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Modulating R-loop triplex nucleic acid structures reveals their roles across the genome. However, common approaches cannot ascribe functions to R-loops in a locus-associated manner. This protocol presents the use of a locus-associated R-loop-modulating system (dubbed LasR), which employs an inducible RNaseH1-EGFP-dCas9 chimaera. We detail the in silico design of sgRNAs and their transfection with the chimaera, and outline steps confirming RNaseH1-EGFP-dCas9 expression, localization, locus-targeted association, and R-loop modulation in cis or trans using immunoblotting, microscopy, and chromatin and DNA-RNA immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Abraham et al. (2020)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Survival of motor neuron (SMN) functions in diverse biological pathways via recognition of symmetric dimethylarginine (Rme2s) on proteins by its Tudor domain, and deficiency of SMN leads to spinal muscular atrophy. Here we report a potent and selective antagonist with a 4-iminopyridine scaffold targeting the Tudor domain of SMN. Our structural and mutagenesis studies indicate that both the aromatic ring and imino groups of compound 1 contribute to its selective binding to SMN. Various on-target engagement assays support that compound 1 specifically recognizes SMN in a cellular context and prevents the interaction of SMN with the R1810me2s of RNA polymerase II subunit POLR2A, resulting in transcription termination and R-loop accumulation mimicking SMN depletion. Thus, in addition to the antisense, RNAi and CRISPR/Cas9 techniques, potent SMN antagonists could be used as an efficient tool to understand the biological functions of SMN., (© 2022. The Author(s).)

Low-cost genome-wide single-nucleotide polymorphisms (SNPs) are routinely used in animal breeding programs. Compared to SNP arrays, the use of whole-genome sequence data generated by the next-generation sequencing technologies (NGS) has great potential in livestock populations. However, sequencing a large number of animals to exploit the full potential of whole-genome sequence data is not feasible. Thus, novel strategies are required for the allocation of sequencing resources in genotyped livestock populations such that the entire population can be imputed, maximizing the efficiency of whole genome sequencing budgets. We present two applications of linear programming for the efficient allocation of sequencing resources. The first application is to identify the minimum number of animals for sequencing subject to the criterion that each haplotype in the population is contained in at least one of the animals selected for sequencing. The second application is the selection of animals whose haplotypes include the largest possible proportion of common haplotypes present in the population, assuming a limited sequencing budget. Both applications are available in an open source program LPChoose. In both applications, LPChoose has similar or better performance than some other methods suggesting that linear programming methods offer great potential for the efficient allocation of sequencing resources. The utility of these methods can be increased through the development of improved heuristics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cheng, Xu, Li and Abraham.)

Intrauterine growth restriction (IUGR) is related to a higher risk of neonatal mortality, minor cognitive deficit, metabolic syndrome, and cardiovascular disease in adulthood. In previous studies, genetic variants in the FTO (fat mass and obesity-associated) and PPARγ (peroxisome proliferator-activated receptor-gamma) genes have been associated with metabolic disease, body mass index, and obesity among other outcomes. We studied the association of selected FTO (rs1421085, rs55682395, rs17817449, rs8043757, rs9926289, and rs9939609) and PPARγ (rs10865710, rs17036263, rs35206526, rs1801282, rs28763894, rs41516544, rs62243567, rs3856806, and rs1805151) single-nucleotide polymorphisms (SNPs) with IUGR, through a case-control study in a cohort of live births that occurred from June 1978 to May 1979 in a Brazilian city. We selected 280 IUGR cases and 256 controls for analysis. Logistic regression was used to jointly analyze the SNPs as well as factors such as maternal smoking, age, and schooling. We found that the PPARγ rs41516544 increased the risk of IUGR for male offspring (OR 27.83, 95%CI 3.65-212.32) as well as for female offspring (OR=8.94, 95%CI: 1.96-40.88). The FTO rs9939609 TA genotype resulted in a reduced susceptibility to IUGR for male offspring only (OR=0.47, 95%CI: 0.26-0.86). In conclusion, we demonstrated that PPARγ SNP had a positive effect and FTO SNP had a negative effect on IUGR occurrence, and these effects were gender-specific.

Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular mechanisms that influence cancer risk and therapeutic benefit or resistance remain only partially understood. BRCA1 and BRCA2 have also been implicated in the suppression of R-loops, triple-stranded nucleic acid structures composed of a DNA:RNA hybrid and a displaced ssDNA strand. Here, we report that loss of RNF168, an E3 ubiquitin ligase and DNA double-strand break (DSB) responder, remarkably protected Brca1-mutant mice against mammary tumorigenesis. We demonstrate that RNF168 deficiency resulted in accumulation of R-loops in BRCA1/2-mutant breast and ovarian cancer cells, leading to DSBs, senescence, and subsequent cell death. Using interactome assays, we identified RNF168 interaction with DHX9, a helicase involved in the resolution and removal of R-loops. Mechanistically, RNF168 directly ubiquitylated DHX9 to facilitate its recruitment to R-loop-prone genomic loci. Consequently, loss of RNF168 impaired DHX9 recruitment to R-loops, thereby abrogating its ability to resolve R-loops. The data presented in this study highlight a dependence of BRCA1/2-defective tumors on factors that suppress R-loops and reveal a fundamental RNF168-mediated molecular mechanism that governs cancer development and vulnerability.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli 1,2 . Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.

Objective: The objective is to determine if suspected concussions in elite football are medically assessed according to the International Conferences on Concussion in Sport consensus statement recommendations., Setting: Men's Union of European Football Association (UEFA) Football Championship., Participants: All professional football players in the UEFA 2016 Championship Tournament., Design: Observational study., Outcome Measures: Potential concussive events (PCEs) were defined as direct head collision incidents resulting in the athlete being unable to immediately resume play following impact. PCEs identified and description of PCE assessment and outcome were accomplished through direct standardised observation of video footage by trained observers in 51 games played in the Men's UEFA European Championship (10 June-10 July 2016)., Results: Sixty-nine total PCEs (1.35 per match) were identified in 51 games played during the 2016 Men's UEFA European Championship. Forty-eight PCEs (69.6%) resulted in two observable signs of concussion, 13 (18.8%) resulted in three signs and 1 (1.4%) resulted in four signs in the injured athletes. Nineteen (27.5%) PCEs were medically assessed by sideline healthcare personnel while 50 (72.5%) were not. Of the 50 PCEs that were not medically assessed, 44 (88%) PCEs resulted in two or more signs of concussion among injured athletes. Of the 19 medically assessed PCEs, 8 resulted in 3 signs of concussion, and 1 resulted in 4 signs; all assessments concluded in the same-game return for the injured athletes., Conclusions: PCEs were frequent events in the 2016 UEFA Euro championship, but were rarely assessed concordant with the International Conferences on Concussion in Sport consensus statement recommendations. There is an imperative need to improve the assessment and management of players suspected of concussion in elite football., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: A population-based study was undertaken to determine the mortality and morbidity for people with hypoparathyroidism compared to the general population., Methods: In this study, patients identified with chronic hypoparathyroidism using data linkage from regional datasets were compared with five age- and gender-matched controls from the general population. Data from biochemistry, hospital admissions, prescribing and the demographic dataset were linked. Outcomes for mortality and specified conditions were examined for all patients and subdivided into post-surgical and non-surgical cases of hypoparathyroidism., Results: All patients had an increased risk of epilepsy (HR 1.65 [95% CI 1.12-2.44]) and cataracts (HR 2.10 [1.30-3.39]) but no increased fracture risk. Only non-surgical hypoparathyroid patients also had increased mortality (HR 2.11 [1.49-2.98]), cardiovascular disease (HR 2.18 [1.41-3.39]), cerebrovascular disease (HR 2.95 [1.46-5.97]), infection (HR 1.87 [1.2-2.92]) and mental illness (HR 1.59 [1.21-2.11]). There was an increased risk of renal failure (HR 10.05 [95% CI 4.71-21.43]) during the first 2000 days (5.5 years) of follow-up. Renal failure and death were associated with increasing serum calcium concentrations., Conclusion: Patients with hypoparathyroidism have an increased risk of cataract and epilepsy. Non-surgical hypoparathyroidism is associated with increased mortality and additional morbidities., (© 2018 John Wiley & Sons Ltd.)

