Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast.

Timely completion of genome replication is a prerequisite for mitosis, genome integrity, and cell survival. A challenge to this timely completion comes from the need to replicate the hundreds of untranscribed copies of rDNA that organisms maintain in addition to the copies required for ribosome biogenesis. Replication of these rDNA arrays is relegated to late S phase despite their large size, repetitive nature, and essentiality. Here, we show that, in Saccharomyces cerevisiae, reducing the number of rDNA repeats leads to early rDNA replication, which results in delaying replication elsewhere in the genome. Moreover, cells with early-replicating rDNA arrays and delayed genome-wide replication aberrantly release the mitotic phosphatase Cdc14 from the nucleolus and enter anaphase prematurely. We propose that rDNA copy number determines the replication time of the rDNA locus and that the release of Cdc14 upon completion of rDNA replication is a signal for cell cycle progression.

Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains.

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two yeast strains challenged by hydroxyurea, an inhibitor of the ribonucleotide reductase. We gained understanding of the impact on origin activation by three factors: S-phase checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Wild type W303 showed a significant reduction of fork progression accompanied by an elevated level of Rad53 phosphorylation as well as physical presence at origins compared to A364a. Moreover, a rad53K227A mutant in W303 activated more origins, accompanied by global reduction of ssDNA across all origins, compared to A364a. Sequence polymorphism in the consensus motifs of origins plays a minor role in determining strain-specific activity. Finally, we identified a new class of origins only active in checkpoint-proficient cells, which we named "Rad53-dependent origins". Our study presents a comprehensive list of differentially used origins and provide new insights into the mechanisms of origin activation.

Emergency department preparation for COVID-19: accelerated care units.

By 11 February 2020 when the WHO named the novel coronavirus (SARS-CoV-2) and the disease it causes (COVID-19), it was evident that the virus was spreading rapidly outside of China. Although San Francisco did not confirm its first locally transmitted cases until the first week of March, our ED and health system began preparing for a potential COVID-19 surge in late February 2020.In this manuscript, we detail how the above responses were instrumental in the rapid deployment of two military-grade negative-pressure medical tents, named accelerated care units (ACU). We describe engagement of our workforce, logistics of creating new care areas, ensuring safety through personal protective equipment access and conservation, and the adaptive leadership challenges that this process posed.We know of no other comprehensive examples of how EDs have prepared for COVID-19 in the peer-reviewed literature. Many other EDs both in and outside of California have requested access to the details of how we operationalised our ACUs to facilitate their own planning. This demonstrates the urgent need to disseminate this information to our colleagues. Below we describe the process of developing and launching our ACUs as a potential model for other EDs around the country.

The Case for Observation Medicine Education and Training in Emergency Medicine.

BACKGROUND: Many hospitals have or will be opening an observation unit (OU), the majority managed by the emergency department (ED). Graduating emergency medicine (EM) residents will be expected to have the knowledge and skills necessary to appropriately identify and manage patients in this setting. Our objective is to examine the current state of observation medicine (OM) education and prevalence in EM training. METHODS: In a follow-up to the 2019 Society for Academic Emergency Medicine (SAEM) OM Interest Group meeting, we convened an expert panel of OM physicians who are members of both the SAEM OM Interest Group and the American College of Emergency Physicians Section of OM. The panel of six emergency physicians representing geographic diversity was formed. A structured literature review was performed yielding 16 educational publications and sources pertaining to OM education and training across all specialties. REPORT ON THE EXISTING LITERATURE: Only a small number of EM residencies have a required or elective OM rotation in an OU. An OM rotation in a protocol-driven ED OU gives residents experience managing patients in this setting and improves skills integral to EM and part of the EM milestones and Accreditation Council for Graduate Medical Education (ACGME) core competencies: reassessment, disposition decision making, risk stratification, team management, and practicing cost-appropriate care. Even without a formal rotation, multiple OM educational resources can be incorporated into EM resident education and didactics. Education research opportunity exists. CONCLUSIONS: This panel believes that OM is an important component of EM that should be incorporated into EM residency as the knowledge and skills learned such as risk stratification, disposition decision making, and team management augment those needed for the practice of EM. There is a distinct opportunity for EM educators to better equip their trainees for a career in EM by including OM education and experience in EM residency training.

