Phosphatidylinositol 3-kinase and Rab5 GTPase inversely regulate the Smad anchor for receptor activation (SARA) protein independently of transforming growth factor-ß1.

SARA has been shown to be a regulator of epithelial cell phenotype, with reduced expression during TGF-ß1-mediated epithelial-to-mesenchymal transition. Examination of the pathways that might play a role in regulating SARA expression identified phosphatidylinositol 3-kinase (PI3K) pathway inhibition as sufficient to reduce SARA expression. The mechanism of PI3K inhibition-mediated SARA down-regulation differs from that induced by TGF-ß1 in that, unlike TGF-ß1, PI3K-dependent depletion of SARA was apparent within 6 h and did not occur at the mRNA or promoter level but was blocked by inhibition of proteasome-mediated degradation. This effect was independent of Akt activity because neither reducing nor enhancing Akt activity modulated the expression of SARA. Therefore, this is likely a direct effect of p85α action, and co-immunoprecipitation of SARA and p85α confirmed that these proteins interact. Both SARA and PI3K have been shown to be associated with endosomes, and either LY294002 or p85α knockdown enlarged SARA-containing endocytic vesicles. Inhibition of clathrin-mediated endocytosis blocked SARA down-regulation, and a localization-deficient mutant SARA was protected against down-regulation. As inhibiting PI3K can activate the endosomal fusion-regulatory small GTPase Rab5, we expressed GTPase-deficient Rab5 and observed endosomal enlargement and reduced SARA protein expression, similar to that seen with PI3K inhibition. Importantly, either interference with PI3K via LY294002 or p85α knockdown, or constitutive activity of the Rab5 pathway, enhanced the expression of smooth muscle α-actin. Together, these data suggest that although TGF-ß1 can induce epithelial-to-mesenchymal transition through reduction in SARA expression, SARA is also basally regulated by its interaction with PI3K.

Study on the treatment of postmenopausal osteoporosis with quercetin in Liuwei Dihuang Pill based on network pharmacology.

BACKGROUND: Liuwei Dihuang Pill (LP) was verified to alleviate postmenopausal osteoporosis (PMOP) development. Nevertheless, the major constituent of LP and the related network pharmacology study remain unexplored. METHODS: Protein-protein interaction was established to identify the downstream target of LP in PMOP, and the related signaling pathway was investigated by bioinformatics analysis. MC3T3-E1 cells were added to ferric ammonium citrate (FAC) to mimic osteoporosis in vitro. The osteoblasts were identified by Alizarin red staining. Western blot was applied to evaluate protein levels. In addition, Cell Counting Kit-8 (CCK8) assay was applied to assess cell viability, and cell apoptosis was assessed by flow cytometry. RESULTS: Quercetin was the major constituent of LP. In addition, quercetin significantly reversed FAC-induced inhibition of osteogenic differentiation in MC3T3-E1 cells. In addition, quercetin notably abolished the FAC-induced upregulation of Bax, Caspase-3, FOS, JUN, TGFB1 and PPARD. In contrast, Bcl-2, p-mTOR/mTOR, p-AKT/AKT and p-PI3K/PI3K levels in MC3T3-E1 cells were reduced by FAC, which was restored by quercetin. Meanwhile, FAC notably inhibited the viability of MC3T3-E1 cells via inducing apoptosis, but this impact was abolished by quercetin. Furthermore, quercetin could reverse pcDNA3.1-FOS-mediated growth of FAC-treated osteoblasts by mediating PI3K/AKT/mTOR signaling. CONCLUSION: Quercetin alleviated the progression of PMOP via activation of PI3K/AKT/mTOR signaling. Hence, this study would shed novel insights into discovering new methods against PMOP.

The anti-fibrotic efficacy of adelmidrol depends on hepatic PPARγ levels.

