Staining Area Research Articles

P162 Biosamples from VEDOSS patients show classic pathological signs of Scleroderma:Opportunity for a biological diagnosis of disease

Abstract Background/Aims The VEDOSS study has recently indicated that more than 50% of patients affected by Raynaud’s phenomenon (RP) and specific SSc anti-nuclear antibodies (ANA) and/or nailfold videocapillaroscopy changes (NVC) (sRP) satisfied ACR/EULAR 2013 criteria within 5 years, indicating a window of opportunity for early detection of SSc. Here we aimed to determine whether sera, skin or dermal fibroblasts cultured from VEDOSS patients showed any biological hallmark of SSc. Methods VEDOSS patients were enrolled in the national inception cohort for patients at risk of Scleroderma (Kennedy Cohort, n = 64) . Sera were tested for IFN-inducible chemokines and biomarker of extracellular matrix turnover (ELF test) and compared to Healthy control (HC) and SSc sera (n = 56, 64, respectively). 3mm skin punch biopsies were taken from the forearms of 12 VEDOSS patients, and 5 healthy controls and 6 SSc patients. Biopsies were analysed for haematoxylin and eosin (H&E), masson trichrome staining (MT) and immunohistological (IHC) staining for CD45. A dermatopathologist blinded to clinical features undertook the analysis. A probabilistic image analysis model was used to detect areas of immunopositivity (CD45) and collagen staining (MT) within the samples, using HeteroGenius Medical Imaging Manager colour analysis. In addition, 7 biopsies along with 2 healthy control and 3 SSc, were used to explant fibroblasts cultures. mRNA and protein were isolated from primary fibroblasts and processed for RT-qPCR and western blotting analyses. Results Skin biopsies from VEDOSS patients showed evidence of fibrosis and increased collagen bundles within the dermis evidenced in H&E and MT staining, as usually seen in SSc, compared to HC. IHC showed increased number of CD45+ cells in VEDOSS. Semiquantitative analysis of histopathological samples indicated a significant correlation between MT and CD45 staining for VEDOSS (R = 0.8828 P = 0.0016). In vitro, fibroblasts from VEDOSS patients showed an average 5-fold higher collagen mRNA levels compared to HC fibroblasts, at a similar range to that seen in SSc fibroblasts, confirmed at protein level via western blotting. Sera from VEDOSS patients showed significantly higher IFN-inducible chemokines compared to HC and an intermediate levels compared to SSc [Mean (STDV) HC = 4.69(0.29) vs. VEDOSS= 4.93 (0.39); SSc= 5.30 (0.54)]. ELF score was within normal range for most patients with SRP and showed no statistical difference to HC. Conclusion Although pilot in nature, this study suggests that patients with no clinical signs of skin fibrosis in their forearms already show biomarker signs of SSc both in their sera, skin biopsies and fibroblast level. Our data indicate that skin thickening is a late manifestation of SSc pathogenesis and early window of opportunity of patients with VEDOSS could be targeted for immune intervention and antifibrotic intervention. Disclosure R.L. ross: None. E. Clarke: None. W. Merchant: None. I. Georgiou: None. P. Mulipa: None. A. Carriero: None. G. Abignano: None. A. herrick: None. C. Denton: None. N. Riobo-Del Galdo: None. F. Del Galdo: Consultancies; FDG received consultancies and research support from Abbvie, AstraZeneca, Boheringer-Ingelheim, Capella, Chemomab, Ergomed, Janssen, Kymab, Mitsubishi-Tanabe.

Targeting Synovial Lymphatic Function as a Novel Therapeutic Intervention for Age-Related Osteoarthritis in Mice.

