Nuclear miR-204-3p mitigates metabolic dysfunction-associated steatotic liver disease in mice.

BACKGROUND & AIMS: Accumulating evidence has indicated the presence of mature microRNAs (miR) in the nucleus, but their effects on steatohepatitis remain elusive. We have previously demonstrated that the intranuclear miR-204-3p in macrophages protects against atherosclerosis, which shares multiple risk factors with metabolic dysfunction-associated steatotic liver disease (MASLD). Herein, we aimed to explore the functional significance of miR-204-3p in steatohepatitis. METHODS: miR-204-3p levels and subcellular localization were assessed in the livers and peripheral blood mononuclear cells of patients with MASLD. Wild-type mice fed high-fat or methionine- and choline-deficient diets were injected with an adeno-associated virus system containing miR-204-3p to determine the effect of miR-204-3p on steatohepatitis. Co-culture systems were applied to investigate the crosstalk between macrophages and hepatocytes or hepatic stellate cells (HSCs). Multiple high-throughput epigenomic sequencings were performed to explore miR-204-3p targets. RESULTS: miR-204-3p expression decreased in livers and macrophages in mice and patients with fatty liver. In patients with MASLD, miR-204-3p levels in peripheral blood mononuclear cells were inversely related to the severity of hepatic inflammation and damage. Macrophage-specific miR-204-3p overexpression reduced steatohepatitis in high-fat or methionine- and choline-deficient diet-fed mice. miR-204-3p-overexpressing macrophages inhibited TLR4/JNK signaling and pro-inflammatory cytokine release, thereby limiting fat deposition and inflammation in hepatocytes and fibrogenic activation in HSCs. Epigenomic profiling identified miR-204-3p as a specific regulator of ULK1 expression. ULK1 transcription and VPS34 complex activation by intranuclear miR-204-3p improved autophagic flux, promoting the anti-inflammatory effects of miR-204-3p in macrophages. CONCLUSIONS: miR-204-3p inhibits macrophage inflammation, coordinating macrophage actions on hepatocytes and HSCs to ameliorate steatohepatitis. Macrophage miR-204-3p may be a therapeutic target for MASLD. IMPACT AND IMPLICATIONS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic inflammatory disease ranging from simple steatosis to steatohepatitis. However, the molecular mechanisms underlying the progression of MASLD remain incompletely understood. Here, we demonstrate that miR-204-3p levels in circulating peripheral blood mononuclear cells are negatively correlated with disease severity in patients with MASLD. Nuclear miR-204-3p activates ULK1 transcription and improves autophagic flux, limiting macrophage activation and hepatic steatosis. Our study provides a novel understanding of the mechanism of macrophage autophagy and inflammation in steatohepatitis and suggests that miR-204-3p may act as a potential therapeutic target for MASLD.

Vascular smooth muscle-specific LRRC8A knockout ameliorates angiotensin II-induced cerebrovascular remodeling by inhibiting the WNK1/FOXO3a/MMP signaling pathway.

Hypertensive cerebrovascular remodeling involves the enlargement of vascular smooth muscle cells (VSMCs), which activates volume-regulated Cl- channels (VRCCs). The leucine-rich repeat-containing family 8 A (LRRC8A) has been shown to be the molecular identity of VRCCs. However, its role in vascular remodeling during hypertension is unclear. In this study, we used vascular smooth muscle-specific LRRC8A knockout (CKO) mice and an angiotensin II (Ang II)-induced hypertension model. The results showed that cerebrovascular remodeling during hypertension was ameliorated in CKO mice, and extracellular matrix (ECM) deposition was reduced. Based on the RNA-sequencing analysis of aortic tissues, the level of matrix metalloproteinases (MMPs), such as MMP-9 and MMP-14, were reduced in CKO mice with hypertension, which was further verified in vivo by qPCR and immunofluorescence analysis. Knockdown of LRRC8A in VSMCs inhibited the Ang II-induced upregulation of collagen I, fibronectin, and matrix metalloproteinases (MMPs), and overexpression of LRRC8A had the opposite effect. Further experiments revealed an interaction between with-no-lysine (K)-1 (WNK1), which is a "Cl--sensitive kinase", and Forkhead transcription factor O3a (FOXO3a), which is a transcription factor that regulates MMP expression. Ang II induced the phosphorylation of WNK1 and downstream FOXO3a, which then increased the expression of MMP-2 and MMP-9. This process was inhibited or potentiated when LRRC8A was knocked down or overexpressed, respectively. Overall, these results demonstrate that LRRC8A knockout in vascular smooth muscle protects against cerebrovascular remodeling during hypertension by reducing ECM deposition and inhibiting the WNK1/FOXO3a/MMP signaling pathway, demonstrating that LRRC8A is a potential therapeutic target for vascular remodeling-associated diseases such as stroke.

