Exploring the relationship between lesion morphology and pathogenesis in acute small subcortical infarction.

Introduction Acute small subcortical infarctions (SSIs) result from occlusions of small penetrating arteries, and the underlying pathological factors can have different clinical implications. The objective of this study was to assess the clinical relevance of acute SSIs based on their sizes and morphologies. Methods This retrospective case-control study analyzed clinical and imaging data of stroke patients with acute SSIs in penetrating artery territories who underwent MRI within 5 days of stroke onset, registered between 2016 and 2020. We categorized these patients into three groups based on size and morphology: diameter < 20mm, diameter ≧ 20mm, and separated lesions. We then evaluated their clinical characteristics and outcomes. Results We analyzed 726 stroke patients with SSIs, among whom 573 had a diameter <20mm, 99 had a diameter ≥20mm, and 54 had separated lesions. The patients had a median age of 70 years and a median National Institutes of Health Stroke Scale (NIHSS) score of 4 on arrival. Patients who experienced early neurological deterioration (END) had a significantly lower chance of good functional outcomes (27.3% vs. 64.4%, p<0.001). Patients with a diameter ≧20mm had the most severe NIHSS on arrival and at day 3, the highest rate of END, and the lowest rate of good outcome at 3 months. The incidence of cardioembolism did not differ between patients with diameters of ≥20mm and <20mm. However, multiple logistic regression analysis revealed that separated lesions were more likely to be associated with cardioembolic stroke (adjusted odds ratio [aOR], 7.6; 95% confidence interval [CI], 2.0-28.5) and parent artery stenosis >50% (aOR, 3.8; 95% CI, 2.1-7.0) than a diameter of <20mm. Moreover, SSIs with a diameter of ≥20mm was found to be associated with an increased risk of END compared to that with a diameter of <20mm (aOR, 2.9; 95% CI, 1.7-5.2). Conclusion Our study suggests that the sizes and morphologies of acute SSIs may indicate different underlying pathologies and be linked to diverse clinical outcomes. Our findings also challenge the current imaging criteria for embolic stroke of undetermined source, as we did not find a link between large subcortical infarction and cardioembolic stroke.

Single-Cell Chromatin and Gene-Regulatory Dynamics of Mouse Nephron Progenitors.

BACKGROUND: We reasoned that unraveling the dynamic changes in accessibility of genomic regulatory elements and gene expression at single-cell resolution will inform the basic mechanisms of nephrogenesis. METHODS: We performed single-cell ATAC-seq and RNA-seq both individually (singleomes; Six2GFP cells) and jointly in the same cells (multiomes; kidneys) to generate integrated chromatin and transcriptional maps in mouse embryonic and neonatal nephron progenitor cells. RESULTS: We demonstrate that singleomes and multiomes are comparable in assigning most cell states, identification of new cell type markers, and defining the transcription factors driving cell identity. However, multiomes are more precise in defining the progenitor population. Multiomes identified a "pioneer" bHLH/Fox motif signature in nephron progenitor cells. Moreover, we identified a subset of Fox factors exhibiting high chromatin activity in podocytes. One of these Fox factors, Foxp1, is important for nephrogenesis. Key nephrogenic factors are distinguished by strong correlation between linked gene regulatory elements and gene expression. CONCLUSION: Mapping the regulatory landscape at single-cell resolution informs the regulatory hierarchy of nephrogenesis. Paired single-cell epigenomes and transcriptomes of nephron progenitors should provide a foundation to understand prenatal programming, regeneration after injury, and ex vivo nephrogenesis.

The polycomb proteins EZH1 and EZH2 co-regulate chromatin accessibility and nephron progenitor cell lifespan in mice.