Adelmidrol, an anti-inflammatory small-molecule compound, can treat inflammatory diseases like arthritis and colitis in a PPARγ-dependent manner. Effective anti-inflammatory therapy is beneficial in delaying the progression of liver fibrosis. This study aimed to investigate the effect and underlying mechanisms of adelmidrol on hepatic fibrosis induced by CCl4 and CDAA-HFD. In the CCl4 model, adelmidrol (10 mg/kg) significantly reduced the incidence of liver cirrhosis from 76.5% to 38.9%, with a reduction of ALT, AST, and extracellular matrix deposition. RNA-seq revealed adelmidrol markedly inhibited the activation of hepatic scar-associated Trem2+ macrophages and PDGFRα+ stellate cells. Adelmidrol exhibited a limited anti-fibrotic effect in CDAA-HFD-induced fibrosis. Further, inconsistencies were observed in the expression trends in liver PPARγ in both models. CCl4 injury led to the continuous decrease in hepatic PPARγ levels, adelmidrol treatment up-regulated hepatic PPARγ expression and down-regulated the expression of pro-inflammatory factor NF-κB and pro-fibrotic factor TGF-ß1. Adelmidrol also inhibited the activation of macrophages and HSCs in a PPARγ-dependent manner in vitro. GW9662, a specific PPARγ antagonist, counteracted the anti-fibrotic effect of adelmidrol. In CDAA-HFD-induced model, hepatic PPARγ expression gradually increased with the progress of modeling. Adelmidrol enhanced steatosis in hepatocytes by the activation of the PPARγ/CD36 pathway in the CDAA-HFD model and FFA-treated HepG2, showing a limited anti-fibrotic effect. GW9662 reversed the pro-steatotic effect of adelmidrol and improved fibrosis. The anti-fibrotic outcomes of adelmidrol were related to hepatic PPARγ levels, which depends on the synergistic effect of PPARγ agonism caused by adelmidrol on hepatocytes, macrophages, and HSCs in different pathological states.

Dynamics of the gut-liver axis in rats with varying fibrosis severity.

The classic carbon tetrachloride (CCl4)-induced liver injury model is widely used to study the pathogenesis of fibrosis and evaluate anti-fibrosis drugs. Here, we investigated the dynamic changes in the gut microbiota, bile acids (BAs) and the gut barrier over different fibrosis severities in a CCl4-based model. 16S rDNA sequencing demonstrated that the beneficial taxon Lactobacillus was always underrepresented, and pathogens including Escherichia_Shigella, Clostridium_sensu_stricto_1, Colidextribacter, and Lachnospiraceae_UCG_010 were significantly overrepresented across liver fibrosis severities. Gut dysbiosis was more severe at the early stage of liver injury and advanced stage of fibrosis. An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis revealed that with the progress of fibrosis, unconjugated BAs in faeces were significantly decreased and conjugated BAs in serum were significantly increased. The FXR-SHP signalling pathway in the liver and ileum was statistically repressed in the fibrosis groups. Determination of lipopolysaccharide (LPS) and fluorescein isothiocyanate (FITC)-dextran levels in plasma showed that the intestinal barrier remained relatively intact in the advanced fibrosis stage. The advances in knowledge of the gut-liver axis provided by this study yield new insights for application in research and drug evaluation.

Revealing potential anti-fibrotic mechanism of Ganxianfang formula based on RNA sequence.

BACKGROUND: Ganxianfang (GXF) formula as a traditional Chinese medicine (TCM) is used for liver fibrosis in clinical practice while its mechanism is unclear. The aim of this study is to explore the molecular mechanism of GXF against CCl4-induced liver fibrosis rats. METHODS: Detected the main compounds of GXF by UPLC-MS/MS. Evaluated the efficacy of GXF (1.58, 3.15, 4.73 g/kg/day) and Fuzheng Huayu (FZHY, positive control, 0.47 g/kg/day) through serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels and histopathological changes. Explored the underlying mechanisms by integrating our total liver RNA sequencing (RNA-seq) data with recent liver single-cell sequencing (scRNA-seq) studies. Verified potential pharmacodynamic substances of GXF by hepatic stellate cell (HSC)-T6 line. RESULTS: Main compounds were identified in GXF by UPLC-MS/MS, including baicalin, wogonoside and matrine etc. With GXF-high dose treatment, the elevation of ALT and AST induced by CCl4 were significantly reduced, and the protective effect of GXF-high dose treatment was better than FZHY. Liver histopathological changes were alleviated by GXF-high dose treatment, the ISHAK scoring showed the incidence of liver cirrhosis (F5/F6) decreased from 76.5 to 55.6%. The results of liver hydroxyproline content were consistent with the histopathological changes. RNA-seq analysis revealed the differential genes (DEGs) were mainly enriched in ECM-receptor interaction and chemokine signaling pathway. GXF effectively inhibited collagen deposition and significantly downregulated CCL2 to inhibit the recruitment of macrophages in liver tissue. Integrating scRNA-seq data revealed that GXF effectively inhibited the expansion of scar-associated Trem2+CD9+ macrophages subpopulation and PDGFRα+PDGFRß+ scar-producing myofibroblasts in the damaged liver, and remodeled the fibrotic niche via regulation of ligand-receptor interactions including TGFß/EGFR, PDGFB/PDGFRα, and TNFSF12/TNFRSF12a signaling. In vitro experiments demonstrated that baicalin, matrine and hesperidin in GXF inhibited the activation of hepatic stellate cells. CONCLUSIONS: This study clarified the potential anti-fibrotic effects and molecular mechanism of GXF in CCl4-induced liver fibrosis rats, which deserves further promotion and application.

