Objective:To explore the feasibility and clinical effectiveness of preoperative positioning grid combined with CTA assisted design of anterolateral thigh perforator flap (ALTPF) in reconstruction of soft tissue defects around foot and ankle.Methods:From May 2018 to December 2021, a total of 18 cases with soft tissue defects around foot and ankle were admitted to the Department of Plastic and Reconstructive Surgery, First Affiliated Hospital of Ningbo University. The patients were 11 males and 7 females, aged from 32 to 78 years old, with an average age of 57.5 years old. Among them, 10 had chronic ulcer wounds, 6 had traumatic wounds, and 2 had postoperative wounds after malignant tumor resection. The sizes of soft tissue defect ranged from 6.0 cm×8.0 cm to 9.0 cm×13.0 cm, and the flap sizes ranged from 8.0 cm×10.0 cm to 11.0 cm×15.0 cm. Preoperative positioning grid combined with CTA three-dimensional reconstruction were used to mark the exit point of the perforator vessels in digital format, in order to restore the course of vessels and calculate the length of the vascular pedicles. ALTPFs were accurately designed based on the digitally reconstructed images, and then the ALTPFs were used to repair the soft tissue defects around the ankle. The flap donor sites were directly closed in stage Ⅰ. After the operation, all the patients were included in scheduled follow-ups at the outpatient department to observe the appearance of the recipient flaps and donor sites. The functional evaluation of the affected feet were assessed according to the American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score.Results:All 18 flaps survived, and the wounds in both the recipient and donor sites healed in stageⅠ. Postoperative follow-up for the 18 patients were 5 to 36 months, with an average of 13 months. At the last follow-up, the appearances of the flaps were good without swelling, the donor sites had good recovery, and there was no obvious scar hypertrophy. The function evaluation of the affected feet were found at excellent in 10 cases, good in 6 cases, and fair in 2 cases.Conclusion:Preoperative position grid combined with CTA three-dimensional reconstruction can digitally mark the exit points and running courses of the perforator vessels. It is an effective method for accurate position of perforator vessels before surgery. It can effectively reduce the operation time, lower the surgical risks, and achieve a high survival rate of the flap, thus holding considerable clinical value.

Alfalfa(Medicago sativa L.)is the most important legume forage crop worldwide with high nutritional value and yield.For a long time,the breeding of alfalfa was hampered by lacking reliable information on the autotetraploid genome and molecular markers linked to important agro-nomic traits.We herein reported the de novo assembly of the allele-aware chromosome-level genome of Zhongmu-4,a cultivar widely cultivated in China,and a comprehensive database of genomic variations based on resequencing of 220 germplasms.Approximate 2.74 Gb contigs(N50 of 2.06 Mb),accounting for 88.39％of the estimated genome,were assembled,and 2.56 Gb contigs were anchored to 32 pseudo-chromosomes.A total of 34,922 allelic genes were identified from the allele-aware genome.We observed the expansion of gene families,especially those related to the nitrogen metabolism,and the increase of repetitive elements including transposable elements,which probably resulted in the increase of Zhongmu-4 genome compared with Medicago truncatula.Population structure analysis revealed that the accessions from Asia and South America had rela-tively lower genetic diversity than those from Europe,suggesting that geography may influence alfalfa genetic divergence during local adaption.Genome-wide association studies identified 101 sin-gle nucleotide polymorphisms(SNPs)associated with 27 agronomic traits.Two candidate genes were predicted to be correlated with fall dormancy and salt response.We believe that the allele-aware chromosome-level genome sequence of Zhongmu-4 combined with the resequencing data of the diverse alfalfa germplasms will facilitate genetic research and genomics-assisted breeding in variety improvement of alfalfa.

