Expression Levels Of Type Research Articles

Treatment of keloids through Runx2 siRNA‑induced inhibition of the PI3K/AKT signaling pathway.

Keloids are a skin fibroproliferative condition characterized by the hyperproliferation of fibroblasts and the excessive deposition of extracellular matrix (ECM) components. Previous studies have determined that Caveolin-1 controlled hyperresponsiveness to mechanical stimuli through Runt-related transcription factor 2 (Runx2) activation in keloids. However, the molecular mechanism of Runx2 regulating the pathological progression of keloids has not been elucidated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that most of the differentially expressed genes (DEGs), including Runx2, were significantly enriched in the biological processes ‘Positive regulation of cell proliferation’, in the cellular components ‘Extracellular matrix’, in the molecular functions ‘Extracellular matrix structural constituents’ and in the KEGG ‘PI3K-Akt signaling pathway’. The aim of the present study was to investigate the expression levels of the Runx2 in human keloid tissues and primary human keloid fibroblasts (HKFs), and to determine the underlying molecular mechanisms involved in the fibrotic roles of Runx2 in keloid formation. Runx2 expression levels were analyzed in patient keloid tissues and HKFs using western blotting, reverse transcription-quantitative PCR (RT-qPCR) and immunofluorescence microscopy. Primary HKFs were transfected with a small interfering RNA (si) specifically targeting Runx2 (si-Runx2). Subsequently, Cell Counting Kit-8, wound healing and Transwell assays, flow cytometry, RT-qPCR and western blotting were applied to evaluate the proliferation, migration, apoptosis, ECM deposition and PI3K/AKT signaling pathway of HKFs, respectively. In addition, western blotting was also used to determine the expression levels of phosphorylated AKT and PI3K in HKFs. The results revealed that Runx2 expression levels were upregulated in keloid tissues and primary HKFs compared with the normal skin tissues and human normal fibroblasts. Following the transfection with si-Runx2, the proliferative and migratory abilities of HKFs were significantly reduced and the apoptotic rate was increased. The expression levels of type I, type III collagen, fibronectin, and α-smooth muscle actin were downregulated in si-Runx2-transfected cells, which was hypothesized to occur through following the downregulation of the phosphorylation levels of PI3K and AKT. In conclusion, the findings of the present study indicated that Runx2 silencing in HKFs might significantly inhibit the cell proliferation, migration and the expression levels of ECM-related proteins, and promote apoptosis via suppressing the PI3K/AKT signaling pathway. Thus, Runx2 siRNA treatment may reverse the pathological phenotype of keloids through the inhibition of PI3K/AKT signaling in patients.

Abstract 2976: Targeting fibroblast growth factor (FGF) signals induced type I IFN regulated response and Immunogenic cell death (ICD) markers in tumor cells with activated FGF signals

Abstract Introduction: The FGF family plays key roles in many developmental and physiological processes. Aberrant activation of FGF signals (receptor mutations, fusions, and amplifications of ligands or receptors) have been reported, and considered to cause malignant cancers. Targeting FGFR suppresses proliferation and survival of tumor cells with activated FGF signal. However, the role of tumor FGFR inhibition in tumor microenvironments (TMEs) is largely still unknown. Lenvatinib (LEN) is a multi-targeted tyrosine kinase inhibitor that mainly inhibits VEGFR1-3 and FGFR1-4. Previously we showed that LEN inhibited tumor FGF signal, and induced cancer cell death, which had a role in antitumor activity. Here, we investigated roles of targeting tumor FGFR in TMEs using LEN or FGFR1-3 specific inhibitor E7090 in tumor cells with activated FGF signals. Methods: To examine effects of FGF signal on gene expression profile (GEP) of tumor cells, mouse breast tumor 4T1 cell lines expressing Luc2 and AcGFP (4T1-Luc), human hepatocellular carcinoma (HCC) Hep3B2.1-7 and HuH-7 cells with FGF19 overexpression, and an endometrial cancer (EC) AN3CA cell line harboring FGFR2 mutation were treated with LEN or E7090. GEP was analyzed by RNA-Seq or qPCR in the 4T1-Luc model. Pathway enrichment analysis using RNA-Seq data was conducted by Ingenuity pathway analysis (IPA). Expression levels of type I IFN-related genes were analyzed in HCC and EC cell lines. Expressions of Cxcl10 and HMGB1 in culture supernatant were measured by ELISA. Cell surface expressions of PD-L1, MHC class I molecules and Calreticulin (CRT) were determined by flow cytometry. Results: Unbiased pathway analysis using RNA-Seq data (fold change >2 or <0.5, TPM in NT-average >1) from 4T1-Luc cells treated with LEN or E7090 identified Interferon (IFN) signal as one of the top 3 most significantly regulated pathways with several genes known to regulate tumor immunity. qPCR analysis showed that LEN and E7090 increased expression of type I IFN-related genes in human HCC and EC cell lines. LEN significantly increased CXCL10 in culture supernatant and expression of MHC class I and PD-L1 on cell surfaces of 4T1-Luc and Hep3B2.1-7 cells. E7090 also increased Cxcl10 in culture sup and expressions of MHC class I and PD-L1 on cell surface of 4T1-Luc cells. In addition to type I IFN-response, we found that LEN and E7090 might regulate ICD evidenced by increased expression levels of cell surface CRT, and Len increased secreted HMGB1 in culture sup of Hep3B2.1-7 cells. Conclusion: Our results suggested that tumor FGF signals modulated immune response in TMEs through regulating interferon signal. Tumor FGF signal inhibition by LEN and E7090 may enhance antitumor immune response in the TMEs of that were immune suppressive with activated FGF signals. Further analyses will be warranted. Citation Format: Taisuke Hoshi, Yu Kato, Yasuhiro Funahashi. Targeting fibroblast growth factor (FGF) signals induced type I IFN regulated response and Immunogenic cell death (ICD) markers in tumor cells with activated FGF signals [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2976.

