R-Loops Promote Antisense Transcription across the Mammalian Genome.

Widespread antisense long noncoding RNA (lncRNA) overlap with many protein-coding genes in mammals and emanate from gene promoter, enhancer, and termination regions. However, their origin and biological purpose remain unclear. We show that these antisense lncRNA can be generated by R-loops that form when nascent transcript invades the DNA duplex behind elongating RNA polymerase II (Pol II). Biochemically, R-loops act as intrinsic Pol II promoters to induce de novo RNA synthesis. Furthermore, their removal across the human genome by RNase H1 overexpression causes the selective reduction of antisense transcription. Consequently, we predict that R-loops act to facilitate the synthesis of many gene proximal antisense lncRNA. Not only are R-loops widely associated with DNA damage and repair, but we now show that they have the capacity to promote de novo transcript synthesis that may have aided the evolution of gene regulation.

Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence.

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.

R-loop formation during S phase is restricted by PrimPol-mediated repriming.

During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three-stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single-stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here, we show in vivo how R-loop formation drives a short purine-rich repeat, (GAA)10, to become a replication impediment that engages the repriming activity of the primase-polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R-loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R-loops promote the creation of replication blocks at susceptible structure-forming sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication.

Linking Workplace-Based Assessment to ACGME Milestones: A Comparison of Mapping Strategies in Two Specialties.

Construct: The construct that is assessed is competency in Pediatrics and Internal Medicine residency training. Background: The Accreditation Council for Graduate Medical Education (ACGME) created milestones to measure learner progression toward competence over time but not as direct assessment tools. Ideal measurement of resident performance includes direct observation and assessment of patient care skills in the workplace. Residency programs have linked these concepts by mapping workplace-based assessments to the milestones of ACGME subcompetencies. Mapping is a subjective process, and little is known about specific techniques or the resulting consequences of mapping program-specific assessment data to larger frameworks of competency. Approach: In this article, the authors compare and contrast the techniques used to link workplace-based assessments called Observable Practice Activities (OPAs) to ACGME subcompetencies in two large academic residency programs from different specialties (Internal Medicine and Pediatrics). Descriptive analysis explored the similarities and differences in the assessment data generated by mapping assessment items to larger frameworks of competency. Results: Each program assessed the core competencies with similar frequencies. The largest discrepancy between the two subspecialties was the assessment of Medical Knowledge, which Internal Medicine assessed twice as often. Pediatrics also assessed the core competency Systems-based Practice almost twice as often as Internal Medicine. Both programs had several subcompetencies that were assessed more or less often than what appeared to be emphasized by the blueprint of mapping. Despite using independent mapping processes, both programs mapped each OPA to approximately three subcompetencies. Conclusions: Mapping workplace-based assessments to the ACGME subcompetencies allowed each program to see the whole of their curricula in ways that were not possible before and to identify existing curricular and assessment gaps. Although each program used similar assessment tools, the assessment data generated were different. The lessons learned in this work could inform other programs attempting to link their own workplace-based assessment elements to ACGME subcompetencies.

Terminate and make a loop: regulation of transcriptional directionality.

Bidirectional promoters are a common feature of many eukaryotic organisms from yeast to humans. RNA Polymerase II that is recruited to this type of promoter can start transcribing in either direction using alternative DNA strands as the template. Such promiscuous transcription can lead to the synthesis of unwanted transcripts that may have negative effects on gene expression. Recent studies have identified transcription termination and gene looping as critical players in the enforcement of promoter directionality. Interestingly, both mechanisms share key components. Here, we focus on recent findings relating to the transcriptional output of bidirectional promoters.

Gastric Cancer Screening in Common Variable Immunodeficiency.

