O-acetyl-L-homoserine (OAH) is a promising platform compound for the production of L-methionine and other valuable compounds, while its low yield and low conversion rate limit the industrial application. To solve these problems, we constructed a strain for high OAH production with the previously constructed L-homoserine producer Escherichia coli HS33 as the chassis by systematic metabolic engineering. Firstly, PEP accumulation, pyruvate utilization, and OAH synthesis pathway (overexpressing aspB, aspA, and thrA^C1034T) were enhanced to obtain an initial strain accumulating 13.37 g/L OAH. Subsequently, the co-factor synthesis genes were integrated to supply reducing power and energy, which increased the yield to 15.79 g/L. The OAH yield of the engineered strain OAH28 was further increased to 17.49 g/L by strengthening the acetic acid reuse pathway, improving the supply of acetyl-CoA, and regulating the expression of MetX from different sources. Finally, in a 5 L fermenter, OAH28 achieved an OAH titer of 47.12 g/L, with a glucose conversion rate of 32% and productivity of 0.59 g/(L·h). The results lay a foundation for increasing the OAH production by metabolic engineering and give insights into the industrial production of OAH.
Metabolic Engineering/methods* ; Escherichia coli/genetics* ; Homoserine/biosynthesis* ; Fermentation

Gene therapy, harnessing the power of CRISPR-Cas9 and/or DNAzyme systems, stands as a pivotal approach in cancer therapy, enabling the meticulous manipulation of genes pivotal to tumorigenesis and immunity. However, the pursuit of precise gene therapy encounters formidable hurdles. Herein, a near-infrared upconversion theranostic nanomachine is devised and tailors for CRISPR-Cas9/DNAzyme systems mediate precise gene therapy. An ingenious logic DNAzyme system consists of Chain 1 (C1)/Chain 2 (C2) and endogenous lncRNA is designed. We employ manganese modified upconversion nanoparticles for carrying ultraviolet-responsive C1-PC linker-C2 (C₂P) chain and Cas9 ribonucleoprotein (RNP), with outermost coats with hyaluronic acid. Upon reaching tumor microenvironment (TME), the released Mn²⁺ ions orchestrate a trifecta: facilitating endosomal escape, activating cGAS-STING signaling, and enabling T1-magnetic resonance imaging. Under near-infrared irradiation, Cas9 RNP/C₂P complex dissociates, releasing Cas9 RNP into the nucleus to perform gene editing of Ptpn2, while C1/C2 chains self-assemble with endogenous lncRNA to form a functional DNAzyme system, targeting PD-L1 mRNA for gene silencing. This strategy remodels the TME by activating cGAS-STING signaling and dual immune checkpoints blockade, thus realizing tumor elimination. Our theranostic nanomachine armed with the CRISPR-Cas9/DNAzyme logic systems, represents a resourceful and promising strategy for advancing cancer systemic immunotherapy and precise gene therapy.

OBJECTIVES: To explore the expression profile of circular RNAs (circRNAs) and their potential roles in prognosis and progression of Wilms' tumor (WT). METHODS: Four pairs of WT and adjacent tissues were collected for high-throughput circRNA sequencing to identify the differentially expressed circular RNAs. RT-qPCR was used to verify the expression levels of the top 6 candidate circRNAs in the clinical samples. hsa_circ_0001900 was selected for analysis of its correlation with clinicopathological features and prognosis in 34 patients with WT. Sanger sequencing and RNase R digestion experiments were used to verify the cycling site and structural stability of hsa_circ_0001900 molecule. RESULTS: A total of 23 978 circular RNA molecules were identified in WT tissues by high-throughput circular RNA sequencing, and among them 614 were differentially expressed in WT. hsa_circ_0001900 showed the highest expression level among the differentially expressed circRNAs, which was consistent with the findings in clinical tumor samples and the sequencing results. Correlation analysis showed that hsa_circ_0001900 expression level was positively correlated with WT volume, and the children with high hsa_circ_0001900 expression had a lowered recurrence-free survival rate. The results of Sanger sequencing verified the circular splice site sequence of the molecule, and Rnase R digestion assay confirmed its stable covalent structure. CONCLUSIONS This study presents a comprehensive expression profile of circular RNAs in WT, and the expression level of hsa_circ_0001900 is related to the size of WT and the patients' prognosis, suggesting its possible role as a key driving gene in WT progression.
Humans ; RNA, Circular ; Wilms Tumor/pathology* ; Prognosis ; High-Throughput Nucleotide Sequencing ; Kidney Neoplasms/genetics* ; Sequence Analysis, RNA ; Male ; Female

