Targeted inhibition of SCFSKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma.

Osteosarcoma(OS) is a highly aggressive bone cancer for which treatment has remained essentially unchanged for decades. Although OS is characterized by extensive genomic heterogeneity and instability, RB1 and TP53 have been shown to be the most commonly inactivated tumor suppressors in OS. We previously generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which largely recapitulates human OS with nearly complete penetrance. SKP2 is a repression target of pRb and serves as a substrate recruiting subunit of the SCFSKP2 complex. In addition, SKP2 plays a central role in regulating the cell cycle by ubiquitinating and promoting the degradation of p27. We previously reported the DKOAA transgenic model, which harbored a knock-in mutation in p27 that impaired its binding to SKP2. Here, we generated a novel p53-Rb1-SKP2 triple-knockout model (TKO) to examine SKP2 function and its potential as a therapeutic target in OS. First, we observed that OS tumorigenesis was significantly delayed in TKO mice and their overall survival was markedly improved. In addition, the loss of SKP2 also promoted an apoptotic microenvironment and reduced the stemness of DKO tumors. Furthermore, we found that small-molecule inhibitors of SKP2 exhibited anti-tumor activities in vivo and in OS organoids as well as synergistic effects when combined with a standard chemotherapeutic agent. Taken together, our results suggest that SKP2 inhibitors may reduce the stemness plasticity of OS and should be leveraged as next-generation adjuvants in this cancer.

SKP2 Knockout in Rb1/p53-Deficient Mouse Models of Osteosarcoma Induces Immune Infiltration and Drives a Transcriptional Program with a Favorable Prognosis.

Osteosarcoma is an aggressive bone malignancy with a poor prognosis. One putative proto-oncogene in osteosarcoma is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase. We previously demonstrated that Skp2 knockout in murine osteosarcoma improved survival and delayed tumorigenesis. Here, we performed RNA sequencing (RNA-seq) on tumors from a transgenic osteosarcoma mouse model with conditional Trp53 and Rb1 knockouts in the osteoblast lineage ("DKO": Osx1-Cre;Rb1lox/lox;p53lox/lox) and a triple-knockout model with additional Skp2 germline knockout ("TKO": Osx1-Cre;Rb1lox/lox;p53lox/lox;Skp2-/-), followed by qPCR and immunohistochemistry validation. To investigate the clinical implications of our results, we analyzed a human osteosarcoma patient cohort ("NCI-TARGET OS") with RNA-seq and clinical data. We found large differences in gene expression after SKP2 knockout. Surprisingly, we observed increased expression of genes related to immune microenvironment infiltration in TKO tumors, especially the signature genes for macrophages and to a lesser extent, T cells, B cells, and vascular cells. We also uncovered a set of relevant transcription factors that may mediate these changes. In osteosarcoma patient cohorts, high expression of genes upregulated in TKO was correlated with favorable overall survival, which was largely explained by the macrophage gene signatures. This relationship was further supported by our finding that SKP2 expression was negatively correlated with macrophage infiltration in the NCI-TARGET osteosarcoma and the TCGA Sarcoma cohorts. Overall, our findings indicate that SKP2 may mediate immune exclusion from the osteosarcoma tumor microenvironment, suggesting that SKP2 modulation in osteosarcoma may induce antitumor immune activation.

Fluoroscopic Percutaneous Fixation of Periacetabular Metastatic Cancer: The Tripod Technique.

There are an increasing number of patients who present with metastatic bone disease as the survival of patients with cancer improves in recent decades. The pelvis is the second most common site for skeletal metastases. Metastatic lesions in the pelvis can be largely divided into periacetabular lesions (Enneking zone II) and non-periacetabular lesions (zones I, III, and IV). Traditionally, patients with a symptomatic zone II lesion are treated with a cemented total hip arthroplasty (THA) using variations on the traditional Harrington method. These open surgeries are accompanied by many inherent risks. Both a prolonged recovery and wide range of potential complications may delay or interrupt the adjuvant radiation and systemic therapy. It was observed that the articular surface of the hip joint was often intact and that the femoral side was frequently not involved in these patients. A novel minimally invasive technique for hip joint preservation has recently been developed. Three large-bore cannulated screws are placed percutaneously under fluoroscopy in a tripod configuration to reinforce the mechanical axis of the acetabulum. Increased stability improves pain control and permits immediate weight bearing. When the disease progresses, this construct can be easily converted to a cemented THA using the tripod screws as rebar to support an acetabular cup, as part of a staged Harrington procedure. This approach is technically demanding. A detailed guide for the tripod technique should encompass indications, preoperative preparation, operating room settings, intraoperative fluoroscopic guidance, modifications, postoperative care, and subsequent conversion to a cemented THA, if needed.