Impact and prognosis of the expression of IFN-α among tuberculosis patients.

Mycobacterium tuberculosis (M.tb) infection stimulates the release of cytokines, including interferons (IFNs). IFNs are initiators, regulators, and effectors of innate and adaptive immunity. Accordingly, the expression levels of Type I (α, β) and II (γ) IFNs, among untreated tuberculosis (TB) patients and household contacts (HHC) clinically free of TB was assessed. A total of 264 individuals (TB patients-123; HHC-86; laboratory volunteers-55; Treated TB patients-36) were enrolled for this study. IFN-α mRNA expression levels predominated compared to IFN-γ and IFN-β among untreated TB patients. IFN-α transcripts were ~3.5 folds higher in TB patients compared to HHC, (p<0.0001). High expression of IFN-α was seen among 46% (56/ 123) of the TB patients and 26%, (22/86) of HHCs. The expression levels of IFN-α correlated with that of IFN transcriptional release factor 7 (IRF) (p<0.0001). In contrast, an inverse relationship exists between PGE2 and IFN-α expression levels; high IFN-α expressers were associated with low levels of PGE2 and vice-versa (Spearman's rho = -0.563; p<0.0001). In-vitro, IFN-α failed to restrict the replication of intracellular M.tb. The anti-mycobacterial activity of IFN-γ was compromised in the presence of IFN-α, but not by IFN-β. The expression of IFN-α and β diminished or is absent, among successfully treated TB patients. These observations suggest the utility of assessment of Type I IFNs expression levels as a prognostic marker to monitor tuberculosis patient response to chemotherapy because changes in Type I IFNs expression are expected to precede the clearance and /reduction in bacterial load.

Isolation of a Leuconostoc mesenteroides Strain With Anti-Porcine Epidemic Diarrhea Virus Activities From Kefir Grains.

Swine grown under commercial conditions are vulnerable to environmental exposure to several viruses, which may cause infectious diseases and spread easily and rapidly, resulting in significant economic losses in animal husbandry. Previous studies have suggested that probiotics seem to be a new and promising alternative to vaccinations to protect animals against potential viral infections. In this study, we used the Vero cell culture model of infection to study porcine epidemic diarrhea virus (PEDV). We screened lactic acid bacteria (LAB) with anti-PEDV potential from kefir grains, which are starter cultures used to ferment milk into kefir. Twenty-nine LAB strains were isolated and identified as Enterococcus durans, Lactobacillus kefiri, Lactococcus lactis, and Leuconostoc mesenteroides, according to 16S ribosomal RNA (rRNA) and rpoA gene sequence analyses. The anti-PEDV activities of the LAB intracellular extracts were compared, and the intracellular extracts of Ln. mesenteroides showed higher anti-PEDV activities than that of the other species. Among the Ln. mesenteroides strains, a strain designated YPK30 showed a higher growth rate than that of the other strains and was further evaluated for its anti-PEDV activity. The results showed that the intracellular extracts of Ln. mesenteroides YPK30 possessed in vitro prophylactic, therapeutic, and direct-inhibitory effects against PEDV in the Vero cell model. The expression levels of Type 1 interferon (IFN)-dependent genes, including Myxovirus resistance 1 (MX1) and interferon-stimulated gene 15 (ISG15), were significantly increased after treatment with intracellular extracts of Ln. mesenteroides YPK30 for 24 h. Such expression suggests that the anti-PEDV activity of Ln. mesenteroides YPK30 could be attributed to its up-regulatory effect on the expression of MX1 and ISG15 genes. These results suggested that Ln. mesenteroides YPK30 has the potential to provide some levels of host protection against PEDV infections.

