PAX6/CXCL14 regulatory axis promotes the repair of corneal injury by enhancing corneal epithelial cell proliferation.

BACKGROUND: Corneal injuries, often leading to severe vision loss or blindness, have traditionally been treated with the belief that limbal stem cells (LSCs) are essential for repair and homeostasis, while central corneal epithelial cells (CCECs) were thought incapable of such repair. However, our research reveals that CCECs can fully heal and maintain the homeostasis of injured corneas in rats, even without LSCs. We discovered that CXCL14, under PAX6's influence, significantly boosts the stemness, proliferation, and migration of CCECs, facilitating corneal wound healing and homeostasis. This finding introduces CXCL14 as a promising new drug target for corneal injury treatment. METHODS: To investigate the PAX6/CXCL14 regulatory axis's role in CCECs wound healing, we cultured human corneal epithelial cell lines with either increased or decreased expression of PAX6 and CXCL14 using adenovirus transfection in vitro. Techniques such as coimmunoprecipitation, chromatin immunoprecipitation, immunofluorescence staining, western blot, real-time PCR, cell colony formation, and cell cycle analysis were employed to validate the axis's function. In vivo, a rat corneal epithelial injury model was developed to further confirm the PAX6/CXCL14 axis's mechanism in repairing corneal damage and maintaining corneal homeostasis, as well as to assess the potential of CXCL14 protein as a therapeutic agent for corneal injuries. RESULTS: Our study reveals that CCECs naturally express high levels of CXCL14, which is significantly upregulated by PAX6 following corneal damage. We identified SDC1 as CXCL14's receptor, whose engagement activates the NF-κB pathway to stimulate corneal repair by enhancing the stemness, proliferative, and migratory capacities of CCECs. Moreover, our research underscores CXCL14's therapeutic promise for corneal injuries, showing that recombinant CXCL14 effectively accelerates corneal healing in rat models. CONCLUSION: CCECs play a critical and independent role in the repair of corneal injuries and the maintenance of corneal homeostasis, distinct from that of LSCs. The PAX6/CXCL14 regulatory axis is pivotal in this process. Additionally, our research demonstrates that the important function of CXCL14 in corneal repair endows it with the potential to be developed into a novel therapeutic agent for treating corneal injuries.

Insect chitosan/pullulan/gallium photo-crosslinking hydrogels with multiple bioactivities promote MRSA-infected wound healing.

Methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant bacteria have become more common in recent years, which has made it extremely difficult to treat and heal many different kinds of wounds and caused enormous financial losses. Because of its unique "Trojan horse" function, Ga3+ has been recognized as a new possible candidate for inhibiting and eradicating drug-resistant bacteria. Furthermore, natural polysaccharide materials with outstanding biological characteristics, such as insect chitosan (CS) and pullulan (PUL), have attracted significant interest. In this study, we used quaternized-catechol chitosan (QDCS-PA), methacrylate-dialdehyde pullulan (DPUL-GMA), and gallium ion (Ga) to create a multi-crosslinked photo-enhanced hydrogel (Q-D/Ga/UV) with antimicrobial, hemostatic, self-healing, and injectable properties for promoting MRSA-infected wound healing. In vitro, the Q-D/Ga/UV hydrogels demonstrated good mechanical properties, antioxidant capabilities, biocompatibility, hemostatic properties, and antibacterial activity. The addition of gallium ions enhanced the hydrogels' mechanical properties, hemostatic capabilities, antibacterial activity, and ability to induce wound healing. Q-D/Ga/UV hydrogel significantly promoted wound contraction, collagen deposition, and angiogenesis while also suppressing inflammation in a whole-skin wound model of MRSA-infected rats. In conclusion, Q-D/Ga/UV hydrogels demonstrate significant promise for healing wounds infected with drug-resistant bacteria.

Multifunctional polysaccharide/metal/polyphenol double-crosslinked hydrogel for infected wound.