The multidisciplinary diagnosis and treatment (MDT) mode plays a positive role in diagnosing and treating difficult and complicated diseases, improving service quality, and promoting the construction of an integrated medical system. In March 2023, Zhejiang Provincial People′s Hospital had launched the " 6 New" MDT management practice to further improve the hospital′s inpatient MDT service level, including new concepts, new architectures, new standards, new processes, new models, and new quality control. This practice had improved the quality and efficiency of MDT management and promoted interdisciplinary integration by strengthening top-level design, establishing MDT leadership and quality control teams, developing relevant standards and specifications, enhancing the level of information management throughout the entire process, implementing diversified MDT models, and implementing quality control measures. As of July 2024, the hospital had established 80 MDT teams covering 30 clinical departments, conduct 120 MDT discussions for hospitalized cases per month, and with the continuous promotion of inpatient MDT work, the number of MDT teams and cases continued to increase. This practice had achieved good results and could provide references for other public hospitals in China to improve the level of inpatient MDT services.

Objectives:To investigate the effect and mechanism of mitochondrial targeting peptide SS-31 on senescence and pyroptosis of nucleus pulposus(NP)cells induced by inflammation.Methods:NP cells were isolated and cultured in vitro from 6-week-old male SD rats,and were divided into control group,lipopolysaccharide(LPS)group,and LPS+SS-31 group;And the control group of cells received no treatment,LPS group of cells were treated with 100μg/mL LPS for 24h,and LPS+SS-31 group of cells were pre-treated with SS-31 30min before LPS intervention.The levels of cellular senescence and pyroptosis were assessed in each group using β-galactosidase(SA-β-Gal)staining and westem blot(WB)assay;Transmission electron mi-croscopy and oxygen consumption rate(OCR)assay were used to analyze the mitochondrial function of NP cells in each group;The expressions of phosphorylated nuclear factor-κB-p65(p-NF-κB-p65),cyclic GMP-AMP synthase(cGAS),and interferon stimulating gene(STING)in each group were detected by WB and im-munofluorescence;Hematoxylin-eosin(HE)staining,immunohistochemistry(IHC)staining and WB assay were performed to analyze the effects of in vivo injection of SS-31 on intervertebral disc degeneration,senescence,and pyroptosis.Results:The positive SA-β-Gal staining of NP cells,expression of senescence marker pro-teins(p53,p21),and pyroptosis-related molecules(NLRP3,Caspase-1 p20)were significantly lower in the LPS+SS-31 group compared with the LPS group(P＜0.05);Transmission electron microscopy(TEM)showed that the mitochondrial swelling of NP cells was round,the matrix was translucent,and the interstitial lumen of the mitochondrial cristae was dilated in the LPS group,while the mitochondrial swelling was subsided,and the morphology of mitochondria tended to be normalized in the LPS+SS-31 group.Meanwhile,the maximum respi-ration level of NP cells was significantly enhanced in the LPS+SS-31 group compared with the LPS group(P＜0.05).The results of WB and immunofluorescence showed that LPS significantly up-regulated the expression of p-NF-κB-p65,cGAS,and STING(P＜0.05),while SS-31 pretreatment significantly inhibited the expression of p-NF-κB-p65,cGAS and STING in NP cells induced by LPS(P＜0.05).Finally,in vivo experiments con-firmed that local injection of SS-31 reduced the senescence and pyroptosis of NP cells,and alleviated the degeneration of intervertebral disc induced by annulus fibropuncture.Conclusions:SS-31 alleviates inflamma-tion-induced senescence and pyroptosis of NP cells,which is related to the inhibition of NF-κB signaling pathway and cGAS/STING signaling pathway.

