Defining a Cancer Dependency Map.

Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.

Implementation of a "threat and error" model in complex neonatal cardiac surgery patients to identify quality improvement opportunities.

INTRODUCTION: Neonates undergoing surgery for congenital heart disease are vulnerable to adverse events. Conventional quality improvement processes centring on mortality and significant morbidity leave a gap in the identification of systematic processes that, though not directly linked to an error, may still contribute to adverse outcomes. Implementation of a multidisciplinary "flight path" process for surgical patients may be used to identify modifiable threats and errors and generate action items, which may lead to quality improvement. METHODS: A retrospective review of our neonatal "flight path" initiative was performed. Within 72 hours of a cardiac surgery, a meeting of the multidisciplinary patient care team occurs. A "flight path" is generated, graphically illustrating the patient's hospital course. Threats, errors, or unintended consequences are identified. Action items are generated, and a working group is formed to address the items. A patient's flight path is updated weekly until discharge. The errors and action items are logged into a database, which is analysed quarterly to identify trends. RESULTS: Thirty one patients underwent flight path review over a 1-year period; 22.5% (N = 7) of patients had an error-free "flight." Eleven action items were generated - four from identified errors and seven from identified threats. Nine action items were completed. CONCLUSIONS: Flight path reviews of congenital cardiac patients can be generated with few resources and aid in the detection of quality improvement opportunities. The regular multidisciplinary meetings that occur as a part of the flight path review process can promote inter-professional teamwork.

Exploring Choke Holds in Brazilian Jiujitsu Athletes: A Demographic Study.

Introduction Brazilian jiujitsu is a relatively new sport that has grown exponentially in popularity along with the growth of the Ultimate Fighting Championship (UFC). In jiujitsu, there are a variety of submissions with a choke hold being one of the most popular. There is a subset of athletes in jiujitsu who believes chokes are safe. However, there have been case reports of relatively young athletes suffering strokes secondary to internal carotid or vertebral artery dissections after being placed in choke holds. There have been manuscripts describing the injury profile in jiujitsu, but none mention stroke or dissections. This study evaluated how frequently chokes happen in jiujitsu and if athletes have ever experienced symptoms consistent with cervical artery dissection (CAD). Additionally, this study aimed to describe the training frequency and baseline demographics of jiujitsu athletes. Methods A survey was distributed throughout social media platforms which asked both quantitative and qualitative questions regarding athlete training. The survey consisted of 28 questions which collected largely baseline grappling information about the participants such as how long they trained, how often they spar, favorite submission, how frequently they are choked, etc. This data was then analyzed using odds ratio and one sample t-test to evaluate for statistical differences. Results A total of 521 participants were included in the analysis. The participants were mostly male (84.7%), trained for four years, four times per week; 99.8% (520) participated in sparring, with an average age of 37; and 55.7% (290) have experienced symptoms consistent with CAD. Descriptive statistics revealed that individuals who were 37 years of age or younger were more likely to experience symptoms consistent with CAD (odds ratio: 1.5337 (95% confidence interval (CI): 1.0827-2.1727). Athletes that were 37 years of age or younger have been training for fewer years (4.7 years vs 8.8 years) but train more days per week (4.03 times per week vs 3.76 time per week), drill for a longer amount of time (46.8 minutes per class vs 38.3 minutes per class), attend longer classes (81.12 minutes vs 72.3 minutes), and train for a longer period of time per week (338.5 minutes vs 274.6 minutes) than athletes over 37 years. All previously mentioned variables were analyzed using a one sample t-test and were significant at the α = 0.05 level. The lone qualitative question regarding the term "train brain" revealed that of those who experienced it, 84.1% (58) described it as a cognitive/physical impairing event. Conclusion Jiujitsu athletes train multiple times per week and are frequently exposed to choke holds. There is no literature to examine the long-term effects of these chokes on the athlete's cervical vasculature. Additional studies should be conducted to evaluate the effects of the repetitive stress placed on these vessels.

Nuclear factor kappa B pathway associated biomarkers in AIDS defining malignancies.

