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This study aimed to assess root canal disinfection through various irrigation protocols, including a novel photothermal system called 'LEAP'. Mandibular premolars were infected with Enterococcus faecalis and divided into five groups for different treatments: Group 1: standard needle irrigation; Group 2: passive ultrasonic irrigation; Group 3: GentleWave; Group 4: LEAP; and Group 5: Group 1 + Group 4. Microbial counts were measured before (S1) and after disinfection (S2) using colony-forming units (CFU) and confocal laser scanning microscopy (CLSM). Results revealed a significant reduction in bacterial counts for all groups (p < 0.05). While the percentage of dead bacteria near the canal wall (0-50 μm) did not differ significantly, at 50-150 μm, LEAP and SNI + LEAP exhibited significantly higher bacterial reduction than other groups (p < 0.05). The findings indicate that LEAP is comparable to existing irrigation devices in the main root canal and notably superior in tubular disinfection., (© 2024 Australian Society of Endodontology Inc.)

Objective: To examine the proportion of healthcare visits are delivered by nurse practitioners and physician assistants versus physicians and how this has changed over time and by clinical setting, diagnosis, and patient demographics., Design: Cross-sectional time series study., Setting: National data from the traditional Medicare insurance program in the USA., Participants: Of people using Medicare (ie, those older than 65 years, permanently disabled, and people with end stage renal disease), a 20% random sample was taken., Main Outcome Measures: The proportion of physician, nurse practitioner, and physician assistant visits in the outpatient and skilled nursing facility settings delivered by physicians, nurse practitioners, and physician assistants, and how this proportion varies by type of visit and diagnosis., Results: From 1 January 2013 to 31 December 2019, 276 million visits were included in the sample. The proportion of all visits delivered by nurse practitioners and physician assistants in a year increased from 14.0% (95% confidence interval 14.0% to 14.0%) to 25.6% (25.6% to 25.6%). In 2019, the proportion of visits delivered by a nurse practitioner or physician assistant varied across conditions, ranging from 13.2% for eye disorders and 20.4% for hypertension to 36.7% for anxiety disorders and 41.5% for respiratory infections. Among all patients with at least one visit in 2019, 41.9% had one or more nurse practitioner or physician assistant visits. Compared with patients who had no visits from a nurse practitioner or physician assistant, the likelihood of receiving any care was greatest among patients who were lower income (2.9% greater), rural residents (19.7%), and disabled (5.6%)., Conclusion: The proportion of visits delivered by nurse practitioners and physician assistants in the USA is increasing rapidly and now accounts for a quarter of all healthcare visits., Competing Interests: Competing interests: All authors have completed the ICMJE uniform disclosure form at www.icmje.org/disclosure-of-interest/ and declare: no support from any organisation for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous three; no other relationships or activities that could appear to have influenced the submitted work., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Policymakers are increasingly interested in using nurse practitioners to provide health care to rural populations, yet little is known about their characteristics and preparation for independent practice., Methods: We obtained data from the 2018 National Sample Survey of Registered Nurses and compared characteristics of family nurse practitioners (FNPs) employed in rural areas versus those employed in non-rural areas. Regression analysis was used to determine the relationship between the outcome variable of interest, preparation for practice and other covariates., Findings: FNPs practicing in a rural setting felt less prepared for independent practice than their counterparts in non-rural settings except for those prepared with a doctoral degree., Discussion: The majority of FNPs working in rural areas believed they were not as well prepared for independent practice. Because rural FNPs often practice autonomously and without medical back up, nursing educators need to educate FNPs with the skills and knowledge necessary to practice effectively in rural settings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Conversion of CO 2 into CO with plasma processing is a potential method to transform intermittent sustainable electricity into storable chemical energy. The main challenges for developing this technology are how to get efficient CO 2 conversion with high energy efficiency and how to prove its feasibility on an industrial scale. In this paper we review the mechanisms and performance of different plasma methodologies used in CO 2 conversion. Mindful of the goals of obtaining efficient conversion and high energy efficiency, as well as industrial feasibility in mind, we emphasize a promising new approach of CO 2 conversion by using a thermal plasma in combination with a carbon co-reactant.

Cystobactamids are myxobacteria-derived topoisomerase inhibitors with potent anti-Gram-negative activity. They are formed by a non-ribosomal peptide synthetase (NRPS) and consist of tailored para-aminobenzoic acids, connected by a unique α-methoxy-L-isoasparagine or a β-methoxy-L-asparagine linker moiety. We describe the heterologous expression of the cystobactamid biosynthetic gene cluster (BGC) in Myxococcus xanthus. Targeted gene deletions produce several unnatural cystobactamids. Using in vitro experiments, we reconstitute the key biosynthetic steps of linker formation and shuttling via CysB to the NRPS. The biosynthetic logic involves a previously uncharacterized bifunctional domain found in the stand-alone NRPS module CysH, albicidin biosynthesis and numerous BGCs of unknown natural products. This domain performs either an aminomutase (AM) or an amide dehydratase (DH) type of reaction, depending on the activity of CysJ which hydroxylates CysH-bound L-asparagine. Furthermore, CysQ O-methylates hydroxyl-L-(iso)asparagine only in the presence of the AMDH domain. Taken together, these findings provide direct evidence for unique steps in cystobactamid biosynthesis.