Individuals with common variable immunodeficiency (CVID) have an increased risk of gastric cancer, and gastrointestinal lymphoma, yet screening for premalignant gastric lesions is rarely offered routinely to these patients. Proposed screening protocols are not widely accepted and are based on gastric cancer risk factors that are not applicable to all CVID patients. Fifty-two CVID patients were recruited for screening gastroscopy irrespective of symptoms or blood results and were compared to 40 controls presenting for gastroscopy for other clinical indications. Overall, 34% of CVID patients had intestinal metaplasia (IM), atrophic gastritis or moderate to severe non-atrophic gastritis, which can increase the risk of gastric cancer, compared to 7.5% of controls (p < 0.01). Focal nodular lymphoid hyperplasia, a precursor lesion for gastrointestinal lymphoma, was seen in eight CVID patients (16%), one of whom was diagnosed with gastrointestinal lymphoma on the same endoscopy. High-risk gastric pathology was associated with increased time since diagnosis of CVID, smoking, Helicobacter pylori, a low-serum pepsinogen I concentration, and diarrhea, but not pepsinogen I/II ratio, iron studies, vitamin B12 levels or upper gastrointestinal symptoms. There was a lower rate of detection of IM when fewer biopsies were taken, and IM and gastric atrophy were rarely predicted by the endoscopist macroscopically, highlighting the need for standardized biopsy protocols. The prevalence of premalignant gastric lesions in patients with CVID highlights the need for routine gastric screening. We propose a novel gastric screening protocol to detect early premalignant lesions and reduce the risk of gastric cancer and gastric lymphoma in these patients.

Gene loops function to maintain transcriptional memory through interaction with the nuclear pore complex.

Inducible genes in yeast retain a "memory" of recent transcriptional activity during periods of short-term repression, allowing them to be reactivated faster when reinduced. This confers a rapid and versatile gene expression response to the environment. We demonstrate that this memory mechanism is associated with gene loop interactions between the promoter and 3' end of the responsive genes HXK1 and GAL1FMP27. The maintenance of these memory gene loops (MGLs) during intervening periods of transcriptional repression is required for faster RNA polymerase II (Pol II) recruitment to the genes upon reinduction, thereby facilitating faster mRNA accumulation. Notably, a sua7-1 mutant or the endogenous INO1 gene that lacks this MGL does not display such faster reinduction. Furthermore, these MGLs interact with the nuclear pore complex through association with myosin-like protein 1 (Mlp1). An mlp1Delta strain does not maintain MGLs, and concomitantly loses transcriptional memory. We predict that gene loop conformations enhance gene expression by facilitating rapid transcriptional response to changing environmental conditions.

Gene loops juxtapose promoters and terminators in yeast.

Mechanistic analysis of transcriptional initiation and termination by RNA polymerase II (PolII) indicates that some factors are common to both processes. Here we show that two long genes of Saccharomyces cerevisiae, FMP27 and SEN1, exist in a looped conformation, effectively bringing together their promoter and terminator regions. We also show that PolII is located at both ends of FMP27 when this gene is transcribed from a GAL1 promoter under induced and noninduced conditions. Under these conditions, the C-terminal domain of the large subunit of PolII is phosphorylated at Ser5. Notably, inactivation of Kin28p causes a loss of both Ser5 phosphorylation and the loop conformation. These data suggest that gene loops are involved in the early stages of transcriptional activation. They also predict a previously unknown structural dimension to gene regulation, in which both ends of the transcription unit are defined before and during the transcription cycle.

Dynamic interactions between the promoter and terminator regions of the mammalian BRCA1 gene.

The 85-kb breast cancer-associated gene BRCA1 is an established tumor suppressor gene, but its regulation is poorly understood. We demonstrate by gene conformation analysis in both human cell lines and mouse mammary tissue that gene loops are imposed on BRCA1 between the promoter, introns, and terminator region. Significantly, association between the BRCA1 promoter and terminator regions change upon estrogen stimulation and during lactational development. Loop formation is transcription-dependent, suggesting that transcriptional elongation plays an active role in BRCA1 loop formation. We show that the BRCA1 terminator region can suppress estrogen-induced transcription and so may regulate BRCA1 expression. Significantly, BRCA1 promoter and terminator interactions vary in different breast cancer cell lines, indicating that defects in BRCA1 chromatin structure may contribute to dysregulated expression of BRCA1 seen in breast tumors.

Transcription and chromatin-based surveillance mechanism controls suppression of cryptic antisense transcription.

Phosphorylation of the RNA polymerase II C-terminal domain Y1S2P3T4S5P6S7 consensus sequence coordinates key events during transcription, and its deregulation leads to defects in transcription and RNA processing. Here, we report that the histone deacetylase activity of the fission yeast Hos2/Set3 complex plays an important role in suppressing cryptic initiation of antisense transcription when RNA polymerase II phosphorylation is dysregulated due to the loss of Ssu72 phosphatase. Interestingly, although single Hos2 and Set3 mutants have little effect, loss of Hos2 or Set3 combined with ssu72Δ results in a synergistic increase in antisense transcription globally and correlates with elevated sensitivity to genotoxic agents. We demonstrate a key role for the Ssu72/Hos2/Set3 mechanism in the suppression of cryptic antisense transcription at the 3' end of convergent genes that are most susceptible to these defects, ensuring the fidelity of gene expression within dense genomes of simple eukaryotes.

