Objective: To identify an optimal back-flush solution for leukocyte-depleted filters that maximizes peripheral blood mononuclear cell (PBMC) recovery with high viability, long-term storage stability, and sterility of the harvested residues, thereby providing a clinically translatable strategy. Methods: Three sterile bag-packaged solutions—Saline, Solvent, and Hanks' balanced salt solution (HBSS)—were used to back-flush randomly assigned leukocyte-depleted filters. Nucleated cell recovery rate and viability of the harvested residues were compared. The optimal solution identified was applied to an expanded sample set. PBMC viability and yield were evaluated after 1h vs 48h storage of the residues. PBMCs isolated from the residues were cryopreserved in liquid nitrogen for 1 month, followed by post-thaw comparisons of viability and T-cell expansion capacity. Results: The Solvent group achieved the highest and most consistent nucleated cell recovery rate. Post-flush recovery rate from filters after 400 mL whole blood processing was (21.3±1.6)% for the Solvent group, significantly higher than Saline group (19.2±6.3)% and HBSS group (11.2±5.0)%, with residues from all groups maintaining viability >90%. No biologically significant difference in residue viability was observed between 48h vs 1h storage groups (93.3±2.3)% vs (95.7±1.8)%). PBMC recovery rates from residues showed no statistical difference between 48h vs 1h storage groups [(48.2%±9.5%)vs (40.41%±8.35%), P>0.05], with (17.7±2.6)×10 cells. After 1-month cryopreservation and 10-day expansion, PBMCs isolated from 48-hour-stored residues retained (91.2±3.2)% viability and achieved a (61.9±15.9)-fold expansion. Conclusion: The bag-packaged Solvent, as a back-flush solution, enables sterile acquisition of leukocyte-depleted filter residues through closed-system tubing connections. These residues maintained PBMC viability and recovery rates after 48h storage at 2℃-8℃, with post-cryopreservation (1-month liquid nitrogen) viability and expansion capacity remaining stable. This protocol complies with blood bank regulatory criteria, addresses the concerns about the infectious window period in cell therapy raw materials, and provides a clinically translatable strategy for PBMC-based applications.

OBJECTIVES: To characterize the biological properties of double-negative T (DNT) cells isolated from leukoreduction filter residues. METHODS: Leukoreduction filters containing residues from 400 mL whole blood units (n=6) were collected from a blood center. Filters were back-flushed with normal saline, and the eluate was concentrated to obtain leukoreduction filter residues. Leukocytes in the residues were counted by dual-fluorescence staining. DNT cells were then isolated from the residues using antibody-mediated adsorption and density gradient centrifugation. Both cryopreserved and fresh unstimulated DNT cells derived from the residues were subjected to in vitro culture. Following culture, cells were assessed for expansion fold, viability, immunophenotype, differentiation status, and cytotoxicity against target cells using dual-fluorescence staining and flow cytometry, with comparisons made to DNT cells derived from whole blood. RESULTS: The leukocyte recovery rate achieved through reverse flushing of the leukocyte reduction filter was (41.9±14.7)%. Compared to whole blood, the DNT cell starting material obtained from filter residues showed no significant difference in total T-cell content (P>0.05). However, the viability and purity of the resulting DNT cell starting materials were significantly lower (both P<0.05). After 17 days of culture, DNT cells from filter residues and whole blood showed no significant differences in expansion fold, immunophenotype, differentiation status, or cytotoxicity toward target cells (all P>0.05). However, the viability of DNT cells from residues was significantly lower than that of whole blood-derived DNT cells [(86.0±4.2)% vs. (92.2±1.2)%, P<0.05]. After thawing (post 3 or 15 days of cryopreservation) and 17 days of culture, DNT cell starting materials from residues showed comparable immunophenotype, expansion fold, and differentiation status to their non-cryopreserved counterparts from the same source (all P>0.05). However, the viability of DNT cells cryopreserved for 3 days [92.4% (91.8%, 92.8%)] and the cytotoxicity against target cells of those cryopreserved for 15 days [91.3% (89.4%, 95.1%)] were significantly higher than those of non-cryopreserved DNT cells [87.8% (82.0%, 89.0%) and 70.9% (67.3%, 80.2%), respectively] (P<0.05). CONCLUSIONS DNT cells derived from leukoreduction filter residues exhibited highly comparable characteristics to those from whole blood in terms of expansion, purity, differentiation, and biological potency. Furthermore, their biological activity post-cryopreservation and revival remained largely similar to non-cryopreserved cells. These findings suggest that leukoreduction filter residues represent a promising alternative source of starting material for manufacturing off-the-shelf, allogeneic DNT cell therapeutics.

