Negative DNA supercoiling induces genome-wide Cas9 off-target activity.

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.

An aptamer-mediated base editing platform for simultaneous knock-in and multiple gene knockout for allogeneic CAR-T cells generation.

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point™ base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

Molecular signatures of alternative reproductive strategies in a facultatively social hover wasp.

Social insect reproductives and non-reproductives represent ideal models with which to understand the expression and regulation of alternative phenotypes. Most research in this area has focused on the developmental regulation of reproductive phenotypes in obligately social taxa such as honey bees, while relatively few studies have addressed the molecular correlates of reproductive differentiation in species in which the division of reproductive labour is established only in plastic dominance hierarchies. To address this knowledge gap, we generate the first genome for any stenogastrine wasp and analyse brain transcriptomic data for non-reproductives and reproductives of the facultatively social species Liostenogaster flavolineata, a representative of one of the simplest forms of social living. By experimentally manipulating the reproductive 'queues' exhibited by social colonies of this species, we show that reproductive division of labour in this species is associated with transcriptomic signatures that are more subtle and variable than those observed in social taxa in which colony living has become obligate; that variation in gene expression among non-reproductives reflects their investment into foraging effort more than their social rank; and that genes associated with reproductive division of labour overlap to some extent with those underlying division of labour in the separate polistine origin of wasp sociality but only explain a small portion of overall variation in this trait. These results indicate that broad patterns of within-colony transcriptomic differentiation in this species are similar to those in Polistinae but offer little support for the existence of a strongly conserved 'toolkit' for sociality.

Symptomatic rib fracture nonunion: a systematic review of the literature.

INTRODUCTION: Rib fractures commonly occur in trauma patients with varying presentations. Though the literature in recent years has moved toward favoring more early intervention of acute rib fractures, little has been reported on the matter of surgical fixation for symptomatic rib fracture nonunions. MATERIALS AND METHODS: We performed a review of PubMed and Cochrane databases for articles published since 2000. Inclusion criteria were studies with greater than six months of follow-up, while case studies were excluded. A thorough analysis was performed on patient outcomes, complications reported, operative techniques utilized, and fixation systems used, among other parameters reported by the articles. RESULTS: One hundred and thirty-nine studies resulted from our review, and a total of nine studies met our inclusion criteria with a combined total of 182 patients who underwent open reduction and internal fixation for symptomatic rib fracture nonunions. All studies reported a significant reduction of pain with increased satisfaction in the majority of patients. There were a total of 71 postoperative complications, the most common of which included surgical site infections, hardware failure, and hematoma. The most serious complications were insulting injury to the lung parenchyma or pleura; however, these were extremely rare based off the current literature. The use of bone grafting was common with eight of the nine studies mentioning the benefits of grafting. CONCLUSION: Surgical stabilization of rib fracture nonunions appears to be an appropriate treatment alternative, and various techniques and approaches may be used with similar success. Further studies with higher level of evidence are recommended on the subject.

Assessing the quality of patient-reported outcome measurements for gynecological cancers: a systematic review.

Aim: To provide perspective on patient-reported outcome measurement (PROM) instruments to adopt in patients diagnosed with gynecological cancers. Methods: A systematic search was conducted to identify PROMs developed for or applied in gynecological cancer populations. PROMs identified in more than one study subsequently underwent assessment according to the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) criteria. Results: Overall, 55 PROMs were identified within the gynecological cancer setting, and 20 were assessed according to COSMIN guidelines. Most PROMs had limited information reported, but a best fit approach was adopted to recommend a number of instruments for use in patients with gynecological cancer. Conclusion: Further study to assess the methodological quality of each PROM utilized in gynecological cancers is warranted to endorse the recommendations of this review.