Ribosomal DNA (rDNA) repeat instability and protein aggregation are thought to be two major and independent drivers of cellular aging. Pbp1, the yeast ortholog of human ATXN2, maintains rDNA repeat stability and lifespan via suppression of RNA-DNA hybrids. ATXN2 polyglutamine expansion drives neurodegeneration causing spinocerebellar ataxia type 2 and promoting amyotrophic lateral sclerosis. Here, molecular characterization of Pbp1 revealed that its knockout or subjection to disease-modeling polyQ expansion represses Ty1 (Transposons of Yeast) retrotransposons by respectively promoting Trf4-depedendent RNA turnover and Ty1 Gag protein aggregation. This aggregation, but not its impact on retrotransposition, compromises rDNA repeat stability and shortens lifespan by hyper-activating Trf4-dependent turnover of intergenic ncRNA within the repeats. We uncover a function for the conserved Pbp1/ATXN2 proteins in the promotion of retrotransposition, create and describe powerful yeast genetic models of ATXN2-linked neurodegenerative diseases, and connect the major aging mechanisms of rDNA instability and protein aggregation., Competing Interests: The authors declare no competing interests.

Karan ("Josh") Abraham is a physician-scientist trainee in the University of Toronto MD-PhD program. He is a Vanier Scholar, Ruggle's Innovation Award winner, Adel S. Sedra Distinguished Graduate Award recipient, and is currently president of the Clinician-Investigator Trainee Association of Canada (CITAC). In his PhD work, he uses molecular, genetic and cell biological approaches to uncover mechanisms that preserve the integrity of the genetic code and sustain the protein synthesis capacity of cells. He hopes to lead a basic research program that will advance scientific knowledge to better understand and treat human disease, and to one day become a leading ambassador for Canadian biomedical research.

Damaged DNA shows increased mobility, which can promote interactions with repair-conducive nuclear pore complexes (NPCs). This apparently random mobility is paradoxically abrogated upon disruption of microtubules or kinesins, factors that typically cooperate to mediate the directional movement of macromolecules. Here, we resolve this paradox by uncovering DNA damage-inducible intranuclear microtubule filaments (DIMs) that mobilize damaged DNA and promote repair. Upon DNA damage, relief of centromeric constraint induces DIMs that cooperate with the Rad9 DNA damage response mediator and Kar3 kinesin motor to capture DNA lesions, which then linearly move along dynamic DIMs. Decreasing and hyper-inducing DIMs respectively abrogates and hyper-activates repair. Accounting for DIM dynamics across cell populations by measuring directional changes of damaged DNA reveals that it exhibits increased non-linear directional behavior in nuclear space. Abrogation of DIM-dependent processes or repair-promoting factors decreases directional behavior. Thus, inducible and dynamic nuclear microtubule filaments directionally mobilize damaged DNA and promote repair.

Purpose: The fusion gene BCR-ABL has an important role to the progression of chronic myeloid leukemia (CML) and several signaling pathways have been characterized as responsible for the terminal blastic phase (BP). However, the initial phase, the chronic phase (CP), is long lasting and there is much yet to be understood about the critical role of BCR-ABL in this phase. This study aims to evaluate transcriptional deregulation in CD34+ hematopoietic cells (CD34+ cells) from patients with untreated newly diagnosed CML compared with CD34+HC from healthy controls., Methods: Gene expression profiling in CML-CD34 cells and CD34 cells from healthy controls were used for this purpose with emphasis on five main pathways important for enhanced proliferation/survival, enhanced self-renewal and block of myeloid differentiation., Results: We found 835 genes with changed expression levels (fold change ≥ ±2) in CML-CD34 cells compared with CD34 cells. These include genes belonging to PI3K/AKT, WNT/b-catenin, SHH, NOTCH and MAPK signaling pathways. Four of these pathways converge to MYC activation. We also identified five transcripts upregulated in CD34-CML patients named OSBPL9, MEK2, p90RSK, TCF4 and FZD7 that can be potential biomarkers in CD34-CML-CP., Conclusion: We show several mRNAs up- or downregulated in CD34-CML during the chronic phase.

A decade of CITAC Annual General Meetings: 2007-2017 In 2007, the Clinician Investigator Trainee Association of Canada (CITAC) launched its inaugural Annual General Meeting (AGM). The AGM has since become a major annual event, jointly organized by CITAC and leaders from the Canadian Society for Clinical Investigation (CSCI), and continues to provide a forum for clinician investigator (CI) trainees to exchange ideas, advance career prospects and engage with the broader community. Indeed, since its inception, all Canadian institutions with medical doctor and clinician investigator (MD+CI) training programs have participated in the AGM, while more than 1,000 trainees have registered as CITAC members. The 10th CITAC-CSCI AGM was recently held in Toronto (November 20-22, 2017). There were nearly 200 attendees, including CI trainees, faculty member and physician leaders from across Canada (Figure 1A, 1B). Trainees spanning diverse career stages had opportunities to participate in interactive poster sessions, workshops and lectures by leading physician-scientists. These exercises were designed to encourage and enhance networking, career development and mentorship for prospective physician-scientists.

