Search

ImportanceHigh-risk neuroblastoma is a complex genetic disease that is lethal in 50% of patients despite intense multimodal therapy. Our genome-wide association study (GWAS) identified single-nucleotide polymorphisms (SNPs) within theBARD1gene showing the most significant enrichment in neuroblastoma patients, and also discovered pathogenic (P) or likely pathogenic (LP) rare germline loss-of-function variants in this gene. The functional implications of these findings remain poorly understood.ObjectiveTo define the functional relevance ofBARD1germline variation in children with neuroblastoma.DesignWe correlatedBARD1genotype withBARD1expression in normal and tumor cells and the cellular burden of DNA damage in tumors. To validate the functional consequences of rare germline P-LPBARD1variants, we generated isogenic cellular models harboring heterozygousBARD1loss-of-function (LOF) variants and conducted multiple complementary assays to measure the efficiency of DNA repair.Setting(N/A)Participants(N/A)Interventions/Exposures(N/A)Main Outcomes and MeasuresBARD1expression, efficiency of DNA repair, and genome-wide burden of DNA damage in neuroblastoma tumors and cellular models harboring disease-associatedBARD1germline variants.ResultsBoth common and rare neuroblastoma associatedBARD1germline variants were significantly associated with lower levels ofBARD1mRNA and an increased burden of DNA damage. Using neuroblastoma cellular models engineered to harbor disease-associated heterozygousBARD1LOF variants, we functionally validated this association with inefficient DNA repair. TheseBARD1LOF variant isogenic models exhibited reduced efficiency in repairing Cas9-induced DNA damage, ineffective RAD51 focus formation at DNA doublestrand break sites, and enhanced sensitivity to cisplatin and poly-ADP ribose polymerase (PARP) inhibition.Conclusions and RelevanceConsidering that at least 1 in 10 children diagnosed with cancer carry a predicted pathogenic mutation in a cancer predisposition gene, it is critically important to understand their functional relevance. Here, we demonstrate that germlineBARD1variants disrupt DNA repair fidelity. This is a fundamental molecular mechanism contributing to neuroblastoma initiation that may have important therapeutic implications, and these findings may also extend to other cancers harboring germline variants in genes essential for DNA damage repair.Key PointsQuestionHow do neuroblastoma patient BRCA1-associated RING domain 1 (BARD1) germline variants impact DNA repair?FindingsNeuroblastoma-associated germlineBARD1variants disrupt DNA repair fidelity. Common risk variants correlate with decreasedBARD1expression and increased DNA double-strand breaks in neuroblastoma tumors and rare heterozygous loss-of-function variants induceBARD1haploinsufficiency, resulting in defective DNA repair and genomic instability in neuroblastoma cellular models.MeaningGermline variation inBARD1contributes to neuroblastoma pathogenesis via dysregulation of critical cellular DNA repair functions, with implications for neuroblastoma treatment, risk stratification, and cancer predisposition.

Beneficial mutations that arise in an evolving asexual population may compete or interact in ways that alter the overall rate of adaptation through mechanisms such as clonal or functional interference. The application of multiple selective pressures simultaneously may allow for a greater number of adaptive mutations, increasing the opportunities for competition between selectively advantageous alterations, and thereby reducing the rate of adaptation. We evolved a strain of Saccharomyces cerevisiae that could not produce its own histidine or uracil for ~500 generations under one or three selective pressures: limitation of the concentration of glucose, histidine, and/or uracil in the media. The rate of adaptation was obtained by measuring evolved relative fitness using competition assays. Populations evolved under a single selective pressure showed a statistically significant increase in fitness on those pressures relative to the ancestral strain, but the populations evolved on all three pressures did not show a statistically significant increase in fitness over the ancestral strain on any single pressure. Simultaneously limiting three essential nutrients for a population of S. cerevisiae effectively slows the rate of evolution on any one of the three selective pressures applied, relative to the single selective pressure cases. We identify possible mechanisms for fitness changes seen between populations evolved on one or three limiting nutrient pressures by high‐throughput sequencing. Adding multiple selective pressures to evolving disease like cancer and infectious diseases could reduce the rate of adaptation and thereby may slow disease progression, prolong drug efficacy and prevent deaths.

Recent advances in DNA sequencing have expanded our understanding of the molecular basis of genetic disorders and increased the utilization of clinical genomic tests. Given the paucity of evidence to accurately classify each variant and the difficulty of experimentally evaluating its clinical significance, a large number of variants generated by clinical tests are reported as variants of unknown clinical significance. Population-scale variant databases can improve clinical interpretation. Specifically, pathogenicity prediction for novel missense variants can use features describing regional variant constraint. Constrained genomic regions are those that have an unusually low variant count in the general population. Computational methods have been introduced to capture these regions and incorporate them into pathogenicity classifiers, but these methods have yet to be compared on an independent clinical variant data set. Here, we introduce one variant data set derived from clinical sequencing panels and use it to compare the ability of different genomic constraint metrics to determine missense variant pathogenicity. This data set is compiled from 17,071 patients surveyed with clinical genomic sequencing for cardiomyopathy, epilepsy, or RASopathies. We further use this data set to demonstrate the necessity of disease-specific classifiers and to train PathoPredictor, a disease-specific ensemble classifier of pathogenicity based on regional constraint and variant-level features. PathoPredictor achieves an average precision >90% for variants from all 99 tested disease genes while approaching 100% accuracy for some genes. The accumulation of larger clinical variant training data sets can significantly enhance their performance in a disease- and gene-specific manner.

