High-resolution genome-wide mapping of chromosome-arm-scale truncations induced by CRISPR-Cas9 editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a powerful tool for introducing targeted mutations in DNA, but recent studies have shown that it can have unintended effects such as structural changes. However, these studies have not yet looked genome wide or across data types. Here we performed a phenotypic CRISPR-Cas9 scan targeting 17,065 genes in primary human cells, revealing a 'proximity bias' in which CRISPR knockouts show unexpected similarities to unrelated genes on the same chromosome arm. This bias was found to be consistent across cell types, laboratories, Cas9 delivery methods and assay modalities, and the data suggest that it is caused by telomeric truncations of chromosome arms, with cell cycle and apoptotic pathways playing a mediating role. Additionally, a simple correction is demonstrated to mitigate this pervasive bias while preserving biological relationships. This previously uncharacterized effect has implications for functional genomic studies using CRISPR-Cas9, with applications in discovery biology, drug-target identification, cell therapies and genetic therapeutics.

Optimal conditions for high-power, short-duration radiofrequency ablation using a novel, flexible-tipped, force-sensing catheter.

Background: High-power, short-duration (HPSD) radiofrequency ablation (RFA) reduces procedure time; however, safety and efficacy thresholds vary with catheter design. Objective: The study sought to determine optimal HPSD ablation conditions with a novel flexible-tipped, contact force-sensing RFA catheter. Methods: RFA lesions were created in thigh muscle (16 swine) over a range of conditions (51-82 W, 2-40 g, 8-40 mL/min irrigation). An intracardiac study was performed (12 swine) to characterize steam pop thresholds. Lesions were created in a second intracardiac study (14 swine, n = 290 pulmonary vein isolation [PVI] lesions) with combinations of radiofrequency power, duration, and contact force. PVI was tested, animals were sacrificed, and lesions were measured. Results: The likelihood of coagulation formation in the thigh model was <20% when power was ≤79 W, when contact force was ≤40 g, when duration was ≤11 seconds, and when irrigation rates were 8 to 40 mL/min. The impact of contact force on lesion safety and efficacy was more pronounced using HPSD (60 W/8 seconds) compared with conventional ablation (30 W/45 seconds) (P = .038). During PVI, focal atrial lesions ranged in width from 4.2 to 12.5 mm and were transmural 80.8% of the time. PVI was achieved in 13 of 14 veins. Logistic regression identified that the optimal parameters for radiofrequency application were 60 to 70 W with a duration <8 seconds and <15 g contact force. Conclusions: Optimal HPSD lesions with this this flexible-tipped, force-sensing RFA catheter were created at 60 to 70 W for <8 seconds with <15 g contact force. Chronic studies are ongoing to assess radiofrequency parameter refinements and long-term lesion durability using these conditions.

mTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.

The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

Safe and effective delivery of high-power, short-duration radiofrequency ablation lesions with a flexible-tip ablation catheter.

Background: High-power, short-duration (HPSD) radiofrequency ablation (RFA) may reduce ablation time. Concerns that catheter-mounted thermocouples (TCs) can underestimate tissue temperature, resulting in elevated risk of steam pop formation, potentially limit widespread adoption of HPSD ablation. Objective: The purpose of this study was to compare the safety and efficacy of HPSD and low-power, long-duration (LPLD) RFA in the context of pulmonary vein isolation (PVI). Methods: An open-irrigated ablation catheter with a contact force sensor and a flexible-tip electrode containing a TC at its distal end (TactiFlexTM Ablation Catheter, Sensor EnabledTM, Abbott) was used to isolate the left pulmonary veins (PVs) in 12 canines with HPSD RFA (50 W for 10 seconds) and LPLD RFA (30 W for a maximum of 60 seconds). PVI was assessed at 30 minutes and 28 ± 3 days postablation. Computed tomographic scans were performed to assess PV stenosis after RFA. Lesions were evaluated with histopathology. Results: A total of 545 ablations were delivered: 252 with LPLD (0 steam pops) and 293 with HPSD RFA (2 steam pops) (P = .501). Ablation time required to achieve PVI was >3-fold shorter for HPSD than for LPLD RFA (P = .001). All 24 PVs were isolated 30 minutes after ablation, with 12/12 LPLD-ablated and 11/12 HPSD-ablated PVs still isolated at follow-up. Histopathology revealed transmural ablations for HPSD and LPLD RFA. No major adverse events occurred. Conclusion: An investigational ablation catheter effectively delivered RFA lesions. Ablation time required to achieve PVI with HPSD with this catheter was >3-fold shorter than with LPLD RFA.

