Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors
The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.
A Large-Scale RNAi-Based Mouse Tumorigenesis Screen Identifies New Lung Cancer Tumor Suppressors that Repress FGFR Signaling
To discover new tumor suppressor genes (TSGs), we developed a functional genomics approach in which immortalized but non-tumorigenic cells were stably transduced with large-scale short hairpin RNA (shRNA) pools and tested for tumor formation in mice. Identification of shRNAs in resulting tumors revealed candidate TSGs, which were validated experimentally and by analyzing expression in human tumor samples. Using this approach, we identified 24 TSGs that were significantly down-regulated in human lung squamous cell carcinomas (hLSCCs). Amplification of fibroblast growth factor receptor 1 (FGFR1), which aberrantly increases FGFR signaling, is a common genetic alteration in hLSCCs. Remarkably, we found that 17 of the TSGs encode repressors of FGFR signaling. Knockdown of 14 of these TSGs transformed immortalized human bronchial epithelial cells and, in most cases, rendered them sensitive to FGFR inhibitors. Our results indicate that increased FGFR signaling promotes tumorigenesis in many hLSCCs that lack FGFR1 amplification or activating mutations.
Mammalian embryonic stem cells (ESCs) and sperm exhibit unusual chromatin packaging that plays important roles in cellular function. Here, we extend a recently developed technique, based on deep paired-end sequencing of lightly digested chromatin, to assess footprints of nucleosomes and other DNA-binding proteins genome-wide in murine ESCs and sperm. In ESCs, we recover well-characterized features of chromatin such as promoter nucleosome depletion and further identify widespread footprints of sequence-specific DNA-binding proteins such as CTCF, which we validate in knockdown studies. We document global differences in nuclease accessibility between ESCs and sperm, finding that the majority of histone retention in sperm preferentially occurs in large gene-poor genomic regions, with only a small subset of nucleosomes being retained over promoters of developmental regulators. Finally, we describe evidence that CTCF remains associated with the genome in mature sperm, where it could play a role in organizing the sperm genome.
Adenosine triphosphate (ATP) synthase beta, the catalytic subunit of mitochondrial complex V, synthesizes ATP. We show that ATP synthase beta is deacetylated by a human nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase, sirtuin 3, and its Drosophila melanogaster homologue, dSirt2. dsirt2 mutant flies displayed increased acetylation of specific Lys residues in ATP synthase beta and decreased complex V activity. Overexpression of dSirt2 increased complex V activity. Substitution of Lys 259 and Lys 480 with Arg in human ATP synthase beta, mimicking deacetylation, increased complex V activity, whereas substitution with Gln, mimicking acetylation, decreased activity. Mass spectrometry and proteomic experiments from wild-type and dsirt2 mitochondria identified the Drosophila mitochondrial acetylome and revealed dSirt2 as an important regulator of mitochondrial energy metabolism. Additionally, we unravel a ceramide-NAD(+)-sirtuin axis wherein increased ceramide, a sphingolipid known to induce stress responses, resulted in depletion of NAD(+) and consequent decrease in sirtuin activity. These results provide insight into sirtuin-mediated regulation of complex V and reveal a novel link between ceramide and Drosophila acetylome.
A separable domain of the p150 subunit of human Chromatin Assembly Factor-1 promotes protein and chromosome associations with nucleoli
Chromatin Assembly Factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here, we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Notably, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. Additionally, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces alpha-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex, because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore, these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.
Tumor suppressor p53 plays an important role in mediating growth inhibition upon telomere dysfunction. Here, we show that loss of the p53 target gene cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21WAF1/CIP1) increases apoptosis induction following telomerase inhibition in a variety of cancer cell lines and mouse xenografts. This effect is highly specific to p21, as loss of other checkpoint proteins and CDK inhibitors did not affect apoptosis. In telomerase, inhibited cell loss of p21 leads to E2F1- and p53-mediated transcriptional activation of p53-upregulated modulator of apoptosis, resulting in increased apoptosis. Combined genetic or pharmacological inhibition of telomerase and p21 synergistically suppresses tumor growth. Furthermore, we demonstrate that simultaneous inhibition of telomerase and p21 also suppresses growth of tumors containing mutant p53 following pharmacological restoration of p53 activity. Collectively, our results establish that inactivation of p21 leads to increased apoptosis upon telomerase inhibition and thus identify a genetic vulnerability that can be exploited to treat many human cancers containing either wild-type or mutant p53.