Deep learning extended depth-of-field microscope for fast and slide-free histology.

Microscopic evaluation of resected tissue plays a central role in the surgical management of cancer. Because optical microscopes have a limited depth-of-field (DOF), resected tissue is either frozen or preserved with chemical fixatives, sliced into thin sections placed on microscope slides, stained, and imaged to determine whether surgical margins are free of tumor cells-a costly and time- and labor-intensive procedure. Here, we introduce a deep-learning extended DOF (DeepDOF) microscope to quickly image large areas of freshly resected tissue to provide histologic-quality images of surgical margins without physical sectioning. The DeepDOF microscope consists of a conventional fluorescence microscope with the simple addition of an inexpensive (less than $10) phase mask inserted in the pupil plane to encode the light field and enhance the depth-invariance of the point-spread function. When used with a jointly optimized image-reconstruction algorithm, diffraction-limited optical performance to resolve subcellular features can be maintained while significantly extending the DOF (200 µm). Data from resected oral surgical specimens show that the DeepDOF microscope can consistently visualize nuclear morphology and other important diagnostic features across highly irregular resected tissue surfaces without serial refocusing. With the capability to quickly scan intact samples with subcellular detail, the DeepDOF microscope can improve tissue sampling during intraoperative tumor-margin assessment, while offering an affordable tool to provide histological information from resected tissue specimens in resource-limited settings.

Selective N-glycan editing on living cell surfaces to probe glycoconjugate function.

Cell surfaces are glycosylated in various ways with high heterogeneity, which usually leads to ambiguous conclusions about glycan-involved biological functions. Here, we describe a two-step chemoenzymatic approach for N-glycan-subtype-selective editing on the surface of living cells that consists of a first 'delete' step to remove heterogeneous N-glycoforms of a certain subclass and a second 'insert' step to assemble a well-defined N-glycan back onto the pretreated glyco-sites. Such glyco-edited cells, carrying more homogeneous oligosaccharide structures, could enable precise understanding of carbohydrate-mediated functions. In particular, N-glycan-subtype-selective remodeling and imaging with different monosaccharide motifs at the non-reducing end were successfully achieved. Using a combination of the expression system of the Lec4 CHO cell line and this two-step glycan-editing approach, opioid receptor delta 1 (OPRD1) was investigated to correlate its glycostructures with the biological functions of receptor dimerization, agonist-induced signaling and internalization.

Structure and dynamics of a water/methanol mixture confined in zeolitic imidazolate framework ZIF-8 from atomistic simulations.

A classical atomistic simulation study is reported for the microscopic structure and dynamics of a water/methanol mixture confined in flexible nanoporous zeolitic imidazolate framework ZIF-8. Both the radial density distribution and vivid two-dimensional density profile demonstrate that methanol molecules can roughly be viewed as "embedded" between two layers of water molecules to form a "sandwich" structure. The reason for the formation of such a specific structure is explained based on the hydrogen-bonding state and the strength of various hydrogen bonds. The investigation of guest molecular diffusion shows that the self-diffusion coefficient of confined water is generally one to two orders of magnitude smaller than that of bulk water. In addition, the dependence of the self-diffusion coefficient on loading is non-monotonic: the self-diffusion coefficient firstly shows a significant increase and then decreases at higher loading. Moreover, both the structure and dynamics of the hydrogen bond (HB) network of confined water molecules are investigated in a spatially resolved manner. The results indicate that both the HB structure and dynamics of water molecules near the ZIF-8 surface deviate significantly from those of bulk water. However, while water molecules located at the pore center are relatively similar to bulk water molecules with respect to the HB structure, they exhibit strong slowdown in HB dynamics when compared with bulk water. This simulation study elucidates in detail the structural and dynamical properties of a water/methanol mixture in nanoscopic ZIF-8 confinement, which is expected to provide a deep insight into the role of porous fillers, such as ZIF-8, in improving the performance of the dehydration of alcohols via pervaporation and other related processes.