Rh alloimmunization remains a challenge for patients with sickle cell disease despite transfusion of serologic Rh C, E and K antigen-matched red cells. Inheritance of altered RH alleles contributes to the prevalence of Rh antibodies following blood transfusion in patients with SCD and explains approximately one-third of cases. The remainder appear to be stimulated by altered Rh proteins on African-American donor red cells. Matching of patients with donors based on RH genotype may mitigate Rh alloimmunization, but the feasibility and resources required are not known. We compared RH allele frequencies between patients with SCD (n=857) and African-American donors (n=587) and show that RH allele frequencies are similar. Overall, 29% of RHD and 53% of RHCE alleles are altered in patients and African-American donors. We modeled RH genotype matching compared to serologic Rh D, C, and E, along with K antigen-matching, and found that approximately twice the number of African-American donors would be required for RH genotype versus Rh serologic matching at our institution. We demonstrate that African-American donor recruitment is necessary to maintain an adequate supply of C, E, and K negative donor units to avoid depleting the Rh negative (RhD-) blood supply. Our results suggest that prophylactic RH genetic matching for patients with SCD is feasible with a donor pool comprised primarily of African-Americans and would optimize utilization of our existing minority donor inventory. The current cost of RH genotyping all minority donors and management of the data remain limiting factors.

Exome-based panels are becoming the preferred diagnostic strategy in clinical laboratories. This approach enables dynamic gene content update and, if needed, cost-effective reflex to whole-exome sequencing. Currently, no guidelines or appropriate resources are available to support the clinical implementation of exome-based panels. Here, we highlight principles and important considerations for the clinical development and validation of exome-based panels. In addition, we developed ExomeSlicer, a novel, web-based resource, which uses empirical exon-level next-generation sequencing quality metrics to predict and visualize technically challenging exome-wide regions in any gene or genes of interest. Exome sequencing data from 100 clinical epilepsy cases were used to illustrate the clinical utility of ExomeSlicer in predicting poor-quality regions and its impact on streamlining the ad hoc Sanger sequencing fill in burden. With the use of ExomeSlicer, >2100 low complexity and/or high-homology regions affecting >1615 genes across the exome were also characterized. These regions can be a source of false-positive or false-negative variant calls, which can lead to misdiagnoses in tested patients and/or inaccurate functional annotations. We provide important considerations and a novel resource for the clinical development of exome-based panels.

Accumulation of aggregated α-synuclein into Lewy bodies is thought to contribute to the onset and progression of dopaminergic neuron degeneration in Parkinson's disease (PD) and related disorders. Although protein aggregation is associated with perturbation of proteostasis, how α-synuclein aggregation affects the brain proteome and signaling remains uncertain. In a mouse model of α-synuclein aggregation, 6% of 6215 proteins and 1.6% of 8183 phosphopeptides changed in abundance, indicating conservation of proteostasis and phosphorylation signaling. The proteomic analysis confirmed changes in abundance of proteins that regulate dopamine synthesis and transport, synaptic activity and integrity, and unearthed changes in mRNA binding, processing and protein translation. Phosphorylation signaling changes centered on axonal and synaptic cytoskeletal organization and structural integrity. Proteostatic responses included a significant increase in the levels of Lmp7, a component of the immunoproteasome. Increased Lmp7 levels and activity were also quantified in postmortem human brains with PD and dementia with Lewy bodies. Functionally, the immunoproteasome degrades α-synuclein aggregates and generates potentially antigenic peptides. Expression and activity of the immunoproteasome may represent testable targets to induce adaptive responses that maintain proteome integrity and modulate immune responses in protein aggregation disorders., Highlights • Relative quantification of 6215 proteins and 8183 phosphopeptides in a mouse model of endogenous aggregation of α-synuclein • The immunoproteasome component Lmp7, is increased in the mouse model and postmortem human brains with Parkinson's and DLB • Lmp7 may represent an adaptive response to protein aggregation through aggregate elimination and innate immunity activation Aggregated a-synuclein is a component of Lewy bodies a hallmark of Parkinson's disease. Changes in the brain proteome and phosphorylated peptides were quantified upon aggregation of α-synuclein in a mouse model. The levels and activity of LMP7, a component of the immunoproteasome, which is linked to proteostasis were increased indicating a potential adaptive response to α-synuclein aggregation.

MYCN amplification and 11q deletion are two inversely correlated prognostic factors of poor outcome in neuroblastoma. Here we identify common variants at 11q22.2 within MMP20 that associate with neuroblastoma cases harboring 11q deletion (rs10895322), using GWAS in 113 European-American cases and 5109 ancestry-matched controls. The association is replicated in 44 independent cases and 1902 controls. Our study yields novel insights into the genetic underpinnings of neuroblastoma, demonstrating that the inherited common variants reported contribute to the origin of intra-tumor genetic heterogeneity in neuroblastoma., Chromosomal abnormalities such as 11q deletion are associated with poor prognosis in neuroblastoma. Here, the authors perform a genome-wide association study and identify an association between a variant within a Matrix metalloproteinase (MMP) gene member, MMP20, and 11q-deletion subtype neuroblastoma.