Aneuploidy causes severe developmental defects and is a near universal feature of tumor cells. Despite its profound effects, the cellular processes affected by aneuploidy are not well characterized. Here, we examined the consequences of aneuploidy on the proteome of aneuploid budding yeast strains. We show that although protein levels largely scale with gene copy number, subunits of multi-protein complexes are notable exceptions. Posttranslational mechanisms attenuate their expression when their encoding genes are in excess. Our proteomic analyses further revealed a novel aneuploidy-associated protein expression signature characteristic of altered metabolism and redox homeostasis. Indeed aneuploid cells harbor increased levels of reactive oxygen species (ROS). Interestingly, increased protein turnover attenuates ROS levels and this novel aneuploidy-associated signature and improves the fitness of most aneuploid strains. Our results show that aneuploidy causes alterations in metabolism and redox homeostasis. Cells respond to these alterations through both transcriptional and posttranscriptional mechanisms.
This is a review of the book, "Drupal in Libraries" by Kenneth J. Varnum. Published by ALA TechSource, 2012.
Objectives: The Lamar Soutter Library at the University of Massachusetts Medical School seeks to evaluate medical students’ awareness of and comfort with data handling and data management concepts. This study will help to triangulate populations and topics for integration of data management curriculum modules. Background: A medical student’s work life is unique due to the demands of their curriculum. In addition, expectations for the stewardship of research data require that students manage their data appropriately. However, data literacy is not a formal component in most undergraduate and graduate student curricula. Libraries have filled this gap by creating educational resources and training opportunities for their communities. For example, the Lamar Soutter Library has developed a comprehensive data management curriculum for students in the sciences. Before piloting this curriculum in the medical school environment, an assessment of medical students’ attitudes toward and comfort levels with data management will isolate receptive populations and curriculum modules. Methods: In Winter 2014, the Lamar Soutter Library will issue a 20-question survey to the students of the School of Medicine, Graduate School of Nursing, and Graduate School of Biomedical Sciences. Critique: The demands of the medical school curricula create a challenge for introducing effective data management training. Needs assessments can identify how data management training can best be integrated into the medical students’ work life. In addition, they may facilitate elective participation in training.
A train-the-trainer presentation about selecting and using the case studies of the New England Collaborative Data Management Curriculum (NECDMC) to teach research data management to diverse audiences.
Presentation on using the research cases in the New England Collaborative Data Management Curriculum to teach data management best practices. Demonstration of how a biomedical research engineering case could be presented to students to teach research data management concepts in a disciplinary context.
Building a Literature Review: A Citation Analysis of Medical Educator’s Research Patterns in Balint Group Studies
This study analyzes how medical educators search literature, using as an example Balint Groups. Balint Group theory is rooted in psychiatry/psychoanalysis. Drawing from literature on medical educator’s search skills, the authors hypothesize that they have not used a systematic approach in their pre-intervention reviews. Instead, it is expected that researchers use literature conveniently found and readily available. Using a citation analysis, this hypothesis will be explored.
Balint Groups began in England in 1950s as a means of teaching students and residents “patient-centered” communication skills. In the U.S., it was first adopted in Family Medicine, then later in different specialties. Due to its international and cross-discipline scope, it is hypothesized that searching for existing literature on Balint Groups can pose a challenge to medical educators. In this study, an exhaustive literature review on Balint Groups will be conducted using the MEDLINE, CINAHL, PsycINFO, EMBASE and ERIC databases. 334 citations were retrieved. A validated inclusion criteria (Robinson et. al., 2011) will be used to select papers from this cohort of results. The authors will then create a comprehensive list of citations used by the selected papers. The analysis will focus on identifying and examining citation patterns to explore factors such as origin of publication and level of evidence of the most highly cited references.
