Objective Utilizing a specially engineered miR-144-GFP-H1 human embryonic stem cell(hESC)reporter line,this study leverages GFP fluorescence as an indicator of miR-144 expression to gauge the progression of erythropoiesis.The investigation is aimed at elucidating the potential roles of lncRNAs within the erythropoietic framework and conducting an initial assessment of their functional impact.Methods The miR-144/451-GFP-H1 cell line(hereafter referred to as 144-H1)was utilized for in vitro erythrocyte induction culture.The subpopulations of cells entering the erythropoiesis stage were characterized by the surface molecules CD71 and GPA.The GFP reporter gene of miR-144 served as a critical determi-nant to distinguish between GFP-positive cells(with a high propensity for erythropoiesis)and GFP-negative cells(with a low propensity for erythropoiesis).Transcriptome sequencing was performed on both groups to identify differentially ex-pressed long non-coding RNAs(lncRNAs).LncRNA entries with potential for validation were selected for preliminary func-tional verification.The CRISPR/Cas9 gene editing technique was employed to design functional interference strategies for the targeted lncRNAs,obtaining 144-H1 cell lines with knocked-out function of the specific lncRNAs.These knockout cell lines,along with non-knockout 144-H1 cell lines,were used for parallel erythrocyte induction culture to identify differential nodes.This approach preliminarily verified their impact on erythropoiesis in an in vitro development model.Results 1)The constructed 144-H1 cell line was capable of expressing GFP fluorescence upon entering the stage of in vitro erythrocyte in-duction,indicating the activation of miR-144/451.2)Within the CD71,GPA double-positive group,significant differences in lncRNA expression were observed between the GFP-positive and GFP-negative subpopulations.3)Gene editing strategies involving the deletion of sequence segments capable of effectively interfering with the function of multiple lncRNA entries were designed and verified for successful editing.In the knockout cell lines,parts of the lncRNA sequences were directly de-leted.4)In parallel validation experiments of erythrocyte induction culture,cell lines with LINC01569 knocked out exhibited significant differences in flow cytometric subpopulations and cell proliferation capabilities compared to the non-knockout cell lines:①The knockout cell lines showed sustained high expression of GFP fluorescence.②The proportion of the CD71-GPA double-positive group in the knockout cell lines continuously decreased during erythrocyte maturation.③No significant ex-pression of hemoglobin was observed in the knockout cell lines,lacking the characteristic red color.④The cell proliferation capability of the knockout cell lines was significantly lower than that of the non-knockout cell lines(P＜0.05).Conclusion The successful employment of the 144-H1 cell line facilitated an exploration into the potential functions of lncRNAs in e-rythropoiesis.This enables the design of more refined in vitro developmental experiments to enhance the precision in captu-ring lncRNA functions.Among the differentially expressed lncRNA entries,LINC01569 was preliminarily validated to play a regulatory role in erythropoiesis.The functional absence of LINC01569 severely impacts the normal differentiation and prolif-eration of erythrocytes.The specific regulatory mechanism of LINC01569 in erythropoiesis warrants further investigation and research.

Cholesterol is an important precursor of many endogenous molecules. Disruption of cholesterol homeostasis can cause many pathological changes, leading to liver and cardiovascular diseases. CYP1A is widely involved in cholesterol metabolic network, but its exact function has not been fully elucidated. Here, we aim to explore how CYP1A regulates cholesterol homeostasis. Our data showed that CYP1A1/2 knockout (KO) rats presented cholesterol deposition in blood and liver. The serum levels of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and total cholesterol were significantly increased in KO rats. Further studies found that the lipogenesis pathway (LXRα-SREBP1-SCD1) of KO rats was activated, and the key protein of cholesterol ester hydrolysis (CES1) was inhibited. Importantly, lansoprazole can significantly alleviate rat hepatic lipid deposition in hypercholesterolemia models by inducing CYP1A. Our findings reveal the role of CYP1A as a potential regulator of cholesterol homeostasis and provide a new perspective for the treatment of hypercholesterolemia.

