Nonhomologous tails direct heteroduplex rejection and mismatch correction during single-strand annealing in Saccharomyces cerevisiae.

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.6 kb of spacer DNA (phage lambda) in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3'-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3, Saw1 and Slx4. A natural 3% divergence of unequally spaced heterologies between these repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutS (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutL), are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the divergent repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain showed a gradient of correction favoring the sequence opposite each 3' end of the annealed strand. Mismatches located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, possibly by 3' nonhomologous tail removal that includes part of the homologous sequence. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, different from the spectrum seen in a msh3Δ msh6Δ double mutant. Previous work has shown that SSA is Rad51-independent but dependent on the strand annealing activity of Rad52. However Rad52 becomes dispensable in a Tailless construct where the DSB is induced by Cas9 or in transformation of a plasmid where SSA occurs in the absence of nonhomologous tails.

Is There a Role for Intravenous Subdissociative-Dose Ketamine Administered as an Adjunct to Opioids or as a Single Agent for Acute Pain Management in the Emergency Department?

BACKGROUND: Whether acute or chronic, emergency physicians frequently encounter patients reporting pain. It is the responsibility of the emergency physician to assess and evaluate, and if appropriate, safely and effectively reduce pain. Recently, analgesics other than opioids are being considered in an effort to provide safe alternatives for pain management in the emergency department (ED). Opioids have significant adverse effects such as respiratory depression, hypotension, and sedation, to say nothing of their potential for abuse. Although ketamine has long been used in the ED for procedural sedation and rapid sequence intubation, it is used infrequently for analgesia. Recent evidence suggests that ketamine use in subdissociative doses proves to be effective for pain control and serves as a feasible alternative to traditional opioids. This paper evaluates ketamine's analgesic effectiveness and safety in the ED. METHODS: This is a literature review of randomized controlled trials, systematic reviews, meta-analyses, and observational studies evaluating ketamine for pain control in the ED setting. Based on these search parameters, eight studies were included in the final analysis and graded based on the American Academy of Emergency Medicine Clinical Practice Committee manuscript review process. RESULTS: A total of eight papers were reviewed in detail and graded. Recommendations were given based upon this review process. CONCLUSIONS: Subdissociative-dose ketamine (low-dose ketamine) is effective and safe to use alone or in combination with opioid analgesics for the treatment of acute pain in the ED. Its use is associated with higher rates of minor, but well-tolerated adverse side effects.

[Survey of Dental Treatment Situation and Needs for Prostheses in Veterinary Practice].

PURPOSE: In today's society, pets have become important members of families since they give mental peace and healing to families. Although veterinary dentistry is recognized to be essential for animal health, there are few reports on dental treatments of animals and the relationship between veterinarians and dental technicians. The purpose of the present study was to clarify the current situation of dental treatments of animals and to discuss the involvement of dental technicians in veterinary dental treatments and their collaboration with veterinarians. METHODS: Anonymous self-administered questionnaires were mailed to 16 university hospitals for animals, 17 animal clinics, and 87 zoological gardens, and handed out to 36 participants at the oral disease seminar organized by Nippon Animal Hospital Association. The questionnaires included questions on veterinary dental treatments, ways to learn veterinary dentistry, and details of prosthodontic treatments. RESULTS: Eighty-two valid responses (51.3%) were obtained. While 93.8% of veterinarians recognized the need for veterinary dental treatments, 67.9% were actually implementing dental treatments. Only 23.5% of veterinarians who conducted dental treatments experienced prosthodontic treatments, and the major prostheses used for treatments were fillings and crowns. Most veterinarians had fewer opportunities to acquire knowledge and skills about dental treatments. In addition, the recognition of dental technicians and their specialties was low among veterinarians. CONCLUSION: The results suggested that the dental technician, as a member of a multi-disciplinary team, can contribute to animal health by providing prosthetic appliances and should make efforts to enhance awareness of their specialty.

Environmental stresses induce misfolded protein aggregation in plant cells in a microtubule-dependent manner.