The nuclear factor kappa B (NFκB) pathway is essential for many human cancers. Therapeutics such as bortezomib (Velcade™) that interfere with NFκB signaling are of great clinical interest. NFκB signaling, however, is multifaceted and variable among tissues, developmental and disease entities. Hence, targeted biomarkers of NFκB pathways are of prime importance for clinical research. We developed a novel real-time qPCR-based NFκB array. Only mechanistically validated NFκB targets were included. We then used random-forest classification to define individual genes and gene combinations within the NFκB pathways that define viral lymphoma subclasses as well as Kaposi sarcoma (KS). Few NFκB targets emerged that were universally present in all tumor types tested, underscoring the need for additional tumor-type specific biomarker discovery. (i) We uncovered tissue of origin-specific tumor markers, specifically CD69, CSF-1 and complement factor B (C1QBP) for primary effusion lymphoma (PEL); IL1-beta, cyclinD3 and CD48 for KS. We found that IL12, jun-B, msx-1 and thrombospondin 2 were associated with EBV co-infection in PEL. (ii) We defined the NFκB signature of Epstein-Barr virus (EBV) positive AIDS-associated Burkitt lymphoma (BL). This signature identified CCR5 as the key marker. (iii) This signature differed from EBV negative BL consistent with the idea that EBV not only activates NFκB activity but that this virus also reprograms NFκB signaling toward different targets.

Adult T-cell leukemia/lymphoma development in HTLV-1-infected humanized SCID mice.

The molecular and genetic factors induced by human T-lymphotropic virus type-1 (HTLV-1) that initiate adult T-cell leukemia/lymphoma (ATLL) remain unclear, in part from the lack of an animal model that accurately recapitulates leukemogenesis. HTLV-1-infected humanized nonobese diabetic severe combined immunodeficiency (HU-NOD/SCID) mice were generated by inoculation of NOD/SCID mice with CD34(+) hematopoietic progenitor and stem cells (CD34(+) HP/HSCs) infected ex vivo with HTLV-1. HTLV-1-HU-NOD/SCID mice exclusively developed CD4(+) T-cell lymphomas with characteristics similar to ATLL and elevated proliferation of infected human stem cells (CD34(+)CD38(-)) in the bone marrow were observed in mice developing malignancies. Purified CD34(+) HP/HSCs from HTLV-1-infected patient peripheral blood mononuclear cells revealed proviral integrations suggesting viral infection of human bone marrow-derived stem cells. NOD/SCID mice reconstituted with CD34(+) HP/HSCs transduced with a lentivirus vector expressing the HTLV-1 oncoprotein (Tax1) also developed CD4(+) lymphomas. The recapitulation of a CD4(+) T-cell lymphoma in HU-NOD/SCID mice suggests that HSCs provide a viral reservoir in vivo and act as cellular targets for cell transformation in humans. This animal model of ATLL will provide an important tool for the identification of molecular and cellular events that control the initiation and progression of the lymphoma and potential therapeutic targets to block tumor development.

Six-month outcomes of the HOPE smartphone application designed to support treatment with medications for opioid use disorder and piloted during an early statewide COVID-19 lockdown.

BACKGROUND: Morbidity and mortality related to opioid use disorder (OUD) in the U.S. is at an all-time high. Innovative approaches are needed to address gaps in retention in treatment with medications for opioid use disorder (MOUD). Mobile health (mHealth) approaches have shown improvement in engagement in care and associated clinical outcomes for a variety of chronic diseases, but mHealth tools designed specifically to support patients treated with MOUD are limited. METHODS: Following user-centered development and testing phases, a multi-feature smartphone application called HOPE (Heal. Overcome. Persist. Endure) was piloted in a small cohort of patients receiving MOUD and at high risk of disengagement in care at an office-based opioid treatment (OBOT) clinic in Central Virginia. Outcomes were tracked over a six-month period following patient enrollment. They included retention in care at the OBOT clinic, usage of various features of the application, and self-rated measures of mental health, substance use, treatment and recovery. RESULTS: Of the 25 participants in the HOPE pilot study, a majority were retained in care at 6 months (56%). Uptake of bi-directional features including messaging with providers and daily check-ins of mood, stress and medication adherence peaked at one month, and usage persisted through the sixth month. Patients who reported that distance to clinic was a problem at baseline had higher loss to follow up compared to those without distance as a reported barrier (67% vs 23%, p = 0.03). Patients lost to in-person clinic follow up continued to engage with one or more app features, indicating that mHealth approaches may bridge barriers to clinic visit attendance. Participants surveyed at baseline and 6 months (N = 16) scored higher on scales related to overall self-control and self-efficacy related to drug abstinence. CONCLUSIONS: A pilot study of a novel multi-feature smartphone application to support OUD treatment showed acceptable retention in care and patient usage at 6 months. Further study within a larger population is needed to characterize 'real world' uptake and association with outcomes related to retention in care, relapse prevention, and opioid-associated mortality.

Convalescent Plasma for Preventing Critical Illness in COVID-19: a Phase 2 Trial and Immune Profile.