The synovial lymphatic system (SLS) removes catabolic factors from the joint. Vascular endothelial growth factor C (VEGF-C) and its receptor, VEGFR-3, are crucial for lymphangiogenesis. However, their involvement in age-related osteoarthritis (OA) is unknown. This study was undertaken to determine whether the SLS and the VEGF-C/VEGFR-3 pathway contribute to the development and progression of age-related OA, using a murine model of naturally occurring joint disease. SLS function was assessed in the knees of young (3-month-old) and aged (19-24-month-old) male and female C57BL/6J mice via a newly established in vivo IVIS-dextran imaging approach, which, in addition to histology, was used to assess the effects of VEGF-C treatment on SLS function and OA pathology in aged mice. RNA-sequencing of synovial tissue was performed to explore molecular mechanisms of the disease in the mouse knee joints. Results showed that aged mice had impaired SLS function, including decreases in joint clearance (mean T1/2 of signal intensity clearance, 2.8 hours in aged mice versus 0.5 hours in young mice; P < 0.0001), synovial influx (mean ± SD 1.7 ± 0.8% in aged mice versus 4.1 ± 1.9% in young mice; P=0.0004), and lymph node draining capacity (mean ± SD epifluorescence total radiant intensity ([photons/second]/[μW/cm2 ]) 1.4 ± 0.8 in aged mice versus 3.7 ± 1.2 in young mice; P < 0.0001). RNA-sequencing of the synovial tissue showed that Vegf-c and Vegfr3 signaling genes were decreased in the synovium of aged mice. VEGF-C treatment resulted in improvements in SLS function in aged mice, including increased percentage of signal intensity joint clearance (mean ± SD 63 ± 9% in VEGF-C-treated aged mice versus 52 ± 15% in vehicle-treated aged mice; P=0.012), increased total articular cartilage cross-sectional area (mean ± SD 0.38 ± 0.07 mm2 in VEGF-C-treated aged mice versus 0.26 ± 0.07 mm2 in vehicle-treated aged mice; P < 0.0001), and decreased percentage of matrix metallopeptidase 13-positive staining area within total synovial area in 22-month-old VEGF-C-treated mice versus 22-month-old vehicle-treated mice (mean ± SD decrease 7 ± 2% versus 4 ± 1%; P=0.0004). SLS function is reduced in the knee joints of aged mice due to decreased VEGF-C/VEGFR-3 signaling. VEGF-C treatment attenuates OA joint damage and improves synovial lymphatic drainage in aged mice. The SLS and VEGF-C/VEGFR-3 signaling represent novel physiopathologic mechanisms that could potentially be used as therapeutic targets for age-related OA.

Klippel-Trenaunay syndrome: A case report of orthodontic-surgical treatment

Introduction: Klippel-Trenaunay syndrome (KTS) is a congenital disorder characterized by a clinical triad of capillary malformation; varicose veins; and soft tissue and/or bony hypertrophy. This rare syndrome has an estimated incidence of about 2–5/100,000 live births. KTS usually affects the extremities but occasionally can manifest in the craniofacial region, including the oral cavity. Case Presentation: Our patient, a 17-year-old girl, presented with large areas of port wine stain, unilateral hypertrophy of the facial region, and varicose veins. She was diagnosed with KTS. Orthodontic treatment followed by orthognathic surgery was performed to address her complaint of facial asymmetry and poor chewing efficacy. Treatment resulted in significant improvement in her appearance and dental occlusion and our patient was very satisfied with the results. Conclusion: This report illustrates an orthodontic and surgical treatment approach of a case with KTS. Effective management of orthodontic and facial manifestations of KTS considerably enhances the patient’s function, appearance, and overall quality of life.

Expression of RANKL in breast cancer tissue in patients with fibrous dysplasia/McCune-Albright syndrome.

In fibrous dysplasia/McCune-Albright syndrome (FD/MAS), mosaic mutations in the GNAS gene lead to locally abnormal bone turnover. Additionally, patients with FD/MAS, particularly with thoracic lesions, have an increased risk for breast cancer. Development and progression of breast cancer has been associated with expression of Receptor Activator of NF-κB ligand (RANKL) in mammary tissue, and due to the GNAS mutation, RANKL is systemically increased in patients with FD/MAS. Yet it is unknown whether breast cancer in FD/MAS is also dependent on RANKL. We hypothesized that the GNAS mutation might induce RANKL overproduction and an oncogenic niche in mammary tissue, and examined RANKL expression in breast cancer tissue of patients with FD/MAS compared to controls. Nine patients with FD/MAS and breast cancer were included and clinical data were retrieved. Patients were matched to controls with breast cancer without FD/MAS based on age and tumor type. Three pregnant breast cancer patients were included as positive controls. Immunohistochemical detection of RANKL was performed on formalin-fixed paraffin-embedded breast cancer specimens. Staining intensity was classified as weak, moderate or intense. The area of positive RANKL staining divided by the total ductal-lobular area was assessed (positive area percentage, PAP). Number of patients with RANKL expression was compared between FD/MAS and control group by chi-square (χ2) test, the PAP by Mann-Whitney U test (MWU). RANKL expression was observed in 3 patients with FD/MAS (38%), mainly in healthy tissue, and none of the control patients (χ2p=0.055). The FD/MAS group demonstrated considerably more intense staining than the control group, comparable to positive controls. The median PAP was 0.64% (range 0.14-2.04%) in the 3 FD/MAS patients with RANKL expression, 0.01% (Q1-Q3: 0.0003-0.514%) in the entire FD/MAS group, 0.006% (Q1-Q3: 0.001-0.012%) in the control group (MWU=0.574), and 0.19% (0.08-0.32%) in the pregnant patients. All patients with FD/MAS and RANKL expression had thoracic bone lesions, but no correlation was observed between RANKL expression and presence of the GNAS mutation or FD disease burden. The triad of a higher number of patients, higher positive area percentage and stronger intensity in the FD/MAS compared to the control group indicates that RANKL may be upregulated in mammary tissue in a subset of patients with FD/MAS, which may explain the increased risk for breast cancer, although the clinical significance remains unclear. Further research is needed to establish risk profiles for the development of RANKL-positive breast cancer and to improve early screening and treatment.

