L-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand. Although microbial cell factories have been widely used for biosynthesis, there are still challenges such as genetic instability and low efficiency in the biosynthesis of L-citrulline. In this study, an efficient, plasmid-free, non-inducible L-citrulline-producing strain of Escherichia coli BL21(DE3) was engineered by combined strategies. Firstly, a chassis strain capable of synthesizing L-citrulline was constructed by block of L-citrulline degradation and removal of feedback inhibition, with the L-citrulline titer of 0.43 g/L. Secondly, a push-pull-restrain strategy was employed to enhance the L-citrulline biosynthesis, which realized the L-citrulline titer of 6.0 g/L. Thirdly, the NADPH synthesis and L-citrulline transport were strengthened to promote the synthesis efficiency, which achieved the L-citrulline titer of 11.6 g/L. Finally, fed-batch fermentation was performed with the engineered strain in a 3 L fermenter, in which the L-citrulline titer reached 44.9 g/L. This study lays the foundation for the industrial production of L-citrulline and provides insights for the modification of other amino acid metabolic networks.
Citrulline/biosynthesis* ; Escherichia coli/genetics* ; Metabolic Engineering/methods* ; Fermentation ; NADP/biosynthesis*

To screen and identify the key host proteins interacting with the non-structural protein 15 (Nsp15) of porcine epidemic diarrhea virus (PEDV). The IP/pull-down assay and mass spectrometry were employed to screen and identify the host proteins interacting with Nsp15. The interaction between the host protein and Nsp15 was studied by co-immunoprecipitation and laser scanning confocal microscopy. Finally, Western blotting and RT-qPCR were employed to examine the interaction between SLC25a3 and PEDV. The recombinant eukaryotic expression vector pcDNA3.1(+)-Flag-Nsp15 was successfully constructed, and the host protein SLC25a3 interacting with PEDV Nsp15 was screened out. An interaction existed between SLC25a3 and Nsp15, and SLC25a3 significantly inhibited PEDV replication in a dose-dependent manner. SLC25a3 inhibits PEDV replication. The results of this study provide a basis for deciphering the role and mechanism of SLC25a3 in the host immune response to PEDV infection.
Porcine epidemic diarrhea virus/genetics* ; Viral Nonstructural Proteins/metabolism* ; Animals ; Swine ; Virus Replication ; Coronavirus Infections/veterinary* ; Swine Diseases/metabolism*

Objective:To investigate the surgical methods and clinical effects of free superficial peroneal artery perforator flap in repairing small and medium-sized thermal crush injury wounds in the hand.Methods:A retrospective observational study was conducted. From August 2018 to December 2021, 12 patients (19 wounds) with small and medium-sized thermal crush injury in the hand who met the inclusion criteria were hospitalized in Suzhou Ruihua Orthopaedic Hospital, including 5 males and 7 females, aged from 30 to 54 years. The area of the wound was from 2.5 cm×2.0 cm to 14.0 cm×3.5 cm, and all the wounds were repaired by using free superficial peroneal artery perforator flaps from lower leg on one side (including single flap, multiple flaps, and multiple flaps with one pedicle resected from the same donor site). The area of the flap was from 3.5 cm×3.0 cm to 16.0 cm×4.0 cm. The wound in the donor site was sutured directly. The vascular crisis and survival of the flap were observed after operation. The texture, appearance, color, hyperpigmentation, sensation, and two-point discrimination of the flap repaired area were followed up, as well as the hyperplasia of scar and pain condition in the donor and recipient sites. At the last follow-up, the curative effect of flap repair was evaluated by the comprehensive evaluation scale, and the extension and flexion functions of the reserved digital joint were evaluated by the total active movement systematic evaluation method recommended by American Academy for Surgery of Hand.Results:One flap developed arterial crisis on the first day after operation but survived after timely exploration. The other 18 flaps survived successfully after operation. Follow-up of 4 to 24 months after operation showed good texture and appearance in the flap repaired area; the color of the flap repaired area was similar to that of the normal skin around the recipient site, without pigmentation; the protective sensation was restored in all cases, but there was no two-point discrimination; there was no obvious hypertrophic scarring or pain in the donor or recipient site. At the last follow-up, the curative effect of flap repair was evaluated with 3 flaps being excellent and 16 flaps being good; the extension and flexion functions of the reserved digital joint were also assessed, being excellent in 8 fingers, good in 9 fingers, and fair in 2 fingers.Conclusions:The blood supply of superficial peroneal artery perforator flap is sufficient and reliable, and multiple flaps of this type or multiple flaps with one pedicle can be resected from one donor site. The use of this flap to repair small and medium-sized thermal crush injury wounds in the hand results in minimal damage to the donor area, and good postoperative appearance and texture of the flap.

