ObjectiveAt present, the matching reagents of commercially available rapid DNA instruments based on microfluidics chip technology are autosome short tandem repeat (STR) individual identification reagents. The non-recombining part of the human Y chromosome is widely used in forensic DNA analysis, particularly in cases where standard autosomal DNA profile is uninformative. Y-STR loci are useful markers to identify males and male lineages in forensic practice. In order to achieve rapid and fully integrated detection ofY-STR loci, this study constructed the RTyper Y27 microfluidic chip rapid detection system and validated the performance of this system. MethodsThe system was verified and evaluated by sensitivity, success rate, typing accuracy, peak height balance, sizing precision and accuracy, mock case sample tests, mixture detection ability, and inhibition tolerance. ResultsComplete Y-STR profiles can be obtained when the template amount of DNA standard 9948 was ≥8 ng, the number of blood cards was ≥3 pieces, and the number of oral swab scrapings was≥7 times. The success rate of fully integrated detection was 91.52%, and the concordance rates was 99.74% for 165 testing samples. The success rate of 115 blood spots in these samples was 90.43%, with a typing accuracy of 99.65%, the success rate of 50 buccal swabs was 94%, with a typing accuracy of 99.92%. There was no significant difference in typing accuracy between blood spots and buccal swab samples. The peak height ratio between different fluorescence channels was 89.81%. The standard deviation of allelic ladder for 10 runs was within 0.5 bp. The size differences between allele and corresponding allele in allelic ladder was within 0.5 bp. The maximum precision CV values within and between batches were 0.48% and 0.68%, respectively, which were lower than 15%. These data indicate that the system has good accuracy and precision. The system was capable of accurately typing oral swabs, blood cards, saliva cards, cigarette butts, blood swabs and seminal stains. Complete Y-STR profiles can be obtained and distinguish at the 1∶3 ratio of minor and major contributors in artificial male DNA mixtures. Complete Y-STR genotyping can be obtained under the interference of inhibitors, such as different concentrations of humic acid (50-400 mg/L), indigotin (20-100 nmol/L) and hemoglobin (100-500 μmol/L). ConclusionIn this study, the RTyper Y27 microfluidic chip rapid amplification system is combined with the Quick TargSeq 1.0 integrated system, and the Y-STR profile can be obtained in approximately 2 h. Through a series of verification experiments, the results show that the system has good repeatability, accuracy and stability, can meet the on-site Y-STR detection requirements, and can be used in forensic practice.

Objective To investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]

OBJECTIVE: To explore whether casticin (CAS) suppresses stemness in cancer stem-like cells (CSLCs) obtained from human cervical cancer (CCSLCs) and the underlying mechanism. METHODS: Spheres from HeLa and CaSki cells were used as CCSLCs. DNA methyltransferase 1 (DNMT1) activity and mRNA levels, self-renewal capability (Nanog and Sox2), and cancer stem cell markers (CD133 and CD44), were detected by a colorimetric DNMT activity/inhibition assay kit, quantitative real-time reverse transcription-polymerase chain reaction, sphere and colony formation assays, and immunoblot, respectively. Knockdown and overexpression of DNMT1 by transfection with shRNA and cDNA, respectively, were performed to explore the mechanism for action of CAS (0, 10, 30, and 100 nmol/L). RESULTS: DNMT1 activity was increased in CCSLCs compared with HeLa and CaSki cells (P<0.05). In addition, HeLa-derived CCSLCs transfected with DNMT1 shRNA showed reduced sphere and colony formation abilities, and lower CD133, CD44, Nanog and Sox2 protein expressions (P<0.05). Conversely, overexpression of DNMT1 in HeLa cells exhibited the oppositive effects. Furthermore, CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in HeLa-derived CCSLCs (P<0.05). Interestingly, DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness. As expected, DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in HeLa cells. CONCLUSION CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation, suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.
Female ; Humans ; Cell Line, Tumor ; HeLa Cells ; Neoplastic Stem Cells/metabolism* ; RNA, Small Interfering/metabolism* ; Uterine Cervical Neoplasms/metabolism*

