Objective To explore the distribution and localization of dopamine receptor D3-D5 in the small intestine of different species.Methods The distribution and expression of D3-D5 in the small intestine of mice,rats and rhesus monkeys were detected by immunohistochemistry and Western blotting.The expression of D3-D5 in immunoglobulin A positive plasma cells(IgA+PC)located in the lamina propria(LP)were detected by immunofluorescence double labeling.Results D3 and D5 were widely distributed in the epithelium,LP,submucosal plexus(SMP)and intermuscular plexus(MP)of the small intestine in mice,rats and rhesus monkeys.The distribution of D4 in the small intestinal of mice and rhesus monkeys were consistent with the result of D3 and D5.D4 was distributed only within the epithelium and LP of rat small intestine.D3 and D5 were expressed in the IgA+PC in the LP of mice and rats,whereas D4 was not.Conclusion The distribution and localization pattern of D3 and D5 are similar in the small intestine of mice,rats and rhesus monkeys,whereas those of D4 vary between different species.Dopamine may be involved in regulating the functions of IgA+PC.

Objective To investigate the impact of iron overload on apoptosis in primary mouse liver cells via a synchronous separation technology.Methods Hepatocyte(HC),liver sinusoidal endothelial cell(LSEC),and Kupffer cell(KC)were isolated and purified with collagenase,percoll density gradient centrifugation,and CD146 magnetic beads.Cell types were identified using flow cytometry and immunofluorescence staining.Cells of different types were cultured in vitro,and an iron overload model was established by treating the mice with 0,25,50 and 100 μmol/L ferric ammonium citrate(FAC)for 24 h.The iron content was quantified using Prussian blue staining,while cell viability and mitochondrial membrane potential were assessed by flow cytometry.Results The synchronous separation technology of primary liver cells exhibited stable efficiency.The yield of HC was(4.0±0.5)×107 cells per mouse,exhibiting an effective survival rate of(76.33±0.67)％.The yield of LSEC was(5.0±1.0)×106 cells per mouse,with a survival rate of(93.63±0.25)％and a purity level of(93.40±0.46)％.The yield of KC was(1.5±0.5)×106 cells per mouse while a high survival rate of(98.33±0.12)％and a purity level of(88.30±2.02)％were maintained.The obtained cells were large in number,with good vitality and high purity,which could meet the requirements of subsequent experiments.Treatment with FAC significantly elevated iron contents in different types of cells when compared with the control group.Upon stimulation of FAC,the survival rate of HC decreased from(73.97±5.54)％to(54.10±1.68)％,the mean fluorescence intensity of JC-1 aggregates decreased from 326.33±30.37 to 155.00±6.56,JC-1 monomer increased from 1700.00±1 44.04 to 3713.33±81.82.The survival rate of LSEC decreased from(90.60±1.74)％to(78.03±2.15)％,the mean fluorescence intensity of JC-1 aggregates decreased from 502.33±5.51 to 372.33±4.04,and JC-1 monomer increased from 750.00±67.51 to 1340.00±36.39.The survival rate of KC decreased from(94.23±1.44)％to(88.37±1.56)％,the mean fluorescence intensity of JC-1 aggregates decreased from 652.67±25.66 to 478.00±12.49,and JC-1 monomer increased from 1984.33±80.65 to 3062.33±245.20.Conclusion A robust and reliable simultaneous isolation technique of primary mouse HC,LSEC,and KC has been established.Moreover,our finding demonstrates that iron overload significantly enhances apoptosis levels in HC,LSEC and KC.