BReast CAncer susceptibility gene 2 deficiency exacerbates oxidized LDL-induced DNA damage and endothelial apoptosis.

Mutations in the tumor suppressor gene BRCA2 (BReast CAncer susceptibility gene 2) predispose carriers to breast, ovarian, and other cancers. In response to DNA damage, BRCA2 participates in homology-directed DNA damage repair to maintain genome stability. Genome-wide association studies have identified an association between BRCA2 single nucleotide polymorphisms and plasma-lipid levels and lipid deregulation in humans. To date, DNA damage, apoptosis, and lipid deregulation are recognized as central pathways for endothelial dysfunction and atherosclerosis; however, the role of BRCA2 in endothelial dysfunction remains to be elucidated. To determine the role of BRCA2 in endothelial dysfunction, BRCA2 was silenced in human umbilical vein endothelial cells (ECs) and assessed for markers of DNA damage, apoptosis, and endothelial function following oxidized low-density lipoprotein (oxLDL) treatment. OxLDL was found to induce significant reactive oxygen species (ROS) production in BRCA2-silenced ECs. This increase in ROS production was associated with exacerbated DNA damage evidenced by increased expression and activation of DNA double-stranded break (DSB) marker γH2AX and reduced RAD51-foci formation-an essential regulator of DSB repair. Increased DSBs were associated with enhanced expression and activation of pro-apoptotic p53 and significant apoptosis in oxLDL-treated BRCA2-silenced ECs. Loss of BRCA2 in ECs was further associated with oxLDL-induced impaired tube-forming potential and eNOS expression. Collectively, the data reveals, for the first time, a novel role of BRCA2 as a regulator of EC survival and function in the setting of oxLDL treatment in vitro. Additionally, the data provide important clues regarding the potential susceptibility of BRCA2 mutation carriers to endothelial dysfunction, atherosclerosis, and other cardiovascular diseases.

Endothelial-specific Loss of IFT88 Promotes Endothelial-to-Mesenchymal Transition and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Intraflagellar transport protein 88 (Ift88) is required for ciliogenesis and shear stress-induced dissolution of cilia in embryonic endothelial cells coincides with endothelial-to-mesenchymal transition (EndMT) in the developing heart. EndMT is also suggested to underlie heart and lung fibrosis, however, the mechanism linking endothelial Ift88, its effect on EndMT and organ fibrosis remains mainly unexplored. We silenced Ift88 in endothelial cells (ECs) in vitro and generated endothelial cell-specific Ift88-knockout mice (Ift88endo) in vivo to evaluate EndMT and its contribution towards organ fibrosis, respectively. Ift88-silencing in ECs led to mesenchymal cells-like changes in endothelial cells. The expression level of the endothelial markers (CD31, Tie-2 and VE-cadherin) were significantly reduced with a concomitant increase in the expression level of mesenchymal markers (αSMA, N-Cadherin and FSP-1) in Ift88-silenced ECs. Increased EndMT was associated with increased expression of profibrotic Collagen I expression and increased proliferation in Ift88-silenced ECs. Loss of Ift88 in ECs was further associated with increased expression of Sonic Hedgehog signaling effectors. In vivo, endothelial cells isolated from the heart and lung of Ift88endo mice demonstrated loss of Ift88 expression in the endothelium. The Ift88endo mice were born in expected Mendelian ratios without any adverse cardiac phenotypes at baseline. Cardiac and pulmonary endothelial cells isolated from the Ift88endo mice demonstrated signs of EndMT and bleomycin treatment exacerbated pulmonary fibrosis in Ift88endo mice. Pressure overload stress in the form of aortic banding did not reveal a significant difference in cardiac fibrosis between Ift88endo mice and control mice. Our findings demonstrate a novel association between endothelial cilia with EndMT and cell proliferation and also show that loss of endothelial cilia-associated increase in EndMT contributes specifically towards pulmonary fibrosis.

