Gartanin is a novel NEDDylation inhibitor for induction of Skp2 degradation, FBXW2 expression, and autophagy.

Gartanin, a 4-prenylated xanthone, has been identified from the purple mangosteen fruit as a potent growth inhibitor of various cancer cell lines, including prostate cancer. However, much of Gartanin's anticancer mechanism remains unknown. We have discovered that Gartanin docked onto the regulatory subunit of the precursor cell-expressed developmentally downregulated 8 (NEDD8)-activating enzyme (NAE) complex and next to the NEDD8 binding complex, which leads to inhibit NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 NEDDylation in an in vitro assay. The S phase kinase-associated protein (Skp2) and F-box and WD-repeat domain-containing 2 (FBXW2), the NEDD8 family members of E3 ubiqutin ligases, were also downregulated and upregulated by Gartainin, respectively. Knock-down of NEDD8 expression by short harpin (Sh) RNAs blocked or attenuated these effects of Gartainin. Finally, Gartanin demonstrated its ability to inhibit growth of prostate cancer lines via autophagy initiation. Our data support that Gartanin is a naturally occurring NEDDylation inhibitor and deserves further investigation for prostate cancer prevention and treatment.

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.

Development and Initial Evaluation of a Cost-Effective Force Sensor for Ureteroscopic Application.

Purpose: Retrograde intrarenal surgery is the gold-standard treatment for most kidney stones. During ureteroscopy, ureteral access sheath insertion at forces greater than 8.0 Newtons (N) risks high-grade ureteral injury. To monitor force, our institution utilizes a unique, Bluetooth-equipped device (i.e., the University of California-Irvine Force Sensor). Given the unique nature of the force sensor, we sought to develop an inexpensive and accessible force sensor based on Boyle's law and the specific amount of force required to compress an occluded 1.0 mL syringe. Materials and Methods: We evaluated three brands of 1.0 mL syringes. After setting the plunger at 1.0 mL, the syringe was occluded, and the syringe plunger was compressed. The syringe volume was recorded when the applied force on the plunger reached 4.0 N, 6.0 N, and 8.0 N. Multiple trials were performed to assess reliability and reproducibility. A method for applying this clinically was also developed. Results: The precise force thresholds identified for a 1.0 mL Luer-Lok™ Syringe (Becton Dickinson, Franklin Lakes, NJ) were 0.30 mL for 4.00 N, 0.20 mL for 6.00 N, and 0.15 mL for 8.00 N. The 1.0 mL Tuberculin Syringe and 1.0 mL Luer Slip Syringe were less precise, but compression from 1.0 to 0.40 mL, 0.25 mL, and 0.20 mL corresponded to force sensor readings that did not exceed 4.00 N, 6.00 N, and 8.00 N, respectively. Conclusions: Based on volume changes, 4.00 N, 6.00 N, and 8.00 N of force can be reliably and reproducibly achieved using an occluded 1.0 mL syringe.

Flavokawain A Reduces Tumor-Initiating Properties and Stemness of Prostate Cancer.

We have previously demonstrated the in vivo chemopreventive efficacy of flavokawain A (FKA), a novel chalcone from the kava plant, in prostate carcinogenesis models. However, the mechanisms of the anticarcinogenic effects of FKA remain largely unknown. We evaluated the effect of FKA on prostate tumor spheroid formation by prostate cancer stem cells, which were sorted out from CD44+/CD133+ prostate cancer cells 22Rv1 and DU145. FKA treatment significantly decreased both the size and numbers of the tumor spheroids over different generations of spheroid passages. In addition, the dietary feeding of FKA-formulated food to Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice bearing CD44+/CD133+ 22Rv1 xenograft tumors resulted in a significant reduction of tumor growth compared to those fed with vehicle control food-fed mice. Furthermore, the expression of stem cell markers, such as Nanog, Oct4, and CD44, were markedly downregulated in both tumor spheroids and tumor tissues. We also observed that FKA inhibits Ubc12 neddylation, c-Myc, and keratin-8 expression in both CD44+/CD133+ prostate tumor spheroids and xenograft tumors. Our results suggest that FKA can reduce the tumor-initiating properties and stemness of prostate cancer, which provides a new mechanism for the chemoprevention efficacy of FKA.

Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders.