A drug-induced hypotensive challenge to verify catheter-based radiofrequency renal denervation in an obese hypertensive swine model.

OBJECTIVE: Sham-controlled trials provided proof-of-principle for the blood pressure-lowering effect of catheter-based renal denervation (RDN). However, indicators for the immediate assessment of treatment success are lacking. This study sought to investigate the impact of RDN on renal renin arteriovenous difference (renal renin AV-Δ) following a hypotensive challenge (HC). METHODS: Twelve hypertensive Ossabaw swine underwent either combined surgical and chemical (n = 3) or catheter-based RDN (n = 9). A telemetry monitor was implanted to acquire hemodynamic data continuously. Before and after RDN, a sodium nitroprusside-induced HC was performed. Renal renin AV-Δ was calculated as the difference of plasma renin concentrations drawn from the renal artery and vein. RESULTS: In total, complete renal renin AV data were obtained in eight animals at baseline and six animals at baseline and 3 months of follow-up. Baseline renal renin AV-Δ correlated inversely with change in 24-h minimum systolic (- 0.764, p = 0.02), diastolic (r = - 0.679, p = 0.04), and mean (r = - 0.663, p = 0.05) blood pressure. In the animals with complete renin secretion data at baseline and follow-up, the HC increased renal renin AV-Δ at baseline, while this effect was attenuated following RDN (0.55 ± 0.34 pg/ml versus - 0.10 ± 0.16 pg/ml, p = 0.003). Renin urinary excretion remained unchanged throughout the study (baseline 0.286 ± 0.187 pg/ml versus termination 0.305 ± 0.072 pg/ml, p = 0.789). CONCLUSION: Renin secretion induced by HC was attenuated following RDN and may serve as an indicator for patient selection and guide successful RDN procedures.

In Vitro Cell Selectivity of Reversible and Irreversible: Electroporation in Cardiac Tissue.

[Figure: see text].

Absence of (sub-)acute cerebral events or lesions after electroporation ablation in the left-sided canine heart.

BACKGROUND: Irreversible electroporation (IRE) is a nonthermal ablation modality. A 200-J application can create deep myocardial lesions, but gas bubbles are created at the ablation electrode. Cerebral effects of these bubbles are unknown. OBJECTIVE: The purpose of this study was to investigate gas microemboli-induced brain lesions after IRE and radiofrequency (RF) ablation to the left side of the canine heart, using magnetic resonance imaging (MRI) and histopathology. METHODS: In 11 canines, baseline cerebral MRI scans were performed. In 9 animals, after retrograde femoral artery access, 12 ± 4 200-J IRE applications were administered in the ascending aorta. In 2 animals, 30 minutes of irrigated 30-W RF ablation using 10-30g of contact force was applied in the left ventricle. At days 1 and 5 after ablation, MRI was repeated. The brain tissue then was histopathologically examined. RESULTS: All ablations and follow-up were uneventful. Intracardiac echography confirmed gas bubble formation after each IRE application. Neurologic examination was normal. MRI scans were normal in all animals at day 1 and were normal in 10 of 11 animals at day 5. In 1 animal, a single <2-mm-diameter lesion in the right temporal region could not be excluded as a small infarct or early hemorrhagic site. Histopathologic analysis of the same region showed no pathologic changes. In all other animals, gross and microscopic pathology were normal. CONCLUSION: MRI images alone or in combination with histologic follow-up did not reveal treatment-related embolic events. Gross and microscopic pathology did not reveal evidence of treatment-related embolic events. IRE seems to be a safe ablation modality for the brain.