Objective:To investigate the urinary virology and clinical characteristics of female overactive bladder (OAB) patients.Methods:Catheterized urine samples were collected from 55 women with OAB and 18 control individuals between January 2021 and August 2021. Inclusion criteria were: female with age>18, diagnosed as OAB, OABSS total score≥3 and item Urgency score≥2, informed consent signed. Exclusion criteria were: Urine culture positive, urinary catheter indwelling status, antibiotic usage in recent 30 days, other disease leading to OAB-like symptoms, pelvic organ prolapse and current pregnancy, immunosuppressive therapy or status. Clinical characteristic and history were collected. OAB symptoms were assessed via both OABSS (overactive bladder symptom score) and OAB-V8 (8-item overactive bladder questionnaire). The urine specimens were analyzed using mNGS for identifying viral infections. The correlation between the disease and JC virus infection was analyzed by t test, chi-square test, binary logistic regression analysis and Spearman correlation matrix, and the Nomogram map for predicting the risk of viral infection was constructed. Results:In total, 55 women with OAB and 18 healthy controls were recruited in the study. There are significant difference in terms of UTI history, pelvic surgery history and the habit of holding urine [60.0%(n=33)to 16.7%(n=3), P=0.002; 43.6%(n=24)to 0.0%(n=0), P<0.01; 36.4%( n=20)to 5.6%( n=1), P=0.015]. Based on mNGS results, OAB patients were identified with more positive viral infection [47.3%(n=26)to 33.3%(n=6)] and more JC virus infection. In the OAB group, subtype 7B of JCV ( n=8) was identified, while in the control group, subtype 7A(n=2) was identified. Pairwise Spearman correlation analysis indicated high correlations between viral infection and OABSS ( r=0.58), age and pausimenia ( r=0.68), hypertension and age ( r=0.53), respectively. Estimates from binary logistic regression model indicated risk factors for virus infection in OAB patients including age ( OR=1.99, 95% CI 0.02-2.61), holding urine habit( OR=2.16, 95% CI 0.18-3.85) and pelvic surgery ( OR=2.53, 95% CI 0.54-4.27). Conclusions:Urinary viral infections appear to be associated with more severe OAB symptoms and JC virus may be a potential therapeutic target for OAB.

How to improve the long-term prognosis of transplant kidney and solve the shortage of donor kidney are still two major problems that plague clinicians. Among them, ischemia-reperfusion injury (IRI), rejection, infection, and immunosuppressive therapy are important issues in the research field of renal transplantation. Therefore, strengthening the literature study in the field of renal transplantation and understanding the nature of transplant kidney related diseases and international frontier research hotspots, help to further improve the function and prolong the survival time of the transplant kidney in clinic. This article interpreted literatures on the research hotspots and new progress in the field of renal transplantation in the third quarter of 2020, combined with the meeting minutes of the 12th Lingnan Reading Club, and reviewed from the three aspects of IRI, rejection and infection.

Objective To investigate the application value of Multi-Latex polygranular technique joint detection of kidney injury-related urinary microproteins in noninvasive diagnosis after renal transplantation. Methods Clinical data of 72 recipients undergoing renal transplantation were retrospectively analyzed. According to the level of serum creatinine (Scr), the recipients were divided into normal renal function group (group A, n=14), mild kidney injury (group B, n=37), and severe kidney injury group (group C, n=21). 20 healthy volunteers were selected as the healthy control group (HC group). The contents of urinary retinol binding protein (RBP), microalbumin (mAlb), IgG, transferrin (TRF), α₁-microglobulin (MG), and β₂-MG of subjects in each group were detected using the Multi-Latex polygranular technique. The correlation between urinary microproteins and Scr, blood urea nitrogen (BUN) was analyzed. The differences of urinary microproteins in each group were compared. And the diagnostic value of single and joint detection of urinary microproteins was evaluated. Results Six kinds of urinary microproteins in HC group and group A were significantly lower than those in group B and group C, and six kinds of urinary microproteins in group B were significantly lower than those in group C (all P < 0.01). Six kinds of urinary microproteins in renal transplant recipients were positively correlated with BUN. RBP, mAlb, α₁-MG, and β₂-MG were positively correlated with Scr. The correlations were statistically significant (P < 0.001-0.05). The diagnostic value of joint detection of urinary microproteins is better than the detection of single index, among which TRF+mAlb+RBP+α₁-MG quadruple detection had the highest diagnostic value. Conclusions Six kinds of urinary microproteins can be used as specific indicators to reflect graft renal function. The polygranular technique can simultaneously detect its contents and achieve noninvasive diagnosis. The diagnosis based on TRF+mAlb+RBP+α₁-MG quadruple detection is expected to further improve the noninvasive diagnosis system after renal transplantation.

