Death-seq identifies regulators of cell death and senolytic therapies.

Selectively ablating damaged cells is an evolving therapeutic approach for age-related disease. Current methods for genome-wide screens to identify genes whose deletion might promote the death of damaged or senescent cells are generally underpowered because of the short timescales of cell death as well as the difficulty of scaling non-dividing cells. Here, we establish "Death-seq," a positive-selection CRISPR screen optimized to identify enhancers and mechanisms of cell death. Our screens identified synergistic enhancers of cell death induced by the known senolytic ABT-263. The screen also identified inducers of cell death and senescent cell clearance in models of age-related diseases by a related compound, ABT-199, which alone is not senolytic but exhibits less toxicity than ABT-263. Death-seq enables the systematic screening of cell death pathways to uncover molecular mechanisms of regulated cell death subroutines and identifies drug targets for the treatment of diverse pathological states such as senescence, cancer, and fibrosis.

Elotuzumab Plus Pomalidomide and Dexamethasone for Relapsed/Refractory Multiple Myeloma: Final Overall Survival Analysis From the Randomized Phase II ELOQUENT-3 Trial.

PURPOSE: In the phase II ELOQUENT-3 trial (ClinicalTrials.gov identifier: NCT02654132), elotuzumab combined with pomalidomide/dexamethasone (EPd) significantly improved progression-free survival (PFS) versus pomalidomide/dexamethasone (Pd) in patients with relapsed/refractory multiple myeloma (RRMM) previously treated with lenalidomide and a proteasome inhibitor (PI). Here, we present the final overall survival (OS) results. METHODS: Patients with RRMM who had received ≥ 2 prior lines of therapy, with disease refractory to last therapy and either refractory or relapsed and refractory to lenalidomide and a PI were randomly assigned (1:1) to receive EPd or Pd. The primary end point was PFS per investigator assessment. ORR and OS were secondary end points planned to be tested hierarchically. RESULTS: A total of 117 patients were randomly assigned to EPd (n = 60) and Pd (n = 57). Among treated patients (EPd 60, Pd 55), there were 37 (61.7%) deaths in the EPd group and 41 (74.5%) in the Pd group, most commonly because of disease progression (EPd 41.7%, Pd 49.1%). Median (95% CI) OS was significantly improved with EPd (29.8 [22.9 to 45.7] months) versus Pd (17.4 [13.8 to 27.7] months), with a hazard ratio of 0.59 (95% CI, 0.37 to 0.93; P = .0217). OS benefit with EPd was observed in most patient subgroups. The safety profile of EPd was consistent with prior reports with no new safety signals detected. CONCLUSION: EPd demonstrated a statistically significant improvement in OS versus Pd in patients with RRMM previously treated with lenalidomide and a PI who had disease refractory to last therapy. In this setting, ELOQUENT-3 is the first randomized study of a triplet regimen incorporating a monoclonal antibody and Pd to improve both PFS and OS significantly.

Inter-cellular CRISPR screens reveal regulators of cancer cell phagocytosis.

Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.

CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.

Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.

Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements.

Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.

A Multiplexed Assay for Exon Recognition Reveals that an Unappreciated Fraction of Rare Genetic Variants Cause Large-Effect Splicing Disruptions.

Mutations that lead to splicing defects can have severe consequences on gene function and cause disease. Here, we explore how human genetic variation affects exon recognition by developing a multiplexed functional assay of splicing using Sort-seq (MFASS). We assayed 27,733 variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,198 human exons in the MFASS minigene reporter and found that 3.8% (1,050) of variants, most of which are extremely rare, led to large-effect splice-disrupting variants (SDVs). Importantly, we find that 83% of SDVs are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate extant, rare genetic variants can have large functional effects on splicing at appreciable rates, even outside the context of disease, and MFASS enables their empirical assessment at scale.

Assessing Intensity of Nursing Care Needs Using Electronically Available Data.

Although previous research has confirmed that nurse staffing affects patient outcomes, some potentially important factors have not been accounted for in tools to assess relationships between staffing and outcomes. The aim of this project was to develop and test a Nursing Intensity of Care Index using electronically available data from 152 072 patient discharges from three hospitals. Initially, 1765 procedure codes were reviewed; 69 were confirmed as directly increasing nursing workload by at least 15 minutes per shift. Two research staff independently reviewed a random sample of 5 patient days to assess interrater reliability with complete scoring agreement. To assess face validity, eight nurse clinician experts reviewed factors included in the Nursing Intensity of Care Index to assess the accuracy of the nursing time estimates in the tool. To examine concurrent validity, Nursing Intensity of Care Index scores for a random sample of 28 patients from four clinical units were compared with assessments made by a unit-based clinical nurse (low/medium/high intensity) for the same patients on the same day with a Spearman correlation of 0.94. In preliminary testing, data for the Nursing Intensity of Care Index, which accurately reflect nursing care intensity, can be obtained electronically in real time. The next steps will be a discrete-event simulation model and large-scale field trials.

