Hydroxyectoine, a vital compatible solute, is widely utilized in cosmetics, food, pharmaceutical industries, and biologics. However, the current microbial fermentation methods for hydroxyectoine production face challenges including insufficient precursor supply and low yields. Therefore, developing engineering microbial strains capable of efficiently synthesizing hydroxyectoine is of great significance. In this study, we first constructed a high-yield ectoine-producing strain ECT04 by multi-copy integration of the ectoine synthesis genes ectABC into the pseudogene loci of Escherichia coli MG1655(DE3), achieving an ectoine titer of 6.03 g/L. Subsequently, we employed plasmids with varying copy numbers to express ectD from Chromohalobacter salexigens to enable the conversion for hydroxyectoine production. We further investigated the effects of promoter, co-substrate ɑ-ketoglutarate, Fe²⁺ concentration, and dissolved oxygen on hydroxyectoine synthesis. Through fed-batch fermentation in a 7-L bioreactor, we significantly enhanced the hydroxyectoine production efficiency, attaining a final titer of 8.58 g/L and a productivity of 0.24 g/(L·h). This work successfully achieved the de novo synthesis of hydroxyectoine in E. coli, laying a foundation for the efficient bioproduction of this compound.
Escherichia coli/genetics* ; Fermentation ; Amino Acids, Diamino/biosynthesis* ; Bioreactors/microbiology* ; Metabolic Engineering/methods* ; Chromohalobacter/genetics* ; Plasmids/genetics*

In the context of the complex and ever-changing spectrum of diseases, the traditional Chinese medicine compatibility of prescription and drugs is no longer able to fully meet the needs of clinical diagnosis and treatment. Therefore, this article is based on the diagnosis and treatment model of the combination of disease, syndrome, and symptom, combined with the development achievements of Western medicine, and explores the principles of formulating traditional prescriptions based on the combination of chief, deputy, assistant, and envoy. This article proposes a formulation principle of composing prescriptions with the diagnosis of syndrome as the chief, the diagnosis of disease as the deputy, the treatment of symptoms as the assistant, and the harmonization of medicine as the envoy. This forms a treatment plan with the core link of syndrome differentiation and treatment, disease differentiation and treatment, symptomatic treatment, detoxification, and efficacy enhancement. The purpose of this article is to address the current clinical challenges such as an increasing disease spectrum and the complexity of syndrome patterns and symptom clusters. It aims to provide new insights into traditional Chinese medicine clinical treatment plans and herbal formulation strategies, with the ultimate goal of improving the clinical effectiveness of traditional Chinese medicine.

Objective:To investigate the endoscopic combined serological diagnosis strategy for G1 and G2 gastric neuroendocrine neoplasms (G-NENs), and to evaluate the safety, short-term, and long-term efficacy of two endoscopic treatment procedures: endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD).Methods:This study retrospectively analyzed the clinical data of 100 consecutive patients with G-NENs who were hospitalized at the Cancer Hospital of the Chinese Academy of Medical Sciences from January 2011 to October 2023. These patients underwent endoscopic treatment, and propensity score matching (PSM) was used to compare clinicopathological characteristics, as well as short-term and long-term efficacy of lesions in the EMR group and ESD group before and after treatment.Results:Among the 100 patients with G-NENs, the median age was 54 years old. Before surgery, 29 cases underwent endoscopic combined serological examination, and 24 of them (82.2%) had abnormally elevated plasma chromogranin A. The combined diagnostic strategy for autoimmune atrophic gastritis (AIG) achieved a diagnostic accuracy of 100%(22/22). A total of 235 G-NEN lesions were included, with 84 in the ESD group and 151 in the EMR group. The median size of the lesions in the ESD group (5.0 mm) was significantly larger than that in the EMR group (2.0 mm, P＜0.001). Additionally, the ESD group had significantly more lesions with pathological grade G2[23.8%(20/84) vs. 1.3%(2/151), P＜0.001], infiltration depth reaching the submucosal layer [78.6%(66/84) vs. 51.0%(77/151), P＜0.001], and more T2 stage compared to the EMR group[15.5%(13/84) vs. 0.7%(1/151), P＜0.001]. After PSM, 49 pairs of lesions were successfully matched between the two groups. Following PSM, there were no significant differences in the en bloc resection rate [100.0%(49/49) vs. 100.0%(49/49)], complete resection rate [93.9%(46/49) vs. 100.0%(49/49)], and complication rate [0(0/49) vs. 4.1%(2/49)] between the two groups. During the follow-up period, no recurrence or distant metastasis was observed in any of the lesions in both groups. Conclusions:The combination of endoscopy and serology diagnostic strategy has the potential to enhance the accuracy of diagnosing G1 and G2 stage G-NENs and their background mucosa. Endoscopic resection surgery (EMR, ESD) is a proven and safe treatment approach for G1 and G2 stage G-NENs.

