CD248 interacts with ECM to promote hypertrophic scar formation and development.

Hypertrophic scar (HS) presents a significant clinical challenge, frequently arising as a fibrotic sequela of burn injuries and trauma. Characterized by the aberrant activation and proliferation of myofibroblasts, HS lacks a targeted therapeutic approach to effectively reduce this dysregulation. This study offers novel evidence of upregulated expression of CD248 in HS tissues compared to normal skin (NS) tissues. Specifically, the expression of CD248 was predominantly localized to α-SMA+-myofibroblasts in the dermis. To explain the functional role of CD248 in dermal myofibroblast activity, we employed a targeted anti-CD248 antibody, IgG78. Both CD248 intervention and IgG78 treatment effectively suppressed the proliferative, migratory, and ECM-synthesizing activities of myofibroblasts isolated from HS dermis. In addition, IgG78 administration significantly attenuated HS formation in an in vivo rabbit ear model. The LC/MS analysis coupled with co-immunoprecipitation of HS tissues indicated a direct interaction between CD248 and the ECM components Fibronectin (FN) and Collagen I (COL I). These findings collectively suggest that CD248 may function as a pro-fibrotic factor in HS development through its interaction with ECM constituents. The utilization of an anti-CD248 antibody, such as IgG78, represents a promising novel therapeutic strategy for the treatment of HS.

[Screening and promoting effect of grow-promoting fungi in rhizosphere of Angelica dahurica var. formosana].

Excessive application of chemical fertilizer has caused many problems in Angelica dahurica var. formosana planting, such as yield decline and quality degradation. In order to promote the green cultivation mode of A. dahurica var. formosana and explore rhizosphere fungus resources, the rhizosphere fungi with nitrogen fixation, phosphorus solubilization, potassium solubilization, iron-producing carrier, and IAA-producing properties were isolated and screened in the rhizosphere of A. dahurica var. formosana from the genuine and non-genuine areas, respectively. The strains were identified comprehensively in light of the morphological characteristics and ITS rDNA sequences, and the growth-promoting effect of the screened strains was verified by pot experiment. The results showed that 37 strains of growth-promoting fungi were isolated and screened from the rhizosphere of A. dahurica var. formosana, mostly belonging to Fusarium. The cultured rhizosphere growth-promoting fungi of A. dahurica var. formosana were more abundant and diverse in the genuine producing areas than in the non-genuine producing areas. Among all strains, Aspergillus niger ZJ-17 had the strongest growth promotion potential. Under the condition of no fertilization outdoors, ZJ-17 inoculation significantly promoted the growth, yield, and accumulation of effective components of A. dahurica var. formosana planted in the soil of genuine and non-genuine producing areas, with yield increases of 73.59% and 37.84%, respectively. To a certain extent, it alleviated the restriction without additional fertilization on the growth of A. dahurica var. formosana. Therefore, A. niger ZJ-17 has great application prospects in increasing yield and quality of A. dahurica var. formosana and reducing fertilizer application and can be actually applied in promoting the growth of A. dahurica var. formosana and producing biofertilizer.

Extracellular vesicles from 3D cultured dermal papilla cells improve wound healing via Krüppel-like factor 4/vascular endothelial growth factor A -driven angiogenesis.

