High-resolution epitope mapping of commercial antibodies to ANCA antigens by yeast surface display.

Epitope mapping provides critical insight into antibody-antigen interactions. Epitope mapping of autoantibodies from patients with autoimmune diseases can help elucidate disease immunogenesis and guide the development of antigen-specific therapies. Similarly, epitope mapping of commercial antibodies targeting known autoantigens enables the use of those antibodies to test specific hypotheses. Anti-Neutrophil Cytoplasmic Autoantibody (ANCA) vasculitis results from the formation of autoantibodies to multiple autoantigens, including myeloperoxidase (MPO), proteinase-3 (PR3), plasminogen (PLG), and peroxidasin (PXDN). To perform high-resolution epitope mapping of commercial antibodies to these autoantigens, we developed a novel yeast surface display library based on a series of >5000 overlapping peptides derived from their protein sequences. Using both FACS and magnetic bead isolation of reactive yeast, we screened 19 commercially available antibodies to the ANCA autoantigens. This approach to epitope mapping resulted in highly specific, fine epitope mapping, down to single amino acid resolution in many cases. Our study also identified cross-reactivity between some commercial antibodies to MPO and PXDN, which suggests that patients with apparent autoantibodies to both proteins may be the result of cross-reactivity. Together, our data validate yeast surface display using maximally overlapping peptides as an excellent approach to linear epitope mapping.

Development and validation of a postoperative pulmonary infection prediction model for patients with primary hepatic carcinoma.

BACKGROUND: There are factors that significantly increase the risk of postoperative pulmonary infections in patients with primary hepatic carcinoma (PHC). Previous reports have shown that over 10% of patients with PHC experience postoperative pulmonary infections. Thus, it is crucial to prioritize the prevention and treatment of postoperative pulmonary infections in patients with PHC. AIM: To identify the risk factors for postoperative pulmonary infection in patients with PHC and develop a prediction model to aid in postoperative management. METHODS: We retrospectively collected data from 505 patients who underwent hepatobiliary surgery between January 2015 and February 2023 in the Department of Hepatobiliary and Pancreaticospleen Surgery. Radiomics data were selected for statistical analysis, and clinical pathological parameters and imaging data were included in the screening database as candidate predictive variables. We then developed a pulmonary infection prediction model using three different models: An artificial neural network model; a random forest model; and a generalized linear regression model. Finally, we evaluated the accuracy and robustness of the prediction model using the receiver operating characteristic curve and decision curve analyses. RESULTS: Among the 505 patients, 86 developed a postoperative pulmonary infection, resulting in an incidence rate of 17.03%. Based on the gray-level co-occurrence matrix, we identified 14 categories of radiomic data for variable screening of pulmonary infection prediction models. Among these, energy, contrast, the sum of squares (SOS), the inverse difference (IND), mean sum (MES), sum variance (SUV), sum entropy (SUE), and entropy were independent risk factors for pulmonary infection after hepatectomy and were listed as candidate variables of machine learning prediction models. The random forest model algorithm, in combination with IND, SOS, MES, SUE, SUV, and entropy, demonstrated the highest prediction efficiency in both the training and internal verification sets, with areas under the curve of 0.823 and 0.801 and a 95% confidence interval of 0.766-0.880 and 0.744-0.858, respectively. The other two types of prediction models had prediction efficiencies between areas under the curve of 0.734 and 0.815 and 95% confidence intervals of 0.677-0.791 and 0.766-0.864, respectively. CONCLUSION: Postoperative pulmonary infection in patients undergoing hepatectomy may be related to risk factors such as IND, SOS, MES, SUE, SUV, energy, and entropy. The prediction model in this study based on diffusion-weighted images, especially the random forest model algorithm, can better predict and estimate the risk of pulmonary infection in patients undergoing hepatectomy, providing valuable guidance for postoperative management.

An Effective Surgical Strategy for Anteromedial Osteoarthritis of the Knee with Simultaneous Popliteal Cysts Drainage.

Unicompartmental knee arthroplasty (UKA) is an established treatment option for anteromedial osteoarthritis, and popliteal cysts are a common finding in the knee among patients with chronic osteoarthritis pain. The two are so closely related that popliteal cysts are commonly discovered during the unicompartmental knee arthroplasty preoperative examination. However, only a few reports exist on the management and outcome of popliteal cysts in the patients receiving UKA for knee osteoarthritis (OA) and popliteal cysts. As such, it is crucial to evaluate different treatment strategies and their management of popliteal cysts. In this paper, we evaluate a surgical strategy for patients with knee anteromedial osteoarthritis and symptomatic popliteal cysts. These patients were treated with UKA and internal drainage of the popliteal cyst. The results shown here, spanning 1-year post-operation follow-up, demonstrated that UKA and internal drainage is an effective surgical protocol for treating anteromedial osteoarthritis with symptomatic popliteal cysts.

Personal predictive model based on systemic inflammation markers for estimation of postoperative pancreatic fistula following pancreaticoduodenectomy.