Objectives:To develop and validate predictive models for esophageal squamous cell carcinoma (ESCC) using circulating cell-free DNA (cfDNA) terminal motif analysis. The goal was to improve the non-invasive detection of early-stage ESCC and its precancerous lesions.Methods:Between August 2021 and November 2022, we prospectively collected plasma samples from 448 individuals at the Department of Endoscopy, Cancer Hospital, Chinese Academy of Medical Sciences for cfDNA extraction, library construction, and sequencing. We analyzed 201 cases of ESCC, 46 high-grade intraepithelial neoplasia (HGIN), 46 low-grade intraepithelial neoplasia (LGIN), 176 benign esophageal lesions, and 29 healthy controls. Participants, including ESCC patients and control subjects, were randomly assigned to a training set ( n=284) and a validation set ( n=122). The training cohort underwent z-score normalization of cfDNA terminal motif matrices and a selection of distinctive features differentiated ESCC cases from controls. The random forest classifier, Motif-1 (M1), was then developed through principal component analysis, ten-fold cross-validation, and recursive feature elimination. M1's efficacy was then validated in the validation and precancerous lesion sets. Subsequently, individuals with precancerous lesions were included in the dataset and participants were randomly allocated to newly formed training ( n=243), validation ( n=105), and test ( n=150) cohorts. Using the same procedure as M1, we trained the Motif-2 (M2) random forest model with the training cohort. The M2 model's accuracy was then confirmed in the validation cohort to establish the optimal threshold and further tested by performing validation in the test cohort. Results:We developed two cfDNA terminal motif-based predictive models for ESCC and associated precancerous conditions. The first model, M1, achieved a sensitivity of 90.0%, a specificity of 77.4%, and an area under the curve (AUC) of 0.884 in the validation cohort. For LGIN, HGIN, and T1aN0 stage ESCC, M1's sensitivities were 76.1%, 80.4%, and 91.2% respectively. Notably, the sensitivity for jointly predicting HGIN and T1aN0 ESCC reached 85.0%. Both the predictive accuracy and sensitivity increased in line with the cancer's progression ( P＜0.001). The second model, M2, exhibited a sensitivity of 87.5%, a specificity of 77.4%, and an AUC of 0.857 in the test cohort. M2's sensitivities for detecting precancerous lesions and ESCC were 80.0% and 89.7%, respectively, and it showed a combined sensitivity of 89.4% for HGIN and T1aN0 stage ESCC. Conclusions:Two predictive models based on cfDNA terminal motif analysis for ESCC and its precancerous lesions are developed. They both show high sensitivity and specificity in identifying ESCC and its precancerous stages, indicating its potential for early ESCC detection.

Based on the"You Gu Wu Yun"theory in traditional Chinese medicine(TCM),this paper believes that"Gu"in"You Gu Wu Yun"is extended to"state"from the perspective of"TCM state".In order to avoid the adverse reactions of TCM,the macro,meso,and micro three views should be used together,and macro,meso,and micro parameters should be integrated.We should also carefully identify the physiological characteristics,pathological characteristics,constitution,syndrome,and disease of human body by combining qualitative and quantitative method,highlighting the relationship between the prescription and the"state".The correspondence between prescription and the"state"will reduce the risk of adverse reactions of TCM.In this paper,we hope to focus on the guiding role of the"You Gu Wu Yun"theory in TCM research,to give full play to the characteristics and advantages of TCM,and to dialectically treat the role of TCM.