SIX2 (SIX homeobox 2)-positive nephron progenitor cells (NPCs) give rise to all epithelial cell types of the nephron, the filtering unit of the kidney. NPCs have a limited lifespan and are depleted near the time of birth. Epigenetic factors are implicated in the maintenance of organ-restricted progenitors such as NPCs, but the chromatin-based mechanisms are incompletely understood. Here, using a combination of gene targeting, chromatin profiling, and single-cell RNA analysis, we examined the role of the murine histone 3 Lys-27 (H3K27) methyltransferases EZH1 (enhancer of zeste 1) and EZH2 in NPC maintenance. We found that EZH2 expression correlates with NPC growth potential and that EZH2 is the dominant H3K27 methyltransferase in NPCs and epithelial descendants. Surprisingly, NPCs lacking H3K27 trimethylation maintained their progenitor state but cycled slowly, leading to a smaller NPC pool and formation of fewer nephrons. Unlike Ezh2 loss of function, dual inactivation of Ezh1 and Ezh2 triggered overexpression of the transcriptional repressor Hes-related family BHLH transcription factor with YRPW motif 1 (Hey1), down-regulation of Six2, and unscheduled activation of Wnt4-driven differentiation, resulting in early termination of nephrogenesis and severe renal dysgenesis. Double-mutant NPCs also overexpressed the SIX family member Six1 However, in this context, SIX1 failed to maintain NPC stemness. At the chromatin level, EZH1 and EZH2 restricted accessibility to AP-1-binding motifs, and their absence promoted a regulatory landscape akin to differentiated and nonlineage cells. We conclude that EZH2 is required for NPC renewal potential and that tempering of the differentiation program requires cooperation of both EZH1 and EZH2.

Histone deacetylases 1 and 2 regulate the transcriptional programs of nephron progenitors and renal vesicles.

Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a, as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles.

Relationship between MTHFR gene polymorphism and susceptibility to bronchial asthma and glucocorticoid efficacy in children. / MTHFRåŸºå› å¤šæ€æ€§ä¸Žå„¿ç«¥æ”¯æ°”ç®¡å“®å–˜æ˜“æ„Ÿæ€§åŠç³–çš®è´¨æ¿€ç´ ç–—æ•ˆçš„ç›¸å ³æ€§.

OBJECTIVES: To study the association of methylenetetrahydrofolate reductase (MTHFR) gene polymorphism with susceptibility to bronchial asthma and glucocorticoid (GC) efficacy in children. METHODS: A total of 173 children with bronchial asthma who were hospitalized between June 2018 and December 2020 were selected as the observation group. The children received aerosol inhalation of GC for three consecutive months. A total of 178 healthy children who underwent physical examination during the same period were selected as the control group. PCR was used to detect the genotypes of the MTHFR C677T for the two groups. The differences in genotype distribution between the two groups were analyzed. Children with different genotypes in the observation group were compared in terms of immunoglobulin E (IgE), interleukin-8 (IL-8), leukotriene B4 (LTB4), lung function, and clinical outcome before and after treatment. RESULTS: TT genotype and T allele were significantly more frequent in the observation group than in the control group (P<0.001). TT/CT genotypes and T allele were independent risk factors for bronchial asthma (OR=6.615 and 7.055 respectively; P<0.001). After GC treatment, the children with CC, CT or TT genotypes experienced significantly decreased levels of IgE, IL-8, and LTB4 and significantly increased forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), and FEV1/FVC ratio (P<0.001). The children with TT genotype showed significantly lower levels of IL-8 and LTB4 than those with CC genotype, a significantly lower level of LTB4 than those with CT genotype, significantly higher FVC than those with CT genotype, and a significantly higher FEV1/FVC ratio than those with CC genotype (P<0.05). The children with TT genotype had better GC efficacy compared with those with CC genotype (P<0.05). TT genotype was an independent factor for good GC efficacy (OR=2.111, P=0.018). CONCLUSIONS: MTHFR gene polymorphism is associated with asthma susceptibility and GC efficacy in children. Children carrying TT/CT genotypes have a higher risk of developing asthma, and those with TT genotype are more sensitive to GC treatment.