Thermostability detection and optimization of glycoengineered antibodies and antibody-drug conjugates based on differential scanning flouremitry analysis.

Thermostability of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), as a critical property of biotherapeutics, is important for their physicochemical processes, pharmacodynamics, and pharmacokinetics. Fc glycosylation of mAbs plays a crucial role in antibody functions including thermostability, however, due to the lack of homogeneous glycosylation for comparison, the precise impact of glycoforms on thermostability of mAbs and ADCs remains challenging to elucidate. In this paper, we employed the technique of differential scanning fluorimetry (DSF) to investigate the thermostability of Fc domains, glycoengineered mAbs, and ADCs, carrying well-defined N-glycan structures for comparison. The results revealed that high-mannose-type N-glycans dramatically reduce the Tm value of Fc, compared to complex-type N-glycans. We also found that core-fucose contributes to the thermostability of mAbs, and the unnatural modification on non-reducing end of biantennary N-glycan can compensate the reduced stability of afucosylated mAbs and maintain the advantage of enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). DSF analysis of lysine-linked and glycosite-specific ADCs indicated that thermostability of glycan-linked ADCs is reduced, but it could be improved by using an optimized linkage. This work provides an in-depth analysis on thermostability of mAbs and ADCs with homogeneous glycoforms, and also proposes new strategies for optimizing glycoengineered mAbs and glycosite-specific ADCs using unnatural glycan and stabilized linkage.

Manipulating the Click Reactivity of Dibenzoazacyclooctynes: From Azide Click Component to Caged Acylation Reagent by Silver Catalysis.

Strain-promoted azide-alkyne cycloaddition using dibenzoazacyclooctyne (DBCO) is widely applied in copper-free bioorthogonal reactions. Reported here is the efficient acid-promoted rearrangement and silver-catalyzed amidation of DBCO, which alters its click reactivity robustly. In the switched click reaction, DBCO, as a caged acylation reagent, enables rapid peptide/protein modification after decaging facilitated by silver catalysts, rendering site-specific conjugation of an IgG antibody by a Fc-targeting peptide.

Simple differential digital confocal aperture to improve axial response of line-scanning confocal microendoscopes.

Line-scanning confocal microendoscopy offers video-rate cellular imaging of scattering tissue with relatively simple hardware, but its axial response is inferior to that of point-scanning systems. Based on Fourier optics theory, we designed differential confocal apertures with a simple subtraction technique to improve the line-scanning sectioning performance. Taking advantage of digital slit apertures on a digital light projector and a CMOS rolling shutter, we demonstrate real-time optical sectioning performance comparable to point scanning in a dual-camera microendoscope (<$6,000). We validate the background rejection capability when imaging porcine columnar epithelium stained with fluorescent contrast agents with different uptake mechanisms and staining properties.

Improving nuclear morphometry imaging with real-time and low-cost line-scanning confocal microendoscope.

Fiber-optic endomicroscopy is a minimally invasive tool to probe disease progression with subcellular resolution. In this Letter, we demonstrate a low-cost and compact fluorescence microendoscope capable of line-scanning confocal imaging by synchronizing a digital light projector with a CMOS camera. We present the digital aperture design to enable real-time confocal imaging, and we implement parallel illumination to improve the optical sectioning performance. Furthermore, we show that the confocal microendoscope can enhance visualization of disease-associated features when imaging highly scattering esophageal specimens.

Reprogramming Tumor-Associated Macrophages To Reverse EGFRT790M Resistance by Dual-Targeting Codelivery of Gefitinib/Vorinostat.