Objective:To explore the surgical method and clinical effect of harvesting 2 ipsilateral free pedicled perforator flaps from a single donor site of superficial peroneal artery in reconstruction of 2 defects in same or adjacent digits.Methods:From November 2017 to August 2021, 12 patients with 2 defects in same or adjacent digits were treated in the Department of Hand Surgery, Suzhou Ruihua Orthopaedic Hospital with 2 ipsilateral free pedicled perforator flaps from a single donor site of superficial peroneal artery. Among the patients, 1 had the defect in dorsal and palmar of index finger, 1 in thumb and index finger, 6 in index and middle fingers, 3 in middle and ring fingers, and 1 in ring and little fingers. The size of digit defects was 1.5 cm×0.8 cm-6.0 cm×3.5 cm. The size of flaps was 2.0 cm× 1.2 cm-8.0 cm×4.0 cm. All the patients were included in postoperative monthly follow-up to assess the recovery of recipient and donor sites at outpatient service, by telephone or WeChat.Results:All 24 flaps in 12 patients survived without vascular compromise and achieved 100% of survival rate. The follow-up period ranged from 4 to 18 months, with an average of 10 months. Six patients were treated with additional flap thinning and plastic surgery at 4 months after the primary surgery due to slightly bloated flaps. Otherwise, all the flaps in the recipient site had neither pigmentation, obvious hyperplasia nor scar pain. All flaps gained the protective sensations, however the assessment of TPD was not conducted. The flaps were wear-resist and had no ulceration. The texture of the flaps was soft with good elasticity, and the flap did not turn to purple or swelling when in cold. The functional recovery of 23 digits in 12 patients was evaluated according to the total active mobility (TAM) of the digits. It achieved excellent in 3 digits, good in 15 digits, and fair in 5 digits, with an excellent and good rate of 78.26%. A linear scar appeared at the donor site without obvious hyperplasia or scar pain. There were normal sensations around the scar and at the digit-tips. The blood supply to the digit-tips was normal.Conclusion:Harvest of multiple free pedicled perforator flaps from a single donor site of superficial peroneal artery is an effective method in reconstruction of 2 defects in same or adjacent digits at the same time. It has advantages of being a simple surgery procedure by sacrificing only one donor site. It achieves a minimal damage to the donor site and a reliable blood supply of the flap.

Fermentative production of amino acids is one of the pillars of the fermentation industry in China. Recently, with the fast development of metabolic engineering and synthetic biology technologies, the metabolic engineering for production of amino acids has been flourishing. Conventional forward metabolic engineering, reversed metabolic engineering based on omics data and in silico simulation, and evolutionary metabolic engineering mimicking the natural evolution, have shown increasingly promising applications. A series of highly efficient and robust amino acids-producing strains have been developed and applied in the industrial production of amino acids. The increasingly fierce market competition has put forward new requirements for strain breeding and selection, such as developing high value-added amino acids, dynamic regulation of cellular metabolism, and adapting to the requirements of new process. This review summarizes the advances and prospects in metabolic engineering for the production of amino acids.
Amino Acids ; China ; Corynebacterium glutamicum/genetics* ; Metabolic Engineering ; Synthetic Biology

