Search

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-bpURA3sequences separated by 2.3-kb of phage lambda DNA 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 and Slx4. A natural 3% divergence between 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 MutSα (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 (MutLα) are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the identical 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, where the F variant carries six single-base mutations and one +1(T) insertion within a run of 10 Ts showed a gradient of correction favoring the sequence opposite each 3’ end of the annealed strand. Mismatches (MMs) 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, using either 3’ nonhomologous tail removal or 3’ to 5’ “proofreading” resection by DNA polymerase δ followed by synthesis to fill in the gap. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat, with a bias in favor of the “left” copy. A distinctive pattern of correction was found in the absence ofMSH2, in both Tailed and Tailless strains, while the deletion ofMSH6resulted in unrepaired MMs.

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.

Intravenous immunoglobulin (IVIG) is a well-established treatment for various autoimmune and inflammatory diseases. However, the standard dose prescribed for autoimmune diseases, including immune thrombocytopenic purpura (ITP), is 2 g/kg, which is markedly high and leads to a high treatment burden. In this study, we generated fragment crystallizable (Fc)-modified anti-haptoglobin (Hp) monoclonal antibodies with non-inferior efficacy compared to IVIG at considerably lower doses than IVIG, as shown by in vitro experiments. We evaluated binding activity of anti-Hp antibodies to Fc gamma receptors (FcγRs) with ELISA and inhibitory activity against the ADCC reaction. Furthermore, we successfully established a novel cynomolgus monkey ITP model and demonstrated that the anti-Hp antibody exerted its effect in this model with only a single dose. This Fc-modified anti-Hp monoclonal antibody could be a valuable therapeutic replacement for IVIG for the treatment of ITP.

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.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.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.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.

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.

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.

We report here the relationship between pedicel development and grape SISTER OF RAMOSA3 (VvSRA) expression in grapevine. The increase in VvSRA expression, but not grape RAMOSA3 expression, in young grape inflorescence decreased the number of pedicels on the lateral branch of grape inflorescence. This suggests that VvSRA negatively regulates pedicel development on the lateral branch of grape inflorescence, thereby decreasing berry number on a bunch in cultivars expressing VvSRA abundantly. Similarly, VvSRA overexpression decreased the number of flower buds on secondary branch in Arabidopsis plants. In addition, VvSRA overexpression had notable effects on Arabidopsis plant phenotypes, such as smaller flowers, shorter inflorescence, and expanded rosette leaves. As VvSRA encodes a trehalose 6-phosphate phosphatase, VvSRA overexpression might decrease trehalose 6-phosphate in the transgenic Arabidopsis plants, thereby causing the phenotypical alternations. These findings may provide new information on grapevine morphogenesis including pedicel development by SISTER OF RAMOSA3 and trehalose metabolism.

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.

We herein report the molecular characterization of grape expansins, VvEXPA14 and VvEXPA18. VvEXPA14 and VvEXPA18 transcription was detected in grape berries at 2 weeks post flowering and their expression was upregulated during growth phase I, the period of rapid berry expansion. Experiments using VvEXPA14:GFP or VvEXPA18:GFP fusion proteins demonstrated that both expansins were localized at cell wall, suggesting that both expansins may function on cell wall. Transgenic tobacco cells overexpressing VvEXPA14 or VvEXPA18 exhibited cell expansion. In addition, VvEXPA14- or VvEXPA18-overexpressing Arabidopsis plants showed primary root elongation associated with cell expansion. VvEXPA14 overexpression enlarged rosette leaves in Arabidopsis transgenic plants, although the height of the plants remained unchanged. Since plant expansin play role in cell wall loosing and softening, these results underscore the possibility that VvEXPA14 and VvEXPA18 are involved in cell expansion and plant growth.