Background: Non-healing wounds are an intractable problem of major clinical relevance. Evidence has shown that dermal papilla cells (DPCs) may regulate the wound-healing process by secreting extracellular vesicles (EVs). However, low isolation efficiency and restricted cell viability hinder the applications of DPC-EVs in wound healing. In this study, we aimed to develop novel 3D-DPC spheroids (tdDPCs) based on self-feeder 3D culture and to evaluate the roles of tdDPC-EVs in stimulating angiogenesis and skin wound healing. Methods: To address the current limitations of DPC-EVs, we previously developed a self-feeder 3D culture method to construct tdDPCs. DPCs and tdDPCs were identified using immunofluorescence staining and flow cytometry. Subsequently, we extracted EVs from the cells and compared the effects of DPC-EVs and tdDPC-EVs on human umbilical vein endothelial cells (HUVECs) in vitro using immunofluorescence staining, a scratch-wound assay and a Transwell assay. We simultaneously established a murine model of full-thickness skin injury and evaluated the effects of DPC-EVs and tdDPC-EVs on wound-healing efficiency in vivo using laser Doppler, as well as hematoxylin and eosin, Masson, CD31 and α-SMA staining. To elucidate the underlying mechanism, we conducted RNA sequencing (RNA-seq) of tdDPC-EV- and phosphate-buffered saline-treated HUVECs. To validate the RNA-seq data, we constructed knockdown and overexpression vectors of Krüppel-like factor 4 (KLF4). Western blotting, a scratch-wound assay, a Transwell assay and a tubule-formation test were performed to detect the protein expression, cell migration and lumen-formation ability of KLF4 and vascular endothelial growth factor A (VEGFA) in HUVECs incubated with tdDPC-EVs after KLF4 knockdown or overexpression. Dual-luciferase reporter gene assays were conducted to verify the activation effect of KLF4 on VEGFA. Results: We successfully cultured tdDPCs and extracted EVs from DPCs and tdDPCs. The tdDPC-EVs significantly promoted the proliferation, lumen formation and migration of HUVECs. Unlike DPC-EVs, tdDPC-EVs exhibited significant advantages in terms of promoting angiogenesis, accelerating wound healing and enhancing wound-healing efficiency both in vitro and in vivo. Bioinformatics analysis and further functional experiments verified that the tdDPC-EV-regulated KLF4/VEGFA axis is pivotal in accelerating wound healing. Conclusions: 3D cultivation can be utilized as an innovative optimization strategy to effectively develop DPC-derived EVs for the treatment of skin wounds. tdDPC-EVs significantly enhance wound healing via KLF4/VEGFA-driven angiogenesis.

[Proanthocyanidins alleviate lipopolysaccharide-induced inflammatory response by up-regulating SIRT1 expression and inhibiting NF-κB pathway in mouse RAW264.7 macrophages].

Objective To investigate the role of proanthocyanidins (PC) in lipopolysaccharide (LPS)-induced inflammatory response and its possible mechanism in RAW264.7 macrophages. Methods RAW264.7 macrophages were cultured and treated with PBS and different concentrations of PC for 24 hours, followed by 1 µg/mL LPS for 6 hours. Real-time PCR was used to detect the mRNA expression of interleukin1ß (IL-1ß), IL-6, monocyte chemoattractant protein 1 (MCP-1), tumor necrotic factor α (TNF-α), IL-4 and arginase 1 (Arg1) in RAW264.7 macrophages. Flow cytometry was used to detect the effects of PBS group, LPS group and PC combined with LPS group on M1 and M2 polarization of macrophages. The protein expressions of silenced information regulator 1 (SIRT1), nuclear factor kappa B p65(NF-κB p65) and acetylated NF-κB p65 (Ace-p65) were detected by Western blot analysis after different concentrations of PC treatment. Co-immunoprecipitation assay was used to detect the binding effect of SIRT1 to NF-κB p65 in macrophages treated with PC. Results Compared with PBS group, the mRNA expression of macrophage pro-inflammatory cytokines IL-1ß, IL-6, MCP-1 and TNF-α decreased and the mRNA expression of anti-inflammatory factors IL-4 and Arg1 increased in PC group. Compared with LPS group, PC combined with LPS group could significantly inhibit M1 polarization and promote M2 polarization of macrophages. With the increase of PC concentration, the expression of SIRT1 was up-regulated, and NF-κB p65 protein did not change significantly. The expression of Ace-p65 protein decreased significantly when treated with high concentration of PC. Conclusion PC can significantly alleviate the LPS-induced inflammatory response by up-regulating the expression of SIRT1 and inhibiting NF-κB pathway in RAW264.7 macrophages.

Analysis of the rhizosphere bacterial diversity of Angelica dahurica var. formosana from different experimental sites and varieties (strains).

