Functional Rescue Research Articles

M6A-Methylated NUTM2B-AS1 Promotes Hepatocellular Carcinoma Stemness Feature via Epigenetically Activating BMPR1A Transcription.

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies in the world. Oncofetal proteins are the optimal diagnostic biomarkers and therapeutic targets for HCC. As the most abundant modification in RNA, N6-methyladenosine (m6A) has been reported to be involved in HCC initiation and progression. However, whether m6A has oncofetal characteristics remains unknown. Gene expression in HCC tissues and cells was detected using qPCR. The level of m6A methylation was determined using methylated RNA immunoprecipitation assay. The biological roles of NUTM2B-AS1 in HCC were detected using Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine incorporation, and spheroid formation assays. The mechanisms underlying the roles of NUTM2B-AS1 were explored using RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), chromatin immunoprecipitation (ChIP), and assay for transposase-accessible chromatin (ATAC). NUTM2B-AS1 was identified as a novel oncofetal long noncoding RNA that was upregulated in the fetal liver and HCC and silenced in adult liver tissues. METTL3 and METTL16 induce m6A hypermethylation of NUTM2B-AS1. The m6A methylation levels of NUTM2B-AS1 exhibit oncofetal characteristics. m6A methylation upregulates NUTM2B-AS1 expression by increasing NUTM2B-AS1 transcript stability. m6A-methylated NUTM2B-AS1 promotes HCC cell proliferation and stemness via epigenetically activating BMPR1A expression. NUTM2B-AS1 specifically binds to BMPR1A promoter. m6A-methylated NUTM2B-AS1 is recognized by the m6A reader YTHDC2, which further binds to the H3K4 methyltransferase MLL1. m6A-methylated NUTM2B-AS1 recruits YTHDC2 and MLL1 to BMPR1A promoter, leading to increased H3K4me3 and chromatin accessibility at BMPR1A promoter. Functional rescue assays suggest that BMPR1A is a critical mediator of the oncogenic role of m6A-methylated NUTM2B-AS1 in HCC. METTL3- and METTL16-mediated m6A methylation of NUTM2B-AS1 is a novel oncofetal molecular event in HCC that promotes HCC stemness via epigenetically activating BMPR1A transcription.

Delayed functional rescue of section of the flexor tendons of the last four (4) fingers at a gold miner in a Sahelian hospital in Niamey (Niger)

Introduction: Secondary reconstructions of flexor tendon section have become rare nowadays. We report the result of delayed functional rescue of a traumatic section of the hand flexor tendons in a young adult. Clinical case: This is a 20-year-old patient, admitted for inability to flex the last 4 fingers of the right hand. The interrogation revealed a history of open trauma to the hand from a stab dating back 3 months following a fight. The physical examination found a transverse scar located on the palmar aspect of the right hand measuring 6 cm in zone three (3) of Verdan. Exploration in the operating room revealed a complete section of the superficial and deep flexor tendons of the last four (4) fingers. An end-to-end suture of the tendons was performed using Kessler stitches using Nylon thread 4.0. After one (1) year and eight (8) months of follow up, we found an excellent result according to the IFSSH (International Federation of Societies for Surgery of the Hand) classification with a score of 88/100. Conclusion: Secondary repair of late flexor tendon ruptures by end-to-end suture gives good results and remains an alternative when a tendon graft is impossible. Keywords: Secondary suture, Flexor tendons, Hand, Niger.