To dissect therapeutic mechanisms of transplanted stem cells and develop exosome-based nanotherapeutics in treating autoimmune and neurodegenerative diseases, we assessed the effect of exosomes secreted from human mesenchymal stem cells (MSCs) in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model. We found that intravenous administration of exosomes produced by MSCs stimulated by IFNγ (IFNγ-Exo) (i) reduced the mean clinical score of EAE mice compared to PBS control, (ii) reduced demyelination, (iii) decreased neuroinflammation, and (iv) upregulated the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice. Co-culture of IFNγ-Exo with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive cytokine indoleamine 2,3-dioxygenase. IFNγ-Exo could also induce Tregs in vitro in a murine splenocyte culture, likely mediated by a third-party accessory cell type. Further, IFNγ-Exo characterization by deep RNA sequencing suggested that IFNγ-Exo contains anti-inflammatory RNAs, where their inactivation partially hindered the exosomes potential to induce Tregs. Furthermore, we found that IFNγ-Exo harbors multiple anti-inflammatory and neuroprotective proteins. These results not only shed light on stem cell therapeutic mechanisms but also provide evidence that MSC-derived exosomes can potentially serve as cell-free therapies in creating a tolerogenic immune response to treat autoimmune and central nervous system disorders.

Bioactive heterocycles containing a 3,4,5-trimethoxyphenyl fragment exerting potent antiproliferative activity through microtubule destabilization.

Novel bioactive heterocycles containing a 3,4,5-trimethoxyphenyl fragment as antiproliferative agents by targeting tubulin were synthesized and their preliminary structure activity relationships (SARs) were explored. Among all these chemical agents, 2-(Benzo[d]oxazol-2-ylthio)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (4d) exhibited the potent antiproliferative activity against MGC-803 cells with an IC50 value of 0.45 µM by induction of G2/M pahse arrest and cell apoptosis. In addition, 4d could change the membrane potential (ΔΨ) of the mitochondria against MGC-803 cells. Importantly, 4d acted as a novel tubulin polymerization inhibitor binding to colchicine site with an IC50 value of 3.35 µM.

Rhodiola rosea L.: an herb with anti-stress, anti-aging, and immunostimulating properties for cancer chemoprevention.

PURPOSE OF REVIEW: Rhodiola rosea extracts have been used as a dietary supplement in healthy populations, including athletes, to non-specifically enhance the natural resistance of the body to both physical and behavior stresses for fighting fatigue and depression. We summarize the information with respect to the new pharmacological activities of Rhodiola rosea extracts and its underlying molecular mechanisms in this review article. RECENT FINDINGS: In addition to its multiplex stress-protective activity, Rhodiola rosea extracts have recently demonstrated its anti-aging, anti-inflammation, immunostimulating, DNA repair and anti-cancer effects in different model systems. Molecular mechanisms of Rhodiola rosea extracts's action have been studied mainly along with one of its bioactive compounds, salidroside. Both Rhodiola rosea extracts and salidroside have contrast molecular mechanisms on cancer and normal physiological functions. For cancer, Rhodiola rosea extracts and salidroside inhibit the mTOR pathway and reduce angiogenesis through down-regulation of the expression of HIF-1α/HIF-2α. For normal physiological functions, Rhodiola rosea extracts and salidroside activate the mTOR pathway, stimulate paracrine function and promote neovascularization by inhibiting PHD3 and stabilizing HIF-1α proteins in skeletal muscles. In contrast to many natural compounds, salidroside is water-soluble and highly bioavailable via oral administration and concentrated in urine by kidney excretion. SUMMARY: Rhodiola rosea extracts and salidroside can impose cellular and systemic benefits similar to the effect of positive lifestyle interventions to normal physiological functions and for anti-cancer. The unique pharmacological properties of Rhodiola rosea extracts or salidroside deserve further investigation for cancer chemoprevention, in particular for human urinary bladder cancer.

Mesenchymal stem cells engineered to express selectin ligands and IL-10 exert enhanced therapeutic efficacy in murine experimental autoimmune encephalomyelitis.

