A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response.

Helix-destabilizing DNA lesions induced by environmental mutagens such as UV light cause genomic instability by strongly blocking the progression of DNA replication forks (RFs). At blocked RF, single-stranded DNA (ssDNA) accumulates and is rapidly bound by Replication Protein A (RPA) complexes. Such stretches of RPA-ssDNA constitute platforms for recruitment/activation of critical factors that promote DNA synthesis restart. However, during periods of severe replicative stress, RPA availability may become limiting due to inordinate sequestration of this multifunctional complex on ssDNA, thereby negatively impacting multiple vital RPA-dependent processes. Here, we performed a genome-wide screen to identify factors that restrict the accumulation of RPA-ssDNA during UV-induced replicative stress. While this approach revealed some expected "hits" acting in pathways such as nucleotide excision repair, translesion DNA synthesis, and the intra-S phase checkpoint, it also identified SCAI, whose role in the replicative stress response was previously unappreciated. Upon UV exposure, SCAI knock-down caused elevated accumulation of RPA-ssDNA during S phase, accompanied by reduced cell survival and compromised RF progression. These effects were independent of the previously reported role of SCAI in 53BP1-dependent DNA double-strand break repair. We also found that SCAI is recruited to UV-damaged chromatin and that its depletion promotes nascent DNA degradation at stalled RF. Finally, we (i) provide evidence that EXO1 is the major nuclease underlying ssDNA formation and DNA replication defects in SCAI knockout cells and, consistent with this, (ii) demonstrate that SCAI inhibits EXO1 activity on a ssDNA gap in vitro. Taken together, our data establish SCAI as a novel regulator of the UV-induced replicative stress response in human cells.

A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21.

Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.

A potent nuclear export mechanism imposes USP16 cytoplasmic localization during interphase.

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper.

A Pilot Study towards the Impact of Type 2 Diabetes on the Expression and Activities of Drug Metabolizing Enzymes and Transporters in Human Duodenum.

To characterize effects of type 2 diabetes (T2D) on mRNA expression levels for 10 Cytochromes P450 (CYP450s), two carboxylesterases, and three drug transporters (ABCB1, ABCG2, SLCO2B1) in human duodenal biopsies. To compare drug metabolizing enzyme activities of four CYP450 isoenzymes in duodenal biopsies from patients with or without T2D. mRNA levels were quantified (RT-qPCR) in human duodenal biopsies obtained from patients with (n = 20) or without (n = 16) T2D undergoing a scheduled gastro-intestinal endoscopy. CYP450 activities were determined following incubation of biopsy homogenates with probe substrates for CYP2B6 (bupropion), CYP2C9 (tolbutamide), CYP2J2 (ebastine), and CYP3A4/5 (midazolam). Covariables related to inflammation, T2D, demographic, and genetics were investigated. T2D had no major effects on mRNA levels of all enzymes and transporters assessed. Formation rates of metabolites (pmoles mg protein-1 min-1) determined by LC-MS/MS for CYP2C9 (0.48 ± 0.26 vs. 0.41 ± 0.12), CYP2J2 (2.16 ± 1.70 vs. 1.69 ± 0.93), and CYP3A (5.25 ± 3.72 vs. 5.02 ± 4.76) were not different between biopsies obtained from individuals with or without T2D (p > 0.05). No CYP2B6 specific activity was measured. TNF-α levels were higher in T2D patients but did not correlate with any changes in mRNA expression levels for drug metabolizing enzymes or transporters in the duodenum. T2D did not modulate expression or activity of tested drug metabolizing enzymes and transporters in the human duodenum. Previously reported changes in drug oral clearances in patients with T2D could be due to a tissue-specific disease modulation occurring in the liver and/or in other parts of the intestines.

Validation and refinement of a clinical decision rule for the use of computed tomography in children with minor head injury in the emergency department.