The disruption of protein expression is a major approach used for investigating protein function in mammalian oocytes. This is often achieved with RNAi/morpholino-mediated knockdown or gene knockout, leading to long-term loss of proteins of interest. However, these methods have noteworthy limitations, including (a) slow protein turnover can prohibit use of these approaches; (b) essential roles in early events precludes characterization of functions in subsequent events; (c) extended protein loss can allow time for compensatory mechanisms and other unanticipated events that confound interpretation of results. The work presented here examines the use of auxin-inducible degradation, a powerful new approach that overcomes these limitations through the depletion of one's protein of interest through controllable ubiquitin-mediated degradation. This method has been employed in yeast and mammalian cell lines, and here we demonstrate the utility of auxin-inducible degradation in mouse oocytes at multiple stages of meiosis, through degradation of exogenously expressed EGFP. We also evaluate important parameters for experimental design for use of this system in oocytes. This study thus expands the toolkit of researchers in oocyte biology, establishing the use of this unique and versatile approach for depleting proteins in oocytes, and providing researchers with valuable information to make use of this system., Summary Sentence Auxin-inducible degradation can be used to deplete proteins in oocytes throughout meiosis (prophase I to metaphase II), providing a valuable alternative to longer-term and post-transcriptional depletion methods such as knockdown and knockout.

BACKGROUND Red blood cell (RBC) alloimmunization is a concern for patients who receive multiple or chronic transfusions. Alloimmunization prevalence in transfused patients with bone marrow failure syndrome (BMFS) is unknown. This study aimed to determine physician practice for RBC antigen matching, immunization rates, and antibody specificities in patients with BMFS. STUDY DESIGN AND METHODS The clinical records of all patients with BMFS seen at the Children's Hospital of Philadelphia between 2001 and 2015 were reviewed. Immunization rate was determined per 100 units transfused. RESULTS ABO/D, C, E, and K (CEK) RBC matching was requested for 21.8% of patients. A total of 3782 RBC units were transfused to 87 patients, of which 2551 (67.5%) were CEK matched and 1231 (32.5%) were ABO/D only matched. The majority of units transfused to patients on a chronic transfusion regimen were CEK matched (89.6% of 2728 units). No anti-C, -E, or -K antibodies formed in any patient during the 14-year study period. Two alloantibodies and two autoantibodies formed, resulting in a rate of 0.05 alloantibodies and 0.05 autoantibodies per 100 units transfused. The prevalence of alloimmunization was 2.3%. CONCLUSION The rate and prevalence of RBC alloimmunization were low in patients with BMFS. CEK matching avoided alloimmunization to these antigens in chronically transfused patients.

Reproductive biologists are well-versed in many types of biochemical signaling, and indeed, there are almost innumerable examples in reproduction, including steroid and peptide hormone signaling, receptor-ligand and secondary messenger-mediated signaling, signaling regulated by membrane channels, and many others. Among reproductive scientists, a perhaps lesser-known but comparably important mode of signaling is mechanotransduction: the concept that cells can sense and respond to externally applied or internally generated mechanical forces. Given the cell shape changes and tissue morphogenesis events that are components of many phenomena in reproductive function, it should be no surprise that mechanotransduction has major impacts in reproductive health and pathophysiology. The conference on "Mechanotransduction in the Reproductive Tract" was a valuable launch pad to bring this hot issue in development, cell biology, biophysics, and tissue regeneration to the realm of reproductive biology. The goal of the meeting was to stimulate interest and increased mechanotransduction research in the reproductive field by presenting a broad spectrum of responses impacted by this process. The meeting highlighted the importance of convening expert investigators, students, fellows, and young investigators from a number of research areas resulting in cross-fertilization of ideas and suggested new avenues for study. The conference included talks on tissue engineering, stem cells, and several areas of reproductive biology, from uterus and cervix to the gametes. Specific reproductive health-relevant areas, including uterine fibroids, gestation and parturition, and breast tissue morphogenesis, received particular attention.