Metformin has been demonstrated to attenuate hyperglycaemia by modulating the gut microbiota. However, the mechanisms through which the microbiome mediates metformin monotherapy failure (MMF) are unclear. Herein, in a prospective clinical cohort study of newly diagnosed type 2 diabetes mellitus (T2DM) patients treated with metformin monotherapy, metagenomic sequencing of faecal samples revealed that Phocaeicola vulgatus abundance was approximately 12 times higher in nonresponders than in responders. P. vulgatus rapidly hydrolysed taurine-conjugated bile acids, leading to ceramide accumulation and reversing the improvements in glucose intolerance conferred by metformin in high-fat diet-fed mice. Interestingly, C22:0 ceramide bound to mitochondrial fission factor to induce mitochondrial fragmentation and impair hepatic oxidative phosphorylation in P. vulgatus-colonized hyperglycaemic mice, which could be exacerbated by metformin. This work suggests that metformin may be unsuitable for P. vulgatus-rich T2DM patients and that clinicians should be aware of metformin toxicity to mitochondria. Suppressing P. vulgatus growth with cefaclor or improving mitochondrial function using adenosylcobalamin may represent simple, safe, effective therapeutic strategies for addressing MMF.

Although a single nucleotide polymorphism for N-acetyltransferase 10 (NAT10) has been identified in patients with early-onset stroke, the role of NAT10 in ischemic injury and the related underlying mechanisms remains elusive. Here, we provide evidence that NAT10, the only known RNA N4-acetylcytidine (ac4C) modification "writer", is increased in the damaged cortex of patients with acute ischemic stroke and the peri-infarct cortex of mice subjected to photothrombotic (PT) stroke. Pharmacological inhibition of NAT10 with remodelin on Days 3-7 post-stroke or astrocytic depletion of NAT10 via targeted virus attenuates ischemia-induced infarction and improves functional recovery in PT mice. Mechanistically, NAT10 enhances ac4C acetylation of the inflammatory cytokine tissue inhibitor of metalloproteinase 1 (Timp1) mRNA transcript, which increases TIMP1 expression and results in the accumulation of microtubule-associated protein 1 light chain 3 (LC3) and progression of astrocyte autophagy. These findings demonstrate that NAT10 regulates astrocyte autophagy by targeting Timp1 ac4C after stroke. This study highlights the critical role of ac4C in the regulation of astrocyte autophagy and proposes a promising strategy to improve post-stroke outcomes via NAT10 inhibition.

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

Synthetic biology is a crucial tool for the development of the bio-industry and bio-economy, representing a significant aspect of new quality productive forces. As a core course for graduate students in bioengineering, Synthetic Biology plays a vital role in ensuring the supply of essential talents for the development of the bio-industry in the new era. To better serve regional economic development and provide high-level talents for China's progress in the bio-industry, we analyzed typical issues encountered in the past teaching activities, set up a multi-disciplinary teaching team, optimized the course contents, adjusted the teaching mode, and mobilized students' learning interest. With the application of scientific research project as the starting point, we guided students to think and discuss deeply through the simulation of application writing and project defense, which improved students' critical thinking and innovative thinking. With industrialization as a focus, we explored a new training model combining production, education, and research through the joint practice base of the university and enterprises introduced typical cases of biomanufacturing to encourage students to engage in scientific research. The teaching reform significantly enhances the comprehensive abilities and national sentiments of graduate students. This paper hopes to serve as a reference for colleagues engaged in teaching in this field.
Synthetic Biology/education* ; Teaching ; China ; Humans

