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Dravet syndrome (DS) is a devastating developmental epileptic encephalopathy marked by treatment-resistant seizures, developmental delay, intellectual disability, motor deficits, and a 10-20% rate of premature death. Most DS patients harbor loss-of-function mutations in one copy of , which has been associated with inhibitory neuron dysfunction. Here we developed an interneuron-targeting AAV human gene replacement therapy using cell class-specific enhancers. We generated a split-intein fusion form of to circumvent AAV packaging limitations and deliver via a dual vector approach using cell class-specific enhancers. These constructs produced full-length Na 1.1 protein and functional sodium channels in HEK293 cells and in brain cells . After packaging these vectors into enhancer-AAVs and administering to mice, immunohistochemical analyses showed telencephalic GABAergic interneuron-specific and dose-dependent transgene biodistribution. These vectors conferred strong dose-dependent protection against postnatal mortality and seizures in two DS mouse models carrying independent loss-of-function alleles of at two independent research sites, supporting the robustness of this approach. No mortality or toxicity was observed in wild-type mice injected with single vectors expressing either the N-terminal or C-terminal halves of , or the dual vector system targeting interneurons. In contrast, nonselective neuronal targeting of conferred less rescue against mortality and presented substantial preweaning lethality. These findings demonstrate proof-of-concept that interneuron-specific AAV-mediated gene replacement is sufficient for significant rescue in DS mouse models and suggest it could be an effective therapeutic approach for patients with DS.

Mitogen- and stress-activated protein kinase 1 (MSK1), a Ser/Thr kinase, phosphorylates nuclear proteins to increase their stability and DNA-binding affinity. Despite the role of MSK1 in promoting cancer progression in colorectal cancer (CRC), the precise molecular mechanisms remain unelucidated. Here we show that MSK1 expression induces the epithelial–mesenchymal transition (EMT) process and increases CRC cell metastasis. Furthermore, we discovered that MSK1 interacts with Snail, a key EMT regulator, and increases its stability by inhibiting ubiquitin-mediated proteasomal degradation. Importantly, MSK1 increased Snail protein stability by promoting deubiquitination rather than inhibiting its ubiquitination. Finally, we identified USP5 as an essential deubiquitinase that binds to Snail protein phosphorylated by MSK1. Based on the experimental data, in CRC, MSK1–Snail–USP5 axis can promote EMT and metastasis of CRC. Together, our findings provide potential biomarkers and novel therapeutic targets for further research in CRC. MSK1 enhances EMT and metastasis in colorectal cancer Colorectal cancer (CRC) is a major health concern worldwide, often leading to death due to its spread to other body parts. To find better treatments, researchers are trying to understand how CRC spreads and have focused on a protein called MSK1, which might play a role in this process. The study used human cancer cell lines and mice to explore how MSK1 affects another protein called Snail, which is involved in cancer spread. They found that MSK1 helps to stabilize Snail by preventing its breakdown, which, in turn, promotes the spread of cancer cells. This was done by examining the interaction between MSK1 and Snail in cells and observing changes in cancer cell behavior. The results showed that high levels of MSK1 are linked to increased cancer spread and lower survival rates in patients with CRC. This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.

Breast cancer remains a significant health concern, with triple-negative breast cancer (TNBC) being an aggressive subtype with poor prognosis. Epithelial-mesenchymal transition (EMT) is important in early-stage tumor to invasive malignancy progression. Snail, a central EMT component, is tightly regulated and may be subjected to proteasomal degradation. We report a novel proteasomal independent pathway involving chaperone-mediated autophagy (CMA) in Snail degradation, mediated via its cytosolic interaction with HSC70 and lysosomal targeting, which prevented its accumulation in luminal-type breast cancer cells. Conversely, Snail predominantly localized to the nucleus, thus evading CMA-mediated degradation in TNBC cells. Starvation-induced CMA activation downregulated Snail in TNBC cells by promoting cytoplasmic translocation. Evasion of CMA-mediated Snail degradation induced EMT, and enhanced metastatic potential of luminal-type breast cancer cells. Our findings elucidate a previously unrecognized role of CMA in Snail regulation, highlight its significance in breast cancer, and provide a potential therapeutic target for clinical interventions.

