[Objective] To explore the feasibility of using whole blood as a source of NK cells for allogeneic CAR NK cell therapy and activated NK cell reinfusion therapy, and initially construct a technical system for the separation and purification of NK cells from whole blood. [Methods] All peripheral blood mononuclear cells (PBMCs) were enriched from 400 mL of whole blood by manual separation and machine separation, respectively. The erythrocyte loss rate, PBMCs number, NK cell purity of the two methods were compared. NK cells were sorted from PBMCs by three separation and enrichment methods as immunomagnetic bead negative selection method, platelet lysate culture expansion and PERCOLL density gradient separation method, and the purity and yield of NK cells, the activity of NK cells and the tumor-killing ability of the three separation and enrichment methods were compared. [Results] The proportion of NK cells in the lymphocyte population was higher in the manual separation method than in the machine separation method[(13.16±5.16)% vs (8.56±3.92)%, P<0.05]; the number PBMCs was lower in the manual separation method than in the machine separation method[(4.09±1.80)×10⁸vs (6.49±2.16)×10⁸, P<0.05], and there was no difference in the red blood cell loss between the two methods (P>0.05). The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomagnetic was (96.77±2.31)%; the yield was (56.27±10.47)%; the inhibition of tumor proliferation was (38.67±14.05)%; and the tumor killing rate was (19.90±8.05)%. The purity of NK cells isolated and enriched from PBMCs by manual separation method using platelet lysis culture expansion method was the highest at day 7, which was (54.84±15.80)%; the cell expansion multiple could reach 16.92±6.28 at day 7; the in vitro tumor killing rate of NK cells was (15.83±5.5)%; the tumor inhibition rate was (44.33±13.5)%; and there was no difference in the toxicity and activity of NK cells between the two methods (P>0.05). The purity of NK cells isolated and enriched by PERCOLL density gradient separation method was (15.83±5.82)%, and the yield was (14±6.25)%, which was significantly lower than the other two methods. [Conclusion] PBMCs isolated from whole blood by manual separation and NK cells enriched by negative selection with immunomagnetic beads have the potential to provide NK cell materials for CAR-NK cell therapy, and NK cells enriched by platelet lysate-conditioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.

Objective To establish a method for determination of pyriclobenzuron (PBU) residues in rice and paddy environments, and to determine the residual amounts and observe the degradation dynamics of PBU. Methods In July 2022, the paddies of Zhejiang Academy of Agricultural Sciences were selected as experimental fields, and were divided into the blank control group (no pesticide application), the 1-fold-concentration pesticide group (1 kg/667 m²), and the 5-fold-concentration pesticide group (5 kg/667 m²), with a 100 m² area in each group. At the early tillering stage of rice, 20% suspension of PBU sulfate was sprayed once in the 1-fold-concentration and 5-fold-concentration pesticide groups, and rice plants, paddy water and soil samples were collected 2 h, and 1, 2, 3, 5, 7, 11, 14, 21, 28, 35, 49 d and 63 d following spraying PBU, while rice straw, field soil, brown rice and rice husk samples were collected 98 d following spraying. PBU was extracted and purified in samples using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) pretreatment technique, and the PBU contents were determined in samples using ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The solvent standard working solution and matrix standard working solution were prepared. A linear regression equation was fitted between PBU concentration (x-axis) and peak area (y-axis), and the ratio of the slope (k) of the matrix standard curve to the slope (K) of the solvent standard curve was calculated to evaluate the matrix effect of PBU in samples. According to the Guidelines for Pesticide Residue Testing in Crops (NY/T 788—2018), the addition levels of PBU were set at 0.005, 0.050, 5.000, 1 000.000 mg/kg in rice plants, 0.005, 0.050, 2.000, 10.000 mg/kg in paddy water, 0.005, 0.050, 2.000 mg/kg in soil, and 0.005, 