Improved gait recognition by gait dynamics normalization.

Potential sources for gait biometrics can be seen to derive from two aspects: gait shape and gait dynamics. We show that improved gait recognition can be achieved after normalization of dynamics and focusing on the shape information. We normalize for gait dynamics using a generic walking model, as captured by a population Hidden Markov Model (pHMM) defined for a set of individuals. The states of this pHMM represent gait stances over one gait cycle and the observations are the silhouettes of the corresponding gait stances. For each sequence, we first use Viterbi decoding of the gait dynamics to arrive at one dynamics-normalized, averaged, gait cycle of fixed length. The distance between two sequences is the distance between the two corresponding dynamics-normalized gait cycles, which we quantify by the sum of the distances between the corresponding gait stances. Distances between two silhouettes from the same generic gait stance are computed in the linear discriminant analysis space so as to maximize the discrimination between persons, while minimizing the variations of the same subject under different conditions. The distance computation is constructed so that it is invariant to dilations and erosions of the silhouettes. This helps us handle variations in silhouette shape that can occur with changing imaging conditions. We present results on three different, publicly available, data sets. First, we consider the HumanlD Gait Challenge data set, which is the largest gait benchmarking data set that is available (122 subjects), exercising five different factors, i.e., viewpoint, shoe, surface, carrying condition, and time. We significantly improve the performance across the hard experiments involving surface change and briefcase carrying conditions. Second, we also show improved performance on the UMD gait data set that exercises time variations for 55 subjects. Third, on the CMU Mobo data set, we show results for matching across different walking speeds. It is worth noting that there was no separate training for the UMD and CMU data sets.

The humanID gait challenge problem: data sets, performance, and analysis.

Identification of people by analysis of gait patterns extracted from video has recently become a popular research problem. However, the conditions under which the problem is "solvable" are not understood or characterized. To provide a means for measuring progress and characterizing the properties of gait recognition, we introduce the HumanID Gait Challenge Problem. The challenge problem consists of a baseline algorithm, a set of 12 experiments, and a large data set. The baseline algorithm estimates silhouettes by background subtraction and performs recognition by temporal correlation of silhouettes. The 12 experiments are of increasing difficulty, as measured by the baseline algorithm, and examine the effects of five covariates on performance. The covariates are: change in viewing angle, change in shoe type, change in walking surface, carrying or not carrying a briefcase, and elapsed time between sequences being compared. Identification rates for the 12 experiments range from 78 percent on the easiest experiment to 3 percent on the hardest. All five covariates had statistically significant effects on performance, with walking surface and time difference having the greatest impact. The data set consists of 1,870 sequences from 122 subjects spanning five covariates (1.2 Gigabytes of data). The gait data, the source code of the baseline algorithm, and scripts to run, score, and analyze the challenge experiments are available at http://www.GaitChallenge.org. This infrastructure supports further development of gait recognition algorithms and additional experiments to understand the strengths and weaknesses of new algorithms. The more detailed the experimental results presented, the more detailed is the possible meta-analysis and greater is the understanding. It is this potential from the adoption of this challenge problem that represents a radical departure from traditional computer vision research methodology.

Effect of silhouette quality on hard problems in Gait recognition.