Effect of different concentrations of collagen, ceramides, N-acetyl glucosamine, or their mixture on enhancing the proliferation of keratinocytes, fibroblasts and the secretion of colagen and/or the expression of mRNA of type I collagen

The objectives of the study are to survey the effects of different concentrations of collagen, ceramides, N-acetyl glucosamine (NAG), or their mixture on the proliferation of keratinocytes, fibroblasts and on the secretion of collagen and/or the expression of mRNA of type I collagen. The optimal concentrations that facilitated the proliferation of fibroblasts were 49, 2.0, and 24.4 μg/mL, respectively for collagen, ceramides, and NAG. The proliferation percentages were 191%, 170%, and 223%, respectively at the same order in compare to the control. The concentration of the mixture containing 6.1, 0.12 and 6.1 μg/mL, respectively of collagen, ceramides, and NAG facilitated the proliferation of fibroblasts. The optimal concentrations that had highest keratinocytes proliferation were 0.76, 0.001, and 0.76 μg/mL, respectively for collagen, ceramides, and NAG. The proliferation percentages were 242%, 203% and 230%, respectively at the same order in compare to the control. The optimal concentration that effects the highest keratinocytes proliferation by the mixture were 0.38, 0.008 and 0.38 μg/mL for collagen, ceramides and NAG, respectively. The concentrations for fibroblasts to express higher amount of type I collagen mRNA were 6.1, 0.49, and 6.1 μg/mL, respectively for collagen, ceramides, and NAG. The expression levels of type I collagen mRNA were 152%, 160% and 220%, respectively at the same order in compare to the control. However, the expressions of type I collagen mRNA were 180% in compare to the control at the concentration of mixture containing 0.76, 0.015 and 0.76 μg/mL, respectively of collagen, ceramides, and NAG.

Negative pressure wound therapy affects circulating plasma microRNAs in patients with diabetic foot ulceration

AimsNegative pressure wound therapy (NPWT) is commonly used in diabetic foot ulceration (DFU). The molecular mechanisms of NPWT action, particularly outside of the wound site, have not been described. We assessed NPWT’s effect on circulating miRNA expression levels in type 2 diabetes (T2DM) patients with DFU. MethodsWe examined 34 T2DM patients treated with either NPWT (n = 24) or standard therapy (ST, n = 10). The group assignment was based on clinical criteria and local practice. Next-generation sequencing-based microRNA expression was determined on the patient’s plasma collected before therapy and after 8 days. ResultsNPWT patients were similar to the ST group in terms of age, BMI, and HbA1c level; however, they differed by mean wound area (12.6 cm2 vs. 1.1 cm2 p = 0.0005). First, we analyzed the change of miRNA after NPWT or ST and observed an upregulation of let-7f-2 only in the NPWT group. Then, we analyzed the differential expression between NPWT and ST groups, looking at possible wound size effects. We found 12 differentially expressed miRNAs in pre-treatment comparison, including let-7f-2, while in post-treatment analysis we identified 28 miRNAs. The pathway enrichment analysis suggests that identified miRNAs may be involved in wound healing, particularly through angiogenesis. ConclusionWe found initial evidence that NPWT in T2DM patients with DFU affects miRNA expression in plasma. Additionally, some differences in plasma miRNA expression may be related to wound size.

Abstract PR05: Blockade of AHR activation by IDO/TDO-derived kynurenine restricts cancer immune suppression