Detecting Harmful Activity in Hajj Plagiarism Using Deep Learning

CCTV surveillance is the most extensively used intelligent latest innovation. The use of surveillance cameras has risen dramatically because of the convenience of monitoring from anywhere and the reduction of crime rates in public areas. In this paper, we introduce the idea of bad vibe activity detection from live videos to enhance the security and safety of pilgrims. The proposed bad vibes activity recognition model is intended to be addressed in the most efficient manner possible using cutting-edge technologies such as TensorFlow and Keras. TensorFlow was chosen because the project could be deployed to a mobile environment in the future with the possibility of extension of other areas such as airport security, bus stain, and public areas that may deserve special attention for security checks. We choose MediaPipe Holistic for employee bad vibe recognition in the model.&#x0D; &#x0D;

Efficacy of Cellular Therapy for Azoospermia in Animal Models: A Systematic Review and Meta-Analysis

This study investigated the efficacy of stem cell transplantation for azoospermia, a major cause of male infertility. We conducted a systematic meta- analysis to assess the therapeutic effectiveness of stem cell transplant, using different transplant methods, injection sites, and stem cell types, and the reliability of this approach in different animal species. PubMed, the Cochrane Library, and Embase were searched for studies published from January 2006 to February 2022 that evaluated the use of stem cell transplant to treat azoospermia. We included 18 studies and conducted the analyses using Review Manager 5.2 software. Expression of the meiosis-related genes Vasa, Scp3, and Dazl and the average hematoxylin and eosin- positive staining area were improved after stem cell transplant. Subgroup analyses by mode of transplant showed higher expression of Scp3 and Dazl in the xenotransplant group. Although subgroup analyses by injection site showed that the seminiferous tubule group showed the most significant effect on Scp3 expression, spermatogenesis and repair of damaged testis were induced in the tunica albuginea group. The testicular torsion group also induced high levels of Scp3. Another subgroup analysis by stem cell type showed that umbilical cord mesenchymal stem cells promoted the highest expression of meiosis-related genes and successfully induced spermatogenesis and the repair of damaged testis. Urine-derived stem cells, spermatogonial stem cells, and amniotic fluid-derived stem cells showed significantly therapeutic effects; however, more studies are needed for definitive conclusions. Subgroup analyses by type of azoospermia animal model indicated that the use of stem cell transplant in rat or mouse models had an obvious therapeutic effect, but no significant therapeutic effect was seen in azoospermia hamsters. The meta-analysis confirmed that stem cell transplant can effectively treat azoospermia in animal models. Xenotransplant is shown to enhance the therapeutic effects of stem cell transplant on azoospermia.

Effect of electroacupuncture on transforming growth factor-β1 and renal tubular epithelial mesenchymal transformation in renal tissue of spontaneously hypertensive rats

To observe the effects of electroacupuncture (EA) on renal fibrosis, the expression of transforming growth factor-β1 (TGF-β1) and epithelial mesenchymal transition (EMT) marker proteins in renal tissue of spontaneously hypertensive rats (SHR), so as to explore the underlying mechanism on EA alleviating hypertensive renal impairment. Twenty-four male SHR were randomly divided into model group, losartan group and EA group, with 8 rats in each group, and eight male Wistar-Kyoto rats were taken as the normal group. Rats in the losartan group received gavage of losartan potassium solution (3 mg/mL, 30 mg·kg-1·d-1),once every other day for 12 weeks. Rats in the EA group received EA stimulation at bilateral "Shenshu" (BL23) and "Geshu" (BL17) (2 Hz/15 Hz, 1.0 mA), 15 min each time, once every other day for 12 weeks. The systolic blood pressure of caudal artery was measured before, and 4, 8 and 12 weeks after the intervention. The 24-hour urinary protein was measured before, and 6 and 12 weeks after the intervention. Histopathological changes of the left renal tissue were observed under light mircoscope after H.E. stain. Extracellular matrix (ECM) in renal tissues was observed after periodate Schiff staining. Basement membrane and collagen fibers were observed after Masson staining with collagen volume fraction (CVF) evaluated. The expression of TGF-β1 mRNA in the right renal was detected by real-time fluorescence quantitative PCR. The expression of TGF-β1 and EMT marker E-cadherin, α-SMA and fibronectin (FN) proteins in the left renal tissue was measured by immunohistochemistry. In the model group, irregular arrangement of nephrocytes, renal tubule atrophy, lumen stenosis, ECM hyperplasia and deposition, scar and sclerosis were observed, which were relatively milder in the EA and losartan groups. Compared with the normal group, tubulointerstitium CVF, systolic blood pressure of caudal artery before, and at 4, 8 and 12 weeks after the intervention, 24-hour urinary protein before, and at 6 and 12 weeks after the intervention, the expression of TGF-β1 mRNA, area of TGF-β1, α-SMA and FN positive staining in renal tissues were significantly increased (P<0.01), while the area of E-cadherin positive staining was significantly decreased (P<0.01) in the model group. Compared with the model group, tubulointerstitium CVF, systolic blood pressure of caudal artery at 4, 8 and 12 weeks after the intervention, 24-hour urinary protein at 6 and 12 weeks after the intervention, the expression of TGF-β1 mRNA, area of TGF-β1, α-SMA and FN positive staining in renal tissues were significantly decreased (P<0.01，P<0.05), while area of E-cadherin positive staining was significantly increased (P<0.01) in the losartan and EA groups. Compared with the losartan group, the area of E-cadherin was conside-rately increased (P<0.01), while the area of α-SMA protein decreased (P<0.01) in the EA group. EA could effectively alleviate hypertension and renal interstitial fibrosis in SHR, the mechanism of which may be related to its function in reducing the expression of TGF-β1 and inhibiting EMT in renal tissue.