0.050, 5.000 mg/kg in brown rice and rice husks. The recovery and relative standard deviation (RSD) of PBU addition were calculated to evaluate the effectiveness of UPLC-MS/MS for determination of PBU contents. The first-order kinetic equation of PBU concentration was fitted in samples at different sampling time points to analyze the trends in PBU degradation in rice plants, paddy water, and soil, and the half-life of PBU was calculated in different samples. Results There was a good linear relationship between the mass concentration and peak area of PBU at concentrations of 0.000 1 to 0.020 0 mg/kg under solvent and matrix conditions (R² = 0.985 8 to 0.999 7, t = -0.47 to 1.62, all P values < 0.01). The matrix effects of PBU were 70.26%, 65.42% and 65.12% in rice plants, brown rice and rice husks, indicating a matrix-inhibitory effect, and the matrix effect was 87.06% in soils, indicating a weak matrix effect. The recovery of PBU addition was 77.61% to 100.12% in different samples, with RSD of 1.43% to 6.74%, and a limit of quantification (LOQ) of 0.005 mg/kg, and the addition recovery and RSD met the requirements of the Guidelines for Pesticide Residue Testing in Crops (NY/T 788—2018), validating the effectiveness of UPLC-MS/MS assay. Following spraying PBU at a dose of 1 kg/667 m², the half-life of PBU was 6.24 d in rice plants and 3.43 d in paddy water samples, respectively. The final residues of PBU were lower than the LOQ of 0.005 mg/kg in brown rice and rice husk samples 98 d following spraying PBU. Following spraying PBU at a dose of 5 kg/667 m², the half-life of PBU was 15.75 d in rice plants and 7.62 d in paddy water samples, respectively. The final residue of PBU was lower than the LOQ of 0.005 mg/kg in brown rice 98 d following spraying PBU, and the final residue of PBU was 0.049 mg/kg in rice husks. Conclusions A simple, and highly accurate and precise UPLC-MS/MS assay has been developed for determination of PBU residues in rice plants and paddy environments through extraction and purification of PBU from matrix samples using QuEChERS pretreatment. After spraying PBU in paddies, the concentration of PBU gradually decreases in rice plants and paddy water over time, and the final residual concentration is low.

BACKGROUND: Several randomized controlled studies have suggested that the prophylactic use of proton pump inhibitors (PPIs) in intensive care unit (ICU) patients could not reduce the incidence of gastrointestinal bleeding (GIB) and may increase adverse events such as intestinal infection and pneumonia. Gut microbiota may play a critical role in the process. PPIs have been widely prescribed for GIB prophylaxis in patients with acute coronary syndrome (ACS). This study aimed to determine the short-term effects of PPI and histamine-2 receptor antagonist (H2RA) treatment on gut microbiota of ACS patients. METHODS: The study was designed as a single-blind, multicenter, three-parallel-arm, randomized controlled trial conducted at three centers in Beijing, China. We enrolled ACS patients at low-to-medium risk of GIB and randomized (2:2:1) them to either PPI ( n = 40), H2RA ( n = 31), or control group ( n = 21). The primary outcomes were the alterations in gut microbiota after 7 days of acid suppressant therapy. Stool samples were collected at baseline and 7 days and analyzed by 16S ribosomal RNA (rRNA) gene sequencing. RESULTS: There were no significant changes in the diversity of gut microbiota after the short-term use of acid suppressants, but the abundance of Fusobacterium significantly increased and that of Bifidobacterium significantly decreased, especially in PPI users. In addition, the abundance of some pathogenic bacteria, including Enterococcus and Desulfovibrio, was significantly elevated in the PPI users. The fecal microbiota of the PPI users included more arachidonic acid metabolism than that of control group. CONCLUSIONS: PPIs may increase the risk of infection by adversely altering gut microbiota and elevating arachidonic acid metabolism, which may produce multiple proinflammatory mediators. For ACS patients at low-to-medium risk of GIB, sufficient caution should be paid when acid-suppressant drugs are prescribed, especially PPIs. REGISTRATION www.chictr.org.cn (ChiCTR2000029552).