Abstract Immune checkpoint blockade (ICB) has been shown to convey significant clinical activity across a spectrum of malignancies, yet there is now recognition that multiple mechanisms of resistance can impair response. The catabolism of tryptophan into metabolites known as kynurenines (Kyn) by enzymes such as indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO) plays a major suppressive role. Recently it was shown that Kyn acts as an endogenous agonist of the Aryl hydrocarbon receptor (AhR). In order to gain a better understanding of this pathway, we sought to characterize the mechanisms of immunosuppression associated with AhR and evaluate its potential as therapeutic target. Gene-expression analysis of IDO-overexpressing melanomas (B16-IDO vs. B16-WT) demonstrated reduced expression levels of Type 1 inflammatory genes, including IFNy, TNF, GzmB, and CD40. In addition, B16-IDO presents higher infiltration of tumor-associated macrophages TAMs, which upregulate the AHR as well as classic AhR-regulated genes (Cyp1a1 and Cyp1b1) and are differentially skewed towards an immunosuppressive M2 phenotype. Tumor-antigen specific CD8+T cells show reduced expression of activation markers (GzmB and CD44) and proliferation rate when primed by Kyn-treated antigen-presenting cells. In addition, TAMs from B16-IDO tumors suppressed activation of CD8+T cells in vitro and their depletion delayed tumor growth. When B16-IDO cells are implanted in mice depleted of Foxp3 expressing cells, TAMs do not accumulate. Treatment of B16-IDO tumors with an AhR-specific antagonist (CH-223191) upregulates MHC II in APCs, activation markers in CD8s, and reduced frequency of T-regs in B16-IDO tumors. AhR inhibition slows tumor growth and prolongs survival of tumors with active IDO/TDO/Kyn pathway (B16-IDO and B16-TDO), and this is enhanced when PD-1 blockade is used in combination. In summary, our findings demonstrate that targeting the Kyn pathway through AhR inhibition could overcome key suppressive mechanisms and sensitize tumors to ICB. This abstract is also being presented as Poster A57. Citation Format: Luis F. Campesato, Sadna Budhu, Mathieu Gigoux, Jeremy Tchaicha, Stephane Pourpe, Cailian Liu, Dmitriy Zamarin, Mark G. Manfredi, Karen McGovern, Jedd D. Wolchok, Taha Merghoub. Blockade of AHR activation by IDO/TDO-derived kynurenine restricts cancer immune suppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR05.

Nanoliposomes codelivering bioactive peptides produce enhanced anti-aging effect in human skin

The present study aimed to develop nanoliposomes codelivering multiple bioactive peptides to achieve improved and comprehensive anti-aging effects in human skin. The nanoliposomes were simultaneously loaded with carnosine, palmitoyl tripeptide-5 and acetyl hexapeptide-3 (CPA-NLPs) for topical delivery. The CPA-NLPs were nano-scaled with narrow size distribution, and showed high encapsulation efficiency, high loading capacity and sustained release of bioactive peptides. Flow cytometry and confocal laser scanning microscopy showed that the nanoliposomes significantly enhanced the cellular uptake of the loaded cargo by human skin fibroblasts (HSF) cells. The CPA-NLPs also protected HSF cells from H2O2-induced oxidative stress damage and increased their expression levels of type Ι collagen and hyaluronic acid. In addition, treatment with CPA-NLPs inhibited catecholamine release in rat adrenal pheochromocytoma (PC12) cells. In a clinical trial, application of a lotion containing 2% CPA-NLPs in face led to a remarkable reduction of the volume, area and area ratio of wrinkles, and an improvement of skin elasticity. These results suggest that loading a combination of bioactive peptides into nanoliposomes is a promising strategy to enhance the topical delivery efficiency and the anti-aging efficacy.

Vitamin C and eggshell membrane facilitate orthodontic tooth movement and induce histological changes in the periodontal tissue

ObjectivesCollagen remodeling of the periodontal tissue is an important mechanism that involves several biologically active substances to accelerate orthodontic tooth movement. It is known that Vitamin C (VC) enhances collagen production and induces tooth movement. Moreover, the eggshell membrane (ESM) is an integral component of various formulations used to promote wound healing. The purpose of our study was to determine the effects of combined treatment with VC and ESM on periodontal tissues during tooth movement. MethodsNine-week-old male osteogenic disorder Shionogi rats were randomized into four groups: control, VC, ESM, and VC + ESM. The control group was given tap water, and the VC, ESM, and VC + ESM groups were orally administered 0.1% VC solution, 1 wt% ESM solution, and a combination of 0.1 wt% VC and 1 wt% ESM solutions, respectively. A force of 25 or 75 g was applied for 10 days to produce orthodontic tooth movement. Distances of tooth movement were measured on days 3, 7, and 10 of treatment. Histological examination of the periodontal ligament was performed to determine the increase in type I and III collagen levels in response to treatment. ResultsDistances of tooth movement were significantly greater in the VC + ESM group than in the control group. The compression area of the alveolar bone showed increased osteoclastic activity and higher levels of bone resorption in the VC + ESM group. Expression levels of type I and III collagen in the tension area of the alveolar bone were higher in the VC + ESM group than in the control group. ConclusionsThis study revealed that the combined administration of VC and ESM accelerated tooth movement by protecting the periodontal tissue during orthodontic treatment. The combined clinical application of VC and ESM could potentially shorten orthodontic treatment time.