The possible protective role of melatonin versus garlic on monosodium Glutamate-induced changes in rat cerebellar cortex: histological, immunohistochemical and electron microscope study

ABSTRACT Monosodium glutamate (MSG) is widely used as a flavor enhancer. Melatonin and garlic are well known as antioxidant. The present study was performed to evaluate the microscopic changes in the cerebellar cortex of rats after the administration of MSG and the possible protective effect of melatonin versus garlic on those changes. The rats were divided into four main groups. Group I (control group). Group II received MSG (4 mg/ g/day). Group III received MSG+ melatonin (10 mg/kg bw/day). Group IV received MSG+garlic (300 mg/kg bw/day). Immunohistochemical staining for glial fibrillary acidic protein (GFAP) was done as a marker for astrocyte demonstration. Morphometric study was done to assess the mean number and diameter of Purkinje cells, the number of astrocytes and the percentage area of positive GFAP immune stain. MSG group demonstrated congested blood vessels, vacuolations in the molecular layer, and Purkinje cells appeared irregular with nuclear degeneration. Granule cells appeared shrunken with darkly stained nuclei. The immunohistochemical stain for GFAP was less than expected in the three layers of the cerebellar cortex. Purkinje cells and granule cells appeared irregular in shape with dark small heterochromatic nuclei. The myelinated nerve fibers showed splitting and loss of the lamellar structure of their myelin sheath. Melatonin group showed that the cerebellar cortex was nearly similar to that of control group. Garlic treated group showed partial improvement. In conclusion, melatonin and garlic could partially protect against MSG induced changes and the protective effect of melatonin was better than garlic.

Characterization of expression and prognostic implications of transforming growth factor beta, programmed death-ligand 1, and T regulatory cells in canine histiocytic sarcoma

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm in dogs. Expression and prognostic significance of transforming growth factor beta (TGF-β), programmed death-ligand 1 (PD-L1), and T regulatory cells (Tregs) in HS is unknown. The goal of this study was to investigate the expression and prognostic significance of TGF-β, PD-L1, and FoxP3/CD25 in canine HS utilizing RNA in situ hybridization (RNAscope®). After validation was performed, RNAscope® on formalin-fixed paraffin-embedded (FFPE) patient HS tissue samples was performed for all targets and expression quantified with HALO® software image analysis. Cox proportional hazard model was conducted to investigate the association between survival time and each variable. Additionally, for categorical data, the Kaplan-Meier product-limit method was used to generate survival curves. TGF-β and PD-L1 mRNA expression was confirmed in the DH82 cell line by reverse transcription polymerase chain reaction (RT-PCR) and CD25 + FoxP3 + cells were detected by flow cytometry in peripheral blood. Once the RNAscope® method was validated, TGF-β H-score and dots/cell and FoxP3 dots/cell were assessed in HS samples and found to be significantly correlated with survival. Moderate positive correlations were found between FoxP3 and PD-L1 H-score, percent staining area, and dots/cell, and FoxP3 and TGF-β dots/cell. In summary, RNAscope® is a valid technique to detect TGF-β and PD-L1 expression and identify Tregs in canine HS FFPE tissues. Furthermore, canine HS expresses TGF-β and PD-L1. Increased TGF-β and FoxP3 correlated with worse prognosis. Prospective studies are warranted to further investigate TGF-β, PD-L1, and Tregs effect on prognosis.