Objective To evaluate the preventive effects of Saccharomyces boulardii powder and tetragenous viable Bifidobacterium tablets on antibiotic-associated diarrhea(AAD)in infants and young children.Methods Children under three years old admitted to the Department of Pediatrics,Affiliated Hospital of Xuzhou Medical University due to non-gastrointestinal infections and requiring antibiotic treatment from July to December 2023 were enrolled.The children were randomly divided into a control group(n=47),a Saccharomyces boulardii group(n=70),and a Bifidobacterium group(n=65)using a random number table method.The control group received antibiotics and symptomatic supportive treatment according to relevant clinical guidelines.In addition to the treatment given to the control group,the Saccharomyces boulardii group and the Bifidobacterium group were respectively administered with Saccharomyces boulardii powder and tetragenous viable Bifidobacterium tablets.Based on the duration of probiotic use(7 days,14 days,and 21 days),the Saccharomyces boulardii group was further divided into 7 d,14 d,and 21 d subgroups,and similarly for the Bifidobacterium group.The incidence of AAD and ratio of cocci to bacilli in feces were compared among the groups after treatment.Results The incidence rate of AAD in both the Saccharomyces boulardii group and the Bifidobacterium group was lower than that in the control group(P<0.017).The duration of AAD and the length of hospital stay were shorter in the Saccharomyces boulardii and Bifidobacterium groups compared to the control group(P<0.05).In the control group,the ratio of cocci to bacilli in feces on days 7,14,and 21 was higher than on day 1(P<0.05).Within-group comparisons showed that the ratio of cocci to bacilli in feces on day 14 in the Bifidobacterium 14 d and 21 d groups were lower than on day 1(P<0.05);and the ratios on day 14 in the control group,Saccharomyces boulardii 14 d group,Saccharomyces boulardii 21 d group,Bifidobacterium 14 d group,and Bifidobacterium 21 d group were lower than on day 7(P<0.05).The ratios on day 21 in the control group and the Saccharomyces boulardii 21 d group were lower than on days 7 and 14(P<0.05).Between-group comparisons indicated that on day 7,the ratios of cocci to bacilli in feces in the Saccharomyces boulardii 7 d,14 d,21 d groups,and Bifidobacterium 7 d,14 d,21 d groups were all lower than in the control group(P<0.05);on day 14,the ratios of cocci to bacilli in feces 14 d and 21 d groups were lower than in the control group and the Bifidobacterium 7 d group(P<0.05).Conclusions Both Saccharomyces boulardii and tetragenous viable Bifidobacterium can effectively improve gut microbiota and prevent the occurrence of AAD in infants and young children.Compared to short-term treatment,appropriately extending the duration of probiotic therapy can further improve the structure of gut microbiota.

Objective A comparison of two method of establishing pressure ulcer rat models to determine which is the most suitable for experimental use.Methods 18 male SD rats were randomly divided into control(n=6),model A(n=6)and model B(n=6)groups.In the control group,iodophor treatment was given after hair removal at the simulated modeling site.In model group A,longitudinal compression was performed by simple deep-tissue foreign body implantation.In model group B,transverse compression was performed via the magnet compression method.The times required to complete the process and for each stage of pressure ulcer model establishment in each group were recorded.The general condition of the rats was observed,and the modeling rate,mortality rate,and infection rate were compared.Results By naked eye,we observed that the model A and model B groups gradually developed redness and swelling,ulceration,bleeding,exudation,and necrosis.Comparison of the whole time to produce pressure uler between model A and model B groups:the difference between the two groups was statitically significant(P＜0.05).Comparison of the time to produce pressure injury between Model A and Model B:The difference between the two groups at stage Ⅰ was not statistically significant(P＞0.05);the difference between the two groups at stage Ⅱ was statistically significant(P＜0 05);the difference between the two groups at stage Ⅲ was statistically significant(P＜0 05);the difference between the two groups at stage Ⅳ was statistically significant(P＜0 05).The mental and sports scores of the rats in the control group were significantly different from those in the model A and model B groups(P＜0.05).The general state of rats in the model group A was significantly different from that in the model B group,and coat color was dimer and activity decreased in the model group A.The modelling rate of rats in both model A and model B groups was 100％.The mortality and infection rates of the model group A were higher than those of the model group B,which were 33.34％and 16.70％,respectively.Conclusions Successful preparation of a four-stage model of pressure ulers in both modalities.The two method have both commonalities and distinct characteristics.The magnet compression method required less time,the rats were generally in good condition,and the mortality and infection rates were low;thus it is suitable for short-term intervention research.The simple deep-tissue foreign body implantation method took longer,required rats to have a certain level of tolerance,had high infection and mortality rates,and is more suitable for use for long-term observations of pressure ulcers.

