Objective: To investigate the role of connective tissue growth factor (CTGF) and PI3K/Akt signaling pathways in paraquat (PQ) -induced alterations in alveolar epithelial cell mesenchymalization (EMT) . Methods: In February 2023, RLE-6TN cells were divided into 2 groups, which were set as uncontaminated group and contaminated group (200 μmol/L PQ), and cellular EMT alteration, CTGF and PI3K/Akt signaling pathway related molecules expression were detected by cell scratch assay, qRT-PCR and western-blot assay. Using shRNA interference technology to specifically inhibit the expression of CTGF, RLE-6TN cells were divided into four groups: control group, PQ group (200 μmol/L PQ), interference group (transfected with a plasmid with shRNA-CTGF+200 μmol/L PQ), and null-loaded group (transfected with a plasmid with scramble- CTGF+200 μmol/L PQ), qRT-PCR and western blot were used to examine the alteration of the cellular EMT and the expression of molecules related to the activity of PI3K/Akt pathway. The PI3K/Akt signaling pathway was blocked by the PI3K inhibitor LY294002, and the expression of EMT-related molecules in cells of the control group, PQ group (200 μmol/L PQ), and inhibitor group (200 μmol/L PQ+20 μmol/L LY294002) was examined by qRT-PCR and western blot.The t-test was used to compare the differences between the two groups, while the analysis of variance (ANOVA) was applied to compare the differences among multiple groups. For further pairwise comparisons, the Bonferroni method was adopted. Results: The results of cell scratch test showed that compared with the uncontaminated group, RLE-6TN cells in the contaminated group had faster migration rate, lower mRNA and protein expression levels of E-Cadherin, and higher mRNA and protein expression levels of α-SMA, CTGF, PI3K and Akt, with statistical significance (P<0.05). After specific inhibition of CTGF expression, the mRNA and protein expression of CTGF, PI3K, Akt, and α-SMA in the cells of the interference group were significantly lower than that of the PQ group and the null-loaded group (P<0.05/6), whereas that of E-Cadherin was higher than that of the PQ group and the null-loaded group (P<0.05/6). Specifically blocking the PI3K/Akt signaling pathway, the mRNA and protein expression of PI3K, Akt and α-SMA in the cells of the inhibitor group was decreased compared with that of the PQ group (P<0.05/3), while the expression of E-Cadherin was elevated compared with that of the PQ group (P<0.05/3) . Conclusion: CTGF may promote PQ-induced alveolar epithelial cell EMT through activation of the PI3K/Akt signaling pathway. Inhibition of CTGF expression or blockade of PI3K/Akt signaling pathway activity can alleviate the extent of PQ-induced alveolar epithelial cell EMT.
目的： 探讨结缔组织生长因子（CTGF）及磷脂酰肌醇3-激酶/丝氨酸/苏氨酸激酶（PI3K/Akt）信号通路在百草枯（PQ）致肺泡上皮细胞上皮-间充质化（EMT）改变的作用。 方法： 于2023年2月，将RLE-6TN细胞分成2组，设为未染毒组和染毒组（200 μmol/L PQ），采用细胞划痕实验、qRT-PCR和Western-blot法检测细胞EMT改变、CTGF及PI3K/Akt信号通路相关分子表达情况。利用shRNA干扰技术特异性抑制CTGF的表达，将RLE-6TN细胞分成4组，分别为对照组、PQ组（200 μmol/L PQ）、干扰组（转染含shRNA-CTGF质粒+200 μmol/L PQ）和空载组（转染含CTGF-scramble质粒+200 μmol/L PQ），qRT-PCR和Western-blot法检测细胞EMT改变及PI3K/Akt通路活性相关分子的表达情况。使用PI3K抑制剂LY294002阻断PI3K/Akt信号通路，运用qRT-PCR和Western-blot法检测对照组、PQ组（200 μmol/L PQ）、抑制剂组（200 μmol/L PQ+20 μmol/L LY294002）细胞EMT相关分子表达情况。两组间差异比较采用t检验，多组间差异比较采用方差分析，进一步两两比较采用Bonferroni法。 结果： 细胞划痕实验结果显示，与未染毒组比较，PQ染毒组RLE-6TN细胞迁移速度更快，E-钙黏蛋白（E-Cadherin）的mRNA和蛋白表达量更低，α-平滑肌肌动蛋白（α-SMA）、CTGF、PI3K、Akt的mRNA和蛋白表达量更高，差异均有统计学意义（P<0.05）。特异性抑制CTGF表达后，干扰组细胞CTGF、PI3K、Akt、α-SMA的mRNA和蛋白表达量明显低于PQ组和空载组（P<0.05/6），E-Cadherin的mRNA和蛋白表达量高于PQ组和空载组（P<0.05/6）。特异性阻断PI3K/Akt信号通路，抑制剂组细胞PI3K、Akt和α-SMA的mRNA和蛋白表达量较PQ组降低（P<0.05/3），E-Cadherin表达量较PQ组升高（P<0.05/3）。 结论： CTGF可能通过激活PI3K/Akt信号通路促进PQ致肺泡上皮细胞EMT改变，抑制CTGF的表达或阻断PI3K/Akt信号通路活性，可减轻PQ所致肺泡上皮细胞EMT的程度。.