Gynecological cancers are cancers which occur in the reproductive system of women. The cervical cancer screening program and development of new treatments mean that women with gynecological cancers are now living longer than before. However, these new treatments may have side effects that can affect the quality of life of women with cancer. Many care providers now agree that looking at women's quality of life during their gynecological cancer journey is an important part of their treatment. Patient-reported outcome measurements (PROMs) are questionnaires that the patient completes to measure their symptoms and quality of life. There are a lot of PROMs available to choose from, and it can be difficult to select one that is relevant and understandable for all women with gynecological cancer. This article searched the literature to find all PROMs that can be completed by women with gynecological cancer and then measured each of the PROM's quality. PROM quality was measured by looking at validity (whether the questionnaire measures what it is supposed to measure), reliability (that the questionnaire is not subject to different errors in measuring), and sensitivity (that the questionnaire can measure changes in questionnaire scores over time). Overall, this study found that there were a few PROMs that were of good enough quality to be completed by women with gynecological cancers. These questionnaires are called the European Organisation for Research and Treatment of Cancer Quality-of-Life Questionnaire Cervical Cancer Module (EORTC QLQ-CX24), the Functional Assessment of Cancer Therapy - General (FACT-G), European Organisation for Research and Treatment of Cancer Quality-of-Life Questionnaire Endometrial Cancer (EORTC QLQ-EN24), Functional Assessment of Cancer Therapy Gynecologic Oncology Group ­ Neurotoxicity (FACT-GOG/Ntx), Functional Assessment of Cancer Therapy ­ Ovarian (FACT-O) and Female Sexual Function Index (FSFI). Each questionnaire can be filled out by women with different types of gynecological cancer, and the FSFI measures sexual problems that women may experience after cancer treatment.

Operative fixation of rib fracture nonunions.

INTRODUCTION: Rib fractures are common injuries in trauma patients that often heal without intervention. Infrequently, symptomatic rib fracture nonunions are a complication after rib fractures. There is a paucity of literature on the surgical treatment of rib fracture nonunion. The purpose of this study was to describe the efficacy of rib fracture nonunion operative fixation with particular focus on surgical technique, healing rates, and complications. MATERIALS AND METHODS: Patients aged ≥ 18 years with symptomatic rib fracture nonunions treated with open reduction and internal fixation (ORIF) with locking plates at a single urban level 1 trauma center were retrospectively reviewed. Pertinent demographic, clinical, radiographic, and surgical data were collected and analyzed. RESULTS: A total of 18 patients met inclusion criteria. The mean time from injury to undergoing ORIF for rib fracture nonunion was just under a year and the number of ribs plated was 2.95 ± 1.16 (1-5 ribs) with bone grafting used in six cases. All patients (100%) showed evidence of healing at an average of 2.65 ± 1.50 months (2-8 months). All patients reported a decrease in pain. No narcotic pain medication was used at an average of 3.88 ± 3.76 weeks (0-10 weeks) post-operatively. Intraoperative and postoperative complications were found in 4 (22.2%) patients. CONCLUSION: This study concluded that operative fixation of symptomatic rib fracture nonunion demonstrated favorable outcomes with reduction in preoperative pain levels, decreased use of narcotic pain medication, minimal complications, and a high rate of fracture union. This described method provides symptomatic relief, reduction in pain, and promotes bony healing of the fracture nonunion without development of major complications. We suggest that operative fixation should be considered as the primary method of treatment of symptomatic rib nonunions.

Reverse shoulder arthroplasty for proximal humerus fractures: a comparison of the deltoid split and deltopectoral approaches.

INTRODUCTION: Reverse shoulder arthroplasty (RSA) is a common treatment for three- and four-part proximal humerus fractures. RSA is commonly performed through a deltopectoral (DP) surgical approach; however, a deltoid split (DS) approach has been described. The purpose of this study was to compare the intraoperative and postoperative outcomes of patients undergoing RSA for proximal humerus fractures through a DP and DS approach. MATERIALS AND METHODS: A retrospective study of patients who underwent RSA for proximal humerus fractures at our level 1 trauma center was performed. Patients were evaluated at a minimum 1-year follow-up. Intraoperative outcomes as well as patient-reported outcome measures (PROMs) were analyzed. Postoperative complications, reoperation rates, and radiographic outcomes were evaluated. RESULTS: Sixteen patients were included in the DP group and 13 patients in the DS group. The average follow-up was 28.6 months in the DP group and 21.1 months in the DS group. There were no significant differences in patient demographics or injury characteristics. Operative time was significantly shorter in the DS group (103 min) compared to the DP group (123 min). Tuberosity repair was performed in 81.3% of patients in the DP group and 92.3% of patients in the DS group. No significant differences were found in PROMs at final follow-up. There were no significant differences in postoperative complications. Three patients in the DP group and one patient in the DS group required reoperation. Lastly, 81.8% of patients achieved tuberosity union in the DS group and 66.7% in the DP group; however, this did not reach statistical significance. CONCLUSION: This study demonstrates that RSA for proximal humerus fractures performed through a DS or DP approach may result in similar intraoperative and postoperative outcomes, with no difference in reoperation or complication rates.

Intramedullary Screw Fixation Versus Traditional Plating for Distal Fibula Fractures.