STUDY HYPOTHESIS Cellular aging of the egg following ovulation, also known as post-ovulatory aging, is associated with aberrant cortical mechanics and actomyosin cytoskeleton functions. STUDY FINDING Post-ovulatory aging is associated with dysfunction of non-muscle myosin-II, and pharmacologically induced myosin-II dysfunction produces some of the same deficiencies observed in aged eggs. WHAT IS KNOWN ALREADY Reproductive success is reduced with delayed fertilization and when copulation or insemination occurs at increased times after ovulation. Post-ovulatory aged eggs have several abnormalities in the plasma membrane and cortex, including reduced egg membrane receptivity to sperm, aberrant sperm-induced cortical remodeling and formation of fertilization cones at the site of sperm entry, and reduced ability to establish a membrane block to prevent polyspermic fertilization. STUDY DESIGN, SAMPLES/MATERIALS, METHODS Ovulated mouse eggs were collected at 21-22 h post-human chorionic gonadotrophin (hCG) (aged eggs) or at 13-14 h post-hCG (young eggs), or young eggs were treated with the myosin light chain kinase (MLCK) inhibitor ML-7, to test the hypothesis that disruption of myosin-II function could mimic some of the effects of post-ovulatory aging. Eggs were subjected to various analyses. Cytoskeletal proteins in eggs and parthenogenesis were assessed using fluorescence microscopy, with further analysis of cytoskeletal proteins in immunoblotting experiments. Cortical tension was measured through micropipette aspiration assays. Egg membrane receptivity to sperm was assessed in in vitro fertilization (IVF) assays. Membrane topography was examined by low-vacuum scanning electron microscopy (SEM). MAIN RESULTS AND THE ROLE OF CHANCE Aged eggs have decreased levels and abnormal localizations of phosphorylated myosin-II regulatory light chain (pMRLC; P = 0.0062). Cortical tension, which is mediated in part by myosin-II, is reduced in aged mouse eggs when compared with young eggs, by ∼40% in the cortical region where the metaphase II spindle is sequestered and by ∼50% in the domain to which sperm bind and fuse (P < 0.0001). Aging-associated parthenogenesis is partly rescued by treating eggs with a zinc ionophore (P = 0.003), as is parthenogenesis induced by inhibition of mitogen-activated kinase (MAPK) 3/1 [also known as extracellular signal-regulated kinase (ERK)1/2] or MLCK. Inhibition of MLCK with ML-7 also results in effects that mimic those of post-ovulatory aging: fertilized ML-7-treated eggs show both impaired fertilization and increased extents of polyspermy, and ML-7-treated young eggs have several membrane abnormalities that are shared by post-ovulatory aged eggs. LIMITATIONS, REASONS FOR CAUTION These studies were done with mouse oocytes, and it remains to be fully determined how these findings from mouse oocytes would compare with other species. For studies using methods not amenable to analysis of large sample sizes and data are limited to what images one can capture (e.g. SEM), data should be interpreted conservatively. WIDER IMPLICATIONS OF THE FINDINGS These data provide insights into causes of reproductive failures at later post-copulatory times. LARGE SCALE DATA Not applicable. STUDY FUNDING AND COMPETING INTERESTS This project was supported by R01 HD037696 and R01 HD045671 from the NIH to J.P.E. Cortical tension studies were supported by R01 GM66817 to D.N.R. The authors declare there are no financial conflicts of interest.

Alterations in the synthesis and bioavailability of NO are central to the pathogenesis of cardiovascular and metabolic disorders. Although endothelial NO synthase–derived (eNOS-derived) NO affects mitochondrial long-chain fatty acid β-oxidation, the pathophysiological significance of this regulation remains unclear. Accordingly, we determined the contributions of eNOS/NO signaling in the adaptive metabolic responses to fasting and in age-induced metabolic dysfunction. Four-month-old eNOS–/– mice are glucose intolerant and exhibit serum dyslipidemia and decreased capacity to oxidize fatty acids. However, during fasting, eNOS–/– mice redirect acetyl-CoA to ketogenesis to elevate circulating levels of β-hydroxybutyrate similar to wild-type mice. Treatment of 4-month-old eNOS–/– mice with nitrite for 10 days corrected the hypertension and serum hyperlipidemia and normalized the rate of fatty acid oxidation. Fourteen-month-old eNOS–/– mice exhibited metabolic derangements, resulting in reduced utilization of fat to generate energy, lower resting metabolic activity, and diminished physical activity. Seven-month administration of nitrite to eNOS–/– mice reversed the age-dependent metabolic derangements and restored physical activity. While the eNOS/NO signaling is not essential for the metabolic adaptation to fasting, it is critical for regulating systemic metabolic homeostasis in aging. The development of age-dependent metabolic disorder is prevented by low-dose replenishment of bioactive NO.

Clinical exome sequencing (CES) has become the preferred diagnostic platform for complex pediatric disorders with suspected monogenic etiologies. Despite rapid advancements, the major challenge still resides in identifying the casual variants among the thousands of variants detected during CES testing, and thus establishing a molecular diagnosis. To improve the clinical exome diagnostic efficiency, we developed Phenoxome, a robust phenotype-driven model that adopts a network-based approach to facilitate automated variant prioritization. Phenoxome dissects the phenotypic manifestation of a patient in concert with their genomic profile to filter and then prioritize variants that are likely to affect the function of the gene (potentially pathogenic variants). To validate our method, we have compiled a clinical cohort of 105 positive patient samples that represent a wide range of genetic heterogeneity. Phenoxome identifies the causative variants within the top 5, 10, or 25 candidates in more than 50%, 71%, or 88% of these exomes, respectively. Furthermore, we show that our method is optimized for clinical testing by outperforming the current state-of-art method. We have demonstrated the performance of Phenoxome using a clinical cohort and showed that it enables rapid and accurate interpretation of clinical exomes. Phenoxome is available at https://phenoxome.chop.edu/ .

