Glucocorticoid use may cause elevated intraocular pressure, leading to the development of glucocorticoid-induced glaucoma (GIG). However, the mechanism of GIG development remains incompletely understood. In this study, we subjected primary human trabecular meshwork cells (TMCs) and mice to dexamethasone treatment to mimic glucocorticoid exposure. The myofibroblast transdifferentiation of TMCs was observed in cellular and mouse models, as well as in human trabecular mesh specimens. This was demonstrated by the cytoskeletal reorganization, alterations in cell morphology, heightened transdifferentiation markers, increased extracellular matrix deposition, and cellular dysfunction. Knockdown of Rho guanine nucleotide exchange factor 26 (ARHGEF26) expression ameliorated dexamethasone-induced changes in cell morphology and upregulation of myofibroblast markers, reversed dysfunction and extracellular matrix deposition in TMCs, and prevented the development of dexamethasone-induced intraocular hypertension. And, this process may be related to the TGF-β pathway. In conclusion, glucocorticoids induced the myofibroblast transdifferentiation in TMCs, which played a crucial role in the pathogenesis of GIG. Inhibition of ARHGEF26 expression protected TMCs by reversing myofibroblast transdifferentiation. This study demonstrated the potential of reversing the myofibroblast transdifferentiation of TMCs as a new target for treating GIG.

Understanding the impact of splicing and nonsense variants on RNA is crucial for the resolution of variant classification as well as their suitability for precision medicine interventions. This is primarily enabled through RNA studies involving transcriptomics followed by targeted assays using RNA isolated from clinically accessible tissues (CATs) such as blood or skin of affected individuals. Insufficient disease gene expression in CATs does however pose a major barrier to RNA based investigations, which we show is relevant to 1,436 Mendelian disease genes. We term these \"silent\" Mendelian genes (SMGs), the largest portion (36%) of which are associated with neurological disorders. We developed two approaches to induce SMG expression in human dermal fibroblasts (HDFs) to overcome this limitation, including CRISPR-activation-based gene transactivation and fibroblast-to-neuron transdifferentiation. Initial transactivation screens involving 40 SMGs stimulated our development of a highly multiplexed transactivation system culminating in the 6- to 90,000-fold induction of expression of 20/20 (100%) SMGs tested in HDFs. Transdifferentiation of HDFs directly to neurons led to expression of 193/516 (37.4%) of SMGs implicated in neurological disease. The magnitude and isoform diversity of SMG expression following either transactivation or transdifferentiation was comparable to clinically relevant tissues. We apply transdifferentiation and/or gene transactivation combined with short- and long-read RNA sequencing to investigate the impact that variants in USH2A, SCN1A, DMD, and PAK3 have on RNA using HDFs derived from affected individuals. Transactivation and transdifferentiation represent rapid, scalable functional genomic solutions to investigate variants impacting SMGs in the patient cell and genomic context.

Mucosal enrichment of the Adherent-Invasive E. coli (AIEC) pathotype and the expansion of pathogenic IFNγ-producing Th17 (pTh17) cells have been linked to Crohn\'s Disease (CD) pathogenesis. However, the molecular pathways underlying the AIEC-dependent pTh17 cell transdifferentiation in CD patients remain elusive. To this aim, we created and functionally screened a transposon AIEC mutant library of 10.058 mutants to identify the virulence determinants directly implicated in triggering IL-23 production and pTh17 cell generation. pTh17 cell transdifferentiation was assessed in functional assays by co-culturing AIEC-infected human dendritic cells (DCs) with autologous conventional Th17 (cTh17) cells isolated from blood of Healthy Donors (HD) or CD patients. AIEC triggered IL-23 hypersecretion and transdifferentiation of cTh17 into pTh17 cells selectively through the interaction with CD-derived DCs. Moreover, the chronic release of IL-23 by AIEC-colonized DCs required a continuous IL-23 neutralization to significantly reduce the AIEC-dependent pTh17 cell differentiation. The multi-step screenings of the AIEC mutant\'s library revealed that deletion of ybaT or rfaP efficiently hinder the IL-23 hypersecretion and hampered the AIEC-dependent skewing of protective cTh17 into pathogenic IFNγ-producing pTh17 cells. Overall, our findings indicate that ybaT (inner membrane transport protein) and rfaP (LPS-core heptose kinase) represent novel and attractive candidate targets to prevent chronic intestinal inflammation in CD.

Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by genomic integration of the Merkel cell polyomavirus (MCPyV). MCPyV-negative cases often present as combined MCCs, which represent a distinctive subset of tumors characterized by association of an MCC with a second tumor component, mostly squamous cell carcinoma. Up to now, only exceptional cases of combined MCC with neuroblastic differentiation have been reported. Herein we describe two additional combined MCCs with neuroblastic differentiation and provide comprehensive morphologic, immunohistochemical, transcriptomic, genetic and epigenetic characterization of these tumors, which both arose in elderly men and appeared as an isolated inguinal adenopathy. Microscopic examination revealed biphasic tumors combining a poorly differentiated high-grade carcinoma with a poorly differentiated neuroblastic component lacking signs of proliferation. Immunohistochemical investigation revealed keratin 20 and MCPyV T antigen (TA) in the MCC parts, while neuroblastic differentiation was confirmed in the other component in both cases. A clonal relation of the two components can be deduced from 20 and 14 shared acquired point mutations detected by whole exome analysis in both combined tumors, respectively. Spatial transcriptomics demonstrated a lower expression of stem cell marker genes such as SOX2 and MCM2 in the neuroblastic component. Interestingly, although the neuroblastic part lacked TA expression, the same genomic MCPyV integration and the same large T-truncating mutations were observed in both tumor parts. Given that neuronal transdifferentiation upon TA repression has been reported for MCC cell lines, the most likely scenario for the two combined MCC/neuroblastic tumors is that neuroblastic transdifferentiation resulted from loss of TA expression in a subset of MCC cells. Indeed, DNA methylation profiling suggests an MCC-typical cellular origin for the combined MCC/neuroblastomas. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Death of mechanosensory hair cells in the inner ear is a common cause of auditory and vestibular impairment in mammals, which have a limited ability to regrow these cells after damage. In contrast, non-mammalian vertebrates, including zebrafish, can robustly regenerate hair cells after severe organ damage. The zebrafish inner ear provides an understudied model system for understanding hair cell regeneration in organs that are highly conserved with their mammalian counterparts. Here, we quantitatively examine hair cell addition during growth and regeneration of the larval zebrafish inner ear. We used a genetically encoded ablation method to induce hair cell death and we observed gradual regeneration with correct spatial patterning over a 2-week period following ablation. Supporting cells, which surround and are a source of new hair cells, divide in response to hair cell ablation, expanding the possible progenitor pool. In parallel, nascent hair cells arise from direct transdifferentiation of progenitor pool cells temporally uncoupled from supporting cell division. These findings reveal a previously unrecognized mechanism of hair cell regeneration with implications for how hair cells may be encouraged to regenerate in the mammalian ear.