Potential Role of Integrin α₅β₁/Focal Adhesion Kinase (FAK) and Actin Cytoskeleton in the Mechanotransduction and Response of Human Gingival Fibroblasts Cultured on a 3-Dimension Lactide-Co-Glycolide (3D PLGA) Scaffold.

BackgroundThe stability of orthodontic treatment is thought to be significantly affected by the compression and retraction of gingival tissues, but the underlying molecular mechanism is not fully elucidated. The objectives of our study were to explore the effects of mechanical force on the ECM-integrin-cytoskeleton linkage response in human gingival fibroblasts (HGFs) cultured on 3-dimension (3D) lactide-co-glycolide (PLGA) biological scaffold and to further study the mechanotransduction pathways that could be involved.Material/MethodsA compressive force of 25 g/m2 was applied to the HGFs-PLGA 3D co-cultured model. Rhodamine-phalloidin staining was used to evaluate the filamentous actin (F-actin) cytoskeleton. The expression level of type I collagen (COL-1) and the activation of the integrin α5β1/focal adhesion kinase (FAK) signaling pathway were determined by using real-time PCR and Western blotting analysis. The impacts of the applied force on the expression levels of FAK, phosphorylated focal adhesion kinase (p-FAK), and COL-1 were also measured in cells treated with integrin α5β1 inhibitor (Ac-PHSCN-NH 2, ATN-161).ResultsMechanical force increased the expression of integrin α5β1, FAK (p-FAK), and COL-1 in HGFs, and induced the formation of stress fibers. Blocking integrin α5β1 reduced the expression of FAK (p-FAK), while the expression of COL-1 was not fully inhibited.ConclusionsThe integrin α5β1/FAK signaling pathway and actin cytoskeleton appear to be involved in the mechanotransduction of HGFs. There could be other mechanisms involved in the promotion effect of mechanical force on collagen synthesis in addition to the integrin α5β1 pathway.

Chloroquine Treatment Suppresses Mucosal Inflammation in a Mouse Model of Eosinophilic Chronic Rhinosinusitis.

PurposeThe Toll-like receptor 9 (TLR9) signaling pathway is involved in the pathogenesis of chronic rhinosinusitis (CRS) with nasal polyposis. The aim of this study was to assess the therapeutic potential of the TLR9 pathway inhibitor chloroquine in CRS mice.MethodsThe expression of type I interferons (IFNs) in human CRS tissues was evaluated using quantitative polymerase chain reaction (qPCR), western blotting, and immunofluorescence. Mice were divided into 4 treatment groups: the control, nasal polyp (NP), chloroquine treatment (NP + Chlq), and dexamethasone treatment (NP + Dexa) groups. The effects of chloroquine on polyp formation and mucosal inflammation were examined by hematoxylin and eosin staining. The expression levels of type I IFN, B-cell activating factor (BAFF), TLR9, high mobility group box 1 (HMGB1), and proinflammatory cytokine expression levels were assessed using qPCR, western blot, or enzyme-linked immunosorbent assay.ResultsIFN-α and IFN-β mRNA levels were significantly higher in patients with eosinophilic NPs (EPs) than in healthy individuals or non-EP patients. The polyp score, epithelial thickness, mucosal thickness, and the number of eosinophils in nasal mucosa were significantly higher in the NP group compared with the control, NP + Chlq, and NP + Dexa groups. NP + Chlq or NP + Dexa significantly suppressed the induction of type I IFN and BAFF expression in the NP group; these treatments also significantly suppressed the induction of TLR9, HMGB1, interferon regulatory factors, interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and Th cytokine expression in the NP group. The secreted levels of anti-dsDNA Immunoglobulin G (IgG) were significantly higher in the NP group than in the control, NP + Chlq, and NP + Dexa groups. There were significant positive correlations between BAFF and mRNA levels of IFN-α/β/the protein levels of anti-dsDNA IgG.ConclusionsChloroquine may be used for the treatment of patients with eosinophilic CRS.

The interferon-gamma pathway is selectively up-regulated in the liver of patients with secondary hemophagocytic lymphohistiocytosis.