Bacterial-infected wounds present a significant challenge in the medical field, posing a severe threat to public health. Traditional wound dressings have limited efficacy in treating bacterial-infected wounds, and antibiotics suffer from cytotoxicity and drug resistance. Consequently, an urgent requirement exists for developing multifunctional wound dressings capable of providing superior antimicrobial activity and expediting wound repair. In recent years, chitosan-based natural polysaccharide hydrogels have garnered attention for their biocompatibility, antimicrobial properties, and ability to aid in hemostasis. This study presents the development of a multi-functional, bi-dynamic network hydrogel for the treatment of wounds infected with bacteria. The hydrogel consists of a backbone of chitosan grafted with chlorogenic acid (CA-ECS), oxidized pullulan polysaccharides (OP), and zinc ions (Zn2+). The CA-ECS/OP/Zn2+ hydrogel displayed strong adhesion, good injectability, and high mechanical strength and was biodegradable and biocompatible. Furthermore, adding Zn2+ and CA enhanced the hydrogel's mechanical properties and antioxidant and antimicrobial activities. In a rat model of full-thickness skin wounds infected with S. aureus, the CA-ECS/OP/Zn2+ hydrogel demonstrated great anti-inflammatory, angiogenic, and folliculogenic properties, resulting in accelerated wound healing. The CA-ECS/OP/Zn2+ hydrogel has great potential for treating bacterial-infected wounds.

Surface Proteome of Extracellular Vesicles and Correlation Analysis Reveal Breast Cancer Biomarkers.

Breast cancer (BC) is the second most frequently diagnosed cancer and accounts for approximately 25% of new cancer cases in Canadian women. Using biomarkers as a less-invasive BC diagnostic method is currently under investigation but is not ready for practical application in clinical settings. During the last decade, extracellular vesicles (EVs) have emerged as a promising source of biomarkers because they contain cancer-derived proteins, RNAs, and metabolites. In this study, EV proteins from small EVs (sEVs) and medium EVs (mEVs) were isolated from BC MDA-MB-231 and MCF7 and non-cancerous breast epithelial MCF10A cell lines and then analyzed by two approaches: global proteomic analysis and enrichment of EV surface proteins by Sulfo-NHS-SS-Biotin labeling. From the first approach, proteomic profiling identified 2459 proteins, which were subjected to comparative analysis and correlation network analysis. Twelve potential biomarker proteins were identified based on cell line-specific expression and filtered by their predicted co-localization with known EV marker proteins, CD63, CD9, and CD81. This approach resulted in the identification of 11 proteins, four of which were further investigated by Western blot analysis. The presence of transmembrane serine protease matriptase (ST14), claudin-3 (CLDN3), and integrin alpha-7 (ITGA7) in each cell line was validated by Western blot, revealing that ST14 and CLDN3 may be further explored as potential EV biomarkers for BC. The surface labeling approach enriched proteins that were not identified using the first approach. Ten potential BC biomarkers (Glutathione S-transferase P1 (GSTP1), Elongation factor 2 (EEF2), DEAD/H box RNA helicase (DDX10), progesterone receptor (PGR), Ras-related C3 botulinum toxin substrate 2 (RAC2), Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), Aconitase 2 (ACO2), UTP20 small subunit processome component (UTP20), NEDD4 binding protein 2 (N4BP2), Programmed cell death 6 (PDCD6)) were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. In total, 846 surface proteins were identified from the second approach, of which 11 were already known as BC markers. This study supports the proposition that Evs are a rich source of known and novel biomarkers that may be used for non-invasive detection of BC. Furthermore, the presented datasets could be further explored for the identification of potential biomarkers in BC.

Molecular characterization of a novel cytorhabdovirus infecting Plumbago indica L.

