Enhancer of zeste homolog 2(EZH2)is a histone methyltransferase It mediates trimethylation of lysine 27 on histone H3,thereby facilitating the epigenetic silencing of downstream genes.In conjunc-tion with SUZ12,EED,and other components,it constitutes the polycomb repressive complex 2(PRC2)complex.While EZH2 is intricately involved in cellular proliferation and cardiac development,the chan-ges in its expression during cardiac terminal differentiation remain elusive.In this study,we employed differential gene expression analysis of embryonic and adult myocardial cells using the GEO database,and found that EZH2 is highly expressed in embryonic myocardium,but is present at very low levels in adult myocardium(P＜0.0001).Conversely,the expression changes of PRC2 members SUZ12 and EED are not as pronounced.Online analysis through the Tabula Muris database indicates that under physiological conditions,various cell subpopulations in the adult mouse heart exhibit negligible expression of EZH2.Immunohistochemical staining of mouse cardiac tissues shows that EZH2 is highly expressed in embryonic and neonatal myocardium but declines progressively from the first day after birth(P＜0.0001),becoming almost undetectable by the third day.Western blotting further confirms the rapid disappearance of EZH2 expression post-birth(P＜0.05),with EZH1 compensating for the downregulation of EZH2 to maintain H3K27me3 modification levels.Additionally,using the P19 teratocarcinoma stem cell model for cardio-myocyte differentiation,it is observed that EZH2 is significantly upregulated during the transition from cardiac progenitor cells to spontaneously beating cardiomyocytes,correlating with the expression of the cardiomyocyte transcription factor Gata4(P＜0.01).Targeted degradation of EZH2 using the small mole-cule drug MS1943 significantly inhibits the proliferation of induced cardiomyocytes,as evidenced by 5-e-thynyl-2'-deoxyuridine(EdU)incorporation assays(P＜0.01),and RT-qPCR reveals a marked in-crease in the expression of the proliferation inhibitor CDKN1A(P＜0.01).In summary,the high expres-sion of EZH2 in embryonic myocardial cells is associated with enhanced cell proliferation.The rapid loss of EZH2 expression postnatally correlates with the loss of proliferative capacity in cardiomyocytes,mark-ing it as a key indicator of cardiac terminal differentiation.

The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose-catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16 ℃ in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni-NTA affinity column.Size-exclusion chromatography and SDS-PAGE analysis demonstrated that the puri-fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD+as a coenzyme to NADP+.The optimal temperature and pH of the TeXDH were 40 ℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26％of the initial enzyme activity,while the divalent metal ions Mg2+or Ca2+could recover the enzyme activity of TeXDH,reaching 103.32％and 110.69％of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn2+,Ni2+,Cu2+,Zn2+,Co2+,and Cd2+significantly inhibited the activity of TeXDH.ICP-MS and molecular doc-king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn2+ions and 1 mol/L Mg2+ion.Further-more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.

Enhancer of zeste homolog 2(EZH2)is a histone methyltransferase It mediates trimethylation of lysine 27 on histone H3,thereby facilitating the epigenetic silencing of downstream genes.In conjunc-tion with SUZ12,EED,and other components,it constitutes the polycomb repressive complex 2(PRC2)complex.While EZH2 is intricately involved in cellular proliferation and cardiac development,the chan-ges in its expression during cardiac terminal differentiation remain elusive.In this study,we employed differential gene expression analysis of embryonic and adult myocardial cells using the GEO database,and found that EZH2 is highly expressed in embryonic myocardium,but is present at very low levels in adult myocardium(P＜0.0001).Conversely,the expression changes of PRC2 members SUZ12 and EED are not as pronounced.Online analysis through the Tabula Muris database indicates that under physiological conditions,various cell subpopulations in the adult mouse heart exhibit negligible expression of EZH2.Immunohistochemical staining of mouse cardiac tissues shows that EZH2 is highly expressed in embryonic and neonatal myocardium but declines progressively from the first day after birth(P＜0.0001),becoming almost undetectable by the third day.Western blotting further confirms the rapid disappearance of EZH2 expression post-birth(P＜0.05),with EZH1 compensating for the downregulation of EZH2 to maintain H3K27me3 modification levels.Additionally,using the P19 teratocarcinoma stem cell model for cardio-myocyte differentiation,it is observed that EZH2 is significantly upregulated during the transition from cardiac progenitor cells to spontaneously beating cardiomyocytes,correlating with the expression of the cardiomyocyte transcription factor Gata4(P＜0.01).Targeted degradation of EZH2 using the small mole-cule drug MS1943 significantly inhibits the proliferation of induced cardiomyocytes,as evidenced by 5-e-thynyl-2'-deoxyuridine(EdU)incorporation assays(P＜0.01),and RT-qPCR reveals a marked in-crease in the expression of the proliferation inhibitor CDKN1A(P＜0.01).In summary,the high expres-sion of EZH2 in embryonic myocardial cells is associated with enhanced cell proliferation.The rapid loss of EZH2 expression postnatally correlates with the loss of proliferative capacity in cardiomyocytes,mark-ing it as a key indicator of cardiac terminal differentiation.