Male infertility: lifestyle factors and holistic, complementary, and alternative therapies.

While we may be comfortable with an allopathic approach to male infertility, we are also responsible for knowledge about lifestyle modifications and holistic, complementary, and alternative therapies that are used by many of our patients. This paper provides an evidence-based review separating fact from fiction for several of these therapies. There is sufficient literature to support weight reduction by diet and exercise, smoking cessation, and alcohol moderation. Supplements that have demonstrated positive effects on male fertility on small randomized controlled trial (RCT) include aescin, coenzyme Q 10 , glutathione, Korean red ginseng, L-carnitine, nigella sativa, omega-3, selenium, a combination of zinc and folate, and the Menevit antioxidant. There is no support for the use of Vitamin C, Vitamin E, or saffron. The data for Chinese herbal medications, acupuncture, mind-body practice, scrotal cooling, and faith-based healing are sparse or inconclusive.

Inflammatory expression profiles in monocyte-to-macrophage differentiation in patients with systemic lupus erythematosus and relationship with atherosclerosis.

INTRODUCTION: Our objectives were to examine mononuclear cell gene expression profiles in patients with systemic lupus erythematosus (SLE) and healthy controls and to compare subsets with and without atherosclerosis to determine which genes' expression is related to atherosclerosis in SLE. METHODS: Monocytes were obtained from 20 patients with SLE and 16 healthy controls and were in vitro-differentiated into macrophages. Subjects also underwent laboratory and imaging studies to evaluate for subclinical atherosclerosis. Whole-genome RNA expression microarray was performed, and gene expression was examined. RESULTS: Gene expression profiling was used to identify gene signatures that differentiated patients from controls and individuals with and without atherosclerosis. In monocytes, 9 out of 20 patients with SLE had an interferon-inducible signature compared with 2 out of 16 controls. By looking at gene expression during monocyte-to-macrophage differentiation, we identified pathways which were differentially regulated between SLE and controls and identified signatures based on relevant intracellular signaling molecules which could differentiate SLE patients with atherosclerosis from controls. Among patients with SLE, we used a previously defined 344-gene atherosclerosis signature in monocyte-to-macrophage differentiation to identify patient subgroups with and without atherosclerosis. Interestingly, this signature further classified patients on the basis of the presence of SLE disease activity and cardiovascular risk factors. CONCLUSIONS: Many genes were differentially regulated during monocyte-to-macrophage differentiation in SLE patients compared with controls. The expression of these genes in mononuclear cells is important in the pathogenesis of SLE, and molecular profiling using gene expression can help stratify SLE patients who may be at risk for development of atherosclerosis.

Latissimus dorsi tendon transfer for massive, irreparable posterosuperior rotator cuff tears: surgical technique.

Massive rotator cuff tears remain a complex and challenging problem for both the patient and the surgeon. Although significant advancements in surgical techniques as well as technology for arthroscopic and mini-open rotator cuff repairs have been made, many massive tears result in failed repair with continued progressive tendon retraction and degeneration. In cases when primary tendon to bone healing is impractical, latissimus dorsi tendon transfer provides promising and reproducible clinical results. Herein, we present a latissimus tendon transfer surgical technique, a procedure we have used as a salvage operation for failed arthroscopic/mini-open primary rotator cuff repair.

Sequence-dependent anesthesia-controlled times: a retrospective study in an ophthalmology department of a single-site hospital.