In 2021, Plumbago indica plants with necrotic spots on their leaves were observed in Beijing, China. Through high-throughput sequencing, we discovered a putative novel member of the genus Cytorhabdovirus, which was provisionally named "plumbago necrotic spot-associated virus" (PNSaV). The full-length negative-sense single-stranded RNA genome of this virus is 13,180 nucleotides in length and contains eight putative open reading frames (ORFs), in the order 3' leader-N-(P')-P-P3-M-G-P6-L-5' trailer. Phylogenetic analysis and pairwise comparisons suggested that PNSaV is most closely related to pastinaca cytorhabdovirus 1, with 59.2% nucleotide sequence identity in the complete genome and 56.4% amino acid sequence identity in the L protein. These findings suggest that PNSaV should be considered a new member of the genus Cytorhabdovirus.

Integrated analysis of tumor microenvironment features to establish a diagnostic model for papillary thyroid cancer using bulk and single-cell RNA sequencing technology.

BACKGROUND: Characterizing tumor microenvironment using single-cell RNA sequencing has been a promising strategy for cancer diagnosis and treatment. However, a few studies have focused on diagnosing papillary thyroid cancer (PTC) through this technology. Therefore, our study explored tumor microenvironment (TME) features and identified potential biomarkers to establish a diagnostic model for papillary thyroid cancer. METHODS: The cell types were identified using the markers from the CellMarker database and published research. The CellChat package was conducted to analyze the cell-cell interaction. The SCEVAN package was used to identify malignant thyroid cells. The SCP package was used to perform multiple single-cell downstream analyses, such as GSEA analysis, enrichment analysis, pseudotime trajectory analysis, and differential expression analysis. The diagnostic model of PTC was estimated using the calibration curves, receiver operating characteristic curves, and decision curve analysis. RT-qPCR was performed to validate the expression of candidate genes in human papillary thyroid samples. RESULTS: Eight cell types were identified in the scRNA-seq dataset by published cell markers. Extensive cell-cell interactions like FN1/ITGB1 existed in PTC tissues. We identified 26 critical genes related to PTC progression. Further, eight subgroups of PTC tumor cells were identified and exhibited high heterogeneity. The MDK/LRP1, MDK/ALK, GAS6/MERTK, and GAS6/AXL were identified as potential ligand-receptor pairs involved in the interactions between fibroblasts/endothelial cells and tumor cells. Eventually, the diagnostic model constructed by TRPC5, TENM1, NELL2, DMD, SLC35F3, and AUTS2 showed a good efficiency for distinguishing the PTC and normal tissues. CONCLUSIONS: Our study comprehensively characterized the tumor microenvironment in papillary thyroid cancer. Through combined analysis with bulk RNA-seq, six potential diagnostic biomarkers were identified and validated. The diagnostic model we constructed was a promising tool for PTC diagnosis. Our findings provide new insights into the heterogeneity of thyroid cancer and the theoretical basis for diagnosing thyroid cancer.

CAVIN2/SDPR Functioned as a Tumor Suppressor in Lung Adenocarcinoma from Systematic Analysis of Caveolae-Related Genes and Experimental Validation.

Background: Caveolae-Related Genes include caveolins and cavins, which are the main component of the fossa and, play important roles in a variety of physiological and pathological processes. Although increasing evidence indicated that caveolins (CAVs) and cavins (CAVINs) are involved in carcinogenesis and progression, their clinical significance and biological function in lung cancer are still limited. Methods: We investigated the expression of CAVs and CAVINs at transcriptional levels using Oncomine and Gene Expression Profiling Interactive Analysis. The protein and mRNA expression levels of CAVs and CAVINs were determined by the human protein atlas website and our surgically resected samples, respectively. The clinical value of prognostic prediction based on the expression of CAVs and CAVINs was also assessed. cBioPortal, GeneMANIA and STRING were used to analyze the molecular characteristics of CAVs and CAVINs in lung adenocarcinoma (LUAD) comprehensively. Finally, we investigated the effect of CAVIN2/SDPR (serum deprivation protein response) on LUAD cells with biological experiments in vitro. Results: The expression of CAV1/2 and CAVIN1/2/3 were significantly downregulated in LUAD and lung squamous cell carcinoma (LUSC). The patients with high expression of CAV1, CAV2, CAV3, CAVIN1 and CAVIN2/SDPR were tightly correlated with a better prognosis in LUAD, while no statistical significances in LUSC. Further, our results found that CAVIN2/SDPR can be identified as a prognostic biomarker independent of other CAVINs in patients with LUAD. Mechanically, the overexpression of CAVIN2/SDPR inhibited cell proliferation and migration owing to the cell apoptosis induction and cell cycle arrest at S phase in LUAD cells. Conclusions: CAVIN2/SDPR functioned as a tumor suppressor, and was able to serve as prognostic biomarkers in precision medicine of LUAD. Mechanically, overexpression of CAVIN2/SDPR inhibited cell proliferation by inducing cell apoptosis and S phase arrest in LUAD cells.

