Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2.

OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC). DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice. RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer. CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.

Selective optogenetic control of Gq signaling using human Neuropsin.

Gq proteins are universally important for signal transduction in mammalian cells. The underlying kinetics and transformation from extracellular stimuli into intracellular signaling, however could not be investigated in detail so far. Here we present the human Neuropsin (hOPN5) for specific and repetitive manipulation of Gq signaling in vitro and in vivo with high spatio-temporal resolution. Properties and G protein specificity of hOPN5 are characterized by UV light induced IP3 generation, Ca2+ transients and inhibition of GIRK channel activity in HEK cells. In adult hearts from a transgenic animal model, light increases the spontaneous beating rate. In addition, we demonstrate light induced contractions in the small intestine, which are not detectable after pharmacological Gq protein block. All-optical high-throughput screening for TRPC6 inhibitors is more specific and sensitive than conventional pharmacological screening. Thus, we demonstrate specific Gq signaling of hOPN5 and unveil its potential for optogenetic applications.

Osteomyelitis and antibiotic treatment in patients with grade IV pressure injury and spinal cord lesion-a retrospective cohort study.

STUDY DESIGN: Retrospective cohort study. OBJECTIVES: To analyze characteristics and treatment of osteomyelitis (OM) in the treatment of grade IV pressure injury (PI) in patients with spinal cord injury/disorder (SCI/D) following the Basel Decubitus Concept. SETTING: Acute care and rehabilitation clinic specialized in SCI/D. METHODS: Patients with SCI/D were admitted for grade IV PI treatment between 1st January 2010 and 28th February 2015. Patients, SCI/D, and PI characteristics were collected from chart reviews. Descriptive statistics and differences between groups with and without OM were evaluated. RESULTS: In total, 117 patients (87 male, 30 female) with 130 PI grade IV were included. In 95 patients (81%), OM was diagnosed histologically. In 87 cases, more than one bacterial species was involved. Out of 49 different bacterial species, Enterococcus faecalis and Staphylococus aureus were most frequently observed. Amoxicillin/clavulanic acid and ciprofloxacin were the most frequently used out of 24 different antibiotics. Length of antibiotic treatment varied between <8 days and >91 days with 31 patients receiving antibiotics for about 8 weeks. Complications occurred in all groups of antibiotic duration. Having a paraplegia, no OM and sacral PI was associated with increased complication rates, but the number of patients did not allow comprehensive risk factor analysis. CONCLUSION: Because the variety of patients concerning SCI/D, PI, and OM characteristics did not show a conclusive relation between length of antibiotic treatment and complication rates, the development of a subgroup specific treatment concept for PI in patients with SCI/D would be favorable to further optimize antibiotic treatment.

Mismatch Repair Protein Expression and Microsatellite Instability in Cutaneous Squamous Cell Carcinoma.

There exist relatively sparse and conflicting data on high-level microsatellite instability (MSI-H) and deficient mismatch repair (dMMR) in cutaneous malignancies. We aimed to determine the expression profiles of MMR proteins (MSH2, MSH6, MLH1, and PMS2) in different progression stages of cutaneous squamous cell carcinoma (cSCC, 102 patients in total) by immunohistochemistry, and search for MSI-H in patients with low-level MMR or dMMR using multiplex-PCR. Low-level MMR protein expression was observed in five patients: One patient with primary cSCC < 2 mm thickness and low-level MLH1, three patients with primary cSCC > 6 mm (including one with low-level MSH2, as well as MSH6 expression, and two with low-level PMS2), and one patient with a cSCC metastasis showing low-level MSH2 as well as MSH6. Intergroup protein expression analysis revealed that MLH1 and MSH2 expression in actinic keratosis was significantly decreased when compared to Bowen's disease, cSCC < 2 mm, cSCC > 6 mm, and cSCC metastasis. In cases with low-level MMR, we performed MSI-H tests revealing three cases with MSI-H and one with low-level MSI-L. We found low-level MMR expression in a small subset of patients with invasive or metastatic cSCC. Hence, loss of MMR expression may be associated with tumour progression in a small subgroup of patients with non-melanoma skin cancer.

Expression of Mismatch Repair Proteins in Merkel Cell Carcinoma.