BACKGROUND: Anesthesia-controlled time (ACT) generally refers to the time durations before and after the period of surgery. The ACT is typically dependent on the sequence of 2 consecutive surgeries and thus adds to the complexity of operating room scheduling. We report a study on sequence-dependent ACTs at the West China Hospital (WCH), focusing on elective surgeries (also referred to as "procedures" below) performed by the ophthalmology department of WCH over a 5-year period, 2007 to 2012. METHODS: ACTs associated with 4 high-volume procedures: phacoemulsification, vitrectomy, strabismus correction, and glaucoma filtration. A total of 29,452 cases were studied, classified into 4 groups according to the sequence of the procedures involved. Specifically, P-P plots were used to determine the distributions of the ACTs, Kruskal-Wallis H test, Nemenyi test, and Student t test were performed to examine the sequence-dependent nature of the ACTs, and the t test was also applied to examine the advantage of sequencing the same procedures consecutively. Permutations were enumerated to identify the best sequence when different procedures were involved. Monte Carlo simulation was used to compute the total completion time, ACTs plus surgical periods, of any given sequence of procedures. RESULTS: We confirm via statistical tests that the ACTs follow lognormal distributions, and identify their corresponding means and variances. Furthermore, we verify that the ACTs are statistically different in means: they are sequence dependent in general. Using statistical tests, we conclude that it is best to sequence identical procedures consecutively, and we also identify the best sequence involving different procedures. Using Monte Carlo simulation, we compared the daily completion times using the best sequences we have identified against actual data from WCH over a 2-year period; the average reduction is 4.7% (with a standard error (SE) of ± ± 0.5%). CONCLUSIONS: ACTs are usually sequence dependent and hence should be considered in operating room scheduling. Although identifying the best sequence in general is a difficult optimization problem, in certain departments (such as the ophthalmology department of WCH) where a set of high-volume small-variety procedures is present, the best sequences can be systematically identified using a combination of statistical tests and Monte Carlo simulation as illustrated in this study.

Diagnosis of relevant prostate cancer using supplementary cores from magnetic resonance imaging-prompted areas following multiple failed biopsies.

OBJECTIVES: To establish the value of MRI in targeting re-biopsy for undiagnosed prostate cancer despite multiple negative biopsies and determine clinical relevance of detected tumors. MATERIALS AND METHODS: Thirty-eight patients who underwent MRI after 2 or more negative biopsies due to continued clinical suspicion and later underwent TRUS-guided biopsy supplemented by biopsy of suspicious areas depicted by MRI were identified. Diagnostic performance of endorectal 3T MRI in diagnosing missed cancer foci was assessed using biopsy results as the standard of reference. Ratio of positive biopsies using systematic versus MRI-prompted approaches was compared. Gleason scores of detected cancers were used as surrogate for clinical relevance. RESULTS: Thirty-four percent of patients who underwent MRI before re-biopsy had prostate cancer on subsequent biopsy. The positive biopsy yield with systematic sampling was 23% versus 92% with MRI-prompted biopsies(p<0.0001). Seventy-seven percent of tumors were detected exclusively in the MRI-prompted zones. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of MRI to provide a positive biopsy were 92%, 60%, 55%, 94% and 71%, respectively. The anterior gland and apical regions contained most tumors; 75% of cancers detected by MRI-prompted biopsy had Gleason score≥7. CONCLUSIONS: Clinically relevant tumors missed by multiple TRUS-guided biopsies can be detected by a MRI-prompted approach.

Diving for drugs: tunicate anticancer compounds.

The marine biosphere boasts tremendous biodiversity replete with structurally unique, active and selective secondary metabolites. Bioprospecting for antitumor compounds has been rewarding, and tunicates have been especially successful in yielding prospective cancer therapies. These compounds are now subjected to clinical trials in Europe and the USA. With the ongoing search for potent and specific anticancer drugs, in this article we discuss the unique perspectives, compounds and opportunities afforded by this rich source of potential pharmaceuticals. We discuss marine-derived antitumor drugs, their structures, and their various types and levels of antitumor activities in bench and bedside efforts.

Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions.

Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions.

O-fucose modulates Notch-controlled blood lineage commitment.

Notch receptors are cell surface molecules essential for cell fate determination. Notch signaling is subject to tight regulation at multiple levels, including the posttranslational modification of Notch receptors by O-linked fucosylation, a reaction that is catalyzed by protein O-fucosyltransferase-1 (Pofut1). Our previous studies identified a myeloproliferative phenotype in mice conditionally deficient in cellular fucosylation that is attributable to a loss of Notch-dependent suppression of myelopoiesis. Here, we report that hematopoietic stem cells deficient in cellular fucosylation display decreased frequency and defective repopulating ability as well as decreased lymphoid but increased myeloid developmental potential. This phenotype may be attributed to suppressed Notch ligand binding and reduced downstream signaling of Notch activity in hematopoietic stem cells. Consistent with this finding, we further demonstrate that mouse embryonic stem cells deficient in Notch1 (Notch1(-/-)) or Pofut1 (Pofut1(-/-)) fail to generate T lymphocytes but differentiate into myeloid cells while coculturing with Notch ligand-expressing bone marrow stromal cells in vitro. Moreover, in vivo hematopoietic reconstitution of CD34(+) progenitor cells derived from either Notch1(-/-) or Pofut1(-/-) embryonic stem cells show enhanced granulopoiesis with depressed lymphoid lineage development. Together, these results indicate that Notch signaling maintains hematopoietic lineage homeostasis by promoting lymphoid development and suppressing overt myelopoiesis, in part through processes controlled by O-linked fucosylation of Notch receptors.