Lysine Acetylome of Breast Cancer-Derived Small Extracellular Vesicles Reveals Specific Acetylation Patterns for Metabolic Enzymes.

Cancer-derived small extracellular vesicles have been proposed as promising potential biomarkers for diagnosis and prognosis of breast cancer (BC). We performed a proteomic study of lysine acetylation of breast cancer-derived small extracellular vesicles (sEVs) to understand the potential role of the aberrant acetylated proteins in the biology of invasive ductal carcinoma and triple-negative BC. Three cell lines were used as models for this study: MCF10A (non-metastatic), MCF7 (estrogen and progesterone receptor-positive, metastatic) and MDA-MB-231 (triple-negative, highly metastatic). For a comprehensive protein acetylation analysis of the sEVs derived from each cell line, acetylated peptides were enriched using the anti-acetyl-lysine antibody, followed by LC-MS/MS analysis. In total, there were 118 lysine-acetylated peptides, of which 22, 58 and 82 have been identified in MCF10A, MCF7 and MDA-MB-231 cell lines, respectively. These acetylated peptides were mapped to 60 distinct proteins and mainly identified proteins involved in metabolic pathways. Among the acetylated proteins identified in cancer-derived sEVs from MCF7 and MDA-MB-231 cell lines are proteins associated with the glycolysis pathway, annexins and histones. Five acetylated enzymes from the glycolytic pathway, present only in cancer-derived sEVs, were validated. These include aldolase (ALDOA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK1), enolase (ENO) and pyruvate kinase M1/2 (PKM). For three of these enzymes (ALDOA, PGK1 and ENO) the specific enzymatic activity was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study reveals that sEVs contain acetylated glycolytic metabolic enzymes that could be interesting potential candidates for early BC diagnostics.

Identification of novel immune subtypes and potential hub genes of patients with psoriasis.

BACKGROUND: Psoriasis is a common, chronic and relapsing immune-related inflammatory dermal disease. Patients with psoriasis suffering from the recurrences is mainly caused by immune response disorder. Thus, our study is aimed to identify novel immune subtypes and select targeted drugs for the precision therapy in different subtypes of psoriasis. METHODS: Differentially expressed genes of psoriasis were identified from the Gene Expression Omnibus database. Functional and disease enrichment were performed by Gene Set Enrichment Analysis and Disease Ontology Semantic and Enrichment analysis. Hub genes of psoriasis were selected from protein-protein interaction networks using Metascape database. The expression of hub genes was validated in human psoriasis samples by RT-qPCR and immunohistochemistry. Further, novel immune subtypes of psoriasis were identified by ConsensusClusterPlus package and its association with hub genes were calculated. Immune infiltration analysis was performed, and its candidate drugs were evaluated by Connectivity Map analysis. RESULTS: 182 differentially expressed genes of psoriasis were identified from GSE14905 cohort, in which 99 genes were significantly up-regulated and 83 genes were down-regulated. We then conducted functional and disease enrichment in up-regulated genes of psoriasis. Five potential hub genes of psoriasis were obtained, including SOD2, PGD, PPIF, GYS1 and AHCY. The high expression of hub genes was validated in human psoriasis samples. Notably, two novel immune subtypes of psoriasis were determined and defined as C1 and C2. Bioinformatic analysis showed C1 and C2 had different enrichment in immune cells. Further, candidate drugs and mechanism of action that applicable to different subtypes were evaluated. CONCLUSIONS: Our study identified two novel immune subtypes and five potential hub genes of psoriasis. These findings might give insight into the pathogenesis of psoriasis and provide effective immunotherapy regimens for the precise treatment of psoriasis.