Functional Precision Medicine Identifies New Therapeutic Candidates for Medulloblastoma.

Medulloblastoma is among the most common malignant brain tumors in children. Recent studies have identified at least four subgroups of the disease that differ in terms of molecular characteristics and patient outcomes. Despite this heterogeneity, most patients with medulloblastoma receive similar therapies, including surgery, radiation, and intensive chemotherapy. Although these treatments prolong survival, many patients still die from the disease and survivors suffer severe long-term side effects from therapy. We hypothesize that each patient with medulloblastoma is sensitive to different therapies and that tailoring therapy based on the molecular and cellular characteristics of patients' tumors will improve outcomes. To test this, we assembled a panel of orthotopic patient-derived xenografts (PDX) and subjected them to DNA sequencing, gene expression profiling, and high-throughput drug screening. Analysis of DNA sequencing revealed that most medulloblastomas do not have actionable mutations that point to effective therapies. In contrast, gene expression and drug response data provided valuable information about potential therapies for every tumor. For example, drug screening demonstrated that actinomycin D, which is used for treatment of sarcoma but rarely for medulloblastoma, was active against PDXs representing Group 3 medulloblastoma, the most aggressive form of the disease. Functional analysis of tumor cells was successfully used in a clinical setting to identify more treatment options than sequencing alone. These studies suggest that it should be possible to move away from a one-size-fits-all approach and begin to treat each patient with therapies that are effective against their specific tumor. SIGNIFICANCE: These findings show that high-throughput drug screening identifies therapies for medulloblastoma that cannot be predicted by genomic or transcriptomic analysis.

Neoadjuvant chemotherapy and penile conservation in the management of Buschke - Lowenstein tumor, a case report.

Buschke-Lowenstein tumor is a rare form of low-grade penile cancer. Its low prevalence amongst the population bars the establishment of a standardized treatment algorithm. We present a case of BLT that was managed with neoadjuvant chemotherapy followed by phallic sparing surgery.

Proteomics, Post-translational Modifications, and Integrative Analyses Reveal Molecular Heterogeneity within Medulloblastoma Subgroups.

There is a pressing need to identify therapeutic targets in tumors with low mutation rates such as the malignant pediatric brain tumor medulloblastoma. To address this challenge, we quantitatively profiled global proteomes and phospho-proteomes of 45 medulloblastoma samples. Integrated analyses revealed that tumors with similar RNA expression vary extensively at the post-transcriptional and post-translational levels. We identified distinct pathways associated with two subsets of SHH tumors, and found post-translational modifications of MYC that are associated with poor outcomes in group 3 tumors. We found kinases associated with subtypes and showed that inhibiting PRKDC sensitizes MYC-driven cells to radiation. Our study shows that proteomics enables a more comprehensive, functional readout, providing a foundation for future therapeutic strategies.

The Influence of Adalimumab on the Healing of Apical Periodontitis in Ferrets.