Misfolded protein aggregation in mammalian cells is one of the cellular responses to environmental stresses. However, the aggregation of misfolded proteins in plant cells exposed to environmental stresses is still poorly understood. Here, we report the misfolded protein aggregation in plant cells in response to environmental stresses, including ultraviolet (UV) radiation, heat stress and cold stress. Treatment of grape and tobacco cultured cells with MG-132, a proteasome inhibitor, induced misfolded protein aggregation. All of the environmental stresses examined induced the endoplasmic reticulum (ER) stress response in the cells. The cells under ER stress showed aggregation of misfolded proteins. The misfolded protein aggregation was completely inhibited by treatment of the cells with trichostatin A or colchicine, suggesting that the misfolded proteins might be aggregated in plant cells in a microtubule-dependent manner. Detected aggregates were initially observed immediately after exposure to the environmental stresses (1 min after UV radiation, 5 min after heat stress exposure, and 15 min after cold stress exposure). Based on these findings, we hypothesize that environmental stresses induce misfolded protein aggregation in plant cells in a microtubule-dependent manner.

Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms.

The Dam1 kinetochore complex is essential for chromosome segregation in budding yeast. This ten-protein complex self-assembles around microtubules, forming ring-like structures that move with depolymerizing microtubule ends, a mechanism with implications for cellular function. Here we used EM-based single-particle and helical analyses to define the architecture of the Dam1 complex at 30-A resolution and the self-assembly mechanism. Ring oligomerization seems to be facilitated by a conformational change upon binding to microtubules, suggesting that the Dam1 ring is not preformed, but self-assembles around kinetochore microtubules. The C terminus of the Dam1p protein, where most of the Aurora kinase Ipl1 phosphorylation sites reside, is in a strategic location to affect oligomerization and interactions with the microtubule. One of Ipl1's roles might be to fine-tune the coupling of the microtubule interaction with the conformational change required for oligomerization, with phosphorylation resulting in ring breakdown.

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi.

The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.

Trends in EMS-attended out-of-hospital cardiac arrest survival, United States 2015-2019.

AIM: Everyday, nearly 1000 U.S. adults experience out-of-hospital cardiac arrest (OHCA). Survival to hospital discharge varies across many factors, including sociodemographics, location of arrest, and whether bystander intervention was provided. The current study examines recent trends in OHCA survival by location of arrest using a cohort of emergency medical service (EMS) agencies that contributed data to the Cardiac Arrest Registry to Enhance Survival. METHODS: The 2015 CARES cohort (N = 122,613) includes EMS agencies contributing data across five consecutive years, 2015-2019. We assessed trends in EMS-attended OHCA survival for the 2015 CARES cohort by location of arrest - public, residential, nursing home. Unadjusted and adjusted percentages were estimated using 3-level hierarchical logistic regression models among cases aged 18-65 years. RESULTS: Overall, survival from EMS-attended OHCA significantly increased from 12.5% in 2015 to 13.8% in 2019 (p = 0.001). Survival from bystander witnessed arrests also increased significantly from 17.8% in 2015 to 19.7% in 2019 (p = 0.004). The trend for survival increased overall and for bystander witnessed OHCAs occurring in public places and nursing homes. CONCLUSION: Increasing trends for EMS-attended OHCA survival were observed in the overall and bystander witnessed groups. No change in the trend for survival was observed among OHCAs in the groups most likely to have a desirable outcome - bystander witnessed, with a shockable rhythm, and receiving bystander intervention. Reporting and monitoring of OHCA may be an important first step in improving outcomes. Additional community interventions focused on bystander CPR and AED use may be warranted.

Distinct time courses of secondary brain damage in the hippocampus following brain concussion and contusion in rats.