Serum-Derived Exosomal TBX2-AS1 Exacerbates COPD by Altering the M1/M2 Ratio of Macrophages through Regulating the miR-423-5p/miR-23b-3p Axis

ABSTRACT Objective To investigate the mechanism of serum exosomes in chronic obstructive pulmonary disease (COPD), especially the effect of lncRNA TBX2-AS1 on macrophage polarization. Methods Screen differentially expressed genes through bioinformatics analysis, detect the expression of related molecules in clinical samples and cell experiments, construct a mouse model and conduct functional rescue experiments, using various experimental techniques such as RT - qPCR, Western Blot, flow cytometry, ELISA, and luciferase reporter assay. Results TBX2-AS1 is highly expressed in the serum and serum exosomes of COPD patients, and it can promote macrophage M1 polarization and inhibit M2 polarization; it exerts its role by negatively regulating the miR-423-5p/miR-23b - 3p axis, where miR-423-5p inhibits CELSR2 expression to prevent M1 polarization, and miR-23b-3p inhibits NEK6 expression to promote M2 polarization; in vivo experiments, down-regulation of CELSR2/NEK6 can reverse the promoting effect of COPD serum exosomes on lung injury and inflammation. Conclusion COPD serum exosomes deliver TBX2-AS1 to macrophages, regulate the miR-423-5p-CELSR2/miR-23b-3p-NEK6 pathway, affect macrophage polarization, and exacerbate the progression of COPD, providing new directions and potential targets for the diagnosis and treatment of COPD.

Pharmacological rescue of the G85E CFTR variant by preclinical and approved modulators.

Cystic Fibrosis (CF) is a genetic disease due to loss-of-function mutations of the CFTR channel. F508del is the most frequent mutation (70% of alleles in Italy), while other mutations have much lower frequency. Among them, G85E (0.4% frequency globally, 1.13% in Italy) emerges as a mutation characterized by a severe CFTR folding and trafficking defect. To investigate the pharmacological responsiveness of the G85E-CFTR variant, we performed a functional and biochemical characterization in heterologous expression systems and ex vivo models based on patient-derived human nasal epithelial cells (HNEC). Our study demonstrated that treatment of primary airway cells with elexacaftor and tezacaftor causes a significant (although modest) rescue of CFTR function, that reaches 15%-25% of the activity measured in non-CF epithelia. A detrimental effect of chronic treatment with ivacaftor, further limiting G85E rescue, was also observed. A higher rescue of CFTR function, up to 25%-35% of the normal CFTR activity, with no evidence of negative effects upon chronic potentiator treatment, can be achieved by combining elexacaftor with ARN23765, a novel type 1 corrector endowed with very high potency. Importantly, dose-response relationships suggest that G85E might alter the binding of type 1 correctors, possibly affecting their affinity for the target. In conclusion, our studies suggest that novel combinations of modulators, endowed with higher efficacy leading to increased rescue of G85E-CFTR, are needed to improve the clinical benefit in patients for this variant.

Exosomal microRNA-21-5p from gastric cancer cells promotes angiogenesis by targeting LEMD3 in human endothelial cells

Abstract Objectives The effect of exosome-derived miR-21-5p from gastric cancer (GC) on angiogenesis remains unclear. This study aims to examine the angiogenic impact of GC exosome-derived miR-21-5p. Methods Exosomes were isolated from GC cells and co-cultured with human umbilical vein endothelial cells (HUVECs). miR-21-5p levels in HUVECs were measured by qPCR. Flow cytometry was used to assess apoptosis, and the Cell Counting Kit-8 was used to assess cell growth. Bioinformatics analysis was used to identify the miR-21-5p target genes, which were then verified by dual-luciferase gene reporter experiments. qPCR and western blotting were employed to assess the expression of genes and proteins, respectively. Functional rescue assays were conducted to verify that miR-21-5p regulates endothelial cell function by targeting the LEM domain containing 3 (LEMD3). Additionally, cell migration was assessed using a scratch assay. Results Co-cultivation with GC-derived exosomes improved cell survival, decreased apoptosis, and raised miR-21-5p levels in HUVECs. Increases in vascular endothelial growth factor A (VEGFA) and the TGF-β/Smad signaling pathway were seen. It was shown that miR-21-5p targets LEMD3. The biological effects of miR-21-5p were lessened when miR-21-5p was inhibited, or LEMD3 was overexpressed. Conclusions By targeting LEMD3, miR-21-5p in GC cell exosomes stimulates angiogenesis by triggering the TGF-β/Smad signaling cascade and upregulating VEGFA expression. This leads to increased proliferation, survival, and migration of HUVECs, underscoring the potential of targeting this pathway in cancer therapy.