Functional redundancy between human SHOX and mouse Shox2 genes in the regulation of sinoatrial node formation and pacemaking function.

The homeodomain transcription factor Shox2 plays a crucial regulatory role in the development of sinoatrial node (SAN) by repressing the expression of Nkx2.5, as demonstrated by failed differentiation of SAN in Shox2 null mice. The SHOX (short stature homeobox) gene family consists of two closely related members, SHOX and SHOX2 in humans, but a SHOX ortholog does not exist in the mouse genome. These two genes exhibit overlapping and distinct expression patterns in many developing organs but whether they share functional redundancy is not known. In this study, we set to investigate possible functional redundancy between SHOX and SHOX2 in vitro and in vivo. We first showed that human SHOX and SHOX2 and mouse Shox2 possess similar transcriptional repressive activities in cell cultures, particularly the repressive effects on the Nkx2.5 promoter activity. We further created an SHOX/Shox2 knock-in mouse line (replacement of Shox2 with SHOX, referred as Shox2(KI/KI)). Mice carrying the hypomorphic Shox2(KI+Neo/KI+Neo) allele exhibit bradycardia and arrhythmia and die a few days after birth. However, mice carrying the Shox2(KI/KI) allele grow to adulthood. Physiological, histological, and molecular analyses demonstrate a fully developed SAN and normal pacemaking function in Shox2(KI/KI) mice. Our results demonstrate a functional redundancy between human SHOX and mouse Shox2 in the regulation of SAN formation and pacemaking function in addition to several other organs. The SHOX/Shox2 dose appears to be critical for normal pacemaking function.

The specific binding of chlorogenic acid to human serum albumin.

Chlorogenic acid (CGA) is one of the most abundant polyphenol compounds in human diet. It is also an active component in traditional Chinese medicines which are used to treat various diseases. In this study, fluorescence spectroscopy in combination with UV-Vis absorption spectroscopy was employed to investigate the specific binding of CGA to human serum albumin (HSA) under the physiological conditions. In the mechanism discussion, it was proved that the fluorescence quenching of HSA by CGA is a result of the formation of CGA-HSA complex. Binding parameters calculating from Stern-Volmer method and Scatchard method showed that CGA bind to HSA with the binding affinities of the order 10(4) l mol(-1). The thermodynamic parameters studies revealed that the binding was characterized by negative enthalpy and positive entropy changes and the electrostatic interactions play a major role for CGA-HSA association. Site marker competitive displacement experiments demonstrated that CGA specific bind to site I (subdomain IIA) of HSA. The binding distance r (3.10 nm) between donor (Trp-214) and acceptor (CGA) was obtained according to fluorescence resonance energy transfer. Furthermore, the effect of metal ions on CGA-HSA system was studied.

Histone deacetylases 1 and 2 target gene regulatory networks of nephron progenitors to control nephrogenesis.

Our studies demonstrated the critical role of Histone deacetylases (HDACs) in the regulation of nephrogenesis. To better understand the key pathways regulated by HDAC1/2 in early nephrogenesis, we performed chromatin immunoprecipitation sequencing (ChIP-Seq) of HDAC1/2 on isolated nephron progenitor cells (NPCs) from mouse E16.5 kidneys. Our analysis revealed that 11,802 (40.4%) of HDAC1 peaks overlap with HDAC2 peaks, further demonstrates the redundant role of HDAC1 and HDAC2 during nephrogenesis. Common HDAC1/2 peaks are densely concentrated close to the transcriptional start site (TSS). GREAT Gene Ontology analysis of overlapping HDAC1/2 peaks reveals that HDAC1/2 are associated with metanephric nephron morphogenesis, chromatin assembly or disassembly, as well as other DNA checkpoints. Pathway analysis shows that negative regulation of Wnt signaling pathway is one of HDAC1/2's most significant function in NPCs. Known motif analysis indicated that Hdac1 is enriched in motifs for Six2, Hox family, and Tcf family members, which are essential for self-renewal and differentiation of nephron progenitors. Interestingly, we found the enrichment of HDAC1/2 at the enhancer and promoter regions of actively transcribed genes, especially those concerned with NPC self-renewal. HDAC1/2 simultaneously activate or repress the expression of different genes to maintain the cellular state of nephron progenitors. We used the Integrative Genomics Viewer to visualize these target genes associated with each function and found that HDAC1/2 co-bound to the enhancers or/and promoters of genes associated with nephron morphogenesis, differentiation, and cell cycle control. Taken together, our ChIP-Seq analysis demonstrates that HDAC1/2 directly regulate the molecular cascades essential for nephrogenesis.