Objective:To investigate the relationship between NOD-like receptors 3 (NLRP3) inflammasomes mediated inflammatory response and the pathogenesis of depression, and to investigate the effect of fluoxetine on this process.Methods:120 male C57BL/6J (wild-type) mice were randomly divided into control group 1, control group 2, chronic unpredictable mild stress (CUMS) group, and CUMS plus fluoxetine group. Another 30 male C57BL/6J (NLRP3-/-) mice were selected as NLRP3-/- group. Control group 1 and control group 2 had No treatment; CUMS group, CUMS plus fluoxetine group, and NLRP3-/- group were given chronic unpredictable mild stimulation for six weeks. After modeling, mice in control group 2 and CUMS plus fluoxetine group were intraperitoneally injected with fluoxetine (10 mg/(kg·d)). In contrast, mice in the other three groups were injected with the same amount of normal saline every day for four weeks. Behavioral tests were performed once a week before and after stress stimulation. Tail venous blood was drawn immediately before stress stimulation, three weeks later and six weeks after stress stimulation and was centrifuged at 3000 r/min for 10 min. The supernatant was kept and frozen for future use. Serum levels of interleukin-1β(IL-1β) and interleukin-18 (IL-18) were detected by enzyme-linked immunosorbent assay. After drug intervention (10 mg/(kg·d) fluoxetine or the same volume of normal saline), the mice in each group underwent behavioral tests once a week. The results included the sugar water preference test, forced swimming test (FST), and tail suspension test (TST). Tail venous blood was drawn from mice in each group at 1, 3, and 4 weeks after fluoxetine administration, and the supernatant was centrifuged and stored for later use. Serum levels of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay. At the above time points, 5 mice in each group were sacrificed each time, and fresh hippocampal tissues were collected and stored at a low temperature. NLRP3, urinary winter peptidase (caspase-1), the induction of transcription factors-KB (nuclear factor-κB, NF-κB), IL-1β and the IL-18 in the hippocampus brain regions were detected by using Western Blot.Results:(1) Model establishment: After six weeks of CUMS, compared with control group 1 and control group 2, the sugar water consumption of mice in CUMS group and CUMS+fluoxetine group was significantly reduced, and the immobility time of FST and TST was significantly prolonged, which proved that the model establishment was successful. After CUMS, NLRP3-/- group mice did not show depression-like changes in FST, sugar water preference test, and TST, which indicated that the model failed to be established. (2) After intraperitoneal fluoxetine injection, there were no significant differences in sugar water consumption, FST and TST immobility time between control group 2 and control group 1 ( P>0.05), and the sugar water consumption of mice in CUMS plus fluoxetine group was significantly increased, compared with CUMS group ( P<0.05). The immobility time of FST and TST was significantly shortened ( P<0.05). (3) The results of the enzyme-linked immunosorbent assay showed that after stimulation of CUMS, compared with control group 1 and control group 2, the serum levels of IL-1β and IL-18 in CUMS group and CUMS plus fluoxetine group were significantly increased ( P<0.05), while NLRP3-/- group had no significant change ( P>0.05). After fluoxetine administration, the serum levels of IL-1β and IL-18 in CUMS plus fluoxetine group were significantly lower than those in CUMS group ( P<0.05). (4) Western blotting results showed that after stimulation of CUMS, compared with control group 1 and control group 2, mice brain hippocampus NLRP3 expression, caspase-1 activation and induction of transcription factors-κB (nuclear factor-κB, NF-κB), IL-1β, IL-18 expression significantly increased ( P<0.05) in CUMS and CUMS plus fluoxetine group. After fluoxetine treatment, mice brain hippocampus NLRP3 expression, caspase-1 activation, NF-κB, IL-1β, and IL-18 expression in CUMS plus fluoxetine group had a significant reduction (restored to control 1 group by 99%, 91%, 97%, 95%, and 97% respectively). Conclusion:(1) CUMS may bring more NLRP3, caspase-1, NF-κB, IL-1β, and IL-18 expression in mice hippocampus, which cannot be seen, in the NLRP3 gene knockout mice; (2) The fluoxetine treatment may significantly decrease the NLRP3, caspase-1, NF-κB, IL-1β, IL-18 expression on depressive model mice, and improve depressive behavior.