Systemic administration of mesenchymal stem cells (MSCs) affords the potential to ameliorate the symptoms of Multiple Sclerosis (MS) in both preclinical and clinical studies. However, the efficacy of MSC-based therapy for MS likely depends on the number of cells that home to inflamed tissues and on the controlled production of paracrine and immunomodulatory factors. Previously, we reported that engineered MSCs expressing P-selectin glycoprotein ligand-1 (PSGL-1) and Sialyl-Lewis(x) (SLeX) via mRNA transfection facilitated the targeted delivery of anti-inflammatory cytokine interleukin-10 (IL-10) to inflamed ear. Here, we evaluated whether targeted delivery of MSCs with triple PSGL1/SLeX/IL-10 engineering improves therapeutic outcomes in mouse experimental autoimmune encephalomyelitis (EAE), a murine model for human MS. We found PSGL-1/SLeX mRNA transfection significantly enhanced MSC homing to the inflamed spinal cord. This is consistent with results from in vitro flow chamber assays in which PSGL-1/SleX mRNA transfection significantly increased the percentage of rolling and adherent cells on activated brain microvascular endothelial cells, which mimic the inflamed endothelium of blood brain/spinal cord barrier in EAE. In addition, IL-10-transfected MSCs show significant inhibitory activity on the proliferation of CD4(+) T lymphocytes from EAE mice. In vivo treatment with MSCs engineered with PSGL-1/SLeX/IL-10 in EAE mice exhibited a superior therapeutic function over native (unmodified) MSCs, evidenced by significantly improved myelination and decreased lymphocytes infiltration into the white matter of the spinal cord. Our strategy of targeted delivery of performance-enhanced MSCs could potentially be utilized to increase the effectiveness of MSC-based therapy for MS and other central nervous system (CNS) disorders.

Elucidation of Exosome Migration across the Blood-Brain Barrier Model In Vitro.

The delivery of therapeutics to the central nervous system (CNS) remains a major challenge in part due to the presence of the blood-brain barrier (BBB). Recently, cell-derived vesicles, particularly exosomes, have emerged as an attractive vehicle for targeting drugs to the brain, but whether or how they cross the BBB remains unclear. Here, we investigated the interactions between exosomes and brain microvascular endothelial cells (BMECs) in vitro under conditions that mimic the healthy and inflamed BBB in vivo. Transwell assays revealed that luciferase-carrying exosomes can cross a BMEC monolayer under stroke-like, inflamed conditions (TNF-α activated) but not under normal conditions. Confocal microscopy showed that exosomes are internalized by BMECs through endocytosis, co-localize with endosomes, in effect primarily utilizing the transcellular route of crossing. Together, these results indicate that cell-derived exosomes can cross the BBB model under stroke-like conditions in vitro. This study encourages further development of engineered exosomes as drug delivery vehicles or tracking tools for treating or monitoring neurological diseases.

Institutional perspective on the impact of positive blood cultures on the economic and clinical outcomes of patients with complicated skin and skin structure infections: focus on gram-positive infections.

BACKGROUND: Traditionally, skin and skin structure infections (SSSIs) have been viewed as having a lower risk of mortality, morbidity, and cost compared with other types of infection. The influence of secondary bacteremia on the medicoeconomic outcomes of patients with SSSIs has not been well described. OBJECTIVE: The goal of this study was to evaluate the impact of bacteremia complicating SSSIs on length of hospital stay and costs. METHODS: This was a retrospective cohort study involving 579 patients with culture-positive SSSIs who were admitted to Barnes-Jewish Hospital, a major academic medical center, between April 1, 2005, and December 31, 2007. The outcomes evaluated in this analysis included hospital mortality, length of stay, hospital costs, and hospital readmission. RESULTS: Secondary bacteremia was present in 277 (47.8%) patients. Hospital mortality was statistically greater among patients with bacteremia (7.9% vs 1.0%; P < 0.001). The unadjusted median length of stay in bacteremic patients was 7.1 days compared with 2.8 days in those without bacteremia (P < 0.001 by log-rank test). This finding correlated with total hospital costs, which were greater in patients with bacteremia (median values: $14,623 vs $5841.50; P < 0.001). In a Cox model controlling for multiple confounders, bacteremia independently correlated with hospital duration (adjusted hazard ratio [HR], 1.820; 95% CI, 1.654-2.003; P < 0.001) and hospital costs (adjusted HR, 1.895; 95% CI, 1.723-2.083; P < 0.001). Hospital readmission within 30 days of discharge was also significantly more common among patients with SSSIs complicated by bacteremia (24.5% vs 12.9%; P < 0.001). CONCLUSIONS: Bacteremia complicating SSSIs occurred in almost 50% of patients infected with gram-positive bacteria in our institution. Beyond its impact on mortality, bacteremia is associated with increased length of stay, hospital costs, and readmission. However, these data are from a single academic medical center and may not be adjusted for all applicable confounders.