GhGTG1 enhances cold stress tolerance by improving sensitivity to ABA in cotton and Arabidopsis

Cold stress is a potent abiotic stress resulting in cotton yield and fiber quality loss. We hypothesized that GhGTG1 could increase low temperature stress tolerance by improving the sensitivity to ABA. Genome-wide identification, biochemical traits and functional characterization of GhGTG1, were investigated under different levels of ABA and cold stress. GhGTG1 had two highly conserved functional domains (ABA_GPCR, GPHR_N) in all cotton species. Under ABA treatment, over expression of GhGTG1 in Arabidopsis had a lower germination rate and root elongation. While under 4 ℃ stress, transgenic plants had a higher germination rate and root elongation. This indicate that GhGTG1 improved sensitivity to ABA under cold stress. In transgenic plants, CAT and SOD activity was increased, while H2O2 and MDA content decreased. Less trypan blue and DAB staining area suggested that GhGTG1 enhances cold stress resistance. Under 4 ℃ treatment, silence cotton seedlings through VIGS method, resulted in a poor growth, lower relative leaf water content, chlorophyll content but a higher ion leakage and excised leaf water loss. The activity of CAT and SOD decreased, H2O2 and MDA content was increased. Two marker genes (OST1 and ABCG40) are associated with the stomatal movement, but these genes were substantially suppressed. This suggesting that GhGTG1 can induce cold stress tolerance by improving ABA sensitivity Via., maintaining stomatal movement. These data will provide new insight of cold resistance mechanism in cotton and further will help the breeder to develop new cotton cold stress tolerance germplasm.

Effects of differential regional PM2.5 induced hepatic steatosis and underlying mechanism

Emerging evidence suggests that exposure to PM2.5 is associated with a high risk of nonalcoholic fatty liver disease (NAFLD). NAFLD is typically characterised by hepatic steatosis. However, the underlying mechanisms and critical components of PM2.5-induced hepatic steatosis remain to be elucidated. In this study, ten-month-old C57BL/6 female mice were exposed to PM2.5 from four cities in China (Taiyuan, Beijing, Hangzhou, and Guangzhou) via oropharyngeal aspiration every other day for four weeks. After the exposure period, hepatic lipid accumulation was evaluated by biochemical and histopathological analyses. The expression levels of genes related to lipid metabolism and metabolomic profiles were assessed in the mouse liver. The association between biomarkers of hepatic steatosis (hepatic Oil Red O staining area and serum and liver triglyceride contents) and typical components of PM2.5 was identified using Pearson correlation analysis. Oil Red O staining and biochemical results indicated that PM2.5 from four cities significantly induced hepatic lipid accumulation. The most severe hepatic steatosis was observed after Guangzhou PM2.5 exposure. Moreover, Guangzhou PM2.5-induced the most significant changes in gene expression associated with lipid metabolism, including increased hepatic fatty acid uptake and lipid droplet formation and decreased fatty acid synthesis and lipoprotein secretion. Contemporaneously, exposure to Guangzhou PM2.5 significantly perturbed hepatic lipid metabolism. According to metabolomic analysis, disturbed hepatic lipid metabolism was primarily concentrated in linoleic acid, α-linoleic acid, and arachidonic acid metabolism. Finally, correlation analysis revealed that copper (Cu) and other inorganic components, as well as the majority of polycyclic aromatic hydrocarbons (PAHs), were related to changes in biomarkers of hepatic steatosis. These findings showed that PM2.5 exposure caused hepatic steatosis in aged mice, which could be related to the critical chemical components of PM2.5. This study provides critical information regarding the components of PM2.5, which cause hepatic steatosis.

Detecting Harmful Activity in Pilgrimage Using Deep Learning

CCTV surveillance is the most extensively used intelligent latest innovation. The use of surveillance cameras has risen dramatically because of the convenience of monitoring from anywhere and the reduction of crime rates in public areas. In this paper, we introduce the idea of bad vibe activity detection from live videos to enhance the security and safety of pilgrims. The proposed bad vibes activity recognition model is intended to be addressed in the most efficient manner possible using cutting-edge technologies such as TensorFlow and Keras. TensorFlow was chosen because the project could be deployed to a mobile environment in the future with the possibility of extension of other areas such as airport security, bus stain, and public areas that may deserve special attention for security checks. We choose MediaPipe Holistic for employee bad vibe recognition in the model.

Evaluation of the effect of pre-sowing treatment of grain sorghum seeds with fungicides on the development of blotches in the forest-steppe of the Samara Region