Novel targets are needed for treatment of devastating diseases such as cancer. For decades, natural products have guided innovative therapies by addressing diverse pathways. Inspired by the potent cytotoxic bioactivity of myxobacterial vioprolides A-D, we performed in-depth studies on their mode of action. Based on its prominent potency against human acute lymphoblastic leukemia (ALL) cells, we conducted thermal proteome profiling (TPP) and deciphered the target proteins of the most active derivative vioprolide A (VioA) in Jurkat cells. Nucleolar protein 14 (NOP14), which is essential in ribosome biogenesis, was confirmed as a specific target of VioA by a suite of proteomic and biological follow-up experiments. Given its activity against ALL cells compared to healthy lymphocytes, VioA exhibits unique therapeutic potential for anticancer therapy through a novel mode of action., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Social workers (SW) and community health workers (CHW) have emerged as key workforce personnel in efforts to care for elders in the U.S. However, little is known about the presence and roles of SW and CHW in primary care practices. This paper presents findings from a nationally representative survey of geriatrics and primary care practices. Physician and nurse practitioner clinicians were randomly selected within practices, stratifying by practice staffing and presence/absence of geriatric clinicians; our final sample for this analysis included 341 practices. Key findings include: reported challenges in meeting the social service needs of elders, underutilization of SW, and fuller utilization of social work competencies in practices in which both SW and CHW were present. These findings offer a unique perspective of SW on interprofessional teams and have implications for the future of the profession.

Objective: Policies that aim to steer patients from higher to lower cost providers of comparable quality have potential to impact health care cost growth - but their effectiveness depends, in part, on consumer perceptions of value and willingness to make tradeoffs. We sought to understand what was required to shift substantial numbers of consumers to higher-value care settings for several "shoppable" conditions., Methods: A discrete choice experiment (DCE) was conducted to elicit patient preferences for hospital type. We used an Internet panel of 1005 Massachusetts residents to conduct this experiment in 2016. The DCE data were analyzed using alternative-specific conditional logit regression., Results: Consumers reported large influences of out of pocket costs, physician referrals and quality ratings on their choice of hospital. For example, up to a third of consumers would shift from Academic Medical Centers to community hospitals if the latter had higher quality ratings, lower copays or a physician referral. Choice of site for maternity care was most influenced by physician referral; cancer treatment and orthopedic procedures by quality ratings; and MRI by cost, suggesting that patients prioritize quality over cost as perceived risk increases., Conclusions and Implications: Our findings provide guidance for identifying promising policy levers that most influence consumer choice of provider. However, the extent to which potential levers can influence choice is likely to be dependent upon the kind of care being sought., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: To describe the study protocol and baseline characteristics of the African Descent and Glaucoma Evaluation Study (ADAGES) III., Design: Cross-sectional, case-control study., Participants: Three thousand two hundred sixty-six glaucoma patients and control participants without glaucoma of African or European descent were recruited from 5 study centers in different regions of the United States., Methods: Individuals of African descent (AD) and European descent (ED) with primary open-angle glaucoma (POAG) and control participants completed a detailed demographic and medical history interview. Standardized height, weight, and blood pressure measurements were obtained. Saliva and blood samples to provide serum, plasma, DNA, and RNA were collected for standardized processing. Visual fields, stereoscopic disc photographs, and details of the ophthalmic examination were obtained and transferred to the University of California, San Diego, Data Coordinating Center for standardized processing and quality review., Main Outcome Measures: Participant gender, age, race, body mass index, blood pressure, history of smoking and alcohol use in POAG patients and control participants were described. Ophthalmic measures included intraocular pressure, visual field mean deviation, central corneal thickness, glaucoma medication use, or past glaucoma surgery. Ocular conditions, including diabetic retinopathy, age-related macular degeneration, and past cataract surgery, were recorded., Results: The 3266 ADAGES III study participants in this report include 2146 AD POAG patients, 695 ED POAG patients, 198 AD control participants, and 227 ED control participants. The AD POAG patients and control participants were significantly younger (both, 67.4 years) than ED POAG patients and control participants (73.4 and 70.2 years, respectively). After adjusting for age, AD POAG patients had different phenotypic characteristics compared with ED POAG patients, including higher intraocular pressure, worse visual acuity and visual field mean deviation, and thinner corneas (all P < 0.001). Family history of glaucoma did not differ between AD and ED POAG patients., Conclusions: With its large sample size, extensive specimen collection, and deep phenotyping of AD and ED glaucoma patients and control participants from different regions in the United States, the ADAGES III genomics study will address gaps in our knowledge of the genetics of POAG in this high-risk population., (Copyright © 2017 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Bacteria produce a large number of secondary metabolites with extraordinary chemical structures and bioactivities. Vioprolides are promising anticancer and antifungal lead compounds produced by the myxobacterium Cystobacter violaceus Cb vi35, which are initially synthesized as acylated precursors (previoprolides) by nonribosomal peptide synthetases (NRPS). Here, we describe and characterize an unprecedented glycerate esterification process in the biosynthesis of vioprolides. In vitro biochemical investigations revealed that the fatty acyl chain of previoprolides is adenylated by the starting fatty acyl-AMP ligase (FAAL) domain, while the glycerate moiety is incorporated by the FkbH domain. An unusual ester-bond forming condensation domain is shown responsible for the acylation of glycerate. LC-MS analysis and bioactivity assays suggest that the acylation serves for directed membrane transport rather than representing a prodrug mechanism.