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an unprecedented event requiring frequent adaptation to changing clinical circumstances. Convalescent immune plasma (CIP) is a promising treatment that can be mobilized rapidly in a pandemic setting. We tested whether administration of SARS-CoV-2 CIP at hospital admission could reduce the rate of ICU transfer or 28-day mortality or alter levels of specific antibody responses before and after CIP infusion. In a single-arm phase II study, patients >18 years-old with respiratory symptoms with confirmed COVID-19 infection who were admitted to a non-ICU bed were administered two units of CIP within 72 h of admission. Levels of SARS-CoV-2 detected by PCR in the respiratory tract and circulating anti-SARS-CoV-2 antibody titers were sequentially measured before and after CIP transfusion. Twenty-nine patients were transfused high titer CIP and 48 contemporaneous comparable controls were identified. All classes of antibodies to the three SARS-CoV-2 target proteins were significantly increased at days 7 and 14 post-transfusion compared with baseline (P < 0.01). Anti-nucleocapsid IgA levels were reduced at day 28, suggesting that the initial rise may have been due to the contribution of CIP. The groups were well-balanced, without statistically significant differences in demographics or co-morbidities or use of remdesivir or dexamethasone. In participants transfused with CIP, the rate of ICU transfer was 13.8% compared to 27.1% for controls with a hazard ratio 0.506 (95% CI 0.165-1.554), and 28-day mortality was 6.9% compared to 10.4% for controls, hazard ratio 0.640 (95% CI 0.124-3.298). IMPORTANCE Transfusion of high-titer CIP to non-critically ill patients early after admission with COVID-19 respiratory disease was associated with significantly increased anti-SARS-CoV-2 specific antibodies (compared to baseline) and a non-significant reduction in ICU transfer and death (compared to controls). This prospective phase II trial provides a suggestion that the antiviral effects of CIP from early in the COVID-19 pandemic may delay progression to critical illness and death in specific patient populations. This study informs the optimal timing and potential population of use for CIP in COVID-19, particularly in settings without access to other interventions, or in planning for future coronavirus pandemics.

Pre-micro RNA signatures delineate stages of endothelial cell transformation in Kaposi sarcoma.

MicroRNAs (miRNA) have emerged as key regulators of cell lineage differentiation and cancer. We used precursor miRNA profiling by a novel real-time QPCR method (i) to define progressive stages of endothelial cell transformation cumulating in Kaposi sarcoma (KS) and (ii) to identify specific miRNAs that serve as biomarkers for tumor progression. We were able to compare primary patient biopsies to well-established culture and mouse tumor models. Loss of mir-221 and gain of mir-15 expression demarked the transition from merely immortalized to fully tumorigenic endothelial cells. Mir-140 and Kaposi sarcoma-associated herpesvirus viral miRNAs increased linearly with the degree of transformation. Mir-24 emerged as a biomarker specific for KS.

Tumor suppressor microRNAs are underrepresented in primary effusion lymphoma and Kaposi sarcoma.

The presence of tumor-specific microRNAs reflects tissue of origin and tumor stage. We show that the absence of miRNAs likewise can be used to determine tumor origin (miR-155) and proliferation state because tumor suppressor miRNAs (miR-222/221, let-7 family) were significantly down-regulated in primary effusion lymphoma (PEL) and in Kaposi sarcoma (KS), an endothelial cell tumor. PEL and KS are associated with KS-associated herpesvirus infection. We identified 15 virally regulated miRNAs in latently infected, nontumorigenic endothelial cells. MiR-143/145 were elevated only in KS tumors, not virally infected endothelial cells. Thus, they represent tumor-specific, rather than virus-specific, miRNAs. Because many tumor suppressor proteins are wild-type in KS and PEL, down-regulation of multiple tumor suppressor miRNAs provides a novel, alternative mechanism of transformation.

Predicting cell health phenotypes using image-based morphology profiling.

Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug discovery and personalized medicine. We developed two customized microscopy assays, one using four targeted reagents and the other three targeted reagents, to collectively measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species, DNA damage, and cell cycle stage. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts. We provide a web app to browse predictions: http://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting datasets.

A Mobile Health App to Support Patients Receiving Medication-Assisted Treatment for Opioid Use Disorder: Development and Feasibility Study.