Challenges and Approaches to Genotyping Repetitive DNA.

Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.

The effects of manipulating levels of replication initiation factors on origin firing efficiency in yeast.

Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome.

Phenotypic and Genotypic Consequences of CRISPR/Cas9 Editing of the Replication Origins in the rDNA of Saccharomyces cerevisiae.

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, thus the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae, developed independently but similar to one developed by others. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9-mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication have on cellular health. We find that long-term growth of the edited clones results in faster-growing suppressors that have acquired segmental aneusomy of the rDNA-containing region of chromosome XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently, and homogeneously edit the rDNA origin via CRISPR/Cas9.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

rDNA Copy Number Variants Are Frequent Passenger Mutations in Saccharomyces cerevisiae Deletion Collections and de Novo Transformants.

The Saccharomyces cerevisiae ribosomal DNA (rDNA) locus is known to exhibit greater instability relative to the rest of the genome. However, wild-type cells preferentially maintain a stable number of rDNA copies, suggesting underlying genetic control of the size of this locus. We performed a screen of a subset of the Yeast Knock-Out (YKO) single gene deletion collection to identify genetic regulators of this locus and to determine if rDNA copy number correlates with yeast replicative lifespan. While we found no correlation between replicative lifespan and rDNA size, we identified 64 candidate strains with significant rDNA copy number differences. However, in the process of validating candidate rDNA variants, we observed that independent isolates of our de novo gene deletion strains had unsolicited but significant changes in rDNA copy number. Moreover, we were not able to recapitulate rDNA phenotypes from the YKO yeast deletion collection. Instead, we found that the standard lithium acetate transformation protocol is a significant source of rDNA copy number variation, with lithium acetate exposure being the treatment causing variable rDNA copy number events after transformation. As the effects of variable rDNA copy number are being increasingly reported, our finding that rDNA is affected by lithium acetate exposure suggested that rDNA copy number variants may be influential passenger mutations in standard strain construction in S. cerevisiae.

Sirtuins in yeast: phenotypes and tools.

Originally discovered as a transcriptional silencing protein, SIR2 was later linked to yeast replicative aging and the rest was history. Sir2p is now known to be a member of a class of protein deacetylases with a unique enzymatic activity coupling the deacetylation event to NAD(+) hydrolysis. While still incompletely understood, the mechanism by which Sir2p modulates yeast aging is linked to inhibition of rDNA recombination. Here we describe phenotypes associated with yeast Sirtuins and assays used to monitor Sirtuin function in yeast, including the replicative aging assay.

A natural polymorphism in rDNA replication origins links origin activation with calorie restriction and lifespan.

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.

C. elegans BLOC-1 functions in trafficking to lysosome-related gut granules.

The human disease Hermansky-Pudlak syndrome results from defective biogenesis of lysosome-related organelles (LROs) and can be caused by mutations in subunits of the BLOC-1 complex. Here we show that C. elegans glo-2 and snpn-1, despite relatively low levels of amino acid identity, encode Pallidin and Snapin BLOC-1 subunit homologues, respectively. BLOC-1 subunit interactions involving Pallidin and Snapin were conserved for GLO-2 and SNPN-1. Mutations in glo-2 and snpn-1,or RNAi targeting 5 other BLOC-1 subunit homologues in a genetic background sensitized for glo-2 function, led to defects in the biogenesis of lysosome-related gut granules. These results indicate that the BLOC-1 complex is conserved in C. elegans. To address the function of C. elegans BLOC-1, we assessed the intracellular sorting of CDF-2::GFP, LMP-1, and PGP-2 to gut granules. We validated their utility by analyzing their mislocalization in intestinal cells lacking the function of AP-3, which participates in an evolutionarily conserved sorting pathway to LROs. BLOC-1(-) intestinal cells missorted gut granule cargo to the plasma membrane and conventional lysosomes and did not have obviously altered function or morphology of organelles composing the conventional lysosome protein sorting pathway. Double mutant analysis and comparison of AP-3(-) and BLOC-1(-) phenotypes revealed that BLOC-1 has some functions independent of the AP-3 adaptor complex in trafficking to gut granules. We discuss similarities and differences of BLOC-1 activity in the biogenesis of gut granules as compared to mammalian melanosomes, where BLOC-1 has been most extensively studied for its role in sorting to LROs. Our work opens up the opportunity to address the function of this poorly understood complex in cell and organismal physiology using the genetic approaches available in C. elegans.