Vioprolides are a promising class of anticancer and antifungal lead compounds produced by the myxobacterium Cystobacter violaceus Cb vi35. Previously nothing had been reported about their biosynthesis, including the origin of the unusual 4-methylazetidinecarboxylic acid (MAZ) moiety. We describe the vioprolide biosynthetic gene cluster and solve the production obstacle by expression in three heterologous hosts. Starting from unstable production in the wild type at the single-digit mg L -1 scale, we developed a stable host that eventually allowed for yields of up to half a gram per liter in fermenters. Gene inactivations coupled with isotope feeding studies identified an S-adenosylmethionine (SAM)-dependent enzyme and a methyltransferase as being responsible for the generation of the MAZ building block by a proposed mechanism unprecedented in bacteria. Furthermore, nonnatural vioprolide derivatives were generated via rational genetic engineering., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The fascinating ability of myxobacteria to form multicellular spore filled fruiting bodies under starvation conditions was widely studied as a model for cooperative microbial behavior. The potential of a life cycle induced change of secondary metabolism, as a means to discover novel natural products, remains largely underexplored. We therefore studied the model organism Myxococcus xanthus DK1622 under submersed and solid cultivation conditions to find putatively life-cycle related compounds by applying statistical analysis on analytical data. Utilizing the advantageous characteristics of LC-MS, LC-MS/MS, and MALDI-MSI allowed the identification of compounds unambiguously associated with myxobacterial fruiting bodies. Our screening effort resulted in the purification and structure elucidation of a novel compound, the homospermidine lipid, from cultures that had undergone the fruiting process. A combination of molecular networking and targeted LC-MS/MS in conjunction with our in-house metabolomics database subsequently revealed alternative producers of the respective compound as well as a number of compounds belonging to the same structural class. Three further members of this compound class were isolated from an alternative producer and structurally elucidated by NMR. Insights into the biosynthesis of this novel compound class was gained by feeding of isotopically labeled substrates and in silico analysis.

We describe a new instrument that uses ion imaging to study molecular beam-surface scattering and surface desorption kinetics, allowing independent determination of both residence times on the surface and scattering velocities of desorbing molecules. This instrument thus provides the capability to derive true kinetic traces, i.e., product flux versus residence time, and allows dramatically accelerated data acquisition compared to previous molecular beam kinetics methods. The experiment exploits non-resonant multiphoton ionization in the near-IR using a powerful 150-fs laser pulse, making detection more general than previous experiments using resonance enhanced multiphoton ionization. We demonstrate the capabilities of the new instrument by examining the desorption kinetics of CO on Pd(111) and Pt(111) and obtain both pre-exponential factors and activation energies of desorption. We also show that the new approach is compatible with velocity map imaging.

Objectives: Debate continues on whether nurse practitioners (NPs) and physician assistants (PAs) are more likely to order ancillary services, or order more costly services among alternatives, than primary care physicians (PCPs). We compared prescription medication and diagnostic service orders associated with NP/PA versus PCP visits for management of neck or back (N/B) pain or acute respiratory infection (ARI)., Study Design: Retrospective, observational study of visits from January 2006 through March 2008 in the adult primary care practice of Kaiser Permanente in Atlanta, Georgia., Methods: Data were obtained from electronic health records. NP/PA and PCP visits for N/B pain or ARI were propensity score matched on patient age, gender, and comorbidities., Results: On propensity score-matched N/B pain visits (n = 6724), NP/PAs were less likely than PCPs to order a computed tomography (CT)/magnetic resonance image (MRI) scan (2.1% vs 3.3%, respectively) or narcotic analgesic (26.9% vs 28.5%) and more likely to order a nonnarcotic analgesic (13.5% vs 8.5%) or muscle relaxant (45.8% vs 42.5%) (all P ≤.05). On propensity score-matched ARI visits (n = 24,190), NP/PAs were more likely than PCPs to order any antibiotic medication (73.7% vs 65.8%), but less likely to order an x-ray (6.3% vs 8.6%), broad-spectrum antibiotic (41.5% vs 42.5%), or rapid strep test (6.3% vs 9.7%) (all P ≤.05)., Conclusions: In the multidisciplinary primary care practice of this health maintenance organization, NP/PAs attending visits for N/B pain or ARI were less likely than PCPs to order advanced diagnostic radiology imaging services, to prescribe narcotic analgesics, and/or to prescribe broad-spectrum antibiotics.