Gefitinib is a first-line therapy in the EGFR-mutated nonsmall cell lung cancer (NSCLC). However, the development of drug resistance is almost unavoidable, thus leading to an unsustainable regimen. EGFRT790M mutation is the major cause responsible for the molecular-targeting therapy failure in NSCLC. Although the recently approved osimertinib is effective for the EGFRT790M-positive NSCLC, the osimertinib-resistant EGFR mutation is rapidly developed, too. In this study, we proposed a tumor-associated macrophage (TAM) reprogramming strategy for overcoming the EGFRT790M-associated drug resistance via a dual-targeting codelivery system of gefitinib/vorinostat that acted on both TAM with overexpression of mannose receptors and the HER-2 positive NSCLC cells. The trastuzumab-modified, mannosylated liposomal system was able to repolarize the protumor M2 phenotype to the antitumor M1 and cause the elevating ROS in the cancer cells, consequently modulating the intracellular redox balance via ROS/NOX3/MsrA axis. The suppressed MsrA facilitated the EGFRT790M degradation through 790M oxidation by ROS, thus resensitizing the EGFRT790M-positive cells to gefitinib. The dual-targeting codelivery and TAM-reprogramming strategies provided a potential method for rescuing the EGFRT790M-caused resistance to tyrosine kinase inhibitor treatment.

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAIN-truncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from that of other previously reported CD97 isoforms. The EGF5 and GAIN domains are likely associated with each other as CD97 expression and SAXS data revealed a potential interaction between the two domains. Finally, the GAIN and EGF domains are also important for CD97-HeLa adhesion, whereas N-glycosylation of the CD97 GAIN domain and GPS auto-proteolysis are not required for HeLa cell attachment.

Aromatic Hydrocarbon Receptor Suppresses Prostate Cancer Bone Metastasis Cells-Induced Vasculogenesis of Endothelial Progenitor Cells under Hypoxia.

BACKGROUND/AIMS: Hypoxia leads to the development of neovascularization in solid tumor by regulating VEGF expression. Aromatic hydrocarbon receptor (AHR), a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with hypoxia-inducible factors 1ß (HIF-1ß) and inhibits the secretion of vascular endothelial growth factor (VEGF). The purpose of this study was to explore whether AHR can suppress hypoxia-induced VEGF production in prostate bone metastasis cells and repress neovascularization in endothelial progenitor cells (EPCs), and, if so, through what mechanisms. METHODS: PC-3 or LNCaP cells induced angiogenesis was detected by Matrigel-based tube formation assay, mRNA expression levels was measured by qRT-PCR, VEGF secretion level was determined by ELISA assay, respectively. RESULTS: AHR activation inhibits hypoxia-induced adhesiveness and vasculogenesis of EPCs induced by PC-3 or LNCaP cells under hypoxia. Moreover, AHR activation suppressed hypoxia-induced VEGF production in PC-3 and LNCaP cells (48 ± 14% in PC-3, p = 0.000; 41 ± 14% in LNCaP, p = 0.000) by attenuating HIF-1α and HIF-1ß level that in turn diminished the angiogenic ability of EPCs in vitro. Furthermore, we found the mRNA level of hypoxia-inducible factors 1α (HIF-1α) (1.54 ± 0.13 fold in PC-3, p = 0.002, 1.62 ± 0.12 fold in LNCaP, p = 0.001) and HIF-1ß (1.67 ± 0.23 fold in PC-3, p = 0.007; 1.75 ± 0.26 fold in LNCaP, p=0.008) were upregulated in prostate cancer bone metastasis PC-3 and LNCaP cell lines in response to hypoxia, and revealed that the regulation of VEGF by HIF-1α and HIF-1ß was possibly mediated by the activation of phosphatidylinositol 3-kinase pathway. CONCLUSION: By providing a mechanistic insight into the modulation of neovascularization by AHR ligand, we suggest that AHR ligand has a strong potential of being a new therapeutic agent with applications in the field of bone metastatic prostate cancer.

Icariin promotes angiogenic differentiation and prevents oxidative stress-induced autophagy in endothelial progenitor cells.