The droplet breakup model and characteristics of pH-shifted peanut protein isolate-high methoxyl pectin stabilised emulsions under ultrasound.

The effect of pH on the occurrence states of peanut protein isolate (PPI) and high methoxyl pectin (HMP), and droplet breakup model of the emulsions under ultrasound were studied. Particle size distribution and scanning electron microscopy results showed that PPI-HMP existed a soluble complex at pH 5.0, had no interaction at pH 7.0, and was co-soluble at pH 9.0. Droplet breakup model results revealed that the characteristics of emulsion stabilised by PPI-HMP treated at pH 5.0 was different from that at pH 7.0 and 9.0. The average diameter of the droplet well satisfied the model. According to rheological properties, interface tension, and microstructure, the formation mechanism and characteristics of emulsion stabilised by PPI-HMP treated at pH 5.0 was different from that at pH 7.0 and pH 9.0. The research provided a reference for constructing emulsions using pH-shifted PPI-HMP under ultrasound.

Qiangjing tablets ameliorate asthenozoospermia via mitochondrial ubiquitination and mitophagy mediated by LKB1/AMPK/ULK1 signaling.

CONTEXT: Therapeutic effects of Qiangjing tablets (QJT) on sperm vitality and asthenozoospermia (AZS) have been confirmed. However, the mechanism of action remains unclear. OBJECTIVE: This study investigates the effects of QJT on AZS and the underlying mechanism of action. MATERIALS AND METHODS: Sixty Sprague-Dawley rats were randomly divided into six groups: Control, ORN (ornidazole; 200 mg/kg), ORN + QJT-low (0.17 g/mL), ORN + QJT-middle (0.33 g/mL), ORN + QJT-high (0.67 g/mL), and ORN + QJT + Radicicol (0.67 g/mL QJT and 20 mg/kg radicicol) groups. Pathological evaluation and analysis of mitophagy were conducted by H&E staining and transmission electron microscopy, respectively. Reactive oxygen species were detected by flow cytometry. Protein expression was determined by Western blotting. RESULTS: QJT significantly improved ORN-treated sperm motility and kinematic parameters, as well as the pathological symptoms of testicular and epididymal tissues. In particular, QJT mitigated impaired mitochondrial morphology, and increased the PHB, Beclin-1, LC3-II protein, and ROS levels (p < 0.05), and reduced the protein expression levels of LC3-I and p62 (p < 0.05). Mechanistically, QJT antagonized the downregulation of SCF and Parkin protein levels (p < 0.05). Furthermore, QJT significantly increased the protein expressions levels of LKB1, AMPKα, p-AMPKα, ULK1 and p-ULK1 (p < 0.05). The ameliorative effect of QJT on pathological manifestations, mitochondrial morphology, and the expressions of mitophagy and mitochondrial ubiquitination-related proteins was counteracted by radicicol. DISCUSSION AND CONCLUSIONS: QJT improved AZS via mitochondrial ubiquitination and mitophagy mediated by the LKB1/AMPK/ULK1 signaling pathway. Our study provides a theoretical basis for the treatment of AZS and male infertility.

Generation and Physiology of Hydrogen Sulfide and Reactive Sulfur Species in Bacteria.

Sulfur is not only one of the most abundant elements on the Earth, but it is also essential to all living organisms. As life likely began and evolved in a hydrogen sulfide (H2S)-rich environment, sulfur metabolism represents an early form of energy generation via various reactions in prokaryotes and has driven the sulfur biogeochemical cycle since. It has long been known that H2S is toxic to cells at high concentrations, but now this gaseous molecule, at the physiological level, is recognized as a signaling molecule and a regulator of critical biological processes. Recently, many metabolites of H2S, collectively called reactive sulfur species (RSS), have been gradually appreciated as having similar or divergent regulatory roles compared with H2S in living organisms, especially mammals. In prokaryotes, even in bacteria, investigations into generation and physiology of RSS remain preliminary and an understanding of the relevant biological processes is still in its infancy. Despite this, recent and exciting advances in the fields are many. Here, we discuss abiotic and biotic generation of H2S/RSS, sulfur-transforming enzymes and their functioning mechanisms, and their physiological roles as well as the sensing and regulation of H2S/RSS.