Relevance. It is important to evaluate the effectiveness of modern fungicides and their effect on the development of leaf infections of grain sorghum in order to optimize plant protection against diseases. Methods. The studies were carried out in the fields of the Volga Scientific Research Institute of Selection and Seed-Growing named after P.N. Konstantinov on grain sorghum variety Premiere in 2014–2016. Leaf diseases were estimated by eye in the field during the phases of flowering, milky, waxy, and full grain ripeness. The average lesion of each plant was evaluated, and the prevalence and intensity of disease development was established. Seeds were treated immediately before sowing. Biological effectiveness of the studied fungicides was determined by prevalence and intensity of disease development. Results. Analysis of leaves showed that quite a wide complex of pathogens belonging to the kingdom of fungi caused blotches on sorghum leaves. Mycelium and spores of fungi Alternaria sp., Fusarium sp., Helminthosporium sp. and others were detected on the affected leaves in the stain area. The obtained results allow us to conclude that the development of the fungal leaf blotches in sorghum in the conditions of forest-steppe of the Samara region considerably depends on hydrothermal conditions of the year: the more arid are conditions of May and June, the higher amount of precipitation and thelower temperature is in July, the higher is leaf blotch infestation of sorghum. Seed pretreatment with fungicides suppressed fungal leaf blotch development in the Premiere variety by 13.6–47.0 % in 2014, 6.9–62.2 % – in 2015, and 3.5–53.7 % – in 2016. At the same time, the highest biological efficacy showed treatment of seeds with fungicides of contact-system action “Fundazol” and “Vitaros”; was 25.0–75.0 % in prevalence and 26.2–62.2 % in the intensity of leaf blotch development.

β-catenin/TCF4 inhibitors ICG-001 and LF3 alleviate BDL-induced liver fibrosis by suppressing LECT2 signaling

Liver fibrosis can be characterized by the over-deposition of extracellular matrix (ECM). It has been reported that β-catenin/TCF4 interaction was enhanced in bile duct ligation (BDL) model, which implicated the critical role of β-catenin/TCF4 interaction during the progression of fibrosis. However, whether inhibiting β-catenin/TCF4 signaling attenuates liver fibrosis remains unknown. In the current study, we used ICG-001, an inhibitor that disrupts the interaction between CREB binding protein (CBP) and β-catenin, to inhibit β-catenin/TCF4 transcriptional activity. We also used LF3, a small molecule antagonist, to inhibit β-catenin/TCF4 interaction. The antifibrotic effect of ICG-001 and LF3 was assessed on BDL-induced liver fibrosis model. The results indicated both ICG-001 and LF3 significantly reduced the positive staining area of Sirius Red and α-SMA. The protein expression levels of α-SMA, Collagen Ⅰ and CD31 were also significantly downregulated in BDL + ICG-001 and BDL + LF3 groups. Besides, ICG-001 and LF3 promoted portal angiogenesis and inhibited sinusoids capillarization in fibrotic livers. For mechanistic study, we measured the level of leukocyte cell-derived chemotaxin 2 (LECT2), a direct target of β-catenin/TCF4, which was recently reported to participate in hepatic fibrosis by regulating angiogenesis. The results showed that both ICG-001 and LF3 reduced LECT2 expression in BDL mice. LF3 also downregulated pSer 675 β-catenin and nuclear β-catenin. In conclusion, this study demonstrated that inhibiting β-catenin/TCF4 signaling by ICG-001 or LF3 mitigated liver fibrosis by downregulating LECT2, promoting portal angiogenesis and inhibiting sinusoids capillarization, which provided new evidence that β-catenin/TCF4 signaling might be a target for the treatment of liver fibrosis.

Icariin Alleviates Nonalcoholic Fatty Liver Disease in Polycystic Ovary Syndrome by Improving Liver Fatty Acid Oxidation and Inhibiting Lipid Accumulation.

(1) Background: Icariin is the main component of the Chinese herb Epimedium. A number of studies have shown that it alleviates abnormal lipid metabolism. However, it is not clear whether and how icariin can ameliorate hepatic steatosis with polycystic ovary syndrome (PCOS). This study was designed to explore the anti-hepatosteatosis effect of icariin in rats with polycystic ovary syndrome. (2) Methods: Female Sprague Dawley(SD)rats were treated with a high-fat diet and letrozole for 21 days to make nonalcoholic fatty liver disease (NAFLD) in the polycystic ovary syndrome model. Then model rats were treated with icariin (by gavage, once daily) for 28 days. Serum hormones and biochemical variables were determined by ELISA or enzyme. RNA-sequence analysis was used to enrich related target pathways. Then, quantitative Real-time PCR (qRT-PCR) and Western blot were performed to verify target genes and proteins. (3) Results: Icariin treatment reduced excess serum levels of Testosterone (T), Estradiol (E2), Luteinizing hormone (LH), Follicle-stimulating hormone (FSH), LH/FSH ratio, insulin, triglycerides (TG), and aspartate aminotransferase (AST) in high-fat diet (HFD) and letrozole fed rats. Meanwhile, icariin ameliorated HFD and letrozole-induced fatty liver, as evidenced by a reduction in excess triglyceride accumulation, vacuolization, and Oil Red O staining area in the liver of model rats. Results of RNA-sequencing, western blotting, and qRT-PCR analyses indicated that icariin up-regulated fatty acid translocase (CD36), in mitochondria, and peroxisome proliferator-activated receptor α (PPARα) expression, which led to the enhancement of fatty acid oxidation molecules, such as cytochrome P450, family 4, subfamily a, polypeptide 3 (CYP4A3), carnitine palmitoyltransferase 1 α (CPT1α), acyl-CoA oxidase 1 (ACOX1), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD). Besides, icariin reduced lipid synthesis, which elicited stearoyl-Coenzyme A desaturase 1 (SCD1), fatty acid synthase (FASN), and acetyl-CoA (ACC). (4) Conclusion: Icariin showed an ameliorative effect on hepatic steatosis induced by HFD and letrozole, which was associated with improved fatty acid oxidation and reduced lipid accumulation in the liver.