BACKGROUND: Opioid use disorder (OUD) is a public health crisis with more than 2 million people living with OUD in the United States. Medication-assisted treatment (MAT) is an evidence-based approach for the treatment of OUD that relies on a combination of behavioral therapy and medication. Less than half of those living with OUD are accessing this treatment. Mobile technology can enhance the treatment of chronic diseases in readily accessible and cost-effective ways through self-monitoring and support. OBJECTIVE: The aim of this study is to describe the adaptation of a mobile platform for patients undergoing treatment for OUD and preliminary pilot testing results. METHODS: Our study was conducted with patient and provider participants at the University of Virginia MAT clinic and was approved by the institutional review board. The formative phase included semistructured interviews to understand the needs of patients with OUD, providers' perspectives, and opportunities for MAT support via a mobile app. A second round of formative interviews used mock-ups of app features to collect feedback on feature function and desirability. Formative participants' input from 16 interviews then informed the development of a functional smartphone app. Patient participants (n=25) and provider participants (n=3) were enrolled in a 6-month pilot study of the completed platform. Patient app use and usability interviews, including a system usability score and open-ended questions, were completed 1 month into the pilot study. Open-ended responses were analyzed for prevalent themes. RESULTS: Formative interviews resulted in the development of a mobile app, named HOPE, which includes both evidence-based and participant-suggested features. The features included daily prompts for monitoring mood, stress, treatment adherence, and substance use; patient tracking of goals, reminders, and triggering or encouraging experiences; informational resources; an anonymous community board to share support with other patients; and secure messaging for communication between patients and providers. All patient participants engaged with at least one app feature during their first month of pilot study participation, and the daily self-monitoring prompts were the most used. Patients and providers reported high levels of system usability (mean 86.9, SD 10.2 and mean 83.3, SD 12.8, respectively). Qualitative analysis of open-ended usability questions highlighted the value of self-monitoring, access to support through the app, and perceived improvement in connection to care and communication for both patient and provider participants. CONCLUSIONS: The use of the HOPE program by pilot participants, high usability scoring, and positive perceptions from 1-month interviews indicate successful program development. By engaging with end users and eliciting feedback throughout the development process, we were able to create an app and a web portal that was highly usable and acceptable to study participants. Further work is needed to understand the program's effect on clinical outcomes, patient linkage, and engagement in care.

Convalescent plasma for preventing critical illness in COVID-19: A phase 2 trial and immune profile.

RATIONALE: The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an unprecedented event requiring rapid adaptation to changing clinical circumstances. Convalescent immune plasma (CIP) is a promising treatment that can be mobilized rapidly in a pandemic setting. OBJECTIVES: We tested whether administration of SARS-CoV-2 CIP at hospital admission could reduce the rate of ICU transfer or 28 day mortality. METHODS: In a single-arm phase II study, patients >18 years-old with respiratory symptoms documented with COVID-19 infection who were admitted to a non-ICU bed were administered two units of CIP within 72 hours of admission. Detection of respiratory tract SARS-CoV-2 by polymerase chain reaction and circulating anti-SARS-CoV-2 antibody titers were measured before and at time points after CIP transfusion. MEASUREMENTS AND MAIN RESULTS: Twenty-nine patients were transfused CIP and forty-eight contemporaneous controls were identified with comparable baseline characteristics. Levels of anti-SARS-CoV-2 IgG, IgM, and IgA anti-spike, anti-receptor-binding domain, and anti-nucleocapsid significantly increased from baseline to post-transfusion for all proteins tested. In patients transfused with CIP, the rate of ICU transfer was 13.8% compared to 27.1% for controls with a hazard ratio 0.506 (95% CI 0.165-1.554), and 28-day mortality was 6.9% compared to 10.4% for controls, hazard ratio 0.640 (95% CI 0.124-3.298). CONCLUSIONS: Transfusion of high-titer CIP to patients early after admission with COVID-19 respiratory disease was associated with reduced ICU transfer and 28-day mortality but was not statistically significant. Follow up randomized trials may inform the use of CIP for COVID-19 or future coronavirus pandemics.

The D1/D5 Dopamine Partial Agonist PF-06412562 in Advanced-Stage Parkinson's Disease: A Feasibility Study.

BACKGROUND: Current drug treatments have little efficacy in advanced-to-end-stage Parkinson's disease (advPD), yet there are no reports of interventional trials in advPD. D1 dopamine agonists have the potential to provide benefit. OBJECTIVE: To determine the feasibility and safety of the selective D1/D5 dopamine partial agonist PF 06412562 in advPD. METHODS: A two-week, randomized, double blind, crossover phase Ib study in advPD patients compared standard-of-care (SoC) carbidopa/levodopa with PF 06412562. Each week, there was a Day 1 baseline evaluation with overnight levodopa washout, then treatment on Days 2 and 3 with either SoC or PF-06412562 (split dose 25 + 20 mg), followed by discharge on Day 4. Primary endpoints were safety and tolerability. Secondary endpoints were global clinical impression of change (GCI-C) rated by clinicians and caregivers. RESULTS: Eight advPD patients and their caregivers consented to participate and six were randomized (average disease duration: 22 y). None withdrew voluntarily. One participant with baseline Day 1 dehydration, pre-renal kidney injury, and autonomic dysfunction experienced symptomatic and serious hypotension after receiving PF-06412562 in Week 1 and was discontinued from the study. All other adverse events were rated mild (PF-06412562: n = 1, SoC: n = 0), moderate (PF-06412562: n = 1, SoC: n = 1), or severe but non-serious (PF-06412562: n = 3, SoC: n = 2). No clinically meaningful laboratory changes were observed. Among the five participants who completed the study, GCI-C favored PF-06412562 in two per clinicians' and four participants per caregivers' rating. CONCLUSION: PF-06412562 was tolerated in advPD patients. This study provides the feasibility for future safety and efficacy studies in this population with unmet needs.