ObjectiveTo evaluate the clinical efficacy of Huayu Tongluo Formula （化瘀通络方， HTF） in patients with mid-stage diabetic kidney disease of blood stasis syndrome and explore its potential mechanisms. MethodsA multi-center， randomized， double-blind， placebo-controlled clinical trial was conducted. Ninety patients of mid-stage diabetic kidney disease of blood stasis syndrome were divided into a control group of 46 cases and a treatment group of 44 cases. Both groups received conventional western medicine treatment， the treatment group additionally taking HTF， while the control group taking a placebo of the formula. The treatment was administered once daily for 24 weeks. The primary outcomes included 24-hour urine total protein （24 h-UTP）， serum albumin （Alb）， glycated hemoglobin （HbA1c）， and serum creatinine （Scr）.The secondary outcomes included changes in levels of endothelin-1 （ET-1）， nitric oxide （NO）， vascular endothelial growth factor （VEGF）， and traditional Chinese medicine （TCM） syndrome scores before and after treatment. Clinical efficacy was evaluated based on TCM syndrome scores and overall disease outcomes. Adverse reactions and endpoint events were recorded. ResultsIn the treatment group after treatment， 24 h-UTP， ET-1， and VEGF levels significantly decreased （P＜0.05）， Alb and NO levels significantly increased （P＜0.05）； while the TCM syndrome scores for edema， lumbar pain， numbness of limbs， dark purple lips， dark purple tongue or purpura， and thin， rough pulse all significantly decreased （P＜0.05）. In the control group， no significant changes were observed in any of the indicators after treatment （P＞0.05）.Compared with the control group， the treatment group showed significant reductions in 24 h-UTP， ET-1， and VEGF levels， and increases in Alb and NO levels （P＜0.05）. The TCM syndrome scores for edema， lumbar pain， dark purple tongue or purpura， and thin， rough pulse were all lower in the treatment group than in the control group （P＜0.05）. The total effective rate of TCM syndrome in the treatment group was 59.09% （26/44）， and the overall clinical effective rate was 45.45% （20/44）. In the control group， these rates were 15.22% （7/46） and 8.7% （4/46）， respectively， with the treatment group showing significantly better outcomes （P＜0.05）. A total of 7 adverse events occurred across both groups， with no significant difference （P＞0.05）. No endpoint events occurred during the study. ConclusionOn the basis of conventional treatment of Western medicine， HTF can further reduce urinary protein levels and improve clinical symptoms in patients with mid-stage diabetic kidney disease of blood stasis syndrome. The mechanism may be related to its effects on endothelial function.

Objective:To investigate the reliability and validity of the China Nanjing consciousness recovery scale(CNCRS)in assessing patients with acute disorder of consciousness(DOC).Method:A total of 306 patients with acute DOC admitted to Nanjing Zijin Hospital between August 2023 and August 2024 were enrolled.Each patient was assessed using both the Glasgow Coma Scale(GCS)and CNCRS.The correlation between GCS and CNCRS scores were analyzed,and the reliability and validity of these two scales were compared.Result:The DOC degree of enrolled patients was severe,with a tracheotomy rate of 61.8%.The median GCS score was 7(5,10)for males and 6(5,9)for females,while the median CNCRS score was 3(1,8)for males and 3(1,7)for females.Scores from both scales were statistically significant correlated across gender groups(P<0.001).The Cron-bach's α coefficient of CNCRS(male 0.918,female 0.889)was higher than that of GCS(male 0.687,female 0.658),indicating superior internal consistency.The validity index Kaiser-Meyer-Olkin(KMO)value(male 0.891,female 0.857)was also higher than the GCS scale(male 0.600,female 0.602),demonstrating better construct validity.Conclusion:The CNCRS demonstrates good reliability and validity and can be effectively applied for the evalu-ation of patients with acute disturbance of consciousness.