Background: Rhizosphere bacteria play important roles in plant growth and secondary metabolite accumulation. Moreover, only with favorable production areas and desirable germplasm can high-yield and high-quality medicinal materials be produced. However, whether origin and germplasm indirectly affect the yield and quality of Angelica dahurica var. formosana through rhizosphere bacterial effects are not known. Methods: In this study, a high-throughput sequencing strategy was used to explore the relationship between the rhizosphere bacterial community and the cultivation of A. dahurica var. formosana from different production areas and germplasm for the first time. Results: (1) Proteobacteria was the dominant bacterial phylum in the rhizosphere soil of A. dahurica var. formosana, and these bacteria were stable and conserved to a certain extent. (2) High abundance of Proteobacteria was an important rhizospheric indicator of high yield, and high abundance of Firmicutes was an important indicator of high quality. Proteobacteria and Firmicutes might have an important relationship with the yield and quality of A. dahurica var. formosana, respectively. (3) PCoA cluster analysis demonstrated that both production area and germplasm affected the bacterial community structure in the rhizosphere of A. dahurica var. formosana to a certain extent, and production area had the greatest effect. In addition to available potassium, the rhizosphere soil nutrient levels of different production areas strongly affected the bacterial diversity and community. These findings provide a theoretical basis for the exploitation and utilization of rhizosphere microbial resources of A. dahurica var. formosana and offer a novel approach for increasing the yield and quality of this crop.

Portal Fibrotic Cord is Associated with Transjugular Intrahepatic Portosystemic Shunt Failure and Death in Cirrhotic Patients.

Background and Aims: Occlusive portal vein thrombosis (PVT) often causes portal hypertension-related complications in cirrhotic patients. Transjugular intrahepatic portosystemic shunt (TIPS) is an effective treatment for this difficult problem. However, the factors influencing TIPS success and overall survival in patients with occlusive PVT are unknown. This study investigated the factors influencing TIPS success and overall survival in cirrhotic patients with occlusive PVT. Methods: Cirrhotic patients with occlusive PVT were selected from a prospective database of consecutive patients treated with TIPS in Xijing Hospital between January 2015 and May 2021. Baseline characteristics, TIPS success rate, complications, and survival were collected, and the factors associated with the TIPS success rate and transplant-free survival were analyzed. Results: A total of 155 cirrhotic patients with occlusive PVT were enrolled. TIPS succeeded in 126 (81.29%) cases. The 1-year survival rate was 74%. Compared with those without, patients with portal fibrotic cord had a lower TIPS success rate (39.02% vs. 96.49%, p<0.001), shorter median overall survival (300 vs. 1,730 days, p<0.001) and more operation-related complications (12.20% vs. 1.75%, p<0.01). Logistic regression analysis found that portal fibrotic cord (odds ratio 0.024) was a risk factor for TIPS failure. Univariate and multivariate analysis showed that portal fibrotic cord was an independent predictor of death (hazard ratio 2.111; 95% CI: 1.094-4.071, p=0.026). Conclusions: Portal fibrotic cord increased the TIPS failure rate and is a risk factor for poor prognosis in cirrhotic patients.

The deacetylation of Akt by SIRT1 inhibits inflammation in macrophages and protects against sepsis.

Sepsis is characterized by uncontrolled inflammatory response and altered polarization of macrophages at the early phase. Akt is known to drive macrophage inflammatory response. However, how macrophage inflammatory response is fine-tuned by Akt is poorly understood. Here, we found that Lys14 and Lys20 of Akt is deacetylated by the histone deacetylase SIRT1 during macrophage activation to suppress macrophages inflammatory response. Mechanistically, SIRT1 promotes Akt deacetylation to inhibit the activation of NF-κB and pro-inflammatory cytokines. Loss of SIRT1 facilitates Akt acetylation and thus promotes inflammatory cytokines in mouse macrophages, potentially worsen the progression of sepsis in mice. By contrast, the upregulation of SIRT1 in macrophages further contributes to the inhibition of pro-inflammatory cytokines via Akt activation in sepsis. Taken together, our findings establish Akt deacetylation as an essential negative regulatory mechanism that curtails M1 polarization.

Engineering the maturation of stem cell-derived cardiomyocytes.

The maturation of human stem cell-derived cardiomyocytes (hSC-CMs) has been a major challenge to further expand the scope of their application. Over the past years, several strategies have been proven to facilitate the structural and functional maturation of hSC-CMs, which include but are not limited to engineering the geometry or stiffness of substrates, providing favorable extracellular matrices, applying mechanical stretch, fluidic or electrical stimulation, co-culturing with niche cells, regulating biochemical cues such as hormones and transcription factors, engineering and redirecting metabolic patterns, developing 3D cardiac constructs such as cardiac organoid or engineered heart tissue, or culturing under in vivo implantation. In this review, we summarize these maturation strategies, especially the recent advancements, and discussed their advantages as well as the pressing problems that need to be addressed in future studies.