Cardiomyocyte-specific disruption of soluble epoxide hydrolase limits inflammation to preserve cardiac function.

Endotoxemia elicits a multiorgan inflammatory response that results in cardiac dysfunction and often leads to death. Inflammation-induced metabolism of endogenous N-3 and N-6 polyunsaturated fatty acids generates numerous lipid mediators, such as epoxy fatty acids (EpFAs), which protect the heart. However, EpFAs are hydrolyzed by soluble epoxide hydrolase (sEH), which attenuates their cardioprotective actions. Global genetic disruption of sEH preserves EpFA levels and attenuates cardiac dysfunction in mice following acute lipopolysaccharide (LPS)-induced inflammatory injury. In leukocytes, EpFAs modulate the innate immune system through the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. However, the mechanisms by which both EpFAs and sEH inhibition exert their protective effects in the cardiomyocyte are still elusive. This study investigated whether cardiomyocyte-specific sEH disruption attenuates inflammation and cardiac dysfunction in acute LPS inflammatory injury via modulation of the NLRP3 inflammasome. We use tamoxifen-inducible CreER recombinase technology to target sEH genetic disruption to the cardiomyocyte. Primary cardiomyocyte studies provide mechanistic insight into inflammasome signaling. For the first time, we demonstrate that cardiomyocyte-specific sEH disruption preserves cardiac function and attenuates inflammatory responses by limiting local cardiac inflammation and activation of the systemic immune response. Mechanistically, inhibition of cardiomyocyte-specific sEH activity or exogenous EpFA treatment do not prevent upregulation of NLRP3 inflammasome machinery in neonatal rat cardiomyocytes. Rather, they limit downstream activation of the pathway leading to release of fewer chemoattractant factors and recruitment of immune cells to the heart. These data emphasize that cardiomyocyte sEH is vital for mediating detrimental systemic inflammation.NEW & NOTEWORTHY The cardioprotective effects of genetic disruption and pharmacological inhibition of sEH have been demonstrated in a variety of cardiac disease models, including acute LPS inflammatory injury. For the first time, it has been demonstrated that sEH genetic disruption limited to the cardiomyocyte profoundly preserves cardiac function and limits local and systemic inflammation following acute LPS exposure. Hence, cardiomyocytes serve a critical role in the innate immune response that can be modulated to protect the heart.

Analysis of Clinicopathological Characteristics and Prognosis of Carcinosarcoma of the Breast.

Background: Few cases of carcinosarcoma of the breast have been reported because of its low incidence rate and rapid progression. Seeking effective therapeutic methods becomes urgent in clinical practice. This study was aimed to investigate the clinical characteristics of carcinosarcoma of the breast and to explore proper therapeutic methods for patients with this rare tumor. Methods: We conducted a retrospective analysis on 47 patients with carcinosarcoma of the breast receiving treatment in our hospital from 2003 to 2020. Most of these patients received primary surgery followed by adjuvant chemotherapy, while four patients had lumpectomy only. Statistics showed no preference in age and menopausal status of patients. Results: The overall survival rate and progression-free survival rate of all patients at a median follow-up time of 33 months were 63.8% and 57.4%, respectively. Tumor size at diagnosis and chemotherapy strategies were both significant prognostic factors in reference to disease-free survival (DFS) and overall survival (OS) of the patients (tumor size: p=0.023 for DFS and p=0.021 for OS; therapeutic method: p=0.041 for DFS and p=0.024 for OS). N stage at diagnosis was significant only with reference to overall survival of the patients (p=0.009). EGFR expression was positive in some patients. Conclusions: Our results elucidated that the patients received comprehensive therapy, especially adjuvant chemotherapy was indispensable for better outcomes. Early detection and treatment were necessary for a higher survival rate when the tumor size was less than 5 cm without lymph node metastasis. Prospective outcomes with novel strategies targeting EGFR need to be further investigated.