Medulloblastoma (MB) is a malignant childhood brain tumor which at molecular level is classified into at least four major subtypes: WNT, SHH, group C and group D differing in response to treatment. Previous studies have associated changes in expression levels and activation of certain HOX genes with MB development. In the present study, we investigate the role of HOX genes in two attributes acquired by tumor cells: migration and proliferation potential, as well as, in vivo tumorigenic potential. We analyzed UW402, UW473, DAOY and ONS-76 human pediatric MB cell lines and cerebellum primary cultures. Two-color microarray-based gene expression analysis was used to identify differentially expressed HOX genes. Among the various HOX genes significantly overexpressed in DAOY and ONS-76 cell lines compared to UW402 and UW473 cell lines, HOXA10 and HOXB4 were selected for further analysis. The expression levels of these HOX genes were validated by real-time PCR. A mouse model was used to study the effect of the HOXA10 and HOXB4 genes on the in vivo tumorigenic potential and the in vitro proliferative and migration potential of MB cell lines. Our results show that the inhibition of HOXA10 in DAOY cell line led to increased in vitro cell migration while in vitro cell proliferation or in vivo tumorigenic potential were unaffected. We also observed that induced expression of HOXB4 in the UW473 cell line significantly reduced in vitro cell proliferation and migration capability of UW473 cells with no effect on the in vivo tumorigenicity. This suggests that HOXA10 plays a role in migration events and the HOXB4 gene is involved in proliferation and migration processes of medulloblastoma cells, however, it appears that these genes are not essential for the tumorigenic process of these cells.

Calorie restriction (CR) is a broadly effective environmental intervention that extends life by operating through numerous biological processes. Here, we discuss how non-coding RNA (ncRNA) molecules act as mediators and targets of lifespan-extending CR. We also highlight how these RNA molecules connect CR to its effects on genome stability, cell metabolism, programmed cell death, senescence, cancer, and neurodegeneration. We anticipate that an advanced understanding of the connections between CR and non-coding RNA will provide unique insights into aging mechanisms while pointing to novel approaches aimed at modulating aging and age-related diseases., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The 2016 Annual General Meeting of the Canadian Society of Clinician Investigators (CSCI) and Clinician Investigator Trainee Association of Canada/Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was a national conference held in Toronto November 21-23, 2016, in conjunction with The University of Toronto Clinician Investigator Program Research Day. The theme for this year's meeting was "Mapping Your Career as a Clinician-Scientist"; emphasizing essential skills for developing a fruitful career as clinician-scientist. The meeting featured an opening presentation by Dr. Alan Underhill, Dr. Nicola Jones and Alexandra Kuzyk. The keynote speakers were Dr. Nada Jabado (McGill University), who discussed the association between cancer and histones, Dr. Norman Rosenblum (University of Toronto), who addressed the career path and the "calling" of the Clinician Scientist, Dr. Martin Schmeing (McGill University), who was the 2016 Joe Doupe Award recipient, and Dr. Linda Rabeneck (Cancer Care Ontario and University of Toronto), who received the Friends of CIHR lectureship. The workshops, focusing on career development for clinician scientists, were hosted by Drs. Alan Underhill, Nicola Jones, Lynn Raymond, Michael Schlossmacher and Norman Rosenblum, as well as University of Toronto communication specialists, Caitlin Johannesson and Suzanne Gold. In addition, the Young Investigators' Forum included presentations from clinician investigator trainees from across the country. The research topics were diverse and comprehensive: from basic sciences to clinical practice; from epidemiology to medical engineering. All scientific abstracts are summarized in this review. Over 70 abstracts were showcased at this year's meeting during two poster sessions, with six outstanding abstracts selected for oral presentations during the President's Forum.