The correspondence between prescription and syndrome is the advantage and characteristic of traditional Chinese medicine(TCM)treatment.However,the pathogenesis of clinical diseases is complex and the condition is changeable,and the clinical application is difficult to achieve the maximum effect under the existing cognition of the correspondence between prescription and syndrome.In this paper,the five categories of physiological characteristics,pathological characteristics,constitution,syndrome,and disease of the longitudinal classification of"TCM state"are introduced into the correspondence of prescription and syndrome.Under the vertical perspective of"TCM state",the theoretical connotation of the correspondence between prescription and syndrome is interpreted as"correspondence between prescription and state",namely correspondence of"prescription-physiological characteristics",correspondence of"prescription-pathological characteristics",correspondence of"prescription-constitution",correspondence of"prescription-syndrome",and correspondence of"prescription-disease".It is hoped to accurately grasp the corresponding connotation of the correspondence between prescription and syndrome,in order to deepen the clinical thinking mode of TCM.

Objective To establish a coronavirus(CoV)infection model using human nasal mucosa organoids for testing antiviral drugs and evaluate the feasibility of using human nasal mucosa organoids with viral infection as platforms for viral research and antiviral drug development.Methods Human nasal mucosa organoids were tested for susceptibility to SARS-CoV-2 and HCoV-OC43 pseudoviruses.In a P3 laboratory,nasal mucosa organoids were infected with the original strain of SARS-CoV-2 and 4 variant strains,and the infection conditions were optimized.The viral loads in the culture supernatants were measured at different time points using RT-qPCR,and immunofluorescence assay was employed to localize SARS-CoV-2 nucleocapsid protein to determine the type of the infected cells.In the optimized nasal mucosa viral infection model,the antiviral effects of camostat and bergamot extract(which were known to inhibit SARS-CoV-2)were tested and the underlying molecular mechanisms were explored.Results In the optimized nasal mucosa organoid models infected with SARS-CoV-2 and HCoV-OC43 pseudoviruses,the viral load in the culture supernatants increased significantly during the period of 2 to 24 h following the infection,which confirmed infection of the organoids by both of the pseudoviruses.The nasal mucosa organoids could be stably infected by the original SARS-CoV-2 strain and its 4 variant strains,validating successful establishment of the viral infection model,in which both camostat and bergamot extract exhibited dose-dependent antiviral effects.Conclusions Human nasal mucosa organoids with SARS-CoV-2 infection can serve as platforms for screening and testing antiviral drugs,particularly those intended for nasal administration.

Objective:To investigate the effect of iron deficiency on the prognosis of elderly patients with ejection fraction preserved heart failure (HFpEF).Methods:The clinical data of old patients (>75 years) with HFpEF from November 2021 to May 2023 in General Hospital of Eastern Theater of the Chinese People′s Liberation Army were retrospectively analyzed. The patients were divided into iron deficiency group (65 cases) and non-iron deficiency group (90 cases) according to serum ferritin (SF) and transferrin saturation (TSAT) at admission. The first hematological indexes and echocardiogram examination results after admission were compared between two groups. The patients were followed up until November 2023, the poor prognosis was recorded. The correlation between iron deficiency, iron metabolism indexes and poor prognosis in elderly patients with HFpEF was analyzed by Spearman correlation analysis. The Kaplan-Meier survival curve was drawn to analyze the effect of iron deficiency on the cumulative survival in elderly patients with HFpEF.Results:There were no statistical difference in triglyceride, total cholesterol, low density lipoprotein cholesterol, C-reactive protein, hemoglobin and echocardiogram indexes between the two groups ( P>0.05). The N-terminal pro-brain natriuretic peptide (NT-proBNP), creatinine, procalcitonin (PCT) and interleukin-6 (IL-6) in iron deficiency group were significantly higher than those in non-iron deficiency group: 427.23 (281.00, 736.90) pmol/L vs. 313.50 (182.47, 363.25) pmol/L, (167.93 ± 51.22) μmol/L vs. (121.71 ± 11.99) μmol/L, 0.12 (0.05, 0.22) μg/L vs. 0.07 (0.04, 0.16) μg/L and 25.60 (8.38, 47.01) ng/L vs. 10.15 (4.75, 19.89) ng/L, the SF, serum iron (SI) and TSAT were significantly lower than those in non-iron deficiency group: 75.40 (42.30, 198.00) μg/L vs. 207.00 (281.00, 736.90) μg/L, (6.49 ± 2.66) μmol/L vs. (12.75 ± 4.24) μmol/L and (16.65 ± 6.26)% vs. (33.78 ± 11.16)%, and there were statistical differences ( P<0.01 or <0.05). The patients were followed up for (12.06 ± 7.58) months, the all-cause mortality, cardiovascular mortality, readmission rate and heart failure readmission rate in iron deficiency group were significantly higher than those in non-iron deficiency group: 40.0% (26/65) vs. 20.0% (18/90), 18.5% (12/65) vs. 4.4% (4/90), 90.8% (59/65) vs. 70.0% (63/90) and 66.2% (43/65) vs. 17.8% (16/90), and there were statistical differences ( P<0.01). Spearman correlation analysis result showed that the iron deficiency was positive correlation with all-cause death, cardiovascular death, readmission and heart failure readmission in elderly patients with HFpEF ( P<0.01); the SI and TSAT were negative correlation with all-cause death, cardiovascular death, readmission and heart failure readmission ( P<0.01 or <0.05); and the SF was not correlation with the indexes ( P>0.05). Kaplan-Meier survival analysis result showed that the risk of all-cause death was significantly increased in elderly HFpEF patients with iron deficiency, and the cumulative survival rate was significantly reduced (log-rank χ2 = 6.48, P<0.05). Conclusions:The elderly HFpEF patients with iron deficiency have poor prognosis with high mortality and readmission rate.

