Cloning of terminal transferase cDNA by antibody screening

A cDNA library was prepared from a terminal deoxynucleotidyltransferase-containing thymoma in the phage vector λgt11. By screening plaques with anti-terminal transferase antibody, positive clones were identified of which some had β-galactosidase-cDNA fusion proteins identifiable after electrophoretic fractionation by immunoblotting with anti-terminal transferase antibody. The predominant class of cross-hybridizing clones was determined to represent cDNA for terminal transferase by showing that one representative clone hybridized to a 2200-nucleotide mRNA in close-matched enzyme-positive but not to enzyme-negative cells and that the cDNA selected a mRNA that translated to give a protein of the size and antigenic characteristics of terminal transferase. Only a small amount of genomic DNA hybridized to the longest available clone, indicating that the sequence is virtually unique in the mouse genome

The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation1. Mutations in DDX3 are linked to tumorigenesis2-4 and intellectual disability5, and the enzyme is targeted by a range of viruses6. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions

Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates

Nitrogen-containing-bisphosphonates (N-BPs) are widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance

Neural connectivity biotypes: associations with internalizing problems throughout adolescence.

BackgroundNeurophysiological patterns may distinguish which youth are at risk for the well-documented increase in internalizing symptoms during adolescence. Adolescents with internalizing problems exhibit altered resting-state functional connectivity (RSFC) of brain regions involved in socio-affective processing. Whether connectivity-based biotypes differentiate adolescents' levels of internalizing problems remains unknown.MethodSixty-eight adolescents (37 females) reported on their internalizing problems at ages 14, 16, and 18 years. A resting-state functional neuroimaging scan was collected at age 16. Time-series data of 15 internalizing-relevant brain regions were entered into the Subgroup-Group Iterative Multi-Model Estimation program to identify subgroups based on RSFC maps. Associations between internalizing problems and connectivity-based biotypes were tested with regression analyses.ResultsTwo connectivity-based biotypes were found: a Diffusely-connected biotype (N = 46), with long-range fronto-parietal paths, and a Hyper-connected biotype (N = 22), with paths between subcortical and medial frontal areas (e.g. affective and default-mode network regions). Higher levels of past (age 14) internalizing problems predicted a greater likelihood of belonging to the Hyper-connected biotype at age 16. The Hyper-connected biotype showed higher levels of concurrent problems (age 16) and future (age 18) internalizing problems.ConclusionsDifferential patterns of RSFC among socio-affective brain regions were predicted by earlier internalizing problems and predicted future internalizing problems in adolescence. Measuring connectivity-based biotypes in adolescence may offer insight into which youth face an elevated risk for internalizing disorders during this critical developmental period

CRISPRi-based radiation modifier screen identifies long non-coding RNA therapeutic targets in glioma.

BackgroundLong non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients.ResultsWe use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy.ConclusionsThese studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy

FORCE-TJR: Innovative design for a national TJR comparative effectiveness research database

BACKGROUND: Joint replacement (TJR) registries have traditionnally focused on collecting implant data and analyzing time-to-revision. Sub-optimal outcomes short of revision are important to surgeons and patients. In 2010, the US federal Agency for Healthcare Research and Quality funded Function and Outcomes Research for Comparative Effectiveness in TJR (FORCE-TJR), a research consortium and database to collect comprehensive TJR outcomes, including patient-reported pain and function and post-operative sequelae. This $12 million research award will provide new information about post-TJR adverse events, patient-reported functional gain, and implant longevity. We developed novel methods to assure critical data collection and sustainability. METHODS: FORCE-TJR developed methods to (1) assemble a research consortium that includes a national sample of diverse surgeons and practices who agree to invite all patients to participate, (2) implement a virtual model for patient consent and data entry of consistent, validated patient-reported surveys, (3) conduct efficient screening for post-TJR sequelae and validated chart review and adjudication, and (4) document implant details. RESULTS: In the first 8 months, FORCE-TJR enrolled more than 90 surgeons in urban and rural settings, across 21 states; with academic, private, and HMO ownership; performing varied annual volumes of TJR surgery. Across practices, 80-95% of patients enrolled and more than 3250 patients consented to complete standardized surveys. More than 150 patients are enrolled each week, and enrollment rates will grow as additional surgeons join. CONCLUSION: FORCE-TJR employs innovative strategies to collect comprehensive post-TJR data from a national cohort of more than 30,000 patients. Comparative effectiveness research emerging from these data will include patient, implant, health system predictors of post-TJR adverse events, pain relief, functional gain, and revision. These TJR analyses will offer novel and important new evidence to guide patient and surgeon decisions, and are possible only because of this comprehensive research design

Polymer-based paclitaxel-eluting stents reduce in-stent neointimal tissue proliferation A serial volumetric intravascular ultrasound analysis from the TAXUS-IV trial