Objective:To investigate the expression and clinical significance of long noncoding RNA (lncRNA) AP006284.1 in bladder cancer tissues, and to analyze the regulatory effect and downstream mechanism of AP006284.1 knockdown on the proliferation and migration of bladder cancer cells. Methods:The expression of AP006284.1 in bladder cancer tissues, and the relationship between AP006284.1 expression and tumor stage and disease-free survival of bladder cancer patients were analyzed in the gene expression omnibus (GEO) database. AP006284.1 gene expression in bladder cancer cell lines, including MGH-U3, T24, UMUC-3, J82 and 5637, and bladder epithelial immortalized SV-HUC-1 cell were detected by real-time reverse transcription-PCR (RT-qPCR) assay. J82 cells were divided into the control group and the transfection group, and transfected with control plasmid and AP006284.1 knockdown plasmid, respectively. The effect of AP006284.1 knockdown on the proliferation of J82 cells was detected by RT-qPCR and cell counting kit-8 (CCK-8) assay. The effect of AP006284.1 knockdown on the migration of J82 cells was determined by the scratch healing assay. The target gene of AP006284.1 was predicted by LncRNA2Target and LncRNome databases. The target fragment of wild-type AP006284.1 ( AP006284.1-Wt) or mutant AP006284.1 ( AP006284.1-Mut) was constructed into pGL3 plasmid by dual luciferase gene reporter assay. J82 cells were co-transfected with miR-205-3p or miR-negtive control (miR-NC) to validate the targeting relationship between AP006284.1 and miR-205-3p. The correlation between miR-205-3p and AP006284.1 expression in bladder cancer tissues was further analyzed by the GEO database. The effect of AP006284.1 knockdown on the expression of miR-205-3p gene in J82 cells was detected by RT-qPCR assay. The effect of AP006284.1 knockdown on the expression of phosphorylated Ras protein (p-Ras), phosphorylated Raf protein (p-Raf), phosphorylated mitogen-activated protein kinase kinase (p-MEK), and phosphorylated extracellular signal-regulated kinase (p-ERK) of the ERK pathway was detected by Western blotting in J82 cells. Results:GEO database analysis showed that the relative expression of AP006284.1 in bladder cancer tissues ( n=304) was significantly higher than that in normal tissues ( n=28, P<0.01). The relative expression of AP006284.1 was positively correlated with the tumor stage of the bladder cancer patients ( P<0.01). Compared with bladder cancer patients with low expression of AP006284.1, patients with high expression had a lower disease-free survival ( P<0.01). Compared with the SV-HUC-1 cell (1.02±0.34), the expression level of AP006284.1 gene was upregulated in MGH-U3 cell (5.77±0.37), T24 cell (3.02±0.40), UMUC-3 cell (3.62±0.59), J82 cell (7.19±0.24) and 5637 cell (5.59±0.30) (all P<0.01). The expression level of the AP006284.1 gene was the highest in J82 cells, therefore, the J82 cells were selected for the study. The expression level of AP006284.1 gene in the control group (7.20±0.26) was 6.92 times higher than that in the transfection group (1.04±0.28, t=16.16, P<0.01). Compared with the control group (0.74±0.11, 1.35±0.09, 1.63±0.14, 1.74±0.11), the absorbance ( A) values of J82 cells in the transfection group (0.49±0.06, 0.95±0.14, 1.09±0.08, 1.13±0.11) were reduced than those in the control group at the 24, 36, 48 and 60 h after AP006284.1 knockdown (all P<0.05). The migration distance of J82 cells in the control group was significantly longer than that in the transfection group. The migration rate of the control group [(65.03±6.20)%], which was 2.58 times higher than that of the transfection group [(25.22±3.45)%, t=5.61, P<0.01]. The target site of miR-205-3p containing AP006284.1 was predicted by LncRNA2Target and LncRNome databases. Compared with miR-NC group (1.00±0.11), the relative activity of dual luciferase of AP006284.1-Wt gene was significantly downregulated in the miR-205-3p group (0.31±0.07, t=5.47, P<0.01). Compared with the miR-NC group (0.97±0.14), the relative activity of dual luciferase of AP006284.1-Mut vector (0.98±0.07) was not significantly change ( t=0.09, P>0.05). The GEO database analysis showed that the expression of AP006284.1 in bladder cancer tissues was negatively correlated with the expression of miR-205-3p ( P<0.01). The expression level of miR-205-3p gene in the transfection group (5.42±0.24) was 5.21 times higher than that in the control group (1.04±0.40, t=9.40, P<0.01). Compared with the control group (3.22±0.17, 5.56±0.19, 4.38±0.17, 5.74±0.36), the expressions of p-Ras (2.33±0.12), p-Raf (1.61±0.20), p-MEK (1.57±0.25), and p-ERK (2.40±0.28) of the ERK pathway were decreased in the transfected J82 cells (all P<0.01). Conclusions:AP006284.1 is highly expressed in bladder cancer tissues. Knockdown of AP006284.1 can inhibit the proliferation and migration of bladder cancer cells by regulating the miR-205-3p and ERK pathway proteins.