BACKGROUND: Postoperative pancreatic fistula (PF) is a serious life-threatening complication after pancreaticoduodenectomy (PD). Our research aimed to develop a machine learning (ML)-aided model for PF risk stratification. AIM: To develop an ML-aided model for PF risk stratification. METHODS: We retrospectively collected 618 patients who underwent PD from two tertiary medical centers between January 2012 and August 2021. We used an ML algorithm to build predictive models, and subject prediction index, that is, decision curve analysis, area under operating characteristic curve (AUC) and clinical impact curve to assess the predictive efficiency of each model. RESULTS: A total of 29 variables were used to build the ML predictive model. Among them, the best predictive model was random forest classifier (RFC), the AUC was [0.897, 95% confidence interval (CI): 0.370-1.424], while the AUC of the artificial neural network, eXtreme gradient boosting, support vector machine, and decision tree were between 0.726 (95%CI: 0.191-1.261) and 0.882 (95%CI: 0.321-1.443). CONCLUSION: Fluctuating serological inflammatory markers and prognostic nutritional index can be used to predict postoperative PF.

Pharmacokinetic Analysis of Diosgenin in Rat Plasma by a UPLC-MS/MS Approach.

Diosgenin, a steroidal sapogenin, has attracted attention worldwide owing to its pharmacological properties, including antitumor, cardiovascular protective, hypolipidemic, and anti-inflammatory effects. The current diosgenin analysis methods have the disadvantages of long analysis time and low sensitivity. The aim of the present study was to establish an efficient, sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach for pharmacokinetic analysis of diosgenin amorphous solid dispersion (ASD) using tanshinone IIA as an internal standard (IS). Male Sprague-Dawley rats were orally administered diosgenin ASD, and orbital blood samples were collected for analysis. Protein precipitation was performed with methanol-acetonitrile (50 : 50, v/v), and the analytes were separated under isocratic elution by applying acetonitrile and 0.03% formic acid aqueous solution at a ratio of 80 : 20 as the mobile phase. MS with positive electron spray ionization in multiple reaction monitoring modes was applied to determine diosgenin and IS with m/z 415.2⟶271.2 and m/z 295.2⟶277.1, respectively. This approach showed a low limit of quantification of 0.5 ng/ml for diosgenin and could detect this molecule at a concentration range of 0.5 to 1,500 ng/ml (r = 0.99725). The approach was found to have intra- and inter-day precision values ranging from 1.42% to 6.91% and from 1.25% to 3.68%, respectively. Additionally, the method showed an accuracy of -6.54 to 4.71%. The recoveries of diosgenin and tanshinone IIA were 85.81-100.27% and 98.29%, respectively, with negligible matrix effects. Diosgenin and IS were stable under multiple storage conditions. Pharmacokinetic analysis showed that the C max and AUC0⟶t of diosgenin ASD were significantly higher than those of the bulk drug. A sensitive, simple, UPLC-MS/MS analysis approach was established and used for the pharmacokinetic analysis of diosgenin ASD in rats after oral administration.

Cellular angiofibroma of the vagina: A case report and literature review.

BACKGROUND: Cellular angiofibroma (CAF), a rare benign mesenchymal tumor, is histologically characterized by abundant thick-walled vessels with a spindle cell component. As one of the female reproductive system tumors, its clinical and pathological features are not well characterized. METHODS: A 47-year-old woman presented for the removal of intrauterine device on October 28, 2021, as she had achieved menopause one year back. The patient had no discomfort or awareness of any mass in her vagina. She has history of breast cancer and papillary thyroid cancer. Till date, no progression of thyroid cancer or breast cancer has been observed. Her menstrual cycle was regular, and she had one child delivered vaginally. RESULTS: Pelvic examination revealed a mass sized 2.5 × 2.0 cm located near the fornix in the upper segment of the left vaginal wall. Thin prep cytologic test (TCT) revealed negative intraepithelial lesion or malignancy (NILM). HPV test was negative and leucorrhea routine inspection cleanliness II degree. No cervical mass was detected by ultrasound examination. The patients underwent the operation for intrauterine device removal plus vaginal tumor resection on November 1, 2021. Postoperative antibiotics (intravenous cefuroxime sodium 0.75 g bid for 1 day) were administered to prevent infection. The patient showed no signs of recurrence at one-month follow-up. CONCLUSION: In summary, CAF is a rare benign soft tissue tumor. Surgery is the only treatment method, and the definitive diagnosis of CAF is based on histopathological examination of surgical specimen. Long-term follow-up is needed for surveillance of recurrence.

Survival outcome assessment for triple-negative breast cancer: a nomogram analysis based on integrated clinicopathological, sonographic, and mammographic characteristics.