In selecting citations, the authors excluded articles that were a) older than 2003, b) bibliographies only, c) opinion-based letters to the editor (with no citations), and d) meeting abstracts. 112 papers were selected. Citations from these papers were reviewed and Balint-specific citations were selected. The resulting list contained 314 citations, 283 from journal literature and 31 from books. References to primary Balint literature (e.g., books originally published by Michael & Enid Balint who defined Balint Group process) equaled just under 25% of the total citations. The top ten cited journal articles equaled 30% of the total citations. Of these top ten, five were published in the 2000’s, three in the 1990’s and two in the 1980’s. Psychiatry, primary care and doctor-patient relationships where the areas most widely studied using Balint Group practice.
The authors conclude that the hypothesis is correct. Of the 334 total citations retrieved in the initial search, the cited output equals approximately 30% of the available research on Balint Groups. Of this, only 6% is from the top primary resources (Balint-authored books) and top ten cited papers.
Eukaryotic cell division is often regulated by extracellular signals. In budding yeast, signaling from mating pheromones arrests the cell cycle in G1 phase. This arrest requires the protein Far1, which is thought to antagonize the G1/S transition by acting as a Cdk inhibitor (CKI), although the mechanisms remain unresolved. Recent studies found that G1/S cyclins (Cln1 and Cln2) recognize Cdk substrates via specific docking motifs, which promote substrate phosphorylation in vivo. Here, we show that these docking interactions are inhibited by pheromone signaling and that this inhibition requires Far1. Moreover, Far1 mutants that cannot inhibit docking are defective at cell-cycle arrest. Consistent with this arrest function, Far1 outcompetes substrates for association with G1/S cyclins in vivo, and it is present in large excess over G1/S cyclins during the precommitment period where pheromone can impose G1 arrest. Finally, a comparison of substrates that do and do not require docking suggests that Far1 acts as a multimode inhibitor that antagonizes both kinase activity and substrate recognition by Cln1/2-Cdk complexes. Our findings uncover a novel mechanism of Cdk regulation by external signals and shed new light on Far1 function to provide a revised view of cell-cycle arrest in this model system.
Gestational Diabetes and Hypertensive Disorders of Pregnancy Among Women Veterans Deployed in Service of Operations in Afghanistan and Iraq
Objective: To determine the prevalence of gestational diabetes (GDM) and hypertensive disorders of pregnancy (HDP) among women Veterans using Department of Veterans Affairs (VA) maternity benefits previously deployed in service of Operation Enduring Freedom/Operation Iraqi Freedom/Operation New Dawn (OEF/OIF/OND), and whether pregnancy complications were associated with VA use following delivery.
Methods: We identified the study population through linkage with the Department of Defense roster and VA administrative and clinical data. GDM and HDP were identified by International Classification of Diseases, Ninth Revision codes in VA inpatient or outpatient files. Similarly, we constructed a nationally representative sample of deliveries from the Nationwide Inpatient Sample. We calculated standardized incidence ratios (SIR) adjusted for age and year of delivery to compare rates of GDM and HDP. Proportional hazards regression was used to determine whether pregnancy complications were associated with use of VA following delivery.
Results: Between 2001 and 2010, 2,288 women OEF/OIF/OND Veterans used VA maternity benefits; 5.2% had GDM and 9.6% had HDP. Compared with women delivering in the United States, women OEF/OIF/OND Veterans using VA maternity benefits had higher risk of developing GDM (SIR: 1.40; 95% confidence interval [CI] 1.16, 1.68) and HDP (SIR: 1.32; 95% CI 1.15, 1.51). Among women OEF/OIF/OND Veterans using VA maternity benefits, GDM (HR 1.01, 95% CI 0.83, 1.24) and HDP (HR 1.07, 95% CI 0.92, 1.25) were not associated with use of VA following delivery.
Conclusions: Non-VA providers should be aware of their patients' Veteran status and the associated elevated risk for pregnancy complications. Within VA, focused efforts to optimize Veterans' preconception and postpartum health are needed.
State of Reproductive Health In Women Veterans – VA Reproductive Health Diagnoses and Organization of Care
Reproductive health (RH) is a critical part of health. For women, RH encompasses gynecological health throughout life, preconception care, maternity care, cancer care, and the interaction of RH with other mental and medical conditions. Reproductive Health is defined as a state of complete physical, mental, and social well-being and not merely the absence of reproductive disease or infirmity. This definition highlights the importance of taking a health systems approach that integrates RH care issues and services with other aspects of care needed across the life course. The RH needs of women are shaped by their stages of life and life experiences. For women Veterans, their military experiences may influence their RH in important ways. Given the increasing numbers of women in the military and women Veterans, it is critical to understand key aspects of RH in this unique population of women. This first report of the State of Reproductive Health in Women Veterans provides an overview of the RH diagnoses of women Veterans utilizing the Department of Veterans Affairs (VA) health care services, VA delivery of RH care, and a vision for RH in VA.