Post-traumatic osteoarthritis progression is diminished by early mechanical unloading and anti-inflammatory treatment in mice.

OBJECTIVE: Post-traumatic osteoarthritis (PTOA) is a degenerative joint disease initiated by injury. Early phase (0-7 days) treatments often include rest (unloading) and anti-inflammatory medications, but how those early interventions impact PTOA progression is unknown. We hypothesized that early unloading and anti-inflammatory treatment would diminish joint inflammation and slow PTOA progression. DESIGN: Mice were injured with non-invasive ACL rupture followed by hindlimb unloading (HLU) or normal cage activity (ground control: GC) for 7 days, after which all mice were allowed normal cage activity. HLU and GC mice were treated with daily celecoxib (CXB; 10 mg/kg IP) or vehicle. Protease activity was evaluated using in vivo fluorescence imaging, osteophyte formation and epiphyseal trabecular bone were quantified using micro-computed tomography, and synovitis and articular cartilage were evaluated using whole-joint histology at 7, 14, 21, and 28 days post-injury. RESULTS: HLU significantly reduced protease activity (-22-30% compared to GC) and synovitis (-24-50% relative to GC) at day 7 post-injury (during unloading), but these differences were not maintained at later timepoints. Similarly, trabecular bone volume was partially preserved in HLU mice at during unloading (-14-15% BV/TV for HLU mice, -21-22% for GC mice relative to uninjured), but these differences were not maintained during reloading. Osteophyte volume was reduced by both HLU and CXB, but there was not an additive effect of these treatments (HLU: -46%, CXB: -30%, HLU + CXB: -35% relative to vehicle GC at day 28). CONCLUSIONS: These data suggest that early unloading following joint injury can reduce inflammation and potentially slow PTOA progression.

Prognostic and Therapeutic Utility of Variably Expressed Cell Surface Receptors in Osteosarcoma.

BACKGROUND: Six cell surface receptors, human epidermal growth factor receptor-2 (Her-2), platelet-derived growth factor receptor-ß (PDGFR-ß), insulin-like growth factor-1 receptor (IGF-1R), insulin receptor (IR), c-Met, and vascular endothelial growth factor receptor-3 (VEGFR-3), previously demonstrated variable expression across varying patient-derived and standard osteosarcoma (OS) cell lines. The current study sought to validate previous expression patterns and evaluate whether these receptors offer prognostic and/or therapeutic value. METHODS: Patient-derived OS cell lines (n = 52) were labeled with antibodies to Her-2, PDGFR-ß, IGF-1R, IR, c-Met, and VEGFR-3. Expression was characterized using flow cytometry. The difference in geometric mean fluorescent intensity (geoMFIdiff = geoMFIpositive - geoMFInegative) was calculated for each receptor across all cell lines. Receptor expression was categorized as low (Q1), intermediate (Q2, Q3), or high (Q4). The event-free survival (EFS) and overall survival for the six cell surface receptors were estimated by the Kaplan-Meier method. Differences in hazard for EFS event and overall survival event for patients in each of the three expression levels in each of the six cell surface receptors were assessed using the log-rank test. RESULTS: All 6 receptors were variably expressed in the majority of cell lines. IR and PDGFR-ß expressions were found to be significant predictors for EFS amongst patients with nonmetastatic disease (p=0.02 and 0.01, respectively). The hazard ratio for EFS was significantly higher between high IR and intermediate IR expression (HR = 2.66, p=0.02), as well as between high PDGFR-ß and intermediate PDGFR-ß expression (HR = 5.68, p=0.002). Her-2, c-Met, IGF-1R, and VEGFR-3 were not found to be significant predictors for either EFS or overall survival. CONCLUSION: The six cell surface receptors demonstrated variable expression across the majority of patient-derived OS cell lines tested. Limited prognostic value was offered by IR and PDGFR-ß expression within nonmetastatic patients. The remaining receptors do not provide clear prognostic utility. Nevertheless, their consistent, albeit variable, surface expression across a large panel of patient-derived OS cell lines maintains their potential use as future therapeutic targets.