B-cell maturation defects in common variable immunodeficiency and association with clinical features.

AIMS: Patients with common variable immunodeficiency (CVID) often have defects in post-antigenic B-cell differentiation with fewer memory B cells and impaired isotype switching. We aimed to classify CVID patients according to these defects and determine whether this predicted clinical manifestations. METHODS: We analysed the memory marker CD27, maturation marker CD21, and IgD on peripheral blood B cells from 31 CVID patients and 23 controls using a whole-blood lysis technique, allocated patients according to two classifications ('Freiburg' and 'Paris') and correlated results with clinical manifestations. RESULTS: CVID patients had fewer memory (CD27(+)) B cells and isotype-switched (IgD(-)) memory B cells in absolute number and proportion. Many CVID patients had increased immature (CD21(-)) B cells. Lymphoproliferation and autoimmune cytopenias were found almost exclusively in these patients, including Freiburg group Ia (decreased switched memory and increased immature B cells), but also those with normal switched memory and increased immature B cells. The Paris classification was less useful in predicting clinical manifestations. CONCLUSIONS: CVID is associated with defects in memory B-cell differentiation. Subclassification helps identify patients with clinical manifestations, particularly lymphoproliferation and autoimmune cytopenias in those with impaired B-cell maturation and isotype switching. Routine B-cell phenotyping may assist clinicians in predicting these clinical features.

Altered pesticide use on transgenic crops and the associated general impact from an environmental perspective.

The large-scale commercial cultivation of transgenic crops has undergone a steady increase since their introduction 10 years ago. Most of these crops bear introduced traits that are of agronomic importance, such as herbicide or insect resistance. These traits are likely to impact upon the use of pesticides on these crops, as well as the pesticide market as a whole. Organizations like USDA-ERS and NCFAP monitor the changes in crop pest management associated with the adoption of transgenic crops. As part of an IUPAC project on this topic, recent data are reviewed regarding the alterations in pesticide use that have been observed in practice. Most results indicate a decrease in the amounts of active ingredients applied to transgenic crops compared with conventional crops. In addition, a generic environmental indicator -- the environmental impact quotient (EIQ) -- has been applied by these authors and others to estimate the environmental consequences of the altered pesticide use on transgenic crops. The results show that the predicted environmental impact decreases in transgenic crops. With the advent of new types of agronomic trait and crops that have been genetically modified, it is useful to take also their potential environmental impacts into account.

Effects of surface treatment and aging on the bond strength of orthodontic brackets to provisional materials.

PURPOSE: The aim of this study was to evaluate the effects of different surface treatments and aging on the bond strength of orthodontic brackets bonded to provisional materials (autopolymerizing polymethylmethacrylate [PMMA] resins and bis-acryl composite). The mode of failure was also compared. MATERIAL: One hundred twenty flat-surfaced disks of each provisional material were fabricated and embedded in acrylic molds. The specimens were divided randomly into 3 groups of 40, according to the surface treatment rendered: control, polished with greenstone, and sandblasted. Brackets were bonded, and specimens were stored in water at 35 degrees C. Half the specimens in each group were debonded after 1 week, and the other half were debonded after 1 month with a shear-peel load on a testing system with a crosshead speed of 0.5 mm/min. The amount of composite resin left on the specimen surfaces was analyzed and classified with the adhesive remnant index. RESULTS: The bond strengths of brackets to bis-acryl composite resin for all 3 surfaces were clinically acceptable (9-12 MPa) when compared with PMMA (3-5 MPa). The bond strengths of both provisional materials were generally influenced by the kind of surface treatment and aging. The mode of failure was adhesive for PMMA and predominantly cohesive for bis-acryl composite provisional materials. CONCLUSIONS: The bond strength of orthodontic brackets to provisional restorations might depend on material, surface treatment, and time. Brackets should be bonded to bis-acryl composite provisional restorations within 1 week of fabrication.

Quantification of patient fears regarding dental injections and patient perceptions of a local noninjectable anesthetic gel.