Objective To explore the neuroprotective effects and mechanisms of asiaticoside(AS)in rats with transient cerebral ischemia.Methods One hundred male Sprague-Dawley(SD)rats were randomly divided into five groups:sham-operated(Sham)group,transient middle cerebral artery occlusion(tMCAO)group,and low-,medium-,and high-dose AS(AS-L,AS-M,AS-H)groups,with 20 rats in each group.Except for the Sham group,rats in the other four groups under-went tMCAO surgery.Rats in the AS-L,AS-M,and AS-H groups received intragastric administration of 20,40 and 80 mg/kg AS respectively,once daily for 7 days starting 1 hour post-surgery.Rats in the Sham and tMCAO groups received equivalent volumes of saline.Neurological deficit score,brain water content,and TTC staining were used to evaluate neurological impairment,cerebral edema,and infarct volume.HE staining and Nissl staining wereused to assess histopathological changes and neu-ronal damage.Autophagy was detected via transmission electron microscopy.Immunofluorescence staining was usedto analyze the expression and localization of microtubule-associated protein light chain 3B(LC3B).TUNEL staining was used to evaluate apoptosis,and Western blot was used to measure protein expression.Results Compared with the Sham group,rats in the tMCAO group ex-hibited significantly increased neurological deficit score,brain water content,infarct volume,and histopathological damage,as well as significantly decreased Nissl body counts(P＜0.05).AS dose-dependently reduced neurological deficits,brain water content,infarct volume,and histopatho-logical damage while increased Nissl body numbers.The tMCAO group showed significantly higher numbers of autophagosomes,lysosomes,and LC3B-positive cells,along with significantly elevated LC3-Ⅱ/LC3-Ⅰ,Beclin-1,Bax,cleaved caspase-3 compared to the Sham group;in contrast,p-PI3K,p-Akt,and p-mTOR protein levels,intact mitochondria count and p62 and Bcl-2 protein levels were significantly lower inthe tMCAO group(P＜0.05).Compared with the tMCAO group,AS treatment dose-dependently significantly decreased autophagosomes,lysosomes,LC3B-positive cells,and the expression of LC3-Ⅱ/LC3-Ⅰ,Beclin-1,Bax,cleaved caspase-3 while significantly increased p-PI3K,p-Akt,and p-mTOR protein,intact mitochondria and p62 and Bcl-2 levels(P＜0.05).Conclusion AS exerts neuroprotective effects by inhibiting excessive autophagy and apopto-sis in rats with transient cerebral ischemia via activation of the PI3K/Akt/mTOR signaling pathway.

Objective To analyze the distribution and drug resistance patterns of pathogenic bacteria in bile and blood cultures obtained from patients with biliary stones accompanied by acute biliary tract infection,to evaluate the clinical appropriate-ness of antibiotic use based on drug sensitivity results,and to provide evidence for empirical antibiotic treatment in such patients.Methods The clinical data of 161 patients with biliary calculi complicated by acute biliary tract infection who were admitted to the First People's Hospital of Neijiang from 2017 to 2023 were retrospectively analyzed.The results of microbial culture,drug sensitivity analysis,and patient characteristics were assessed to evaluate the appropriateness of clinical antimicrobial therapy.Results Among the 161 patients with positive cultures,212 strains of pathogenic bacteria were detected.The predominant patho-gens were Escherichia coli,Klebsiella pneumoniae subspecies,and Enterococcus faecium.Age and underlying diseases significantly affected the distribution of Escherichia coli and Klebsiella pneumoniae subspecies.Within the gram-negative bacterial group,Esche-richia coli and Klebsiella pneumoniae subspecies exhibited higher drug resistance to commonly used broad-spectrum penicillin,third-generation cephalosporin and quinolones but lower resistance rates to piperacillin and tazobactam;furthermore,elderly indi-viduals aged ≥65 years showed higher resistance rates to ceftriaxone than those under age 65 while people with drug exposure history had higher ceftazidime resistance rates that were statistically significant.In contrast to Enterococcus faecalis which displayed low antimicrobial resistance rates for most drugs tested in this study,Enterococcus faecium demonstrated high levels of antibiotic resistance;however,both Enterococcus faecalis and Enterococcus faecium exhibited zero-resistance rates against vancomycin and tigecycline although this may be attributed to their small sample size in our study cohort.Finally,we found that empirical anti-in-fective drugs,as well as target anti-infective drugs,were not prescribed rationally among these patients due mainly to inappropriate combinations of antibiotics or incorrect dosages.Conclusions The predominant pathogens in patients with acute biliary tract infection are gram-negative bacteria,Gram-positive bacteria,and fungi;however,the potential involvement of anaerobic bacteria should not be overlooked.Vancomycin exhibits sensitivity against gram-positive bacteria,yet the overall rationality of antibiotic usage remains suboptimal.Enhanced clinical testing for pathogenic microorganisms is imperative in the management of biliary stones accompanied by acute biliary tract infection.In contrast,clinical pharmacists should provide comprehensive training on anti-infective drugs to clinicians to facilitate their judicious selection of antibiotics based on drug sensitivity results and prevent the e-mergence of multidrug-resistant bacteria.