Natural polymorphism in BUL2 links cellular amino acid availability with chronological aging and telomere maintenance in yeast.

Aging and longevity are considered to be highly complex genetic traits. In order to gain insight into aging as a polygenic trait, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard strain RM11 and a laboratory strain S288c, to identify quantitative trait loci that control chronological lifespan. Among the major loci that regulate chronological lifespan in this cross, one genetic linkage was found to be congruent with a previously mapped locus that controls telomere length variation. We found that a single nucleotide polymorphism in BUL2, encoding a component of an ubiquitin ligase complex involved in trafficking of amino acid permeases, controls chronological lifespan and telomere length as well as amino acid uptake. Cellular amino acid availability changes conferred by the BUL2 polymorphism alter telomere length by modulating activity of a transcription factor Gln3. Among the GLN3 transcriptional targets relevant to this phenotype, we identified Wtm1, whose upregulation promotes nuclear retention of ribonucleotide reductase (RNR) components and inhibits the assembly of the RNR enzyme complex during S-phase. Inhibition of RNR is one of the mechanisms by which Gln3 modulates telomere length. Identification of a polymorphism in BUL2 in this outbred yeast population revealed a link among cellular amino acid availability, chronological lifespan, and telomere length control.

Differential use of diagnostic ultrasound in u.s. Emergency departments by time of day.

BACKGROUND: Survey data over the last several decades suggests that emergency department (ED) access to diagnostic ultrasound performed by the radiology department is unreliable, particularly outside of regular business hours. OBJECTIVE: To evaluate the association between the time of day of patient presentation and the use of diagnostic ultrasound services in United States (U.S.) EDs. METHODS: This was a cross-sectional study of ED patient visits using the National Hospital Ambulatory Medical Care Survey for the years 2003 to 2005. Our main outcome measure was the use of diagnostic ultrasound during the ED patient visit as abstracted from the medical record. We performed multivariate analyses to identify any association between ultrasound use and time of presentation for all patients, as well as for two subgroups who are more likely to need ultrasound as part of their routine workup: patients at risk of deep venous thrombosis, and patients at risk for ectopic pregnancy. RESULTS: During the three-year period, we analyzed 110,447 patient encounters, representing 39 million national visits. Of all ED visits, 2.6% received diagnostic ultrasound. Presenting to the ED "off hours" (defined as Monday through Friday 7pm to 7am and weekends) was associated with a lower rate of ultrasound use independent of potential confounders (odds ratio [OR] 0.73, 95% confidence interval [CI]: 0.65 - 0.82). Patients at increased risk of deep venous thrombosis who presented to the ED during "off hours" were also less likely to undergo diagnostic ultrasound (OR 0.34, 95% CI: 0.15 - 0.79). Similarly, patients at increased risk of ectopic pregnancy received fewer diagnostic ultrasounds during "off hours" (OR 0.56, 95% CI 0.35 - 0.91). CONCLUSION: In U.S. EDs, ultrasound use was lower during "off hours," even among patient populations where its use would be strongly indicated.

glo-3, a novel Caenorhabditis elegans gene, is required for lysosome-related organelle biogenesis.