Phosphoproteomic Analysis of Breast Cancer-Derived Small Extracellular Vesicles Reveals Disease-Specific Phosphorylated Enzymes.

Small membrane-derived extracellular vesicles have been proposed as participating in several cancer diseases, including breast cancer (BC). We performed a phosphoproteomic analysis of breast cancer-derived small extracellular vesicles (sEVs) to provide insight into the molecular and cellular regulatory mechanisms important for breast cancer tumor progression and metastasis. We examined three cell line models for breast cancer: MCF10A (non-malignant), MCF7 (estrogen and progesterone receptor-positive, metastatic), and MDA-MB-231 (triple-negative, highly metastatic). To obtain a comprehensive overview of the sEV phosphoproteome derived from each cell line, effective phosphopeptide enrichment techniques IMAC and TiO2, followed by LC-MS/MS, were performed. The phosphoproteome was profiled to a depth of 2003 phosphopeptides, of which 207, 854, and 1335 were identified in MCF10A, MCF7, and MDA-MB-231 cell lines, respectively. Furthermore, 2450 phosphorylation sites were mapped to 855 distinct proteins, covering a wide range of functions. The identified proteins are associated with several diseases, mostly related to cancer. Among the phosphoproteins, we validated four enzymes associated with cancer and present only in sEVs isolated from MCF7 and MDA-MB-231 cell lines: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6). With the exception of PFKM, the specific activity of these enzymes was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study demonstrates that sEVs contain functional metabolic enzymes that could be further explored for their potential use in early BC diagnostic and therapeutic applications.

MED1 Downregulation Contributes to TGFß-Induced Metastasis by Inhibiting SMAD2 Ubiquitination Degradation in Cutaneous Melanoma.

Metastasis is the main reason for the high mortality of patients and indeed a difficult task in the treatment of cutaneous melanoma. Therefore, it is of great clinical value to explore the molecular mechanism of cutaneous metastatic melanoma and develop novel therapies. MED1, acting as a factor required for activator-dependent transcription, is reported to be involved in carcinogenesis and progression. In this study, we found that MED1 was highly expressed in patients with cutaneous melanoma. MED1 downregulation could induce cellular epithelial-to-mesenchymal transition and promote migration, invasion, and metastasis of cutaneous melanoma in vivo and in vitro. Further analysis showed that in Med1 knockdown cells, the TGFß/SMAD2 signaling pathway mediated an increase in epithelial-to-mesenchymal transition phenotype and migration. The opposite results were observed after treatment with TGFß inhibitors. To further explore the mechanism, we found that MED1 interacted with SMAD2, and MED1 downregulation could protect SMAD2 from degradation by inhibiting SMAD2 ubiquitination. Together, these results suggest that MED1 inhibited TGFß signaling pathway to reduce cell epithelial-to-mesenchymal transition phenotype and migration through SMAD2 ubiquitination in the metastasis of cutaneous melanoma. Our findings elucidated the role of MED1 in the metastasis of cutaneous melanoma and provided a target for the therapeutic strategies of cutaneous melanoma.

Aberrant HO-1/NQO1-Reactive Oxygen Species-ERK Signaling Pathway Contributes to Aggravation of TPA-Induced Irritant Contact Dermatitis in Nrf2-Deficient Mice.