Secondary brain damage (SBD) is caused by apoptosis after traumatic brain injury that is classified into concussion and contusion. Brain concussion is temporary unconsciousness or confusion caused by a blow on the head without pathological changes, and contusion is a brain injury with hemorrhage and broad extravasations. In this study, we investigated the time-dependent changes of apoptosis in hippocampus after brain concussion and contusion using rat models. We generated the concussion by dropping a plumb on the dura from a height of 3.5 cm and the contusion by cauterizing the cerebral cortex. SBD was evaluated in the hippocampus by histopathological analyses and measuring caspase-3 activity that induces apoptotic neuronal cell death. The frequency of abnormal neuronal cells with vacuolation or nuclear condensation, or those with DNA fragmentation was remarkably increased at 1 hr after concussion (about 30% for each abnormality) from the pre-injury level (0%) and reached the highest level (about 50% for each) by 48 hrs, whereas the frequency of abnormal neuronal cells was increased at 1 hr after contusion (about 10%) and reached the highest level (about 40%) by 48 hrs. In parallel, caspase-3 activity was increased sevenfold in the hippocampus at 1 hr after concussion and returned to the pre-injury level by 48 hrs, whereas after contusion, caspase-3 activity was continuously increased to the highest level at 48 hrs (fivefold). Thus, anti-apoptotic-cell-death treatment to prevent SBD must be performed by 1 hr after concussion and at latest by 48 hrs after contusion.

A protein interaction map of the mitotic spindle.

The mitotic spindle consists of a complex network of proteins that segregates chromosomes in eukaryotes. To strengthen our understanding of the molecular composition, organization, and regulation of the mitotic spindle, we performed a system-wide two-hybrid screen on 94 proteins implicated in spindle function in Saccharomyces cerevisiae. We report 604 predominantly novel interactions that were detected in multiple screens, involving 303 distinct prey proteins. We uncovered a pattern of extensive interactions between spindle proteins reflecting the intricate organization of the spindle. Furthermore, we observed novel connections between kinetochore complexes and chromatin-modifying proteins and used phosphorylation site mutants of NDC80/TID3 to gain insights into possible phospho-regulation mechanisms. We also present analyses of She1p, a novel spindle protein that interacts with the Dam1 kinetochore/spindle complex. The wealth of protein interactions presented here highlights the extent to which mitotic spindle protein functions and regulation are integrated with each other and with other cellular activities.

Retrospective cohort study of the efficacy and safety of dabigatran: real-life dabigatran use including very low-dose 75 mg twice daily administration.

BACKGROUND: Dabigatran is a direct thrombin inhibitor and an anticoagulant that is prescribed to prevent ischemic stroke and systemic embolism in non-valvular atrial fibrillation. Dabigatran (150 mg twice daily) is non-inferior to warfarin for the prevention of stroke and systemic embolism. A dose reduction to 110 mg twice daily should be considered for patients with decreased renal function, elderly patients, and those with a history of gastrointestinal bleeding. A small number of patients are prescribed 75 mg twice daily; however, excessive dose reduction below that indicated on the package insert may decrease the effectiveness of dabigatran. In this study, we investigated the incidence of thromboembolic events and hemorrhagic complications in patients receiving different doses of dabigatran, including patients receiving the very low-dose of 75 mg twice daily. METHODS: Five hospitals in Meguro and Setagaya areas of Tokyo were included in this study. The subjects were patients receiving dabigatran in the hospitals from March 2011 to February 2014. Thromboembolic events (stroke, systemic embolism, and transient cerebral ischemic attack) and hemorrhagic complications occurring before December 2014 were retrospectively evaluated. RESULTS: A total of 701 subjects received dabigatran during the study period: 187 patients (26.7%) received 150 mg twice daily (normal dose), 488 patients (69.6%) received 110 mg twice daily (low-dose), and 26 patients (3.7%) received 75 mg twice daily (very low-dose). Thromboembolism occurred in 4 (2.1%), 11 (2.3%), and 3 patients (11.5%), in the normal dose, low-dose, and very low-dose groups, respectively. The odds ratio of the 75 mg dose to the 150 and 110 mg doses was 5.73 (95% CI, 1.55-21.2; p = 0.009), and the incidence with the 75 mg dose was higher than that with the other doses. Although the number of events was limited, it should be noted that 3 patients in the very low-dose group had thromboembolic events. CONCLUSIONS: The results suggest that sufficient anticoagulation efficacy may not be maintained when the dabigatran dose is excessively reduced to 75 mg twice daily.

Out-of-hospital cardiac arrest survival in international airports.