Wound healing involves the collaboration of multiple cells, including macrophages and fibroblasts, and requires the coordination of cytokines, growth factors, and matrix proteins to regulate the repair response. In this study, we investigated how M2 macrophages regulate expression of the anti-fibrotic and anti-inflammatory regulator tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6) secreted by adipose tissue-derived stem cells (ASCs) during wound healing. Interleukin (IL)-4/IL-13, which is used to differentiate macrophage M2 phenotypes, increases TSG-6 in ASCs; however, M2 macrophages significantly decrease TSG-6 in ASCs. Transforming growth factor (TGF)-β expression was increased, and TNF-α expression was decreased in M2 macrophages. TGF-β inhibited IL-4/IL-13-induced ASC TSG-6 expression. In addition, TSG-6 suppressed TGF-β-triggered wound closure and fibrogenic responses in LX-2 cells. Collectively, TSG-6 inhibited wound healing, but M2 macrophage-expressed TGF-β prevented TSG-6 production from ASCs, which ultimately helped wound healing. Our results indicate that the balance of TNF-α and TGF-β levels during wound healing regulates TSG-6 production from ASCs, which may ultimately modulate the healing process. Our study findings could contribute to novel therapeutic strategies that manipulate the delicate balance between TNF-α and TGF-β to enhance wound repair and mitigate fibrosis.

Haptoglobin (Hp) scavenges cell-free hemoglobin and correlates with the prognosis of human sepsis, a life-threatening systemic inflammatory condition. Despite extensive research on Hp glycosylation as a glyco-biomarker for cancers, understanding glycosylated modifications of Hp in sepsis patients (SPs) remains limited. Our study reveals elevated levels of terminal fucosylation at Asn207 and Asn211 of Hp in SP plasma, along with heightened inflammatory responses, compared to healthy controls (trial registration NCT05911711). Fucosylated (Fu)-Hp purified from SPs upregulates inflammatory cytokines and chemokines, along with NLRP3 inflammasome activation. Single-cell RNA sequencing identifies a distinct macrophage-like cell population with increased expressions of inflammatory mediators and FUT4 in response to Fu-Hp. Additionally, Mincle, a C-type lectin receptor, interacts with Fu-Hp to amplify the inflammatory responses and signaling. Moreover, the Hp fucosylation (AAL) level significantly correlates with the levels of inflammatory cytokines in sepsis patients, suggesting that Fu-Hp is clinically relevant. Finally, Fu-Hp treatment significantly enhances the levels of inflammatory cytokines in the plasma and various tissues of mice. Together, our findings reveal a role of Fu-Hp, derived from sepsis patients, in driving inflammation, and suggest that targeting Fu-Hp could serve as a promising intervention for combating sepsis. Trial registration NCT05911711 Haptoglobin (Hp) scavenges cell-free haemoglobin and is associated with the prognosis of human sepsis. Here, the authors characterize the association of fucosylated Hp in sepsis progression and identify a macrophage population expressing a receptor for fucosylated Hp, highlighting its inflammation-amplifying effects in in vitro human cell cultures and in vivo using a mouse model.

Malignant melanoma is a fatal disease that rapidly spreads to the whole body. Treatments have limited efficiency owing to drug resistance and various side effects. Pseudomonas syringae pv. tomato ( Pto ) is a model bacterial pathogen capable of systemic infection in plants. Pto injects the effector protein HopQ into the plant cytosol via a type III secretion machinery and suppresses the host immunity. Intriguingly, host plant proteins regulated by HopQ are conserved even in humans and conferred in tumor metastasis. Nevertheless, the potential for HopQ to regulate human cancer metastasis was unknown. In this study, we addressed the suitability of HopQ as a possible drug against melanoma metastasis. In melanoma cells, overexpressed HopQ is phosphorylated and bound to 14-3-3 through its N-terminal domain, resulting in stronger interaction between HopQ and vimentin. The binding of HopQ to vimentin allowed for degradation of vimentin via p62-dependent selective autophagy. Attenuation of vimentin expression by HopQ inhibited melanoma motility and in vivo metastasis. These findings demonstrated that HopQ directly degraded vimentin in melanoma cells and could be applied to an inhibitor of melanoma metastasis.

Response surface methodology (RSM) is one of the most effective design of experiments (DoE) methods for analyzing and optimizing experiments with limited data. However, the performance of RSM is highly dependent on the quality of the experimental data (e.g., measurement error and bias). In this work, we introduce a coefficient clipping technique based on prior knowledge to address this problem in RSM. To maintain the simplicity of RSM, the representative prior knowledge of monotonically increasing/decreasing and convex/concave relationships is considered as constraints. The proposed method uses the same experimental data as typical RSM, but can more accurately analyze the relationship between the independent variable and the output response. The performance of the proposed method is verified via various case studies, including the experiment of antibiotic adsorption in wastewater.