Recent advances in DNA sequencing technologies have expanded our understanding of the molecular underpinnings for several genetic disorders, and increased the utilization of genomic tests by clinicians. Given the paucity of evidence to assess each variant, and the difficulty of experimentally evaluating a variant’s clinical significance, many of the thousand variants that can be generated by clinical tests are reported as variants of unknown clinical significance. However, the creation of population-scale variant databases can significantly improve clinical variant interpretation. Specifically, pathogenicity prediction for novel missense variants can now utilize features describing regional variant constraint. Constrained genomic regions are those that have an unusually low variant count in the general population. Several computational methods have been introduced to capture these regions and incorporate them into pathogenicity classifiers, but these methods have yet to be compared on an independent clinical variant dataset. Here we introduce one variant dataset derived from clinical sequencing panels, and use it to compare the ability of different genomic constraint metrics to determine missense variant pathogenicity. This dataset is compiled from 17,071 patients surveyed with clinical genomic sequencing for cardiomyopathy, epilepsy, or RASopathies. We further utilize this dataset to demonstrate the necessity of disease-specific classifiers, and to train PathoPredictor, a disease-specific ensemble classifier of pathogenicity based on regional constraint and variant level features. PathoPredictor achieves an average precision greater than 90% for variants from all 99 tested disease genes while approaching 100% accuracy for some genes. Accumulation of larger clinical variant datasets and their utilization to train existing pathogenicity metrics can significantly enhance their performance in a disease and gene-specific manner.

Exome-based panels are becoming the preferred diagnostic strategy in clinical laboratories. This approach enables dynamic gene content update and, if needed, cost-effective reflex to whole-exome sequencing. Currently, no guidelines or appropriate resources are available to support the clinical implementation of exome-based panels. Here, we highlight principles and important considerations for the clinical development and validation of exome-based panels. In addition, we developed ExomeSlicer, a novel, web-based resource, which uses empirical exon-level next-generation sequencing quality metrics to predict and visualize technically challenging exome-wide regions in any gene or genes of interest. Exome sequencing data from 100 clinical epilepsy cases were used to illustrate the clinical utility of ExomeSlicer in predicting poor-quality regions and its impact on streamlining the ad hoc Sanger sequencing fill in burden. With the use of ExomeSlicer,2100 low complexity and/or high-homology regions affecting1615 genes across the exome were also characterized. These regions can be a source of false-positive or false-negative variant calls, which can lead to misdiagnoses in tested patients and/or inaccurate functional annotations. We provide important considerations and a novel resource for the clinical development of exome-based panels.

Exome-based panels (exome slices) are becoming the preferred diagnostic strategy in clinical laboratories, especially for genetically heterogeneous disorders. The advantages of this approach include enabling frequent updates to gene content without the need for re-designing, reflexing to exome analysis bioinformatically without requiring additional sequencing, and streamlining laboratory operation by using established exome kits and protocols. Despite their increasing use, there are currently no guidelines or appropriate resources to support their clinical implementation. Here, we highlight principles and important considerations for the clinical development and validation of exome-based panels, guided by clinical data from a diagnostic epilepsy panel using this approach. We also present a novel, publically accessible web-based resource, ExomeSlicer, and demonstrate its clinical utility in predicting gene-specific and exome-wide technically challenging regions that are not amenable to Next Generation Sequencing (NGS), and that might significantly lead to increased post hoc Sanger fill in burden. Using this tool, we also characterize > 2000 low complexity, GC-rich and/or high homology, regions across the exome that can be a source of false positive or false negative variant calls thus potentially leading to misdiagnoses in tested patients.

Just as it is important to understand the cell biology of signaling pathways, it is valuable also to understand mechanical forces in cells. The field of mechanobiology has a rich history, including study of cellular mechanics during mitosis and meiosis in echinoderm oocytes and zygotes dating back to the 1930s. This chapter addresses the use of micropipette aspiration (MPA) to assess cellular mechanics, specifically cortical tension, in mammalian oocytes.

Bromodomain and extraterminal motif (BET) proteins are pharmacologic targets for the treatment of diverse diseases, yet the roles of individual BET family members remain unclear. We find that BRD2, but not BRD4, co-localizes with the architectural/insulator protein CCCTC-binding factor (CTCF) genome-wide. CTCF recruits BRD2 to co-bound sites whereas BRD2 is dispensable for CTCF occupancy. Disruption of a CTCF/BRD2-occupied element positioned between two unrelated genes enables regulatory influence to spread from one gene to another, suggesting that CTCF and BRD2 form a transcriptional boundary. Accordingly, single-molecule mRNA fluorescence in situ hybridization (FISH) reveals that, upon site-specific CTCF disruption or BRD2 depletion, expression of the two genes becomes increasingly correlated. HiC shows that BRD2 depletion weakens boundaries co-occupied by CTCF and BRD2, but not those that lack BRD2. These findings indicate that BRD2 supports boundary activity, and they raise the possibility that pharmacologic BET inhibitors can influence gene expression in part by perturbing domain boundary function.

During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis–G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis–G1 transition, ∼50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer–promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry but do not spike. Of the chromatin-associated features examined, histone H3 Lys27 acetylation levels at individual loci in mitosis best predict the mitosis–G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis–G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis–G1 transition might predispose cells to diverge in gene expression states.