OBJECTIVE: This study aimed to incorporate clinicopathological, sonographic, and mammographic characteristics to construct and validate a nomogram model for predicting disease-free survival (DFS) in patients with triple-negative breast cancer (TNBC). METHODS: Patients diagnosed with TNBC at our institution between 2011 and 2015 were retrospectively evaluated. A nomogram model was generated based on clinicopathological, sonographic, and mammographic variables that were associated with 1-, 3-, and 5-year DFS determined by multivariate logistic regression analysis in the training set. The nomogram model was validated according to the concordance index (C-index) and calibration curves in the validation set. RESULTS: A total of 636 TNBC patients were enrolled and divided into training cohort (n = 446) and validation cohort (n = 190). Clinical factors including tumor size > 2 cm, axillary dissection, presence of LVI, and sonographic features such as angular/spiculated margins, posterior acoustic shadows, and presence of suspicious lymph nodes on preoperative US showed a tendency towards worse DFS. The multivariate analysis showed that no adjuvant chemotherapy (HR = 6.7, 95% CI: 2.6, 17.5, p < 0.0005), higher axillary tumor burden (HR = 2.7, 95% CI: 1.0, 7.1, p = 0.045), and ≥ 3 malignant features on ultrasound (HR = 2.4, CI: 1.1, 5.0, p = 0.021) were identified as independent prognostic factors associated with poorer DFS outcomes. In the nomogram, the C-index was 0.693 for the training cohort and 0.694 for the validation cohort. The calibration plots also exhibited excellent consistency between the nomogram-predicted and actual survival probabilities in both the training and validation cohorts. CONCLUSIONS: Clinical variables and sonographic features were correlated with the prognosis of TNBCs. The nomogram model based on three variables including no adjuvant chemotherapy, higher axillary tumor load, and more malignant sonographic features showed good predictive performance for poor survival outcomes of TNBC. KEY POINTS: • The absence of adjuvant chemotherapy, heavy axillary tumor load, and malignant-like sonographic features can predict DFS in patients with TNBC. • Mammographic features of TNBC could not predict the survival outcomes of patients with TNBC. • The nomogram integrating clinicopathological and sonographic characteristics is a reliable predictive model for the prognostic outcome of TNBC.

Short-term outcomes of Oxford unicompartmental knee arthroplasty with coronal subluxation of the knee: a retrospective case-control study.

BACKGROUND: The goal of this study was to assess short-term outcomes in single compartment osteoarthritis patients associated with the coronal tibiofemoral subluxation (CTFS) of the knee joint after Oxford unicompartmental knee arthroplasty (OUKA), and to establish the potential impact of the degree of CTFS on operative outcomes. METHODS: Data pertaining to 183 patients with medial compartment osteoarthritis that underwent OUKA treatment between February 2016 and June 2019 were retrospectively analyzed. The presence and degree of severity of CTFS were assessed using preoperative weight-bearing anteroposterior X-ray images of the knee. Patients were stratified into three subgroups based upon the observed degree of subluxation: a normal group, a mild subluxation group (CTFS < 0.5 cm), and a severe subluxation group (CTFS ≥ 0.5 cm). Anterior and posterior X-ray examination of the knee was conducted at the time of most recent follow-up for each patient to assess the degree of CTFS correction following OUKA. Clinical function was assessed using Oxford knee score (OKS) and Hospital for Special Surgery score (HSS) values, while pain was rated using visual-analog scale (VAS) scores. The mechanical femoral tibial angle (mFTA), range of motion (ROM), and complication rates in these three groups were additionally compared. RESULTS: The average follow-up duration for patients in this study was 24.1 months (range: 17-32 months). There were no significant differences in patient age, sex, body mass index (BMI), follow-up duration, mFTA, ROM, OKS, HSS, or VAS scores among these three groups (P > 0.05). After surgery, OKS and HSS scores declined significantly, but no differences in these scores were observed among groups (P > 0.05). Of these patients, 135 (73.8%) were satisfied with the operation, of whom 80 (43.7%) were very satisfied. There were no significant differences in ROM or VAS scores among groups (P > 0.05). The degree of CTFS for patients in the mild and severe subluxation groups was significantly improved following OUKA relative to preoperative values such that the degree of postoperative CTFS did not differ significantly among these groups (P > 0.05). Postoperative mFTA was also significantly improved in these three patient subgroups (P < 0.05). No patients experienced operative complications over the follow-up period. CONCLUSIONS: OUKA can successfully improve clinical symptoms in patients with single compartmental osteoarthritis. Moreover, OUKA can effectively correct CTFS of the knee in these patients, and the degree of preoperative CTFS has no impact on surgical efficacy. LEVEL OF EVIDENCE: III.

Arthroscopic Internal Drainage with Cyst Wall Resection and Arthroscopic Internal Drainage with Cyst Wall Preservation to Treat Unicameral Popliteal Cysts: A Retrospective Case-Control Study.