Aim of this study was to investigate the activation of the IFNγ pathway in the affected liver and in the blood of patients with secondary hemophagocytic lymphohistiocytosis (sHLH). To this purpose, the mRNA expression levels of IFNG and IFNγ-inducible genes as well as Tyrosine (701)-phosphorylated signal transducer and activator of transcription 1 (STAT1) protein levels were evaluated in the liver and in peripheral blood mononuclear cells (PBMCs) of three patients with sHLH with predominant liver involvement. The mRNA expression levels of IFNG and IFNγ-inducible genes were markedly higher in patient livers compared to control livers and to one disease control liver. Conversely, slight differences in the expression levels of Type I IFN-inducible genes and other classical inflammatory cytokine genes were found. Further supporting the activation of the IFNγ pathway, higher protein levels of phosphorylated and total STAT1 were detected in patient livers compared to control livers. When the expression of the same genes analysed in liver tissues was evaluated in PBMCs collected from 2 out of 3 patients before the liver biopsy, we found that mRNA levels of IFNγ-inducible genes were markedly increased. Accordingly, high circulating levels of IFNγ-inducible CXCL9 were observed in patients. Altogether, these data demonstrate the selective and marked up-regulation of the IFNγ pathway in the liver tissue and blood of patients with active sHLH. Finally, we show that measurement of circulating CXCL9 levels and evaluation of IFNγ–inducible gene expression levels in PBMCs may represent a new valid tool to better identify patients with suspected HLH with predominant liver involvement.

Function of leukaemia inhibitory factor in spermatogenesis of a teleost fish, the medaka Oryzias latipes.

In response to gonadotropins and androgens, testicular cells produce various molecules that control proper proliferation and differentiation of spermatogenic cells through their paracrine and autocrine actions. However, molecules functioning downstream of the hormonal stimulation are poorly understood. Leukaemia inhibitory factor (Lif) is known to maintain the pluripotency of stem cells including embryonic stem cells and primordial germ cells at least in vitro, but its actual roles in vivo remain to be elucidated. To clarify the function of Lif in teleost (medaka) testes, we examined the effects of Lif on spermatogenesis in a newly established cell culture system using a cell line (named Mtp1) derived from medaka testicular somatic cells as feeder cells. We found that addition of baculovirus-produced recombinant medaka Lif to the culture medium or co-culture with Lif-overexpressing Mtp1 cells increased the number of spermatogonia. In situ hybridization and immunohistochemical analyses of the medaka testes showed that mRNAs and proteins of Lif are expressed in spermatogonia and the surrounding Sertoli cells, with higher expression levels in type A (undifferentiated) spermatogonia than in type B (differentiated) spermatogonia. Our findings suggest that Lif regulates spermatogonial cell proliferation in the medaka.

High expression of VEGFA in MSCs promotes tendon-bone healing of rotator cuff tear via microRNA-205-5p.

To explore the role of vascular endothelial growth factor A (VEGFA) in tendon-bone healing of rotator cuff tear (RCT) and to investigate its possible mechanism. Mesenchymal stem cells (MSCs) were transfected with pcDNA-VEGFA. The viability of MSCs was detected by cell counting kit-8 (CCK-8) assay. Expression levels of type I and type II collagen in MSCs were detected by quantitative Real time-polymerase chain reaction (qRT-PCR). RCT was constructed in rats. Meanwhile, all rats were divided into MSCs group and MSCs-pcDNA-VEGFA group, respectively. Biomechanical test was performed to detect ultimate load of failure and stiffness in RCT rats. Dual-luciferase reporter gene assay was conducted to analyze the binding condition between microRNA-205-5p and VEGFA, which was further verified by Western blot and qRT-PCR. VEGFA overexpression significantly promoted viability and proliferation of MSCs. Expression levels of type I and type II collagen were significantly upregulated after VEGFA overexpression in MSCs. Biomechanical test showed that VEGFA overexpression in RCT rats remarkably elevated ultimate load of failure and stiffness. Dual-luciferase reporter gene assay elucidated that VEGFA was the target gene of microRNA-205-5p. Furthermore, VEGFA negatively regulated microRNA-205-5p expression. VEGFA promotes tendon-bone healing of RCT via inhibiting microRNA-205-5p expression.

Mechanical force promotes the proliferation and extracellular matrix synthesis of human gingival fibroblasts cultured on 3D PLGA scaffolds via TGF‑β expression.