We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like CT somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1(P29S)) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1(P29S) showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

PurposeThe purpose of this paper is to outline what it takes to be a successful interim manager in today's pharmaceutical industry, based on the experiences of two people currently in the role.Design/methodology/approachThe paper explains when it is and is not appropriate to employ an interim manager, and details the main advantages and disadvantages from the points of view of both the company and interim manager involved.FindingsThe paper describes the main challenges as: working across a range of time zones; traveling between countries; juggling different and demanding clients; recognizing that one has been brought in to solve a specific problem, not to become a permanent employee; guiding without interfering; managing client expectations; and coping with down‐time when the contract finishes.Practical implicationsThe paper demonstrates how interns may help companies to solve a particular problem quickly and easily.Social implicationsThe paper explains that, when companies are feeling the pinch and may have frozen their recruitment of permanent employees, interims offer immediate support and expertise but are dispensable.Originality/valueThe paper contains much to interest companies thinking of taking on an interim and individuals considering interim management as a career.

Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells - oocyte and polar body - with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.

One important result of egg activation is the establishment of blocks to prevent polyspermic fertilization; these blocks are established on the zona pellucida and the egg plasma membrane. This study examines what the sperm brings to the egg to induce the establishment of the membrane block to polyspermy, building on past evidence that membrane block establishment does not occur in response to parthenogenetic stimuli that induce a single transient increase in cytosolic Ca2+ or intracytoplasmic sperm injection (ICSI). We test the hypotheses that (i) sperm-associated Ca2+ release activity triggers membrane block establishment; (ii) introduction of sperm contents via variations on ICSI protocols (resulting in improved Ca2+ transients, egg activation and embryo development over traditional ICSI protocols) triggers membrane block establishment and (iii) sperm adhesion [binding of an extracellular sperm ligand(s) to an egg receptor(s)] combined with sperm-associated Ca2+ release activity triggers membrane block establishment. Interestingly, none of these stimuli induced establishment of the membrane block to polyspermy in mouse eggs. However, the sperm-associated remodeling of the egg cortical cytoskeleton differs between conventionally fertilized and ICSI-fertilized eggs; taken with our previous data implicating actin microfilaments in membrane block establishment, this raises the possibility that cortical reorganization may be a contributing factor. In sum, fertilization-like Ca2+ transients, either alone or combined with sperm-egg binding, are not sufficient for membrane block establishment, but that an event(s) associated with gamete interaction plays a role in this membrane function change.

One crucial result of egg activation is the establishment of blocks on the zona pellucida and the egg plasma membrane to prevent fertilization by additional sperm. The mechanism(s) by which a mammalian egg regulates the establishment of the membrane block to polyspermy is largely unknown. Since Ca2+signaling regulates several egg activation events, this study investigates how sperm-induced Ca2+transients affect the membrane block to polyspermy, building on our previous work (Biology of Reproduction 67:1342). We demonstrate that mouse eggs that experience only one sperm-induced Ca2+transient establish a membrane block that is less effective, than in eggs that experience normal sperm-induced Ca2+transients but that is more effective than in eggs with completely suppressed [Ca2+]cytincreases. Sperm-induced increases in [Ca2+]cytregulate the timing of membrane block establishment, as this block is established more slowly in eggs that experience one or no sperm-induced Ca2+transients. Finally, our studies produce the intriguing discovery that there is also a Ca2+-independent event that is associated with fertilization in the pathway leading to membrane block establishment. Taken together, these data indicate that Ca2+plays a role in facilitating membrane block establishment by regulating the timing with which this change in egg membrane function occurs, and also that the membrane block differs from other post-fertilization egg activation responses as Ca2+is not the only stimulus. The membrane block to polyspermy in mammalian eggs is likely to be the culmination of multiple post-fertilization events that together modify the egg membrane’s receptivity to sperm.

Red blood cell (RBC) alloimmunization is a concern for patients who receive multiple or chronic transfusions. Alloimmunization prevalence in transfused patients with bone marrow failure syndrome (BMFS) is unknown. This study aimed to determine physician practice for RBC antigen matching, immunization rates, and antibody specificities in patients with BMFS.The clinical records of all patients with BMFS seen at the Children's Hospital of Philadelphia between 2001 and 2015 were reviewed. Immunization rate was determined per 100 units transfused.ABO/D, C, E, and K (CEK) RBC matching was requested for 21.8% of patients. A total of 3782 RBC units were transfused to 87 patients, of which 2551 (67.5%) were CEK matched and 1231 (32.5%) were ABO/D only matched. The majority of units transfused to patients on a chronic transfusion regimen were CEK matched (89.6% of 2728 units). No anti-C, -E, or -K antibodies formed in any patient during the 14-year study period. Two alloantibodies and two autoantibodies formed, resulting in a rate of 0.05 alloantibodies and 0.05 autoantibodies per 100 units transfused. The prevalence of alloimmunization was 2.3%.The rate and prevalence of RBC alloimmunization were low in patients with BMFS. CEK matching avoided alloimmunization to these antigens in chronically transfused patients.