ZSCAN16 expedites hepatocellular carcinoma progression via activating TBC1D31

ObjectiveHepatocellular carcinoma (HCC) is fatal and poses great challenges to early diagnosis and effective treatment. This paper sought to expound the function of Zinc finger and SCAN domain-containing protein 16 (ZSCAN16) and TBC1 domain family member 31 (TBC1D31) in HCC progression.MethodsZSCAN16 and TBC1D31 levels were detected by RT-qPCR, Western blot, and immunohistochemistry. The transcriptional regulation of TBC1D31 by ZSCAN16 was demonstrated by ChIP-qPCR and dual-luciferase assay. Colony formation assay, migration and invasion assays, TUNEL staining, CCK-8 assay, flow cytometry, and western blot analysis were adopted to evaluate the biological activity of HCC cells. The role of the ZSCAN16/TBC1D31 axis in HCC was demonstrated by lentiviral gene intervention combined with functional rescue experiments. Hep3B cells were used to establish a nude mouse xenograft tumor model to study the role of the ZSCAN16/TBC1D31 axis in vivo.ResultsZSCAN16 and TBC1D31 were highly expressed in HCC. Downregulation of ZSCAN16 repressed the proliferation, migration, and invasion of HCC cells while promoting apoptosis, as well as curbing tumor growth in vivo. Mechanistic studies showed that ZSCAN16 mediated the transcriptional activation of TBC1D31, which in turn led to tumor development. TBC1D31 overexpression reversed the inhibitory effect of ZSCAN16 knockdown on the malignant behavior and tumor growth of HCC cells and accelerated tumor development.ConclusionZSCAN16 mediates transcriptional activation of TBC1D31 and promotes HCC progression.

Deep functional measurements of Fragile X syndrome human neurons reveal multiparametric electrophysiological disease phenotype

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by hypermethylation of expanded CGG repeats (>200) in the FMR1 gene leading to gene silencing and loss of Fragile X Messenger Ribonucleoprotein (FMRP) expression. FMRP plays important roles in neuronal function, and loss of FMRP in mouse and human FXS cell models leads to aberrant synaptic signaling and hyperexcitability. Multiple drug candidates have advanced into clinical trials for FXS, but no efficacious treatment has been identified to date, possibly as a consequence of poor translation from pre-clinical animal models to human. Here, we use a high resolution all-optical electrophysiology platform applied to multiple FXS patient-derived and CRISPR/Cas9-generated isogenic neuronal cell lines to develop a multi-parametric FXS disease phenotype. This neurophysiological phenotype was optimized and validated into a high throughput assay based on the amount of FMRP re-expression and the number of healthy neurons in a mosaic network necessary for functional rescue. The resulting highly sensitive and multiparameter functional assay can now be applied as a discovery platform to explore new therapeutic approaches for the treatment of FXS.

Identification and Validation of circDOCK1/miR-138-5p/GRB7 Axis for Promoting Breast Cancer Progression.