Human gingival fibroblasts (HGFs) are responsible for connective tissue repair and scarring, and are exposed to mechanical forces under physiological and pathological conditions. The exact mechanisms underlying gingival tissue reconstruction under mechanical forces remain unclear. The present study aimfed to investigate the effects of mechanical forces on the proliferation and extracellular matrix synthesis in HGFs by establishing a 3-dimensional (3D) HGF culture model using poly(lactide-co-glycolide) (PLGA) scaffolds. HGFs were cultured in 3D PLGA scaffolds and a mechanical force of 0, 5, 15, 25 or 35 g/cm2 was applied to HGFs for 24 h. A mechanical force of 25 g/cm2 induced the highest proliferation rate, and thus was selected for subsequent experiments. Cell viability was determined using the MTT assay at 0, 24, 48 and 72 h. The expression levels of type I collagen (COL-1) and matrix metallopeptidase (MMP)-1 were examined by reverse transcription-quantitative polymerase chain reaction and ELISA, and transforming growth factor (TGF)-β expression was evaluated by ELISA. The application of mechanical force on HGFs cultured on the 3D PLGA scaffolds resulted in a significant increase in cell proliferation and COL-1 expression, as well as a decrease in MMP-1 expression. A TGF-β1 inhibitor was also applied, which attenuated the effects of mechanical force on HGF proliferation, and COL-1 and MMP-1 expression, thus suggesting that TGF-β signaling pathways may mediate the mechanical force-induced alterations observed in HGFs. In conclusion, these findings helped to clarify the mechanisms underlying mechanical force-induced HGF proliferation and ECM synthesis, which may promote the development of targeted therapeutics to treat various diseases, including gingival atrophy caused by orthodontic treatment.

Salidroside inhibits platelet-derived growth factor-induced proliferation and migration of airway smooth muscle cells.

Abnormal proliferation and migration of airway smooth muscle cells (ASMCs) have been found to be important for the airway remodeling during the pathogenesis of asthma. Salidroside a bioactive glucoside that exerts antitumor activity via inhibiting the cell proliferation and migration of cancer cells. The aim of the current study was to evaluate the effects of salidroside on the proliferation and migration of ASMCs. Our results showed that salidroside inhibited the proliferation and migration of ASMCs in response to platelet-derived growth factor (PDGF) stimulation. Salidroside markedly attenuated the PDGF-induced production of matrix metalloproteinase 2 (MMP-2) and MMP-9 in ASMCs. The levels of contractile phenotype markers including smooth muscle α-actin and calponin were reduced in response to PDGF stimulation, which was attenuated by salidroside pretreatment. Salidroside diminished the increase in the expression levels of type I collagen and fibronectin in PDGF-stimulated ASMCs. Furthermore, salidroside blocked the PDGF-induced activation of the nuclear factor-κB (NF-κB) pathway in ASMCs. The results suggested that salidroside functionally regulated the proliferation, migration, phenotype plasticity, and extracellular matrix deposition in PDGF-induced ASMCs and the NF-κB pathway might be implicated in the effects of salidroside on ASMCs induced by PDGF.

Low-protein diet improves meat quality of growing and finishing pigs through changing lipid metabolism, fiber characteristics, and free amino acid profile of the muscle.

The objective of the study was to investigate the effect of feeding reduced CP, AA-supplemented diets on meat quality in growing and finishing pigs as well as the related mechanism. In experiment 1, 18 growing pigs (36.5 kg BW) were assigned randomly and fed 1 of 3 corn-soybean meal diets containing either 18% CP (normal protein, NP), 15% CP (low protein, LP), or 12% CP (very low protein, VLP). In experiment 2, 18 finishing pigs (62.3 kg BW) were allotted randomly into 1 of the following diets: 16% CP (NP), 13% CP (LP), or 10% CP (VLP). In both experiments, the LP and VLP diets were supplemented with crystalline AA to achieve equal content of standardized ileal digestible lysine, methionine, threonine, and tryptophan. At the end of each experiment, all pigs were slaughtered to collect longissimus dorsi muscle (LM) samples. Samples were used for determining meat quality, intramuscular fat (IMF) content, fatty acid composition, free AA profile, and expression of genes for myosin heavy chain isoforms. Results showed that growing and finishing pigs fed the LP diets increased (P < 0.05) redness value of LM, while finishing pigs fed the LP and VLP diets decreased (P < 0.05) the shear force values. Compared with the NP diet, growing and finishing pigs fed lower CP diets had higher (P < 0.05) contents of IMF and MUFA, and lower (P < 0.05) contents of PUFA. Besides, higher (P < 0.05) expression levels of type I and/or IIa muscle fibers were observed in LP diet-fed growing and finishing pigs, and greater concentrations of taurine and tasty AA in VLP diet-fed growing and finishing pigs. Taken together, our results indicate that low-protein diets could positively affect meat quality of growing and finishing pigs, and likely through regulation of IMF content and fatty acid composition, fiber characteristics, and free AA profile in the muscle.