Viral studies in burkitt lymphoma: association with Epstein-Barr virus but not HHV-8.

Burkitt lymphoma (BL) is a highly aggressive non-Hodgkin lymphoma, composed of a monomorphic population of medium-sized B cells with a high proliferation rate and a consistent MYC translocation. Epstein-Barr virus (EBV) has been associated with BL with different frequencies depending on the clinical variant. Kaposi sarcoma-associated herpesvirus, or human herpesvirus 8 (HHV-8), infects a wide range of normal cells, having a well-established role in the pathogenesis of various neoplasms, including Kaposi sarcoma, primary effusion lymphoma, multicentric Castleman disease (MCD) and MCD-associated plasmablastic lymphoma. In secondary immunodeficiencies, such as HIV-1 infection and organ transplantation, HHV-8 is considered an opportunistic pathogen linked to the development of lymphomas in patients with AIDS and HIV + patients. We studied the association of EBV and HHV-8 by immunohistochemical analysis, in situ hybridization, and polymerase chain reaction in a large number of well-characterized BLs. EBV was present in 45.0% of all BL cases with higher incidence in the pediatric group; most cases were EBV type A. We found no association of BL with HHV-8 in EBV + BL or in EBV-cases, including the HIV + BL group.

A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing.

The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2.

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

Estrogen dendrimer conjugates that preferentially activate extranuclear, nongenomic versus genomic pathways of estrogen action.

Estrogenic hormones are classically thought to exert their effects by binding to nuclear estrogen receptors and altering target gene transcription, but estrogens can also have nongenomic effects through rapid activation of membrane-initiated kinase cascades. The development of ligands that selectively activate only the nongenomic pathways would provide useful tools to investigate the significance of these pathways. We have prepared large, abiotic, nondegradable poly(amido)amine dendrimer macromolecules that are conjugated to multiple estrogen molecules through chemically robust linkages. Because of their charge and size, these estrogen-dendrimer conjugates (EDCs) remain outside the nucleus. They stimulate ERK, Shc, and Src phosphorylation in MCF-7 breast cancer cells at low concentrations, yet they are very ineffective in stimulating transcription of endogenous estrogen target genes, being approximately 10,000-fold less potent than estradiol in genomic actions. In contrast to estradiol, EDC was not effective in stimulating breast cancer cell proliferation. Because these EDC ligands activate nongenomic activity at concentrations at which they do not alter the transcription of estrogen target genes, they should be useful in studying extranuclear initiated pathways of estrogen action in a variety of target cells.

Spinal Kaposi sarcoma presenting without cutaneous manifestations. Case report.

Kaposi sarcoma (KS) is one of the most common tumors in patients with human immunodeficiency virus (HIV), which characteristically presents with cutaneous lesions. The authors report a rare case of spinal KS with no cutaneous manifestation in a 32-year-old man with the acquired immunodeficiency syndrome who presented with abdominal pain. A computed tomography scan revealed incidental lesions in his lumbar spine, and additional imaging studies revealed numerous lesions in the lumbosacral spine and pelvis. An open biopsy was performed, and histopathological examination of the lesion confirmed the diagnosis of KS. At the time of presentation, the patient had no skin lesion or any other manifestation indicative of KS. The authors suggest that in HIV-positive patients who present with spinal lesions, KS should be included in the differential diagnosis.

Complementary information derived from CRISPR Cas9 mediated gene deletion and suppression.

CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes. CRISPRc identified 98% of previously defined cell essential genes. After optimizing library construction by analysing transcriptional start sites (TSS), CRISRPi identified 92% of core cell essential genes and did not show a bias to regions involved in copy number alterations. However, bidirectional promoters scored as false positives in CRISRPi. We conclude that CRISPRc and CRISPRi have different off-target effects and combining these approaches provides complementary information in loss-of-function genetic screens.