The interaction of SKP2 with p27 enhances the progression and stemness of osteosarcoma.

Osteosarcoma is a highly aggressive malignancy for which treatment has remained essentially unchanged for years. Our previous studies found that the F-box protein SKP2 is overexpressed in osteosarcoma, acting as a proto-oncogene; p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases and a downstream substrate of SKP2-mediated ubiquitination. Overexpression of SKP2 and underexpression of p27 are common characteristics of cancer cells. The SCFSKP2 E3 ligase ubiquitinates Thr187-phosphorylated p27 for proteasome degradation, which can be abolished by a Thr187Ala knock-in (p27T187A KI) mutation. RB1 and TP53 are two major tumor suppressors commonly coinactivated in osteosarcoma. We generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which developed osteosarcoma with full penetrance. When p27T187A KI mice were crossed on to the DKO background, p27T187A protein was found to accumulate in osteosarcoma tumor tissues. Furthermore, p27T187A promoted apoptosis in DKO tumors, slowed disease progression, and significantly prolonged overall survival. RNA sequencing analysis also linked the SCFSKP2 -p27T187A axis to potentially reduced cancer stemness. Given that RB1 and TP53 loss or coinactivation is common in human osteosarcoma, our study suggests that inhibiting the SKP2-p27 axis may represent a desirable therapeutic strategy for this cancer.

Keeping Each Other Accountable: Social Strategies for Smoking Cessation and Healthy Living in Vietnamese American Men.

Vietnamese American males have high smoking rates. This study explored social support mechanisms provided by lay health workers (LHWs) and family members through a smoking cessation intervention. Eight focus groups (N = 54) were conducted in Vietnamese stratified by intervention arms (Tobacco [experimental] and healthy living [control]) with 18 smokers, 18 family members, and 18 LHWs. Smokers reported feeling more accountable for their health behaviors, and smoking changes were reinforced by family members, peers, and LHWs through conversations facilitated during and outside the program. Culturally appropriate interventions with multiple social support mechanisms may reduce smoking in minority populations.

Wnt Signaling in Osteosarcoma.

Wnt molecules are a class of cysteine-rich secreted glycoproteins that participate in various developmental events during embryogenesis and adult tissue homeostasis. Since its discovery in 1982, the roles of Wnt signaling have been established in various key regulatory systems in biology. Wnt signals exert pleiotropic effects, including mitogenic stimulation, cell fate specification, and differentiation. The Wnt signaling pathway in humans has been shown to be involved in a wide variety of disorders including colon cancer, sarcoma, coronary artery disease, tetra-amelia, Mullerian duct regression, eye vascular defects, and abnormal bone mass. The canonical Wnt pathway functions by regulating the function of the transcriptional coactivator ß-catenin, whereas noncanonical pathways function independent of ß-catenin. Although the role of Wnt signaling is well established in epithelial malignancies, its role in mesenchymal tumors is more controversial. Some studies have suggested that Wnt signaling plays a pro-oncogenic role in various sarcomas by driving cell proliferation and motility; however, others have reported that Wnt signaling acts as a tumor suppressor by committing tumor cells to differentiate into a mature lineage. Wnt signaling pathway also plays an important role in regulating cancer stem cell function. In this review, we will discuss Wnt signaling pathway and its role in osteosarcoma.

Characterization of bony anatomic regions in pediatric and adult healthy volunteers using diffuse optical spectroscopic imaging.