Gait as a behavioral biometric has been the subject of recent investigations. However, understanding the limits of gait-based recognition and the quantitative study of the factors effecting gait have been confounded by errors in the extracted silhouettes, upon which most recognition algorithms are based. To enable us to study this effect on a large population of subjects, we present a novel model based silhouette reconstruction strategy, based on a population based hidden Markov model (HMM), coupled with an eigen-stance model, to correct for common errors in silhouette detection arising from shadows and background subtraction. The model is trained and benchmarked using manually specified silhouettes for 71 subjects from the recently formulated HumanID Gait Challenge database. Unlike other essentially pixel-level silhouette cleaning methods, this method can remove shadows, especially between feet for the legs-apart stance, and remove parts due to any objects being carried, such as briefcase or a walking cane. After quantitatively establishing the improved quality of the silhouette over simple background subtraction, we show on the 122 subjects HumanID Gait Challenge Dataset and using two gait recognition algorithms that the observed poor performance of gait recognition for hard problems involving matching across factors such as surface, time, and shoe are not due to poor silhouette quality, beyond what is available from statistical background subtraction based methods.

Creation and initial assessment of a second-trimester uterine model.

INTRODUCTION: Training for obstetrics and gynecology residents in second-trimester dilation and evacuation (D&E) procedures is extremely limited despite the Accreditation Council for Graduate Medical Education mandating all residents to receive abortion training. Simulation-based training improves surgical competence, but no second-trimester uterine models exist. The purposes of this study were to create a realistic, low-cost model and to assess the prototype. METHODS: A uterine model was created with 6 silicone cervixes of varying texture and dilations that are interchangeable. The uterus is neoprene and opens to allow for objects to be placed within it for extraction. At a national meeting, experienced D&E surgeons assessed the prototype by using the model and then completing a questionnaire. RESULTS: Twenty-one expert surgeons completed the questionnaires. Participants rated the prototype as "useful" or "very useful" for teaching extraction skills and for training in general. Subjects agreed this represented a clinical scenario they were likely to encounter and the model allowed for practicing the necessary steps for performing D&Es. The model cost approximately $35 to fabricate. CONCLUSIONS: Expert surgeons believe that this model accurately and realistically replicates a second-trimester uterus and cervix. This prototype may be used in simulation environments to train obstetrics and gynecology residents.

[Reverse island flap of digital artery parallel for repairing degloved injuries of fingertip].

OBJECTIVE: To investigate the effectiveness of reverse island flaps of digital artery parallel for repairing degloved injuries of the fingertip. METHODS: Between June 2008 and January 2010, 13 cases of degloved injuries of the fingertip were treated. There were 8 males and 5 females with an average age of 34 years (range, 19-62 years). The causes of injuries were as follow: impact and press injury in 5 cases, wringer injury in 7 cases, and crush injury in 1 case. The injured fingers were comprised of index finger in 6 cases, middle finger in 4 cases, ring finger in 2 cases, and little finger in 1 case. The size of skin and soft tissue defect ranged from 2.0 cm x 1.8 cm to 3.0 cm x 2.5 cm. Three cases complicated by fracture of the distal phalanx, 1 case by rupture of the insertion of extensor tendon, and 1 case by rupture of the insertion of flexor tendon. The average time from injure to surgery was 4 hours (range, 1 hour and 30 minutes-12 hours). Two neighboring skin flaps located in the same course of digital artery were adopted to repair defect of the fingertip. The size of proximal skin flap ranged from 1.2 cm x 1.0 cm to 2.0 cm x 1.5 cm and the size of distal skin flap ranged from 1.1 cm x 1.0 cm to 1.5 cm x 1.3 cm. The free skin grafts were used to repair the donor sites. RESULTS: Circulation crisis occurred in 1 case at 2 hours after operation and was eliminated by interval disconnecting. The other flaps and skin grafts survived and the wounds healed by first intention. The patients were followed up 6-18 months (mean, 10 months). All flaps presented the satisfactory appearance and texture, and the flexion and extension function of wounded fingers recovered to normal. Two-point discrimination ranged from 7 to 11 mm at last follow-up. According to the functional assessment criteria of upper limb formulated by the Hand Surgery Branch of Chinese Medical Association, the results were excellent in 9 cases, good in 3 cases, and fair in 1 case with an excellent and good rate of 92.3%. CONCLUSION: Based on the anatomical features of communicating branches of distal interphalangeal joint, two neighboring flaps located in the same course of digital artery are adopted to repair soft tissue defect of the fingertip. This surgical method is a simple and effective method.