Objective:To investigate the relationship between NOD-like receptors 3 (NLRP3) inflammasomes mediated inflammatory response and the pathogenesis of depression, and to investigate the effect of fluoxetine on this process.Methods:120 male C57BL/6J (wild-type) mice were randomly divided into control group 1, control group 2, chronic unpredictable mild stress (CUMS) group, and CUMS plus fluoxetine group. Another 30 male C57BL/6J (NLRP3-/-) mice were selected as NLRP3-/- group. Control group 1 and control group 2 had No treatment; CUMS group, CUMS plus fluoxetine group, and NLRP3-/- group were given chronic unpredictable mild stimulation for six weeks. After modeling, mice in control group 2 and CUMS plus fluoxetine group were intraperitoneally injected with fluoxetine (10 mg/(kg·d)). In contrast, mice in the other three groups were injected with the same amount of normal saline every day for four weeks. Behavioral tests were performed once a week before and after stress stimulation. Tail venous blood was drawn immediately before stress stimulation, three weeks later and six weeks after stress stimulation and was centrifuged at 3000 r/min for 10 min. The supernatant was kept and frozen for future use. Serum levels of interleukin-1β(IL-1β) and interleukin-18 (IL-18) were detected by enzyme-linked immunosorbent assay. After drug intervention (10 mg/(kg·d) fluoxetine or the same volume of normal saline), the mice in each group underwent behavioral tests once a week. The results included the sugar water preference test, forced swimming test (FST), and tail suspension test (TST). Tail venous blood was drawn from mice in each group at 1, 3, and 4 weeks after fluoxetine administration, and the supernatant was centrifuged and stored for later use. Serum levels of IL-1β and IL-18 were detected by enzyme-linked immunosorbent assay. At the above time points, 5 mice in each group were sacrificed each time, and fresh hippocampal tissues were collected and stored at a low temperature. NLRP3, urinary winter peptidase (caspase-1), the induction of transcription factors-KB (nuclear factor-κB, NF-κB), IL-1β and the IL-18 in the hippocampus brain regions were detected by using Western Blot.Results:(1) Model establishment: After six weeks of CUMS, compared with control group 1 and control group 2, the sugar water consumption of mice in CUMS group and CUMS+fluoxetine group was significantly reduced, and the immobility time of FST and TST was significantly prolonged, which proved that the model establishment was successful. After CUMS, NLRP3-/- group mice did not show depression-like changes in FST, sugar water preference test, and TST, which indicated that the model failed to be established. (2) After intraperitoneal fluoxetine injection, there were no significant differences in sugar water consumption, FST and TST immobility time between control group 2 and control group 1 ( P>0.05), and the sugar water consumption of mice in CUMS plus fluoxetine group was significantly increased, compared with CUMS group ( P<0.05). The immobility time of FST and TST was significantly shortened ( P<0.05). (3) The results of the enzyme-linked immunosorbent assay showed that after stimulation of CUMS, compared with control group 1 and control group 2, the serum levels of IL-1β and IL-18 in CUMS group and CUMS plus fluoxetine group were significantly increased ( P<0.05), while NLRP3-/- group had no significant change ( P>0.05). After fluoxetine administration, the serum levels of IL-1β and IL-18 in CUMS plus fluoxetine group were significantly lower than those in CUMS group ( P<0.05). (4) Western blotting results showed that after stimulation of CUMS, compared with control group 1 and control group 2, mice brain hippocampus NLRP3 expression, caspase-1 activation and induction of transcription factors-κB (nuclear factor-κB, NF-κB), IL-1β, IL-18 expression significantly increased ( P<0.05) in CUMS and CUMS plus fluoxetine group. After fluoxetine treatment, mice brain hippocampus NLRP3 expression, caspase-1 activation, NF-κB, IL-1β, and IL-18 expression in CUMS plus fluoxetine group had a significant reduction (restored to control 1 group by 99%, 91%, 97%, 95%, and 97% respectively). Conclusion:(1) CUMS may bring more NLRP3, caspase-1, NF-κB, IL-1β, and IL-18 expression in mice hippocampus, which cannot be seen, in the NLRP3 gene knockout mice; (2) The fluoxetine treatment may significantly decrease the NLRP3, caspase-1, NF-κB, IL-1β, IL-18 expression on depressive model mice, and improve depressive behavior.

Objective: To investigate the clinical effect of free medical sural artery perforator myocutaneous flap for repairingof the woundwith osteomyelitis. Methods: 17 patients suffered from the wound with osteomyelitis were treated in the Ningbo First People′s Hospital, There were 11 males and 6 females with an mean age of 53.2 years (range, 21-76 years). The sizes of the defect ranged 5 cm×4 cm to 13 cm×6 cm. All patients underwent debridement and used antibiotic-loaded bonecement to cover the wound. Meanwhile, patients were treated with sensitive antibiotics, operation and free medical sural artery perforator myocutaneous flap were used to treat the wound. Preoperative use ultrasound and CT angiography to positioning perforator, The flap area ranged from 6 cm×4 cm to 13 cm×7 cm and the donor sites were closed directly. The author provided the patients with the treatment of anti-inflammatory, anti-spasmodic and anti-coagulantin the postoperative. Used infrared thermograms to assess the flap blood supply. Results: One flap skin margin was non union due to poor blood supply.All of the other 16 flaps success survived and the donor sites were closed directly. Postoperative follow-up period was 4 to 23 months and the flaps had satisfied texture and appearance.All the donor sites had a good healing with no pain and complications, also the osteomyelitis was controlled. Conclusions The free medical sural artery perforator myocutaneous flap is reliable for reconstruction of the wound with osteomyelitis.