Gut granules are specialized lysosome-related organelles that act as sites of fat storage in Caenorhabditis elegans intestinal cells. We identified mutations in a gene, glo-3, that functions in the formation of embryonic gut granules. Some glo-3(-) alleles displayed a complete loss of embryonic gut granules, while other glo-3(-) alleles had reduced numbers of gut granules. A subset of glo-3 alleles led to mislocalization of gut granule contents into the intestinal lumen, consistent with a defect in intracellular trafficking. glo-3(-) embryos lacking gut granules developed into adults containing gut granules, indicating that glo-3(+) function may be differentially required during development. We find that glo-3(+) acts in parallel with or downstream of the AP-3 complex and the PGP-2 ABC transporter in gut granule biogenesis. glo-3 encodes a predicted membrane-associated protein that lacks obvious sequence homologs outside of nematodes. glo-3 expression initiates in embryonic intestinal precursors and persists almost exclusively in intestinal cells through adulthood. GLO-3GFP localizes to the gut granule membrane, suggesting it could play a direct role in the trafficking events at the gut granule. smg-1(-) suppression of glo-3(-) nonsense alleles indicates that the C-terminal half of GLO-3, predicted to be present in the cytoplasm, is not necessary for gut granule formation. Our studies identify GLO-3 as a novel player in the formation of lysosome-related organelles.

Function of the Caenorhabditis elegans ABC transporter PGP-2 in the biogenesis of a lysosome-related fat storage organelle.

Caenorhabditis elegans gut granules are intestine specific lysosome-related organelles with birefringent and autofluorescent contents. We identified pgp-2, which encodes an ABC transporter, in screens for genes required for the proper formation of gut granules. pgp-2(-) embryos mislocalize birefringent material into the intestinal lumen and are lacking in acidified intestinal V-ATPase-containing compartments. Adults without pgp-2(+) function similarly lack organelles with gut granule characteristics. These cellular phenotypes indicate that pgp-2(-) animals are defective in gut granule biogenesis. Double mutant analysis suggests that pgp-2(+) functions in parallel with the AP-3 adaptor complex during gut granule formation. We find that pgp-2 is expressed in the intestine where it functions in gut granule biogenesis and that PGP-2 localizes to the gut granule membrane. These results support a direct role of an ABC transporter in regulating lysosome biogenesis. Previously, pgp-2(+) activity has been shown to be necessary for the accumulation of Nile Red-stained fat in C. elegans. We show that gut granules are sites of fat storage in C. elegans embryos and adults. Notably, levels of triacylglycerides are relatively normal in animals defective in the formation of gut granules. Our results provide an explanation for the loss of Nile Red-stained fat in pgp-2(-) animals as well as insight into the specialized function of this lysosome-related organelle.

Prevalence of acute myocardial infarction and other serious diagnoses in patients presenting to an urban emergency department with chest pain.

A retrospective cohort study and chart review were performed to estimate the absolute and relative prevalence of the serious diagnoses that might cause a patient to present to the Emergency Department (ED) with a chief complaint of chest pain. In this study, we queried a database of 347,229 complete visits to the San Francisco General Hospital Emergency Department between July 1, 1993 and June 30, 1998 for visits by patients > 35 years old with a chief complaint of chest pain and no history of trauma. Visits for chest pain that resulted in hospitalization were assigned to one of nine diagnostic groups according to final diagnoses as coded in the database. Manual chart review by trained abstractors using explicit criteria was done when group assignment based on coded diagnoses was unclear and in all diagnoses of pulmonary embolism and aortic dissection. Of 8,711 visits (2.5% of all visits) with a chief complaint of non-traumatic chest pain, 3,271 (37.6%) resulted in hospitalization. Of the 3,078 (94.1% of those hospitalized) assigned a final diagnosis, 329 (10.7% of hospitalizations, 3.8% of all visits) had acute myocardial infarction, 693 (22.5%) had either unstable angina or stable coronary artery disease, and 345 (11.2%) had pulmonary causes (mainly bacterial pneumonia) deemed serious enough to require hospitalization. Pulmonary embolism and aortic dissection were diagnosed in only 12 (0.4%) and 8 (0.3%) patients, respectively. In 905 (29.4%) hospitalizations for chest pain, myocardial infarction was "ruled out" and no cardiac ischemia or other serious etiology for the chest pain was diagnosed. Among patients presenting with chest pain, those in older age groups had dramatically increased risk of acute myocardial infarction. Women presenting with chest pain had a lower risk of acute myocardial infarction than men. In conclusion, the prevalence of acute myocardial infarction in the undifferentiated ED patient with a chief complaint of chest pain is only about 4%. An equal number of patients will have a serious pulmonary cause as the etiology of their pain. Pulmonary embolism and aortic dissection are important but extremely rare causes of a chest pain presentation to the ED.