BACKGROUND: There is uncertainty about which children with minor head injury need to undergo computed tomography (CT). We sought to prospectively validate the accuracy and potential for refinement of a previously derived decision rule, Canadian Assessment of Tomography for Childhood Head injury (CATCH), to guide CT use in children with minor head injury. METHODS: This multicentre cohort study in 9 Canadian pediatric emergency departments prospectively enrolled children with blunt head trauma presenting with a Glasgow Coma Scale score of 13-15 and loss of consciousness, amnesia, disorientation, persistent vomiting or irritability. Phys icians completed standardized assessment forms before CT, including clinical predictors of the rule. The primary outcome was neurosurgical intervention and the secondary outcome was brain injury on CT. We calculated test characteristics of the rule and used recursive partitioning to further refine the rule. RESULTS: Of 4060 enrolled patients, 23 (0.6%) underwent neurosurgical intervention, and 197 (4.9%) had brain injury on CT. The original 7-item rule (CATCH) had sensitivities of 91.3% (95% confidence interval [CI] 72.0%-98.9%) for neurosurgical intervention and 97.5% (95% CI 94.2%-99.2%) for predicting brain injury. Adding "≥ 4 episodes of vomiting" resulted in a refined 8-item rule (CATCH2) with 100% (95% CI 85.2%-100%) sensitivity for neurosurgical intervention and 99.5% (95% CI 97.2%-100%) sensitivity for brain injury. INTERPRETATION: Among children presenting to the emergency department with minor head injury, the CATCH2 rule was highly sensitive for identifying those children requiring neurosurgical intervention and those with any brain injury on CT. The CATCH2 rule should be further validated in an implementation study designed to assess its clinical impact.

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair.

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1-3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response.

Characterization of hMTr1, a human Cap1 2'-O-ribose methyltransferase.

Cellular eukaryotic mRNAs are capped at their 5' ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2'-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2'-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo.

Impact of GSTA1 Polymorphisms on Busulfan Oral Clearance in Adult Patients Undergoing Hematopoietic Stem Cell Transplantation.

BACKGROUND: Busulfan pharmacokinetics exhibit large inter-subject variability. Our objective was to evaluate the influence of glutathione S-transferase A1 (GSTA1) gene variants on busulfan oral clearance (CLo) in a population of patients undergoing hematopoietic stem cell transplantation. METHODS: This is a quasi-experimental retrospective study in adult patients (n = 87 included in the final analyses) receiving oral busulfan. Pharmacokinetics data (area under the plasma concentration-time curve (AUC) determined from 10 blood samples) were retrieved from patients' files and GSTA1 *A and *B allele polymorphisms determined from banked DNA samples. Three different limited sampling methods (LSM) using four blood samples were also compared. RESULTS: Carriers of GSTA1*B exhibited lower busulfan CLo than patients with an *A/*A genotype (p < 0.002): Busulfan CLo was 166 ± 31, 187 ± 37 vs. 207 ± 47 mL/min for GSTA1*B/*B, *A/*B and *A/*A genotypes, respectively. Similar results were obtained with the tested LSMs. Using the standard AUC method, distribution of patients above the therapeutic range after the first dose was 29% for GSTA1*A/*A, 50% for *A/*B, and 65% for *B/*B. The LSMs correctly identified ≥91% of patients with an AUC above the therapeutic range. The misclassified patients had a mean difference less than 5% in their AUCs. CONCLUSION: Patients carrying GSTA1 loss of function *B allele were at increased risk of overdosing on their initial busulfan oral dose. Genetic polymorphisms associated with GSTA1 explain a significant part of busulfan CLo variability which could be captured by LSM strategies.

An open-source algorithm for rapid unbiased determination of DNA fiber length.

DNA fiber fluorography is widely employed to study the kinetics of DNA replication, but the usefulness of this approach has been limited by the lack of freely-available automated analysis tools. Quantification of DNA fibers usually relies on manual examination of immunofluorescence microscopy images, which is laborious and prone to inter- and intra-operator variability. To address this, we developed an unbiased, fully automated algorithm that quantifies length and color of DNA fibers from fluorescence microscopy images. Our fiber quantification method, termed FiberQ, is an open-source image processing tool based on edge detection and a novel segment splicing approach. Here, we describe the algorithm in detail, validate our results experimentally, and benchmark the analysis against manual assessments. Our implementation is offered free of charge to the scientific community under the General Public License.

Opto-magnetic capture of individual cells based on visual phenotypes.

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes.