OBJECTIVE: To compare the clinical efficacy and safety of arthroscopic internal drainage for the treatment of unicameral popliteal cysts with or without cyst wall resection. METHODS: This was a retrospective case-control study of 73 patients diagnosed with unicameral popliteal cysts from January 2012 to January 2019 who received arthroscopic treatment. The study included 38 cases with cyst wall resection (CWR group) and 35 cases with cyst wall preservation (CWP group). The CWR group consisted of 14 men and 24 women with an average age of 51.8 years, while the CWP group consisted of 13 men and 22 women with an average age of 52.0 years. All patients were examined for intra-articular lesions and communicating ports by magnetic resonance imaging (MRI) prior to surgery, and recurrence of cysts was evaluated at the last follow-up examination. Rauschning and Lindgren grade (R-L grade) and Lysholm score were used to evaluate clinical outcomes. In addition, operation time and complications were recorded. RESULTS: The average length of follow-up was 24.2 months (range, 16 to 32 months). There were no considerable differences in age, gender, cyst size, Lysholm score, R-L grade and concomitant intra-articular cases between the CWR group and CWP group prior to surgery (P > 0.05). The last follow-up MRI scans showed that in the CWR group, the cyst disappeared in 25 cases and shrunk in 13 cases. In the CWP group, the cyst disappeared in 22 cases, shrunk in 12 cases and persisted in one case. There was no obvious difference in recurrence rate between the two groups (0% vs 2.9%, P = 0.899). At the last follow-up, there were no differences in the R-L grade (P = 0.630) and Lysholm score (88.3 ± 5.6 points vs 90.1 ± 3.8 points, P = 0.071) between the two groups. Compared with the CWP group, operation time was significantly prolonged in the CWR group (38.3 ± 3.1 min vs 58.3 ± 4.4 min, P < 0.05). In the CWR group, three cases occurred fluid infiltration under the gastrocnemius muscle, which improved after pressure bandaging and cold compress. In another three cases, hematoma was found. The incidence of complications in the CWR group was markedly higher than that in the CWP group (15.8% vs 0%, P < 0.05). During the follow-up period, none of the patients developed serious complications such as neurovascular injury, deep venous thrombosis, or infection. CONCLUSION: For unicameral popliteal cysts, arthroscopic internal drainage combined with resection of the cyst wall did not further improve the clinical outcomes or reduce the recurrence rate, while prolonging the operation time and increasing the possibility of complications.

Soluplus-Mediated Diosgenin Amorphous Solid Dispersion with High Solubility and High Stability: Development, Characterization and Oral Bioavailability.

BACKGROUND AND PURPOSE: The traditional Chinese medicine, diosgenin (Dio), has attracted increasing attention because it possesses various therapeutic effects, including anti-tumor, anti-infective and anti-allergic properties. However, the commercial application of Dio is limited by its extremely low aqueous solubility and inferior bioavailability in vivo. Soluplus, a novel excipient, has great solubilization and capacity of crystallization inhibition. The purpose of this study was to prepare Soluplus-mediated Dio amorphous solid dispersions (ASDs) to improve its solubility, bioavailability and stability. METHODS: The crystallization inhibition studies were firstly carried out to select excipients using a solvent shift method. According to solubility and dissolution results, the preparation methods and the ratios of drug to excipient were further optimized. The interaction between Dio and Soluplus was characterized by differential scanning calorimetry (DSC), fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), powder X-ray diffraction (PXRD) and molecular docking. The pharmacokinetic study was conducted to explore the potential of Dio ASDs for oral administration. Furthermore, the long-term stability of Dio ASDs was also investigated. RESULTS: Soluplus was preliminarily selected from various excipients because of its potential to improve solubility and stability. The optimized ASDs significantly improved the aqueous solubility of Dio due to its amorphization and the molecular interactions between Dio and Soluplus, as evidenced by dissolution test in vitro, DSC, FT-IR spectroscopy, SEM, PXRD and molecular docking technique. Furthermore, pharmacokinetic studies in rats revealed that the bioavailability of Dio from ASDs was improved about 5 times. In addition, Dio ASDs were stable when stored at 40°C and 75% humidity for 6 months. CONCLUSION: These results indicated that Dio ASDs, with its high solubility, high bioavailability and high stability, would open a promising way in pharmaceutical applications.

One-Stage Repair of Posterior Oblique Ligament Avulsion Fracture Combined with Medial Collateral Ligament Injury.

OBJECTIVE: To evaluate the clinical effect of the one-stage repair of a posterior oblique ligament avulsion fracture combined with a medial collateral ligament injury. METHODS: This study was a retrospective trial. From February 2007 to May 2017, five patients with posterior oblique ligament avulsion fracture combined with medial collateral ligament injury were included in this study. The patients were aged 37-58 years old with a mean of 45.2 years. All patients underwent the primary repair of a posterior oblique ligament avulsion fracture and medial collateral ligament injury. The main observational index included Lysholm score, International Knee Documentation Committee (IKDC) score, Visual Analogue Scale (VAS) score, and range of motion (ROM). RESULTS: The results showed that the average time of follow-up was 53.6 months (range, 20-86 months). When compared to preoperative scores, the preoperative Lysholm score was significantly increased (47.8 ± 5.1 vs 95.0 ± 3.7, P < 0.05), the IKDC score was significantly increased (51.2 ± 5.6 vs 88.6 ± 4.2, P < 0.05), the VAS score was significantly decreased (7.0 ± 0.7 vs 0.4 ± 0.5, P < 0.05), and the ROM was significantly increased (91.6° ± 8.4° vs 129.9° ± 4.4°, P < 0.05). CONCLUSION: Our study found that with the combination of the one-stage repair of a posterior oblique ligament (POL) avulsion fracture and medial collateral ligament injury, the patient's postoperative function recovered well, their pain was relieved, and their knee joint stability was reliable.