INTRODUCTION: Given the increasing use of anti-tumor necrosis factor α (anti-TNFα) biologic medications, and their interferences with the immune-inflammatory response, this study evaluated the effect of adalimumab (anti-TNFα), on healing and healing time of apical periodontitis (AP) in ferrets. METHODS: Twelve male ferrets received cone beam computed tomography of the jaws at baseline health (T0); AP confirmation (T1); and 30 (T2), 60 (T3), and 90 (T4) days after root canal treatment (RCT) to monitor healing. All animals had AP induced in the canines; 3 ferrets (12 teeth) provided the positive controls for the histologic evaluation; 9 ferrets were randomly divided into 3 treatment groups with 12 teeth each in the following manner: Systemic: conventional RCT and systemic anti-TNFα; Local: RCT and periapical administration of anti-TNFα before canal obturation; conventional RCT only (control). Two calibrated radiologists assessed the cone beam computed tomography images independently and blindly for AP identification and quantification. Rank-based analysis of covariance was used for statistical analysis of lesion size. RESULTS: AP was induced in all teeth. Following RCT, all AP lesions in the 3 groups showed a significant reduction in size. Specific pairwise comparisons of the related samples (Friedman's 2-way analysis of variance by ranks within each group) demonstrated a decreasing trend in lesion size with healing time in all 3 groups, most pronounced for local group (local adalimumab). No statistical difference was noticed between groups. CONCLUSIONS: Both systemic and local anti-TNFα did not hinder AP healing in this animal model and a faster healing response may also be anticipated. These findings encourage follow-up studies with larger sample sizes.

Comparison of branch and distally focused main renal artery denervation using two different radio-frequency systems in a porcine model.

OBJECTIVES: Anatomic placement of lesions may impact efficacy of radio-frequency (RF) catheter renal denervation (RDN). However, it is unclear if it is necessary to perform treatments post bifurcation with systems that may provide deeper penetration to achieve successful RDN. METHODS: Sixteen domestic swine (n=16) were randomly assigned to 4 groups: 1) 8 lesions created in the branch arteries using the Spyral catheter (SP8); 2) 8 lesions created in the branch arteries plus 4 lesions created in the main artery using the SP catheter (SP12); 3) 8 lesions created in the main artery using the EnligHTN catheter with the distal position as close as possible to the bifurcation (EN8); and 4) 12 lesions created in the main artery using the EN catheter with the distal position as close as possible to the bifurcation (EN12). RESULTS: Each arm showed statistically significant changes in kidney norepinephrine (NE, ng/g) between treated kidneys vs. untreated contralateral control. There were no statistically significant differences in tissue NE% reductions across each arm based on catheter, anatomic location, & number of lesions (p=0.563): EN8 -74±34%, EN12 -95±3%, SP8 -76±16%, SP12 -82±17% (p=0.496). A total of 46 lesions were measured for lesion depth: EN main (3.3±2.8mm) vs. SP branch (2.0±1.0mm, p=0.039), SP main (2.9±1.6mm) vs. SP branch (p=0.052), and EN main vs. SP main (p=0.337). CONCLUSIONS: Distally-focused main renal artery treatment using the EN system appears to be equally efficacious in reducing tissue NE levels compared with SP treatment in the branches plus main renal arteries, advocating for device-specific procedure execution.

Interaction Landscape of Inherited Polymorphisms with Somatic Events in Cancer.

Recent studies have characterized the extensive somatic alterations that arise during cancer. However, the somatic evolution of a tumor may be significantly affected by inherited polymorphisms carried in the germline. Here, we analyze genomic data for 5,954 tumors to reveal and systematically validate 412 genetic interactions between germline polymorphisms and major somatic events, including tumor formation in specific tissues and alteration of specific cancer genes. Among germline-somatic interactions, we found germline variants in RBFOX1 that increased incidence of SF3B1 somatic mutation by 8-fold via functional alterations in RNA splicing. Similarly, 19p13.3 variants were associated with a 4-fold increased likelihood of somatic mutations in PTEN. In support of this association, we found that PTEN knockdown sensitizes the MTOR pathway to high expression of the 19p13.3 gene GNA11 Finally, we observed that stratifying patients by germline polymorphisms exposed distinct somatic mutation landscapes, implicating new cancer genes. This study creates a validated resource of inherited variants that govern where and how cancer develops, opening avenues for prevention research.Significance: This study systematically identifies germline variants that directly affect tumor evolution, either by dramatically increasing alteration frequency of specific cancer genes or by influencing the site where a tumor develops. Cancer Discovery; 7(4); 410-23. ©2017 AACR.See related commentary by Geeleher and Huang, p. 354This article is highlighted in the In This Issue feature, p. 339.