Grapevine leaf has three unique sinuses. This study examined the relationship between the formation of the upper lateral sinus and the expression of VvNAC21/22-like , a member of grape NAC transcription factor family. VvNAC21/22-like expression was not upregulated in young leaves of all the cultivars tested and in mature leaves of cultivars having shallow sinuses but was abundant in mature leaves of cultivars having deep sinuses. Simple linear regression analysis demonstrated that in mature leaves, VvNAC21/22-like expression, but not VvNAC1 , VvCUC2 , and VvCUC3 expression, showed strong positive correlation with the depth of upper lateral sinus, suggesting that the increase in VvNAC21/22-like expression at the late stage of leaf maturation was related to the formation of deep upper lateral sinus in grapevine leaves. VvNAC21/22-like mRNAs were distributed in the upper and lower lateral sinuses of grapevine leaves. From these results, we hypothesize that VvNAC21/22-like plays a role in the formation of the upper lateral sinus in grapevine leaves. The findings indicate that sinuses in grapevine leaves may serve as a breeding target to enhance adaptation to environmental conditions.

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.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.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.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.

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.

山梨県では平成18年から山梨県合同輸血療法委員会I&A委員会による輸血医療の点検視察が開始された．施設の自発的受審ではなく県が視察病院を指定して，10施設の中小規模病院に対して点検視察が実施された．点検視察の結果「改善すべき」と指摘された事項は輸血検査や輸血用血液製剤の管理に関する項目が多く，「改善が望ましい」と指摘された事項は輸血管理体制や製剤の保管管理に関する項目が多かった．点検視察の指摘事項に関する改善状況のアンケート調査の結果では病院間で改善状況に差があることが示され，改善が進まない背景には輸血責任医師が兼任で実質的に不在であることや検査技師の人員不足等の問題があると考えられた．I&A活動の目的は輸血実施手順や輸血管理体制の標準化により病院の規模に関係なく安全で適正な輸血が実施されることである．山梨県I&A委員会の継続的な取り組みによって県内の中小規模病院の問題点の改善と輸血医療の向上を目指し，将来的には中小規模病院の学会I&A受審につなげたいと考える．

CK2 associates with kinetochore protein Mif2/CENP-C. Loss of CK2 results in defects in chromosome segregation, short mitotic spindle, and activation of the spindle assembly checkpoint. CK2 phosphorylates Mif2 and Ndc10. CK2 phosphorylation plays antagonistic and synergistic roles with Aurora B phosphorylation of Mif2 and Ndc10, respectively., Protein kinase CK2 is one of the most conserved kinases in eukaryotic cells and plays essential roles in diverse processes. While we know that CK2 plays a role(s) in cell division, our understanding of how CK2 regulates cell cycle progression is limited. In this study, we revealed a regulatory role for CK2 in kinetochore function. The kinetochore is a multi-protein complex that assembles on the centromere of a chromosome and functions to attach chromosomes to spindle microtubules. To faithfully segregate chromosomes and maintain genomic integrity, the kinetochore is tightly regulated by multiple mechanisms, including phosphorylation by Aurora B kinase. We found that a loss of CK2 kinase activity inhibits anaphase spindle elongation and results in chromosome missegregation. Moreover, a lack of CK2 activates the spindle assembly checkpoint. We demonstrate that CK2 associates with Mif2, the Saccharomyces cerevisiae homologue of human CENP-C, which serves as an important link between the inner and outer kinetochore. Furthermore, we show Mif2 and the inner kinetochore protein Ndc10 are phosphorylated by CK2, and this phosphorylation plays antagonistic and synergistic roles with Aurora B phosphorylation of these targets, respectively.

Recent data suggest a decreased prevalence of IFN-γ-producing T lymphocytes (Type 1 T cells) in tumor-bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti-tumor responses.

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.

It is well known that lymphocytes from patients with advanced-stage cancer have impaired immune responsiveness and that type1 T lymphocyte subsets in tumor bearing hosts are suppressed. Treg have been reported to comprise a subgroup which inhibits T cell mediated immune responses. In the present study, the percentage of Treg, Th1 and Tc1 in the peripheral blood of tumor bearing dogs with or without metastases was evaluated. The percentages of Th1 and Tc1 in dogs with metastatic tumor were significantly less, and that of Treg was significantly greater, than those of dogs without metastatic tumor. The percentage of Treg showed an inverse correlation with that of Th1 and Tc1 in tumor bearing dogs. It was concluded that an increase in Treg in the peripheral blood of dogs with metastatic tumor may induce suppression of tumor surveillance by the Type1 immune response and lead to metastasis of tumor[0][0].[0].