Estrogen Stimulation of Pleiotrophin Enhances Osteoblast Differentiation and Maintains Bone Mass in IGFBP-2 Null Mice.

Insulin-like growth factor binding protein-2 (IGFBP-2) stimulates osteoblast differentiation but only male Igfbp2 null mice have a skeletal phenotype. The trophic actions of IGFBP-2 in bone are mediated through its binding to receptor tyrosine phosphatase beta (RPTPß). Another important ligand for RPTPß is pleiotrophin (PTN), which also stimulates osteoblast differentiation. We determined the change in PTN and RPTPß in Igfbp2-/- mice. Analysis of whole bone mRNA in wild-type and knockout mice revealed increased expression of Ptn. Rptpß increased in gene-deleted animals with females having greater expression than males. Knockdown of PTN expression in osteoblasts in vitro inhibited differentiation, and addition of PTN to the incubation medium rescued the response. Estradiol stimulated PTN secretion and PTN knockdown blocked estradiol-stimulated differentiation. PTN addition to IGFBP-2 silenced osteoblast stimulated differentiation, and an anti-fibronectin-3 antibody, which inhibits PTN binding to RPTPß, inhibited this response. Estrogen stimulated PTN secretion and downstream signaling in the IGFBP-2 silenced osteoblasts and these effects were inhibited with anti-fibronectin-3. Administration of estrogen to wild-type and Igfbp2-/- male mice stimulated an increase in both areal bone mineral density and trabecular bone volume fraction but the increase was significantly greater in the Igfbp2-/- animals. Estrogen also stimulated RPTPß expression in the null mice. We conclude that loss of IGFBP-2 expression is accompanied by upregulation of PTN and RPTPß expression in osteoblasts, that the degree of increase is greater in females due to estrogen secretion, and that this compensatory change may account for some component of the maintenance of normal bone mass in female mice.

Overexpression of interleukin-20 receptor subunit beta (IL20RB) correlates with cell proliferation, invasion and migration enhancement and poor prognosis in papillary renal cell carcinoma.

Papillary renal cell carcinoma (PRCC) accounts for about 10 percent of all renal cell carcinomas, and the prognosis is poor for people with advanced disease. Interleukin-20 receptor subunit beta (IL20RB) is a single-pass type I membrane protein of the type II cytokine receptor family and is related to the pathogenesis of chronic inflammation and autoimmune diseases, including psoriasis, glaucoma, vitiligo, rheumatoid arthritis, and inflammatory bowel disease. However, little has been reported on IL20RB with respect to cancer, especially in PRCC. Thus, we performed this study to explore its biological characteristics in PRCC. Data from the TCGA database were used to analyze the expression and prognosis of IL20RB. qRT-PCR was used to detect the expression of IL20RB in PRCC cells in vitro. After knockdown of IL20RB with small interfering RNA (siRNA) technology, the proliferation, migration, and invasion of Ketr-3 cells and the expression of related proteins in the epithelial-mesenchymal transition (EMT) pathway were measured with Cell Counting Kit-8 (CCK-8), transwell, and western blot assays. The findings demonstrated that the expression of IL20RB was upregulated in both PRCC tissues and cells and that the high expression of IL20RB led to low overall survival (OS). Furthermore, after knockdown of IL20RB in vitro, the proliferation, migration, and invasion of Ketr-3 cells were reduced, and the expression of related proteins in the EMT pathway declined, suggesting that IL20RB plays a vital role in PRCC through the EMT pathway. These results reveal the biological significance of IL20RB in PRCC and provide new insight for future targeted drugs.

Inhibition of Aberrant IGF-I Signaling in Diabetic Male Rat Retina Prevents and Reverses Changes of Diabetic Retinopathy.