Ten renin-angiotensin system-related gene polymorphisms in maximally treated Canadian Caucasian patients with heart failure.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: The progression and pharmacological response of heart failure-affected patients are subject to interindividual variability. It is also acknowledged that the genotype frequency of certain gene polymorphisms varies across different ethnic groups and that a difference in gene polymorphism frequencies between healthy and heart failure patients seems to exist. WHAT THIS STUDY ADDS: This study investigated associations between 10 gene polymorphisms of RAAS-related genes with an individual's susceptibility to heart failure. Our data suggest that the angiotensinogen (AGT) 235 single nucleotide polymorphism (SNP) may be associated with heart failure in our population and that the AGT(M174)-AGT(T235) haplotype, as well as the AGT/angiotensin-converting enzyme (ACE) gene combination, may play an important role in disease predisposition. AIMS: Racial differences in survival outcomes point towards a genetic role in the pathophysiology of heart failure. Furthermore, contemporary evidence links genetics to heart failure (HF) predisposition. We tested for a difference in prevalence of 10 renin-angiotensin-aldosterone system (RAAS)-related gene polymorphisms between a homogenous population of HF patients and healthy controls. METHODS: One hundred and eleven healthy volunteers and 58 HF patients were included in this study. The healthy control group consisted of males aged between 18 and 35 years old. The HF group consisted of patients (89.7% male) who were 63.8 +/- 7.9 years old, were in New York Heart Association (NYHA) class II-III and had a documented left ventricular ejection fraction (LVEF)

Effects of AGTR1 A1166C gene polymorphism in patients with heart failure treated with candesartan.

BACKGROUND: The benefits of angiotensin II receptor blockers (ARBs) in patients with heart failure who are treated with standard pharmacotherapy, including an angiotensin-converting enzyme (ACE) inhibitor, were demonstrated in 2 large randomized trials. It is currently impossible to determine which patient will benefit from the addition of an ARB. OBJECTIVE: To explore the impact of selected candidate genes on the hemodynamic, neurohormonal, and antiinflammatory effects of candesartan in patients with heart failure who are already being treated with an ACE inhibitor. METHODS: We investigated the impact of 10 candidate genetic polymorphisms on the effects of candesartan in patients with heart failure who are treated with an ACE inhibitor. We evaluated their impact on acute (2 wk) and long-term (24 wk) changes in blood pressure and N-terminal proB-type natriuretic peptide (NT-proBNP) and high sensitivity C-reactive protein (hsCRP) during treatment with candesartan. RESULTS: Thirty-one patients were included. Homozygotes of the AGTR1 A1166 allele (n = 13) had a greater decrease in systolic (-9.1 +/- 4.7 vs 1.1 +/- 3.3 mm Hg; p = 0.04 by analysis of variance [ANOVA], adjusting for dose) and diastolic blood pressure (-5.1 +/- 1.5 vs 1.9 +/- 1.9 mm Hg; p = 0.005 by ANOVA, adjusting for dose) compared with C1166 allele carriers (n = 18) following 2 weeks of treatment. After 6 months of treatment, C1166 carriers experienced a greater decrease in NT-proBNP (-151.4 [-207; -19.8] ng/L vs 147.3 [-61.3; 882.9] ng/L; p = 0.03) and hsCRP (-0.8 [-2.2; -0.03] mg/L) vs 0.2 [-1.8; 5.3] mg/L; p = 0.09) compared with patients carrying the AA1166 genotype. No other significant association was found. CONCLUSIONS: The results of this proof-of concept study provide the first evidence that the AGTR1 A1166C polymorphism could influence the response to candesartan in patients with heart failure who are receiving ACE inhibitors. Validation of these exploratory findings in larger populations is required before use of the AGTR1 A1166C genotype can be incorporated into clinical practice.

Dynamics of nascent mRNA folding and RNA-protein interactions: an alternative TAR RNA structure is involved in the control of HIV-1 mRNA transcription.