The Aurora kinase complex, also called the chromosomal passenger complex (CPC), is essential for faithful chromosome segregation and completion of cell division. In Fungi and Animalia, this complex consists of the kinase Aurora B/AIR-2/Ipl1p, INCENP/ICP-1/Sli15p, and Survivin/BIR-1/Bir1p. A fourth subunit, Borealin/Dasra/CSC-1, is required for CPC targeting to centromeres and central spindles and has only been found in Animalia. Here we identified a new core component of the CPC in budding yeast, Nbl1p. NBL1 is essential for viability and nbl1 mutations cause chromosome missegregation and lagging chromosomes. Nbl1p colocalizes and copurifies with the CPC, and it is essential for CPC localization, stability, integrity, and function. Nbl1p is related to the N-terminus of Borealin/Dasra/CSC-1 and is similarly involved in connecting the other CPC subunits. Distant homology searching identified nearly 200, mostly unannotated, Borealin/Dasra/CSC-1–related proteins from nearly 150 species within Fungi and Animalia. Analysis of the sequence of these proteins, combined with comparative protein structure modeling of Bir1p-Nbl1p-Sli15p using the crystal structure of the human Survivin–Borealin–INCENP complex, revealed a striking structural conservation across a broad range of species. Our biological and computational analyses therefore establish that the fundamental design of the CPC is conserved from Fungi to Animalia.

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.

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.

