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*

Objective:To evaluate the epidemiological characteristics and explore the associated factors of breakthrough cases (BC) from Public Health Emergency Events (PHEEs) of varicella in Zhejiang Province from 2019 to 2022.Methods:Data on cases were obtained from the China Information System for Disease Control and Prevention and the PHEEs Reporting Information Database of Varicella in Zhejiang Province. History records were matched through the Zhejiang Provincial Immunization Information System. Descriptive analysis and multiple logistic regression model with a bidirectional stepwise selection method were performed to explore associated factors for BC during 2019-2022.Results:A total of 144 276 varicella cases were reported from 2019 to 2022, with the annual reported incidence of 47.35-82.80 cases per 100 000 population. Among these cases, 109 172 were non-breakthrough cases (NBC, accounting for 75.67%), 34 517 were BC (23.92%), and the rest 587 cases had unclear vaccination history on varicella (0.41%). A total of 214 PHEEs of varicella were reported, of which 99.07% occurred in school settings. The proportion of PHEEs that occurred in high school increased significantly as time went on ( χ2trend=5.742, P=0.017). Multiple logistic regression model which focused on "BC vs. NBC (as the reference)" indicated that the year of onset ( OR=1.585, 95% CI:1.343-1.878), the month of onset (taking January as the reference, OR=2.311-15.652), city (taking Hangzhou as the reference, Jiaxing OR=2.370, Jinhua OR=2.197, Lishui OR=0.134), age ( OR=0.887, 95% CI: 0.826-0.944), PHEEs setting (taking "primary school and below" as the reference, "high school and above" OR=0.516, 95% CI: 0.305-0.897), and the number of rashes ( OR=0.569, 95% CI: 0.458-0.703) were associated factors. Multiple logistic regression model which focused on "two-dose BC vs. one-dose BC (as the reference)" showed that the age of initial vaccination ( OR=0.045, 95% CI: 0.014-0.107), the time interval from onset to the last dose ( OR=0.037, 95% CI: 0.011-0.087) and the age of onset ( OR=20.724, 95% CI: 8.383-72.485) were associated factors. Conclusion:During 2019-2022, the reported high-risk group of varicella in Zhejiang Province has shifted to adolescents and young adults. Although vaccination could not completely prevent the onset of VZV, it could relieve clinical symptoms and delay the age of onset.

Objective:To investigate the impact of non-high-density lipoprotein cholesterol (non-HDL-C) level on major adverse cardiovascular and cerebrovascular events (MACCE) and all-cause mortality in the Kailuan Study cohort undergoing revascularization.Methods:This is a prospective cohort study, with participants from the Kailuan Study cohort who participated in physical examinations from 2006 to 2020 and received revascularization therapy for the first time. According to the level of non-HDL-C, the study subjects were divided into 3 groups:<2.6 mmol/L group, 2.6-<3.4 mmol/L group, and≥3.4 mmol/L group. Annual follow-up was performed, and the endpoint events were MACCE and all-cause mortality. Cox proportional regression model was implemented to estimate the impact on MACCE and all-cause mortality associated with the different non-HDL-C groups. The partial distributed risk model was used to analyze the impact of different non-HDL-C levels on MACCE event subtypes, and death was regarded as a competitive event. The restricted cubic spline regression model was used to explore the dose-response relationship between non-HDL-C level and all-cause mortality, MACCE and its subtypes.Results:A total of 2 252 subjects were enrolled in the study, including 2 019 males (89.65%), aged (62.8±8.3) years, the follow-up time was 5.72 (3.18, 8.46) years. There were 384 cases(17.05%) of MACCE and 157 cases(6.97%) of all-cause mortality. Compared with patients with non-HDL-C≥3.4 mmol/L, patients with non-HDL-C<2.6 mmol/L were associated with a 38% reduced risk of MACCE after revascularization [ HR=0.62(95% CI: 0.48-0.80)]. Every 1 mmol/L decrease in non-HDL-C was associated with a 20% reduction in the risk of MACCE [ HR=0.80(95% CI: 0.73-0.88)]. The results of restricted cubic spline also showed that non-HDL-C levels after revascularization therapy were positively correlated with MACCE events (overall association P<0.001, non-linear association P=0.808). For all-cause mortality, compared to the non-HDL-C≥3.4 mmol/L group, the HR for all-cause mortality after revascularization in non-HDL-C<2.6 mmol/L group was 0.67(95% CI: 0.46-1.01). Every 1 mmol/L decrease in non-HDL-C was associated with a 15% reduction in the risk of all-cause mortality [ HR=0.85(95% CI: 0.73-0.99)]. The restricted cubic spline results showed a linear association between non-HDL-C levels after revascularization therapy and the risk of all-cause mortality (overall association P=0.039, non-linear association P=0.174). Conclusion:The decrease in non-HDL-C levels after revascularization were significantly associated with a reduced risk of MACCE and all-cause mortality.