Identification of magnetic resonance imaging features for the prediction of unrecognized atrial fibrillation in acute ischemic stroke.

Background and purpose: The early identification of cardioembolic stroke is critical for the early initiation of anticoagulant treatment. However, it can be challenging to identify the major cardiac source, particularly since the predominant source, paroxysmal atrial fibrillation (AF), may not be present at the time of stroke. In this study, we aimed to evaluate imaging predictors for unrecognized AF in patients with acute ischemic stroke. Methods: We performed a cross-sectional analysis of data and magnetic resonance imaging (MRI) scans from two prospective cohorts of patients who underwent serial 12-lead electrocardiography and 24-h Holter monitoring to detect unrecognized AF. The imaging patterns in diffusion-weighted imaging and imaging characteristics were assessed and classified. A logistic regression model was used to identify predictive factors for newly detected AF in patients with acute ischemic stroke. Results: A total of 734 patients were recruited for analysis, with a median age of 72 (interquartile range: 65-79) years and a median National Institutes of Health Stroke Scale score of 4 (interquartile range: 2-6). Of these patients, 64 (8.7%) had newly detected AF during the follow-up period. Stepwise multivariate logistic regression revealed that age ≥75 years [adjusted odds ratio (aOR) 5.66, 95% confidence interval (CI) 2.98-10.75], receiving recombinant tissue plasminogen activator treatment (aOR 4.36, 95% CI 1.65-11.54), congestive heart failure (aOR 6.73, 95% CI 1.85-24.48), early hemorrhage in MRI (aOR 3.62, 95% CI 1.52-8.61), single cortical infarct (aOR 6.49, 95% CI 2.35-17.92), and territorial infarcts (aOR 3.54, 95% CI 1.06-11.75) were associated with newly detected AF. The C-statistic of the prediction model for newly detected AF was 0.764. Conclusion: Initial MRI at the time of stroke may be useful to predict which patients have cardioembolic stroke caused by unrecognized AF. Further studies are warranted to verify these findings and their application to high-risk patients.

Targeted disruption of the histone lysine 79 methyltransferase Dot1L in nephron progenitors causes congenital renal dysplasia.

The epigenetic regulator Dot1, the only known histone H3K79 methyltransferase, has a conserved role in organismal development and homoeostasis. In yeast, Dot1 is required for telomeric silencing and genomic integrity. In Drosophila, Dot1 (Grappa) regulates homoeotic gene expression. Dysregulation of DOT1L (human homologue of Dot1) causes leukaemia and is implicated in dilated cardiomyopathy. In mice, germline disruption of Dot1L and loss of H3K79me2 disrupt vascular and haematopoietic development. Targeted inactivation of Dot1L in principal cells of the mature collecting duct affects terminal differentiation and cell type patterning. However, the role of H3K79 methylation in mammalian tissue development has been questioned, as it is dispensable in the intestinal epithelium, a rapidly proliferating tissue. Here, we used lineage-specific Cre recombinase to delineate the role of Dot1L methyltransferase activity in the mouse metanephric kidney, an organ that develops via interactions between ureteric epithelial (Hoxb7) and mesenchymal (Six2) cell lineages. The results demonstrate that Dot1LHoxb7 is dispensable for ureteric bud branching morphogenesis. In contrast, Dot1LSix2 is critical for the maintenance and differentiation of Six2+ progenitors into epithelial nephrons. Dot1LSix2 mutant kidneys exhibit congenital nephron deficit and cystic dysplastic kidney disease. Molecular analysis implicates defects in key renal developmental regulators, such as Lhx1, Pax2 and Notch. We conclude that the developmental functions of Dot1L-H3K79 methylation in the kidney are lineage-restricted. The link between H3K79me and renal developmental pathways reaffirms the importance of chromatin-based mechanisms in organogenesis.