G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Ripostatin is a promising antibiotic that inhibits RNA polymerase by binding to a novel binding site. In this study, the characterization of the biosynthetic gene cluster of ripostatin, which is a peculiar polyketide synthase (PKS) hybrid cluster encoding cis- and trans-acyltransferase PKS genes, is reported. Moreover, an unprecedented mechanism for phenyl acetic acid formation and loading as a starter unit was discovered. This phenyl-C2 unit is derived from phenylpyruvate (phenyl-C3) and the mechanism described herein explains the mysterious loss of one carbon atom in ripostatin biosynthesis from the phenyl-C3 precursor. Through in vitro reconstitution of the whole loading process, a pyruvate dehydrogenase like protein complex was revealed that performs thiamine pyrophosphate dependent decarboxylation of phenylpyruvate to form a phenylacetyl-S-acyl carrier protein species, which is supplied to the subsequent biosynthetic assembly line for chain extension to finally yield ripostatin., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: There has been concern that greater use of nurse practitioners (NP) and physician assistants (PA) in face-to-face primary care may increase utilization and spending., Objective: To evaluate a natural experiment within Kaiser Permanente in Georgia in the use of NP/PA in primary care., Study Design: From 2006 through early 2008 (the preperiod), each NP or PA was paired with a physician to manage a patient panel. In early 2008, NPs and PAs were removed from all face-to-face primary care. Using the 2006-2010 data, we applied a difference-in-differences analytic approach at the clinic level due to patient triage between a NP/PA and a physician. Clinics were classified into 3 different groups based on the percentage of visits by NP/PA during the preperiod: high (over 20% in-person primary care visits attended by NP/PAs), medium (5%-20%), and low (<5%) NP/PA model clinics., Measures: Referrals to specialist physicians; emergency department visits and inpatient admissions; and advanced diagnostic imaging services., Results: Compared with the low NP/PA model, the high NP/PA model and the medium NP/PA model were associated with 4.9% and 5.1% fewer specialist referrals, respectively (P<0.05 for both estimates); the high NP/PA model and the medium NP/PA model also showed fewer hospitalizations and emergency department visits and fewer advanced diagnostic imaging services, but none of these was statistically significant., Conclusions: We find no evidence to support concerns that under a physician's supervision, NPs and PAs increase utilization and spending.

Myotonic dystrophy type 1 (DM1) is a genetic disorder in which dominant-active DM protein kinase (DMPK) transcripts accumulate in nuclear foci, leading to abnormal regulation of RNA processing. A leading approach to treat DM1 uses DMPK-targeting antisense oligonucleotides (ASOs) to reduce levels of toxic RNA. However, basal levels of DMPK protein are reduced by half in DM1 patients. This raises concern that intolerance for further DMPK loss may limit ASO therapy, especially since mice with Dmpk gene deletion reportedly show cardiac defects and skeletal myopathy. We re-examined cardiac and muscle function in mice with Dmpk gene deletion, and studied post-maturity knockdown using Dmpk-targeting ASOs in mice with heterozygous deletion. Contrary to previous reports, we found no effect of Dmpk gene deletion on cardiac or muscle function, when studied on two genetic backgrounds. In heterozygous knockouts, the administration of ASOs reduced Dmpk expression in cardiac and skeletal muscle by > 90%, yet survival, electrocardiogram intervals, cardiac ejection fraction and muscle strength remained normal. The imposition of cardiac stress by pressure overload, or muscle stress by myotonia, did not unmask a requirement for DMPK. Our results support the feasibility and safety of using ASOs for post-transcriptional silencing of DMPK in muscle and heart., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objectives: The Institute of Medicine has recently argued against a value index as a mechanism to address geographic variation in spending and instead promoted payment reform targeted at individual providers. It is unknown whether such provider-focused payment reform reduces geographic variation in spending., Study Design: We estimated the potential impact of 3 Medicare provider-focused payment policies-pay-for-performance, bundled payment, and accountable care organizations-on geographic variation in Medicare spending across Hospital Referral Regions (HRRs). We compared geographic variation in spending, measured using the coefficient of variation (CV) across HRRs, between the baseline case and a simulation of each of the 3 policies., Methods: Policy simulation based on 2008 national Medicare data combined with other publicly available data., Results: Compared with the baseline (CV, 0.171), neither pay-for-performance nor accountable care organizations would change geographic variation in spending (CV, 0.171), while bundled payment would modestly reduce geographic variation (CV, 0.165)., Conclusions: In our models, the bundled payment for inpatient and post acute care services in Medicare would modestly reduce geographic variation in spending, but neither accountable care organizations nor pay-for-performance appear to have an impact.