HIV-1 Tat protein regulates transcription elongation by binding to the 59 nt TAR RNA stem-loop structure transcribed from the HIV-1 5' long terminal repeat (5'-LTR). This established Tat-TAR interaction was used to investigate mRNA folding and RNA-protein interactions during early transcription elongation from the HIV-1 5'-LTR. Employing a new site-specific photo-cross-linking strategy to isolate transcription elongation complexes at early steps of elongation, we found that Tat interacts with HIV-1 transcripts before the formation of full-length TAR (TAR59). Analysis of RNA secondary structure by free energy profiling and ribonuclease digestion indicated that nascent transcripts folded into an alternative TAR RNA structure (TAR31), which requires only 31 nt to form and includes an analogous Tat-binding bulge structure. Functionally, TAR31, similar to TAR59, acts as a transcriptional terminator in vitro, and mRNA expression from TAR31-deficient HIV-1 5'-LTR mutant promoters is significantly decreased. Our results support a role for TAR31 in the control of HIV-1 mRNA transcription and we propose that this structure is important to stabilize the short early transcripts before the transcription complex commits for processive elongation. Overall, this study demonstrates that RNA folding during HIV-1 transcription is dynamic and that as the nascent RNA chain grows during transcription, it folds into a number of conformations that function to regulate gene expression. Finally, our results provide a new experimental strategy for studying mRNA conformation changes during transcription that can be applied to investigate the folding and function of nascent RNA structures transcribed from other promoters.

Replication Protein A Availability during DNA Replication Stress Is a Major Determinant of Cisplatin Resistance in Ovarian Cancer Cells.

Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines.Significance: The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance. Cancer Res; 78(19); 5561-73. ©2018 AACR.

Effects of a Series of Acidic Drugs on L-Lactic Acid Transport by the Monocarboxylate Transporters MCT1 and MCT4.

BACKGROUND: Drug-induced myopathy is a serious side effect that often requires removal of a medication from a drug regimen. For most drugs, the underlying mechanism of drug-induced myopathy remains unclear. Monocarboxylate transporters (MCTs) mediate L-lactic acid transport, and inhibition of MCTs may potentially lead to perturbation of L-lactic acid accumulation and muscular disorders. Therefore, we hypothesized that L-lactic acid transport may be involved in the development of drug-induced myopathy. The aim of this study was to assess the inhibitory potential of 24 acidic drugs on L-lactic acid transport using breast cancer cell lines Hs578T and MDA-MB-231, which selectively express MCT1 and MCT4, respectively. METHODS: The influx transport of L-lactic acid was minimally inhibited by all drugs tested. The efflux transport was next examined: loratadine (IC50: 10 and 61 µM) and atorvastatin (IC50: 78 and 41 µM) demonstrated the greatest potency for inhibition of L-lactic acid efflux by MCT1 and MCT4, respectively. Acidic drugs including fluvastatin, cerivastatin, simvastatin acid, lovastatin acid, irbesartan and losartan exhibited weak inhibitory potency on L-lactic acid efflux. RESULTS: Our results suggest that some acidic drugs, such as loratadine and atorvastatin, can inhibit the efflux transport of L-lactic acid. CONCLUSION: This inhibition may cause an accumulation of intracellular L-lactic acid leading to acidification and muscular disorders.

Aligning coding sequences with frameshift extension penalties.

BACKGROUND: Frameshift translation is an important phenomenon that contributes to the appearance of novel coding DNA sequences (CDS) and functions in gene evolution, by allowing alternative amino acid translations of gene coding regions. Frameshift translations can be identified by aligning two CDS, from a same gene or from homologous genes, while accounting for their codon structure. Two main classes of algorithms have been proposed to solve the problem of aligning CDS, either by amino acid sequence alignment back-translation, or by simultaneously accounting for the nucleotide and amino acid levels. The former does not allow to account for frameshift translations and up to now, the latter exclusively accounts for frameshift translation initiation, not considering the length of the translation disruption caused by a frameshift. RESULTS: We introduce a new scoring scheme with an algorithm for the pairwise alignment of CDS accounting for frameshift translation initiation and length, while simultaneously considering nucleotide and amino acid sequences. The main specificity of the scoring scheme is the introduction of a penalty cost accounting for frameshift extension length to compute an adequate similarity score for a CDS alignment. The second specificity of the model is that the search space of the problem solved is the set of all feasible alignments between two CDS. Previous approaches have considered restricted search space or additional constraints on the decomposition of an alignment into length-3 sub-alignments. The algorithm described in this paper has the same asymptotic time complexity as the classical Needleman-Wunsch algorithm. CONCLUSIONS: We compare the method to other CDS alignment methods based on an application to the comparison of pairs of CDS from homologous human, mouse and cow genes of ten mammalian gene families from the Ensembl-Compara database. The results show that our method is particularly robust to parameter changes as compared to existing methods. It also appears to be a good compromise, performing well both in the presence and absence of frameshift translations. An implementation of the method is available at https://github.com/UdeS-CoBIUS/FsePSA.