Objective:To establish an HPLC method to simultaneously determine the contents of 7 components, including Cardol triene, Cardol diene, (8'Z,11'Z,14'Z)-5-(Heptadeca-8',11',14'-trienyl)benzene-1,3-diol, Alkylresorcinol B, 5-(8'Z,11'Z-heptadecadienyl)-1,3-benzenediol, Adipostatin A, and 5-(11'Z-heptadecenyl)-resorcinol in Embelia laeta (L.) Mez. Methods:Chromatographic column was Arcus EP-C18(4.6 mm×250 mm, 5 μm); mobile phase was methanol-water (90∶10); column temperature was 25 ℃; flow rate was 1 ml/min; detection wavelength was 220 nm; the injection volume was 10 μl.Results:The 7 compounds in Embelia laeta (L.) Mez showed good linear relationships, which sample recovery rate ranges from 97.69%- 100.62%. The average contents of the 7 components from 9 origins were 3.099 9, 6.246 3, 9.942 8, 4.093 7, 2.180 3, 0.960 2, 1.855 9 mg/g. Conclusion:The established HPLC method for simultaneous determination of the contents of 7 components in Embelia laeta (L.) Mez is feasible, which can provide references for further development and utilization of Embelia laeta (L.) Mez.

Background Excessive radon exposure is considered the second risk factor for lung cancer. Since the opening of the subway in a city of Zhejiang Province, the exposure level of radioactive gas radon in subway stations and its impact on occupational health have become one of the important issues of public concern. Objective To monitor the radon concentration of subways in a city in Zhejiang Province and explore the effect of radon exposure on chromosome aberration and micronuclei in the working population. Methods A total of 55 vehicle control rooms of 55 stations affiliated to two different subway lines in a city were measured for one year; the 110 ticket offices and 55 security checkpoints from the same 55 stations were measured from 16 March to 14 June. The radon concentrations were compared by job types, subway lines, and seasons referring to Measurement methods for determination of radon in environmental air (HJ 1212-2021). Peripheral blood lymphocyte chromosome aberration and micronucleus analyses were conducted in 165 subway workers from monitoring sites for three different job types, then the influencing factors were analyzed. The detection methods were adopted from the standards of Test and assessment of chromosomal aberrations on occupational health examinations for radiation workers (GBZ/T 248-2014) and Standard for the method of micronucleus detection in lymphocytes on occupational health examination for radiation workers and exposure dose estimation (GBZ/T 328-2023). Results The radon concentration range of the target subways in Zhejiang Province was 10-320 Bq·m⁻³, all lower than the national limit (≤400 Bq·m⁻³). The differences in radon radioactivity levels among different lines, job types, and time segments were statistically significant (P<0.05). The rates of chromosomal aberration and micronucleus formation among the 165 subjects were 0.224% and 0.024%, respectively. There were significant differences in the rates of chromosome aberration and micronuclei among different jobs (vehicle control room, ticket office, security checkpoint) (P<0.05), but the abnormal rates were lower than the limits of the corresponding national standard. No significant correlation was found between jobs and chromosomal aberrations or micronuclei (P>0.05). Chromosome aberration and micronuclei varied by age, subway station seniority, and smoking (P<0.05). No effect of the above factors on chromosome aberration and micronuclei was observed by logistic regression (P>0.05). Conclusion The radon concentration in the target subway system is at a normal level. The rates of chromosomal aberration and micronucleus formation vary by jobs, but both are lower than the corresponding national limits. Therefore, radon exposure has not yet caused outstanding health impact on the subway workers.