Objective To investigate the prevalence and correlates of depression in residents aged 15 and older in Hainan province.Methods Stratified multistage random sampling methods were utilized to identify 12 117 individuals (≥15 years old) from 59 villages (neighborhoods) in 24 towns (streets) within 6 counties (cities) in Hainan Province from September 2010 to November 2011.The subjects were screened with an expanded version of the General Health Questionnaire (GHQ-12) and the respondents were divided into three groups by high risk,moderate risk and low risk of mental disorder,followed by formal diagnosis according to the Structured Clinical Interview for DSM-IV-TR (SCID-I/P) by psychiatrists among 100％ subjects in group with high risk,40％ subjects in group with moderate risk and 10％ subjects in group with low risk.The adjusted rate,standardized rate,and 95％ confidence interval (95％CI) of the one-month and lifetime prevalence were also calculated among the 3 groups of individuals who were diagnosed with depressive disorder.The difference in whether they suffered depression was determined by the chi-squared test.Correlations to depressive disorder and the odds ratio (OR) were explored with multiple regression analysis.Results There were 97 cases of depressive disorders (1-month) and 166 cases of lifetime depressive disorders finally confirmed.The adjusted rate of 1-month prevalence was 1.38％ (95％CI:1.12-1.59) and the life-time adjusted prevalence rate was 2.80％ (95％CI:2.51-3.09).Female (OR=1.55,95％CI:1.12-2.14),mid-age(50-64 y,OR=1.84,95％CI:1.13-2.99),being divorced or living separated (OR=4.87,95％CI:1.86-12.73),suffering chronic diseases (OR=2.19,95％CI:1.56-3.07),and low family income were significantly associated with suffering depressive disorder.Conclusions The prevalence of the depressive disorder among residents aged 15 and older in Hainan province is lower than the nationwide prevalence.People who are female,mid-age,being divorced or not living with the partner,earning a low income,and suffering chronic diseases have more predisposition to develop the depressive disorder in Hainan province.

Objective To investigate the prevalence and correlates of depression in residents aged 15 and older in Hainan province.Methods Stratified multistage random sampling methods were utilized to identify 12 117 individuals (≥15 years old) from 59 villages (neighborhoods) in 24 towns (streets) within 6 counties (cities) in Hainan Province from September 2010 to November 2011.The subjects were screened with an expanded version of the General Health Questionnaire (GHQ-12) and the respondents were divided into three groups by high risk,moderate risk and low risk of mental disorder,followed by formal diagnosis according to the Structured Clinical Interview for DSM-IV-TR (SCID-I/P) by psychiatrists among 100％ subjects in group with high risk,40％ subjects in group with moderate risk and 10％ subjects in group with low risk.The adjusted rate,standardized rate,and 95％ confidence interval (95％CI) of the one-month and lifetime prevalence were also calculated among the 3 groups of individuals who were diagnosed with depressive disorder.The difference in whether they suffered depression was determined by the chi-squared test.Correlations to depressive disorder and the odds ratio (OR) were explored with multiple regression analysis.Results There were 97 cases of depressive disorders (1-month) and 166 cases of lifetime depressive disorders finally confirmed.The adjusted rate of 1-month prevalence was 1.38％ (95％CI:1.12-1.59) and the life-time adjusted prevalence rate was 2.80％ (95％CI:2.51-3.09).Female (OR=1.55,95％CI:1.12-2.14),mid-age(50-64 y,OR=1.84,95％CI:1.13-2.99),being divorced or living separated (OR=4.87,95％CI:1.86-12.73),suffering chronic diseases (OR=2.19,95％CI:1.56-3.07),and low family income were significantly associated with suffering depressive disorder.Conclusions The prevalence of the depressive disorder among residents aged 15 and older in Hainan province is lower than the nationwide prevalence.People who are female,mid-age,being divorced or not living with the partner,earning a low income,and suffering chronic diseases have more predisposition to develop the depressive disorder in Hainan province.