Objective:To evaluate the epidemiological characteristics and explore the associated factors of breakthrough cases (BC) from Public Health Emergency Events (PHEEs) of varicella in Zhejiang Province from 2019 to 2022.Methods:Data on cases were obtained from the China Information System for Disease Control and Prevention and the PHEEs Reporting Information Database of Varicella in Zhejiang Province. History records were matched through the Zhejiang Provincial Immunization Information System. Descriptive analysis and multiple logistic regression model with a bidirectional stepwise selection method were performed to explore associated factors for BC during 2019-2022.Results:A total of 144 276 varicella cases were reported from 2019 to 2022, with the annual reported incidence of 47.35-82.80 cases per 100 000 population. Among these cases, 109 172 were non-breakthrough cases (NBC, accounting for 75.67%), 34 517 were BC (23.92%), and the rest 587 cases had unclear vaccination history on varicella (0.41%). A total of 214 PHEEs of varicella were reported, of which 99.07% occurred in school settings. The proportion of PHEEs that occurred in high school increased significantly as time went on ( χ2trend=5.742, P=0.017). Multiple logistic regression model which focused on "BC vs. NBC (as the reference)" indicated that the year of onset ( OR=1.585, 95% CI:1.343-1.878), the month of onset (taking January as the reference, OR=2.311-15.652), city (taking Hangzhou as the reference, Jiaxing OR=2.370, Jinhua OR=2.197, Lishui OR=0.134), age ( OR=0.887, 95% CI: 0.826-0.944), PHEEs setting (taking "primary school and below" as the reference, "high school and above" OR=0.516, 95% CI: 0.305-0.897), and the number of rashes ( OR=0.569, 95% CI: 0.458-0.703) were associated factors. Multiple logistic regression model which focused on "two-dose BC vs. one-dose BC (as the reference)" showed that the age of initial vaccination ( OR=0.045, 95% CI: 0.014-0.107), the time interval from onset to the last dose ( OR=0.037, 95% CI: 0.011-0.087) and the age of onset ( OR=20.724, 95% CI: 8.383-72.485) were associated factors. Conclusion:During 2019-2022, the reported high-risk group of varicella in Zhejiang Province has shifted to adolescents and young adults. Although vaccination could not completely prevent the onset of VZV, it could relieve clinical symptoms and delay the age of onset.

Objective:To explore the heterogeneity among keloids before and after radiotherapy and identify the changes of key cell subpopulations and their interactions utilizing single cell RNA sequencing technology.Methods:Four patients provided a total of 12 samples, each consisting of keloid tissue before and after radiotherapy and the normal skin tissue adjacent to the untreated keloid. The keloid was divided into left and right sides from the midline, and the left-side keloid was fractionally irradiated with 20 Gy electron beam in total in 4 consecutive days. The right-side keloid was irradiated with 10 Gy in 2 fractions before surgery and 10 Gy in 2 fractions after surgery.Results:A total of 25 573 fibroblasts were analyzed and categorized into nine subgroups (fibroblasts 1-9). The proportion of fibroblast-2 increased after radiotherapy ( t=4.70, P<0.05). The number of classical monocytes and macrophages increased after radiotherapy, but there was no significant difference due to the shorter time of sample taking at 2 d after radiotherapy ( P>0.05). Macrophages (4 723 cells) were further divided into four categories. CellPhoneDB analysis showed that type-3 macrophages interacted significantly more closely with fibroblasts than type-1 and type-2 macrophages. The most prominent signaling pathways for the interactions between type-3 macrophages and major fibroblast subtypes were the collagen signaling pathway and the chemerin signaling pathway. These interactions were more pronounced in the keloid samples after radiotherapy. Conclusions:The interactions between type-3 macrophages and fibroblasts (such as fibroblast-2) may serve as an important point for future studies on radio-sensitization of keloids.