Non-coding RNAs have received increasing attention in human tumors, with RNA interaction networks playing important roles in breast cancer. This study aims to explore novel circular RNAs and their mechanisms of biological function in breast cancer. Six HER2-positive breast cancer tissues and paired normal tissues were obtained for the whole transcriptome RNA sequencing. Differentially expressed (DE) circRNAs, miRNAs and mRNAs were identified and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DERNAs were performed. DECircRNAs- DEmiRNAs- DEmRNAs networks were constructed and further verified by bioinformatics database analyses, luciferase assays and RIP assays. The expression level of circDOCK1 in breast cancer specimens was measured using qRT-PCR. Functional rescue experiments were conducted to explore the role of circDOCK1/miR-138-5p/GRB7 axis in breast cancer cells. The correlation of circDOCK1 expression and clinicopathologic features of 102HER2 positive breast cancer patients was analyzed. A total of 6960 DEmRNAs, 133 DE miRNAs and 1691 DE circRNAs were identified from HER2-positive breast cancer tissues and paracancerous tissues. Enrichment Analysis showed that the differential mRNAs were associated with cell division in biological processes and cell cycle and signaling pathways. GO and KEGG analysis demonstrated that DE circRNAs were mainly enriched in double-strand break repair, positive regulation of transcription by RNA polymerase II, nucleoplasma, nucleus, chromatin binding and protein binding. Forty networks of competing endogenous RNAs (ceRNAs) were constructed and circDOCK1/miR-138-5p/GRB7 axis was verified. Functional experiments revealed that the axis promotes migration and invasion of breast cancer cells. CircDOCK1 expression was elevated in breast cancer patients and correlated with adverse clinicopathologic parameters. Patients with high circDOCK1 level had poor outcomes. A novel circDOCK1/miR-138-5p/GRB7 axis promotes HER2 positive breast cancer metastasis and progression, providing a potential therapeutic target in the treatment of breast cancer.

Ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of CF. The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex, which was previously found to modulate ribosome collisions during "preemptive quality control" of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest that interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.

Pulmonary and extra-pulmonary effects of lung transplantation in an Italian cohort of patients with systemic sclerosis

Abstract Objectives Lung transplantation (LuTx) is a life-saving intervention for SSc patients with end-stage lung disease. The aim of this study was to evaluate patients’ survival and LuTx outcomes on systemic disease manifestations. Methods A retrospective evaluation was conducted on SSc patients who underwent LuTx between 2010 and 2021. Outcomes assessed at baseline, 6, 12 and 24 months post-LuTx included skin involvement by modified Rodnan skin score (mRSS), and global disease activity using a modified EUSTAR index (0–9 scale). Lung function rescue was evaluated by forced vital capacity (FVC). Patient survival was assessed by Kaplan–Meier analysis. Results Thirteen SSc patients were included, with a male/female ratio 9/4 and a median age of 48.7 years. Nine patients were affected by diffuse cutaneous scleroderma (dcSSc) and four by limited cutaneous scleroderma (lcSSc). FVC significantly increased from 56% of the predicted value at baseline to 78% at 2 years (P = 0.003). mRSS decreased from 7.4 ± 3.8 to 3.3 ± 2.5 in patients with dcSSc (P = 0.02). The modified EUSTAR index score decreased from 2.54 ± 1.8 at baseline to 0.49 ± 0.5 at 2 years (P = 0.02). Survival rate was 92.3% at 2 years, and 76.9% at 5 years. No unexpected adverse events were observed. Conclusions In SSc patients undergoing LuTx, an excellent 2-year survival was observed, without any disease-related adverse events. Our study supports LuTx as a viable option in SSc patients with end-stage lung disease. Apart from expected recovery of lung function, LuTx was associated with improvement of mRSS and global systemic disease activity.

The functional rescue of novel epilepsy-linked missense mutations in the human GABA transporter 1 by pharmacochaperoning

The functional rescue of novel epilepsy-linked missense mutations in the human GABA transporter 1 by pharmacochaperoning

Casticin suppresses self-renewal related stemness via miR-342-3p-mediated FoxM1 downregulation in cervical cancer cells