Hyperglycemia results in inhibition of cleavage of integrin-associated protein (IAP) thereby allowing it to bind to SHPS-1 which results in pathophysiologic changes in endothelial function. This study determined if an anti-rat IAP antibody directed against the SHPS-1 binding site which disrupts IAP/SHPS-1 association could inhibit these pathophysiologic changes. The anti-IAP antibody inhibited IGF-I-stimulated SHPS-1, p52Shc, MAP kinase phosphorylation, and proliferation in endothelial cells. To determine if it could reverse established pathophysiologic changes in vivo, this antibody or normal rat IgG F(ab)2 was injected intraperitoneally for 6 weeks into rats that had diabetes for 4 weeks. Optical coherence tomography (OCT) showed that retinal thickness increased at 4 weeks and this increase was maintained in rats treated with the control antibody for an additional 6 weeks. The increase was reversed by anti-IAP antibody treatment (84.6 ± 2.0 compared to 92.3 ± 2.5 µm, p < 0.01). This value was similar to nondiabetic animals (82.2 ± 1.6 µm, p, NS). The anti-IAP antibody also decreased retinal vascular permeability (0.62 ± 0.12 vs. 0.96 ± 0.25%/g/h, p < 0.001). To determine if it was effective after local injection, this antibody or control was administered via intravitreal injection. After 3 weeks, retinal thickness increased to 6.4 ± 2.8% in diabetic rats, and IAP antibody treatment prevented this increase (0.8 ± 2.5%, p < 0.01). It also prevented the increase of retinal vascular permeability (0.92 ± 0.62 vs. 1.63 ± 0.99%/g/h, p < 0.001). Biochemical analyses of retinal extracts showed that the anti-IAP antibody inhibited IAP/SHPS-1 association and SHPS-1 phosphorylation. This resulted in inhibition of AKT activation and VEGF synthesis in the retina: changes associated with increased vascular permeability. We conclude the anti-rat IAP antibody disrupts IAP/SHPS-1 association and attenuates aberrant IGF-I signaling thereby preventing or reversing the progression of retinal pathophysiological changes.

Hyperglycemia induces vascular smooth muscle cell dedifferentiation by suppressing insulin receptor substrate-1-mediated p53/KLF4 complex stabilization.

Hyperglycemia and insulin resistance accelerate atherosclerosis by an unclear mechanism. The two factors down-regulate insulin receptor substrate-1 (IRS-1), an intermediary of the insulin/IGF-I signaling system. We previously reported that IRS-1 down-regulation leads to vascular smooth muscle cell (VSMC) dedifferentiation and that IRS-1 deletion from VSMCs in normoglycemic mice replicates this response. However, we did not determine IRS-1's role in mediating differentiation. Here, we sought to define the mechanism by which IRS-1 maintains VSMC differentiation. High glucose or IRS-1 knockdown decreased p53 levels by enhancing MDM2 proto-oncogene (MDM2)-mediated ubiquitination, resulting in decreased binding of p53 to Krüppel-like factor 4 (KLF4). Exposure to nutlin-3, which dissociates MDM2/p53, decreased p53 ubiquitination and enhanced the p53/KLF4 association and differentiation marker protein expression. IRS-1 overexpression in high glucose inhibited the MDM2/p53 association, leading to increased p53 and p53/KLF4 levels, thereby increasing differentiation. Nutlin-3 treatment of diabetic or Irs1-/- mice enhanced p53/KLF4 and the expression of p21, smooth muscle protein 22 (SM22), and myocardin and inhibited aortic VSMC proliferation. Injecting normoglycemic mice with a peptide disrupting the IRS-1/p53 association reduced p53, p53/KLF4, and differentiation. Analyzing atherosclerotic lesions in hypercholesterolemic, diabetic pigs, we found that p53, IRS-1, SM22, myocardin, and KLF4/p53 levels are significantly decreased compared with their expression in nondiabetic pigs. We conclude that IRS-1 is critical for maintaining VSMC differentiation. Hyperglycemia- or insulin resistance-induced IRS-1 down-regulation decreases the p53/KLF4 association and enhances dedifferentiation and proliferation. Our results suggest that enhancing IRS-1-dependent p53 stabilization could attenuate the progression of atherosclerotic lesions in hyperglycemia and insulin-resistance states.

A peptide containing the receptor binding site of insulin-like growth factor binding protein-2 enhances bone mass in ovariectomized rats.