A Network of Conserved Synthetic Lethal Interactions for Exploration of Precision Cancer Therapy.

An emerging therapeutic strategy for cancer is to induce selective lethality in a tumor by exploiting interactions between its driving mutations and specific drug targets. Here we use a multi-species approach to develop a resource of synthetic lethal interactions relevant to cancer therapy. First, we screen in yeast ∼169,000 potential interactions among orthologs of human tumor suppressor genes (TSG) and genes encoding drug targets across multiple genotoxic environments. Guided by the strongest signal, we evaluate thousands of TSG-drug combinations in HeLa cells, resulting in networks of conserved synthetic lethal interactions. Analysis of these networks reveals that interaction stability across environments and shared gene function increase the likelihood of observing an interaction in human cancer cells. Using these rules, we prioritize ∼10(5) human TSG-drug combinations for future follow-up. We validate interactions based on cell and/or patient survival, including topoisomerases with RAD17 and checkpoint kinases with BLM.

Catheter-based radio-frequency renal nerve denervation lowers blood pressure in obese hypertensive swine model.

OBJECTIVES: Radio-frequency renal denervation (RDN) therapy is under investigation for the treatment of uncontrolled hypertension. Data in hypertensive, drug-naïve large animal models using RDN is limited. METHODS: A cohort of Ossabaw swine (N = 9) was implanted with telemetry monitors, enrolled on a high calorie-feed regimen and randomly assigned to RDN. Blood pressure (BP) data were separated and analyzed according to the following epoch definitions: 24-h (h), most-active-h, light-h, and dark-h. RESULTS: The mean weight increased by 45% from 86.5 ±â€Š2.5 kg at telemetry implant (day 87) to 125.2 ±â€Š4.5 kg at time of RDN therapy (day 227). Hypertension developed in all swine (24-h BP: 169.5/128.3 ±â€Š5.8/5.1 mmHg pre-RDN). RDN resulted in significant reductions in noradrenaline kidney tissue concentration by 63%. Significant BP reductions were documented at 45 days post-RDN in all defined interday epochs, except for the dark-h period. The most pronounced SBP/DBP reduction was 12.4/11.2 mmHg (P < 0.05), observed during the most-active-h period. Animals continued to gain weight after the RDN procedure to the end of the study at 90 days (125.2 ±â€Š4.5-138.5 ±â€Š6.6 kg, P < 0.001). At 90 days post-RDN, the mean 24-h BP returned near pre-RDN baseline values. Given the strong relationship of BP to weight (R = 0.87, P < 0.001), group mean SBP/DBP was normalized by weight resulting in significant and continued reductions at both 45 and 90 days post-RDN across all intradaily epochs. CONCLUSION: Catheter-based RDN, using a multielectrode system, resulted in a significant reduction in 24-h BP in this drug-naïve, hypertensive animal model.

Use of Activated Carbon in Packaging to Attenuate Formaldehyde-Induced and Formic Acid-Induced Degradation and Reduce Gelatin Cross-Linking in Solid Dosage Forms.

Formaldehyde and formic acid are reactive impurities found in commonly used excipients and can be responsible for limiting drug product shelf-life. Described here is the use of activated carbon in drug product packaging to attenuate formaldehyde-induced and formic acid-induced drug degradation in tablets and cross-linking in hard gelatin capsules. Several pharmaceutical products with known or potential vulnerabilities to formaldehyde-induced or formic acid-induced degradation or gelatin cross-linking were subjected to accelerated stability challenges in the presence and absence of activated carbon. The effects of time and storage conditions were determined. For all of the products studied, activated carbon attenuated drug degradation or gelatin cross-linking. This novel use of activated carbon in pharmaceutical packaging may be useful for enhancing the chemical stability of drug products or the dissolution stability of gelatin-containing dosage forms and may allow for the 1) extension of a drug product's shelf-life when the limiting attribute is a degradation product induced by a reactive impurity, 2) marketing of a drug product in hotter and more humid climatic zones than currently supported without the use of activated carbon, and 3) enhanced dissolution stability of products that are vulnerable to gelatin cross-linking.