We aimed to assess for the first time the mismatch repair (MMR) protein expression in Merkel cell carcinoma (MCC). Immunohistochemistry was performed for MLH1, MSH2, MSH6, and PMS2 on patients' tumor tissue (n = 56), including neighbored healthy control tissue. In cases with low-level MMR expression (<10th percentile), we performed multiplex PCR in combination with high-resolution capillary electrophoresis in order to confirm microsatellite instability (MSI). Microscopic evaluation revealed a high median expression for all MMR proteins studied (91.6-96.3%). However, six patients (56/10.7%) had low-level MLH1 expression, six (55/10.9%) had low-level MSH2 expression, five (56/8.9%) had low-level MSH6 expression, and six (54/11.1%) had low-level PMS2 expression. Together, we observed nine (56/16.1%) patients who had low-level MMR expression of at least one protein. Of the patients with low-level MMR expression, MSI evaluation was possible in five cases, revealing one case with high-level MSI. In all MMR proteins assessed, low-level expression was significantly (p = 0.0004 to p < 0.0001) associated with a negative Merkel cell polyomavirus (MCPyV) status. However, the expression profiles of the MMR proteins did not correlate with clinical outcome measures such as disease relapse or death (p > 0.05). MCC appears to be a malignancy characterized by low-level MMR rather than completely deficient MMR in a subset of cases, predominantly affecting MCPyV-negative tumors. Future studies will establish whether this subset of MCC patients respond better to immune checkpoint inhibitor therapy.

Direct optogenetic stimulation of smooth muscle cells to control gastric contractility.

Rationale: Antral peristalsis is responsible for gastric emptying. Its failure is called gastroparesis and often caused by dysfunction of enteric neurons and interstitial cells of Cajal (ICC). Current treatment options, including gastric electrical stimulation, are non-satisfying and may improve symptoms but commonly fail to restore gastric emptying. Herein, we explore direct optogenetic stimulation of smooth muscle cells (SMC) via the light-gated non-selective cation channel Channelrhodopsin2 (ChR2) to control gastric motor function. Methods: We used a transgenic mouse model expressing ChR2 in fusion with eYFP under the control of the chicken-ß-actin promoter. We performed patch clamp experiments to quantify light-induced currents in isolated SMC, Ca2+ imaging and isometric force measurements of antral smooth muscle strips as well as pressure recordings of intact stomachs to evaluate contractile responses. Light-induced propulsion of gastric contents from the isolated stomach preparation was quantified in video recordings. We furthermore tested optogenetic stimulation in a gastroparesis model induced by neuronal- and ICC-specific damage through methylene blue photo-toxicity. Results: In the stomachs, eYFP signals were restricted to SMC in which blue light (460 nm) induced inward currents typical for ChR2. These depolarizing currents led to contractions in antral smooth muscle strips that were stronger than those triggered by supramaximal electrical field stimulation and comparable to those evoked by global depolarization with high K+ concentration. In the intact stomach, panoramic illumination efficiently increased intragastric pressure achieving 239±46% (n=6) of the pressure induced by electrical field stimulation and triggered gastric transport. Within the gastroparesis model, electric field stimulation completely failed but light still efficiently generated pressure waves. Conclusions: We demonstrate direct optogenetic stimulation of SMC to control gastric contractility. This completely new approach could allow for the restoration of motility in gastroparesis in the future.

PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase.

The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.

Reactive mesothelial hyperplasia mimicking mesothelioma in an African green monkey (Chlorocebus aethiops).

A spontaneous reactive mesothelial hyperplasia occurred in a female, 15.7-year-old African green monkey (grivet; Chlorocebus aethiops). At necropsy, massive effusions were found in the abdomen, the thorax, and the pericardium. Additionally, multiple small, beige-gray nodules were detected on the serosal surfaces of the abdominal organs. Histopathologically, the mesothelial cells resembled the epithelioid subtype of a mesothelioma, but no infiltrative or invasive growth could be demonstrated. The mesothelial cells on the thoracis, liver, and intestinal serosa were accompanied by chronic serositis. Mesothelial cells expressed cytokeratin, vimentin, calretinin, desmin, Wilms Tumor 1 (WT-1) protein, and epithelial membrane antigen (EMA). Cells were negative for carcinoembryonic antigen (CEA), cluster of differentiation 15 (CD15), and podoplanin. Ultrastructurally, cells revealed a moderate amount of microvilli of medium length, perinuclear tonofilament bundles, and long desmosomes. In fluorescence in situ hybridization (FISH) for the detection of characteristic gene loss (p16; CDKN2A), NF2, and MTAP, no deletions were detected. No asbestos fibers and no presence of Simian virus 40 antigen (SV40) could be demonstrated.