Aim To screen and study the effects of Tao Hong Si Wu decoction(THSWD)-mediated treat-ment on circular RNA(circRNA)expression profiles in rats with middle cerebral artery occlusion(MCAO),and investigate the possible roles and molecular mecha-nisms of THSWD.Methods Next-generation RNA sequencing was conducted to identify circRNA expres-sion profiles in MCAO rats after treatment with THSWD and compared with the MCAO model group and control group.Bioinformatics analysis was performed to predict the potential target microRNAs and mRNAs.Gene On-tology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses for the potential target mRNAs were applied to explore the potential roles of differentially expressed circRNAs.RT-qPCR was performed to verify circRNAs with significant differences in expression.Results We identified 87 significantly differentially expressed circRNAs between the MCAO group versus the control group,and 86 sig-nificantly differentially expressed circRNAs between the MCAO group versus the THSWD group.respective-ly.Among them,17 circRNAs induced by the MCAO model were reversed via treatment with THSWD.To demonstrate the roles of mRNAs targeted by DECs,the GO and KEGG databases were used.Further analysis revealed that five circRNAs may play important roles in the development of MCAO.Conclusions The com-prehensive expression profile of circRNAs in rats with middle cerebral artery occlusion after THSWD treat-ment is determined for the first time,suggesting that the therapeutic effect of THSWD on MCAO may be a-chieved by regulating the expression of circRNAs.

Objective: To identify the risk factors in mortality of pediatric acute respiratory distress syndrome (PARDS) in pediatric intensive care unit (PICU). Methods: Second analysis of the data collected in the "efficacy of pulmonary surfactant (PS) in the treatment of children with moderate to severe PARDS" program. Retrospective case summary of the risk factors of mortality of children with moderate to severe PARDS who admitted in 14 participating tertiary PICU between December 2016 to December 2021. Differences in general condition, underlying diseases, oxygenation index, and mechanical ventilation were compared after the group was divided by survival at PICU discharge. When comparing between groups, the Mann-Whitney U test was used for measurement data, and the chi-square test was used for counting data. Receiver Operating Characteristic (ROC) curves were used to assess the accuracy of oxygen index (OI) in predicting mortality. Multivariate Logistic regression analysis was used to identify the risk factors for mortality. Results: Among 101 children with moderate to severe PARDS, 63 (62.4%) were males, 38 (37.6%) were females, aged (12±8) months. There were 23 cases in the non-survival group and 78 cases in the survival group. The combined rates of underlying diseases (52.2% (12/23) vs. 29.5% (23/78), χ²=4.04, P=0.045) and immune deficiency (30.4% (7/23) vs. 11.5% (9/78), χ²=4.76, P=0.029) in non-survival patients were significantly higher than those in survival patients, while the use of pulmonary surfactant (PS) was significantly lower (8.7% (2/23) vs. 41.0% (32/78), χ²=8.31, P=0.004). No significant differences existed in age, sex, pediatric critical illness score, etiology of PARDS, mechanical ventilation mode and fluid balance within 72 h (all P>0.05). OI on the first day (11.9(8.3, 17.1) vs.15.5(11.7, 23.0)), the second day (10.1(7.6, 16.6) vs.14.8(9.3, 26.2)) and the third day (9.2(6.6, 16.6) vs. 16.7(11.2, 31.4)) after PARDS identified were all higher in non-survival group compared to survival group (Z=-2.70, -2.52, -3.79 respectively, all P<0.05), and the improvement of OI in non-survival group was worse (0.03(-0.32, 0.31) vs. 0.32(-0.02, 0.56), Z=-2.49, P=0.013). ROC curve analysis showed that the OI on the thind day was more appropriate in predicting in-hospital mortality (area under the curve= 0.76, standard error 0.05,95%CI 0.65-0.87,P<0.001). When OI was set at 11.1, the sensitivity was 78.3% (95%CI 58.1%-90.3%), and the specificity was 60.3% (95%CI 49.2%-70.4%). Multivariate Logistic regression analysis showed that after adjusting for age, sex, pediatric critical illness score and fluid load within 72 h, no use of PS (OR=11.26, 95%CI 2.19-57.95, P=0.004), OI value on the third day (OR=7.93, 95%CI 1.51-41.69, P=0.014), and companied with immunodeficiency (OR=4.72, 95%CI 1.17-19.02, P=0.029) were independent risk factors for mortality in children with PARDS. Conclusions: The mortality of patients with moderate to severe PARDS is high, and immunodeficiency, no use of PS and OI on the third day after PARDS identified are the independent risk factors related to mortality. The OI on the third day after PARDS identified could be used to predict mortality.
Female ; Male ; Humans ; Child, Preschool ; Infant ; Child ; Critical Illness ; Pulmonary Surfactants/therapeutic use* ; Retrospective Studies ; Risk Factors ; Respiratory Distress Syndrome/therapy*