Objective:To investigate the relation between 25 hydroxyvitamin D3 level and global registry of acute coronary event (GRACE) score in elderly patients with acute coronary syndrome (ACS).Methods:The clinical data of 120 elderly male patients with ACS hospitalized in the General Hospital of Eastern Theater Command from January 2020 to June 2022 were retrospectively analyzed. Clinical characteristics of patients were collected and the 25 hydroxyvitamin D3 level was assessed with the chemiluminescent immunoassay method. According to GRACE score, the patients were divided into intermediate-risk group (109 to 140 scores, 46 cases) and high-risk group(>140 scores, 74 cases). The severity of coronary lesion was assessed according to the results of coronary angiography (CAG) and then they were divided into A, B, C group. The independent influential factors of GRACE score were analyzed by Logistic regression analysis.Results:The level of 25 hydroxyvitamin D3 in the high-risk group was lower than that in the intermediate-risk group: (13.84 ± 3.42) μg/L vs. (18.57 ± 5.17) μg/L, the usage rate of angiotensin-converting enzyme inhibitors (ACEI)/angiotonin receptor blocker (ARB) in the high-risk group was lower than that in the intermediate-risk group: 17.6%(13/71) vs. 41.3%(19/46), there were statistical differences ( P<0.05). Logistic regression analysis showed that 25 hydroxyvitamin D3 level was the risk factor for GRACE score ( OR = 0.745, 95% CI 0.657-0.844, P<0.05). The level of 25 hydroxyvitamin D3 had negative correlation with the severity of coronary lesion ( P<0.05). Conclusions:The level of 25 hydroxyvitamin D3 has correlation with GRACE score and the severity of coronary lesion in elderly patients with ACS.

Objective To establish a coronavirus(CoV)infection model using human nasal mucosa organoids for testing antiviral drugs and evaluate the feasibility of using human nasal mucosa organoids with viral infection as platforms for viral research and antiviral drug development.Methods Human nasal mucosa organoids were tested for susceptibility to SARS-CoV-2 and HCoV-OC43 pseudoviruses.In a P3 laboratory,nasal mucosa organoids were infected with the original strain of SARS-CoV-2 and 4 variant strains,and the infection conditions were optimized.The viral loads in the culture supernatants were measured at different time points using RT-qPCR,and immunofluorescence assay was employed to localize SARS-CoV-2 nucleocapsid protein to determine the type of the infected cells.In the optimized nasal mucosa viral infection model,the antiviral effects of camostat and bergamot extract(which were known to inhibit SARS-CoV-2)were tested and the underlying molecular mechanisms were explored.Results In the optimized nasal mucosa organoid models infected with SARS-CoV-2 and HCoV-OC43 pseudoviruses,the viral load in the culture supernatants increased significantly during the period of 2 to 24 h following the infection,which confirmed infection of the organoids by both of the pseudoviruses.The nasal mucosa organoids could be stably infected by the original SARS-CoV-2 strain and its 4 variant strains,validating successful establishment of the viral infection model,in which both camostat and bergamot extract exhibited dose-dependent antiviral effects.Conclusions Human nasal mucosa organoids with SARS-CoV-2 infection can serve as platforms for screening and testing antiviral drugs,particularly those intended for nasal administration.