American Journal of Emergency Medicine xxx (2015) xxx–xxx Contents lists available at ScienceDirect American Journal of Emergency Medicine journal homepage: www.elsevier.com/locate/ajem Controversies Editorial: Ambulance Diversion: the Con Perspective Yuko Nakajima, MD ⁎ , Gary M. Vilke, MD Department of Emergency Medicine, UC San Diego Health System, 200 West Arbor Dr #8676 San Diego, 92103 As the ultimate safety net, emergency departments (EDs) are expected to care for any patient, at any time, under any circumstances. When EDs are overwhelmed in periods of surge, one solution is to redistribute pa- tients. A commonly used method for redistributing patients is ambulance diversion. Ambulance diversion is not a new phenomenon and, over time, has become commonly used by EDs to address the growing problem of ED overcrowding and saturation [1]. As ED visits have increased through the years, ambulance diversion has evolved into standard practice in many health systems. Along with this, ambulance diversion has always involved controversy over whether,overall, it is beneficial or detrimental to the pa- tient, emergency medical services (EMS) systems, and hospitals. In some circumstances, EDs and hospitals may occasionally be overwhelmed and may not be able to provide optimal patient care. Di- version may be viewed as a necessary mechanism to avoid the substan- dard situations in the ED represented by crowding, boarding, and hallway beds. It is used as a way to direct patients away from one’s hos- pital when waiting rooms are crowded and ED beds are full. However, in many circumstances, the patient is being directed to another facility that is just as busy and impacted but now further away, keeping pa- tients in the back of ambulances longer and thus keeping ambulances out of service for longer periods of time. Ambulances being out of ser- vice have been associated with delays in response to the next emergen- cy because there are fewer units in service and the available ambulances have to travel further to reach patients. These delays have also attribut- ed to adverse medical outcomes. Recent data support that ambulance diversion does not work to ease ED overcrowding and may result in the worsening of patient care. Stud- ies have shown that ambulance diversion can lead to delays for patients in obtaining definitive medical care given the increased field time and transport time and distances [2,3] leading to adverse outcomes includ- ing death. Diversion increases traffic accident risks and may malposition EMS resources. In addition, some patients may require specialty re- sources only available at the requested hospital and may not be able to receive them at an alternate destination. There is a possible association between ED diversion and increased mortality in certain populations of patients, such as trauma and acute myocardial infarction patients [4,5]. Studies like these led to American College of Emergency Physicians forming an EMS taskforce to review the topic of ambulance diversion. The taskforce penned a position paper that stated, “Ambulance diversion should occur only after the hospital has exhausted all internal mechanisms to avert a diversion, which includes calling in overtime staff” [6]. The National Association of EMS Physicians also states that “… ambulance diversion has not been shown to improve ED patient throughput” [7]. It has been demonstrated that if one hospital goes into ED diversion status, an oscillatory phenomenon may occur, where the one hospital going on bypass causes a neighboring hospital to receive a dispropor- tionate share of patients and is then forced to go onto bypass itself. This can create a domino effect impacting other hospitals nearby or, if there are no other hospitals to receive the patients, forcing the original hospital to accept more patients until it again gets impacted and goes back onto bypass. The cycle continues ad infinitum [8]. During this time of diversion, the hospital is receiving its own patients as well as pa- tients who originally had requested another hospital as a destination. These patients will arrive to a hospital that does not have immediate ac- cess to medical records, study results, private physicians, and some- times not even an inpatient bed being held for that patient for admission. Countless times, patients arrive to the “wrong” hospital be- cause of diversion and have to be transferred back to the original re- quested destination for admission. And this transfer occurs after possibly an unnecessary workup and evaluation, as well as taking up valuable ED bed time to complete the evaluation, transfer call process, and wait-time for a transport team to remove the patient from the bed and transfer said patient to the hospital that they should have gone in the first place. Hours of physician, nursing, and administrative time are wasted. This does not even quantify the inconvenience and frustration to the patient and families. In the end, the patients ultimately end up at the originally requested hospital, but not until after time and health care dollars are wasted. While this is happening at the receiving hospital, patients requesting said facility end up at the “wrong” hospital, thus creating the same inefficien- cies there. And so the oscillating cycle continues, even leading to defensive ambulance diversion, where the second hospital, though not at saturation status, would go on diversion when hearing that the first hospital just went on diversion. After observing the oscillatory phenomenon, two hos- pitals that collaborated and committed to staying off ambulance diversion for a week were able to demonstrate that this oscillating effect goes away, patients get to the “right” hospital the first time, and still all of the patients get seen without any detriment to patient care [8]. Follow-up work quantified at a more regional level involving multi- ple hospitals working together to collectively stay off ambulance diver- sion demonstrated that this can be done safely with no reported adverse effects to patient care. All of the patients still get seen in the collective group of EDs, but the patients get to the correct hospital the first time [9]. This eliminates the ineffectiveness as described above for patients not being delivered to their requested medical home. San Diego County took the success of these two regional short-trial successes to the next level in 2002. After ambulance diversion was http://dx.doi.org/10.1016/j.ajem.2015.03.005 0735-6757/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Nakajima Y, Vilke GM, Editorial: ambulance diversion: the con perspective, Am J Emerg Med (2015), http://dx.doi.org/ 10.1016/j.ajem.2015.03.005

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.

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.

Gastrectomy or vagotomy may result in reactive hypoglycemia, which, in some cases, can reduce the plasma glucose levels to 30-40 mg/dl due to rapid digestion and absorption of food, especially carbohydrates. It also occurs sometimes in patients on hemodialysis, where it is a potentially lethal complication. Because insulin has a longer half-life due to lack of renal degradation, hypoglycemia can be induced by insulin in patients with renal failure. We treated a patient with frequent episodes of severe hypoglycemia, that were sometimes accompanied by convulsions. He had undergone total gastrectomy 8 years before and had been maintained on hemodialysis for 3 years. Hyperinsulinemia caused by oxyhyperglycemia associated with post-gastrectomy led to severe hypoglycemia in this patient because of the lack of renal insulin degradation. Since nutritional treatment did not successfully manage his reactive hypoglycemia, an alpha-glucosidase inhibitor, acarbose, was administered to treat his oxyhyperglycemia. This therapy was very effective and he has not had any recurrence of reactive hypoglycemia since the initiation of the therapy.