Deacetylation of IRF8 inhibits iNOS expression and inflammation via SIRT1 in macrophages.

AIMS: Dysregulated interferon regulatory factor 8 (IRF8) mediated inducible nitric oxide synthase (iNOS) transcription is crucial to the pathogenesis of several inflammatory disorders. However, the molecular mechanism that control the transcription activity of IRF8 in the regulation of iNOS is not fully elucidated. This study is undertaken to determine whether SIRT1 impacts IRF8 acetylation level in the macrophages. MAIN METHODS: The silver stain, mass spectrum, bone marrow-derived monocytes differentiation, lentiviral transduction, immunoprecipitation and chromatin immunoprecipitation assay were used to investigate the relationship between IRF8 and SIRT1. KEY FINDINGS: We demonstrate that deacetylation of IRF8 is induced by lipopolysaccharide (LPS) and suppresses iNOS expression. Macrophages expressing acetylation-defective iNOS are highly septic upon transfer to macrophages cleaned up mice. Mechanistically, deacetylation IRF8 facilitates the binding of silent information regulator 1 (SIRT1) to the iNOS promoter and restricts iNOS transcription. The expression of iNOS was enhanced in the macrophages from SIRT1 conditional knockout mice and the progression of sepsis is more serious. SIGNIFICANCE: The discovery of the IRF8-SIRT1 interaction that governs iNOS expression may exploit new therapeutic strategies for inflammatory disorders.

MiR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

Background: Gastric cancer (GC) is one of the most malignant and lethal cancers worldwide. Multiple microRNAs (miRNAs) have been identified as key regulators in the progression of GC. However, the underlying pathogenesis that miRNAs govern GC malignancy remains uncertain. Here, we identified a novel miR-585-5p as a key regulator in GC development. Methods: The expression of miR-585-5p in the context of GC tissue was detected by in situ hybridization for GC tissue microarray and assessed by H-scoring. The gain- and loss-of-function analyses comprised of Cell Counting Kit-8 assay and Transwell invasion and migration assay. The expression of downstream microphthalmia-associated transcription factor (MITF), cyclic AMP-responsive element-binding protein 1 (CREB1) and mitogen-activated protein kinase 1 (MAPK1) were examined by Immunohistochemistry, quantitative real-time PCR and western blot. The direct regulation between miR-585-5p and MITF/CREB1/MAPK1 were predicted by bioinformatic analysis and screened by luciferase reporter assay. The direct transcriptional activation of CREB1 on MITF was verified by luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs). The interaction between MAPK1 and MITF was confirmed by co-immunoprecipitation (Co-IP) and immunofluorescent double-labelled staining. Results: MiR-585-5p is progressively downregulated in GC tissues and low miR-585-5p levels were strongly associated with poor clinical outcomes. Further gain- and loss-of-function analyses showed that miR-585-5p possesses strong anti-proliferative and anti-metastatic capacities in GC. Follow-up studies indicated that miR-585-5p targets the downstream molecules CREB1 and MAPK1 to regulate the transcriptional and post-translational regulation of MITF, respectively, thus controlling its expression and cancer-promoting activity. MiR-585-5p directly and negatively regulates MITF together with CREB1 and MAPK1. According to bioinformatic analysis, promotor reporter gene assays, ChIP and EMSAs, CREB1 binds to the promotor region to enhance transcriptional expression of MITF. Co-IP and immunofluorescent double-labelled staining confirmed interaction between MAPK1 and MITF. Protein immunoprecipitation revealed that MAPK1 enhances MITF activity via phosphorylation (Ser73). MiR-585-5p can not only inhibit MITF expression directly, but also hinder MITF expression and pro-cancerous activity in a CREB1-/MAPK1-dependent manner indirectly. Conclusions: In conclusion, this study uncovered miR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

The interaction between autophagy and the epithelial-mesenchymal transition mediated by NICD/ULK1 is involved in the formation of diabetic cataracts.