Male Igfbp2-/- mice have a significant reduction in bone mass and administration of a peptide that contains the insulin-like growth factor binding protein-2(IGFBP-2) receptor-binding domain stimulates bone formation in these animals. Female Igfbp2-/- mice do not have this phenotype but following ovariectomy (OVX) lose more bone than OVX wild-type mice. This suggests that in the absence of estrogen, IGFBP-2 is required to maintain bone mass. Therefore these studies were undertaken to determine if this peptide could stimulate bone acquisition in OVX rats. OVX rats were divided into seven treatment groups: sham animals, OVX animals, OVX animals receiving a control scrambled peptide, or one of three doses of the active peptide termed PEG-HBD-1 (0.7, 2, and 6 mg·kg-1) and an OVX group receiving parathyroid hormone (PTH) (50 µg·kg-1 per day). The peptides were administered for 8 weeks. DXA revealed a significant reduction in femoral and tibial areal bone mineral density (aBMD) after OVX, whereas treatment with the high-dose peptide increased aBMD by 6.2% ± 2.4% (P < 0.01) compared to control peptide; similar to the increase noted with PTH (5.6% ± 3.0%, P < 0.01). Similar increases were noted with two lower doses of the peptide (3.8% ± 1.5%, P < 0.05 for low dose; 3.1% ± 1.6%, P = 0.07 for middle dose). Micro CT showed that the OVX control peptide animals had reductions of 41% and 64% in femoral trabecular BV/TV and trabecular number, respectively. All three doses of the peptide increased bone volume/total volume (BV/TV) significantly, while the low and middle doses increased trabecular number. Cortical BV/TV and thickness at the midshaft increased significantly with each dose of peptide (18.9% ± 9.8%, P < 0.01 and 14.2% ± 7.9%, P < 0.01 for low dose; 23.7% ± 10.7%, P < 0.001 and 15.8% ± 6.1%, P < 0.001 for middle dose; 19.0% ± 6.9%, P < 0.01 and 16.2% ± 9.7%, P < 0.001 for high dose) and with PTH (25.8% ± 9.2%, P < 0.001 and 19.4% ± 8.8%, P < 0.001). Histomorphometry showed that the lowest dose of peptide stimulated BV/TV, trabecular thickness, mineral apposition rate (MAR), bone formation rate/bone surface (BFR/BS), number of osteoblasts/bone perimeter (N.ob/B.pm), and decreased osteoclast surface/bone perimeter (Oc.S/B.Pm). The highest dose stimulated each of these parameters except MAR and BFR/BS. Thus, the heparin-binding domain receptor region of IGFBP-2 accounts for its anabolic activity in bone. Importantly, this peptide enhances bone mass in estrogen-deficient animals.

Effects of long noncoding RNA-ROR on tamoxifen resistance of breast cancer cells by regulating microRNA-205.

PURPOSE: To explore how long noncoding RNA-ROR (lncRNA-ROR) affects the tamoxifen resistance of breast cancer cells. METHODS: Breast epithelial (MCF10A), breast cancer (MCF7), and natural tamoxifen-resistant breast cancer (MDA-MB-231) cell lines were selected, and the relative lncRNA-ROR expressions were detected using quantitative real-time polymerase chain reaction (qRT-PCR). In vitro induction of TR5 cell line was performed. There were six groups: MCF7, MCF7/TR5, MDA-MB-231, MCF7-ROR, MCF7/TR5 ROR-siRNA, and the MDA-MB-231 ROR-siRNA groups. CCK-8 assay was conducted to assess the changes in drug resistance of each group. The expression of the epithelial mesenchymal transition (EMT) marker was detected using Western blotting. Transwell was selected to test the invasive ability of the cells. Expressions of microRNA-205 (miR-205) and ZEB1/2 were detected using qRT-PCR . RESULTS: Compared with the MCF7 cells, the proliferation rates of the MCF7/TR5 and MDA-MB-231 cells were significantly increased. Compared with the MCF7 cells, the MCF7-ROR cells had remarkably higher proliferation rates, down-regulated E-cadherin and miR-205 expressions, as well as increased vimentin, invasive ability, and mRNA expression of ZEB1 and ZEB2. Compared with the MCF7/TR5 and MDA-MB-231 cells, up-regulated E-cadherin and miR-205 expression, down-regulated expression of vimentin, ZEB1, and ZEB2 mRNA, and decreased invasive ability were observed in the MCF7/TR5 ROR-siRNA and MDA-MB-231 ROR-siRNA cells. CONCLUSION: In MDA-MB-231 cells, down-regulated lncRNA-ROR could inhibit the EMT of breast cancer cells and enhance the sensibility of breast cancer cells to tamoxifen by increasing miR-205 expression and suppressing the expressions of ZEB1 and ZEB2.

In Vitro Effects of HAS-2 Gene Silencing on the Proliferation and Apoptosis of the MCF-7 Human Breast Cancer Cell Line.

BACKGROUND: Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. This study is aimed to investigate the effects of silencing the HAS-2 gene on the proliferation and apoptosis of human breast cancer cells. METHODS: MCF-7 cells were collected and assigned into control, scrambled siRNA and HAS-2- siRNA groups. After transfection, the morphological changes in the MCF-7 cells were observed using phase contrast microscopy. qRT-PCR and Western blot assays were used to detect the mRNA and protein expression of apoptosis-related proteins. CCK-8 and flow cytometry were performed to evaluate cell proliferation, the cell cycle and apoptosis. RESULTS: In the control and the scrambled siRNA groups, cells grew adhered to the wall and mainly showed a spindle shape with a clear nucleolus. Compared with the control and scrambled siRNA groups, increases in the number of cells in early apoptosis and metaphase cells in apoptosis were observed in the HAS-2-siRNA group. The HAS-2-siRNA group showed decreased expression of HAS-2 relative to that in the control and scrambled siRNA groups. No significant differences in cell proliferation, cell cycle distribution or apoptosis were noted between the control and scrambled siRNA groups. In the HAS-2-siRNA group, the cell proliferation ability decreased significantly, but the number of cells in the G0/G1 stage, the number of apoptotic cells and the expression of caspase-3 and caspase-9 increased significantly. CONCLUSION: Our findings indicate that HAS-2 gene silencing may inhibit proliferation and promote apoptosis in the MCF-7 human breast cancer cell line.