MYC Recruits SPT5 to RNA Polymerase II to Promote Processive Transcription Elongation.

The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.

Optimized Multi-Ion Cavity Coupling.

Recent technological advances in cavity quantum electrodynamics (CQED) are paving the way to utilize multiple quantum emitters confined in a single optical cavity. In such systems, it is crucially important to control the quantum mechanical coupling of individual emitters to the cavity mode. In this regard, combining ion trap technologies with CQED provides a particularly promising approach due to the well-established motional control over trapped ions. Here, we experimentally demonstrate coupling of up to five trapped ions in a string to a high-finesse optical cavity. By changing the axial position and spacing of the ions in a fully deterministic manner, we systematically characterize their coupling to the cavity mode through visibility measurements of the cavity emission. In good agreement with the theoretical model, the results demonstrate that the geometrical configuration of multiple trapped ions can be manipulated to obtain optimal cavity coupling. Our system presents a new ground for exploring CQED with multiple quantum emitters, enabled by the highly controllable collective light-matter interaction.

Antibiotic Prophylaxis During Dental Procedures in Patients with Prosthetic Joints.

In patients with artificial joints, the need for antimicrobial prophylaxis during dental procedures is often raised. The present document describes the pathogenic mechanisms and epidemiological data on the subject of periprosthetic joint infections (PJI) after dental procedures. The document reflects the opinion and recommendations of the expert group 'Infection' of Swiss Orthopaedics. Microorganisms belonging to oral flora can seed haematogenously to an artificial joint. The proof of a causative relation with dental procedures is not possible, because the responsible bacteraemia can originate from the oral cavity at any time, irrespective of when the dental procedure occurs. Good oral hygiene is associated with a lower risk for PJI. Transient bacteraemia occurs during daily oral hygiene activity (e.g., tooth brushing) and thus the cumulative risk for a haematogenous PJI from tooth brushing is higher than that from a dental procedure. PJI after a dental procedure are rarely reported. On the basis of an epidemiological model, several thousand patients with artificial joints must receive antimicrobial prophylaxis to prevent a single PJI. Considering this ratio, the number of adverse events due to the antimicrobial compound exceeds the benefit of administering it by a large magnitude. Therefore, as a rule for the vast majority of cases, antimicrobial prophylaxis during dental procedures is not recommended. It is important that a patient has a good oral health status before joint implantation and that good oral hygiene is continuously maintained in patients with artificial joints.

Salinomycin increases chemosensitivity to the effects of doxorubicin in soft tissue sarcomas.

BACKGROUND: Chemotherapy for soft tissue sarcomas remains unsatisfactory due to their low chemosensitivity. Even the first line chemotherapeutic agent doxorubicin only yields a response rate of 18-29%. The antibiotic salinomycin, a potassium ionophore, has recently been shown to be a potent compound to deplete chemoresistant cells like cancer stem like cells (CSC) in adenocarcinomas. Here, we evaluated the effect of salinomycin on sarcoma cell lines, whereby salinomycin mono- and combination treatment with doxorubicin regimens were analyzed. METHODS: To evaluate the effect of salinomycin on fibrosarcoma, rhabdomyosarcoma and liposarcoma cell lines, cells were drug exposed in single and combined treatments, respectively. The effects of the corresponding treatments were monitored by cell viability assays, cell cycle analysis, caspase 3/7 and 9 activity assays. Further we analyzed NF-κB activity; p53, p21 and PUMA transcription levels, together with p53 expression and serine 15 phosphorylation. RESULTS: The combination of salinomycin with doxorubicin enhanced caspase activation and increased the sub-G1 fraction. The combined treatment yielded higher NF-κB activity, and p53, p21 and PUMA transcription, whereas the salinomycin monotreatment did not cause any significant changes. CONCLUSIONS: Salinomycin increases the chemosensitivity of sarcoma cell lines - even at sub-lethal concentrations - to the cytostatic drug doxorubicin. These findings support a strategy to decrease the doxorubicin concentration in combination with salinomycin in order to reduce toxic side effects.