BACKGROUND: The 2019 novel coronavirus disease (COVID-19) has had a massive impact on public health, resulting in sudden dietary and behavioral habit changes. Frontline epidemic prevention workers play a pivotal role against COVID-19. They must face high-risk infection conditions, insufficient anti-epidemic material supplies, mental pressure, and so on. COVID-19 seriously affects their dietary and behavioral habits, and poor habits make them more susceptible to COVID-19. However, their baseline dietary and behavioral habits before COVID-19 and their willingness to change these habits after the outbreak of COVID-19 remain unclear for these workers in China. This study aimed to explore the baseline dietary and behavioral habits of frontline workers and their willingness to change these habits after the outbreak of the epidemic; in addition, susceptible subgroups were identified by stratified analyses as targets of protective measures to keep them from being infected with COVID-19. METHODS: A cross-sectional study was conducted through an online questionnaire using a sample of 22,459 valid individuals living in China, including 9402 frontline epidemic prevention workers. RESULTS: Before COVID-19, 23.9% of the frontline epidemic prevention workers reported a high-salt diet, 46.9% of them reported a high frequency of fried foods intake, and 50.9% of them smoked cigarettes. After the outbreak of COVID-19, 34.6% of them expressed a willingness to reduce salt intake, and 43.7% of them wanted to reduce the frequency of pickled vegetables intake. A total of 37.9% of them expressed a willingness to decrease or quit smoking, and 44.5% of them wanted to increase sleep duration. Significant differences in the baseline dietary and behavioral habits and the willingness to change their habits were observed between frontline epidemic prevention workers and other participants. Among the frontline epidemic prevention workers with poor dietary and behavioral habits before COVID-19, frontline epidemic prevention experience was a promoting factor for adopting worse dietary and behavioral habits, including those in the high-salt intake subgroup (OR, 2.824; 95% CI, 2.341-3.405) and the 11-20 cigarettes/day subgroup (OR, 2.067; 95% CI, 1.359-3.143). CONCLUSIONS The dietary and behavioral habits of frontline epidemic prevention workers were worse than that those of other participants before COVID-19. They had a greater willingness to adopt healthy dietary and behavioral habits after experiencing the outbreak of COVID-19. However, frontline epidemic prevention workers with poor dietary and behavioral habits before COVID-19 continued in engage in these poor habits. Dietary and behavioral intervention policies should be drafted to protect their health, especially frontline epidemic prevention workers with poor habits at baseline.
Adult ; COVID-19/psychology* ; China/epidemiology* ; Cross-Sectional Studies ; Diet/standards* ; Female ; Health Behavior ; Health Knowledge, Attitudes, Practice ; Health Personnel/psychology* ; Humans ; Male ; Risk Reduction Behavior ; SARS-CoV-2 ; Surveys and Questionnaires

Objective    To explore the practical feasibility of the weaving technique for pectus carinatum. Methods    From January 2011 to December 2018, a total of 51 patients with pectus carinatum, including 47 males and 4 females at age of 9-29 (13.7±2.9) years, were applied with minimally invasive waving technique for the correction. The steel plate was inserted through the subcutaneous layer, intercostal space and over the sternal surface under direct thoracoscopic vision. The number of implanted steel plates was determined by the degree of chest wall deformity. The steel plate was removed 2 years after surgery. Results    All the operations were successfully completed, the average operation time was 63.9±15.8 min, the amount of bleeding was 19.8±8.8 mL, and the duration of postoperative hospitalization was 4.6±1.6 d. The adverse events included intercostal artery injury (n=2), pneumothorax (n=4), pleural effusion (n=3) and skin rupture (n=1). And there were 29 patients of moderate pain (numerical rating scale 4-6 points) on the first day after surgery, but no patient was asked to remove the steel palate due to intolerable discomfort. All patients were followed up after plate placement. Of the 51 patients, the plates were removed in 37 patients until 2 years after placement, and the duration of postoperative hospitalization was 1.4±0.5 d. After 33 (1-48) months of routine follow-up after the removal of the plate, 22 patients achieved excellent outcomes and 9 patients with good outcomes. Besides, there were 5 patients with fair outcome and 1 patient with poor outcome. No adverse effect was found in growth and development after the steel plate placement. Conclusion    Minimally invasive weaving technique is a safe, feasible, effective and individualized operation for pectus carinatum with substantial thoracic reconstruction.