IRS-1 Functions as a Molecular Scaffold to Coordinate IGF-I/IGFBP-2 Signaling During Osteoblast Differentiation.

Insulin like growth factor I (IGF-I) and insulin like growth factor binding protein-2 (IGFBP-2) function coordinately to stimulate AKT and osteoblast differentiation. IGFBP-2 binding to receptor protein tyrosine phosphatase ß (RPTPß) stimulates polymerization and inactivation of phosphatase activity. Because phosphatase and tensin homolog (PTEN) is the primary target of RPTPß, this leads to enhanced PTEN tyrosine phosphorylation and inactivation. However RPTPß inactivation also requires IGF-I receptor activation. The current studies were undertaken to determine the mechanism by which IGF-I mediates changes in RPTPß function in osteoblasts. IGFBP-2/IGF-I stimulated vimentin binding to RPTPß and this was required for RPTPß polymerization. Vimentin serine phosphorylation mediated its binding to RPTPß and PKCζ was identified as the kinase that phosphorylated vimentin. To determine the mechanism underlying IGF-I stimulation of PKCζ-mediated vimentin phosphorylation, we focused on insulin receptor substrate-1 (IRS-1). IGF-I stimulated IRS-1 phosphorylation and recruitment of PKCζ and vimentin to phospho-IRS-1. IRS-1 immunoprecipitates containing PKCζ and vimentin were used to confirm that activated PKCζ directly phosphorylated vimentin. PKCζ does not contain a SH-2 domain that is required to bind to phospho-IRS-1. To determine the mechanism of PKCζ recruitment we analyzed the role of p62 (a PKCζ binding protein) that contains a SH2 domain. Exposure to differentiation medium plus IGF-I stimulated PKCζ/p62 association. Subsequent analysis showed the p62/PKCζ complex was co-recruited to IRS-1. Peptides that disrupted p62/PKCζ or p62/IRS-1 inhibited IGF-I/IGFBP-2 stimulated PKCζ activation, vimentin phosphorylation, PTEN tyrosine phosphorylation, AKT activation, and osteoblast differentiation. The importance of these signaling events for differentiation was confirmed in primary mouse calvarial osteoblasts. These results demonstrate the cooperative interaction between RPTPß and the IGF-I receptor leading to a coordinated series of signaling events that are required for osteoblast differentiation. Our findings emphasize the important role IRS-1 plays in modulating these signaling events and confirm its essential role in facilitating osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.

IGFBP-2 directly stimulates osteoblast differentiation.

Insulin-like growth factor binding protein 2 (IGFBP-2) is important for acquisition of normal bone mass in mice; however, the mechanism by which IGFBP-2 functions is not defined. These studies investigated the role of IGFBP-2 in stimulating osteoblast differentiation. MC-3T3 preosteoblasts expressed IGFBP-2, and IGFBP-2 knockdown resulted in a substantial delay in osteoblast differentiation, reduced osteocalcin expression and Alizarin red staining. These findings were replicated in primary calvarial osteoblasts obtained from IGFBP-2(-/-) mice, and addition of IGFBP-2 rescued the differentiation program. In contrast, overexpression of IGFBP-2 accelerated the time course of differentiation as well as increasing the total number of differentiating cells. By day 6, IGFBP-2-overexpressing cells expressed twice as much osteocalcin as control cultures and this difference persisted. To determine the mechanism by which IGFBP-2 functions, the interaction between IGFBP-2 and receptor tyrosine phosphatase ß (RPTPß) was examined. Disruption of this interaction inhibited the ability of IGFBP-2 to stimulate AKT activation and osteoblast differentiation. Knockdown of RPTPß enhanced osteoblast differentiation, whereas overexpression of RPTPß was inhibitory. Adding back IGFBP-2 to RPTPß-overexpressing cells was able to rescue cell differentiation via enhancement of AKT activation. To determine the region of IGFBP-2 that mediated this effect, an IGFBP-2 mutant that contained substitutions of key amino acids in the heparin-binding domain-1 (HBD-1) was prepared. This mutant had a major reduction in its ability to stimulate differentiation of calvarial osteoblasts from IGFBP-2(-/-) mice. Addition of a synthetic peptide that contained the HBD-1 sequence to calvarial osteoblasts from IGFBP-2(-/-) mice rescued differentiation and osteocalcin expression. In summary, the results clearly demonstrate that IGFBP-2 stimulates osteoblast differentiation and that this effect is mediated through its heparin-binding domain-1 interacting with RPTPß. The results suggest that stimulation of differentiation is an important mechanism by which IGFBP-2 regulates the acquisition of normal bone mass in mice.