The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose-catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16 ℃ in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni-NTA affinity column.Size-exclusion chromatography and SDS-PAGE analysis demonstrated that the puri-fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD+as a coenzyme to NADP+.The optimal temperature and pH of the TeXDH were 40 ℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26％of the initial enzyme activity,while the divalent metal ions Mg2+or Ca2+could recover the enzyme activity of TeXDH,reaching 103.32％and 110.69％of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn2+,Ni2+,Cu2+,Zn2+,Co2+,and Cd2+significantly inhibited the activity of TeXDH.ICP-MS and molecular doc-king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn2+ions and 1 mol/L Mg2+ion.Further-more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.

Nonobstructive azoospermia (NOA), one of the most severe types of male infertility, etiology often remains unclear in most cases. Therefore, this study aimed to detect four biallelic detrimental variants (0.5%) in the minichromosome maintenance domain containing 2 ( MCMDC2 ) genes in 768 NOA patients by whole-exome sequencing (WES). Hematoxylin and eosin (H&E) demonstrated that MCMDC2 deleterious variants caused meiotic arrest in three patients (c.1360G>T, c.1956G>T, and c.685C>T) and hypospermatogenesis in one patient (c.94G>T), as further confirmed through immunofluorescence (IF) staining. The single-cell RNA sequencing data indicated that MCMDC2 was substantially expressed during spermatogenesis. The variants were confirmed as deleterious and responsible for patient infertility through bioinformatics and in vitro experimental analyses. The results revealed four MCMDC2 variants related to NOA, which contributes to the current perception of the function of MCMDC2 in male fertility and presents new perspectives on the genetic etiology of NOA.
Humans ; Male ; Azoospermia/genetics* ; Meiosis/genetics* ; Spermatogenesis/genetics* ; Adult ; Exome Sequencing ; Microtubule-Associated Proteins/genetics* ; Alleles ; Infertility, Male/genetics*

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.

Platinum-based chemotherapy resistance is a key factor of poor prognosis and recurrence in hepatocellular carcinoma (HCC). Herein, RNAseq analysis revealed that elevated tubulin folding cofactor E (TBCE) expression is associated with platinum-based chemotherapy resistance. High expression of TBCE contributes to worse prognoses and earlier recurrence among liver cancer patients. Mechanistically, TBCE silencing significantly affects cytoskeleton rearrangement, which in turn increases cisplatin-induced cycle arrest and apoptosis. To develop these findings into potential therapeutic drugs, endosomal pH-responsive nanoparticles (NPs) were developed to simultaneously encapsulate TBCE siRNA and cisplatin (DDP) to reverse this phenomena. NPs (siTBCE + DDP) concurrently silenced TBCE expression, increased cell sensitivity to platinum treatment, and subsequently resulted in superior anti-tumor effects both in vitro and in vivo in orthotopic and patient-derived xenograft (PDX) models. Taken together, NP-mediated delivery and the co-treatment of siTBCE + DDP proved to be effective in reversing chemotherapy resistance of DDP in multiple tumor models.

OBJECTIVE: To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies. METHODS: Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy. RESULTS: After a 14-day's culture, the contents of CD3^-CD56⁺ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3⁺CD4⁺ T cells and CD3⁺CD56⁺ NKT cells in M-NK group decreased significantly. The percentages of CD16⁺, NKG2D⁺, NKp44⁺, CD25⁺ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a⁺ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05). CONCLUSION The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.
Humans ; Fetal Blood ; Killer Cells, Natural ; T-Lymphocytes ; Leukocytes, Mononuclear/metabolism* ; Cell Proliferation ; CD56 Antigen/metabolism*

Humans ; Burkitt Lymphoma/therapy* ; Hematopoietic Stem Cell Transplantation ; HIV Infections/complications*