Key Points: Na(+) current (INa) results from the integrated function of a molecular aggregate (the voltage-gated Na(+) channel complex) that includes the β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including Brugada syndrome and sudden unexpected death in patients with epilepsy. We used Scn1b null mice to understand better the relation between Scn1b expression, and cardiac electrical function. Loss of Scn1b caused, among other effects, increased amplitude of tetrodotoxin-sensitive INa, delayed after-depolarizations, triggered beats, delayed Ca(2+) transients, frequent spontaneous calcium release events and increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nM tetrodotoxin. We propose that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel α subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis., Abstract: Na(+) current (INa) is determined not only by the properties of the pore-forming voltage-gated Na(+) channel (VGSC) α subunit, but also by the integrated function of a molecular aggregate (the VGSC complex) that includes the VGSC β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including cases of Brugada syndrome and sudden unexpected death in patients with epilepsy. Here, we have used Scn1b null mouse models to understand better the relation between Scn1b expression, and cardiac electrical function. Using a combination of macropatch and scanning ion conductance microscopy we show that loss of Scn1b in juvenile null animals resulted in increased tetrodotoxin-sensitive INa but only in the cell midsection, even before full T-tubule formation; the latter occurred concurrent with increased message abundance for the neuronal Scn3a mRNA, suggesting increased abundance of tetrodotoxin-sensitive NaV 1.3 protein and yet its exclusion from the region of the intercalated disc. Ventricular myocytes from cardiac-specific adult Scn1b null animals showed increased Scn3a message, prolonged action potential repolarization, presence of delayed after-depolarizations and triggered beats, delayed Ca(2+) transients and frequent spontaneous Ca(2+) release events and at the whole heart level, increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nM tetrodotoxin. Our results suggest that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel α subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis in ventricular myocytes., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Background: Inpatient quality deficits have important implications for the health and well-being of patients. They also have important financial implications for payers and hospitals by leading to longer lengths of stay and higher intensity of treatment. Many of these costly quality deficits are particularly sensitive to nursing care., Objective: To assess the effect of nurse staffing on quality of care and inpatient care costs., Design: Longitudinal analysis using hospital nurse staffing data and the Healthcare Cost and Utilization Project State Inpatient Databases from 2008 through 2011., Subjects: Hospital discharges from California, Nevada, and Maryland (n=18,474,860)., Methods: A longitudinal, hospital-fixed effect model was estimated to assess the effect of nurse staffing levels and skill mix on patient care costs, length of stay, and adverse events, adjusting for patient clinical and demographic characteristics., Results: Increases in nurse staffing levels were associated with reductions in nursing-sensitive adverse events and length of stay, but did not lead to increases in patient care costs. Changing skill mix by increasing the number of registered nurses, as a proportion of licensed nursing staff, led to reductions in costs., Conclusions: The study findings provide support for the value of inpatient nurse staffing as it contributes to improvements in inpatient care; increases in staff number and skill mix can lead to improved quality and reduced length of stay at no additional cost.

Differential ion mobility spectrometry (DMS) coupled to mass spectrometry is increasingly used in both quantitative analyses of biological samples and as a means of removing background interferences for enhanced selectivity and improved quality of mass spectra. However, DMS separation efficiency using dry inert gases often lacks the required selectivity to achieve baseline separation. Polar gas-phase modifiers such as alcohols are therefore frequently employed to improve selectivity via clustering/declustering processes. The choice of an optimal modifier currently relies on trial and error experiments, making method development a tedious activity. It was the goal of this study to establish a means of CV prediction for compounds using a homologous series of alcohols as gas-phase modifiers. This prediction was based on linear regression of compensation voltages of two calibration runs for the alcohols with the lowest and the highest molecular weights and readily available descriptors such as proton affinity and gas phase acidity of the modifier molecules. All experiments were performed on a commercial quadrupole linear ion trap mass spectrometer equipped with a DMS device between electrospray ionization source and entrance quadrupole lens. We evaluated our approach using a homologous series of 4-alkylbenzoic acids and a selection of 23 small molecules of high chemical diversity. Predicted CV values typically deviated from the experimentally determined values by less than 0.5 V. Several test compounds changed their ion mobility behavior for the investigated gas phase modifiers (e.g., from type B to type A) and thus could thus not be evaluated.