BACKGROUND: Cataracts are the leading cause of blindness and a common ocular complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) and altered autophagic activity occur during the development of diabetic cataracts. The disturbed interaction of autophagy with EMT in LECs stimulated by high glucose levels may participate in cataract formation. METHODS: A rat diabetic cataract model induced by streptozotocin (STZ) and human lens epithelial cells (HLE-B3) stimulated with a high glucose concentration were employed in the study. These models were treated with rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)), and N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT, an inhibitor of γ-secretase) alone or in combination. Lens opacity was observed and photographed under a slit-lamp microscope. Histological changes in paraffin sections of lenses were detected under a light microscope after hematoxylin and eosin staining. Alterations of autophagosomes in LECs were counted and evaluated under a transmission electron microscope. The expression levels of proteins involved in the EMT, autophagy, and the signaling pathways in LECs were measured using Western blotting and immunofluorescence staining. Cell migration was determined by performing transwell and scratch wound assays. Coimmunoprecipitation (Co-IP) was performed to verify protein-protein interactions. Proteins were overexpressed in transfected cells to confirm their roles in the signaling pathways of interest. RESULTS: In LECs, a high glucose concentration induces the EMT by activating Jagged1/Notch1/Notch intracellular domain (NICD)/Snail signaling and inhibits autophagy through the AKT/mTOR/unc 51-like kinase 1 (ULK1) signaling pathway in vivo and in vitro, resulting in diabetic cataracts. Enhanced autophagic activity induced by rapamycin suppressed the EMT by inducing Notch1 degradation by SQSTM1/p62 and microtubule-associated protein light chain 3 (LC3) in LECs, while inhibition of the Notch signaling pathway with DAPT not only prevented the EMT but also activated autophagy by decreasing the levels of NICD, which bound to ULK1, phosphorylated it, and then inhibited the initiation of autophagy. CONCLUSIONS: We describe a new interaction of autophagy and the EMT involving NICD/ULK1 signaling, which mediates crosstalk between these two important events in the formation of diabetic cataracts. Activating autophagy and suppressing the EMT mutually promote each other, revealing a potential target and strategy for the prevention of diabetic cataracts.

Functionalizing multi-component bioink with platelet-rich plasma for customized in-situ bilayer bioprinting for wound healing.

In-situ three-dimensional (3D) bioprinting has been emerging as a promising technology designed to rapidly seal cutaneous defects according to their contour. Improvements in the formulations of multi-component bioink are needed to support cytocompatible encapsulation and biological functions. Platelet-rich plasma (PRP), as a source of patient-specific autologous growth factors, exhibits capabilities in tissue repair and rejuvenation. This study aimed to prepare PRP-integrated alginate-gelatin (AG) composite hydrogel bioinks and evaluate the biological effects in vitro and in vivo. 3D bioprinted constructs embedded with dermal fibroblasts and epidermal stem cells were fabricated using extrusion strategy. The integration of PRP not only improved the cellular behavior of seeded cells, but regulate the tube formation of vascular endothelial cells and macrophage polarization in a paracrine manner, which obtained an optimal effect at an incorporation concentration of 5%. For in-situ bioprinting, PRP integration accelerated the high-quality wound closure, modulated the inflammation and initiated the angiogenesis compared with the AG bioink. In conclusion, we revealed the regenerative potential of PRP, readily available at the bedside, as an initial signaling provider in multi-component bioink development. Combined with in-situ printing technology, it is expected to accelerate the clinical translation of rapid individualized wound repair.

KLF4 Alleviates Hypertrophic Scar Fibrosis by Directly Activating BMP4 Transcription.