Data have shown that 30% of all Americans do not seek dental care and/or treatment unless a problem arises that causes them severe pain. Similar study results have been found in Europe as well. While some studies indicate that cost concerns prevent people from seeking dental care, the fear of pain has been identified as a factor in keeping people from seeing a dentist. A random sample of US and European patients who had recently undergone a scaling and root planing procedure was surveyed via telephone interview to quantify data on patient concerns and fears regarding anesthesia administered by injection, as well as to determine patient interest and price perception of an anesthetic gel product. The survey also provided data on the patient's experience and perception about the scaling and root planing procedure. Responses from the study population showed that patients find the injection painful and do not like the prolonged numbness. Additionally, based on the patients surveyed, they experience injection anxiety before appointments, and a significant number of them cancel appointments or simply do not seek treatment because they are afraid of the injection. Finally, the study also demonstrated that, while not eliminating dental anxiety completely, the availability of a new noninjectable anesthetic would assist in relieving patient fear, with almost half of the patients surveyed being more likely to seek treatment if only the new noninjectable anesthetic was used. Additionally, most patients surveyed would be willing to pay for the noninjectable anesthetic out of their own pockets if it was not covered by their health insurance.

Improving resident handoffs for children transitioning from the intensive care unit.

BACKGROUND AND OBJECTIVE: Handoffs ensure patient safety during patient care transitions in the hospital setting. At our institution, verbal handoffs communicated by resident physicians are suggested practice for patients transferring from the PICU to the hospital medicine (HM) service. Despite their importance, these verbal handoffs occurred only 76% of the time before patient arrival on HM units. Our goal was to increase the completion rate of verbal handoffs to 100% within 5 months. METHODS: Baseline data were collected in a daily survey of HM residents. Interventions were developed and tested on small, incremental change cycles. Key interventions included education about the importance of handoffs, standardization of the handoff process, standardization of handoff documentation, and identification and mitigation of handoff documentation failures. We tracked handoff completion rates by using statistical control charts. After success with improving the completion rate of patient handoffs to the HM service, we applied our process to handoffs from the PICU to all inpatient services. RESULTS: Median completion of verbal patient handoff increased from 76% to 100% within 6 weeks, with improvement sustained for 15 months. Physician compliance with electronic medical record documentation increased from 58% to 94% within 8 months. After spreading to all patients transferring out of the PICU, documentation of patient handoffs increased from 76% to 94% in 5 months. CONCLUSIONS: A system using improvement science methods was successful in increasing the reliability of resident verbal patient handoffs. Consistent documentation and internal redundancy with checklists were associated with sustained improvement.

Chemokine receptor expression in B-cell lymphoproliferative disorders.

Chemokine receptors are expressed by many cells, including lymphoid cells, and function to mediate cell trafficking and localization. Normal B-cells have been reported to express CXCR3, CXCR4, CXCR5, CCR6 and CCR7, however changes in chemokine receptor expression during B-cell development and in B-cell lymphoproliferative disorders (BCLPDs) are incompletely understood, and could provide important information about normal B-cell development and about behavior of neoplastic B-cells. The objective was to perform a systematic study of chemokine receptor expression on B cells from normal subjects and from patients with a range of BCLPDs. Expression of the above chemokine receptors, and CCR5, were analyzed by flow cytometry on lymphocytes from normal controls (n=20), and samples of follicular centre cell lymphoma (FCCL, n=16), precursor B-acute lymphoblastic leukemia (Prec-B-ALL, n=16), chronic lymphocytic leukemia (CLL, n=21), small cell lymphocytic lymphoma (SLL, n=9), hairy cell leukemia (HCL, n=10) and other miscellaneous disorders (n=9). Normal B cells were typically positive for CXCR4, CXCR5 and CCR6, negative for CCR5 and variable for CCR7. Prec-B-ALL cells expressed CXCR4 but were negative for the other receptors. B-CLL cells lost expression of CCR6 but showed strong expression of CCR7. In contrast, SLL cells failed to express CCR7, but were otherwise similar to CLL cells. HCL cells showed absence of CXCR5 and CCR7, but interestingly all but one case expressed CCR5, whilst CD25-negative "variant" HCL cells did not express CCR5. FCCL cells down-regulated CXCR4 and CXCR5 expression and lost expression of CCR6 and CCR7. CXCR3 expression was highly variable on normal B-cells and on cells from BCLPDs, possibly due to its lability during processing. Distinct changes in chemokine receptor expression accompany B-cell development, whilst some BCLPDs show characteristic alterations that may be useful in phenotyping and in understanding biological behavior.

Pesticide residues in food--acute dietary exposure.