Objective: To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat-treated Lewis lung cancer cell lysates for treatment of lung cancer in mice. Methods: Bone marrow cells were induced by the recombinant mouse fms-like tyrosine kinase receptor 3 ligand (rmFlt3-L) in vitro, myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC∶pDC=1∶1 were stimulated with heat-treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ (MHC-Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin-12 (IL-12), interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Results: The lymphocyte proliferation in mice stimulated with mDC+ pDC group loaded with heat-treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively (P<0.05). When the ratio of effector cells: target cells (E∶T) was 10∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P<0.05). When the ratio of E∶T was 20∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 54.77%±3.28%, significantly higher than 35.25%±1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P<0.05). When the ratio of E∶T was 40∶1, the killing rate of the mDC+ pDC group loaded with heat-treated tumor cell lysate was 73.01%±0.91%, significantly higher than 51.36%±0.58% of mDC group and 22.65%±1.28% of pDC group, respectively (P<0.05). With the rate of E∶T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC-Ⅱ of mDC: pDC=1 group pulsed with heat-treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL-6 cytokine concentrations of mDC+ pDC group, mDC group and pDC group loaded with heat-treated Lewis lung cancer cell lysates were (586.67±52.52) pg/ml, (323.33±67.14) pg/ml and (166.67±16.07) pg/ml, respectively. The concentrations of IL-12 in each group were (2 568.75±119.24) pg/ml, (2 156.25±120.55) pg/ml and (672.92±31.46) pg/ml, respectively. The concentrations of TNF-α in each group were (789.33±48.08) pg/ml, (584.89±116.49) pg/ml and (291.56±40.73) pg/ml, respectively. The concentrations of IL-6, IL-12 and TNF-α secreted by mDC+ pDC group were much higher than those of mDC group and pDC group under the same culture conditions (P<0.05). Conclusions The mDCs and pDCs combined vaccines pulsed with heat-treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti-tumor effect is related with up-regulation of co-stimulatory molecules and increased secretion of cytokines.

To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat?treated Lewis lung cancer cell lysates for treatment of lung cancer in mice. Methods Bone marrow cells were induced by the recombinant mouse fms?like tyrosine kinase receptor 3 ligand ( rmFlt3?L) in vitro, myeloid dendritic cells ( mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC ∶ pDC＝1 ∶ 1 were stimulated with heat?treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ( MHC?Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin?12 (IL?12), interlukin?6 (IL?6), and tumor necrosis factor α ( TNF?α) were detected by enzyme?linked immunosorbent assay ( ELISA). Results The lymphocyte proliferation in mice stimulated with mDC+pDC group loaded with heat?treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively ( P＜0.05). When the ratio of effector cells:target cells (E ∶ T) was 10 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P＜0.05). When the ratio of E ∶ T was 20 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 54.77%± 3.28%, significantly higher than 35.25%± 1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P＜0.05). When the ratio of E ∶ T was 40 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 73.01%± 0.91%, significantly higher than 51.36%± 0.58% of mDC group and 22.65%± 1.28% of pDC group, respectively (P＜0.05 ). With the rate of E ∶ T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC?Ⅱ of mDC:pDC＝1 group pulsed with heat?treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL?6 cytokine concentrations of mDC+pDC group, mDC group and pDC group loaded with heat?treated Lewis lung cancer cell lysates were (586.67±52.52) pg／ml, (323.33±67.14) pg／ml and (166.67± 16.07) pg／ml, respectively. The concentrations of IL?12 in each group were ( 2 568.75± 119.24) pg／ml, (2 156.25±120.55) pg／ml and (672.92±31.46) pg／ml, respectively. The concentrations of TNF?α in each group were (789.33±48.08) pg／ml, (584.89±116.49) pg／ml and (291.56±40.73) pg／ml, respectively. The concentrations of IL?6, IL?12 and TNF?α secreted by mDC+pDC group were much higher than those of mDC group and pDC group under the same culture conditions ( P＜0.05). Conclusions The mDCs and pDCs combined vaccines pulsed with heat?treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti?tumor effect is related with up?regulation of co?stimulatory molecules and increased secretion of cytokines.