SIGNIFICANCE: Diffuse optical spectroscopic imaging (DOSI) measures quantitative functional and molecular information in thick tissue in a noninvasive manner using near-infrared light. DOSI may be useful for diagnosis and prognosis of bone pathologies including osteosarcoma and Ewing's sarcoma, but little is currently known about DOSI-derived parameters in bony anatomic locations where this disease occurs. AIM: Our goal is to quantify the optical characteristics and chromophore content of bony anatomic locations of healthy volunteers and assess differences due to anatomic region, age, sex, ethnicity, race, and body fat. APPROACH: Fifty-five healthy volunteers aged 4 to 72 were enrolled in the study. The optical properties and quantitative tissue concentrations of oxyhemoglobin, deoxyhemoglobin, water, and lipids were assessed at the distal humerus, distal femur, and proximal tibia. Body fat was assessed using skinfold calipers. One volunteer underwent a more comprehensive body scan from neck to foot to explore chromophore distributions within an individual. Regression analysis was used to identify the most important sources of variation in the measured data set. RESULTS: Statistical differences between bony locations were found for scattering, water, and lipids, but not for hemoglobin. All chromophores had statistical differences with sex, but there were no significant age-related correlations. Regression analysis revealed that body fat measured with skinfold calipers was the most important predictor of oxy-, deoxy-, total hemoglobin, and lipids. Hemoglobin and lipid levels were highly correlated (ρ ≥ 0.7) over the subject population and within the single-subject body scan. CONCLUSIONS: DOSI can successfully measure bony anatomic sites where osteosarcomas and Ewing's sarcomas commonly occur. Future studies of bone pathology using DOSI should account for the variation caused by anatomic region, sex, race and ethnicity, and body fat as these cause substantial variations in DOSI-derived metrics.

Skp2 depletion reduces tumor-initiating properties and promotes apoptosis in synovial sarcoma.

Synovial sarcoma (SS) is an aggressive soft-tissue cancer with a poor prognosis and a propensity for local recurrence and distant metastasis. In this study, we investigated whether S phase kinase-associated protein (Skp2) plays an oncogenic role in tumor initiation, progression, and metastasis of SS. Our study revealed that Skp2 is frequently overexpressed in SS specimens and SS18-SSX transgenic mouse tumors, as well as correlated with clinical stages. Next, we identified that genetic depletion of Skp2 reduced mesenchymal and stemness markers, and inhibited the invasive and proliferative capacities of SS cell lines. Furthermore, Skp2 depletion markedly suppressed the growth of SS xenografts tumors. Treatment of SS cell lines with the skp2 inhibitor flavokawain A (FKA) reduced Skp2 expression in a dose-dependent manner and resulted in cell cycle arrest and apoptosis. FKA also suppressed the invasion and tumor-initiating properties in SS, similar to the effects of Skp2 knockdown. In addition, a combination of FKA and conventional chemotherapy showed a synergistic therapeutic efficacy. Taken together, our results suggest that Skp2 plays an essential role in the biology of SS by promoting the mesenchymal state and cancer stemness. Given that chemotherapy resistance is often associated with cancer stemness, strategies of combining Skp2 inhibitors with conventional chemotherapy in SS may be desirable.

A Cluster Randomized Controlled Trial of a Lay Health Worker Intervention to Increase Healthy Eating and Physical Activity Among Vietnamese Americans.

INTRODUCTION: Americans have low levels of knowledge of and adherence to recommendations for healthy eating of fruits and vegetables and for physical activity (HEPA). We conducted a cluster randomized controlled trial of a lay health worker intervention to increase HEPA among Vietnamese Americans. METHODS: We randomized 64 lay health workers to 2 intervention arms. Each lay health worker recruited 10 participants aged 50 to 74. From 2008 to 2013, using flip charts, lay health workers led 2 educational sessions on HEPA (intervention) or colorectal cancer (comparison). We assessed HEPA knowledge and self-reported behaviors by preintervention and postintervention surveys 6 months apart. RESULTS: Of the 640 participants, 50.0% were female, 38.4% had lived in the United States for 10 years or fewer, and 71.4% reported limited English proficiency. Knowledge of the recommended intake of fruits and vegetables (≥5 servings daily) increased from 2.6% to 60.5% in the intervention group (n = 311) and from 2.9% to 6.7% in the comparison group (n = 316) (intervention vs comparison change, P < .001). Knowledge of the physical activity recommendation (≥150 minutes weekly) increased from 2.6% to 62.4% among intervention participants and from 1.0% to 2.5% among comparison participants (P < .001). Consumption of 5 or more daily servings of fruits and vegetables increased more in the intervention group (8.4% to 62.1%) than in the comparison group (5.1% to 12.7%) (P < .001). Participants reporting 150 minutes or more of physical activity weekly increased from 28.9% to 54.0% in the intervention group and from 38.0% to 46.8% in the comparison group (intervention vs comparison change, P = .001). CONCLUSION: A lay health worker intervention increased both healthy eating and physical activity knowledge and self-reported behaviors among older Vietnamese Americans.

Antibodies and B cells recognising citrullinated proteins display a broad cross-reactivity towards other post-translational modifications.

OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.

Overdispersion models for correlated multinomial data: Applications to blinding assessment.