Lesion Size and Safety Comparison Between the Novel Flex Tip on the FlexAbility Ablation Catheter and the Solid Tips on the ThermoCool and ThermoCool SF Ablation Catheters.

INTRODUCTION: Next-generation catheters have been developed to reduce irrigation volume and preserve power delivery. A novel design uses a flexible tip (FlexAbility™ catheter) that directs flow to the contact surface. Because of recent safety issues with new catheters, we undertook a study in a canine heart with 3 irrigated catheters to compare efficacy and safety. METHODS: Endocardial ablation was performed by 2 independent operators in 12 anesthetized canines with the FlexAbility (St. Jude Medical), ThermoCool™ (Biosense Webster), and ThermoCool™ SF (Biosense Webster) catheters. Endocardial RF lesions were delivered with each catheter in all 4 chambers of each animal for 52 ± 16 seconds. Each chamber was randomized to receive ablation from one catheter with recording of safety events. Cardiac pathology was performed with triphenyl tetrazolium chloride stain. RESULTS: Average lesion dimensions were not significantly different between the 3 catheters. FlexAbility™ demonstrated a lower risk of steam pops relative to ThermoCool SF (P-value = 0.013) despite equal mean power and radiofrequency time. High-temperature generator shutdowns were observed with FlexAbility™ but not with either ThermoCool catheter. High-temperature shutdowns were associated with larger average impedance drops (28.5 ohms vs. 19 ohms) without compromising lesion size. CONCLUSIONS: The FlexAbility™ tip is safe and effective with no significant difference in lesion sizes compared to both standard ThermoCool and ThermoCool SF. FlexAbility™ has a significantly lower risk of steam pops compared to ThermoCool SF in a beating heart as defined predominantly by an abrupt rise of impedance.

DISPOSITION OF CEFTIOFUR AND ITS ACTIVE METABOLITES IN FALLOW DEER (DAMA DAMA) FOLLOWING SINGLE-DOSE INTRAVENOUS AND INTRAMUSCULAR ADMINISTRATION.

Septicemia and foot infections associated with Fusobacterium necrophorum , Pasturella multocida, and Streptococcus suis in captive fallow deer (Dama dama) are reasonably treated with ceftiofur hydrochloride. This study describes the disposition of ceftiofur after single-dose intravenous and intramuscular administration of 3.65±0.1678 mg/kg in six female adult fallow deer using a nonrandomized crossover design and a 7-day washout period. Serial blood samples were collected for 12 hr postdrug administration. Ceftiofur bioactivity, including its active metabolite desfuroylceftiofur, was quantitated in serum using a microbiologic assay. After i.v. administration, the extrapolated serum drug concentration reported as median (range) was 52.83 (43.32-57.49) µg/ml and elimination half-life was 178.36 (19.75-217.22) min. The volume of distribution at steady-state was 0.171 (0.101-0.229) L/kg and serum clearance was 0.97 (0.48-4.3) ml/min per kg. After i.m. administration, median peak plasma concentration (Cmax) was 14.37 (9.00-32.00) µg/ml at 54.5 (11.00-95.00) min. The median elimination half-life and mean residence time were 128.32 (38.03-242.40) and 203.65 (62.48-347.15) min, respectively. The median absorption time after i.m. administration was 14.77 (-57.74 to 94.79) min. Bioavailability of ceftiofur following i.m. administration was 78.00 (58.00-137.00) percent. Based on this study, a mean i.m. dose of ceftiofur of 3.65±0.1678 mg/kg every 12 hr is recommended for maintaining serum concentrations above MIC90 levels for infections associated with F. necrophorum, P. multocida, and S. suis, in addition to other susceptible infectious bacteria.