Nitric oxide (NO) is a signaling intermediate during glutamatergic neurotransmission in the central nervous system (CNS). NO signaling is in part accomplished through cysteine S-nitrosylation, a posttranslational modification by which NO regulates protein function and signaling. In our investigation of the protein targets and functional impact of S-nitrosylation in the CNS under physiological conditions, we identified 269 S-nitrosocysteine residues in 136 proteins in the wild-type mouse brain. The number of sites was significantly reduced in the brains of mice lacking endothelial nitric oxide synthase (eNOS(-/-)) or neuronal nitric oxide synthase (nNOS(-/-)). In particular, nNOS(-/-) animals showed decreased S-nitrosylation of proteins that participate in the glutamate/glutamine cycle, a metabolic process by which synaptic glutamate is recycled or oxidized to provide energy. (15)N-glutamine-based metabolomic profiling and enzymatic activity assays indicated that brain extracts from nNOS(-/-) mice converted less glutamate to glutamine and oxidized more glutamate than those from mice of the other genotypes. GLT1 [also known as EAAT2 (excitatory amino acid transporter 2)], a glutamate transporter in astrocytes, was S-nitrosylated at Cys(373) and Cys(561) in wild-type and eNOS(-/-) mice, but not in nNOS(-/-) mice. A form of rat GLT1 that could not be S-nitrosylated at the equivalent sites had increased glutamate uptake compared to wild-type GLT1 in cells exposed to an S-nitrosylating agent. Thus, NO modulates glutamatergic neurotransmission through the selective, nNOS-dependent S-nitrosylation of proteins that govern glutamate transport and metabolism.

Vertebrate eggs are arrested at metaphase of meiosis II, a state classically known as cytostatic factor arrest. Maintenance of this arrest until the time of fertilization and then fertilization-induced exit from metaphase II are crucial for reproductive success. Another key aspect of this meiotic arrest and exit is regulation of the metaphase II spindle, which must be appropriately localized adjacent to the egg cortex during metaphase II and then progress into successful asymmetric cytokinesis to produce the second polar body. This study examined the mitogen-activated protein kinases MAPK3 and MAPK1 (also known as ERK1/2) as regulators of these two related aspects of mammalian egg biology, specifically testing whether this MAPK pathway affected myosin-II function and whether myosin-II perturbation would produce some of the same effects as MAPK pathway perturbation. Inhibition of the MEK1/2-MAPK pathway with U0126 leads to reduced levels of phosphorylated myosin-regulatory light chain (pMRLC) and causes a reduction in cortical tension, effects that are mimicked by treatment with the myosin light chain kinase (MLCK) inhibitor ML-7. These data indicate that one mechanism by which the MAPK pathway acts in eggs is by affecting myosin-II function. We further show that MAPK or MLCK inhibition induces loss of normal cortical spindle localization or parthenogenetic egg activation. This parthenogenesis is dependent on cytosolic and extracellular calcium and can be rescued by hyperloading eggs with zinc, suggesting that these effects of inhibition of MLCK or the MAPK pathway are linked with dysregulation of ion homeostasis.

SummaryS-Acylation, S-glutathionylation, S-nitrosylation, and S-sulfenylation are prominent, chemically distinct modifications that regulate protein function, redox sensing, and trafficking. Although the biological significance of these modifications is increasingly appreciated, their integration in the proteome remains unknown. Novel mass spectrometry-based technologies identified 2,596 predominately unique sites in 1,319 mouse liver proteins under physiological conditions. Structural analysis localized the modifications in unique, evolutionary conserved protein segments, outside commonly annotated functional regions. Contrary to expectations, propensity for modification did not correlate with biophysical properties that regulate cysteine reactivity. However, the in vivo chemical reactivity is fine-tuned for specificity, demonstrated by the nominal complementation between the four modifications and quantitative proteomics which showed that a reduction in S-nitrosylation is not correlated with increased S-glutathionylation. A comprehensive survey uncovered clustering of modifications within biologically related protein networks. The data provide the first evidence for the occurrence of distinct, endogenous protein networks that undergo redox signaling through specific cysteine modifications.

Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2(+/-) mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2(-/-) mice have moderately delayed growth but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1(-/-) males that have normal fertility, Tpst2(-/-) males are infertile. Tpst2(-/-) sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida-intact eggs. Adhesion of Tpst2(-/-) sperm to the egg plasma membrane is reduced compared with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility.

We report on whole-exome sequencing (WES) of 213 melanomas. Our analysis established NF1, encoding a negative regulator of RAS, as the third most frequently mutated gene in melanoma, after BRAF and NRAS. Inactivating NF1 mutations were present in 46% of melanomas expressing wild-type BRAF and RAS, occurred in older patients and showed a distinct pattern of co-mutation with other RASopathy genes, particularly RASA2. Functional studies showed that NF1 suppression led to increased RAS activation in most, but not all, melanoma cases. In addition, loss of NF1 did not predict sensitivity to MEK or ERK inhibitors. The rebound pathway, as seen by the induction of phosphorylated MEK, occurred in cells both sensitive and resistant to the studied drugs. We conclude that NF1 is a key tumor suppressor lost in melanomas, and that concurrent RASopathy gene mutations may enhance its role in melanomagenesis.