BackgroundCasticin (CAS), a natural flavonoid found in Viticis Fructus, Viticis Cannabifoliae Fructus, and Semen Euphorbiae, shows anti-inflammatory activity and efficacy against various cancers. However, its effect on stemness associated with self-renewal in cervical cancer (CC) cells remains unclear, as well as the underlying mechanism. PurposeThe primary objective of this study was to examine the effect of CAS on CC stemness and to explore the underpinning regulatory mechanism. MethodsHeLa cells underwent treatment with varying concentrations of CAS (0, 10, 30, 100 nM). To evaluate the impacts of CAS on CC stemness and tumorigenicity, sphere- and colony-formation assays and a xenograft model were employed. The study involved screening for changes in miRNAs and their target genes. The miRNA array identified an upregulation in miRNAs, whereas the mRNA array detected a downregulation of specific target genes. The latter genes were found to regulate stem cell-related genes through miR-342-3p in HeLa cells administered CAS. Next, whether miR-342-3p directly targets FOXM1 when upregulated by CAS was assessed by the luciferase reporter assay. qRT-PCR was performed to analyze miR-342-3p expression. Additionally, immunoblotting was conducted to assess the protein amounts of FoxM1 and stemness-related factors (CD133, CD49f, Nanog, and Sox2). Function rescue experiments were conducted to determine the mechanism of CAS in stemness regulation. These experiments involved utilizing a miR-342-3p inhibitor and overexpressing FOXM1 in HeLa cells. ResultsCAS decreased in vitro stemness, suppressing sphere- and colony-formation capabilities of CC. It also dose-dependently downregulated the expression of stemness-associated proteins, including CD133, CD49f, Nanog, and Sox2. Moreover, CAS inhibited in vivo carcinogenesis, remarkably reducing tumor growth in mice bearing HeLa cell xenografts. Analysis revealed downregulated FOXM1 expression in HeLa cells treated with CAS. In the luciferase reporter assay, miR-342-3p was found to directly target FOXM1 in CAS-treated HeLa cells. Additionally, miR-342-3p inhibitor transfection successfully rescued CAS’ suppressive impact on stemness. Furthermore, overexpression of FOXM1 did not induce changes in miR-342-3p expression. However, it effectively rescued CAS’ suppressive effects on stemness. Moreover, CAS also inhibited stemness, upregulated miR-342-3p, and lowered FOXM1 expression in the SiHa cell line. ConclusionCAS suppresses self-renewal-associated stemness by targeting FOXM1 via miR-342-3p upregulation. These findings suggest CAS is promising as a novel therapeutic candidate in CC.

RBM8A, a new target of TEAD4, promotes breast cancer progression by regulating IGF1R and IRS-2

BackgroundBreast cancer (BC) is the most common malignant tumor in women worldwide, and further elucidation of the molecular mechanisms involved in BC pathogenesis is essential to improve the prognosis of BC patients. RNA Binding Motif Protein 8 A (RBM8A), with high affinity to a myriad of RNA transcripts, has been shown to play a crucial role in genesis and progression of multiple cancers. We attempted to explore its functional significance and molecular mechanisms in BC.MethodsBioinformatics analysis was performed on publicly available BC datasets. qRT-PCR was used to determine the expression of RBM8A in BC tissues. MTT assay, clone formation assay and flow cytometry were employed to examine BC cell proliferation and apoptosis in vitro. RNA immunoprecipitation (RIP) and RIP-seq were used to investigate the binding of RBM8A/EIF4A3 to the mRNA of IGF1R/IRS-2. RBM8A and EIF4A3 interactions were determined by co-immunoprecipitation (Co-IP) and immunofluorescence. Chromatin immunoprecipitation (Ch-IP) and dual-luciferase reporter assay were carried out to investigate the transcriptional regulation of RBM8A by TEAD4. Xenograft model was used to explore the effects of RBM8A and TEAD4 on BC cell growth in vivo.ResultsIn this study, we showed that RBM8A is abnormally highly expressed in BC and knockdown of RBM8A inhibits BC cell proliferation and induces apoptosis in vitro. EIF4A3, which phenocopy RBM8A in BC, forms a complex with RBM8A in BC. Moreover, EIF4A3 and RBM8A complex regulate the expression of IGF1R and IRS-2 to activate the PI3K/AKT signaling pathway, thereby promoting BC progression. In addition, we identified TEAD4 as a transcriptional activator of RBM8A by Ch-IP, dual luciferase reporter gene and a series of functional rescue assays. Furthermore, we demonstrated the in vivo pro-carcinogenic effects of TEAD4 and RBM8A by xenograft tumor experiments in nude mice.ConclusionCollectively, these findings suggest that TEAD4 novel transcriptional target RBM8A interacts with EIF4A3 to increase IGF1R and IRS-2 expression and activate PI3K/AKT signaling pathway, thereby further promoting the malignant phenotype of BC cells.