Gastrogastric fistula following Roux-en-Y bypass is attributed to both surgical technique and experience.

BACKGROUND: The stomach can either be divided or undivided in performing Roux-en-Y gastric bypass (RGB) for morbid obesity. We evaluated whether surgical technique is the sole contributing factor to the formation of gastrogastric fistula (GGF). METHODS: A retrospective analysis of 1,036 consecutive patients was evaluated. RGB was performed as open undivided, open divided, and laparoscopic (divided). Incidence of GGF was identified for each technique and its relationship to surgical experience was assessed. RESULTS: Overall incidence of GGF was 1.3%. All fistulae occurred in patients who received undivided open RGB. There was a significant difference between the undivided open group and the divided open+laparoscopic groups (2.1% vs 0%, P<.01). Incidence of GGF decreased over time with increasing open undivided RGB volume. CONCLUSIONS: GGF was only identified in undivided RGB. The occurrence decreased with increasing surgical experience. Together, overall surgical technique in addition to gastric division must play a role in fistula formation.

Should bariatric revisional surgery be avoided secondary to increased morbidity and mortality?

BACKGROUND: Revisional bariatric surgery may be necessary due to inadequate weight loss or postoperative complications of the primary operation. We sought to identify the reasons for revision, characteristics of the surgery, and outcomes. We hypothesize that revisional surgery, although technically challenging, can produce desirable outcomes. METHODS: Patients undergoing bariatric surgery at our institution between 1998 and 2007 were reviewed from a prospective database. Patients who had revisional surgery were compared to those who had primary surgery. RESULTS: We have identified 46 of 1,038 patients who underwent revisional surgery. Twenty of 46 had a primary Roux-en-Y gastric bypass. The most common indication for revisions is inadequate weight loss secondary to gastrogastric fistula (15/20). Leaks occurred more frequently following revisional surgeries (11% vs 1.2%), but intensive care unit (ICU) utilization was less (11% vs 4.4%) and mortality was lower (0% vs .3%) with bariatric revision surgery. CONCLUSIONS: Although we saw a 9-fold increase in leaks, a 2-5 fold increase in ICU utilization, and 1.5-fold increase in length of stay, our mortality rate was zero. In experienced hands, bariatric revision surgery can be performed to produce desirable outcomes.

Preoperative findings predict conversion from laparoscopic to open cholecystectomy.

BACKGROUND: Previous studies evaluating predictive factors for conversion from laparoscopic to open cholecystectomy have drawn conflicting conclusions. We evaluated objective preoperative variables to create an accurate, accessible risk score to predict conversion. METHODS: A retrospective review was performed of laparoscopic cholecystectomy patients at an urban tertiary care center. Seventy characteristics were subjected to bivariate and multivariate logistic regression analysis to identify parameters that independently predict conversion to open cholecystectomy. A model was created based on this analysis. RESULTS: Laparoscopic cholecystectomy was performed on 1377 patients for benign gallbladder disease over a 71-month period. There were 112 (8.1%) conversions to open cholecystectomy. The correlation between the preoperative clinical diagnosis and pathologic diagnosis for acute and chronic cholecystitis was 48.6% and 94.6%, respectively. Multivariate analysis identified male gender, elevated white blood cell count, low serum albumin, ultrasound finding of pericholecystic fluid, diabetes mellitus, and elevated total bilirubin as independent predictors of conversion. These 6 factors were also associated with the pathologic diagnosis of acute cholecystitis. A model to calculate the risk for conversion was created with an area under the receiver operator curve of 0.83. The risk for conversion also can be estimated based on the number of factors identified present and ranged from 2% when 1 factor was present to 89% with 6 factors. CONCLUSIONS: These results demonstrate that conversion to open cholecystectomy can be predicted based on parameters available preoperatively. Conversion is more likely in patients who have acute cholecystitis; however, the correlation between its clinical and pathologic diagnosis is poor. Improvements in the ability to determine the risk for conversion have important implications for surgical care.