Best practices: implementation of a glucose screening program based on diffusion of innovation theory methods.

There is public health interest in the identification and treatment of modifiable cardiometabolic risk factors among patients treated with antipsychotic medications. However, best-practice screening recommendations endorsed by multiple medical organizations have not translated into real-world clinical practice. Quality improvement strategies may help to address the gap between policy and implementation. This column describes the successful implementation of a best-practice glucose screening program in a large network of community mental health centers that was based on Six Sigma and diffusion of innovation theory.

Development and validation of a Clostridium difficile infection risk prediction model.

OBJECTIVE: To develop and validate a risk prediction model that could identify patients at high risk for Clostridium difficile infection (CDI) before they develop disease. DESIGN AND SETTING: Retrospective cohort study in a tertiary care medical center. PATIENTS: Patients admitted to the hospital for at least 48 hours during the calendar year 2003. METHODS: Data were collected electronically from the hospital's Medical Informatics database and analyzed with logistic regression to determine variables that best predicted patients' risk for development of CDI. Model discrimination and calibration were calculated. The model was bootstrapped 500 times to validate the predictive accuracy. A receiver operating characteristic curve was calculated to evaluate potential risk cutoffs. RESULTS: A total of 35,350 admitted patients, including 329 with CDI, were studied. Variables in the risk prediction model were age, CDI pressure, times admitted to hospital in the previous 60 days, modified Acute Physiology Score, days of treatment with high-risk antibiotics, whether albumin level was low, admission to an intensive care unit, and receipt of laxatives, gastric acid suppressors, or antimotility drugs. The calibration and discrimination of the model were very good to excellent (C index, 0.88; Brier score, 0.009). CONCLUSIONS: The CDI risk prediction model performed well. Further study is needed to determine whether it could be used in a clinical setting to prevent CDI-associated outcomes and reduce costs.

Bacteremia increases the risk of death among patients with soft-tissue infections.

BACKGROUND: Soft-tissue infections traditionally have been viewed as carrying a lower risk of death than other types of infection such as pneumonia, blood stream, and intra-abdominal. The influence of secondary bacteremia on the outcomes of patients with soft-tissue infections is not well described. OBJECTIVES: To describe the risk factors for bacteremia among patients admitted to an urban medical center with soft-tissue infections and the influence of bacteremia on outcomes. METHODS: A retrospective cohort study of 717 patients with culture-positive non-necrotizing soft-tissue infections admitted between April 1, 2005, and December 31, 2007. RESULTS: Bacteremia was present in 52% of the patients. Increasing age, previous hospitalization, decubitus or lower-extremity ulcers, device-related soft-tissue infection, and polymicrobial infection were independent predictors of bacteremia. Intensive care unit admission (adjusted odds ratio [AOR] 3.57; 95% confidence interval [CI] 2.17, 5.86), lower-extremity ulcer (AOR 3.43; 95% CI 2.07, 5.70), and bacteremia (AOR 6.37; 95% CI 3.34, 12.12) were independent predictors of in-hospital death. When patients with device-related soft-tissue infections were excluded, the rate of secondary bacteremia was 37.6% (201/535), and it remained an independent predictor of in-hospital death. CONCLUSIONS: The occurrence of bacteremia in soft-tissue infections is associated with a greater risk of death. Health care providers should be aware of the risk factors for bacteremia in patients with soft-tissue infections in order to provide more appropriate initial antimicrobial therapy and to ascertain its presence as a prognostic indicator.

Hospitalizations with healthcare-associated complicated skin and skin structure infections: impact of inappropriate empiric therapy on outcomes.