Background: Hypertrophic scars (HS) often occur after burns, surgery and extensive trauma. Krüppel-like factor 4 (KLF4) is a member of the Krüppel-like factor family, a group of conserved zinc finger transcription factors that regulate diverse cellular processes. KLF4 can participate in the regulation of fibrotic diseases in many organs, such as the lung, liver, and heart. However, the antifibrotic effect of KLF4 in skin HS remains elusive. Result: This study observed the inhibition of KLF4 on fibrosis in vivo and in vitro. Our results revealed that KLF4 expression was decreased in HS tissue and fibroblasts. The results of KLF4 transfection confirmed its ability to alleviate the transdifferentiation of fibroblasts into myofibroblasts both in vitro and in vivo, thereby inhibiting the development of fibrosis. In addition, ChIP assays showed that BMP4 was the target gene of KLF4 for inhibiting skin fibrosis. Conclusions: Collectively, this evidence indicates that KLF4 is associated with BMP4 and could play an important regulatory role in HS formation by downregulating myofibroblast transdifferentiation. Our study provides a new target for the prevention and treatment of hypertrophic scars.

[Bioinformatics and expression analysis on MYB-related family in Angelica dahurica var. formosana].

In recent years, the MYB-related gene family has been found pivotal in plant growth and development. MYB-related gene family in Angelica dahurica var. formosana was systematically investigated based on "Chuanzhi No. 2" through transcriptome database search and bioinformatics and the temporal and spatial expression patterns were analyzed through real-time fluorescence-based quantitative polymerase chain reaction(PCR). The results showed that 122 MYB-related proteins family were identified, mainly including the unstable hydrophilic proteins with good thermal stability. Most of the proteins were located in nuclei. The majority of the proteins had the structures of random coil and α-helix. Five MYB-related proteins family of A. dahurica var. formosana had membrane-binding domains. The conserved domain analysis of MYB-related proteins family of A. dahurica var. formosana showed that the MYB domains of genes in five subgroups, similar to 2 R-, 3 R-, and 4 R-MYB proteins, contained three evenly distributed Trp(W) residues in the MYB repeat sequence. The phylogenetic analysis of MYB-related proteins family in A. dahurica var. formosana and Arabidopsis thaliana showed that the MYB-related members were unevenly distributed in five subgroups, and A. thaliana and A. dahurica var. formosana had almost the same number of genes in the CCA1-like subgroup. There were differences in the number, type, and distribution of motifs contained in 122 encoded proteins. Transcription factors with similar branches had similar domains and motifs. The expression pattern analysis showed that the transcription factors AdMYB53, AdMYB83, and AdMYB89 responded to hormones to varying degrees, and they were highly expressed in leaves and responded quickly in roots. This study lays a foundation for further investigating the function of MYB-related transcription factors of A. dahurica var. formosana and solving the corresponding biological problems such as bolting early.

Regulation of SIRT1 and Its Roles in Inflammation.

The silent information regulator sirtuin 1 (SIRT1) protein, a highly conserved NAD+-dependent deacetylase belonging to the sirtuin family, is a post-translational regulator that plays a role in modulating inflammation. SIRT1 affects multiple biological processes by deacetylating a variety of proteins including histones and non-histone proteins. Recent studies have revealed intimate links between SIRT1 and inflammation, while alterations to SIRT1 expression and activity have been linked to inflammatory diseases. In this review, we summarize the mechanisms that regulate SIRT1 expression, including upstream activators and suppressors that operate on the transcriptional and post-transcriptional levels. We also summarize factors that influence SIRT1 activity including the NAD+/NADH ratio, SIRT1 binding partners, and post-translational modifications. Furthermore, we underscore the role of SIRT1 in the development of inflammation by commenting on the proteins that are targeted for deacetylation by SIRT1. Finally, we highlight the potential for SIRT1-based therapeutics for inflammatory diseases.

Circular RNA expression profiles following negative pressure wound therapy in burn wounds with experimental Pseudomonas aeruginosa infection.

Infections of burn wounds, especially those caused by Pseudomonas aeruginosa, could trigger sepsis or septic shock, which is the main cause of death after burn injury. Compared with traditional saline-wet-to-dry dressings, negative pressure wound therapy (NPWT) is more effective for the prevention and treatment of wound infections. However, the mechanism by which NPWT controls infection and accelerates wound healing remains unclear. Accordingly, in this study, the molecular mechanisms underlying the effects of NPWT were explored using a murine model of P. aeruginosa-infected burn wounds. NPWT significantly reduced P. aeruginosa levels in wounds, enhanced blood flow, and promoted wound healing. Additionally, NPWT markedly alleviated wound inflammation and increased the expression of wound healing-related molecules. Recent evidence points to a role of circular RNAs (circRNAs) in wound healing; hence, whole-transcriptome sequencing of wound tissues from NPWT and control groups was performed to evaluate circRNA expression profiles. In total, 12 up-regulated and 25 down-regulated circRNAs were identified between groups. Among these, five significant differentially expressed circRNAs acting as microRNA sponges were identified, and their predicted targets were verified by reverse transcription-quantitative polymerase chain reaction. These results further support the roles of circRNAs in wound healing by NPWT and the prevention of P. aeruginosa infection, providing key molecular targets for further functional analyses.