Aim To explore the key targets of d-borneol combined with eisplatin for sensitization of cisplatin-resistant NCSLC cells by RNA-Seq and verify its mechanism. Methods Cisplatin-resistant human large cell lung cancer cells (H460/CDDP) were inoculated into the right armpit of male BALB/c nude mice (4 weeks old) to construct a xenograft tumor model. Then they were randomly divided into control group, vehicle group, eisplatin group, and combination group (d-borneol + eisplatin) with 6 nude mice and treated for 14 d. After last administration of 24 h, the tumor tissue was taken for RNA-Seq. And then real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were used to verify the expression of cell cycle-related molecules. Results RNA-seq analysis showed that there were significant differences in gene expression between the eisplatin group and combined group, and they were significantly enriched in cell cycle. RT-PCR and IHC results showed that d-borneol combined with eisplatin could significantly inhibit the expressions of cyclins (cyclin A2, cyclin D3) and cyclin-dependent kinases (CDK2, CDK6) and promote the expression of its upstream molecular cyclin-dependent kinase inhibitor CD-KI (P21, P27) (P<0. 05, P<0.01). Conclusions d-Borneol increases the sensitivity of eisplatin by increasing the expression of P21 and P27 and inhibiting the expression of cyclinA2/D3 and CDK2/6 to induce cell cycle arrest and inhibit the malignant proliferation of H460/CDDP cells, thereby achieving the effect of anti-drug sensitization.

Objective: To analyze the clinical presentation and progression risk factors of patients with monoclonal gammopathy of undetermined significance (MGUS) in China. Methods: We retrospectively assessed the clinical features and disease progression of 1 037 patients with monoclonal gammopathy of undetermined significance between January 2004 and January 2022 at Peking Union Medical College Hospital. Results: A total of 1 037 patients were recruited in the study, including 636 males (63.6%) , with a median age of 58 (18-94) years. The median concentration of serum monoclonal protein was 2.7 (0-29.4) g/L. The monoclonal immunoglobulin type was IgG in 380 patients (59.7%) , IgA in 143 patients (22.5%) , IgM in 103 patients (16.2%) , IgD in 4 patients (0.6%) , and light chain in 6 patients (0.9%) . 171 patients (31.9%) had an abnormal serum-free light chain ratio (sFLCr) . According to the Mayo Clinic model for risk of progression, the proportion of patients in the low-risk, medium-low-risk, medium-high risk, and high-risk groups were 254 (59.5%) , 126 (29.5%) , 43 (10.1%) , and 4 (0.9%) , respectively. With a median follow-up of 47 (1-204) months, 34 of 795 patients (4.3%) had disease progression, and 22 (2.8%) died. The overall progression rate was 1.06 (0.99-1.13) /100 person-years. Patients with non-IgM MGUS have a markedly higher disease progression rate per 100 person-years than IgM-MGUS (2.87/100 person-years vs 0.99/100 person-years, P=0.002) . The disease progression rate per 100 person-years in non-IgM-MGUS patients of Mayo classification low-risk, medium-low risk and medium-high risk groups were 0.32 (0.25-0.39) /100 person-years, 1.82 (1.55-2.09) /100 person-years, and2.71 (1.93-3.49) /100 person-years, which had statistically difference (P=0.005) . Conclusion: In comparison to non-IgM-MGUS, IgM-MGUS has a greater risk of disease progression. The Mayo Clinic progression risk model applies to non-IgM-MGUS patients in China.
Male ; Humans ; Middle Aged ; Aged ; Aged, 80 and over ; Monoclonal Gammopathy of Undetermined Significance ; Retrospective Studies ; Risk Factors ; Immunoglobulin Light Chains ; Disease Progression

Humans ; Thyroid Neoplasms/pathology* ; Carcinoma, Papillary/pathology* ; World Health Organization ; Adenocarcinoma, Follicular/pathology*

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.
Isatis/genetics* ; Plant Proteins/metabolism* ; Phylogeny ; Arabidopsis/genetics* ; Flavonoids ; Cloning, Molecular