TP53AIP1 induce autophagy via the AKT/mTOR signaling pathway in the breast cancer cells

ABSTRACT Breast cancer ranks the first in the incidence of female cancer and is the most common cancer threatening the life and health of women worldwide.Tumor protein p53-regulated apoptosis-inducing protein 1 (TP53AIP1) is a pro-apoptotic gene downstream of p53. However, the role of TP53AIP1 in BC needs to be investigated. In vitro and in vivo experiments were conducted to assess the biological functions and associated mechanisms. Several bioinformatics analyses were made, CCK8 assay, wound healing, transwell assays, colony formation assay, EDU, flow cytometry, Immunofluorescence, qRT-PCR and Western-blotting were performed. In our study, we discovered that BC samples had low levels of TP53AIP1 expression, which correlated with a lower survival rate in BC patients. When TP53AIP1 was up-regulated, it caused a decrease in cell proliferation, migration, and invasion. It also induced epithelial-to-mesenchymal transition (EMT) and protective autophagy. Furthermore, the over-expression of TP53AIP1 suppressed tumor growth when tested in vivo. We also noticed that TP53AIP1 up-regulation resulted in decreased levels of phosphorylation in AKT and mTOR, suggesting a mechanistic role. In addition, we performed functional rescue experiments where the activation of AKT was able to counteract the impact of TP53AIP1 on the survival and autophagy in breast cancer cell lines. This suggests that TP53AIP1 acts as an oncogene by controlling the AKT/mTOR pathway. These findings reveal TP53AIP1 as a gene that suppresses tumor growth and triggers autophagy through the AKT/mTOR pathway in breast cancer cells. As a result, TP53AIP1 presents itself as a potential target for novel therapeutic approaches in treating breast cancer.

The RhoGEF protein Plekhg5 self-associates via its PH domain to regulate apical cell constriction.

RhoGEFs are critical activators of Rho family small GTPases and regulate diverse biological processes, such as cell division and tissue morphogenesis. We reported previously that the RhoGEF gene plekhg5 controls apical constriction of bottle cells at the blastopore lip during Xenopus gastrulation, but the detailed mechanism of plekhg5 action is not understood in depth. In this study, we show that localization of Plekhg5 in the apical cortex depends on its N-terminal sequences and intact guanine nucleotide exchange activity, whereas the C-terminal sequences prevent ectopic localization of the protein to the basolateral compartment. We also reveal that Plekhg5 self-associates via its PH domain, and this interaction leads to functional rescue of two mutants that lack the N-terminal region and the guanine nucleotide exchange factor activity, respectively, in trans. A point mutation in the PH domain corresponding to a variant associated with human disease leads to loss of self-association and failure of the mutant to induce apical constriction. Taken together, our results suggest that PH-mediated self-association and N-terminal domain-mediated subcellular localization are both crucial for the function of Plekhg5 in inducing apical constriction.

Activation of SIRT3 by Tanshinone IIA ameliorates renal fibrosis by suppressing the TGF-β/TSP-1 pathway and attenuating oxidative stress