CAEP 2015 Academic Symposium: Current State and Recommendations to Achieve Adequate and Sustainable Funding for Emergency Medicine Academic Units.

OBJECTIVES: To describe the current state of academic emergency medicine (EM) funding in Canada and develop recommendations to grow and establish sustainable funding. METHODS: A panel of eight leaders from different EM academic units was assembled. Using mixed methods (including a literature review, sharing of professional experiences, a survey of current EM academic heads, and data previously collected from an environmental scan), 10 recommendations were drafted and presented at an academic symposium. Attendee feedback was incorporated, and the second set of draft recommendations was further distributed to the Canadian Association Emergency Physicians (CAEP) Academic Section for additional comments before being finalized. RESULTS: Recommendations were developed around the funding challenges identified and solutions developed by academic EM university-based units across Canada. A strategic plan was seen as integral to achieving strong funding of an EM unit, especially when it aligned with departmental and institutional priorities. A business plan, although occasionally overlooked, was deemed an important component for planning and sustaining the academic mission. A number of recommendations surrounding philanthropy consisted of creating partnerships with existing foundations and engaging multiple stakeholders and communities. Synergy between academic and clinical EM departments was also viewed as an opportunity to ensure integration of common missions. Education and networking for current and future leaders were also viewed as invaluable to ensure that opportunities are optimized through strong leadership development and shared experiences to further the EM academic missions across the country. CONCLUSIONS: These recommendations were designed to improve the financial circumstances for many Canadian EM units. There is a considerable wealth of resources that can contribute to financial stability for an academic unit, and an annual networking meeting and continuing education on these issues will facilitate more rapid implementation of these recommendations.

Study of the functional interaction of the 900 Tetraloop of 16S ribosomal RNA with helix 24 within the bacterial ribosome.

The 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) is amongst the most conserved regions of rRNA. This tetraloop forms a GNRA motif that docks into the minor groove of three base-pairs at the bottom of helix 24 of 16S rRNA in the 30S subunit. Both the tetraloop and its receptor in helix 24 contact the 23S rRNA, forming the intersubunit bridge B2c. Here, we investigated the interaction between the 900 tetraloop and its receptor by genetic complementation. We used a specialized ribosome system in combination with an in vivo instant evolution approach to select mutations in helix 24 compensating for a mutation in the 900 tetraloop (A900G) that severely decreases ribosomal activity, impairing subunit association and translational fidelity. We selected two mutants where the G769-C810 base-pair of helix 24 was substituted with either U-A or C x A. When these mutations in helix 24 were investigated in the context of a wild-type 900 tetraloop, the C x A but not the U-A mutation severely impaired ribosome activity, interfering with subunit association and decreasing translational fidelity. In the presence of the A900G mutation, both mutations in helix 24 increased the ribosome activity to the same extent. Subunit association and translational fidelity were increased to the same level. Computer modeling was used to analyze the effect of the mutations in helix 24 on the interaction between the tetraloop and its receptor. This study demonstrates the functional importance of the interaction between the 900 tetraloop and helix 24.

Functional studies of the 900 tetraloop capping helix 27 of 16S ribosomal RNA.

The 900 tetraloop (positions 898-901) of Escherichia coli 16S rRNA caps helix 27, which is involved in a conformational switch crucial for the decoding function of the ribosome. This tetraloop forms a GNRA motif involved in intramolecular RNA-RNA interactions with its receptor in helix 24 of 16S rRNA. It is involved also in an intersubunit bridge, via an interaction with helix 67 in domain IV of 23S rRNA. Using a specialized ribosome system and an instant-evolution procedure, the four nucleotides of this loop were randomized and 15 functional mutants were selected in vivo. Positions 899 and 900, responsible for most of the tetraloop/receptor interactions, were found to be the most critical for ribosome activity. Functional studies showed that mutations in the 900 tetraloop impair subunit association and decrease translational fidelity. Computer modeling of the mutations allows correlation of the effect of mutations with perturbations of the tetraloop/receptor interactions.