The traditional method of treating fibular fractures in unstable ankle injuries involves open reduction and internal fixation with a plate and screw construct. Less invasive percutaneous fixation techniques with intramedullary fibular screws have been utilized for many years to reduce wound and implant complications while maintaining a stable ankle mortise. However, there have been no direct case-control studies comparing percutaneous intramedullary fibular screw fixation to the traditional open reduction and internal fixation with plates and screws. In our study, we compared radiographic and clinical outcomes for unstable ankle fractures in which the fibula fracture was treated with either a percutaneous intramedullary screw or by open reduction and internal fixation with a plate and screw construct. We retrospectively reviewed 69 consecutive patients from 2011 to 2019 with unstable ankle fractures treated with intramedullary fibular screws and compared them to 216 case-control patients treated with traditional plate and screw construct over the same time period. The average follow-up for the intramedullary screw group was 11.5 months and 15.2 months for the plate and screw group. We collected general demographic data, measured intraoperative and final follow-up talocrural angles, Kellgren-Lawrence osteoarthritis grade, union rates, implant removal rates, infection rates, and American Orthopedic Foot and Ankle Society ankle-hindfoot scores. The intramedullary screw group had a statistically significant lower rate of delayed implant removal (8.7% vs 23.6%) and there was no detectable difference in other measures.

Correlation of Lisfranc Injuries With Regional Bone Density.

Lisfranc injuries present a challenge due to their ubiquity though frequent missed diagnoses. A paucity of data exists associating the contribution of bone density to injury type. This investigation compares the regional bone density between Lisfranc injury types using computed-tomography (CT)-derived Hounsfield units. A retrospective chart review identified patients with gross ligamentous and avulsion-type Lisfranc injuries determined by CT examination of the second metatarsal base and medial cuneiform. Regional bone density was assessed by averaging the Hounsfield units of the first metatarsal base, navicular, cuboid, calcaneus, and talus between 2 reviewers. Density was compared between injury type, isolated concomitant forefoot, and mid/hindfoot fractures. One hundred thirty-four patients were separated into avulsion (n = 85) and ligamentous (n = 49) groups. No statistically significant difference in patient body mass index, age, smoking status, or Quenu and Kuss injury pattern was observed between groups. The regional bone density of the cuboid (p = .03) and talus (p = .04) was greater in the ligamentous group. Lower extremity concomitant mid/hindfoot fracture patients exhibited greater regional bone density in the ligamentous group in all assessed bones (p ≤ .04) except the calcaneus. Ligamentous injuries of the Lisfranc complex are associated with increased regional bone density among patients sustaining concomitant mid/hindfoot fractures. This study expands the utility of regional bone density analysis in foot and ankle trauma.

18F FDG PET/CT and Novel Molecular Imaging for Directing Immunotherapy in Cancer.

Immunotherapy has transformed the treatment landscape of many cancers, with durable responses in disease previously associated with a poor prognosis. Patient selection remains a challenge, with predictive biomarkers an urgent unmet clinical need. Current predictive biomarkers, including programmed death-ligand 1 (PD-L1) (measured with immunohistochemistry), are imperfect. Promising biomarkers, including tumor mutation burden and tumor infiltrating lymphocyte density, fail to consistently predict response and have yet to translate to routine clinical practice. Heterogeneity of immune response within and between lesions presents a further challenge where fluorine 18 fluorodeoxyglucose PET/CT has a potential role in assessing response, stratifying treatment, and detecting and monitoring immune-related toxicities. Novel radiopharmaceuticals also present a unique opportunity to define the immune tumor microenvironment to better predict which patients may respond to therapy, for example by means of in vivo whole-body PD-L1 and CD8+ T cell expression imaging. In addition, longitudinal molecular imaging may help further define dynamic changes, particularly in cases of immunotherapy resistance, helping to direct a more personalized therapeutic approach. This review highlights current and emerging applications of molecular imaging to stratify, predict, and monitor molecular dynamics and treatment response in areas of clinical need.

Gastral Drumming in Vespula germanica (Hymenoptera: Vespidae): Vibrational Communication at Night Suggests Additional Roles in Colony Organization.