Defining the dynamic chromatin landscape of mouse nephron progenitors.

Six2+ cap mesenchyme cells, also called nephron progenitor cells (NPC), are precursors of all epithelial cell types of the nephron, the filtering unit of the kidney. Current evidence indicates that perinatal 'old' NPC have a greater tendency to exit the progenitor niche and differentiate into nascent nephrons than their embryonic 'young' counterpart. Understanding the underpinnings of NPC development may offer insights to rejuvenate old NPC and expand the progenitor pool. Here, we compared the chromatin landscape of young and old NPC and found common features reflecting their shared lineage but also intrinsic differences in chromatin accessibility and enhancer landscape supporting the view that old NPC are epigenetically poised for differentiation. Annotation of open chromatin regions and active enhancers uncovered the transcription factor Bach2 as a potential link between the pro-renewal MAPK/AP1 and pro-differentiation Six2/b-catenin pathways that might be of critical importance in regulation of NPC fate. Our data provide the first glimpse of the dynamic chromatin landscape of NPC and serve as a platform for future studies of the impact of genetic or environmental perturbations on the epigenome of NPC.

Protective effects of pogostone against LPS-induced acute lung injury in mice via regulation of Keap1-Nrf2/NF-κB signaling pathways.

Pogostone, a major component of Pogostemon cablin, has been demonstrated to possess antibacterial, anti-fungal, immunosuppressive and anti-inflammatory properties. To investigate the potential therapeutic effect of pogostone on lipopolysaccharide (LPS)-induced acute lung injury (ALI), mice were pretreated with pogostone prior to LPS exposure. After LPS challenge, the lungs were excised and the histological changes, wet to dry weight ratios, MPO activity reflecting neutrophil infiltration, and MDA activity reflecting oxidative stress were examined. The inflammatory cytokines in the BALF were determined by ELISA assay. Moreover, the expressions of p65 and phosphorylated p65 subunit of NF-κB, and Nrf2 in the nucleus in lung tissues were measured by Western blot analysis, and meanwhile the dependent genes of NF-κB and Nrf2 were assessed by RT-qPCR. The results showed that pretreatment with pogostone markedly improved survival rate, attenuated the histological alterations in the lung, reduced the MPO and MDA levels, decreased the wet/dry weight ratio of lungs, down-regulated the level of pro-inflammatory mediators including TNF-a, IL-1ß and IL-6. Furthermore, pretreatment with pogostone enhanced the Nrf2 dependent genes including NQO-1, GCLC and HO-1 but suppressed NF-κB regulated genes including TNF-α, IL-1ß and IL-6. The mechanism behind the protective effect was correlated with its regulation on the balance between Keap1-Nrf2 and NF-κB signaling pathways. Therefore, pogostone may be considered as a potential therapeutic agent for preventing and treating ALI.

Maple syrup urine disease in the Austronesian aboriginal tribe Paiwan of Taiwan: a novel DBT (E2) gene 4.7 kb founder deletion caused by a nonhomologous recombination between LINE-1 and Alu and the carrier-frequency determination.