Primary familial brain calcification (PFBC) is an inherited neurodegenerative disorder mainly characterized by progressive calcium deposition bilaterally in the brain, accompanied by various symptoms, such as dystonia, ataxia, parkinsonism, dementia, depression, headaches, and epilepsy. Currently, the etiology of PFBC is largely unknown, and no specific prevention or treatment is available. During the past 10 years, six causative genes (SLC20A2, PDGFRB, PDGFB, XPR1, MYORG, and JAM2) have been identified in PFBC. In this review, considering mechanistic studies of these genes at the cellular level and in animals, we summarize the pathogenesis and potential preventive and therapeutic strategies for PFBC patients. Our systematic analysis suggests a classification for PFBC genetic etiology based on several characteristics, provides a summary of the known composition of brain calcification, and identifies some potential therapeutic targets for PFBC.
Animals ; Brain Diseases/therapy* ; Xenotropic and Polytropic Retrovirus Receptor ; Brain/pathology*

BACKGROUND: Few studies have explored the impact of perchlorate, nitrate, and thiocyanate (PNT) on kidney function. This study aimed to evaluate the association of urinary levels of PNT with renal function as well as the prevalence of chronic kidney disease (CKD) among the general population in the United States. METHODS: This analysis included data from 13,373 adults (≥20 years) from the National Health and Nutrition Examination Survey 2005 to 2016. We used multivariable linear and logistic regression, to explore the associations of urinary PNT with kidney function. Restricted cubic splines were used to assess the potentially non-linear relationships between PNT exposure and outcomes. RESULTS: After traditional creatinine adjustment, perchlorate (P-traditional) was positively associated with estimated glomerular filtration rate (eGFR) (adjusted β: 2.75; 95% confidence interval [CI]: 2.25 to 3.26; P  < 0.001), and negatively associated with urinary albumin-to-creatinine ratio (ACR) (adjusted β: -0.05; 95% CI: -0.07 to -0.02; P  = 0.001) in adjusted models. After both traditional and covariate-adjusted creatinine adjustment, urinary nitrate and thiocyanate were positively associated with eGFR (all P values <0.05), and negatively associated with ACR (all P values <0.05); higher nitrate or thiocyanate was associated with a lower risk of CKD (all P values <0.001). Moreover, there were L-shaped non-linear associations between nitrate, thiocyanate, and outcomes. In the adjusted models, for quartiles of PNT, statistically significant dose-response associations were observed in most relationships. Most results were consistent in the stratified and sensitivity analyses. CONCLUSIONS Exposures to PNT might be associated with kidney function, indicating a potential beneficial effect of environmental PNT exposure (especially nitrate and thiocyanate) on the human kidney.
Adult ; Humans ; United States/epidemiology* ; Nitrates/adverse effects* ; Nutrition Surveys ; Thiocyanates/urine* ; Perchlorates/urine* ; Creatinine ; Environmental Exposure ; Renal Insufficiency, Chronic/epidemiology* ; Logistic Models

Objective: To obtain chimeric antigen receptor macrophages ( CAR-M) targeting HER2 stably transfected． Methods : CAR lentivirus vector targeting HER2 was constructed and infected with human monocytic leukemia cell line (THP-1) ．CAR THP-1 cells with green fluorescent labeling were selected by sorting flow cytometry and continued to be cultured in vitro．The CAR THP-1 cells targeting HER2 were co-cultured with the endometrial cancer cell line Ishikawa with negative and positive HER2 expression，and their targeted phagocytosis of CAR-M to HER2 positive tumor cells was detected by imaging flow cytometry ，and the targeted phagocytosis efficiency of CAR-M to HER2 positive tumor cells was detected by flow cytometry. Results : CAR lentivirus infection with THP- 1 cells was less efficient ; After co-culture with cancer cells，flow cytometry and imaging flow cytometry showed that CAR THP-1 cells had enhanced phagocytosis of HER2 positive Ishikawa cells compared with the empty body group (P＜0. 01) ． Conclusion In this experiment，CAR THP-1 cell line targeting HER2 was established by constructing CAR lentivirus vector and transfecting THP-1 cells ，and it was proved that CAR THP-1 could phagocytize HER2 positive Ishikawa cells through specific targeting.