Amyloids play critical roles in human diseases but have increasingly been recognized to also exist naturally. Shared physicochemical characteristics of amyloids and of their smaller oligomeric building blocks offer the prospect of molecular interactions and crosstalk amongst these assemblies, including the propensity to mutually influence aggregation. A case in point might be the recent discovery of an interaction between the amyloid β peptide (Aβ) and somatostatin (SST). Whereas Aβ is best known for its role in Alzheimer disease (AD) as the main constituent of amyloid plaques, SST is intermittently stored in amyloid-form in dense core granules before its regulated release into the synaptic cleft. This review was written to introduce to readers a large body of literature that surrounds these two peptides. After introducing general concepts and recent progress related to our understanding of amyloids and their aggregation, the review focuses separately on the biogenesis and interactions of Aβ and SST, before attempting to assess the likelihood of encounters of the two peptides in the brain, and summarizing key observations linking SST to the pathobiology of AD. While the review focuses on Aβ and SST, it is to be anticipated that crosstalk amongst functional and disease-associated amyloids will emerge as a general theme with much broader significance in the etiology of dementias and other amyloidosis., Competing Interests: Conflict of interests: A provisional US patent (application number 62/451-309) titled ‘Oligomeric Abeta-Binding Polypeptides) that lists HW and GS as inventors was filed on January 27, 2017, by the University of Toronto., (Copyright: © 2017, Lau et al. and Applied Systems.)

Adult honey bee queens and workers drastically differ in ovary size. This adult ovary phenotype difference becomes established during the final larval instar, when massive programmed cell death leads to the degeneration of 95-99% of the ovariole anlagen in workers. The higher juvenile hormone (JH) levels in queen larvae protect the ovaries against such degeneration. To gain insights into the molecular architecture underlying this divergence critical for adult caste fate and worker sterility, we performed a microarray analysis on fourth and early fifth instar queen and worker ovaries. For the fourth instar we found nine differentially expressed genes (DEGs) with log 2 FC > 1.0, but this number increased to 56 in early fifth-instar ovaries. We selected 15 DEGs for quantitative PCR (RT-qPCR) analysis. Nine differed significantly by the variables caste and/or development. Interestingly, genes with enzyme functions were higher expressed in workers, while those related to transcription and signaling had higher transcript levels in queens. For the RT-qPCR confirmed genes we analyzed their response to JH. This revealed a significant up-regulation for two genes, a short chain dehydrogenase reductase (sdr) and a heat shock protein 90 (hsp90). Five other genes, including hsp60 and hexamerin 70b (hex70b), were significantly down-regulated by JH. The sdr gene had previously come up as differentially expressed in other transcriptome analyses on honey bee larvae and heat shock proteins are frequently involved in insect hormone responses, this making them interesting candidates for further functional assays., (Copyright © 2016. Published by Elsevier Ltd.)

Dietary calorie restriction is a broadly acting intervention that extends the lifespan of various organisms from yeast to mammals. On another front, magnesium (Mg 2+ ) is an essential biological metal critical to fundamental cellular processes and is commonly used as both a dietary supplement and treatment for some clinical conditions. If connections exist between calorie restriction and Mg 2+ is unknown. Here, we show that Mg 2+ , acting alone or in response to dietary calorie restriction, allows eukaryotic cells to combat genome-destabilizing and lifespan-shortening accumulations of RNA-DNA hybrids, or R-loops. In an R-loop accumulation model of Pbp1-deficient Saccharomyces cerevisiae, magnesium ions guided by cell membrane Mg 2+ transporters Alr1/2 act via Mg 2+ -sensitive R-loop suppressors Rnh1/201 and Pif1 to restore R-loop suppression, ribosomal DNA stability and cellular lifespan. Similarly, human cells deficient in ATXN2, the human ortholog of Pbp1, exhibit nuclear R-loop accumulations repressible by Mg 2+ in a process that is dependent on the TRPM7 Mg 2+ transporter and the RNaseH1 R-loop suppressor. Thus, we identify Mg 2+ as a biochemical signal of beneficial calorie restriction, reveal an R-loop suppressing function for human ATXN2 and propose that practical magnesium supplementation regimens can be used to combat R-loop accumulation linked to the dysfunction of disease-linked human genes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Dysfunction of key miRNA pathways regulating basic cellular processes is a common driver of many cancers. However, the biological roles and/or clinical applications of such pathways in Merkel cell carcinoma (MCC), a rare but lethal cutaneous neuroendocrine (NE) malignancy, have yet to be determined. Previous work has established that miR-375 is highly expressed in MCC tumors, but its biological role in MCC remains unknown. Herein, we show that elevated miR-375 expression is a specific feature of well-differentiated MCC cell lines that express NE markers. In contrast, miR-375 is strikingly down-regulated in highly aggressive, undifferentiated MCC cell lines. Enforced miR-375 expression in these cells induced NE differentiation, and opposed cancer cell viability, migration, invasion, and survival, pointing to tumor-suppressive roles for miR-375. Mechanistically, miR-375-driven phenotypes were caused by the direct post-transcriptional repression of multiple Notch pathway proteins (Notch2 and RBPJ) linked to cancer and regulation of cell fate. Thus, we detail a novel molecular axis linking tumor-suppressive miR-375 and Notch with NE differentiation and cancer cell behavior in MCC. Our findings identify miR-375 as a putative regulator of NE differentiation, provide insight into the cell of origin of MCC, and suggest that miR-375 silencing may promote aggressive cancer cell behavior through Notch disinhibition., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application., Methods: Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium, Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated., Results: We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF, respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF, respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium, for two cryopreservation systems (P<0.05)., Conclusion: We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated, maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