Background Arsenic, cobalt, barium, and other individual metal exposure have been confirmed to be associated with the incidence of kidney stones. However, there are few studies on the association between mixed metal exposure and kidney stones, especially in occupational groups. Objective To investigate the association between mixed metal exposure and kidney stones in an occupational population from a metal smelting plant. Methods A questionnaire survey was conducted to collect sociodemographic characteristics, medical history, and lifestyle information of 1158 mixed metal-exposed workers in a metal smelting plant in Guangdong Province from July 2021 to January 2022. Midstream morning urine samples were collected from the workers, the concentrations of 18 metals including lithium, vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, strontium, molybdenum, cadmium, cesium, barium, tungsten, titanium, and lead were measured by inductively coupled plasma mass spectrometry, and the urinary mercury levels were measured by cold atomic absorption spectroscopy. Based on predetermined inclusion criteria, a total of 919 mixed metal-exposed workers were included in the study, including 117 workers in the kidney stone group and 802 workers in the non-kidney stone group. With a detection rate of urinary metals greater than 80% as entry criterion, 16 eligible metals were finally included for further analysis. Parametric or non-parametric methods were used to compare the differences between continuous or categorical variables of the non-kidney stone group and the kidney stone group. Logistic regression models were constructed to explore the association between individual metal exposures and kidney stones. Weighted quantile sum (WQS) regression models were used to evaluate the association between mixed metal exposure and kidney stones, as well as the weights of each metal on kidney stones. Then Bayesian kernel machine regression (BKMR) models were used to explore the overall effect of mixed metal exposure on renal calculi and the potential interactions between metals. Results We found that there were significant differences in sex, age, length of service, and body mass Index (BMI) between the non-kidney stone group and the kidney stone group (P<0.05). The urinary concentrations of molybdenum and barium in the kidney stone group were higher than those in the non-kidney stone group, and the differences were statistically significant (P<0.05). The logistic regression models demonstrated that urinary cobalt, arsenic, molybdenum, and barium were positively correlated with the risk of kidney stones (P_trend<0.05). The WQS regression models showed that the mixed exposure to vanadium, cobalt, arsenic, molybdenum, and barium was positively associated with the risk of kidney stones (P<0.05). Among them, molybdenum, arsenic, and barium accounted for 0.391, 0.337, and 0.154, respectively. The BKMR results revealed a positive association between metal mixture exposure and the risk of kidney stones (P<0.05). When other metals were fixed at the 25th, 50th, or 75th percentile, arsenic, molybdenum, cobalt, and barium exhibited significant positive effects on the risk of kidney stones (P<0.05), while vanadium showed a significant negative effect (P<0.05). The interaction analysis demonstrated interactions between barium and cobalt, as well as between vanadium and cobalt (P<0.05). Conclusion In the occupational population of this smelter, occupational mixed metal exposure could increase the risk of kidney stones, and the main metals are molybdenum, arsenic, barium, and cobalt.