Heterozygous variants in USP25 cause genetic generalized epilepsy.

USP25 encodes ubiquitin-specific proteases 25, a key member of deubiquitinating enzyme family and is involved in neural fate determination. Although abnormal expression in Down's syndrome was reported previously, the specific role of USP25 in human diseases has not been defined. In this study, we performed trio-based whole exome sequencing in a cohort of 319 cases (families) with generalized epilepsy of unknown etiology. Five heterozygous USP25 variants including two de novo and three co-segregated variants were determined in eight individuals affected by generalized seizures and/or febrile seizures from five unrelated families. The frequency of USP25 variants showed a significantly high aggregation in this cohort compared to the East Asian population and all populations in the gnomAD database. The mean onset ages of febrile and afebrile seizures were 10 months (infancy) and 11.8 years (juvenile), respectively. The patients achieved seizure freedom except one had occasional nocturnal seizures at the last follow-up. Two patients exhibited intellectual disability. Usp25 was ubiquitously expressed in mouse brain with two peaks on embryonic days (E14‒E16) and postnatal day 21, respectively. Similarly, USP25 expressed in fetus/early childhood stage with a second peak at approximately 12‒20 years old in human brain, consistent with the seizure onset age at infancy and juvenile in the patients. To investigate the functional impact of USP25 deficiency in vivo, we established Usp25 knock-out mice, which showed increased seizure susceptibility compared to wild-type mice in pentylenetetrazol-induced seizure test. To explore the impact of USP25 variants, we employed multiple functional detections. In HEK293T cells, the severe phenotype associated variant (p.Gln889Ter) led to a significant reduction of mRNA and protein expressions but formed a stable truncated dimers with increment of deubiquitinating enzyme activities and abnormal cellular aggregations, indicating a gain-of-function effect. The p.Gln889Ter and p.Leu1045del increased neuronal excitability in mice brain, with a higher firing ability in p.Gln889Ter. These functional impairments align with the severity of the observed phenotypes, suggesting a genotype-phenotype correlation. Hence, a moderate association between USP25 and epilepsy was noted, indicating USP25 is potentially a predisposing gene for epilepsy. Our results from Usp25 null mice and the patient-derived variants indicated that USP25 would play epileptogenic role via loss-of-function or gain-of-function effects. The truncated variant p.Gln889Ter would have profoundly different effect on epilepsy. Together, our results underscore the significance of USP25 heterozygous variants in epilepsy, thereby highlighting the critical role of USP25 in the brain.

Changes in clinical outcomes and alignment of the ipsilateral knee and ankle after supramalleolar osteotomy in patients with varus osteoarthritis of the ankle: a short-term follow-up study.

PURPOSE: The purpose of this study was to investigate the changes in clinical outcomes and alignment of the ipsilateral knee and ankle in patients with varus ankle osteoarthritis after supramalleolar osteotomy (SMO). METHODS: We retrospectively reviewed 23 patients (24 ankles) with Takakura II, IIIa and IIIb ankle osteoarthritis treated with SMO between May 2017 and March 2022. The radiologic parameters of ankles contained medial distal tibial angle (TAS), tibiotalar angle (TT), tibial lateral surface (TLS), tibial plafond inclination (TPI) and talar inclination (TI). The radiologic parameters of knees contained medial proximal tibial angle (MPTA), joint line convergence angle (JLCA), the knee joint line orientation relative to ground (G-KJLO) and WBL. Hip-knee-ankle angle (HKA) was also collected. The Takakura system was used for evaluating the ankle osteoarthritis and the Kellgren-Lawrence (KL) system was used for evaluating the knee osteoarthritis. Clinical evaluation of the ankle joints contained American Orthopedic Foot and Ankle Society (AOFAS), range of motion (ROM) and visual analogue scale (VAS). Clinical evaluation of the knee joints contained Japanese Orthopaedic Association Scores (JOA), ROM, VAS. RESULTS: The mean follow-up times were 20.3 ± 7.3 months (range 12-38). According to the radiologic evaluation, the TAS increased from preoperative 84.7° ± 2.0° to 91.2° ± 1.8° at the last follow-up (P < 0.001). The TPI and TI decreased from 4.4° ± 4.2° and 11.0° ± 5.2° to 0.1° ± 4.7° and 4.1° ± 4.8° (P < 0.001 for both). The TT angel improved from 9.5° ± 4.1° to 4.9° ± 3.3° (P < 0.001). No significant differences were found regarding MPTA, JLCA, G-KJLO, knee WBL and HKA (P > 0.05 for all). The Takakura stage improved after SMO (P < 0.001) whilst the KL stage maintains the similar lever (P > 0.05). According to the clinical evaluation, the AOFAS significantly increased from 67.5 ± 10.6 to 88.5 ± 9.3 and the VAS of the ankle decreased from 4.7 ± 1.6 to 1.2 ± 1.1, whilst there were no changes on VAS and even the JOA and knee ROM after SMO (P > 0.05 for all). CONCLUSIONS: SMO can alleviate the symptoms of varus ankle osteoarthritis and delay the time for ankle replacement or arthrodesis by redistributing the abnormal stress of the ankle and restoring the congruence of the tibiotalar joint. In addition, it did not induce the clinical symptoms of knee without compromising lower limb alignment or knee joint line orientation in the short term. LEVEL OF EVIDENCE: Level IV case series.