Overdispersion models have been extensively studied for correlated normal and binomial data but much less so for correlated multinomial data. In this work, we describe a multinomial overdispersion model that leads to the specification of the first two moments of the outcome and allows the estimation of the global parameters using generalized estimating equations (GEE). We introduce a Global Blinding Index as a target parameter and illustrate the application of the GEE method to its estimation from (1) a clinical trial with clustering by practitioner and (2) a meta-analysis on psychiatric disorders. We examine the impact of a small number of clusters, high variability in cluster sizes, and the magnitude of the intraclass correlation on the performance of the GEE estimators of the Global Blinding Index using the data simulated from different models. We compare these estimators with the inverse-variance weighted estimators and a maximum-likelihood estimator, derived under the Dirichlet-multinomial model. Our results indicate that the performance of the GEE estimators was satisfactory even in situations with a small number of clusters, whereas the inverse-variance weighted estimators performed poorly, especially for larger values of the intraclass correlation coefficient. Our findings and illustrations may be instrumental for practitioners who analyze clustered multinomial data from clinical trials and/or meta-analysis.

High prevalence of colonisation with carbapenem-resistant Enterobacteriaceae among patients admitted to Vietnamese hospitals: Risk factors and burden of disease.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) is an increasing problem worldwide, but particularly problematic in low- and middle-income countries (LMIC) due to limitations of resources for surveillance of CRE and infection prevention and control (IPC). METHODS: A point prevalence survey (PPS) with screening for colonisation with CRE was conducted on 2233 patients admitted to neonatal, paediatric and adult care at 12 Vietnamese hospitals located in northern, central and southern Vietnam during 2017 and 2018. CRE colonisation was determined by culturing of faecal specimens on selective agar for CRE. Risk factors for CRE colonisation were evaluated. A CRE admission and discharge screening sub-study was conducted among one of the most vulnerable patient groups; infants treated at an 80-bed Neonatal ICU from March throughout June 2017 to assess CRE acquisition, hospital-acquired infection (HAI) and treatment outcome. RESULTS: A total of 1165 (52%) patients were colonised with CRE, most commonly Klebsiella pneumoniae (n = 805), Escherichia coli (n = 682) and Enterobacter spp. (n = 61). Duration of hospital stay, HAI and treatment with a carbapenem were independent risk factors for CRE colonisation. The PPS showed that the prevalence of CRE colonisation increased on average 4.2% per day and mean CRE colonisation rates increased from 13% on the day of admission to 89% at day 15 of hospital stay. At the NICU, CRE colonisation increased from 32% at admission to 87% at discharge, mortality was significantly associated (OR 5·5, P < 0·01) with CRE colonisation and HAI on admission. CONCLUSION: These data indicate that there is an epidemic spread of CRE in Vietnamese hospitals with rapid transmission to hospitalised patients.

Histopathologic Findings on Implantation Renal Allograft Biopsies Correlate With Kidney Donor Profile Index and 30-Day Serum Creatinine.

BACKGROUND: The Kidney Donor Profile Index (KDPI) provides a numerical estimate of deceased donor kidney quality. The KDPI uses 10 donor factors but it does not consider histopathologic findings. We examined whether the KDPI and its component donor factors correlate with the degree of histopathologic changes seen in implantation renal allograft biopsies. METHODS: All deceased donor kidney transplants at our institution from July 1, 2016 to March 15, 2017 that had an implantation biopsy were included. The biopsies were graded based on the Banff criteria for interstitial fibrosis, tubular atrophy, arterial intimal fibrosis, and arteriolar hyalinosis, as well as percent glomerulosclerosis. Linear and logistic regression were used to assess the correlation between histopathologic findings and KDPI and the ability of these variables to predict 30-day serum creatinine (SCr) and delayed graft function (DGF). RESULTS: One hundred thirty-four recipients from 107 donors were included. All histopathologic features examined correlated significantly with KDPI, with arteriolar hyalinosis correlating most strongly. Arteriolar hyalinosis was also associated with the most component donor factors of the KDPI. Histopathologic findings alone or in combination with KDPI predicted 30-day SCr but not DGF. Using the KDPI in combination with degree of interstitial fibrosis and tubular atrophy was the best predictor of 30-day SCr. CONCLUSION: Histopathologic changes seen in implantation renal allograft biopsies correlate with KDPI and predict 30-day SCr. Using a combination of donor histopathologic findings and KDPI may be the best predictor of short-term graft function.

Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation.

BACKGROUND: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. METHODS: An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. RESULTS: FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. CONCLUSION: These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

LigaSure Use Decreases Intraoperative Blood Loss Volume and Blood Transfusion Volume in Sarcoma Surgery.

INTRODUCTION: The LigaSure system has been successfully used in thoracic and abdominal surgery. However, to date, its use in the resection of sarcomas has not been systematically studied. We aimed to determine whether the use of the LigaSure system reduces blood loss and blood transfusion volumes in sarcoma surgery. METHODS: One hundred forty-two consecutive patients who underwent sarcoma surgeries between July 2010 and October 2016 were included. Conventional electrocautery alone (n = 91) and with LigaSure (n = 51) were compared. Case-matched samples (n = 46) from each group were additionally compared. RESULTS: The use of the LigaSure system resulted in a significant decrease in mean intraoperative blood loss (P = 0.02) and blood transfusion volume (P = 0.04). Likewise, a significant decrease in both mean and median intraoperative blood loss (P = 0.003; P < 0.0001) was seen with LigaSure in the case-matched analysis. In the soft-tissue sarcoma subgroup, a significant decrease was observed in mean hemoglobin reduction (P = 0.03) and mean intraoperative blood loss with LigaSure (P = 0.04). No adverse perioperative complications attributed to the LigaSure system were identified. CONCLUSIONS: The LigaSure vessel sealing and dividing system is a safe and effective hemostatic tool for deep dissection in bone and soft-tissue sarcoma surgery. LEVEL OF EVIDENCE: Level III, therapeutic study.

Epidemiology, Therapeutic Strategies, Outcomes, and Complications of Pathologic Fractures.

Much can be learned about the epidemiology of metastatic disease of bone through large databases. Secondary data analyses add substantial knowledge of the incidence, prevalence, cost, complications, risk factors, and treatment variability by using modern statistical methods in a large patient cohort. Investigators must be aware of the intentions of these data sources as well as the limitations in any conclusions drawn. Large databases are primarily beneficial in generating hypotheses and will likely continue to be an important source of information. For the general orthopaedist, surgical management of metastatic skeletal disease can be a challenging problem with many potential risks. Complications are often encountered and can be influenced by the patient's medical conditions, stage of disease, and the selected surgical procedure. Patients diagnosed with skeletal metastases are often at higher risk of having perioperative complications, such as infection and thromboembolism, than is the general population. A case-based approach highlights potential risks with examples of common scenarios that can arise.

Novel Enzyme Actions for Sulphated Galactofucan Depolymerisation and a New Engineering Strategy for Molecular Stabilisation of Fucoidan Degrading Enzymes.

Fucoidans from brown macroalgae have beneficial biomedical properties but their use as pharma products requires homogenous oligomeric products. In this study, the action of five recombinant microbial fucoidan degrading enzymes were evaluated on fucoidans from brown macroalgae: Sargassum mcclurei, Fucus evanescens, Fucus vesiculosus, Turbinaria ornata, Saccharina cichorioides, and Undaria pinnatifida. The enzymes included three endo-fucoidanases (EC 3.2.1.-GH 107), FcnA2, Fda1, and Fda2, and two unclassified endo-fucoglucuronomannan lyases, FdlA and FdlB. The oligosaccharide product profiles were assessed by carbohydrate-polyacrylamide gel electrophoresis and size exclusion chromatography. The recombinant enzymes FcnA2, Fda1, and Fda2 were unstable but were stabilised by truncation of the C-terminal end (removing up to 40% of the enzyme sequence). All five enzymes catalysed degradation of fucoidans containing α(1→4)-linked l-fucosyls. Fda2 also degraded S. cichorioides and U. pinnatifida fucoidans that have α(1→3)-linked l-fucosyls in their backbone. In the stabilised form, Fda1 also cleaved α(1→3) bonds. For the first time, we also show that several enzymes catalyse degradation of S. mcclurei galactofucan-fucoidan, known to contain α(1→4) and α(1→3) linked l-fucosyls and galactosyl-ß(1→3) bonds in the backbone. These data enhance our understanding of fucoidan degrading enzymes and their substrate preferences and may assist development of enzyme-assisted production of defined fuco-oligosaccharides from fucoidan substrates.