Consumer risk assessment is a crucial step in the regulatory approval of pesticide use on food crops. Recently, an additional hurdle has been added to the formal consumer risk assessment process with the introduction of short-term intake or exposure assessment and a comparable short-term toxicity reference, the acute reference dose. Exposure to residues during one meal or over one day is important for short-term or acute intake. Exposure in the short term can be substantially higher than average because the consumption of a food on a single occasion can be very large compared with typical long-term or mean consumption and the food may have a much larger residue than average. Furthermore, the residue level in a single unit of a fruit or vegetable may be higher by a factor (defined as the variability factor, which we have shown to be typically x3 for the 97.5th percentile unit) than the average residue in the lot. Available marketplace data and supervised residue trial data are examined in an investigation of the variability of residues in units of fruit and vegetables. A method is described for estimating the 97.5th percentile value from sets of unit residue data. Variability appears to be generally independent of the pesticide, the crop, crop unit size and the residue level. The deposition of pesticide on the individual unit during application is probably the most significant factor. The diets used in the calculations ideally come from individual and household surveys with enough consumers of each specific food to determine large portion sizes. The diets should distinguish the different forms of a food consumed, eg canned, frozen or fresh, because the residue levels associated with the different forms may be quite different. Dietary intakes may be calculated by a deterministic method or a probabilistic method. In the deterministic method the intake is estimated with the assumptions of large portion consumption of a 'high residue' food (high residue in the sense that the pesticide was used at the highest recommended label rate, the crop was harvested at the smallest interval after treatment and the residue in the edible portion was the highest found in any of the supervised trials in line with these use conditions). The deterministic calculation also includes a variability factor for those foods consumed as units (eg apples, carrots) to allow for the elevated residue in some single units which may not be seen in composited samples. In the probabilistic method the distribution of dietary consumption and the distribution of possible residues are combined in repeated probabilistic calculations to yield a distribution of possible residue intakes. Additional information such as percentage commodity treated and combination of residues from multiple commodities may be incorporated into probabilistic calculations. The IUPAC Advisory Committee on Crop Protection Chemistry has made 11 recommendations relating to acute dietary exposure.

The Figure-of-Eight Walk test: reliability and associations with stroke-specific impairments.

OBJECTIVES: To investigate (1) the intra-rater, inter-rater and test-retest reliabilities of the Figure-of-Eight Walk (F8W) test times; (2) its correlation with other stroke-specific impairments; and (3) the cut-off scores best discriminating patients with stroke from the healthy elderly. DESIGN: Cross-sectional study. SETTING: University-based rehabilitation centre. PARTICIPANTS: A convenience sample of 64 subjects: 35 subjects with chronic stroke and 29 healthy elderly. MAIN OUTCOME MEASURES: F8W test times, Fugl-Meyer Motor Assessment for the lower extremities (FMA-LE), hand-held dynamometer measurements of bilateral hip abductor and knee extensor isometric muscle strength, Five times Sit to Stand Test (FTSTST) times, 10-Meter Walk Test (10MWT), Timed Up and Go Test (TUGT) times, Berg Balance Scale (BBS) and Activities-specific Balance Confidence Scale (ABC) scores. RESULTS: Excellent intra-rater, inter-rater and test-retest reliabilities (intra-class correlation coefficient (ICC) range 0.944-0.999) of F8W test times were found. The F8W test times were also found to be significantly associated with FMA-LE, BBS, FTSTST, TUG scores and 10MWT. No significant correlation was found between F8W test times and either leg strength or ABC results. A F8W test time of 8.2 s was found to be the most representative for discriminating between healthy elderly and stroke subjects, with a sensitivity of 100% and a specificity of 89.7%. CONCLUSIONS: The F8W test time is a reliable measurement tool, which is able to differentiate the patients with stroke and healthy elderly subjects and correlated well with stroke-specific impairments and walking tests. The F8W is a reliable measurement tool for assessing the advanced walking performance of subjects with chronic stroke. Implication for Rehabilitation The F8W test times have excellent intra-rater, inter-rater and test-retest reliabilities in patients with chronic stroke. The F8W test times were also found to be significantly associated with FMA-LE, BBS, FTSTST, TUG scores and 10MWT. A F8W test time of 8.2 s was found to be the most representative for discriminating between healthy elderly and stroke subjects, with a sensitivity of 100% and a specificity of 89.7%. The F8W test time is a reliable and valid measure in assessing the advanced walking skill in patients with stroke.

Rad51, friend or foe?

A protein long recognized for its role in DNA repair has now paradoxically been implicated in DNA damage.