Gastral drumming (GD) is a type of vibrational communication that has been reported in several species of yellowjackets and hornets. Despite early claims that it acts as a hunger signal, a more recent study found evidence that it acts as a nest-based food-recruitment signal, the first reported for eusocial wasps. Early studies also claimed, without supporting data, that it is produced most often in the early morning hours when the sun rises. Here, I recorded drumming continuously in colonies of Vespula germanica (Fabricius) to assess whether production was highest in the morning. Although I found no evidence in support of greater early morning production, I found, surprisingly, that it is produced at night, a time when foraging does not occur. When these results are combined with the results from previous studies on this species and similar findings in honey bees, they suggest that GD may be a modulatory signal, which acts by increasing general activity levels and by increasing the rate that individuals come into contact with social cues.

The Intercalary Fragment in Posterior Malleolus Fractures: Characterization and Significance.

Methods of fixation in ankle fractures involving the posterior malleolus have become increasingly scrutinized. With the increase in computed tomography (CT), an intercalary fracture fragment (ICF) adjacent to the posterior malleolus has been oft described. Treatment of the ICF remains controversial and the purpose of this study was to evaluate radiographic and clinical outcomes in patients who had direct reduction and fixation of this fragment compared to those where the ICF was not fixed. This retrospective study included 249 trimalleolar and posterior pilon ankle fractures grouped into those who had the ICF reduced and fixed (n = 74) and those where the ICF was not directly addressed (n = 175). CT scans were evaluated for size and location of the ICF. Demographic, radiographic and intraoperative variables were collected and analyzed. The group which had the ICF reduced and fixed had decreased Kellgren-Lawrence scores (p = .001). There was also a higher rate of repeat surgery in the group who had the ICF fixed, although not meeting statistical significance. There were no differences in size or location of the ICF fragment between groups. We did identify similarities with other studies in regard to size and posterolateral location of the ICF between groups. However, based on worsening radiographic outcomes of the group where the ICF was reduced and fixed, we do not necessarily recommend universal treatment of this fragment. The surgeon's goal should always be a concentric articular reduction and treatment of the ICF should be considered on a case-by-case basis.

Acetylcholine Modulates Cerebellar Granule Cell Spiking by Regulating the Balance of Synaptic Excitation and Inhibition.

Sensorimotor integration in the cerebellum is essential for refining motor output, and the first stage of this processing occurs in the granule cell layer. Recent evidence suggests that granule cell layer synaptic integration can be contextually modified, although the circuit mechanisms that could mediate such modulation remain largely unknown. Here we investigate the role of ACh in regulating granule cell layer synaptic integration in male rats and mice of both sexes. We find that Golgi cells, interneurons that provide the sole source of inhibition to the granule cell layer, express both nicotinic and muscarinic cholinergic receptors. While acute ACh application can modestly depolarize some Golgi cells, the net effect of longer, optogenetically induced ACh release is to strongly hyperpolarize Golgi cells. Golgi cell hyperpolarization by ACh leads to a significant reduction in both tonic and evoked granule cell synaptic inhibition. ACh also reduces glutamate release from mossy fibers by acting on presynaptic muscarinic receptors. Surprisingly, despite these consistent effects on Golgi cells and mossy fibers, ACh can either increase or decrease the spike probability of granule cells as measured by noninvasive cell-attached recordings. By constructing an integrate-and-fire model of granule cell layer population activity, we find that the direction of spike rate modulation can be accounted for predominately by the initial balance of excitation and inhibition onto individual granule cells. Together, these experiments demonstrate that ACh can modulate population-level granule cell responses by altering the ratios of excitation and inhibition at the first stage of cerebellar processing.SIGNIFICANCE STATEMENT The cerebellum plays a key role in motor control and motor learning. While it is known that behavioral context can modify motor learning, the circuit basis of such modulation has remained unclear. Here we find that a key neuromodulator, ACh, can alter the balance of excitation and inhibition at the first stage of cerebellar processing. These results suggest that ACh could play a key role in altering cerebellar learning by modifying how sensorimotor input is represented at the input layer of the cerebellum.

An assessment of the use of patient reported outcome measurements (PROMs) in cancers of the pelvic abdominal cavity: identifying oncologic benefit and an evidence-practice gap in routine clinical practice.