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Objective: To investigate the role and underlying mechanisms of methyltransferase (Mettl) 3 in the process of angiotensin Ⅱ (Ang Ⅱ)-induced pericyte-to-myofibroblast transdifferentiation and renal fibrosis. Methods: C57BL/6J mice were used, in cell experiments, mouse renal pericytes were isolated and cultured using magnetic bead sorting. These pericytes were then induced to transdifferentiate into myofibroblasts with 1×106 mmol/L Ang Ⅱ, which was the Ang Ⅱ group, while pericytes cultured in normal conditions served as the control group. Successful transdifferentiation was verified by immunofluorescence staining, Western blotting, and real-time reverse transcription PCR (RT-qPCR) for α-smooth muscle actin (α-SMA). The levels of m6A modifications and related enzymes (Mettl3, Mettl14), Wilms tumor 1-associated protein (WTAP), fat mass and obesity protein (FTO), ALKBH5, YTHDF1, YTHDF2, YTHDC1, YTHDC2, YTHDC3 were assessed by Dot blot, RT-qPCR and Western blot. Mettl3 expression was inhibited in cells using lentivirus-mediated Mettl3-shRNA transfection, creating sh-Mettl3 and Ang Ⅱ+sh-Mettl3 groups, while lentivirus empty vector transfection served as the negative control (Ang Ⅱ+sh-NC group). The impact of Ang Ⅱ on pericyte transdifferentiation was observed, and the expression of downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway proteins, including PI3K, AKT, phosphorylated AKT at serine 473 (p-AKT (S473)), and phosphorylated AKT at threonine 308 (p-AKT (T308)), were examined. PI3K gene transcription was inhibited by co-culturing cells with actinomycin D, and the half-life of PI3K mRNA was calculated by measuring residual PI3K mRNA expression over different co-culture time. The reversibility of Mettl3 inhibition on Ang Ⅱ-induced pericyte-to-myofibroblast transdifferentiation was assessed by adding the AKT activator SC79 to the Ang Ⅱ+sh-Mettl3 group. In animal experiments, mice were divided into these groups: sham group (administered 0.9% sterile saline), Ang Ⅱ group (infused with Ang Ⅱ solution), sh-Mettl3 group (injected with Mettl3 shRNA lentivirus solution), Ang Ⅱ+sh-Mettl3 group (infused with Ang Ⅱ solution and injected with Mettl3 shRNA lentivirus solution), and Ang Ⅱ+sh-Mettl3+SC79 group (administered Ang Ⅱ solution and Mettl3 shRNA lentivirus, with an additional injection of SC79). Each group consisted of six subject mice. Blood pressure was measured using the tail-cuff method before and after surgery, and serum creatinine, urea, and urinary albumin levels were determined 4 weeks post-surgery. Kidney tissues were collected at 28 days and stained using hematoxylin-eosin (HE) and Masson\'s trichrome to assess the extent of renal fibrosis. Results: Primary renal pericytes were successfully obtained by magnetic bead sorting, and intervened with 1×106 mmol/L Ang Ⅱ for 48 hours to induce pericyte-to-myofibroblast transdifferentiation. Dot blot results indicated higher m6A modification levels in the Ang Ⅱ group compared to the control group (P<0.05). RT-qPCR and Western blot results showed upregulation of Mettl3 mRNA and protein levels in the Ang Ⅱ group compared to the control group (both P<0.05). In the Ang Ⅱ+sh-Mettl3 group, Mettl3 protein expression was lower than that in the Ang Ⅱ group, with reduced expression levels of α-SMA, vimentin, desmin, fibroblast agonist protein (FAPa) and type Ⅰ collagen (all P<0.05). Compared to the control group, PI3K mRNA expression level was elevated in the Ang Ⅱ group, along with increased p-AKT (S473) and p-AKT (T308) expressions. In the Ang Ⅱ+sh-Mettl3 group, PI3K mRNA expression and p-AKT (S473) and p-AKT (T308) levels were decreased (all P<0.05). The half-life of PI3K mRNA was shorter in the Ang Ⅱ+sh-Mettl3 group than that in the Ang Ⅱ+sh-NC group (2.34 h vs. 3.42 h). The ameliorative effect of Mettl3 inhibition on Ang Ⅱ-induced pericyte-to-myofibroblast