Objective To explore the variation trend of natural killer (NK) cell subsets in the recipients infected with cytomegalovirus (CMV) after renal transplantation. Methods Clinical data of 92 renal transplant recipients were retrospectively analyzed. All recipients were divided into the CMV infection group (n=43), CMV infection recovery group (n=13), stable renal function group (n=15), rejection group (n=11) and other infection group (n=10). In addition, healthy adult volunteers were enrolled in the healthy control group (n=15). The proportion of NK cells in peripheral blood, the expression proportion and the mean fluorescence intensity (MFI) of CD226 and CD16 in NK cells were observed and statistically compared among different groups. Results The proportion of NK cells was 4.9% (2.2%, 11.5%) in the CMV infection group and 3.7% (2.3%, 6.5%) in the CMV infection recovery group, which were significantly lower than those in the other groups (all P < 0.05). The expression proportion of CD226 and CD16 in NK cells in the CMV infection group was significantly lower compared with those in the healthy control group and stable renal function group(all P < 0.05). The expression proportion of CD226 and CD16 in NK cells in the CMV infection recovery group was remarkably higher than those in the CMV infection group (both P < 0.05). The MFI of CD226 and CD16 in the CMV infection group was significantly lower than those in the healthy control group (both P < 0.05). The MFI of CD226 and CD16 in the CMV infection recovery group was significantly higher than those in the CMV infection group (both P < 0.05). Conclusions The expression proportion and MFI of CD226 and CD16 in NK cells are down-regulated in CMV infection period, whereas up-regulated during the CMV infection recovery period, prompting that CD226 and CD16 expressed by NK cells are intimately correlated with the course of CMV infection.

Objective: To explore the effect of insulin-like growth factor-Ⅰ (IGF-Ⅰ) receptor on radiosensitivity of HepG2 cells and the underlying mechanism. Methods: HepG2 cells were divided into the following groups: negative control group, siRNA group, irradiation group and combined group. HepG2 cells were transfected with IGF-Ⅰ receptor siRNA combined with irradiation therapy to investigate the effect on cell proliferation by methyl thiazolyl tetrazolium and cell cycle using flow cytometry. Expression of IGF-Ⅰ receptor, proliferating cell nuclear antigen (PCNA), cyclin-dependent kinases 1(CDK1) and Survivin were detected using Western blotting and Q-PCR. Results: The expression of IGF-Ⅰ receptor in HepG2 cells was decreased significantly after siRNA transfection compared with the control group. After the combinational therapy, cell viability was decreased significantly according with control group [(1.02±0.08) vs. (1.08± 0.10) vs. (0.60±0.07)]; In addition, cell cycle was arrested in G2/M[(20.3±0.3)% vs. (22.6±0.4)% vs. (34.7±0.5)%] and CDK1 expression was reduced significantly. The relative expression of Survivin in siRNA group was lower than negative control group, the difference was statistically significant (P<0.05). Conclusion Inhibition of IGF-Ⅰ receptor can enhance the radiosensitivity of HepG2 cells through cell cycle arrest.