OBJECTIVE: To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method. METHODS: All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology. RESULTS: A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database. CONCLUSION The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.
Humans ; Alleles ; Polymerase Chain Reaction ; Genotype ; High-Throughput Nucleotide Sequencing ; Histocompatibility Testing/methods* ; Technology

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.
Panax/genetics* ; Promoter Regions, Genetic ; Agrobacterium tumefaciens/genetics* ; Computational Biology ; Cloning, Molecular

BACKGROUND: Clinical outcomes are poor if patients with acute heart failure (AHF) are discharged with residual congestion in the presence of renal dysfunction. However, there is no single indication to reflect the combined effects of the two related pathophysiological processes. We, therefore, proposed an indicator, congestion and renal index (CRI), and examined the associations between the CRI and one-year outcomes and the incremental prognostic value of CRI compared with the established scoring systems in a multicenter prospective cohort of AHF. METHODS: We enrolled AHF patients and calculated the ratio of thoracic fluid content index divided by estimated glomerular filtration rate before discharge, as CRI. Then we examined the associations between CRI and one-year outcomes. RESULTS: A total of 944 patients were included in the analysis (mean age 63.3 ± 13.8 years, 39.3% women). Compared with patients with CRI ≤ 0.59 mL/min per kΩ, those with CRI > 0.59 mL/min per kΩ had higher risks of cardiovascular death or HF hospitalization (HR = 1.56 [1.13-2.15]) and all-cause death or all-cause hospitalization (HR = 1.33 [1.01-1.74]). CRI had an incremental prognostic value compared with the established scoring system. CONCLUSIONS In patients with AHF, CRI is independently associated with the risk of death or hospitalization within one year, and improves the risk stratification of the established risk models.

Background: and Purpose Orthostatic hypotension (OH) is common in patients with Parkinson’s disease (PD). Early recognition OH is required with sensitive assessments. The purpose of this study was to determine whether blood pressure (BP) changes during exercise can predict the occurrence of OH in PD. Methods: This prospective cohort study included 80 consecutive patients with PD. All patients agreed to participate in a baseline evaluation and cardiopulmonary exercise test (CPET).According to the initial active standing test (AST), those without OH (PD-nonOH) at baseline had their AST results followed up for 6 months. The main outcome was defined as whether patients without OH at baseline would develop OH after 6 months. Logistic regression analysis was applied to identify the relevant variables. A nomogram was constructed based on clinical features and identified variables. The concordance index (C-index) and area under the receiver operating characteristic curve (AUC) were used to evaluate the accuracy and predictive ability of the nomogram, respectively. Results: CPET results indicated that peak load, peak heart rate, heart rate recovery at 1 min, and systolic BP change (ΔSBP) were lower in those with OH than in the PD-nonOH group (p<0.05) at baseline. Logistic regression analysis indicated that peak load and ΔSBP during CPET had significant effects on OH (p<0.05). Age, sex, peak load, and ΔSBP were used to construct the nomogram model (C-index=0.761). The prediction model had an AUC of 0.782 (95% confidence interval=0.649–0.889) and a specificity and sensitivity of 70.0% and 81.8%, respectively. Conclusions This study has identified predictive factors for OH development in patients with PD. CPET could be used as a complementary examination to identify patients at a high risk of OH.