ObjectivesThe aim of this study was to use serial volumetric intravascular ultrasound (IVUS) to evaluate the effects of polymer-based, paclitaxel-eluting stents on in-stent neointima formation and late incomplete stent apposition.BackgroundThe TAXUS-IV trial demonstrated that the slow-release, polymer-based, paclitaxel-eluting stent reduces angiographic restenosis and the need for repeat revascularization procedures. Serial IVUS studies reveal details of the pattern of vascular responses provoked by stent implantation that provide insight into device safety and efficacy.MethodsIn the TAXUS-IV trial, patients were randomized to the slow-release, polymer-based, paclitaxel-eluting TAXUS stent or a bare-metal EXPRESS stent (Boston Scientific Corp., Natick, Massachusetts). As part of a formal substudy, complete volumetric IVUS data were available in 170 patients, including 88 TAXUS patients and 82 controls, at implantation and at nine-month follow-up.ResultsNo baseline differences were present in the clinical characteristics or IVUS parameters between the control and TAXUS groups. At nine-month follow-up, IVUS lumen volumes were larger in the TAXUS group (123 ± 43 mm3vs. 104 ± 44 mm3, p = 0.005), due to a reduction in neointimal volume (18 ± 18 mm3vs. 41 ± 23 mm3, p < 0.001). Millimeter-by-millimeter analysis within the stent demonstrated uniform suppression of neointimal growth along the entire stent length. Late lumen loss was similar at the proximal edge of the stent between the two groups, and reduced with the TAXUS stent at the distal edge (p = 0.004). Incomplete stent apposition at nine months was observed in only 3.0% of control and 4.0% of TAXUS stents (p = 0.12).ConclusionsPolymer-based, paclitaxel-eluting TAXUS stents are effective in inhibiting neointimal tissue proliferation, and do not result in late incomplete stent apposition

A prudent path forward for genomic engineering and germline gene modification

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed

Development and Evaluation of a Palliative Medicine Curriculum for Third-Year Medical Students

Abstract Objective: To assess the impact, retention, and magnitude of effect of a required didactic and experiential palliative care curriculum on third-year medical students' knowledge, confidence, and concerns about end-of-life care, over time and in comparison to benchmark data from a national study of internal medicine residents and faculty. Design: Prospective study of third-year medical students prior to and immediately after course completion, with a follow-up assessment in the fourth year, and in comparison to benchmark data from a large national study. Setting: Internal Medicine Clerkship in a public accredited medical school. Participants: Five hundred ninety-three third-year medical students, from July 2002 to December 2007. Main outcome measures: Pre- and postinstruction performance on: knowledge, confidence (self-assessed competence), and concerns (attitudes) about end-of-life care measures, validated in a national study of internal medicine residents and faculty. Medical student's reflective written comments were qualitatively assessed. Intervention: Required 32-hour didactic and experiential curriculum, including home hospice visits and inpatient hospice care, with content drawn from the AMA-sponsored Education for Physicians on End-of-life Care (EPEC) Project. Results: Analysis of 487 paired t tests shows significant improvements, with 23% improvement in knowledge (F1,486=881, p<0.001), 56% improvement in self-reported competence (F1,486=2,804, p<0.001), and 29% decrease in self-reported concern (F1,486=208, p<0.001). Retesting medical students in the fourth year showed a further 5% increase in confidence (p<0.0002), 13% increase in allaying concerns (p<0.0001), but a 6% drop in knowledge. The curriculum's effect size on M3 students' knowledge (0.56) exceeded that of a national cross-sectional study comparing residents at progressive training levels (0.18) Themes identified in students' reflective comments included perceived relevance, humanism, and effectiveness of methods used to teach and assess palliative care education. Conclusions: We conclude that required structured didactic and experiential palliative care during the clinical clerkship year of medical student education shows significant and largely sustained effects indicating students are better prepared than a national sample of residents and attending physicians.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98455/1/jpm%2E2010%2E0502.pd

Compensation of BRG-1 Function by Brm: INSIGHT INTO THE ROLE OF THE CORE SWI·SNF SUBUNITS IN RETINOBLASTOMA TUMOR SUPPRESSOR SIGNALING

The BRG-1 subunit of the SWI-SNF complex is involved in chromatin remodeling and has been implicated in the action of the retinoblastoma tumor suppressor (RB). Given the importance of BRG-1 in RB function, germ line BRG-1 mutations in tumorigenesis may be tantamount to RB inactivation. Therefore, in this study we assessed the behavior of cells harboring discrete BRG-1 alleles for the RB-signaling pathway. Using p16ink4a, an upstream activator of endogenous RB, or a constitutively active RB construct (PSM-RB), we determined that the majority of tumor lines with germ line defects in BRG-1 were sensitive to RB-mediated cell cycle arrest. By contrast, A427 (lung carcinoma) cells were resistant to expression of p16ink4a and PSM-RB. Analysis of the SWI-SNF subunits in the different tumor lines revealed that A427 are deficient for BRG-1 and its homologue, Brm, whereas RB-sensitive cell lines retained Brm expression. Similarly, the RB-resistant SW13 and C33A cell lines were also deficient for both BRG-1/Brm. Reintroduction of either BRG-1 or Brm into A427 or C33A cells restored RB-mediated signaling to cyclin A to cause cell cycle arrest. Consistent with this compensatory role, we observed that Brm could also drive expression of CD44. We also determined that loss of these core SWI-SNF subunits renders SW13 cells resistant to activation of the RB pathway by the chemotherapeutic agent cisplatin, since reintroduction of either BRG-1 or Brm into SW13 cells restored the cisplatin DNA-damage checkpoint. Together, these data demonstrate that Brm can compensate for BRG-1 loss as pertains to RB sensitivity