NF-erythroid 2-related factor 2 (Nrf2) is a major transcription factor to protect cells against reactive oxygen species (ROS) and reactive toxicants. Meanwhile, Nrf2 can inhibit contact dermatitis through redox-dependent and -independent pathways. However, the underlying mechanisms of how Nrf2 mediates irritant contact dermatitis (ICD) are still unclear. In this article, we elucidated the role of Nrf2 in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced acute ICD. Our study demonstrated that the ear thickness, redness, swelling, and neutrophil infiltration were significantly increased, accompanied by increased expression of inflammatory cytokines (IL-1α, IL-1ß, IL-6, etc.) and decreased expression of antioxidant genes (HO-1 and NQO1) in Nrf2 knockout mice. Moreover, ERK phosphorylation was elevated in mouse embryonic fibroblasts (MEFs) from Nrf2 knockout mouse. Inhibition of ERK significantly alleviated TPA-induced cutaneous inflammation and ROS accumulation in MEFs derived from mouse. Conversely, ROS scavenging inhibited the ERK activation and TPA-induced inflammation in MEFs. Taken together, the findings illustrate the key role of the Nrf2/ROS/ERK signaling pathway in TPA-induced acute ICD.

The functions and prognostic value of Krüppel-like factors in breast cancer.

BACKGROUND: Krüppel-like factors (KLFs) are zinc finger proteins which participate in transcriptional gene regulation. Although increasing evidence indicate that KLFs are involved in carcinogenesis and progression, its clinical significance and biological function in breast cancer are still limited. METHODS: We investigated all the expression of KLFs (KLF1-18) at transcriptional levels by using Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA). The mRNA and protein expression levels of KLFs were also determined by using RT-qPCR and immunohistochemistry, respectively. CBioPortal, GeneMANIA and STRING were used to comprehensive analysis of the molecular characteristics of KLFs. The clinical value of prognostic prediction based on the expression of KLFs was determined by using the KM plotter. The relevant molecular pathways of KLFs were further analyzed by using Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Finally, we investigated the effect of KLF2 and KLF15 on biological behavior of breast cancer cells in vitro. RESULTS: The expression of KLF2/4/6/8/9/11/15 was significantly down-regulated in breast cancer. The patients with high KLF2, KLF4 or KLF15 expression had a better outcome, while patients with high KLF8 or KLF11 had a poor prognosis. Furthermore, our results showed that KLF2 or KLF15 can be used as a prognostic factor independent on the other KLFs in patients with breast cancer. Overexpression of KLF2 or KLF15 inhibited cell proliferation and migration, and blocked cell cycle at G0/G1 phase, resulting in cell apoptosis. CONCLUSIONS: KLF2 and KLF15 function as tumor suppressors in breast cancer and are potential biomarkers for prognostic prediction in patients with breast cancer.

Deficiency of vitamin D receptor in keratinocytes augments dermal fibrosis and inflammation in a mouse model of HOCl-induced scleroderma.

Scleroderma, characterized by extensive fibrosis and vascular alterations, involves excessive fibroblast activation, uncontrolled inflammation, and abnormal collagen deposition. Previous studies showed that administrations of either 1,25(OH)2D3 or vitamin D analog effectively decreased or reversed skin fibrosis by regulating the extracellular matrix homeostasis. The actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR), a transcription regulator crucial for skin homeostasis. Although evidence suggests that keratinocyte-fibroblast interaction influences the development of scleroderma, the role of keratinocytes in scleroderma remains unknown. Here, we demonstrated that the ablation of VDR in keratinocytes greatly exacerbated dermal fibrosis in HOCl-induced scleroderma in mice. The deficiency of VDR in the epidermis marked increased dermal thickness, inflammatory cell infiltration, and severe collagen deposition in comparison to the control group in HOCl-treated skin. Moreover, significant elevations in expression levels of mRNA for collagen overproduction (Col1A1, Col1A2, Col3A1, α-SMA, MMP9, TGF-ß1) and proinflammatory cytokines (IL-1ß, IL-6, CXCL1, CXCL2) were observed in VDR conditional KO versus control mice following HOCl treatment. Collectively, these results suggest that VDR in keratinocytes plays a pivotal role in scleroderma progression, and the interplay between keratinocytes and fibroblasts deserves more attention regarding the exploration of the pathogenesis and treatment for scleroderma.