fertile wheat using one pair of degenerated primer designed based on the conserved domain of MS2 gene in Arabidopsis thaliana and Oryza sativa, and one 1604 bp male sterility gene homology sequence was extended by in silico cloning based on the 134 bp fragment. The amino acids encoded by the male sterility gene homology sequence include a 200 amino acid conserved domain of male sterility, and this sequence expressed only in wheat male fertile anthers; no expression in male sterile anthers, roots and leaves. This research demonstrated that the cloned male sterile homology sequence is specific to wheat anther development.

Objective: To investigate the signification of CTnI in acute myocardial infarction., Methods: The model of myocardial infarction was established by ligating the left ventricle branch. Immunohistochemistry and image analysis were used to detect the change of CTnI in the model, and The sensitivity of immunohistochemistry and HE coloration were also compared., Results: The acute myocardial infarction tissue showed obvious depletion of CTnI, there was no characterization of myocardial infarction in HE coloration., Conclusion: CTnI is sensitive to diagnosis of acute myocardial infarction.

The molecular evolution of cultivated rice Oryza sativa L. has long been a subject of rice evolutionists. To investigate genetic diversity within and differentiation between the indica and japonica subspecies, 22 accessions of indica and 35 of japonica rice were examined by five microsatellite loci from each chromosome totalling 60 loci. Mean gene diversity value in the indica rice (H=0.678) was 1.18 times larger than in the japonica rice (H=0.574). Taking the sampling effect into consideration, average allele number in the indica rice was 1.40 times higher than that in the japonica rice (14.6 vs 10.4 per variety). Chromosome-based comparisons revealed that nine chromosomes (1, 2, 3, 4, 5, 8, 9, 10 and 11) harboured higher levels of genetic diversity within the indica rice than the japonica rice. An overall estimate of F(ST) was 0.084-0.158, indicating that the differentiation is moderate and 8.4-15.8% of the total genetic variation resided between the indica and japonica groups. Our chromosome-based comparisons further suggested that the extent of the indica-japonica differentiation varied substantially, ranging from 7.62% in chromosome 3 to 28.72% in chromosome 1. Cluster analyses found that most varieties formed merely two clusters for the indica and japonica varieties, in which two japonica varieties and five indica varieties were included in the counterpart clusters, respectively. The 12 chromosome-based trees further showed that 57 rice varieties cannot be clearly clustered together into either the indica or japonica groups, but displayed relatively different clustering patterns. The results suggest that the process of indica japonica differentiation may have proceeded through an extensive contribution by the alleles of the majority in the rice genome.

Four hundred and seventy-eight microsatellite markers derived from expressed sequence tags (EST-SSRs) were screened among three mapping populations (W-7984xOpata 85, WOpop; LumaixHanxuan, LHpop; WenmaixShanhongmai, WSpop). The number of polymorphic EST-SSR primer pairs found in WOpop, LHpop and WSpop was 92, 58 and 29 respectively. A total of 101 EST-SSR loci amplified from 88 primer sets were distributed over the 20 chromosomes of the reference maps (no markers were located on chromosome 4B). These 101 mapped EST-SSR markers add to the existing 450 microsatellite loci previously mapped in bread wheat. Seventy-four of the 101 loci showed significant similarities to known genes, including 24 genes involved in metabolism, 4 in cellular structures, 9 in stress resistance, 12 in transcription, 2 in development, 2 transporters and 21 storage proteins. Besides gliadin and glutenin, most of the 53 genes with putative functions were mapped for the first time by EST-SSR markers in bread wheat. Sequence alignment of the mapped wheat EST-SSR loci allowed tentative assignment of functionality to the other members of grasses family. Colinearity combined with homology information offers an attractive approach to comparative genomics.

Objective: To build the trestle of tissue engineering for skin with the collagen., Methods: The collagen was obtained from the baby cattle hide pretreated by Na2S and elastinase and Protease M, then the collagen was dissolved in 0.5 mol/L acetic acid solution. The collagen was treated with Protease N to minimize its immunogenicity. The resulting collagen could be used to build the trestle of tissue engineering for skin because of good biocompatibility. The collagen molecular weight and structure were analyzed by SDS-PAGE. The bioactivity of trestle was tested in the experiment of the mice wound healing and the cell implantation., Results: The SDS-PAGE result of the collagen treated by Protease M showed the typical spectrum of type I collagen. The built trestle was a collagen sponge matrix in which micropore size was 50-200 microns. It could accelerate wound healing and the implanted fibroblasts could proliferate well., Conclusion: The collagen treated by Protease N can get good biocompatibilily and is suitable for building the trestles of tissue engineering for skin with good bioactivity.