OBJECTIVE:To investigate the regulation effects of oleanolic acid on the mitochondrial nitric oxide synthase(mt-NOS) in human umbilical vein endothelial cells (HUVECs) with oxidative damage. METHODS:HUVECs in exponential phase were divided into normal group,model group and oleanolic acid low,medium and high dose groups(5,20 and 35 μmol/L). After drug acting for 24 h,all groups were given culture solution containing 100 μg/ml ox-LDL to reproduce oxidative damage except normal group. CCK-8 was used to detect cell viability. The mitochondria in cells were extrated,enzyme chemical method was used to detect mtNOS activity and mtNO content,fluorescence microplate method was used to detect fluorescence intensity of reactive oxygen species(ROS),and western blot was used to detect expression of cytochrome C(Cyto-C). RESULTS:Compared with nor-mal group,the cell viability in model group was decreased;mtNOS activity,mtNO content,ROS fluorescence intensity and Cy-to-C protein expression were increased,with significant differences (P<0.05). Compared with model group,the cell viability in oleanolic acid low,medium and high dose groups was increased;mtNOS activity,mtNO content,ROS fluorescence intensity and Cyto-C protein expression were decreased,with significant differences(P<0.05),and they had positive correlation with concentra-tions. CONCLUSIONS:Oleanolic acid can decrease the mtNOS activity of HUVECs,reduce the production of mtNO and Cyto-C, by a mechanism that may be related to the decrease of ROS expression.

BACKGROUND:A large number of studies have confirmed that tissue-engineered stem cel therapy is feasible to repair peripheral nerve injury, but the repair mechanism is unclear.
OBJECTIVE:To observe the differentiation and homing of bone marrow mesechnymal stem cel s under local nerve microenvironment by exploring the migration and effect of bone marrow mesenchymal stem cel s in the repair of damaged nerve.
METHODS:Male SD rats, aged 8 weeks, were selected to establish segmental nerve injury models by freezing the sciatic nerve. Thirty-six model rats were randomized into three groups (n=12):frozen nerve injury group, cel injection into the nerve group, cel injection around the nerve group. Before modeling and at 4, 8, 12 weeks after cel implantation, the sciatic nerve function index was measured. Electrophysiological test, contractility recovery rate, wet weight recovery rate of the triceps surae were detected and Masson staining was performed;toluidine blue staining of the distal nerve injury and immunofluorescence staining of the damaged nerve were performed.
RESULTS AND CONCLUSION:At 4, 8, 12 weeks after cel implantation, the sciatic nerve function index was ranked as fol ows:frozen nerve injury group

Objective:To express SARS-CoV nucleocapsid protein in Bac-to-Bac Baculovirus Expression System and analyze the antigenicity of the recombinant protein.Methods: The SARS-CoV nucleocapsid gene was amplified by PCR.The PCR product digested with BamHⅠand SalⅠrestriction endonucleases was cloned into vector pFastBac HTC of Bac-to-Bac Baculovirus expression system.Recombinant plasmid was transformed DH 10Bac cells to obtain the recombinant Bacmid DNA.Recombinant Bacmid DNA was transferred into Sf9 cells which were inducted to express the recombinant protein in High Five cells.After purified by Ni affinity chroma-tography ,the antigenicity of the recombinant protein was analyzed by Western blot and ELISA.Results:Recombinant plasmid was con-structed successfully.The recombinant protein with the relative molecular mass of 48 kD was efficiently expressed in High Five cells and purified successfully by Ni affinity chromatography.Western blot and ELISA analysis showed that the recombinant protein could be spe -cifically recognized by the monoclonal antibody to SARS-CoV N protein and immune serum from rabbits ,respectively.The recombinant protein can specifically reacted with serum from SARS patients ,not with serum from healthy persons and patients infected with hCoV-229 E and hCoV-OC43.Conclusion: SARS-CoV nucleocapsid protein has been expressed successfully in the Bac-to-Bac Baculovirus Expression System ,and obtained good antigenicity.It is preliminary deemed that it can't reacted with serum from patients infected with hCoV-229E and hCoV-OC43.