To investigate the feasibility of myeloid and plasmacytoid dendritic cell combined vaccines loaded with heat?treated Lewis lung cancer cell lysates for treatment of lung cancer in mice. Methods Bone marrow cells were induced by the recombinant mouse fms?like tyrosine kinase receptor 3 ligand ( rmFlt3?L) in vitro, myeloid dendritic cells ( mDC) and plasmacytoid dendritic cells (pDC) were separated by magnetic beads. The mDC, pDC, and mDC ∶ pDC＝1 ∶ 1 were stimulated with heat?treated Lewis lung cancer cell lysates, respectively. The effects of each group on stimulating of lymphocyte proliferation and inducing of T cell to kill tumor cells in vitro were compared. The alternations of the immunophenotypes of CD80, CD86, CD40 and major histocompatibility complex Ⅱ( MHC?Ⅱ) were detected by flow cytometry. The secretion of cytokines including interlukin?12 (IL?12), interlukin?6 (IL?6), and tumor necrosis factor α ( TNF?α) were detected by enzyme?linked immunosorbent assay ( ELISA). Results The lymphocyte proliferation in mice stimulated with mDC+pDC group loaded with heat?treated Lewis lung cancer cell lysates was 10.80±0.66, significantly higher than 8.63±0.65 of mDC group and 7.10±0.46 pDC group under the same culture conditions, respectively ( P＜0.05). When the ratio of effector cells:target cells (E ∶ T) was 10 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 31.68%±2.93%, significantly higher than 17.44%±0.97% of mDC group and 10.29%±1.33% of pDC group, respectively (P＜0.05). When the ratio of E ∶ T was 20 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 54.77%± 3.28%, significantly higher than 35.25%± 1.51% of mDC group and 15.52%±0.73% of pDC group, respectively (P＜0.05). When the ratio of E ∶ T was 40 ∶ 1, the killing rate of the mDC+pDC group loaded with heat?treated tumor cell lysate was 73.01%± 0.91%, significantly higher than 51.36%± 0.58% of mDC group and 22.65%± 1.28% of pDC group, respectively (P＜0.05 ). With the rate of E ∶ T increased, the killing rate also increased. The mean fluorescence intensities of surface molecules including CD80, CD86, CD40 and MHC?Ⅱ of mDC:pDC＝1 group pulsed with heat?treated Lewis lung cancer cell lysates were higher than those of mDC group and pDC group. The IL?6 cytokine concentrations of mDC+pDC group, mDC group and pDC group loaded with heat?treated Lewis lung cancer cell lysates were (586.67±52.52) pg／ml, (323.33±67.14) pg／ml and (166.67± 16.07) pg／ml, respectively. The concentrations of IL?12 in each group were ( 2 568.75± 119.24) pg／ml, (2 156.25±120.55) pg／ml and (672.92±31.46) pg／ml, respectively. The concentrations of TNF?α in each group were (789.33±48.08) pg／ml, (584.89±116.49) pg／ml and (291.56±40.73) pg／ml, respectively. The concentrations of IL?6, IL?12 and TNF?α secreted by mDC+pDC group were much higher than those of mDC group and pDC group under the same culture conditions ( P＜0.05). Conclusions The mDCs and pDCs combined vaccines pulsed with heat?treated Lewis lung cancer cell lysates have synergistic effects on inducing of T lymphocyte proliferation and killing tumor cells in vitro. This synergistic anti?tumor effect is related with up?regulation of co?stimulatory molecules and increased secretion of cytokines.