Background: Law enforcement restraint-related death is frequently associated with excited delirium syndrome (ExDS). Because such deaths are rare, the pathophysiology underlying ExDS deaths remains unknown, making identification of high-risk situations challenging. This study describes the medical conditions and situations surrounding restraint of individuals identified by law enforcement to be experiencing ExDS., Methods: Individuals with ExDS as determined by law enforcement officers during use of force encounters over a 3-year period were identified. For subjects who were brought to the emergency department after restraint, medical records and police narratives were reviewed to identify circumstances surrounding restraint, abnormalities found during evaluation, and final diagnoses., Results: Sixty-six cases were identified, of which 43 had emergency department evaluation. On presentation, 36 (84%) were tachycardic and 3 (7%) were hyperthermic; 35 (77%) had toxicology studies positive for stimulants; 2 (5%) had a pH level less than 7.2, and 5 (12%) had an elevated lactate; and 3 (7%) had a creatinine kinase level higher than 1500 U/L. Two (5%) patients were admitted to the hospital for medical reasons: one had had a field pulseless electrical activity arrest prior to restraint and the other was admitted for rhabdomyolysis., Conclusion: Officer-identified cases of ExDS infrequently involved individuals requiring extensive restraint or with medical conditions that objectively placed them at high risk for sudden death. The low specificity of this syndrome in predicting risk of sudden death may present a challenge to law enforcement and emergency physicians., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Under new bundled payment models, hospitals are financially responsible for post-acute care delivered by providers such as skilled nursing facilities (SNFs) and home health agencies (HHAs). The hope is that hospitals will use post-acute care more prudently and better coordinate care with post-acute providers. However, little is known about existing patterns in hospitals׳ referrals to post-acute providers., Methods: Post-acute provider referrals were identified using SNF and HHA claims within 14 days following hospital discharge. Hospital post-acute care network size and concentration were estimated across hospital types and regions. The 2008 Medicare Provider Analysis and Review claims for acute hospitals and SNFs, and the 100% HHA Standard Analytic Files were used., Results: The mean post-acute care network size for U.S. hospitals included 57.9 providers with 37.5 SNFs and 23.4 HHAs. The majority of these providers (65.7% of SNFs, 60.9% of HHAs) accounted for 1 percent or less of a hospital׳s referrals and classified as "low-volume". Other post-acute providers we classified as routine. The mean network size for routine providers was greater for larger hospitals, teaching hospitals and in regions with higher per capita post-acute care spending., Conclusions: The average hospital works with over 50 different post-acute providers. Moreover, the size of post-acute care networks varies considerably geographically and by hospital characteristics. These results provide context on the complex task hospitals will face in coordinating care with post-acute providers and cutting costs under new bundled payment models., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We present a new photo-fragment imaging spectrometer, which employs a movable repeller in a single field imaging geometry. This innovation offers two principal advantages. First, the optimal fields for velocity mapping can easily be achieved even using a large molecular beam diameter (5 mm); the velocity resolution (better than 1%) is sufficient to easily resolve photo-electron recoil in (2 + 1) resonant enhanced multiphoton ionization of N2 photoproducts from N2O or from molecular beam cooled N2. Second, rapid changes between spatial imaging, velocity mapping, and slice imaging are straightforward. We demonstrate this technique's utility in a re-investigation of the photodissociation of N2O. Using a hot nozzle, we observe slice images that strongly depend on nozzle temperature. Our data indicate that in our hot nozzle expansion, only pure bending vibrations--(0, v2, 0)--are populated, as vibrational excitation in pure stretching or bend-stretch combination modes are quenched via collisional near-resonant V-V energy transfer to the nearly degenerate bending states. We derive vibrationally state resolved absolute absorption cross-sections for (0, v2 ≤ 7, 0). These results agree well with previous work at lower values of v2, both experimental and theoretical. The dissociation energy of N2O with respect to the O((1)D) + N2¹Σ(g)⁺ asymptote was determined to be 3.65 ± 0.02 eV.

The widely used green fluorescent protein (GFP) decarboxylates upon irradiation; this involves removal of the acidic function of the glutamic acid at position 222, thereby resulting in the irreversible photoconversion of GFP. To suppress this phenomenon, the photostable, non-photoconvertible histidine was introduced at position 222 in GFP. The variant E222H shows negligible photodynamic processes and high expression yield. In addition, the stable and bright fluorescence over a wide pH range makes the E222H protein an alternative for GFP in fluorescence imaging and spectroscopy. Other fluorescent proteins are predicted to benefit from replacement of the catalytic glutamic acid by histidine., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To evaluate changes in peripapillary and macular choroidal thickness and volume after the water-drinking test (WDT) using swept-source optical coherence tomography (SS OCT)., Design: Prospective, cross-sectional, observational study., Participants: Fifty-six eyes of 28 healthy volunteers., Methods: Participants underwent a 3-dimensional optic disc and macula scanning protocol with a prototype SS OCT (Topcon, Inc., Tokyo, Japan) at baseline and 15, 30, 45, and 120 minutes after the start of the WDT. The WDT consisted of drinking 1000 ml of water within 5 minutes. Objective measurements of the choroid were obtained with automated segmentation of the choroidal boundaries., Main Outcome Measures: Choroidal thickness and volume., Results: Mean age ± standard deviation of participants was 35.6 ± 9.1 years. Intraocular pressure (IOP) increased from 14.9 ± 2.7 mmHg at baseline to a peak of 16.8 ± 3.0 mmHg 15 minutes after the WDT (P < 0.001). Mean baseline choroidal thickness and volume were 181.3 ± 50.8 μm and 6.19 ± 1.80 mm(3), respectively, at the optic disc and 217.4 ± 43.6 μm and 7.83 ± 1.55 mm(3), respectively, at the macula. After the WDT, peripapillary and macular choroidal thickness increased by a maximum of 5.7% (P<0.001) and 4.3% (P<0.001), respectively. Choroidal volumes increased by 6.4% (P<0.001) and 3.9% (P<0.001), respectively. There was no association between change in IOP and peripapillary (P = 0.27) or macular (P = 0.09) choroidal thickness., Conclusions: Using automated segmentation of SS OCT measurements, significant increases in choroidal thickness and volume are observed after the WDT in healthy subjects., (Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.