Reduced tissue levels of endothelial progenitor cells (EPCs) and functional impairment of endothelium are frequently observed in patients with diabetes and cardiovascular disease. The vascular endothelium is specifically sensitive to oxidative stress, and this is one of the mechanisms that causes widespread endothelial dysfunction in most cardiovascular diseases and disorders. Hence attention has increasingly been paid to enhance mobilization and differentiation of EPCs for therapeutic purposes. The aim of this study was to investigate whether Icariin, a natural bioactive component known from traditional Chinese Medicine, can induce angiogenic differentiation and inhibit oxidative stress-induced cell dysfunction in bone marrow-derived EPCs (BM-EPCs), and, if so, through what mechanisms. We observed that treatment of BM-EPCs with Icariin significantly promoted cell migration and capillary tube formation, substantially abrogated hydrogen peroxide (H2 O2 )-induced apoptotic and autophagic programmed cell death that was linked to the reduced intracellular reactive oxygen species levels and restored mitochondrial membrane potential. Icariin downregulated endothelial nitric oxide synthase 3, as well as nicotinamide-adenine dinucleotide phosphate-oxidase expression upon H2 O2 induction. These antiapoptotic and antiautophagic effects of Icariin are possibly mediated by restoring the loss of mammalian target of rapamycin /p70S6K/4EBP1 phosphorylation as well as attenuation of ATF2 and ERK1/2 protein levels after H2 O2 treatment. In summary, favorable modulation of the angiogenesis and redox states in BM-EPCs make Icariin a promising proangiogenic agent both enhancing vasculogenesis and protecting against endothelial dysfunction.

One-pot N-glycosylation remodeling of IgG with non-natural sialylglycopeptides enables glycosite-specific and dual-payload antibody-drug conjugates.

Chemoenzymatic transglycosylation catalyzed by endo-S mutants is a powerful tool for in vitro glycoengineering of therapeutic antibodies. In this paper, we report a one-pot chemoenzymatic synthesis of glycoengineered Herceptin using an egg-yolk sialylglycopeptide (SGP) substrate. Combining this one-pot strategy with novel non-natural SGP derivatives carrying azido or alkyne tags, glycosite-specific conjugation was enabled for the development of new antibody-drug conjugates (ADCs). The site-specific ADCs and semi-site-specific dual-drug ADCs were successfully achieved and characterized with SDS-PAGE, intact antibody or ADC mass spectrometry analysis, and PNGase-F digestion analysis. Cancer cell cytotoxicity assay revealed that small-molecule drug release of these ADCs relied on the cleavable Val-Cit linker fragment embedded in the structure. These results represent a new approach for glycosite-specific and dual-drug ADC design and rapid synthesis, and also provide the structural requirement for their biologic activities.

Pristimerin Inhibits Prostate Cancer Bone Metastasis by Targeting PC-3 Stem Cell Characteristics and VEGF-Induced Vasculogenesis of BM-EPCs.

BACKGROUND/AIMS: Prostate cancer (PCa) is one of the most common malignant cancers and a major leading cause of cancer deaths in men. Cancer stem-like cells are shown to be highly tumorigenic, pro-angiogenic and can significantly contribute to tumor new vessel formation and bone marrow derived-EPCs (BM-EPCs) are shown to recruit to the angiogenic switch in tumor growth and metastatic progression, suggesting the importance of targeting cancer stem cells (CSCs) and EPCs for novel tumor therapies. Pristimerin, an active component isolated from Celastraceae and Hippocrateaceae, has shown anti-tumor effects in some cell lines in previous studies. However, the effect and mechanism of Pristimerin on CSCs and EPCs in PCa bone metastasis are not well studied. METHODS: The effect of Pristimerin on PC-3 stem cell characteristics and metastasis were detected by spheroid formation, CD133 and CD44 protein expression, matrix-gel invasive assay and colony-formation assay in vitro, VEGF and pro-inflammatory cytokines expression by ELISA assay, and tumor tumorigenicity by X-ray and MR in NOD-SCID mice model in vivo. In addition, we also detected the effect of Pristimerin on VEGF-induced vasculogenesis and protein expression of BM-EPCs. RESULTS: Pristimerin could significantly inhibit spheroid formation and protein expression of CD133 and CD44, reduce VEGF and pro-inflammation cytokines expression of PC-3 cell, and prevent the xenografted PC-3 tumor growth in the bone of nude mice. The present data also showed that Pristimerin significantly inhibited VEGF-induced vasculogenesis of BM-EPCs by suppressing the EPCs functions including proliferation, adhesion, migration, tube formation and inactivation the phosphorylation of VEGFR-2, Akt and eNOS. CONCLUSION: These data provide evidence that Pristimerin has strong potential for development as a novel agent against prostate bone metastasis by suppressing PC-3 stem cell characteristics and VEGF-induced vasculogenesis of BM-EPCs.

A DNA-inspired injectable adhesive hydrogel with dual nitric oxide donors to promote angiogenesis for enhanced wound healing.