OBJECTIVE: The RNA binding protein quaking (QKI) is associated with the development and progression of tumor suppressors in various cancers. However, the clinical implications of QKI expression have not yet been fully elucidated. In this study, we aimed to investigate the clinicopathological and prognostic significance of QKI expression in hepatocellular carcinoma (HCC).
METHODS: We performed QKI, Zinc finger E-box-binding homeobox 1 (ZEB1), E-cadherin, and glutathione peroxidase 4 (GPX4) immunohistochemical staining on 166 HCC patient tissue samples. X-tile bioinformatics software was used to set the cut-off value for high QKI expression. Correlations between QKI expression and various clinicopathological parameters were assessed.
RESULTS: The best cut-off value for high QKI expression was 12.5. High QKI expression was observed in 28 of 166 patients (16.9%) and was an independent prognostic factor for inferior recurrence-free survival (RFS). In addition, high ZEB1 and GPX4 expression correlated with high QKI expression, but not with the loss of E-cadherin expression.
CONCLUSIONS: High QKI expression was identified in HCCs and associated with poor RFS. QKI might be a prognostic biomarker of HCCs associated with epithelial-to-mesenchymal transition and a potential candidate therapeutic target.

Colorectal cancer (CRC) is the third most common malignancy cause of cancer-related mortality worldwide. Epithelial-mesenchymal transition (EMT) promotes cancer metastasis and a tumour-based Glasgow EMT score was associated with adverse clinical features and poor prognosis. In this study, the impact of using the established five tumour-based EMT markers consisting of E-cadherin (E-cad), β-catenin (β-cat), Snail, Zeb-1, and Fascin in combination with the stromal periostin (PN) on the prediction of CRC patients\' prognosis were invesigated. Formalin-fixed paraffin-embedded tissues of 202 CRC patients were studies the expressions of E-cad, β-cat, Snail, Zeb-1, Fascin, and PN by immunohistochemistry. Individually, cytoplasmic Fascin (Fc), cytoplasmic Snail (Sc), nuclear Snail (Sn), stromal Snail (Ss), and stromal PN (Ps) were significantly associated with reduced survival. A combination of Ps with Fc, Fs, and Sn was observed in 2 patterns including combined Fc, Fs, and Ps (FcFsPs) and Fc, Sn, and Ps (FcSnPs). These combinations enhanced the prognostic power compared to individual EMT markers and were independent prognostic markers. As the previously established scoring method required five markers and stringent criteria, its clinical use might be limited. Therefore, using these novel combined prognostic markers, either FcFsPs or FcSnPs, may be useful in predicting CRC patient outcomes.

Approximately half of the cases of chronic kidney disease (CKD) in childhood are caused by congenital anomalies of the kidney and urinary tract (CAKUT). Specific genes were identified as having significant importance in regard to the underlying genetic factors responsible for the CAKUT phenotype, and in our research, we focused on analyzing and comparing the expression levels of ectodysplasin A2 receptor (EDA2R), protocadherin9 (PCDH9), and TNF receptor-associated factor 7 (TRAF7) proteins in the cortex and medulla of healthy control kidneys during developmental phases 2, 3, and 4. We also performed an analysis of the area percentages of the mentioned proteins in the cortical and medullary sections of healthy embryonic and fetal kidneys compared to those affected by CAKUT, including duplex kidneys (DK), horseshoe kidneys (HK), hypoplastic kidneys (HYP), and dysplastic kidneys (DYS). We found that the CAKUT candidate gene proteins EDA2R, PCDH9, and TRAF7 are all expressed during normal human kidney development stages. In DYS, the expression of EDA2R was higher than in normal kidneys, likely due to EDA2R\'s role in apoptosis, which was upregulated in specific cases and could possibly contribute to the formation of DYS. The expression of PCDH9 was lower in HK, which can be attributed to the possible role of PCDH9 in cell migration suppression. Decreased PCDH9 expression is linked to increased cell migration, potentially contributing to the development of HK. The level of TRAF7 expression was reduced in all examined kidney disorders compared to normal kidneys, suggesting that this reduction might be attributed to the crucial role of TRAF7 in the formation of endothelium and ciliogenesis, both of which are essential for normal kidney development. Further research is required to ascertain the function of these proteins in both the typical development of the kidney and in CAKUT.