Glaucoma and age-related macular degeneration are ocular diseases targeted clinically by anecortave acetate (AA). AA and its deacetylated metabolite, anecortave desacetate (AdesA), are intraocular pressure (IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid activity but with an unknown mechanism of action. We used a methotrexate-anchored yeast three-hybrid (Y3H) technology to search for binding targets for AA in human trabecular meshwork (TM) cells, the target cell type that controls IOP, a major risk factor in glaucoma. Y3H hits were filtered by competitive Y3H screens and coimmunoprecipitation experiments and verified by surface plasmon resonance analysis to yield a single target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding protein with additional function outside the PDE6 phototransduction system. Overexpression of PDE6D in mouse eyes caused elevated IOP, and this elevation was reversed by topical ocular application of either AA or AdesA. The identification of PDE6D as the molecular binding partner of AA provides insight into the role of this drug candidate in treating glaucoma.

Introduction: Because of its many participants and thorough records, competitive Masters swimming offers a rich data source for determining the rate of physical decline associated with aging in physically fit individuals. The decline in performance among national champion swimmers, both men and women and in short and long swims, is linear, at about 0.6% per year up to age 70-75, after which it accelerates in quadratic fashion. These conclusions are based primarily on cross-sectional studies, and little is known about individual performance declines with aging. Herein we present performance profiles of 19 male and 26 female national and international champion Masters swimmers, ages 25 to 96 years, participating in competitions for an average of 23 years., Methods and Results: Swimmers' longitudinal data were compared with the fastest times of world record holders across ages 35-100 years by two regression methods. Neither method proved to accurately model this data set: compared with the rates of decline estimated from the world record data, which represent the best recorded times at given ages, there was bias toward shallower rates of performance decline in the longitudinal data, likely owing to a practice effect in some swimmers as they began their Masters programs. In swimmers' later years, once maximum performance had been achieved, individual profiles followed the decline represented in the world records, and a few swimmers became the world record holders. In some instances, the individual profiles indicated performance better than the world record data; these swimmers achieved their times after the world record data were collected in 2005-2006., Conclusion: Declining physiological functional capacity occurs with advancing age, and this is reflected in the performance decrements of aging Masters swimmers. Individual swimmers show different performance trajectories with aging, declines being mitigated by practice, which improves both physiological capacity and swimming technique, particularly in the early years of participation. The longitudinal data of this study indicate that individuals can participate in high-intensity swimming over several decades, competitively improving over those decades until, in some instances, they become world record holders for their age groups.

Cationic polyrotaxanes, obtained by temperature activated threading of cationic cyclodextrin derivatives onto water-soluble cationic polymers (ionenes), form metastable nanometric polyplexes with pDNA and combinations of siRNA with pDNA. Because of their low toxicity, the polyrotaxane polyplexes constitute a very interesting system for the transfection of polynucleotides into mammalian cells. The complexation of Cy3-labeled siRNA within the polyplexes was demonstrated by fluorescence correlation spectroscopy. The uptake of the polyplexes (red) was imaged by confocal fluorescence microscopy using the A549 cell line as a model (blue: nuclei, green: membranes). The results prove the potential of polyrotaxanes for further investigations involving knocking down genes of therapeutic interest., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Gold nanoclusters prepared with a controlled amount of Ag exhibit intense fluorescence with a quantum yield of ~16% and a "quasi-monoexponential" long lifetime of >200 ns. Characterization of the luminescent probes indicates high photostability and easy detection in cells. Additionally, fluorescence enhancement in the presence of proteins was found.

Weibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

The majority of small molecule drugs act on protein targets to exert a therapeutic function. It has become apparent in recent years that many small molecule drugs act on more than one particular target and consequently, approaches which profile drugs to uncover their target binding spectrum have become increasingly important. Classical yeast two-hybrid systems have mainly been used to discover and characterize protein-protein interactions, but recent modifications and improvements have opened up new routes towards screening for small molecule-protein interactions. Such yeast "n"-hybrid systems hold great promise for the development of drugs which interfere with protein-protein interactions and for the discovery of drug-target interactions. In this review, we discuss several yeast two-hybrid based approaches with applications in drug discovery and describe a protocol for yeast three-hybrid screening of small molecules to identify their direct targets., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cystic fibrosis lung disease is caused by reduced Cl(-) secretion along with enhanced Na(+) absorption, leading to reduced airway surface liquid and compromised mucociliary clearance. Therapeutic strategies have been developed to activate cystic fibrosis transmembrane conductance regulator (CFTR) or to overcome enhanced Na(+) absorption by the epithelial Na(+) channel (ENaC). In a split-ubiquitin-based two-hybrid screening, we identified stress-associated ER protein 1 (SERP1)/ribosome-associated membrane protein 4 as a novel interacting partner for the ENaC β-subunit. SERP1 is induced during cell stress and interacts with the molecular chaperone calnexin, thus controlling early biogenesis of membrane proteins. ENaC activity was measured in the human airway epithelial cell lines H441 and A549 and in voltage clamp experiments with ENaC-overexpressing Xenopus oocytes. We found that expression of SERP1 strongly inhibits amiloride-sensitive Na(+) transport. SERP1 coimmunoprecipitated and colocalized with βENaC in the endoplasmic reticulum, together with the chaperone calnexin. In contrast to the inhibitory effects on ENaC, SERP1 appears to promote expression of CFTR. Taken together, SERP1 is a novel cochaperone and regulator of ENaC expression.