Infection risk after orthopedic surgery in patients with inflammatory rheumatic diseases treated with immunosuppressive drugs.

OBJECTIVE: The influence of specific medications on the risk of postoperative infection in patients with rheumatoid arthritis and other inflammatory rheumatic diseases (IRDs) remains unclear. This retrospective study examined the risk of postoperative infection at the site of surgery in patients treated with immunosuppressive drugs (including biologic agents) undergoing different types of orthopedic surgery. METHODS: The study included 50,359 cases of orthopedic surgery performed in our hospital between 2000 and 2008. The primary outcome was operation-related infection. IRD patients were compared with those with degenerative or posttraumatic disorders, and in IRD patients, the effect of immunosuppressive medication, specifically tumor necrosis factor α (TNFα) inhibitors and their preoperative management, was examined. RESULTS: There were 373 operation-related infections (0.8%) of 47,887 cases in the degenerative/posttraumatic group and 49 (2.0%) of 2,472 in the IRD group (higher infection rate in the IRD group; odds ratio [OR] 2.58 [95% confidence interval (95% CI) 1.91-3.48], P < 0.001). In the IRD group, elbow and foot surgery had the highest infection rates. The risk of infection was significantly increased in patients taking multiple conventional disease-modifying antirheumatic drugs (DMARDs; OR 2.49 [95% CI 1.06-5.84], P = 0.036) or TNFα inhibitors (OR 2.54 [95% CI 1.08-5.97], P = 0.032). The risk was especially high (6 [12%] of 49) if the last dose of TNFα inhibitor was given <1 administration interval before surgery. CONCLUSION: The risk of postoperative infection was elevated in patients with IRDs, especially those taking >1 conventional DMARD or TNFα inhibitors. It may be advisable to consider stopping TNFα inhibitors ≥1 administration interval before surgery, since the risk of postoperative infection appears to be higher if the operation occurs within this period.

Increased preoperative serum apoptosis marker fas ligand correlates with histopathology and new-onset of atrial fibrillation in patients after cardiac surgery.

BACKGROUND: We evaluated if preoperative serum apoptosis markers correlate with atrial histological remodeling and postoperative atrial fibrillation (POAF) after cardiac surgery. METHODS AND RESULTS: A total of 33 patients with sinus rhythm (SR) and without history of atrial fibrillation (AF) undergoing cardiac surgery were prospectively enrolled. Serum concentrations of Fas (apoptosis-stimulating fragment ligand) and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) were measured preoperatively. Right atrial appendage (RAA) tissue was obtained during surgery. Atrial apoptosis was assessed via TUNEL assay and degree of atrial fibrosis was categorized histologically by visual quantification. Continuous ECG-Monitoring was used to screen for POAF throughout 10 days after cardiac surgery. POAF occurred in 15 patients (45%). Atrial apoptosis was higher in patients with POAF as compared to those without (35.9 ± 9.8% vs 14.5 ± 7.5%; P < 0.0001) and correlated with the degree of atrial fibrosis (r = 0.69; P < 0.0001). In contrast to TRAIL (87.0 ± 8.2 pg/mL vs 83.3 ± 9.4 pg/mL; P = 0.77), preoperative Fas serum concentration was significantly higher in patients with POAF compared to patients in stable SR (91.3 ± 7.2 pg/mL vs 66.7 ± 3.0 pg/mL; P < 0.01). Serum Fas concentration correlated with the degree of atrial apoptosis (r = 0.63; P < 0.001) and the degree of atrial fibrosis (r = 0.39; P < 0.05). CONCLUSION: Preoperative evaluation of serum apoptosis marker Fas is useful to identify patients at risk for POAF undergoing cardiac surgery. Fas but not TRAIL correlates with the documented degree of atrial apoptosis and atrial fibrosis in RAA tissue. Further studies need to identify the prospective role of Fas in predicting POAF events.

MicroRNA-205, a novel regulator of the anti-apoptotic protein Bcl2, is downregulated in prostate cancer.