Chronic diabetic wounds are a severe complication of diabetes, often leading to high treatment costs and high amputation rates. Numerous studies have revealed that nitric oxide (NO) therapy is a promising option because it favours wound revascularization. Here, base-paired injectable adhesive hydrogels (CAT) were prepared using adenine- and thymine-modified chitosan (CSA and CST). By further introducing S-nitrosoglutathione (GSNO) and binary l-arginine (bArg), we obtained a NO sustained-release hydrogel (CAT/bArg/GSON) that was more suitable for the treatment of chronic wounds. The results showed that the expression of HIF-1α and VEGF was upregulated in the CAT/bArg/GSON group, and improved blood vessel regeneration was observed, indicating an important role of NO. In addition, the research findings revealed that following treatment with the CAT/bArg/GSON hydrogel, the viability of Staphylococcus aureus and Escherichia coli decreased to 14 ± 2 % and 6 ± 1 %, respectively. Moreover, the wound microenvironment was improved, as evidenced by a 60 ± 1 % clearance of DPPH. In particular, histological examination and immunohistochemical staining results showed that wounds treated with CAT/bArg/GSNO exhibited denser neovascularization, faster epithelial tissue regeneration, and thicker collagen deposition. Overall, this study proposes an effective strategy to prepare injectable hydrogel dressings with dual NO donors. The functionality of CAT/bArg/GSON has been thoroughly demonstrated in research on chronic wound vascular regeneration, indicating that CAT/bArg/GSON could be a potential option for promoting chronic wound healing. STATEMENT OF SIGNIFICANCE: This article prepares a chitosan hydrogel utilizing the principle of complementary base pairing, which offers several advantages, including good adhesion, biocompatibility, and flow properties, making it a good material for wound dressings. Loaded GSNO and bArg can steadily release NO and l-arginine through the degradation of the gel. Then, the released l-arginine not only possesses antioxidant properties but can also continue to generate a small amount of NO under the action of NOS. This design achieves a sustained and stable supply of NO at the wound site, maximizing the angiogenesis-promoting and antibacterial effects of NO. More neovascularization and abundant collagen were observed in the regenerated tissues. This study provides an effective repair hydrogel material for diabetic wound.

H3K27ac acts as a molecular switch for doxorubicin-induced activation of cardiotoxic genes.

BACKGROUND: Doxorubicin (Dox) is an effective chemotherapeutic drug for various cancers, but its clinical application is limited by severe cardiotoxicity. Dox treatment can transcriptionally activate multiple cardiotoxicity-associated genes in cardiomyocytes, the mechanisms underlying this global gene activation remain poorly understood. METHODS AND RESULTS: Herein, we integrated data from animal models, CUT&Tag and RNA-seq after Dox treatment, and discovered that the level of H3K27ac (a histone modification associated with gene activation) significantly increased in cardiomyocytes following Dox treatment. C646, an inhibitor of histone acetyltransferase, reversed Dox-induced H3K27ac accumulation in cardiomyocytes, which subsequently prevented the increase of Dox-induced DNA damage and apoptosis. Furthermore, C646 alleviated cardiac dysfunction in Dox-treated mice by restoring ejection fraction and reversing fractional shortening percentages. Additionally, Dox treatment increased H3K27ac deposition at the promoters of multiple cardiotoxic genes including Bax, Fas and Bnip3, resulting in their up-regulation. Moreover, the deposition of H3K27ac at cardiotoxicity-related genes exhibited a broad feature across the genome. Based on the deposition of H3K27ac and mRNA expression levels, several potential genes that might contribute to Dox-induced cardiotoxicity were predicted. Finally, the up-regulation of H3K27ac-regulated cardiotoxic genes upon Dox treatment is conservative across species. CONCLUSIONS: Taken together, Dox-induced epigenetic modification, specifically H3K27ac, acts as a molecular switch for the activation of robust cardiotoxicity-related genes, leading to cardiomyocyte death and cardiac dysfunction. These findings provide new insights into the relationship between Dox-induced cardiotoxicity and epigenetic regulation, and identify H3K27ac as a potential target for the prevention and treatment of Dox-induced cardiotoxicity.

Design and Evaluation of ScanCap: A Low-Cost, Reusable Tethered Capsule Endoscope with Blue-Green Illumination Imaging for Unsedated Screening and Early Detection of Barrett's Esophagus.