Reward-representing D1-type neurons in the medial shell of the accumbens nucleus regulate palatable food intake

Background/objectivesDysfunction in reward-related aspects of feeding, and consequent overeating in humans, is a major contributor to obesity. Intrauterine undernutrition and overnutrition are among the predisposing factors, but the exact mechanism of how overeating develops is still unclear. Consummatory behavior is regulated by the medial shell (mSh) of the accumbens nucleus (Nac) through direct connections with the rostral part of the lateral hypothalamic area (LHA). Our aim was to investigate whether an altered Nac-LHA circuit may underlie hyperphagic behavior.Subjects/methodsIntrauterine protein-restricted (PR) male Wistar rats were used as models for hyperphagia. The experiments were performed using young adult control (normally nourished) and PR animals. Sweet condensed milk (SCM) served as a reward to test consumption and subsequent activation (Fos+) of Nac and LHA neurons. Expression levels of type 1 and 2 dopamine receptors (D1R, D2R) in the Nac, as well as tyrosine hydroxylase (TH) levels in the ventral tegmental area, were determined. The D1R agonist SKF82958 was injected into the mSh-Nac of control rats to test the effect of D1R signaling on SCM intake and neuronal cell activation in the LHA.ResultsA group of food reward-representing D1R+ neurons was identified in the mSh-Nac. Activation (Fos+) of these neurons was highly proportional to the consumed palatable food. D1R agonist treatment attenuated SCM intake and diminished the number of SCM-activated cells in the LHA. Hyperphagic PR rats showed increased intake of SCM, reduced D1R expression, and an impaired response to SCM-evoked neuronal activation in the mSh-Nac, accompanied by an elevated number of Fos+ neurons in the LHA compared to controls.ConclusionsSensitivity of food reward-representing neurons in the mSh-Nac determines the level of satisfaction that governs cessation of consumption, probably through connections with the LHA. D1R signaling is a key element in this function, and is impaired in obesity-prone rats.

Diagnostic Values of Free Triiodothyronine and Free Thyroxine and the Ratio of Free Triiodothyronine to Free Thyroxine in Thyrotoxicosis.

Background The results of previous studies on the usefulness of free triiodothyronine (FT3) to free thyroxine (FT4) are controversial. We investigated the usefulness of FT3, FT4, and FT3/FT4 ratio in differentiating Graves' disease (GD) from destructive thyroiditis. Methods A total of 126 patients with untreated GD, 36 with painless thyroiditis, 18 with painful subacute thyroiditis, and 63 healthy controls, were recruited. The levels of FT3 and FT4 and the FT3/FT4 ratios for the different etiologies of thyrotoxicosis were evaluated separately by receiver operating characteristic (ROC) curve analysis. The expression levels of type 1 and type 2 deiodinase (DIO1 and DIO2) in thyroid tissues were also investigated. Results The optimal cut-off values were 7.215 pmol/L for FT3, 21.71 pmol/L for FT4, and 0.4056 for the FT3/FT4 ratio. The specificity and positive predictive value of the FT3/FT4 ratio were highest for values > 0.4056. DIO1 mRNA expression was significantly higher in the thyroid tissue of patients with GD (P = 0.013). Conclusions We demonstrated that the FT3/FT4 ratio was useful in differentiating GD from destructive thyroiditis. In addition, a relatively high expression of type 1 deiodinase in the thyroid might be responsible for the high FT3/FT4 ratio in patients with GD.

ALK5 transfection of bone marrow mesenchymal stem cells to repair osteoarthritis of knee joint

Previous studies have suggested that the transforming growth factor- $$\upbeta $$ receptor ALK5 is crucial for articular chondrogenesis by bone marrow mesenchymal stem cells. Here, the wild-type ALK5 plasmids were mutated by overlapping extended PCR and transfected into bone marrow mesenchymal stem cells. The knee joint osteoarthritis mouse model was constructed by cutting off the anterior cruciate ligament and divided into three groups: saline group, bone marrow mesenchymal stem cells and ALK5-transfected bone marrow mesenchymal stem cells group. HE staining showed that the articular cartilage lesions were more serious of saline group compared with that of mesenchymal stem cell group, and this trend was more pronounced as time goes on. Immunohistochemical staining showed that although the expression level of type II collagen in all three groups down-regulated gradually upon time, its expression in ALK5-transfected bone marrow mesenchymal stem cells group was significantly enhanced compared with the other two groups. Micro-CT also suggested that ALK5 transfection of mouse bone marrow mesenchymal stem cells would promote repairing the knee cartilage lesions with arthritis of the mice. Although the osteoarthritis mechanism underlying a variety of factors work together, and the appropriate proportion of ALK5/ALK1 was also emphasized for the treatment of osteoarthritis. This work therefore demonstrated that ALK5 transfection of bone marrow mesenchymal stem cells could be a promising stem cell therapy for repair of cartilage lesions.