Exosomes Derived From Adipose-Derived Mesenchymal Stem Cells Ameliorate Radiation-Induced Brain Injury by Activating the SIRT1 Pathway.

OBJECTIVE: Studies have shown that the therapeutic effects of mesenchymal stem cells (MSCs) are mediated in a paracrine manner, mainly through extracellular vesicles such as exosomes. Here, we designed a study to investigate whether exosomes derived from adipose-derived mesenchymal stem cells (ADMSC-Exos) had protective effects in a rat model of radiation-induced brain injury and in microglia. METHODS: Male adult Sprague-Dawley (SD) rats were randomly divided into three groups: the control group, the radiation group (30 Gy), and the radiation + exosomes group (30 Gy + 100 ug exosomes). Meanwhile, microglia were divided into four groups: the control group, the radiation group (10 Gy), the radiation + exosomes group (10 Gy + 4 ug exosomes), and radiation + exosomes + EX527 group (10 Gy + 4 ug exosomes + 100 nM EX527). Tissue samples and the levels of oxidative stress and inflammatory factors in each group were compared. RESULTS: Statistical analysis showed that after irradiation, ADMSC-Exos intervention in vivo significantly reduced the levels of caspase-3, malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), and promoted the recovery of superoxide dismutase (SOD), catalase (CAT), IL-4, and IL-10. Moreover, ADMSC-Exos intervention inhibited microglial infiltration and promoted the expression of SIRT1. Furthermore, the results in vitro showed that the above effects of ADMSC-Exos could be reversed by SIRT-1 inhibitor EX527. CONCLUSION: This study demonstrated that ADMSC-Exos exerted protective effects against radiation-induced brain injury by reducing oxidative stress, inflammation and microglial infiltration via activating the SIRT1 pathway. ADMSC-Exos may serve as a promising therapeutic tool for radiation-induced brain injury.

Identification of the Key Residues of the Uridine Diphosphate Glycosyltransferase 91D2 and its Effect on the Accumulation of Steviol Glycosides in Stevia rebaudiana.

Stevia (Stevia rebaudiana Bertoni) possesses substantial value for its unique sweet compounds-steviol glycosides (SGs). In the metabolic glycosylation grid of SGs, SrUGT91D2 has been shown to catalyze formation of 1,2-ß-d-glucoside linkages at the C13- and C19-positions and play a crucial role in the synthesis of SGs, including the formation of stevioside (ST), rebaudioside E (RE), and rebaudioside D (RD). However, the key residues of the SrUGT91D2 enzyme and how SrUGT91D2 affects the accumulation of SGs in S. rebaudiana remain unclear. In the present study, cloning and functional analysis of full-length SrUGT91D2 gene sequences were performed in 10 different S. rebaudiana genotypes with divergent SG compositions. After sequence analysis, it was found that most of the sequences of this gene (more than 50%) in each genotype were consistent with the UGT91D2e_No.5 allele, which has been reported to exert catalytic activity on 1,2-ß-d-glucoside. Moreover, six variants (UGT91D2e_No.5, SrUGT91D2-11-14, SrUGT91D2-110, SrUGT91D2-023, SrUGT91D2-N01, and SrUGT91D2-N04) of this gene were obtained, and their activities were identified. Although there were some differences among these variants, the only type of mutation was partial base substitution at a very low level. In addition, the expression analysis of SrUGT91D2 in each genotype showed that the expression level of the gene was significantly different among genotypes, and a significant positive correlation was found between the content of RD (which was closely influenced by SrUGT91D2) and the expression level of SrUGT91D2 in each genotype (correlation coefficient = 0.91). Thus, it was indicated that SrUGT91D2 was relatively conserved in S. rebaudiana, and the differential effect of SrUGT91D2 on the accumulation of related SGs mainly derived from its expression level. Furthermore, based on homologous modeling and molecular docking analysis, T84, T144, A194, S284, E285, V286, G365, E369, R404, and G409 were predicted to be key residues in the glucosylation of SGs by SrUGT91D2. After site-mutation and enzyme assays, it was confirmed that T84, T144, R404, A194, and G409 are the key residues in the SrUGT91D2 protein, especially T144 and G409. This work provided valuable information for understanding the structure-activity relationship of the SrUGT91D2 protein and the molecular mechanism of SG accumulation in stevia.