Tunicate orthologs in the human genome comprise just 84 genes of the 19,872 protein-coding genes and 23 of the 16,528 non-coding genes, yet they stand at the base of the Olfactores clade, which radiated to generate thousands of tunicate and vertebrate species. What were the powerful drivers among these genes that enabled this process? Many of these orthologs are present in gene families. We discuss the biological role of each family and the orthologs\' quantitative contribution to the family. Most important was the evolution of a second type of cadherin. This, a Type II cadherin, had the property of detaching the cell containing that cadherin from cells that expressed the Type I class. The set of such Type II cadherins could now detach and move away from their Type I neighbours, a process which would eventually evolve into the formation of the neural crest, \"the fourth germ layer\", providing a wide range of possibilities for further evolutionary invention. A second important contribution were key additions to the broad development of the muscle and nerve protein and visual perception toolkits. These developments in mobility and vision provided the basis for the development of the efficient predatory capabilities of the Vertebrata.

Identifying novel epigenetic biomarkers is a promising way to improve the clinical management of patients with breast cancer. Our study aimed to determine the methylation pattern of 25 tumor suppressor genes (TSG) and select the best methylation biomarker associated with clinicopathological features in the cohort of Slovak patients diagnosed with invasive ductal carcinoma (IDC). Overall, 166 formalin-fixed, paraffin-embedded (FFPE) tissues obtained from patients with IDC were included in the study. The methylation status of the promoter regions of 25 TSG was analyzed using semiquantitative methylation-specific MLPA (MS-MLPA). We identified CDH13 as the most frequently methylated gene in our cohort of patients. Further analysis by ddPCR confirmed an increased level of methylation in the promoter region of CDH13. A significant difference in CDH13 methylation levels was observed between IDC molecular subtypes LUM A versus HER2 (P = 0.0116) and HER2 versus TNBC (P = 0.0234). In addition, significantly higher methylation was detected in HER2+ versus HER2- tumors (P = 0.0004) and PR- versus PR+ tumors (P = 0.0421). Our results provide evidence that alteration in CDH13 methylation is associated with clinicopathological features in the cohort of Slovak patients with IDC. In addition, using ddPCR as a methylation-sensitive method represents a promising approach characterized by higher precision and technical simplicity to measure the methylation of target CpGs in CDH13 compared to other conventional methods such as MS-MLPA.

Circulating leukocytes enter tissue either through endothelial junctions (paracellular) or via a pore through the body of endothelial cells (transcellular). We have previously shown that genetically replacing VE-cadherin with a VE-cadherin-α-catenin (VEC-αC) fusion construct-which binds constitutively to actin-obstructs junctions, and blocks leukocyte extravasation in lung, skin and postcapillary venules of cremaster muscle. However, neutrophil recruitment into the inflamed peritoneal cavity was unimpaired. Investigating reasons for this, here, we visualized neutrophil diapedesis by 3D intravital video microscopy in the cremaster muscle and omentum, the major site of neutrophil recruitment into the peritoneal cavity. We found that 80% of neutrophil-extravasation occurred through HEVs in the omentum, which was unimpaired by VEC-αC. In addition, in larger venules (60-85 µm) of both tissues, less than 15% of neutrophils extravasated transcellularly in WT mice. However, in VEC-α-C mice, transcellular diapedesis increased severalfold in the omentum, but not in the cremaster. In line with this, omental venules expressed higher levels of ICAM-1 and atypical chemokine receptor 1. Furthermore, only in the omentum, VEC-αC expression caused reduced elongation of venular endothelium in flow-direction, suggesting different biomechanical properties. Collectively, VEC-αC does not inhibit paracellular transmigration in all types of venules and can modulate the diapedesis route.