Decreased expression of the microRNA miR-205 has been observed in multiple tumour types due to its role in the epithelial to mesenchymal transition, which promotes metastasis. We determined the expression of miR-205 in 111 archival samples of prostate carcinoma and found it to be strongly reduced in most samples, with a median expression level of 16% in comparison to benign tissue from the same patient. Lower miR-205 expression correlated significantly with tumour size and miR-205 levels decreased with increasing Gleason score from 7a=3+4 to 8=4+4. In addition, we describe the anti-apoptotic protein BCL2 as a target of miR-205, relevant for prostate cancer due to its role in prognosis of primary tumours and in the appearance of androgen independence. The repression of BCL2 by miR-205 was confirmed using reporter assays and western blotting. BCL2 mRNA expression in the same collective of prostate cancer tissue samples was associated with higher Gleason score and extracapsular extension of the tumour (pT3). Consistent with its anti-apoptotic target BCL2, miR-205 promoted apoptosis in prostate cancer cells in response to DNA damage by cisplatin and doxorubicin in the prostate cancer cell lines PC3 and LnCap. MiR-205 also inhibited proliferation in these cell lines.

Correlation between total atrial conduction time estimated via tissue Doppler imaging (PA-TDI Interval), structural atrial remodeling and new-onset of atrial fibrillation after cardiac surgery.

BACKGROUND: Recent studies identified total atrial conduction time (TACT) as an independent and powerful predictor of new-onset atrial fibrillation (AF). The purpose of this study was to assess the association between the degree of atrial fibrosis, TACT, and frequency of postoperative atrial fibrillation (POAF) among patients undergoing cardiac surgery. METHODS AND RESULTS: Sixty patients in sinus rhythm (mean ± SD age 66 ± 10 years; 22% women) and without a history of AF undergoing cardiac surgery were prospectively enrolled. TACT was measured preoperatively in the left atrium by tissue-Doppler Imaging (PA-TDI interval). Holter-ECG/telemetry was used to screen for POAF throughout 10 days after cardiac surgery. Right atrial appendages (RAA) were obtained in 33 patients during surgery; atrial fibrosis was assessed by visual quantification (% area of positive van Gieson elastic staining). POAF occurred in 23 patients (38%). Fibrosis extent of RAA was higher in patients with POAF as compared to those without (27.5 ± 1.93 vs 15.8 ± 0.81% area; mean ± SEM; P < 0.001). PA-TDI interval was longer in patients with POAF versus patients who maintained in sinus rhythm (152.1 ± 3.0 vs 120.8 ± 1.8 milliseconds; P < 0.001) and correlated with the degree of atrial fibrosis (r = 0.73; P < 0.01). At the cut-off value of 133 milliseconds, TACT sensitivity and specificity related to POAF were 100% and 86%, respectively. CONCLUSION: PA-TDI interval is useful to identify patients at risk for POAF undergoing cardiac surgery and correlates with the degree of atrial fibrosis.

Salinomycin induces autophagy in colon and breast cancer cells with concomitant generation of reactive oxygen species.

BACKGROUND: Salinomycin is a polyether ionophore antibiotic that has recently been shown to induce cell death in human cancer cells displaying multiple mechanisms of drug resistance. The underlying mechanisms leading to cell death after salinomycin treatment have not been well characterized. We therefore investigated the role of salinomycin in caspase dependent and independent cell death in colon cancer (SW480, SW620, RKO) and breast cancer cell lines (MCF-7, T47D, MDA-MB-453). METHODOLOGY/PRINCIPAL FINDINGS: We detected features of apoptosis in all cell lines tested, but the executor caspases 3 and 7 were only strongly activated in RKO and MDA-MB-453 cells. MCF-7 and SW620 cells instead presented features of autophagy such as cytoplasmic vacuolization and LC3 processing. Caspase proficient cell lines activated autophagy at lower salinomycin concentrations and before the onset of caspase activation. Salinomycin also led to the formation of reactive oxygen species (ROS) eliciting JNK activation and induction of the transcription factor JUN. Salinomycin mediated cell death could be partially inhibited by the free radical scavenger N-acetyl-cysteine, implicating ROS formation in the mechanism of salinomycin toxicity. CONCLUSIONS: Our data indicate that, in addition to its previously reported induction of caspase dependent apoptosis, the initiation of autophagy is an important and early effect of salinomycin in tumor cells.