BACKGROUND: Patient reported outcome measurements (PROMs) are emerging as an important component of patient management in the cancer setting, providing broad perspectives on patients' quality of life and experience. The use of PROMs is, however, generally limited to the context of randomised control trials, as healthcare services are challenged to sustain high quality of care whilst facing increasing demand and financial shortfalls. We performed a systematic review of the literature to identify any oncological benefit of using PROMs and investigate the wider impact on patient experience, in cancers of the pelvic abdominal cavity specifically. METHODS: A systematic review of the literature was conducted using MEDLINE (Pubmed) and Ovid Gateway (Embase and Ovid) until April 2020. Studies investigating the oncological outcomes of PROMs were deemed suitable for inclusion. RESULTS: A total of 21 studies were included from 2167 screened articles. Various domains of quality of life (QoL) were identified as potential prognosticators for oncologic outcomes in cancers of the pelvic abdominal cavity, independent of other clinicopathological features of disease: 3 studies identified global QoL as a prognostic factor, 6 studies identified physical and role functioning, and 2 studies highlighted fatigue. In addition to improved outcomes, a number of included studies also reported that the use of PROMs enhanced both patient-clinician communication and patient satisfaction with care in the clinical setting. CONCLUSIONS: This review highlights the necessity of routine collection of PROMs within the pelvic abdominal cancer setting to improve patient quality of life and outcomes.

Understanding the causes and consequences of variability in infant ERP editing practices.

The current study examined the effects of variability on infant event-related potential (ERP) data editing methods. A widespread approach for analyzing infant ERPs is through a trial-by-trial editing process. Researchers identify electroencephalogram (EEG) channels containing artifacts and reject trials that are judged to contain excessive noise. This process can be performed manually by experienced researchers, partially automated by specialized software, or completely automated using an artifact-detection algorithm. Here, we compared the editing process from four different editors-three human experts and an automated algorithm-on the final ERP from an existing infant EEG dataset. Findings reveal that agreement between editors was low, for both the numbers of included trials and of interpolated channels. Critically, variability resulted in differences in the final ERP morphology and in the statistical results of the target ERP that each editor obtained. We also analyzed sources of disagreement by estimating the EEG characteristics that each human editor considered for accepting an ERP trial. In sum, our study reveals significant variability in ERP data editing pipelines, which has important consequences for the final ERP results. These findings represent an important step toward developing best practices for ERP editing methods in infancy research.

Associations between Hospital Volume and Outcomes of Robot-Assisted Radical Prostatectomy.

PURPOSE: Robot-assisted radical prostatectomy has become the predominant surgical modality to manage localized prostate cancer in the U.S. However, there are few studies focusing on the associations between hospital volume and outcomes of robot-assisted radical prostatectomy. MATERIALS AND METHODS: We identified robot-assisted radical prostatectomies for clinically localized (cT1-2N0M0) prostate cancer diagnosed between 2010 and 2014 in the National Cancer Database. We categorized annual average hospital robot-assisted radical prostatectomy volume into very low, low, medium, high and very high by most closely sorting the final included patients into 5 equal-sized groups (quintiles). Outcomes included 30-day mortality, 90-day mortality, conversion (to open), prolonged length of stay (more than 2 days), 30-day (unplanned) readmission, positive surgical margin and lymph node dissection rates. RESULTS: A total of 114,957 patients were included in the study, and hospital volume was categorized into very low (3 to 45 cases per year), low (46 to 72), medium (73 to 113), high (114 to 218) and very high (219 or more). Overall 30-day mortality (0.12%), 90-day mortality (0.16%) and conversion rates (0.65%) were low. Multivariable logistic regressions showed that compared with the very low volume group, higher hospital volume was associated with lower odds of conversion to open surgery (OR 0.23, p <0.001 for very high), prolonged length of stay (OR 0.25, p <0.001 for very high), 30-day readmission (OR 0.53, p <0.001 for very high) and positive surgical margins (OR 0.61, p <0.001 for very high). Higher hospital volume was also associated with higher odds of lymph node dissection in the intermediate/high risk cohort (OR 3.23, p <0.001 for very high). CONCLUSIONS: Patients undergoing robot-assisted radical prostatectomy at higher volume hospitals are likely to have improved perioperative and superior oncologic outcomes compared to lower volume hospitals.

Complication rate after cystectomy following pelvic radiotherapy: an international, multicenter, retrospective series of 682 cases.