KRT1 gene silencing ameliorates myocardial ischemia-reperfusion injury via the activation of the Notch signaling pathway in mouse models.

Myocardial ischemia and reperfusion injury (MIRI) includes major drawbacks, such as excessive formation of free radicals and also overload of calcium, which lead to cell death, tissue scarring, and remodeling. The current study aims to explore whether KRT1 silencing may ameliorate MIRI via the Notch signaling pathway in mouse models. Myocardial tissues were used for the determination of the positive rate of KRT1 protein expression, apoptosis of myocardial cells, creatine kinase (CK) and lactate dehydrogenase (LDH) expression, expression of related biomarkers as well as myocardial infarction area. The transfected myocardial cells were treated with KRT1-siRNA, Jagged1, and DAPT (inhibitor of Notch-1 signaling pathway). The expression of KRT1, NICD, Hes1, Bcl-2, and Bax protein was detected. The MTT assay was applied for cell proliferation and flow cytometry was used for cell apoptosis. Mice with MIRI had a higher positive rate of KRT1 protein expression, apoptosis of myocardial cells, CK and LDH expression, myocardial infarction area, increased expression of MDA, NO, SDH, IL-1, IL-6, TNF-α, CRP, KRT1, Bax protein, CK, and LDH, and decreased expression of SOD, NICD, Hes1, and Bcl-2. The downregulation of KRT1 led to decreased expression of KRT1 and Bax protein, increased expression of NICD, Hes1, and Bcl-2, decreased cell apoptosis, and improved cell proliferation. The inhibition of the Notch signaling pathway leads to reduced expression of Bax, increased expression of NICD, Hes1, and Bcl 2, and also decreased cell apoptosis and increased cell proliferation. Our data conclude that KRT1 silencing is able to make MIRI better by activating the Notch signaling pathway in mice.

The role of microRNA-1 targeting of MAPK3 in myocardial ischemia-reperfusion injury in rats undergoing sevoflurane preconditioning via the PI3K/Akt pathway.

Recent studies have uncovered the vital roles played by microRNAs in regulating cardiac injury. Among them, the cardiac enriched microRNA-1 (miR-1) has been extensively studied and proven to be detrimental to cardiac myocytes. Hence, the current study aimed to explore whether miR-1 affects myocardial ischemia-reperfusion injury (MIRI) in rats undergoing sevoflurane preconditioning and the underlying mechanism. After successful model establishment, rats with MIRI were transfected with mimics or inhibitors of miR-1, or siRNA against MAPK3, and then were injected with sevoflurane. A luciferase reporter gene assay was conducted to evaluate the targeting relationship between miR-1 and MAPK3. Reverse transcription quantitative polymerase chain reaction and Western blot analysis were employed to evaluate the expressions of miR-1, MAPK3, phosphatidylinositol 3-kinase (PI3K), and Akt. Additionally, the concentration of lactate dehydrogenase (LDH) was determined. Cell apoptosis and viability were assessed using TUNEL and cell counting kit-8 assays, and the ischemic area at risk and infarct size were detected using Evans blue and triphenyltetrazolium chloride staining. MAPK3 was found to be the target gene of miR-1. miR-1 expressed at a high level whereas MAPK3 expressed at a low level in MIRI rats. Overexpressing miR-1 or silencing MAPK3 blocked the PI3K/Akt pathway to increase cell apoptosis, ischemic area at risk, and infarct area but decreased cell viability and increased LDH concentration. In contrast, miR-1 downregulation abrogated the effects induced by miR-1 mimics or siRNA against MAPK3. These findings indicate that inhibition of miR-1 promotes MAPK3 to protect against MIRI in rats undergoing sevoflurane preconditioning through activation of the PI3K/Akt pathway.