Humans ; Proton Pump Inhibitors/therapeutic use* ; Acute Coronary Syndrome/microbiology* ; Female ; Gastrointestinal Microbiome/drug effects* ; Male ; Middle Aged ; Histamine H2 Antagonists/therapeutic use* ; Aged ; Single-Blind Method

OBJECTIVES: The risk factors and role of mother‒child gut microbiota in pediatric inflammatory bowel disease (PIBD) remain unclear. We aimed to explore the clinical risk factors associated with PIBD, analyze the characteristics of gut microbiota of children and their mothers, and examine the correlation of the microbial composition in mother‒child pairs. METHODS: We conducted a case-control study including children with PIBD and their mothers as the case group, as well as healthy children and their mothers as the control group. Questionnaires were used to collect information such as family illness history and maternal and early-life events. Fecal samples were collected from the children and mothers for microbiota 16S ribosomal RNA (rRNA) sequencing to analyze the composition and its potential association with PIBD. RESULTS: A total of 54 pairs of cases and 122 pairs of controls were recruited. A family history of autoimmune disease and antibiotic use during pregnancy were associated with an increased risk of PIBD, and a higher education level of the father was associated with a decreased risk of PIBD. Children with PIBD and mothers exhibited different gut microbiota compared to healthy children and mothers. Similarities were observed in the gut microbiota of mothers and children in the same groups. Some bacterial biomarkers of mothers discovered in this study had the power to predict PIBD in their offspring. CONCLUSIONS PIBD is influenced by maternal risk factors and has unique gut microbiota characteristics. The mother‒child gut microbiota is closely related, suggesting the transmission and influence of the gut microbiota between mothers and children. This study highlights the potential pathogenesis of PIBD and provides a basis for developing targeted interventions.
Humans ; Gastrointestinal Microbiome ; Female ; Risk Factors ; Case-Control Studies ; Male ; Child ; Inflammatory Bowel Diseases/etiology* ; Adult ; RNA, Ribosomal, 16S/genetics* ; Feces/microbiology* ; Mothers ; Pregnancy ; Child, Preschool

Acute respiratory distress syndrome (ARDS) is a common respiratory emergency, but current clinical treatment remains at the level of symptomatic support and there is a lack of effective targeted treatment measures. Our previous study confirmed that inhalation of hydrogen gas can reduce the acute lung injury of ARDS, but the application of hydrogen has flammable and explosive safety concerns. Drinking hydrogen-rich liquid or inhaling hydrogen gas has been shown to play an important role in scavenging reactive oxygen species and maintaining mitochondrial quality control balance, thus improving ARDS in patients and animal models. Coral calcium hydrogenation (CCH) is a new solid molecular hydrogen carrier prepared from coral calcium (CC). Whether and how CCH affects acute lung injury in ARDS remains unstudied. In this study, we observed the therapeutic effect of CCH on lipopolysaccharide (LPS) induced acute lung injury in ARDS mice. The survival rate of mice treated with CCH and hydrogen inhalation was found to be comparable, demonstrating a significant improvement compared to the untreated ARDS model group. CCH treatment significantly reduced pulmonary hemorrhage and edema, and improved pulmonary function and local microcirculation in ARDS mice. CCH promoted mitochondrial peripheral division in the early course of ARDS by activating mitochondrial thioredoxin 2 (Trx2), improved lung mitochondrial dysfunction induced by LPS, and reduced oxidative stress damage. The results indicate that CCH is a highly efficient hydrogen-rich agent that can attenuate acute lung injury of ARDS by improving the mitochondrial function through Trx2 activation.