An investigation of package inserts was performed for 794 oral prescription drugs, in order to assess the package inserts as an information resource regarding the timing of drug intake which is important for rational usage of medicine. As a result, the number of package inserts which described the proper timing of drug intake was only 157 (20%). The timings of drug intake could be classified into five categories, i.e., before a meal, immediately before a meal, immediately after a meal, after a meal and between meals (at a hunger state). Evidence for the timing of drug intake is written in only 9% of the package inserts. However, such evidence could be provided from interview forms and original articles for 24% and 44% of the drugs, respectively. Furthermore, most of the evidence for the timing of drug intake (94%) was shown to be meant for either the appropriate onset of therapeutic actions (58%) or the prevention of adverse reactions (36%). On the other hand, there was a clear tendency that more evidence is provided for drugs which were put on market more recently. From these results, the information only from package inserts was found to be insufficient for the rational usage of medicine and that the timing of drug intake should thus be explained to patients based on additional investigation from interview forms and original articles as described in this study in order to achieve an improved efficacy and safety of drug administration.

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.

This paper deals with vibration and acoustic emission (AE) measurements evaluating the separation of the balls and raceways with lubricating film in a linear-guideway–type recirculating ball bearing (linear bearing) under grease lubrication. In the experiments, three types of commercial grease, AS2, LG2, and PS2, were used. The vibratory acceleration, AE, temperature, and electric conductivity (contact voltage) in the test bearing were measured, while a carriage of the test bearing was driven at a certain linear velocity. Experimental results showed that the measured vibratory acceleration, AE, and contact voltage of the test bearing were affected by the linear velocity and the base oil viscosity of the grease. Next, the rail side film parameter ΛR and the carriage side film parameter ΛC were examined for the test bearing in operation, and it was shown that the ΛR value was lower than the ΛC value. In addition, a condition for the separation of all the balls and raceways with lubricating film was presented. Finally, it was shown that the measured root-mean-square (RMS) values of vibratory acceleration or AE can be used for evaluating the separation of all the balls and raceways with lubricating film in the test bearing.

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.

Tooth abnormalities occur in microphthalmic (mi/mi) mice. The elongated odontogenic epithelium is interrupted by unresorbed bone at the basal end of the mi/mi incisor, with the epithelium gathered into cell clusters. These clusters develop to odontoma-like masses. To identify the origin of the cell types of these odontoma-like masses, the localization of osteonectin (Osn), osteocalcin (Osc), osteopontin (Osp), matrix Gla protein (MGP) and amelogenin (Am) mRNA in the process of tooth development in mi/mi and +/+ mice was investigated by means of in situ hybridization. Decalcified mandibles of neonatal, 5-, 10-, 14-day-old mice were examined. Osn and Osc mRNA, which localized in osteoblasts and odontoblasts, were also detected in the cells of odontoma-like masses in mi/mi mice. The cells expressing these mRNA were short, columnar and odontoblast-like. Am mRNA was detected in ameloblasts. In mi/mi mice, Am mRNA was also detected in ameloblastic cell clusters, which were formed by the tall columnar cells in the odontoma-like masses. No apparent Osp mRNA expression was detected in the masses. These results indicated that even in odontogenic abnormal cells resulting from physical obstruction in mi/mi mice, the genes that are involved in normal tooth development were still expressed.

Ipl1/Aurora-mediated phosphorylation of the CPC component Sli15/INCENP promotes microtubule dynamics by restricting CPC–spindle association., Dynamic microtubules facilitate chromosome arrangement before anaphase, whereas during anaphase microtubule stability assists chromosome separation. Changes in microtubule dynamics at the metaphase–anaphase transition are regulated by Cdk1. Cdk1-mediated phosphorylation of Sli15/INCENP promotes preanaphase microtubule dynamics by preventing chromosomal passenger complex (CPC; Sli15/INCENP, Bir1/Survivin, Nbl1/Borealin, Ipl1/Aurora) association with spindles. However, whether Cdk1 has sole control over microtubule dynamics, and how CPC–microtubule association influences microtubule behavior, are unclear. Here, we show that Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP modulates microtubule dynamics by preventing CPC binding to the preanaphase spindle and to the central spindle until late anaphase, facilitating spatiotemporal control of microtubule dynamics required for proper metaphase centromere positioning and anaphase spindle elongation. Decreased Ipl1-dependent Sli15 phosphorylation drives direct CPC binding to microtubules, revealing how the CPC influences microtubule dynamics. We propose that Cdk1 and Ipl1/Aurora cooperatively modulate microtubule dynamics and that Ipl1/Aurora-dependent phosphorylation of Sli15 controls spindle function by excluding the CPC from spindle regions engaged in microtubule polymerization.