Optimization of postoperative protocol and diagnosis of inflammatory complications of post-extraction wounds at the stages of prosthetic rehabilitation

Was to optimize the protocol for postoperative orthopedic management and diagnosis of wound healing zones in patients after multiple teeth extractions. At the Department of Orthopedic Dentistry and Orthodontics, Ryazan State Medical University, orthopedic treatment of 30 patients was carried out after the extraction of upper teeth. Patients were divided into 3 groups depending on the used immediate prostheses: (I) traditional, (II) having a shock-absorbing polypropylene mesh in their design, (III) having a drug reservoir made of elastic plastic and a ring made of monomer-free plastic in the region of the closing borders prosthesis. To assess the effectiveness of treatment, patients on days 5, 10, 20 underwent diagnostic supravital staining of the mucous membrane using an iodine-containing solution, planimetric control, and computerized capillaroscopy. In group I by the end of the observation period a pronounced dynamics of inflammation persisted in 30% of cases, the objective signs of which amounted to 12.5±2.06 mm2 of the area of the positive indicator of supravital staining, while in group II it was 7.2±2.09, in group III - 8.3±1.41 mm2 (p≤0.05). According to the results of supravital staining and capillaroscopy at day 20, in group II morphological and objective indicators of inflammation productivity were significantly higher than in group III: the density of the vascular network in group II was 52.5±2.17, in group III - 46.3±2.4 capillary loops/µm2, staining area 7.2±2.09 and 8.3±1.41 mm2, respectively (p≤0.05). More active wound healing in patients of group II was achieved by optimizing the design of the immediate prosthesis. An accessible and objective assessment of the severity of inflammation using vital staining allows you to accurately assess the dynamics of wound healing with a blurred or unexpressed clinical picture and timely suggest the features of inflammation to correct the course of treatment.

Perirenal adipose tissues as a human elastin source, and optimize the extraction process.

Elastin is very rarely repaired extracellular matrix (ECM) in physiological condition. The commercial human elastin for exogenous medical treatment is very expensive, and has a potential for disease transmission. Animal-origin elastin is relatively low price, but has concerns for xenogeneic immune responses. Considering cost and safety, we focused on the perirenal adipose tissue, donated from healthy young people via donor nephrectomy. Until now, all of the perirenal adipose tissues are discarded as a medical waste after kidney transplantation. In the present study, we applied perirenal adipose tissues as the source of human elastin, and optimized the extraction process to get high purified and quantified elastin. Through pre-processing step, the delipidated and decellularized ECM was prepared. Next, with four different elastin extraction process (acidic solvents, neutral salt, organic solvents or hot alkali method), elastin was extracted, and the concentration of amino acid between each product was compared, and bright-field/electron microscopy, Fourier transform infrared (FT-IR) spectroscopy and cytotoxicity analysis were also performed. As controls, bovine neck ligament-derived and human skin-derived elastin were used. Among the elastin extraction methods, the hot alkali insoluble product showed (1) relatively high positive area of Verhoeff's and low Masson's trichrome stain, (2) 64.24% purity, 159.29mg/g quantity, and ∼6.37% yield in amino acid analysis, (3) β-sheet second structure, and (4) thin fiber composed mesh-like sheet structure in SEM image. These values were higher than those of the commercial human skin elastin. When comparing hydrolyzed forms, α-elastin from hot alkali insoluble product showed enhanced cell proliferation and maintained cell properties compared to the κ-elastin. Therefore, we confirmed that the perirenal adipose tissue is an ideal source of human elastin with safety assurance, and the hot alkali process combined with pre-process seems to be the optimal method for elastin extraction with high purity and quantity.

Combination of autologous osteochondral and periosteum transplantation effectively promotes fibrocartilage regeneration at the tendon-bone junction of the rotator cuff in rabbits.