BACKGROUND: The highest achievable survival rate following out-of-hospital cardiac arrest is unknown. Data from airports serving international destinations (international airports) provide the opportunity to evaluate the success of pre-hospital resuscitation in a relatively controlled but real-life environment. METHODS: This retrospective cohort study included all cases of out-of-hospital cardiac arrest at international airports with resuscitation attempted between January 1st, 2013 and December 31st, 2015. Crude incidence, patient, event characteristics and survival to hospital discharge/survival to 30 days (survival) were calculated. Mixed effect logistic regression analyses were performed to identify predictors of survival. Variability in survival between airports/countries was quantified using the median odds ratio. RESULTS: There were 800 cases identified, with an average of 40 per airport. Incidence was 0.024/100,000 passengers per year. Percentage survival for all patients was 32%, and 58% for patients with an initial shockable heart rhythm. In adjusted analyses, initial shockable heart rhythm was the strongest predictor of survival (odds ratio, 36.7; 95% confidence interval [CI], 15.5-87.0). In the bystander-witnessed subgroup, delivery of a defibrillation shock by a bystander was a strong predictor of survival (odds ratio 4.8; 95% CI, 3.0-7.8). Grouping of cases was significant at country level and survival varied between countries. CONCLUSIONS: In international airports, 32% of patients survived an out-of-hospital cardiac arrest, substantially more than in the general population. Our analysis suggested similarity between airports within countries, but differences between countries. Systematic data collection and reporting are essential to ensure international airports continually maximise activities to increase survival.

Th1/Th2 balance in canine peripheral blood lymphocytes--a flow cytometric study.

The canine immune system undergoes continuous remodeling with advancing age. We measured the Th1/Th2 balance in peripheral blood lymphocytes (PBLs) obtained from 23 Beagles ranging in age from 0.5 to 11.8 years by flow cytometric analysis using intracellular cytokine staining. The percentage of CD4 cells producing interferon-gamma (Th1) increased with age. The percentage of CD4 cells producing interleukin-4 (Th2) increased but much less so. In conclusion, we demonstrated that the Th1/Th2 balance in canine peripheral blood could be measured by this flow cytometry technique and the Th1/Th2 balance inclined to dominance of the Th1 subpopulation in PBLs as the dog matured.

Chromosomes at loose ends.

Broken ends of a budding yeast chromosome exhibit increased mobility, presumably to facilitate repair by recombination. A new study reports that increased mobility reflects the untethering of the broken chromosome, triggered by a DNA damage response that phosphorylates the Cep3 kinetochore protein and weakens the association between the centromere and the spindle pole body.

Investigation of mouth washing by patients after inhaling corticosteroids.

We report an effective method for mouth washing after inhalation of corticosteroids for the prevention of local adverse effects such as hoarseness and oropharyngeal candidiasis. This method involves gargling and rinsing immediately after inhalation, repeated at least twice. We performed a questionnaire survey on mouth washing after inhalation of corticosteroids of 19 inpatients who used inhaled corticosteroids at the University of Tokyo Hospital. The questions concerned: 1) awareness of local adverse effects of inhaled corticosteroids; 2) gargling and rinsing habits; 3) repeating mouth washing at least twice; and 4) mouth washing immediately after inhalation. The percentage of patients correctly performing the individual maneuvers were: 1) 63.2%; 2) 36.8%; 3) 36.8%; and 4) 63.2%. The percentage of patients performing our recommended method of mouth washing (all four elements) was 11%. These results suggest that patients receiving inhaled corticosteroids poorly comprehend mouth washing procedures after inhalation of corticosteroids. It is important that pharmacists advise patients on the correct method of mouth washing.

[Surveillance for various injectable antimicrobial susceptibility among clinical isolates of Pseudomonas aeruginosa in hyogo prefecture].