DNA double-strand breaks (DSBs) are often targeted to nuclear pore complexes (NPCs) for repair. How targeting is achieved and the DNA repair pathways involved in this process remain unclear. Here, we show that the kinesin-14 motor protein complex (Cik1-Kar3) cooperates with chromatin remodellers to mediate interactions between subtelomeric DSBs and the Nup84 nuclear pore complex to ensure cell survival via break-induced replication (BIR), an error-prone DNA repair process. Insertion of a DNA zip code near the subtelomeric DSB site artificially targets it to NPCs hyperactivating this repair mechanism. Kinesin-14 and Nup84 mediate BIR-dependent repair at non-telomeric DSBs whereas perinuclear telomere tethers are only required for telomeric BIR. Furthermore, kinesin-14 plays a critical role in telomerase-independent telomere maintenance. Thus, we uncover roles for kinesin and NPCs in DNA repair by BIR and reveal that perinuclear telomere anchors license subtelomeric DSBs for this error-prone DNA repair mechanism.

Ligation-mediated-PCR was performed followed by the mapping of 177 and 150 integration sites from HepG2 and Hek293 transduced with chimera vector carrying recombinant human Factor IX (rhFIX) cDNA, respectively. The sequences were analyzed for chromosome preference, CpG, transcription start site (TSS), repetitive elements, fragile sites and target genes. In HepG2, rhFIX was had an increased preference for chromosomes 6 and 17; the median distance to the nearest CpG islands was 15,240 base pairs and 37 % of the integrations occurred in RefSeq genes. In Hek293, rhFIX had an increased preference for chromosome 5; the median distance to the nearest CpG islands was 209,100 base pairs and 74 % of the integrations occurred in RefSeq genes. The integrations in both cell lines were distant from the TSS. The integration patterns associated with this vector are different in each cell line.

Declining brain and neurobiological function is arguably one of the most common features of human aging. The study of conserved aging processes as well as the characterization of various neurodegenerative diseases using different genetic models such as yeast, fly, mouse, and human systems is uncovering links to non-coding RNAs. These links implicate a variety of RNA-regulatory processes, including microRNA function, paraspeckle formation, RNA-DNA hybrid regulation, nucleolar RNAs and toxic RNA clearance, amongst others. Here we highlight these connections and reveal over-arching themes or questions related to recently appreciated roles of non-coding RNA in neural function and dysfunction across lifespan.

Background: Genetic Analyses in large sample populations are important for a better understanding of the variation between populations, for designing conservation programs, for detecting rare mutations which may be risk factors for a variety of diseases, among other reasons. However these analyses frequently assume that the participating individuals or animals are mutually unrelated which may not be the case in large samples, leading to erroneous conclusions. In order to retain as much data as possible while minimizing the risk of false positives it is useful to identify a large subset of relatively unrelated individuals in the population. This can be done using a heuristic for finding a large set of independent of nodes in an undirected graph. We describe a fast randomized heuristic for this purpose. The same methodology can also be used for identifying a suitable set of markers for analyzing population stratification, and other instances where a rapid heuristic for maximal independent sets in large graphs is needed., Results: We present FastIndep, a fast random heuristic algorithm for finding a maximal independent set of nodes in an arbitrary undirected graph along with an efficient implementation in C++. On a 64 bit Linux or MacOS platform the execution time is a few minutes, even with a graph of several thousand nodes. The algorithm can discover multiple solutions of the same cardinality. FastIndep can be used to discover unlinked markers, and unrelated individuals in populations., Conclusions: The methods presented here provide a quick and efficient method for identifying sets of unrelated individuals in large populations and unlinked markers in marker panels. The C++ source code and instructions along with utilities for generating the input files in the appropriate format are available at http://taurus.ansci.iastate.edu/wiki/people/jabr/Joseph&#95;Abraham.html.

Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Kn(b) allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Kn(b) and KAM(+)) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.

Markov chain Monte Carlo (MCMC) methods have been widely used to overcome computational problems in linkage and segregation analyses. Many variants of this approach exist and are practiced; among the most popular is the Gibbs sampler. The Gibbs sampler is simple to implement but has (in its simplest form) mixing and reducibility problems; furthermore in order to initiate a Gibbs sampling chain we need a starting genotypic or allelic configuration which is consistent with the marker data in the pedigree and which has suitable weight in the joint distribution. We outline a procedure for finding such a configuration in pedigrees which have too many loci to allow for exact peeling. We also explain how this technique could be used to implement a blocking Gibbs sampler.

Dichelobacter nodosus is the causative agent of ovine foot rot, a disease that is a constant economic burden for many Western sheep ranches. Vaccination is one method of treating foot rot. A higher and more specific immune response is observed when monovalent vaccines are used to treat foot rot, as compared to multivalent vaccines, which incorporate all 10 major New Zealand D. nodosus serogroups. There is no single assay for specifically identifying and grouping D. nodosus for the purpose of incorporating only the desired serogroup(s) in a vaccine. A polymerase chain reaction (PCR)-based assay was used to specifically identify and group D. nodosus from a foot rot lesion. Identification and grouping was determined by predicted fragment size analysis and nucleotide sequence information. The PCR approach vastly improves the accuracy in identifying and grouping D. nodosus from a foot rot lesion., (Copyright 1999 Academic Press.)

This is a retrospective, hospital based study of the resuscitative management of 40 consecutive, multitrauma patients (Injury Severity Score (ISS) > 25) admitted directly from an inner metropolitan environment over a one year period. The aim was to identify physiological, anatomical and time variables that correlated with an adverse outcome. Such information would facilitate the development of management protocols to improve future care. The clinical management of airways, breathing, circulation and head injury was reviewed in both the pre-hospital and Emergency Department (ED) phases of care. Eleven patients died during the resuscitative phase, 10 from blood loss and one from head injury. Nine patients died during the definitive care phase, seven from head injury and two from multiple organ failure. Scene hypotension (systolic blood pressure < or = 80 mmHg), ED Glasgow Coma Scale < 9, ISS > or = 50, and Revised Trauma Score < or = 4 were variables that correlated strongly with fatal outcomes. The median pre-hospital time was 33 min for those hypotensive in the field. The median ED time was 70 min for hypotensive patients who went to operating theatres. Survival following severe trauma may be increased by avoiding secondary insults in head injured patients and improving the management of haemorrhagic shock. The time frame from accident to operating theatre should be kept under 90 min. Warmed blood, fresh frozen plasma and platelets should be used early in the resuscitation. An early move to definitive control of bleeding should accompany vigorous volume resuscitation.

This report describes the epidemiology of pedestrian injury in four inner metropolitan local government areas of Sydney. These data were obtained from the Roads and Traffic Authority of New South Wales. The spectrum of injury and clinical outcome was defined in patients with an Injury Severity Score (ISS) > 15 admitted from the study area, during a 1 year period, to the four inner metropolitan teaching hospitals. The incidence of pedestrian death was 3.3 times the state average of 32/10(6)/year. An average of 235 pedestrians, injured in the study area, were hospitalized each year during the period 1987-89. On average 24 pedestrians died each year, seven at the scene and 17 in hospital. Fifty patients (ISS > 15) were admitted to the four teaching hospitals during a 1 year period 1990-91. Forty-five were adults and five children. Multiplicity of injury was seen in 68% of patients. The pelvis and lower extremities were involved in 70%, the head in 66% and chest in 42%. The hospital mortality rate was 30% with five patients dying on the first day from blood loss and nine dying during subsequent days from head injury. This study has important implications for trauma service development. Successful clinical management of the severely injured pedestrian requires close co-operation between pre-hospital and hospital care providers. An integrated hospital trauma team response is mandatory to ensure appropriate management of what is often a shocked, hypoxic, head-injured patient.