Although doxorubicin (DOX) is a common chemotherapeutic drug, the serious nephrotoxicity caused by DOX-induced renal fibrosis remains a considerable clinical problem. Tanshinone IIA (Tan IIA), a compound extracted from Salvia miltiorrhiza, has been reported to have an anti-fibrotic effect. Therefore, this study investigated the molecular pathway whereby Tan IIA protects the kidneys from DOX administration. DOX (3 mg/kg body weight) was intraperitoneally administered every 3 d for a total of 7 injections (cumulative dose of 21 mg/kg) to induce nephrotoxicity. Then, Tan IIA (5 or 10 mg/kg/d) was administered by intraperitoneal injection for 28 d. In an in vitro study, 293 T cells were cultured and treated with DOX and Tan IIA for 24 h. Tan IIA reduced the blood urea nitrogen levels elevated by DOX while increasing superoxide dismutase activity, down-regulating reactive oxygen species, ameliorating renal-tubule thickening, and rescuing mitochondrial morphology. Additionally, Tan IIA reduced the renal collagen deposition, increased ATP production and complex-I activity, down-regulated transforming growth factor-β1 (TGF-β1) and thrombospondin-1 (TSP-1), and up-regulated sirtuin 3 (SIRT3). Tan IIA significantly increased cell viability. Additionally, RNA interference was employed to silence the expression of SIRT3, which eliminated the effect of Tan IIA in suppressing the expression of TGF-β1 and TSP-1. In conclusion, Tan IIA ameliorated DOX-induced nephrotoxicity by attenuating oxidative injury and fibrosis. The Tan IIA-induced rescue of mitochondrial morphology and function while alleviating renal fibrosis may be associated with the activation of SIRT3 to suppress the TGF-β/TSP-1 pathway.

LIM kinase 2 activates cardiac fibroblasts and exacerbates postinfarction left ventricular remodeling via crosstalk between the canonical and non-canonical Wnt pathways

Ischemic heart failure rates rise despite decreased acute myocardial infarction (MI) mortality. Excessive myofibroblast activation post-MI leads to adverse remodeling. LIM kinases (LIMK1 and LIMK2) regulate cytoskeleton homeostasis and are pro-fibrotic markers in atrial fibrillation. However, their roles and mechanisms in postinfarction fibrosis and ventricular remodeling remain unclear. This study found that the expression of LIMKs elevated in the border zone (BZ) in mice MI models. LIMK1/2 double knockout (DKO) restrained pathological remodeling and reduced mortality by suppressing myofibroblast activation. By using adeno-associated virus (AAV) with a periostin promoter to overexpress LIMK1 or LIMK2, this study found that myofibroblast-specific LIMK2 overexpression diminished these effects in DKO mice, while LIMK1 did not. LIMK2 kinase activity was critical for myofibroblast proliferation by using AAV overexpressing mutant LIMK2 lack of kinase activity. According to phosphoproteome analysis, functional rescue experiments, co-immunoprecipitation, and protein-protein docking, LIMK2 led to the phosphorylation of β-catenin at Ser 552. LIMK2 nuclear translocation also played a role in myofibroblast proliferation after MI with the help of AAV overexpressing mutant LIMK2 without nuclear location signal. Chromatin immunoprecipitation sequencing identified that LIMK2 bound to Lrp6 promoter region in TGF-β treated cardiac fibroblasts, positively regulating Wnt signaling via Wnt receptor internalization. This study demonstrated that LIMK2 promoted myofibroblast proliferation and adverse cardiac remodeling after MI, by enhancing phospho-β-catenin (Ser552) and Lrp6 signaling. This suggested that LIMK2 could be a target for the treatment of postinfarction injury.

Partial rescue of the full-field electroretinogram in patients with RPE65-related retinal dystrophy following gene augmentation therapy with voretigene neparvovec-rzyl.

To present a series of patients with RPE65-related retinal dystrophy showing a partial rescue of the full-field electroretinogram (ERG) following gene replacement therapy with voretigene neparovec-rzyl (Luxturna®). This retrospective chart review examined 17 patients treated with voretigene neparovec-rzyl (VN) at the Casey Eye Institute (2018-2022). The last pre-operative ERG and all available post-operative ERGs were analyzed to identify subjects with functional rescue. Measurements of amplitudes and implicit times were compared to data from age-matched controls and the attenuation relative to the lower limit of normal (LLN) was calculated. For comparison with other functional exams, the last pre-operative and all post-treatment best-corrected visual acuity (BCVA) data, visual field (VF) tests and full-field threshold stimulus tests (FST) were also described. Of patients who underwent ERGs, most had unrecordable ERGs that did not change after treatment. However, we identified three patients, treated bilaterally, who demonstrated partial rescue of the full-field ERG in both eyes which was sustained during the course of the study. This is the largest series of patients treated with VN showing a partial rescue of the ERG. This is also the first report of bilateral ERG rescue, as well as the first description of ERG recovery occurring in non-pediatric subjects. Full-field ERG could be used in combination with other psychophysical tests and imaging modalities to detect and deepen our understanding of the response to this gene therapy approach.