Maple syrup urine disease (MSUD) is an autosomal recessive inborn error disorder derived from the accumulation of the branched-chain amino acids (BCAAs) leucine, isoleucine and valine. Either the E1alpha, E1beta or DBT (E2) genes are responsible for this neurometabolic disease. Here, we report the identification and characterization of a novel E2 gene 4.7 kb deletion as a rare nonhomologous recombination of the long interspersed nuclear elements 1 (LINE-1) in intron 10 and the Alu in the 3' UTR of the E2 gene from three classic MSUD patients of the Austronesian aboriginal tribe Paiwan in Taiwan. The E2 gene 4.7 kb deletion accounted for five out of six alleles in the three unrelated Paiwanese MSUD patients, indicating a founder effect. Carrier-frequency study revealed one deleted heterozygote out of 101 normal Paiwanese. As the nine Taiwanese Austronesian aboriginal tribes share a common origin, this E2 4.7 kb deletion may be preserved in some of the other Austronesian aboriginal tribes of Taiwan. This is the first comprehensive genetics study of MSUD in the Austronesian tribal groups as well as in Taiwan.

Urinary brain-derived neurotrophic factor: a potential biomarker for objective diagnosis of overactive bladder.

PURPOSE: To investigate the diagnostic performance of urinary brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) as potential biomarkers for overactive bladder (OAB). METHODS: Ninety women diagnosed with OAB and 45 normal controls without OAB were enrolled. Urine samples were collected from all subjects. Urinary BDNF and NGF levels were measured using enzyme-linked immunosorbent assays. Results normalized by urinary creatinine (Cr) levels were compared between OAB groups and controls. Symptom severity was assessed using overactive bladder symptom score. RESULTS: Urinary BDNF and NGF levels were elevated in OAB groups but not in controls. Mean (SD) baseline BDNF and NGF levels normalized by Cr levels were significantly higher in OAB subjects than in controls (20.609 ± 23.932 vs. 1.779 ± 0.729, p < 0.01) and (0.258 ± 0.264 vs. 0.081 ± 0.028, p < 0.01), respectively. Urinary BDNF/Cr levels were 80-fold higher than NGF/Cr levels in OAB subjects. Receiver operating characteristic curves for assessing urinary BDNF/Cr levels in OAB groups showed sensitivity and specificity of 93.33 and 88.89 %, respectively. Urinary BDNF levels were associated with OAB symptom severity. CONCLUSIONS: Urinary BDNF/Cr levels are elevated in women with OAB and are significantly associated with symptom severity. No elevation of BDNF is found in women without OAB. BDNF analysis has better sensitivity than NGF in detecting OAB in subjects without other lower urinary tract disorders. Results of the present study suggest a potential role for BDNF as an objective biomarker for OAB diagnosis.

Phosphorylation of Shox2 is required for its function to control sinoatrial node formation.

BACKGROUND: Inactivation of Shox2, a member of the short-stature homeobox gene family, leads to defective development of multiple organs and embryonic lethality as a result of cardiovascular defects, including bradycardia and severe hypoplastic sinoatrial node (SAN) and sinus valves, in mice. It has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the fate of the SAN cells. However, the functional mechanism of Shox2 protein as a transcriptional repressor on Nkx2.5 expression remains completely unknown. METHODS AND RESULTS: A specific interaction between the B56δ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2-hybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (p-Shox2a) in cell culture as well as in the developing mouse and human SAN. Site-directed mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signal-regulated kinase 1 and 2. Despite that Shox2a and its phosphorylation mutants possessed similar transcriptional repressive activities in cell cultures when fused with Gal4 protein, the mutant forms exhibited a compromised repressive effect on the activity of the mouse Nkx2.5 promoter in cell cultures, indicating that phosphorylation is required for Shox2a to repress Nkx2.5 expression specifically. Transgenic expression of Shox2a, but not Shox2a-S92AS110A, mutant in the developing heart resulted in down-regulation of Nkx2.5 in wild-type mice and rescued the SAN defects in the Shox2 mutant background. Last, we demonstrated that elimination of both phosphorylation sites on Shox2a did not alter its nuclear location and dimerization, but depleted its capability to bind to the consensus sequences within the Nkx2.5 promoter region. CONCLUSIONS: Our studies reveal that phosphorylation is essential for Shox2a to repress Nkx2.5 expression during SAN development and differentiation.