The sensitive and high resolution method of in situ hybridization technique has been developed using digoxigenin-11 UTP labeled cRNA as a probe. The system was applied to the decalcified mineralized tissues such as bone and dentin. Using the system, the localization of the mRNA of bone extracellular matrix proteins, osteopontin (Osp), osteonectin (Osn), Osteocalcin (Osc) and matrix Gla protein (MGP) was examined in decalcified bone. Decalcified femurs and mandibulae of embryo, neonatal, 2 to 40-week old mice were used for examination. Osn and Osc mRNAs were localized at osteoblast in bone and at odontoblast in dentin. Although the distribution pattern of Osn positive cells and Osc positive cells was partially overlapped, Osc mRNA was detected at matured osteoblast and odontoblast but Osn mRNA was detected not only at matured but differentiating osteoblast such as flat osteoblast in the periosteal layer. Osp mRNA was detected at osteoblast in bone, but no apparent expression of Osp mRNA was found at odontoblast in dentin. MGP mRNA was detected in hypertrophic chondrocytes. These results indicated the usefulness of this system for identifying the cell types in bone and dentin.

Several models of Dam1 complex function at the kinetochore have been proposed. Here we show that removal of the E-hooks of tubulin reduces Dam1 binding 40-fold. We also report the first structure of the Dam1 ring around microtubules and evidence that this ring freely diffuses on microtubules based on imaging individual rings., 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.

Recent data suggest a decreased prevalence of IFN-gamma-producing T lymphocytes (Type 1 T cells) in tumor-bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P0.01, Tc1: P0.05), the percentage of Treg was increased (P0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P0.01), and a significant correlation between that of Treg and the clinical tumor stage (P0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P0.05) or Tc1 (P0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti-tumor responses.

The antitumor effect and tumor levels of 5-fluoro-2'-deoxyuridylate (FdUMP) following oral administration of tetradecyl 2'-deoxy-5-fluoro-5'-uridylate (TT-62) were compared with those attained following intravenous (i.v.) or intraperitoneal (i.p.) administration of 5-fluorouracil (5-FU) or 5-fluoro-2'-deoxyuridine (FUdR) in BDF1 mice bearing murine mammary adenocarcinoma 755 and athymic mice bearing the transplantable human colon adenocarcinoma LS174T. Oral administration of TT-62 showed a stronger antitumor effect against adenocarcinoma 755 than FUdR. The maximum effect of TT-62 was similar to that of 5-FU. However, TT-62 and FUdR treatments were more effective than i.v. administration of 5-FU against LS174T. Thus, oral administration of TT-62 showed marked antitumor activity in both tumor systems. The maximum tolerated dose of FUdR resulted in a much higher level of free FdUMP in the LS174T tumor than that obtained with 5-FU. After oral administration of TT-62 the levels of FdUMP in the tumor were about 10 times those attained with 5-FU, but significantly lower than the levels obtained following i.v. administration of FUdR. With TT-62 the levels of FdUMP in the tumor reached their peak at 60 min following the administration and gradually decreased thereafter. However, FdUMP levels after administration of FUdR decreased rapidly. Three hours after the administration of TT-62 and for up to 24 h the FdUMP levels in the LS174T tumor were almost the same as after administration of FUdR, i.e. effective levels of FdUMP were maintained for a long time with TT-62.

A yellow and new dark red pigments were isolated from Lethariella sernanderi, L. cashmeriana, and L. sinensis as antioxidant components. The yellow pigment was identified as canarione (1), and the others were determined to be 1,2-quinone derivatives, rubrocashmeriquinone (2) and 7-chlororubrocashmeriquinone (3), and 7-chlorocanarione (4) by analysis of their spectroscopic data.

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.