Objective To investigate the correlation between the expression of hsa_circ_0001785 in triple negative breast cancer(TNBC)and the efficacy of neoadjuvant chemotherapy(NAC).Methods A total of 129 patients with triple negative breast cancer who were admitted to our hospital from October 2021 to February 2023 were regarded as the study group,and 125 patients with benign breast lesions who underwent surgery in our hospital were served as the control group.The influencing factors of NAC efficacy in TNBC patients were analyzed by multivariate logistic regression model;receiver operating characteristic(ROC)curve was applied to analyze the predictive value of hsa_circ_0001785 level for NAC efficacy in triple negative breast cancer patients.Results Compared with the control group(1.05±0.16),the expression level of hsa_circ_0001785 in the study group(2.47±0.39)increased(P＜0.05);compared with the effective group(2.34±0.35),the expression level of hsa_circ_0001785 in the ineffective group(3.48±0.56)increased(P＜0.05);hsa_circ_0001785 was highly expressed in triple negative breast cancer patients with tumor diameter＞2 cm,lymph node metastasis and high tissue grade(P＜0.05);high expression of hsa_circ_0001785,tumor diameter＞2 cm,occurrence of lymph node metastasis and high histological grade were risk factors for NAC efficacy in triple negative breast cancer patients(P＜0.05).Hsa_circ_0001785 level has certain predictive value for NAC efficacy in TNBC patients.Conclusion Hsa_circ_0001785 is highly expressed in triple negative breast cancer,and the level of hsa_circ_0001785 has a certain predictive value for the efficacy of NAC in patients.

Objective To investigate the effect and potential mechanism of Desmodium renifolium(Linn.)Schindl on ovariectomized osteoporosis rat model.Methods Sixty 3-month-old female SD rats were randomly divided into Sham group,model group,Xianlin Gubao group(0.24 g·kg-1),Desmodium renifolium low-dose group(1.35 g·kg-1,medium-dose group(2.7 g·kg-1)and high-dose group(5.4 g·kg-1).Osteoporosis was induced by performing double ovariectomy in rats.After 14 weeks treatment,the contents of osteocalcin(BGP),osteoprotectin(OPG)and alkaline phosphatase(ALP)in serum were determined.Hematoxylin-eosin(HE)staining was used to observe the changes of bone trabeculae.The osteoclast progenitor-cell line RAW264.7 was divided into negative control group,Receptor activator of nuclear factor-κB ligand(RANKL)group,Xianlin Gubao group,Desmodium renifolium low-dose,medium-dose and high-dose drug groups.The RANKL group and drug groups were induced with 50 ng·mL-1 RANKL.Meanwhile,Xianlin Gubao group and different concentrations of drug-containing serum of Desmodium renifolium,were added for intervention.Tartrate-resistant acid phosphatase(TRAP)staining was performed 10 days after intervention to observe the differentiation of osteoclasts,and quantitative real-time PCR(qPCR)was used to determine the mRNA expression.Results Compared with Sham group,the contents of OPG in serum of model group were significantly decreased(P<0.01),while ALP and BGP were significantly increased(P<0.01),bone trabeculae were significantly reduced,broken,sparsely arranged,and the space between bone trabeculae was large,the OPG mRNA expression in model group were significantly decreased(P<0.01),and the mRNA expression of receptor activator of nuclear factor-κB ligand(RANKL),TNF receptor associated factor 6(TRAF6),nuclear factor of activated t-cells,cytoplasmic 1(NFATC1),cathepsin K(CTK)and calcitonin receptor(CALCR)in model group were significantly increased(P<0.01),all these aspects showed remarkable improvement after Desmodium renifolium intervention.After 10 days of RAW264.7 culture,no osteoclasts were found in the Control group.Compared with the negative control group,osteoclasts in RANKL group were significantly increased,treatment with Desmodium renifolium markedly decreased osteoclast number,the result of RANKL/RANK/OPG signaling mRNA expression were consistent with the animal experiments.Conclusion Desmodium renifolium exerts effects on osteoporosis in ovariectomized rats,its mechanism might be realized by inhibiting osteoclast proliferation and differentiation,and regulating OPG/RANKL/RANK signaling pathway.