transdifferentiation was reversible by SC79. Animal experiments showed higher blood pressure, serum creatinine, urea, and 24-hour urinary protein levels, and a larger fibrosis area in the Ang Ⅱ group compared to the sham group (all P<0.05). The fibrosis area was smaller in the Ang Ⅱ+sh-Mettl3 group than that in the Ang Ⅱ group (P<0.05), but increased again upon addition of SC79. Conclusion: Mettl3-mediated RNA m6A epigenetic regulation is involved in Ang Ⅱ-induced pericyte-to-myofibroblast transdifferentiation and renal fibrosis, potentially by affecting PI3K stability and regulating the PI3K/AKT signaling pathway.
目的： 探讨甲基转移酶3（Mettl3）在血管紧张素Ⅱ（Ang Ⅱ）诱导周细胞向肌成纤维细胞转分化及肾脏纤维化过程中的作用及相关机制。 方法： 使用C57BL/6J小鼠，（1）细胞实验中，采用磁珠分选法培养纯化小鼠肾脏周细胞，并予以1×106 mmol/L的Ang Ⅱ诱导周细胞-肌成纤维细胞转分化，为Ang Ⅱ组；正常培养的周细胞为对照组。采用免疫荧光染色、蛋白质印迹（Western blot）及实时反转录PCR（RT-qPCR）检测α平滑肌肌动蛋白（α-SMA）以验证转分化成功。采用斑点印迹、RT-qPCR、Western blot检测N6-甲基腺苷（m6A）修饰水平及相关酶［Mettl3、Mettl14、Wilms肿瘤蛋白1相关蛋白（WTAP）、肥胖相关蛋白（FTO）、ALKB同源蛋白5（ALKBH5）、YTH结构域家族蛋白（YTHDF）1、YTHDF2、YTHDC1、YTHDC2、YTHDC3］的表达水平。采用慢病毒转染Mettl3 shRNA的方法抑制细胞中的Mettl3表达，为sh-Mettl3组、Ang Ⅱ+sh-Mettl3组；以转染慢病毒空载体作为阴性对照，为Ang Ⅱ+sh-NC组，观察Ang Ⅱ对周细胞转分化的影响，并检测下游磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）信号通路蛋白的表达，包括PI3K、AKT（丝氨酸磷酸化位点473）［p-AKT（S473）］、AKT（苏氨酸磷酸化位点308）［p-AKT（T308）］等。加入放线菌素D与各组周细胞共培养以抑制PI3K基因的转录，通过检测不同共培养时间残余PI3K mRNA表达量以计算PI3K mRNA半衰期。在Ang Ⅱ+sh-Mettl3组周细胞中加入AKT激动剂SC79，观察是否可逆转Mettl3抑制对Ang Ⅱ诱导周细胞-肌成纤维细胞转分化的作用。（2）动物实验中，分为假手术组（仅给予0.9%无菌生理盐水）、Ang Ⅱ组（泵入Ang Ⅱ溶液）、sh-Mettl3组（注射Mettl3 shRNA慢病毒溶液）、Ang Ⅱ+sh-Mettl3组（泵入Ang Ⅱ溶液+注射Mettl3 shRNA慢病毒溶液）、Ang Ⅱ+sh-Mettl3+SC79组（Ang Ⅱ+sh-Mettl3组处理基础上注射SC79），每组6只。手术前后采用尾夹法测定血压，术后4周测定血清肌酐、尿素含量及尿液白蛋白含量。28 d后取肾脏组织，采用苏木精-伊红（HE）及Masson三色法对组织切片进行染色，检测肾脏纤维化程度。 结果： （1）磁珠分选法可获得原代肾脏周细胞，以1×106 mmol/L的Ang Ⅱ处理周细胞48 h可成功诱导其向肌成纤维细胞转分化。斑点印迹结果显示，Ang Ⅱ组总RNA的m6A修饰水平高于对照组（P<0.05），RT-qPCR及Western blot结果显示，与对照组相比，Ang Ⅱ组中Mettl3 mRNA及蛋白表达水平上调（P均<0.05）。Ang Ⅱ+sh-Mettl3组细胞中的Mettl3蛋白表达水平低于Ang Ⅱ组，α-SMA、波形蛋白、肌间线蛋白、成纤维细胞激活蛋白a（FAPa）以及Ⅰ型胶原蛋白的表达水平亦低于Ang Ⅱ组（P均<0.05）。与对照组相比，Ang Ⅱ组的PI3K mRNA表达水平上调，且p-AKT（S473）和p-AKT（T308）蛋白高表达；Ang Ⅱ+sh-Mettl3组的PI3K mRNA表达水平低于Ang Ⅱ组，p-AKT（S473）和p-AKT（T308）蛋白表达下调（P均<0.05）。Ang Ⅱ+sh-Mettl3组的半衰期短于Ang Ⅱ+sh-NC组（2.34 h比3.42 h）。而Mettl3抑制对Ang Ⅱ诱导周细胞-肌成纤维细胞转分化的改善作用可被SC79逆转。（2）动物实验结果显示，与假手术组比较，Ang Ⅱ组小鼠的血压更高，血清肌酐、尿素及24 h尿蛋白测量值更高，纤维化面积更大（P均<0.05）；而Ang Ⅱ+sh-Mettle3组的纤维化面积小于Ang Ⅱ组（P<0.05），但在加入SC79后肾脏纤维化又加重。 结论： Mettl3介导的RNA m6A表观遗传调控参与Ang Ⅱ诱导的肾脏周细胞-肌成纤维细胞转分化及肾脏纤维化，Mettl3可能通过影响PI3K的稳定性，进而影响PI3K/AKT信号通路发挥调控作用。.

BACKGROUND: Neuroendocrine prostate cancer (NEPC), a lethal subset of prostate cancer (PCa), is characterized by loss of AR signaling and resistance to AR-targeted therapy. While it is well reported that second-generation AR blockers induce neuroendocrine (NE) trans-differentiation of castration-resistant prostate cancer (CRPC) to promote the occurrence of NEPC, and pluripotent transcription factors might be potential regulators, the underlying molecular mechanisms remain unclear.
METHODS: We analyzed the data from public databsets to screen candidate genes and then focused on SOX4, a regulator of NE trans-differentiation. The expression changes of SOX4 and its relationship with tumor progression were validated in clinical tumor tissues. We evaluated malignant characteristics related to NEPC in prostate cancer cell lines with stable overexpression or knockdown of SOX4 in vitro. Tumor xenografts were analyzed after inoculating the relevant cell lines into nude mice. RNA-seq, ATAC-seq, non-targeted metabolomics analysis, as well as molecular and biochemical assays were carried out to determine the mechanism.