Esophageal carcinoma is the sixth-leading cause of cancer death worldwide. A precursor to esophageal adenocarcinoma (EAC) is Barrett's Esophagus (BE). Early-stage diagnosis and treatment of esophageal neoplasia (Barrett's with high-grade dysplasia/intramucosal cancer) increase the five-year survival rate from 10% to 98%. BE is a global challenge; however, current endoscopes for early BE detection are costly and require extensive infrastructure for patient examination and sedation. We describe the design and evaluation of the first prototype of ScanCap, a high-resolution optical endoscopy system with a reusable, low-cost tethered capsule, designed to provide high-definition, blue-green illumination imaging for the early detection of BE in unsedated patients. The tethered capsule (12.8 mm diameter, 35.5 mm length) contains a color camera and rotating mirror and is designed to be swallowed; images are collected as the capsule is retracted manually via the tether. The tether provides electrical power and illumination at wavelengths of 415 nm and 565 nm and transmits data from the camera to a tablet. The ScanCap prototype capsule was used to image the oral mucosa in normal volunteers and ex vivo esophageal resections; images were compared to those obtained using an Olympus CV-180 endoscope. Images of superficial capillaries in intact oral mucosa were clearly visible in ScanCap images. Diagnostically relevant features of BE, including irregular Z-lines, distorted mucosa, and dilated vasculature, were clearly visible in ScanCap images of ex vivo esophageal specimens.

DeepDOF-SE: affordable deep-learning microscopy platform for slide-free histology.

Histopathology plays a critical role in the diagnosis and surgical management of cancer. However, access to histopathology services, especially frozen section pathology during surgery, is limited in resource-constrained settings because preparing slides from resected tissue is time-consuming, labor-intensive, and requires expensive infrastructure. Here, we report a deep-learning-enabled microscope, named DeepDOF-SE, to rapidly scan intact tissue at cellular resolution without the need for physical sectioning. Three key features jointly make DeepDOF-SE practical. First, tissue specimens are stained directly with inexpensive vital fluorescent dyes and optically sectioned with ultra-violet excitation that localizes fluorescent emission to a thin surface layer. Second, a deep-learning algorithm extends the depth-of-field, allowing rapid acquisition of in-focus images from large areas of tissue even when the tissue surface is highly irregular. Finally, a semi-supervised generative adversarial network virtually stains DeepDOF-SE fluorescence images with hematoxylin-and-eosin appearance, facilitating image interpretation by pathologists without significant additional training. We developed the DeepDOF-SE platform using a data-driven approach and validated its performance by imaging surgical resections of suspected oral tumors. Our results show that DeepDOF-SE provides histological information of diagnostic importance, offering a rapid and affordable slide-free histology platform for intraoperative tumor margin assessment and in low-resource settings.

Identification and Validation a Necroptosis-Related Prognostic Signature in Cervical Cancer.

Necroptosis is a promising novel target for cervical cancer therapy. Nevertheless, differentially expressed necroptosis-related genes (NRGs) in cervical cancer and their associations with prognosis are far from fully clarified. In this study, differentially expressed NRGs (DE-NRGs) were screened out and their bio-function was elucidated. Subsequently, a prognostic scoring model based on the regression coefficients of the screened out NRGs and their corresponding mRNA expressions were constructed and validated. Finally, the survival probability of cervical cancer patients based on the constructed prognostic scoring model in 3 and 5 years was predicted and assessed. We found 17 DE-NRGs in cervical cancer tissues which were closely related to cancer progression, and most of them were significantly highly expressed. Furthermore, 3 NRG were confirmed as the prognostic signature genes from 17 DE-NRGs by regression analysis. Overall survival predicted through our prognostic scoring model was lower in the high-risk group than in the low-risk group (p < 0.05) in both the TCGA cohort and the external GEO44001 validation cohort. What's more, the prediction performance of our prognostic scoring models well verified by the ROC curve, and the risk score calculated could act as an independent prognostic factor for cervical cancer patients. The calibration curve and C-index (0.776) of the nomogram analysis suggested that the predictive performance of the nomogram was satisfactory. Our study identified and validated a necroptosis-related prognostic signature in cervical cancer, which could well predict the prognosis for cervical cancer patients.