Decreased pancreatic β-cell volume is a serious problem in patients with type 2 diabetes mellitus, and there is a need to establish appropriate treatments. Increasingly, sodium/glucose cotransporter 2 (SGLT2) inhibitors, which have a protective effect on pancreatic β-cells, are being prescribed to treat diabetes; however, the underlying mechanism is not well understood. We previously administered SGLT2 inhibitor dapagliflozin to a mouse model of type 2 diabetes and found significant changes in gene expression in the early-treated group, which led us to hypothesize that epigenetic regulation was a possible mechanism of these changes. Therefore, we performed comprehensive DNA methylation analysis by methylated DNA immunoprecipitation using isolated pancreatic islets after dapagliflozin administration to diabetic model mice. As a result, we identified 31 genes with changes in expression due to DNA methylation changes. Upon immunostaining, cystic fibrosis transmembrane conductance regulator and cadherin 24 were found to be upregulated in islets in the dapagliflozin-treated group. These molecules may contribute to the maintenance of islet morphology and insulin secretory capacity, suggesting that SGLT2 inhibitors\' protective effect on pancreatic β-cells is accompanied by DNA methylation changes, and that the effect is long-term and not temporary. In future diabetes care, SGLT2 inhibitors may be expected to have positive therapeutic effects, including pancreatic β-cell protection.

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the Wnt/β-catenin signaling pathway and epithelial-mesenchymal transition (EMT)-related proteins in rats with intrauterine adhesions (IUA), so as to explore the possible mechanisms of EA in repairing endometrial damage in IUA.
METHODS: Female SD rats were randomly divided into blank, model, EA, and ICG-001 groups, with 10 rats in each group. The IUA model was established by using mechanical scraping combined with lipopolysaccharide infection for double injury. In the EA group, \"Guanyuan\" (CV4) was needled and EA (2 Hz/15 Hz, 1-2 mA) was applied to \"Zusanli\" (ST36) and \"Sanyinjiao\"(SP6) on both sides. In the ICG-001 group, ICG-001 (5 mg/kg), the inhibitor of β-catenin was intraperitoneally injected. After intervention, samples were taken from 5 rats in each group, and uterine endometrium morphology, endometrial thickness, and gland counts were observed using HE staining. Masson staining was used to assess the degree of fibrosis in the endometrial tissue. Immunohistochemistry was used to detect the positive expression of transforming growth factor β1 (TGF-β1), α-smooth muscle actin (α-SMA), fibronectin (FN), connective tissue growth factor (CTGF), type I collagen (Col- Ⅰ), glycogen synthase kinase-3β (GSK-3β), β-catenin, E-cadherin, N-cadherin, and Vimentin in the endometrial tissue. Western blot was used to detect the relative expression of GSK-3β, β-catenin, E-cadherin, N-cadherin, and Vimentin proteins in the endometrial tissue. Another 5 rats from each group were placed in cages with male rats after intervention to record the number of embryo implantations.
RESULTS: Necrosis and loss of endometrial tissue in the model group observed after HE staining were alleviated in the EA group, better than those in the ICG-001 group. Compared with the blank group, the numbers of glands and endometrial thickness in the uterine endometrial tissue, relative expression and positive expression of E-cadherin and GSK-3β proteins in the uterine endometrial tissue, and embryo implantation numbers were reduced(P<0.000 1, P<0.001, P<0.01) in the model group, while fibrosis area ratio in the uterine endometrial tissue, TGF- β 1, α -SMA, FN, CTGF, Col- Ⅰ positive expressions, N-cadherin, Vimentin, and β-catenin proteins expression and positive expression were increased(P<0.000 1, P<0.001, P<0.01). Compared with the model group, the number of glands and endometrial thickness, E-cadherin and GSK-3β proteins expression and positive expression, and embryo implantation numbers were increased (P<0.001, P<0.05, P<0.01) in the EA and ICG-001 groups, while the fibrosis area ratio in the uterine endometrial tissue, TGF-β1, α-SMA, FN, CTGF, Col- Ⅰ positive expression, and N-cadherin, Vimentin, and β-catenin proteins expression and positive expression were decreased(P<0.001, P<0.01, P<0.05). Compared with the EA group, the differences of the above-mentioned indicators in the ICG-001 group were not statistically significant.
CONCLUSIONS: EA may reverse the EMT process and reduce the degree of fibrosis in endometrial tissue by inhibiting the Wnt/β-catenin signaling pathway, thereby promoting the repair of endometrial damage in IUA.
目的: 观察电针对宫腔粘连（IUA）大鼠子宫内膜Wnt/β-连环蛋白（β-catenin）信号通路与上皮间质转化（EMT）相关蛋白的影响，探讨其修复IUA子宫内膜的可能机制。方法: 雌性SD大鼠随机分为空白组、模型组、电针组、ICG-001组，每组10只。采用机械搔刮联合脂多糖感染双重损伤法制备IUA模型。电针组针刺“关元”，电针双侧“足三里”“三阴交”，20 min/次，1次/d；ICG-001组腹腔注射β-catenin抑制剂ICG-001（5 mg/kg），1次/2 d；以上干预均连续进行3个动情周期。每组5只大鼠干预后取材，HE染色法观察IUA大鼠子宫内膜形态、子宫内膜厚度及腺体数目变化，Masson染色法观察子宫内膜组织纤维化程度，免疫组织化学法检测子宫内膜组织转化生长因子β1（TGF-β1）、α-平滑肌肌动蛋白（α-SMA）、纤维连接蛋白（FN）、结缔组织生长因子（CTGF）、Ⅰ型胶原蛋白（Col-Ⅰ）、糖原合酶激酶3β（GSK-3β）、β-catenin、E-钙粘蛋白（E-cadherin）、N-钙粘蛋白（N-cadherin）及波形蛋白（Vimentin）的阳性表达，Western blot法检测子宫内膜组织中GSK-3β、β-catenin、E-cadherin、N-cadherin及Vimentin蛋白相对表达量；每组剩余5只大鼠，干预后与雄鼠合笼，记录各组大鼠子宫胚胎着床数目。结果: HE染色示模型组子宫内膜组织部分坏死或缺失，电针组有所恢复，且较ICG-001组更好。与空白组相比，模型组大鼠子宫内膜组织腺体数目与子宫内膜厚度，子宫内膜组织中E-cadherin和GSK-3β蛋白表达与阳性表达降低（P<0.000 1，P<0.001，P<0.01），子宫内膜纤维化面积比值，子宫内膜组织中TGF-β1、α-SMA、FN、CTGF、Col-Ⅰ阳性表达，N-cadherin、Vimentin及β-catenin蛋白表达与阳性表达升高（P<0.000 1，P<0.001，P<0.01），胚胎着床数目减少（P<0.000 1）；与模型组相比，电针组与ICG-001组大鼠子宫内膜组织腺体数目与子宫内膜厚度，子宫内膜组织E-cadherin与GSK-3β蛋白表达及阳性表达升高（P<0.001，P<0.05，P<0.01），子宫内膜纤维化面积比值，子宫内膜组织TGF-β1、α-SMA、FN、CTGF、Col-Ⅰ阳性表达，N-cadherin、Vimentin、β-catenin蛋白表达与阳性表达降低（P<0.001，P<0.01，P<0.05），胚胎着床数目增多（P<0.001）；与电针组相比，ICG-001组以上指标差异均无统计学意义。结论: 电针可能通过抑制Wnt/β-catenin信号通路逆转EMT进程，降低内膜组织纤维化程度，从而促进IUA子宫内膜修复。.