Antimicrobial susceptibility of 13 antimicrobial drugs for the injection and O-group antigen serotype were measured for the 766 Pseudomonas aeruginosa strains that had been isolated from various clinical materials in 29 facilities in the Hyogo prefecture from April to September in 2004. Metallo beta-lactamase detection was also performed. The antimicrobial activity was excellent in the order of GM, MEPM, AMK, CPFX and CAZ. Susceptible category of the breakpoint by National Committee for Clinical Laboratory Standards (CLSI/NCCLS) was excellent in the order of AMK, GM, PIPC, CZOP, and MEPM. As for the susceptibility of Carbapenem, it is confirmed that susceptible of MEPM was detected in 47 strains (36.4%) and metallo beta-lactamase producing P. aeruginosa was in 3 strains (0.4%) and multi-drug resistant P. aeruginosa were in 7 strains only (0.9%) among 129 strains of the IPM resistant (I or R). The results of the susceptibility test against P. aeruginosa were different in each facility, but there were several stocks having the identical O-antigen serotype and anti-biogram pattern in some facilities. The nosocomial infection measures including the antimicrobial propriety use are necessary.

Analysis of information submitted by clinical trial sponsors regarding the safety of investigational drugs.

During performance of clinical trials in medical institutions, information regarding the safety of investigational drugs is submitted by trial sponsors according to guidelines for good clinical practice. In the present study, reports of clinical trials conducted at the University of Tokyo Hospital were examined, focusing on the safety information provided to the Institutional Review Board (IRB). Two hundred two reports (52 protocols) of safety information were submitted to the IRB by clinical trial sponsors between April 2000 and March 2001, of which 185 contained a total of 3021 cases of adverse events. Of those, 194 reports were judged by clinical investigators/physicians not to be associated with any significant problems and the trials were continued. For 157 of those 194 reports, it was considered unnecessary to inform the test subjects of the report contents, including the adverse events. The decision of whether or not the test subjects should be informed of such contents tended to depend on the causal relationship between the adverse events and drug intake, as well as the predictability of the adverse events. For 8 of those 194 reports, the IRB recommended that the clinical investigators/ physicians provide information to the test subjects and/or submit detailed information on the status of these subjects to the IRB. From these results, we suggest that establishment of a system to unify and evaluate drug safety information is necessary to provide safe and efficient clinical trials.

Plant DNA-damage repair/toleration 100 protein repairs UV-B-induced DNA damage.

We report the characterization of VvDRT100-L, a grape DNA-damage repair/toleration 100 protein. VvDRT100-L has nine leucine-rich repeats and belongs to the plant DRT100 protein family. VvDRT100-L is expressed abundantly in green organs of grapevines, including tendrils, leaves, and green berry skins. The overexpression of VvDRT100-L in Arabidopsis plants decreased the number of abasic sites and the frequency of DNA single-strand breaks in the DNA damaged by UV-B irradiation, whereas UV-B irradiation markedly increased the number of abasic sites and the frequency of DNA single-strand breaks in T-DNA insertion mutant drt100 plants. VvDRT100-L-overexpressing plants remained viable and noticeably healthy under lethal UV doses, suggesting that VvDRT100-L may enhance UV tolerance in plant. Taken together, we concluded that VvDRT100-L might play an important role in the repair and toleration of UV-B-induced DNA damage. These findings would help us better understand how plants acquire UV stress acclimation, tolerance and DNA repair.

The Dam1 ring binds to the E-hook of tubulin and diffuses along the microtubule.

There has been much effort in recent years aimed at understanding the molecular mechanism by which the Dam1 kinetochore complex is able to couple microtubule depolymerization to poleward movement. Both a biased diffusion and a forced walk model have been proposed, and several key functional aspects of Dam1-microtubule binding are disputed. Here, we investigate the elements involved in tubulin-Dam1 complex interactions and directly visualize Dam1 rings on microtubules in order to infer their dynamic behavior on the microtubule lattice and its likely relevance at the kinetochore. We find that the Dam1 complex has a preference for native tubulin over tubulin that is lacking its acidic C-terminal tail. Statistical mechanical analysis of images of Dam1 rings on microtubules, applied to both the distance between rings and the tilt angle of the rings with respect to the microtubule axis, supports a diffusive ring model. We also present a cryo-EM reconstruction of the Dam1 ring, likely the relevant assembly form of the complex for energy coupling during microtubule depolymerization in budding yeast. The present studies constitute a significant step forward by linking structural and biochemical observations toward a comprehensive understanding of the Dam1 complex.