OBJECTIVE: Inappropriate empiric therapy worsens outcomes in certain healthcare-associated infections (HCAI). We studied the association of inappropriate empiric therapy with outcomes in patients with HCA complicated skin and skin structure infections (cSSSI). DESIGN: A single-center retrospective cohort study. PATIENTS: Hospitalized with a culture-positive cSSSI. MEASUREMENTS: We defined HCA-cSSSI as having ≥1 of these risk factors: (1) recent hospitalization, (2) recent antibiotics, (3) hemodialysis, (4) transfer from a nursing home, and inappropriate treatment as no antimicrobial therapy active against the pathogen(s) within 24 hours of obtaining culture specimen. We performed descriptive and multivariate statistics to compute the impact of inappropriate empiric therapy on outcomes. Hospital length of stay (LOS) served as primary and mortality as secondary outcomes. RESULTS: Of the 717 patients with culture-positive cSSSI, 527 (73.5%) had HCAI, of whom 405 (76.9%) received appropriate treatment. A higher proportion of those receiving inappropriate than appropriate treatment had a decubitus ulcer (29.5% vs. 10.9%, P < 0.001), a device-associated infection (42.6% vs. 28.6%, P = 0.004), or bacteremia (68.9% vs. 57.8%, P = 0.028). The frequency of methicillin-resistant Staphylococcus aureus (MRSA) did not differ between the groups. The low overall unadjusted mortality rate did not vary based on initial treatment. In a multivariable analysis adjusting for potential confounders inappropriate therapy had an attributable increase in hospital LOS of 1.8 days (95% CI, 1.4-2.3). CONCLUSION: Similar to other populations with HCAI, HCA-cSSSI patients are likely to receive inappropriate empiric therapy for their infection. This early exposure is associated with a significant prolongation of the hospitalization by nearly 2 days.

Epidemiology and outcomes of hospitalizations with complicated skin and skin-structure infections: implications of healthcare-associated infection risk factors.

OBJECTIVE: Healthcare-associated infections are likely to be caused by drug-resistant and possibly mixed organisms and to be treated with inappropriate antibiotics. Because prompt appropriate treatment is associated with better outcomes, we studied the epidemiology of healthcare-associated complicated skin and skin-structure infections (cSSSIs). PATIENTS: Persons hospitalized with cSSSI and a positive culture result. METHODS: We conducted a single-center retrospective cohort study from April 2006 through December 2007. We differentiated healthcare-associated from community-acquired cSSSIs by at least 1 of the following risk factors: (1) recent hospitalization, (2) recent antibiotics, (3) hemodialysis, and (4) transfer from a nursing home. Inappropriate treatment was defined as no antimicrobial therapy with activity against the offending pathogen(s) within 24 hours after collection of a culture specimen. Mixed infections were those caused by both a gram-positive and a gram-negative organism. RESULTS: Among 717 hospitalized patients with cSSSI, 527 (73.5%) had healthcare-associated cSSSI. Gram-negative organisms were more common (relative risk, 1.24 [95% confidence interval, 1.14-1.35) and inappropriate treatment trended toward being more common (odds ratio, 1.29 [95% confidence interval, 0.85-1.95]) in healthcare-associated cSSSI than in community-acquired cSSSI. Mixed cSSSIs occurred in 10.6% of patients with healthcare-associated cSSSI and 6.3% of those with community-acquired cSSSI (P = .082) and were more likely to be treated inappropriately than to be nonmixed infections (odds ratio, 2.42 [95% confidence interval, 1.43-4.10]). Both median length of hospital stay (6.2 vs 2.9 days; P < .001) and mortality rate (6.6% vs 1.1%; P = .003) were significantly higher for healthcare-associated cSSSI than community-acquired cSSSI. CONCLUSIONS: Healthcare-associated cSSSIs are common and are likely to be caused by gram-negative organisms. Mixed infections carry a >2-fold greater risk of inappropriate treatment. Healthcare-associated cSSSIs are associated with increased mortality and prolonged length of hospital stay, compared with community-acquired cSSSIs.

Physicians' knowledge and attitudes about coronary heart disease secondary prevention.

We administered a knowledge and attitudes questionnaire regarding a technology assisted pharmacist mediated academic detailing intervention designed to improve physician adherence to coronary heart disease (CHD) secondary-prevention guidelines. Questionnaires were administered in two settings: an academic hospital and a community hospital. More knowledgeable physicians reported being more likely to prescribe a pharmacists' recommended medication and to agree that lipid profiles should be automatically performed for inpatients with elevated troponin.

Voluntary incident reporting tool for a multi-facility environment.

As awareness of medical errors increases, innovative solutions are required to address these errors so that healthcare providers can be proactive rather than reactive. The development of an enterprise-wide voluntary incident reporting application will provide a mechanism to organize and prioritize resources toward correction of processes that could endanger patients.

Physicians' knowledge and attitudes about coronary heart disease prevention guidelines and technology assisted interventions.

BJC Healthcare is conducting a randomized controlled study to evaluate the impact of a technology-assisted pharmacist intervention on physicians' adherence to national coronary heart disease (CHD) prevention guidelines. We surveyed physicians to assess their knowledge of the guidelines and attitudes toward pharmacist-mediated interventions.