OBJECTIVE: To investigate the effect of miR-22 targeting formin-like protein 2 (FMNL2) on the migration and apoptosis of childhood acute myeloid leukemia (AML) cells. METHOD: Peripheral blood samples from 11 children with AML, 10 children with immune thrombocytopenia, human AML cell lines TF-1a, HL-60, THP-1 and human bone marrow stromal cells HS-5 were used as the research objects. UniCel DxH 800 automatic hematology analyzer detected platelet count, hemoglobin, and white blood cell count in peripheral blood samples, and RT-qPCR detected miR-22 expression in peripheral blood samples and AML cells. HL-60 cells were transfected with Lipofectamine^TM 2000 kit, the experiments were divided into seven groups: blank (no cells transfected), miR-NC, miR-22 mimics, si-NC, si-FMNL2 , miR-22 mimics+OE-NC and miR-22 mimics+OE-FMNL2 . RT-qPCR was used to detect the expression of miR-22 in each group. Transwell was used to detect cell migration. Flow cytometry was used to detect cell apoptosis. Dual-luciferase reporter gene detection experiments verified the targeting relationship between miR-22 and FMNL2 . Western blot was used to detect the expression of FMNL2 protein. RESULTS: Compared with the control group, the number of leukocytes in the peripheral blood of children with AML was significantly increased (P <0.001), while the concentration of hemoglobin and the number of platelets were significantly decreased P <0.001). The expression level of miR-22 in peripheral blood of children with AML was significantly lower than that in control group (P <0.001). Compared with HS-5 cells, the expression levels of miR-22 in TF-1a, HL-60, and THP-1 cells were significantly decreased (P <0.05), and in HL-60 cells was the lowest. Therefore, HL-60 cells were selected for subsequent experiments. Up-regulation of miR-22 or silencing of FMNL2 could reduce the number of migrating cells and increase apoptosis rate (P <0.05). MiR-22 targeted and negatively regulated the expression of FMNL2 . FMNL2 overexpression reversed the effects of up-regulated miR-22 on migration and apoptosis of HL-60 cells. CONCLUSION MiR-22 can inhibit the migration and promote apoptosis of HL-60 cells by down regulating the expression of FMNL2 .
Humans ; Child ; MicroRNAs/metabolism* ; Leukemia, Myeloid, Acute/metabolism* ; Cell Proliferation ; Apoptosis ; Myeloproliferative Disorders ; Cell Movement ; Hemoglobins ; Cell Line, Tumor ; Formins

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death among urban and rural residents in China, and elevated low-density lipoprotein cholesterol (LDL-C) is a risk factor for ASCVD. Considering the increasing burden of ASCVD, lipid management is of the utmost importance. In recent years, research on blood lipids has made breakthroughs around the world, hence a revision of China guidelines for lipid management is imperative, especially since the target lipid levels in the general population vary in respect to the risk of ASCVD. The level of LDL-C, which can be regarded as appropriate in a population without frisk factors, can be considered abnormal in people at high risk of developing ASCVD. As a result, the "Guidelines for the prevention and treatment of dyslipidemia" were adapted into the "China Guidelines for Lipid Management" (henceforth referred to as the new guidelines) by an Experts' committee after careful deliberation. The new guidelines still recommend LDL-C as the primary target for lipid control, with CVD risk stratification to determine its target value. These guidelines recommend that moderate intensity statin therapy in adjunct with a heart-healthy lifestyle, be used as an initial line of treatment, followed by cholesterol absorption inhibitors or/and proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, as necessary. The new guidelines provide guidance for lipid management across various age groups, from children to the elderly. The aim of these guidelines is to comprehensively improve the management of lipids and promote the prevention and treatment of ASCVD by guiding clinical practice.

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.
Humans ; Salvia miltiorrhiza/metabolism* ; Biosynthetic Pathways ; Quinones/metabolism* ; Plant Roots/metabolism* ; Gene Expression Regulation, Plant