Arrhythmogenic cardiomyopathy (ACM) is an inherited myocardial disease at risk of sudden death. Genetic testing impacts greatly in ACM diagnosis, but gene-disease associations have yet to be determined for the increasing number of genes included in clinical panels. Genetic variants evaluation was undertaken for the most relevant non-desmosomal disease genes. We retrospectively studied 320 unrelated Italian ACM patients, including 243 cases with predominant right-ventricular (ARVC) and 77 cases with predominant left-ventricular (ALVC) involvement, who did not carry pathogenic/likely pathogenic (P/LP) variants in desmosome-coding genes. The aim was to assess rare genetic variants in transmembrane protein 43 (TMEM43), desmin (DES), phospholamban (PLN), filamin c (FLNC), cadherin 2 (CDH2), and tight junction protein 1 (TJP1), based on current adjudication guidelines and reappraisal on reported literature data. Thirty-five rare genetic variants, including 23 (64%) P/LP, were identified in 39 patients (16/243 ARVC; 23/77 ALVC): 22 FLNC, 9 DES, 2 TMEM43, and 2 CDH2. No P/LP variants were found in PLN and TJP1 genes. Gene-based burden analysis, including P/LP variants reported in literature, showed significant enrichment for TMEM43 (3.79-fold), DES (10.31-fold), PLN (117.8-fold) and FLNC (107-fold). A non-desmosomal rare genetic variant is found in a minority of ARVC patients but in about one third of ALVC patients; as such, clinical decision-making should be driven by genes with robust evidence. More than two thirds of non-desmosomal P/LP variants occur in FLNC.