[Effects of Omi/HtrA2 on expression of anti-apoptotic protein PED/PEA-15 and apoptosis of prostate cancer cell line PC-3].

BACKGROUND & OBJECTIVE: The interaction between pro-apoptotic factors and anti-apoptotic factors is closely related to the genesis and development of tumors. Omi/HtrA2 is a novel gene involved in the regulation of apoptosis. PED/PEA-15 is a widely expressed anti-apoptotic protein. This study was to explore the effects of Omi/HtrA2 on PED/PEA-15 expression and apoptosis of prostate cancer cell line PC-3. METHODS: Omi/HtrA2 expression and specific siRNA vectors were constructed and transiently transfected into PC-3 cells. The effect of Omi/HtrA2 on PED/PEA-15 expression was assayed by Western blot, and its effect on apoptosis of PC-3 cells was analyzed by ELISA. Caspase-8 activity was assayed using Caspase-8 colorimetric assay kit. The effects of Omi/HtrA2-specific siRNA sequence on its transcription and translation were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The sensitivity of PC-3 cells to cisplatin (DDP) after Omi/HtrA2 gene silencing was determined by flow cytometry. RESULTS: Enzyme digestion analysis and DNA sequencing confirmed Omi/HtrA2 expression, and specific siRNA vectors were successfully constructed. After transfection of Omi/HtrA2 expression vector, PED/PEA-15 expression was inhibited, Caspase-8 activity was promoted, and the apoptosis of PC-3 cells was enhanced. The sensitivity of PC-3 cells to DDP was suppressed after Omi/HtrA2 gene silencing. CONCLUSION: Omi/HtrA2 can promote the apoptosis of PC-3 cells through inhibiting PED/PEA-15 expression.

[In vitro cytotoxicity of Helicobacter pylori on hepatocarcinoma HepG2 cells].

BACKGROUND & OBJECTIVE: Helicobacter pylori (HP) is the important pathogen of chronic gastritis and peptic ulcer. HP infection is closely associated with extragastrointestinal disease of human and animals. This study was designed to investigate the effect of HP on hepatocarcinoma HepG2 cells. METHODS: HepG2 cells was cocultured with cagA (+) HP and cagA(-) HP at different concentrations. Then the cell morphology, DNA fragments electrophoresis, transmission electron microscopy, MTT method, and enzyme assays were used to observe the effect of HP on the morphology, growth rate, and enzyme change of HepG2 cells. RESULTS: (1) With the increasing concentrations of cagA (+) HP and prolongation of culture time, the morphology of HepG2 cells was changed from spindle shape to round; the ability of HepG2 cells adhering to wall were decreased; the ability of HepG2 cells in suspension were increased; and debris emitted around the cells; (2) With the increasing concentrations of cagA (+) HP, DNA ladder occurred; (3) With the increasing concentrations of cagA (+) HP and prolongation of culture time, the nuclei of HepG2 cells showed chromatin pyknosis, and clustered on the inner border of karyotheca, condensed cytoplasm with many vacuoles; (4) The MTT values of HepG2 cells were decreased with the increasing concentration of cagA(+) HP (P< 0.01); (5)The values of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in coculture supernatants of cagA (+) Hp and HepG2 were increased (P< 0.01); (6) No influence of cagA (-) Hp on HepG2 was found. CONCLUSIONS: cagA (+) Hp has in vitro cytotoxicity on HepG2 cells, and the cytotoxicity is positively related to the concentration and culture time of cagA(+) HP.