Rotator cuff tendon-bone healing often leads to scarring and low biomechanical strength, resulting in a tendency to re-tear. This study examined whether combining autologous osteochondral transplantation and periosteum transplantation increases fibrocartilage transition zone regeneration and improves biomechanical fixation. A total of 48 New Zealand white rabbits were divided into the periosteum, autologous osteochondral, combination of autologous osteochondral and periosteum, and control groups. The supraspinatus tendon was cut from the greater tuberosity and repaired by different transplants. A total of 12 rabbits were used for histological examination (haematoxylin and eosin staining, Masson's staining and Safranin-O staining) at 4, 8 and 12weeks after the repair, and 36 rabbits were used for biomechanical tests (maximal failure load and stiffness). At 4weeks following the operation, each group had a large tendon-bone gap with a small number of disordered collagen fibres. At 8weeks, the tendon-bone gap was smaller than that before the operation, and the tendon-bone gap in each experimental group was smaller with neater and denser collagen fibres and chondrocytes than in the control group, with the osteochondral combined periosteum group having the best results. At 12weeks, the typical tendon-bone transitional structure was observed in the osteochondral combined periosteum group, and more collagen fibres and chondrocytes were generated in each group. The osteochondral combined periosteum group had the largest staining area and the largest amount of cartilage. The maximum tensile strength and stiffness of each group increased over time. There was no significant difference in each group's maximum tensile strength and stiffness at 4weeks after the operation. However, the maximum tensile strength and stiffness of the osteochondral combined periosteum group at 8 and 12weeks after operation were significantly higher than those of other groups (P < 0.05). Histological and biomechanical results show that autologous osteochondral transplantation combined with periosteum transplantation can effectively promote the regeneration of fibrous cartilage in the tendon-bone junction of the rotator cuff. It is concluded that this technique is a new treatment method to promote tendon-bone healing in the rotator cuff.

Bacillus velezensis BY6 Promotes Growth of Poplar and Improves Resistance Contributing to the Biocontrol of Armillaria solidipes.

To improve the application of endophyte Bacillus velezensis BY6 from the xylem of poplar, the effect of BY6 on the growth of diseased Populus davidiana × Populus. alba var. pyramidalis Louche (Pdpap poplar) seedlings and the biological control effect on the pathogen Armillaria solidipes were tested using a plant split-root experiment. After applying BY6 to the roots of diseased Pdpap poplar seedlings, the results show that plant growth indicators (dry mass, fresh mass, and plant height) were significantly increased (p < 0.05), and genes related to auxin hormone signal transcription were activated. BY6 indicated a surprising control effect after the inoculation of diseased Pdpap poplar seedlings. Compared to the infected control group, the treated disease index of the diseased Pdpap poplar seedlings in the treatment group were reduced by 49.53% on the 20th day. The relative staining areas of diaminobenzidine (DAB) and Trypan blue decreased by 3.37 and 7.31 times, respectively. The physiological indicators (soluble sugar and protein) and oxidase indicators were significantly increased (p < 0.05). The expression levels of defense genes related to salicylic acid (SA) and jasmonic acid (JA) signaling pathways were significantly increased (p < 0.05). Amazingly, the results indicate that BY6 simultaneously activates induced systemic resistance (ISR) and systemic acquired resistance (SAR) in diseased Pdpap poplar seedlings and promotes growth. The results indicate that BY6 is a promising candidate for developing forest tree biofertilizers and biopesticides.

Microglia are implicated in the development of paclitaxel chemotherapy-associated cognitive impairment in female mice

Chemotherapy remains a mainstay in the treatment of many types of cancer even though it is associated with debilitating behavioral side effects referred to as “chemobrain,” including difficulty concentrating and memory impairment. The predominant hypothesis in the field is that systemic inflammation drives these cognitive impairments, although the brain mechanisms by which this occurs remain poorly understood. Here, we hypothesized that microglia are activated by chemotherapy and drive chemotherapy-associated cognitive impairments. To test this hypothesis, we treated female C57BL/6 mice with a clinically-relevant regimen of a common chemotherapeutic, paclitaxel (6 i.p. doses at 30 mg/kg), which impairs memory of an aversive stimulus as assessed via a contextual fear conditioning (CFC) paradigm. Paclitaxel increased the percent area of IBA1 staining in the dentate gyrus of the hippocampus. Moreover, using a machine learning random forest classifier we identified immunohistochemical features of reactive microglia in multiple hippocampal subregions that were distinct between vehicle- and paclitaxel-treated mice. Paclitaxel treatment also increased gene expression of inflammatory cytokines in a microglia-enriched population of cells from mice. Lastly, a selective inhibitor of colony stimulating factor 1 receptor, PLX5622, was employed to deplete microglia and then assess CFC performance following paclitaxel treatment. PLX5622 significantly reduced hippocampal gene expression of paclitaxel-induced proinflammatory cytokines and restored memory, suggesting that microglia play a critical role in the development of chemotherapy-associated neuroinflammation and cognitive impairments. This work provides critical evidence that microglia drive paclitaxel-associated cognitive impairments, a key mechanistic detail for determining preventative and intervention strategies for these burdensome side effects.