Objective To investigate the effects of N-acetyl-D-glucosamine(GlcNAc)on acute pancreatitis in rats.Methods Twenty male SD rats were randomly divided into a control group,AP group,low GlcNAc + AP group and high GlcNAc + AP group,with five rats in each group.Acute pancreatitis was induced in AP group,low GlcNAc + AP group and high GlcNAc + AP group by two intraperitoneal injections of 2.5 g/kg L-arginine with a 1 hour interval.Among them,low GlcNAc + AP group and high GlcNAc + AP group were administered 50 and 200 mg/kg GlcNAc,respectively,by intraperitoneal injection at 24 hours before the first intraperitoneal injection of L-arginine.Group control and AP were intraperitoneally injected with the same volume of normal saline.After 24 h,the rats were sacrificed,and serum and pancreatic tissues were collected.Pancreatic tissue morphology was observed by HE staining,and serum levels of amylase(AMY),lipase(LPS),interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),superoxide dismutase(SOD)and malondialdehyde(MDA)were measured by enzyme-linked immunosorbent assay.Protein expression of nuclear factor E2-related factor 2(NRF2),heme oxygenase-1(HO-1),and peroxisome proliferator-activated receptor-γ(PPAR-γ)in pancreatic tissue was detected by Western Blot.Cluster of differentiation(CD)86,CD206 and macrophage markers(F4/80)were detected by immunofluorescence.Expression of CD86 and CD206 in pancreatic tissue was detected by immunohistochemistry.Results(1)Compared with control group,AMY,LPS,IL-1β,IL-6,TNF-α,and MDA levels and pancreatic CD86 expression in AP group were significantly increased(P<0.05),while SOD activity,protein expression levels of NRF2,HO-1,and PPAR-γ,and pancreatic CD206 expression were significantly decreased(P<0.05).(2)Compared with AP group,serum IL-1β,IL-6,TNF-α,MDA,and LPS and the pancreatic CD86 expression in low GlcNAc + AP group were significantly decreased(P<0.05).The PPAR-γ protein level in the pancreas was significantly increased(P<0.05).(3)Compared with AP group,serum AMY,LPS,IL-1β,IL-6,TNF-α,and MDA and pancreatic CD86 expression in high GlcNAc + AP group were significantly decreased(P<0.05),while serum SOD,and NRF2,HO-1,PPAR-γ,and pancreatic CD206 expression were significantly increased(P<0.05).(4)Compared with low GlcNAc + AP group,serum LPS,IL-1β and IL-6 in high GlcNAc + AP group were significantly decreased(P<0.05).Pancreatic expression of HO-1,PPAR-γ,and pancreatic CD206 were significantly increased(P<0.05).Conclusion GlcNAc treatment attenuates acute pancreatitis injury in AP rats,possibly by activating the NRF2/HO-1 signaling pathway to inhibit oxidative stress and promoting M2 macrophage polarization to attenuate pancreatic injury in AP rats.

Objective: To establish an optimized path for health management of HBV infections among pregnant and lying-in women based on a Delphi method, so as to provide the evidence for intensifying the interruption of the mother-to-child transmission of HBV. Methods: Based on literature review and previous studies, the preliminary framework and contents of the optimized path for health management of HBV infections were constructed. Experts from epidemiology, clinical medicine and maternal and children healthcare were invited to participate in two-round Delphi consultations, and the preliminarily designed indicators were screened and revised. The score for feasibility of each indicator was calculated, and the weight of each indicator was estimated using a proportional distribution method. Results: Sixteen experts participated in the consultation, including 13 women. The participants had a mean age of (45.69±5.71) years, and a mean employment duration of (23.06±7.05) years. All participants had a degree of bachelor and above, and there were 14 experts with vice senior professional titles. The mean positive coefficient was 96.88% and the mean authority coefficient was 0.790 during the two-round expert consultations. There were significant differences in the coordination coefficient of importance, necessity and feasibility of indicators at all levels (P<0.05), and the coefficient of variation of the feasibility was all less than 0.250. The final optimized path for health management of HBV infections among pregnant and lying-in women included 6 primary indicators, 17 secondary indictors and 73 tertiary indicators. Among the primary indicators, delivery management (0.173 4), screening and evaluation (0.172 8) and pregnancy management (0.172 7) had a high weight. Conclusion A scientific and reliable optimized path is created for health management of HBV infections among pregnant and lying-in women, which has a potential value for improving the interruption of mother-to-child transmission of HBV.