Characterizing glycosyltransferases by a combination of sequencing platforms applied to the leaf tissues of Stevia rebaudiana.

BACKGROUND: Stevia rebaudiana (Bertoni) is considered one of the most valuable plants because of the steviol glycosides (SGs) that can be extracted from its leaves. Glycosyltransferases (GTs), which can transfer sugar moieties from activated sugar donors onto saccharide and nonsaccharide acceptors, are widely distributed in the genome of S. rebaudiana and play important roles in the synthesis of steviol glycosides. RESULTS: Six stevia genotypes with significantly different concentrations of SGs were obtained by induction through various mutagenic methods, and the contents of seven glycosides (stevioboside, Reb B, ST, Reb A, Reb F, Reb D and Reb M) in their leaves were considerably different. Then, NGS and single-molecule real-time (SMRT) sequencing were combined to analyse leaf tissue from these six different genotypes to generate a full-length transcriptome of S. rebaudiana. Two phylogenetic trees of glycosyltransferases (SrUGTs) were constructed by the neighbour-joining method and successfully predicted the functions of SrUGTs involved in SG biosynthesis. With further insight into glycosyltransferases (SrUGTs) involved in SG biosynthesis, the weighted gene co-expression network analysis (WGCNA) method was used to characterize the relationships between SrUGTs and SGs, and forty-four potential SrUGTs were finally obtained, including SrUGT85C2, SrUGT74G1, SrUGT76G1 and SrUGT91D2, which have already been reported to be involved in the glucosylation of steviol glycosides, illustrating the reliability of our results. CONCLUSION: Combined with the results obtained by previous studies and those of this work, we systematically characterized glycosyltransferases in S. rebaudiana and forty-four candidate SrUGTs involved in the glycosylation of steviol glucosides were obtained. Moreover, the full-length transcriptome obtained in this study will provide valuable support for further research investigating S. rebaudiana.

ING4 alleviated lipopolysaccharide-induced inflammation by regulating the NF-κB pathway via a direct interaction with SIRT1.

Sepsis is a complex inflammatory disorder in which high mortality is associated with an excessive inflammatory response. Inhibitor of growth 4 (ING4), which is a cofactor of histone acetyltransferase and histone deacetylase complexes, could negatively regulate this inflammation. However, the exact molecular signaling pathway regulated by ING4 remains uncertain. As a pivotal histone deacetylase, Sirtuin1 (SIRT1), which is widely accepted to be an anti-inflammatory molecule, has not been found to be linked to ING4. This study investigated how ING4 is involved in the regulation of inflammation by constructing lipopolysaccharide (LPS)-induced macrophage and mouse sepsis models. Our results revealed that ING4 expression decreased, whereas the levels of proinflammatory cytokines increased in LPS-stimulated cultured primary macrophages and RAW 264.7 cells. ING4 transfection was confirmed to alleviate the LPS-induced upregulation of proinflammatory cytokine expression both in vitro and in vivo. In addition, ING4-overexpressing mice were hyposensitive to an LPS challenge and displayed reduced organ injury. Furthermore, immunoprecipitation indicated a direct interaction between ING4 and the SIRT1 protein. Moreover, ING4 could block nuclear factor-kappa B (NF-κB) P65 nuclear translocation and restrict P65 acetylation at lysine 310 induced by LPS treatment. These results are the first to clarify that the anti-inflammatory role of ING4 is associated with SIRT1, through which ING4 inhibits NF-κB signaling activation. Our studies provide a novel signaling axis involving ING4/SIRT1/NF-κB in LPS-induced sepsis.