Objective To analyze the MTHFR C677T gene polymorphism and homocysteine in women in early pregnancy in Ordos region,to clarify the distribution characteristics of MTHFR C677T gene polymor-phism and the correlation between the two,and to provide genetic basis for scientific guidance on folic acid supplementation during pregnancy and prevention of birth defects.Methods A total of 602 Han women in early pregnancy who were registered and underwent early pregnancy examinations in the gynecology clinic of Ordos Central Hospital from September 2022 to September 2023 were selected as the research subjects.Blood samples were collected from all research objects.MTHFR C677T gene polymorphism was detected by using PCR chip hybridization,and homocysteine level was detected by biochemical enzyme circulation method.Sta-tistical analysis of MTHFR C677T locus genotype and allele frequency,as well as their correlation with homo-cysteine was conducted.Results The detection frequencies of MTHFR C677T gene polymorphism CC,CT,and TT types were 23.6%,47.5%,and 28.9%,respectively.The detection frequencies of alleles C and T were 47.3%and 52.7%,respectively.There were statistically significant differences compared to Han women in Shanghai,Wenzhou,Meishan,Nanning,and other regions(P<0.05),but there was no statistically significant difference compared to Han women in Xi'an(P>0.05).The serum homocysteine level of pregnant women with TT genotype was higher than that of pregnant women with CC and CT genotypes,while the serum ho-mocysteine level of pregnant women with CT genotype was higher than that of pregnant women with CC gen-otype(P<0.05).The CT and TT genotypes of MTHFR C677T were both risk factors for hyperhomocys-teinemia in women in early pregnancy in this region,the risk was 2.80 and 8.07 times higher than that of the CC genotype,respectively,and the differences were statistically significant(P<0.05).Conclusion The distri-bution of MTHFR C677T gene polymorphism Han women in early pregnancy in Ordos region has regional characteristics and is correlated with homocysteine level.Developing personalized folic acid supplementation plans based on different genotypes during pregnancy is of great significance for preventing birth defects.

Objective To investigate the clinical manifestations,molecular genetics and gonadal pathol-ogy characteristics of patients with disorders of sex development(DSD),and to summarize the clinical experi-ence of identifying rare diseases from common symptoms.Methods The clinical data of 416 patients with DSD diagnosed and treated in the multidisciplinary center of DSD of Guangzhou Women and Children's Medical Cen-ter from May 2018 to August 2023 were retrospectively analyzed,summarized and discussed.Results Accord-ing to chromosome karyotype,416 cases of DSD were classified into three types:92 cases(22.1％)of abnormal sex chromosome karyotype,285 cases(68.5％)of 46,XY karyotype and 39 cases(9.4％)of 46,XX karyotype.Among the 92 patients with abnormal sex chromosome karyotype,59 cases were raised as males,18 cases(30.5％)complained of short penis with hypospadias and cryptorchidism.The most common karyotype was 45,X/46,XY(58 cases,63.0％).Among the 285 patients with 46,XY karyotype,238 cases were raised as males,and 63 cases(26.5％)complained of short penis and hypospadias;47 cases were raised as females,and 13 ca-ses(27.7％)complained of inguinal mass.A total of 216 patients with 46,XY karyotype were subjected to whole exome gene detection,and 155 cases(71.8％)were found to have molecular pathogenesis with the clinical phe-notype.Among the 39 patients with 46,XX karyotype,19 cases were raised as males,and 8 cases(42.1％)com-plained of short penis and hypospadias.In the 18 cases of gonad biopsy,17 cases showed testicular tissue in go-nads.Whole exome sequencing was performed in 14 cases.NR5A1 gene heterozygous mutation,SRY gene muta-tion and SOX3 gene mutation were found in 2 cases,respectively(14.3％).Twenty cases were raised as females,and 14 cases(70.0％)complained of clitoral hypertrophy.Gonad biopsy was performed in 8 cases,with 7 cases of ovotestis(87.5％)and 1 case of NR5A1 gene heterozygous mutation(14.3％).Conclusions The etiologies of DSD are complex and diverse,and the clinical manifestations are various,which can be manifested as hypospa-dias,micropenis,cryptorchidism and other common symptoms of the urinary system.Different etiologies have dif-ferent treatment options.Therefore,chromosome karyotype,molecular genetic testing and gonadal pathology can be used to clarify the cause of disease,especially for rare diseases,improve the detection rate,reduce the rate of missed diagnosis,and ensure reasonable treatment,especially sex selection.