Objectives: The goal of this project was to develop the first disease-specific instrument for the evaluation of quality of life in chronic pancreatitis.
Methods: Focus groups and interview sessions were conducted, with chronic pancreatitis patients, to identify items felt to impact quality of life which were subsequently formatted into a paper-and-pencil instrument. This instrument was used to conduct an online survey by an expert panel of pancreatologists to evaluate its content validity. Finally, the modified instrument was presented to patients during precognitive testing interviews to evaluate its clarity and appropriateness.
Results: In total, 10 patients were enrolled in the focus groups and interview sessions where they identified 50 items. Once redundant items were removed, the 40 remaining items were made into a paper-and-pencil instrument referred to as the Pancreatitis Quality of Life Instrument. Through the processes of content validation and precognitive testing, the number of items in the instrument was reduced to 24.
Conclusions: This marks the development of the first disease-specific instrument to evaluate quality of life in chronic pancreatitis. It includes unique features not found in generic instruments (economic factors, stigma, and spiritual factors). Although this marks a giant step forward, psychometric evaluation is still needed prior to its clinical use.
Personal reflection on the interplay between radiation oncology, which has long been on the leading edge of technological advances in treatment planning and delivery systems, and the personal side of medicine, often known as high touch.
Targeted germ line disruptions reveal general and species-specific roles for paralog group 1 hox genes in zebrafish
BACKGROUND: The developing vertebrate hindbrain is transiently segmented into rhombomeres by a process requiring Hox activity. Hox genes control specification of rhombomere fates, as well as the stereotypic differentiation of rhombomere-specific neuronal populations. Accordingly, germ line disruption of the paralog group 1 (PG1) Hox genes Hoxa1 and Hoxb1 causes defects in hindbrain segmentation and neuron formation in mice. However, antisense-mediated interference with zebrafish hoxb1a and hoxb1b (analogous to murine Hoxb1 and Hoxa1, respectively) produces phenotypes that are qualitatively and quantitatively distinct from those observed in the mouse. This suggests that PG1 Hox genes may have species-specific functions, or that anti-sense mediated interference may not completely inactivate Hox function in zebrafish.
RESULTS: Using zinc finger and TALEN technologies, we disrupted hoxb1a and hoxb1b in the zebrafish germ line to establish mutant lines for each gene. We find that zebrafish hoxb1a germ line mutants have a more severe phenotype than reported for Hoxb1a antisense treatment. This phenotype is similar to that observed in Hoxb1 knock out mice, suggesting that Hoxb1/hoxb1a have the same function in both species. Zebrafish hoxb1b germ line mutants also have a more severe phenotype than reported for hoxb1b antisense treatment (e.g. in the effect on Mauthner neuron differentiation), but this phenotype differs from that observed in Hoxa1 knock out mice (e.g. in the specification of rhombomere 5 (r5) and r6), suggesting that Hoxa1/hoxb1b have species-specific activities. We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression.
CONCLUSIONS: We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b. Our analyses indicate that Hoxb1 and hoxb1a have comparable functions in zebrafish and mouse, suggesting a conserved function for these genes. In contrast, while Hoxa1 and hoxb1b share functions in the formation of r3 and r4, they differ with regards to r5 and r6, where Hoxa1 appears to control formation of r5, but not r6, in the mouse, whereas hoxb1b regulates formation of r6, but not r5, in zebrafish. Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.
This book surveys critical aspects of modern military health care in the US and various other Western countries with troops in Iraq and Afghanistan. This book covers health care issues prior to deployment, such as screening for mental health, evaluating long-term consequences of exposure to military service, and provision of insurance; care during a conflict, primarily battlefield clinics, battlefield trauma care, and evacuation procedures; and post- combat care, including serious war injuries, psychiatric, and long-term care. Bringing together research from a wide range of contributors, the volume provides readers with an extensive, up-to-date source of information on military medicine.