Anti-hyperlipidemic, Anti-inflammatory, and Ameliorative Effects of DRP1 Inhibition in Rats with Experimentally Induced Myocardial Infarction.

This study aims to investigate the biological role of DRP1 in myocardial infarction (MI) in concert with hyperlipidemia (HL). Based on the available literatures, 10 genes related to MI with HL (HL-MI) were screened and detected in clinical samples. High-fat diet (HFD) was used to establish HL rat models, after which the rats were subcutaneously injected with PBS or isoproterenol hydrochloride to induce acute MI. Then, rats with HL-MI were injected with pcDNA3.1, pcDNA3.1-DRP1, sh-NC, or sh-DRP1. Serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) were measured. Cardiac function was evaluated by detecting left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF). Infarct size and histopathological changes were assessed as well as myocardial apoptosis and collagen deposition. The concentration of IL-6, IL-1ß, and TNF-α in rat serum and cardiac tissues was also measured by ELISA. Mitochondrial function was shown by measuring the morphology, mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) level. Pro-apoptotic proteins (Bax, caspase-1, and cleaved caspase-1) and NLRP3 inflammasome activation were also assessed. The expressions of the 10 genes were measured in clinical samples and DRP1 was selected for further experiments with significantly upregulated expression in MI patients. HFD-induced rats showed increased body weight, concurrent with higher levels of TG, TC, and LDL-C and lower HDL-C level. Compared with the BD-PBS group, the HFD-PBS group presented higher mRNA and protein expression levels of DRP1, exacerbated cardiac functions, enlarged infarct size, loss of cardiomyocytes, and disordered island cardiomyocytes. In the HL-MI rat model, injection of pcDNA3.1-DRP1 enhanced the levels of serum lipids and inflammation cytokines, induced loss of a number of cardiomyocytes and collagen deposition, and decreased LVFS and LVEF, while injection of sh-DRP1 ameliorated myocardial injuries, inflammation, and cardiomyocyte apoptosis and fibrosis. In coronary artery endothelial cells from the rats, loss of MMP was observed in the HFD-MI, LV-NC, LV-DRP1, and sh-NC groups and concomitantly, the sh-DRP1group showed increased MMP and decreased levels of mitochondrial ROS, cytochrome C, pro-apoptotic proteins, and NLRP3. Inhibition of DRP1 markedly suppressed HL, systematic inflammation, and myocardial injuries induced by HL-MI through partly restoring mitochondrial function and reducing NLRP3 expression.

Exogenous hydrogen sulfide ameliorates high glucose-induced myocardial injury & inflammation via the CIRP-MAPK signaling pathway in H9c2 cardiac cells.

AIMS: Hydrogen sulfide (H2S) is a novel signaling molecule with potent cytoprotective actions. In this study, we hypothesize that exogenous H2S may protect cardiac cells against high glucose (HG)-induced myocardial injury and inflammation with the involvement of the CIRP-MAPK signaling pathway. MAIN METHODS: H9c2 cardiac cells cultured under HG conditions were transfected with siRNA and different inhibitor for detecting the effects of sodium hydrogen sulfide (NaHS) (a H2S donor) on cell biological processes. The cardiac cell viability and LDH activity were determined by CCK-8 and LDH kit. ELISA was employed to measure the levels of inflammatory factors, while 2',7'-dichlorofluorescein diacetate (DCFH-DA) to evaluate reactive oxygen species (ROS). Mitochondrial membrane potential (MMP) was identified by rhodamine 123 staining. TUNEL staining and Hoechst 33258 staining were employed to observe cardiac cell apoptosis. Besides, we determined the expression of CIRP-MAPK signaling pathway- and apoptosis-related factors by protein immunoblot analysis. KEY FINDINGS: HG culturing induced toxicity, LDH, higher level of inflammatory factors, ROS, MMP, and apoptosis in cardiac cells, attenuated the viability of cardiac cells, and activated the CIRP-MAPK signaling pathway. Notably, CIRP silencing aggravated the above condition. H2S or blockade of the MAPK signaling pathway reversed the above conditions induced by HG. SIGNIFICANCE: The present study provides evidence for the protective effect of exogenous H2S on HG-induced myocardial injury and inflammation in H9c2 cardiac cells and suggests that the activation of CIRP-MAPK signaling pathway might be one of the mechanisms underlying the protective effect of H2S.