The epithelial–mesenchymal transition (EMT) plays a pivotal role in the conversion of early‐stage tumors into invasive malignancies. The transcription factor Snail, an extremely unstable protein whose subcellular levels are regulated by many E3 ubiquitin ligases, promotes EMT as well as associated pathological characteristics including migration, invasion, and metastasis. Through yeast two‐hybrid screening, we identified the carboxyl terminus of Hsc70‐interacting protein (CHIP) as a novel Snail ubiquitin ligase that interacts with Snail to induce ubiquitin‐mediated proteasomal degradation. Inhibition of CHIP expression increases Snail protein levels, induces EMT, and enhances in vitro migration and invasion as well as in vivo metastasis of ovarian cancer cells. In turn, Snail depletion abrogates all phenomena induced by CHIP depletion. Finally, Snail and CHIP expression is inversely correlated in ovarian tumor tissues. These findings establish the CHIP–Snail axis as a post‐translational mechanism of EMT and cancer metastasis regulation. In normal or early‐stage tumor cells, CHIP is highly expressed and effectively degrades Snail through ubiquitylation, contributing to reduced epithelial–mesenchymal transition (EMT) (left). In malignant tumor cells, CHIP expression is repressed by promoter hypermethylation, and increased nuclear Snail protein induces EMT (right).

Wound healing involves the collaboration of multiple cells, including macrophages and fibroblasts, and requires the coordination of cytokines, growth factors, and matrix proteins to regulate the repair response. In this study, we investigated how M2 macrophages regulate expression of the anti-fibrotic and anti-inflammatory regulator tumor necrosis factor-[alpha] (TNF-[alpha])-stimulated gene 6 (TSG-6) secreted by adipose tissue-derived stem cells (ASCs) during wound healing. Interleukin (IL)-4/IL-13, which is used to differentiate macrophage M2 phenotypes, increases TSG-6 in ASCs; however, M2 macrophages significantly decrease TSG-6 in ASCs. Transforming growth factor (TGF)-[beta] expression was increased, and TNF-[alpha] expression was decreased in M2 macrophages. TGF-[beta] inhibited IL-4/IL-13-induced ASC TSG-6 expression. In addition, TSG-6 suppressed TGF-[beta]-triggered wound closure and fibrogenic responses in LX-2 cells. Collectively, TSG-6 inhibited wound healing, but M2 macrophage-expressed TGF-[beta] prevented TSG-6 production from ASCs, which ultimately helped wound healing. Our results indicate that the balance of TNF-[alpha] and TGF-[beta] levels during wound healing regulates TSG-6 production from ASCs, which may ultimately modulate the healing process. Our study findings could contribute to novel therapeutic strategies that manipulate the delicate balance between TNF-[alpha] and TGF-[beta] to enhance wound repair and mitigate fibrosis.

Obesity is a complex health condition characterized by excessive adipose tissue accumulation, leading to significant metabolic disturbances such as insulin resistance and cardiovascular diseases. Fatty acid synthase (FAS), a key enzyme in lipogenesis, has been identified as a potential therapeutic target for obesity due to its role in adipocyte differentiation and lipid accumulation. This study employed a multidisciplinary approach involving in silico and in vitro analyses to investigate the anti-adipogenic properties of maclurin, a natural phenolic compound derived from Morus alba. Using SwissDock software (ChEMBL version 23), we predicted protein interactions and demonstrated a high probability (95.6%) of maclurin targeting FAS, surpassing the interaction rates of established inhibitors like cerulenin. Docking simulations revealed maclurin’s superior binding affinity to FAS, with a binding score of −7.3 kcal/mol compared to −6.7 kcal/mol for cerulenin. Subsequent in vitro assays confirmed these findings, with maclurin effectively inhibiting FAS activity in a concentration-dependent manner in 3T3-L1 adipocytes, without compromising cell viability. Furthermore, maclurin treatment resulted in significant reductions in lipid accumulation and the downregulated expression of critical adipogenic genes such as PPARγ, C/EBPα, and FAS, indicating the suppression of adipocyte differentiation. Maclurin shows potential as a novel FAS inhibitor with significant anti-adipogenic effects, offering a promising therapeutic avenue for the treatment and prevention of obesity.