Objective To investigate the clinical characteristics of DCD donor-derived CRKP infection and bleeding in kidney transplantation,and to summarize the experience of diagnosis,treatment and prevention.Methods A retrospective analysis was carried out from July 2016 to December 2017 in hospital,containing clinical data of 4 cases of CRKP-infected DCD donors and 7 cases of kidney transplantation recipients.Results In the CRKP culture of 4 cases of DCD donors,1 case was positive for blood culture,1 case was positive for urine culture,1 case was positive for sputum culture,and 1 case was negative for blood,urine and sputum culture.The corresponding 7 recipients were all positive for blood culture after renal transplantation,4 cases were positive for urine culture,3 cases were positive for sputum culture,and 5 cases were positive for perirenal drainage.Of the 7 patients,4 cases had renal artery hemorrhage,1 of them was died.The average bleeding time was 17.75 days after operation (14-19 days).In 7 patients with renal transplantation,CRP increasd.And in 3 cases of deaths,CRP was stably higher than normal.Meanwhile,CRP in 4 surviving patients gradually decreased to the normal range after effective anti-infection treatment.All 7 patients were treated with carbapenems;2 patients were dead without avibactam therapy;and 5 cases were treated with avibactam and carbapenems and survived,1 case died and 1 case had good renal function recovery.Conclusion Positive CRKP in blood,urine and sputum of DCD donors can lead to CRKP infection in kidney transplant recipients.Even if the body fluids of donors are all negative,the false negative results could not be excluded.Persistent or increased high-level CRP after operation is an early warning on CRKP infection.And CRP can be used as an indicator for evaluating the effectiveness of anti CRKP therapy.The combination of avibactam and carbapenem antibiotics is an effective regimen in the treatment of DCD donor-derived CRKP.

Objective:To establish the HPLC specific chromatogram and provide comprehensive evaluation of Xanthii fructus from different regions.Methods:The HPLC analysis was performed on an Alltima C18 column (250 mm ×4.6 mm,5μm) with acetonitrile-0.1％phosphoric acid solution as the mobile phase with gradient elution at a flow rate of 0.8 ml· min-1 .The detection wavelength was 278 nm.The column temperature was 25℃.The software"Similarity Evaluation System for Chromatographic Fingerprint of TCMs"was employed to carry out the similarity analysis of the samples from Henan , Jilin, Anhui and the other regions .Results:The specific chromatogram was preliminarily constructed and 5 common peaks with chlorogenic acid as the reference were identified .Conclusion:The method is scientific basis of the quality assessment of Xanthii fructus with convenient and reliable properties .

OBJECTIVETo investigate the value of single nucleotide polymorphism array (SNP array) for the identification of de novo mutations in the DMD gene among fetuses.

METHODSG-banded karyotyping and SNP array were performed on a fetus with intrauterine growth restriction but without family history of Duchenne/Becker muscular dystrophy (DMD/BMD). Multiplex ligation-dependent probe amplification (MLPA) was subsequently applied on amniocytes and maternal peripheral blood sample to detect DMD gene deletion/duplication mutations.

RESULTSKaryotyping of amniocytes showed a normal 46, XY karyotype. SNP array on amniocytes detected a 116 kb deletion (chrX: 32 455 741-32 571 504) at Xp21.1 with breakpoints at introns 16 and 30 respectively, encompassing exons 17-29 of the DMD gene. In addition, MLPA analysis of the DMD gene on amniocytes confirmed the deletion of exons 17 to 29 identified by SNP array. However, no deletion/duplication mutation was detected by MLPA in the mother.

CONCLUSIONThe de novo deletion of exons 17 to 29 of the DMD gene detected in the fetus may result in BMD or DMD. SNP array can improve the efficiency for detecting genomic disorders in fetuses with unidentified pathogenic genes, negative family history and nonspecific phenotypes.

Adult ; Dystrophin ; genetics ; Exons ; genetics ; Female ; Fetus ; abnormalities ; Gene Deletion ; Humans ; Muscular Dystrophy, Duchenne ; genetics ; Phenotype ; Polymorphism, Single Nucleotide ; genetics ; Pregnancy