Synergistic rescue of temperature-sensitive p53 mutants by hypothermia and arsenic trioxide.

The p53 tumor suppressor is inactivated by mutations in about 50% of tumors. Rescuing the transcriptional function of mutant p53 has potential therapeutic benefits. Approximately 15% of p53 mutants are temperature sensitive (TS) and regain maximal activity at 32°C. Proof of concept study showed that induction of 32°C hypothermia in mice restored TS mutant p53 activity and inhibited tumor growth. However, 32°C is the lower limit of therapeutic hypothermia procedures for humans. Higher temperatures are preferable but result in suboptimal TS p53 activation. Recently, arsenic trioxide (ATO) was shown to rescue the conformation of p53 structural mutants by stabilizing the DNA binding domain. We examined the responses of 17 frequently observed p53 TS mutants to functional rescue by temperature shift and ATO. The results showed that ATO only rescued mild p53 TS mutants with high basal activity at 37°C. Mild TS mutants showed a common feature of regaining significant activity at the semi-permissive temperature of 35°C and could be further stimulated by ATO at 35°C. TS p53 rescue by ATO was antagonized by the cellular redox mechanism and was rapidly reversible. Inhibition of glutathione (GSH) biosynthesis enhanced ATO rescue efficiency and sustained p53 activity after ATO washout. The results suggest that mild TS p53 mutants are uniquely responsive to functional rescue by ATO due to small thermostability deficits and inherent potential to regain active conformation. Combining mild hypothermia and ATO may provide an effective and safe procedure for targeting tumors with p53 TS mutations.

Knockdown of WISP1/DKK1 restrains phenotypic plasticity in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition and stemness.

Wnt-induced signaling protein 1 (WISP1) and Dickkopf-1 (DKK1) are highly expressed in esophageal squamous cell carcinoma (ESCC), but no direct connection was identified between them. Phenotypic plasticity is a hallmark of ESCC. This research intended to identify the association between WISP1 and DKK1 and their roles in the phenotypic plasticity of ESCC. Genes differentially expressed in esophageal carcinoma were analyzed in the GEO database, followed by analyses of GO and KEGG enrichment to screen the hub gene. WISP1 expression and DKK1 secretion was assessed in ESCC tissues and cells. The tumor xenograft and in vivo metastasis models were established by injecting ESCC cells into nude mice. Functional deficiency and rescue experiments were conducted, followed by assays for cell proliferation, migration/invasion, stemness, epithelial-mesenchymal transition (EMT), and apoptosis, as well as tumor volume, weight, proliferation, stemness, and lung metastasis. The binding relationship and co-expression of WISP1 and DKK1 were determined. WISP1 and DKK1 were upregulated in ESCC cells and tissues, and WISP1 was enriched in the cell stemness and Wnt pathways. WISP1 knockdown subdued proliferation, migration/invasion, EMT activity, and stemness but enhanced apoptosis in ESCC cells. WISP1 knockdown restrained ESCC growth, proliferation, stemness, and metastasis in vivo. WISP1 bound to DKK1 in ESCC. DKK1 overexpression abolished the repressive impacts of WISP1 knockdown on the malignant behaviors of ESCC cells in vitro and of ESCC tumor in vivo. Knockdown of WISP1/DKK1 restrains the phenotypic plasticity in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition and stemness.