This study examines the effects of actin microfilament-disrupting drugs on events of fertilization, with emphasis on gamete membrane interactions. Mouse eggs, freed of their zonae pellucidae, were treated with drugs that perturb the actin cytoskeleton by different mechanisms (cytochalasin B, cytochalasin D, jasplakinolide, latrunculin B) and then inseminated. Cytochalasin B, jasplakinolide, and latrunculin B treatments resulted in a decrease in the percentage of eggs fertilized and the average number of sperm fused per egg. However, cytochalasin D treatment resulted in an increase in the average number of sperm fused per egg and the percentage of polyspermic eggs. This increase in polyspermy occurred despite the observation that cytochalasin D treatment caused a decrease in sperm-egg binding and did not affect spontaneous acrosome reactions or sperm motility. This suggested that cytochalasin D-treated eggs had an impaired ability to establish a block to polyspermy at the level of the plasma membrane. The effect of cytochalasin D on the block to polyspermy was not due to a general disruption of egg activation because sperm-induced calcium oscillations and cortical granule exocytosis were similar in cytochalasin D-treated and control eggs. However, buffering of intracellular calcium levels with the calcium chelator BAPTA-AM resulted in an increase in polyspermy. Together, these data suggest that a postfertilization decrease in egg membrane receptivity to sperm requires functions of the egg actin cytoskeleton that are disrupted by cytochalasin D. Furthermore, egg activation-associated increased intracellular calcium levels are necessary but not sufficient to affect postfertilization membrane dynamics that contribute to a membrane block to polyspermy.

Fertilin β (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin β utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin β binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the α5/α8/αv/αIIb or RGD-binding subfamily (α5β1, α8β1, αVβ1, αVβ3, αVβ5, αVβ6, αVβ8, and αIIbβ3) and the α4/α9 subfamily (α4β1, α9β1, and α4β7). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin β binding. Peptides with the sequence MLDG, which perturb α4/α9 integrin-mediated interactions, signific...

Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.

Exome sequencing in cancer is a powerful tool for identifying mutational events across the coding region of human genes. Here, we describe computational methods that use exome sequencing reads from cancer samples to identify somatic single nucleotide variants (SNVs), copy number alterations, and short insertions and deletions (InDels). We further describe analytical methods to generate lists of driver genes with more mutational events than expected by chance.

Mammalian oocytes in ovarian follicles are arrested in meiosis at prophase I. This arrest is maintained until ovulation, upon which the oocyte exits from this arrest, progresses through meiosis I and to metaphase of meiosis II. The progression from prophase I to metaphase II, known as meiotic maturation, is mediated by signals that coordinate these transitions in the life of the oocyte. ENSA (α-endosulfine) and ARPP19 (cAMP-regulated phosphoprotein-19) have emerged as regulators of M-phase, with function in inhibition of protein phosphatase 2A (PP2A) activity. Inhibition of PP2A maintains the phosphorylated state of CDK1 substrates, thus allowing progression into and/or maintenance of an M-phase state. We show here ENSA in mouse oocytes plays a key role in the progression from prophase I arrest into M-phase of meiosis I. The majority of ENSA-deficient oocytes fail to exit from prophase I arrest. This function of ENSA in oocytes is dependent on PP2A, and specifically on the regulatory subunit PPP2R2D (also known as B55δ). Treatment of ENSA-deficient oocytes with Okadaic acid to inhibit PP2A rescues the defect in meiotic progression, with Okadaic acid-treated, ENSA-deficient oocytes being able to exit from prophase I arrest. Similarly, oocytes deficient in both ENSA and PPP2R2D are able to exit from prophase I arrest to an extent similar to wild-type oocytes. These data are evidence of a role for ENSA in regulating meiotic maturation in mammalian oocytes, and also have potential relevance to human oocyte biology, as mouse and human have genes encoding both Arpp19 and Ensa.

Exome sequencing in cancer is a powerful tool for identifying mutational events across the coding region of human genes. Here, we describe computational methods that use exome sequencing reads from cancer samples to identify somatic single nucleotide variants (SNVs), copy number alterations, and short insertions and deletions (InDels). We further describe analytical methods to generate lists of driver genes with more mutational events than expected by chance.

Inpp5b is an ubiquitously expressed type II inositol polyphosphate 5-phosphatase. We have disrupted the Inpp5b gene in mice and found that homozygous mutant males are infertile. Here we examine the causes for the infertility in detail. We demonstrate that sperm from Inpp5b(-/-) males have reduced motility and reduced ability to fertilize eggs, although capacitation and acrosome exocytosis appear to be normal. In addition, fertilin beta, a sperm surface protein involved in sperm-egg membrane interactions that is normally proteolytically processed during sperm transit through the epididymis, showed reduced levels of processing in the Inpp5b(-/-) animals. Inpp5b was expressed in the Sertoli cells and epididymis and at low levels in the developing germ cells; however, mice lacking Inpp5b in spermatids and not in other cell types generated by conditional gene targeting, were fully fertile. The abnormalities in mutant sperm function and maturation appear to arise from defects in the functioning of Sertoli and epididymal epithelial cells. Our results directly demonstrate a previously unknown role for phosphoinositides in normal sperm maturation beyond their previously characterized involvement in the acrosome reaction. Inpp5b(-/-) mice provide an excellent model to study the role of Sertoli and epididymal epithelial cells in the differentiation and maturation of sperm.