PURPOSE: Conflicting evidence exists on the complication rates after cystectomy following previous radiation (pRTC) with only a few available series. We aim to assess the complication rate of pRTC for abdominal-pelvic malignancies. METHODS: Patients treated with radical cystectomy following any previous history of RT and with available information on complications for a minimum of 1 year were included. Univariable and multivariable logistic regression models were used to assess the relationship between the variable parameters and the risk of any complication. RESULTS: 682 patients underwent pRTC after a previous RT (80.5% EBRT) for prostate, bladder (BC), gynecological or other cancers in 49.1%, 27.4%, 9.8% and 12.9%, respectively. Overall, 512 (75.1%) had at least one post-surgical complication, classified as Clavien ≥ 3 in 29.6% and Clavien V in 2.9%. At least one surgical complication occurred in 350 (51.3%), including bowel leakage in 6.2% and ureteric stricture in 9.4%. A medical complication was observed in 359 (52.6%) patients, with UTI/pyelonephritis being the most common (19%), followed by renal failure (12%). The majority of patients (86%) received an incontinent urinary diversion. In multivariable analysis adjusted for age, gender and type of RT, patients treated with RT for bladder cancer had a 1.7 times increased relative risk of experiencing any complication after RC compared to those with RT for prostate cancer (p = 0.023). The type of diversion (continent vs non-continent) did not influence the risk of complications. CONCLUSION: pRTC carries a high rate of major complications that dramatically exceeds the rates reported in RT-naïve RCs.

Decoding non-random mutational signatures at Cas9 targeted sites.

The mutation patterns at Cas9 targeted sites contain unique information regarding the nuclease activity and repair mechanisms in mammalian cells. However, analytical framework for extracting such information are lacking. Here, we present a novel computational platform called Rational InDel Meta-Analysis (RIMA) that enables an in-depth comprehensive analysis of Cas9-induced genetic alterations, especially InDels mutations. RIMA can be used to quantitate the contribution of classical microhomology-mediated end joining (c-MMEJ) pathway in the formation of mutations at Cas9 target sites. We used RIMA to compare mutational signatures at 15 independent Cas9 target sites in human A549 wildtype and A549-POLQ knockout cells to elucidate the role of DNA polymerase θ in c-MMEJ. Moreover, the single nucleotide insertions at the Cas9 target sites represent duplications of preceding nucleotides, suggesting that the flexibility of the Cas9 nuclease domains results in both blunt- and staggered-end cuts. Thymine at the fourth nucleotide before protospacer adjacent motif (PAM) results in a two-fold higher occurrence of single nucleotide InDels compared to guanine at the same position. This study provides a novel approach for the characterization of the Cas9 nucleases with improved accuracy in predicting genome editing outcomes and a potential strategy for homology-independent targeted genomic integration.

A 9-Point Risk Assessment for Patients Who Inject Drugs and Require Intravenous Antibiotics: Focusing Inpatient Resources on Patients at Greatest Risk of Ongoing Drug Use.

A 9-point risk assessment identified persons with a history of injection drug use who were safe for discharge. "Low-risk" patients were discharged with outpatient antibiotics; others continued inpatient treatment. Use of the assessment reduced the mean length of stay by 20 days and total direct cost by 33%, creating capacity for an additional 333 patients.

Association of Smoking and Death from Genitourinary Malignancies: Analysis of the National Longitudinal Mortality Study.

PURPOSE: We explored the association between tobacco use and genitourinary cancer specific survival in a contemporary, nationally representative sample of the United States civilian population. MATERIALS AND METHODS: A total of 493,282 participants in the National Longitudinal Mortality Study who provided detailed tobacco information from 1993 to 2005 were included in study. Our primary outcome was death from bladder, kidney or prostate cancer. Cause of death was determined from death certificates. Analyzed smoking parameters included smoking status at the time of the survey, age at the start of smoking and home smoking rules. Multivariable Cox regression models were used to assess associations of different smoking parameters with bladder, kidney and prostate cancer specific mortality. RESULTS: During a 5-year followup 5.6% of participants who had ever smoked died compared to 3.1% of those who had never smoked (p <0.0001). Of those who died of bladder, kidney and prostate cancer 62%, 58% and 62%, respectively, were ever smokers. On multivariable analysis ever smoking was associated with bladder and kidney cancer mortality (HR 1.92, 95% CI 1.25-2.97, and HR 1.54, 95% CI 1.01-2.34, respectively). Additionally, starting to smoke during teenage years and smoking at home were associated with bladder cancer specific mortality (HR 2.14, 95% CI 1.28-3.56 and HR 2.99, 95% CI 1.34-6.65) and kidney cancer specific mortality (HR 1.65, 95% CI 1.03-2.66 and HR 2.84, 95% CI 1.54-5.23, respectively). However, only everyday smoking was associated with an increased risk of prostate cancer mortality (HR 1.81, 95% CI 1.30-2.53). CONCLUSIONS: In a nationally representative study we confirmed the association between smoking intensity and mortality from genitourinary malignancies. Starting to smoke at a younger age and smoking at home conferred a significantly higher risk of death from bladder and kidney cancers.