RESULTS: We screened public datasets and identified that expression of SOX4 was significantly elevated in NEPC. Overexpressing SOX4 in C4-2B cells increased cell proliferation and migration, upregulated the expression of NE marker genes, and inhibited AR expression. Consistently, inhibition of SOX4 expression in DU-145 and PC-3 cells reduced the above malignant phenotypes and repressed the expression of NE marker genes. For the in vivo assay, we found that knockdown of SOX4 inhibited tumor growth of subcutaneous xenografts in castrated nude mice which were concomitantly treated with enzalutamide (ENZ). Mechanically, we identified that one of the key enzymes in gluconeogenesis, PCK2, was a novel target of SOX4. The activation of carbohydrate metabolism reprogramming by SOX4 could promote NE trans-differentiation via the SOX4/PCK2 pathway.
CONCLUSIONS: Our findings reveal that SOX4 promotes NE trans-differentiation both in vitro and in vivo via directly enhancing PCK2 activity to activate carbohydrate metabolism reprogramming. The SOX4/PCK2 pathway and its downstream changes might be novel targets for blocking NE trans-differentiation.

PDAC is a typical \"cold tumor\" characterized by low immune cell infiltration and a suppressive immune microenvironment. We previously observed the existence of a rare group of follicular helper T cells (Tfh) that could enhance antitumor immune responses by recruiting other immune cells in PDAC. In this study, we ectopically expressed BCL6 in CD4+ T cells, and successfully induced Tfh-like transdifferentiation in vitro. This strategy provided abundant Tfh-like cells (iTfhs) that can recruit CD8+ T cells like endogenous Tfhs. Subsequently, Chimeric Antigen Receptors (CARs) against both MSL (Mesothelin) and EPHA2 (Ephrin receptor A2) were used to modify iTfh cells, and the CAR-iTfh cells significantly improved infiltration and antitumor cytotoxicity of co-cultured CD8+ T cells. After that, combinatory administration of CAR-iTfh & CAR-CD8 T cell therapy displayed a better effect in repressing the PDAC tumors in xenograft mouse models, compared to conventional CAR-CD4 & CAR-CD8 combinations, and the models received the CAR-iTfh & CAR-CD8 T cells displayed a significantly improved survival rate. Our study revealed the plasticity of Thelper differentiation, expanded the source of Tfh-like cells for cell therapy, and demonstrated a novel and potentially more efficient cellular composition for CAR-T therapy.

UNASSIGNED: Cellular senescence, macrophages infiltration, and vascular smooth muscle cells (VSMCs) osteogenic transdifferentiation participate in the pathophysiology of vascular calcification in chronic kidney disease (CKD). Senescent macrophages are involved in the regulation of inflammation in pathological diseases. In addition, senescent cells spread senescence to neighboring cells via Interferon-induced transmembrane protein3 (IFITM3). However, the role of senescent macrophages and IFITM3 in VSMCs calcification remains unexplored.
UNASSIGNED: To explore the hypothesis that senescent macrophages contribute to the calcification and senescence of VSMCs via IFITM3.
UNASSIGNED: Here, the macrophage senescence model was established using Lipopolysaccharides (LPS). The VSMCs were subjected to supernatants from macrophages (MCFS) or LPS-induced macrophages (LPS-MCFS) in the presence or absence of calcifying media (CM). Senescence-associated β-galactosidase (SA-β-gal), Alizarin red (AR), immunofluorescent staining, and western blot were used to identify cell senescence and calcification.
UNASSIGNED: The expression of IFITM3 was significantly increased in LPS-induced macrophages and the supernatants. The VSMCs transdifferentiated into osteogenic phenotype, expressing higher osteogenic differentiation markers (RUNX2) and lower VSMCs constructive makers (SM22α) when cultured with senescent macrophages supernatants. Also, senescence markers (p16 and p21) in VSMCs were significantly increased by senescent macrophages supernatants treated. However, IFITM3 knockdown inhibited this process.
UNASSIGNED: Our study showed that LPS-induced senescence of macrophages accelerated the calcification of VSMCs via IFITM3. These data provide a new perspective linking VC and aging, which may provide clues for diagnosing and treating accelerated vascular aging in patients with CKD.