Yeast two-hybrid based systems are powerful tools for the detection and characterization of protein-protein interactions (PPIs). However, some important protein classes, e.g., integral membrane proteins and transcription factors, are difficult to study using these technologies. To overcome these limitations, we have employed a novel protein complementation screening platform. Protein interactions are detected by reconstitution of the split-protein sensor TRP1, enabling trp1 cells to grow on medium lacking tryptophan. Since the interaction readout is direct and independent of transcriptional reporter activation the rate of false positives is lowered. Furthermore, the technology allows for detection of protein interactions in their natural setting, e.g., the cytosol, the nucleus, and at cellular or organellar membranes. The protocols used for screening are explained in detail and as an example we describe the isolation of novel binding partners found with APP screened against a human cDNA library.

Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing amino-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 together target an internal degradation signal (degron) of the Mgt1 DNA repair protein. Ufd4 is an E3 enzyme of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we show that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1-Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1-Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately for two decades.

A new technique for manipulation and control of gradient-driven instabilities through nonlinear interaction with Alfvén waves in a laboratory plasma is presented. A narrow, field-aligned density depletion is created in the Large Plasma Device, resulting in coherent, unstable fluctuations on the periphery of the depletion. Two independent shear Alfvén waves are launched along the depletion at separate frequencies, creating a nonlinear beat-wave response at or near the frequency of the original instability. When the beat wave has sufficient amplitude, the original unstable mode is suppressed, leaving only the beat-wave response, generally at lower amplitude.

We have studied the interaction of H(2) on Cu(111) using quasi-classical and quantum dynamics, and a chemically accurate six-dimensional potential energy surface (PES). The PES was computed using the specific reaction parameter (SRP) approach to density functional theory (DFT), in an implementation adapted to molecules interacting with metal surfaces. To perform this study we have applied the Born-Oppenheimer static surface (BOSS) approximation, i.e., we used both the Born-Oppenheimer (BO) and the static surface (SS) approximations. We show that our theoretical approach accurately describes experiments on dissociative adsorption, the effect of molecular vibrational and rotational motion on dissociative (associative) adsorption (desorption), and rotational excitation upon scattering. More specifically, dynamics calculations on reactive scattering of H(2) reproduce reaction probabilities measured in molecular beam experiments, effective barrier heights describing the dependence of reaction on the initial rovibrational state, and data on rotationally inelastic scattering with chemical accuracy (i.e., within 1 kcal mol(-1) approximately 4.2 kJ mol(-1)). These processes are not affected much by surface motion, either because they were measured using a low surface temperature, T(s), or because the computed observable is independent of T(s). However, we show that to account for the dependence of molecular orientation on a reaction the inclusion of surface motion is required. We have also found that vibrational excitation is poorly described within the BOSS approximation, suggesting a breakdown of this approximation.

Methods for accurately computing the interaction of molecules with metal surfaces are critical to understanding and thereby improving heterogeneous catalysis. We introduce an implementation of the specific reaction parameter (SRP) approach to density functional theory (DFT) that carries the method forward from a semiquantitative to a quantitative description of the molecule-surface interaction. Dynamics calculations on reactive scattering of hydrogen from the copper (111) surface using an SRP-DFT potential energy surface reproduce data on the dissociative adsorption probability as a function of incidence energy and reactant state and data on rotationally inelastic scattering with chemical accuracy (within approximately 4.2 kilojoules per mole).

A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided.

Translation initiation in eukaryotes is accomplished by a large set of translation initiation factors, some of which are regulated by signals monitoring intracellular and environmental conditions. Here, we show that Uri1p is required for efficient translation initiation in budding yeast. Indeed, uri1Delta cells are slow growing, sensitive to translation inhibitors and they exhibit an increased 80S peak in polysome profiles. Moreover, GCN4 translation is derepressed in uri1Delta cells, strongly supporting an initiation defect. Genetic and biochemical experiments indicate that Uri1p interacts with the translation initiation factor eIF1A and promotes ternary complex (TC) recruitment to the 40S subunit. Interestingly, we found that Uri1p is also part of a chaperone-network, including the prefoldin Pfd6p and several other proteins involved in cotranslational quality control such as the ribosome-associated Hsp70 chaperone Ssb1p, the Hsp40 Sis1p and the translation elongation factor eEF1A. Together with genetic data, these interactions indicate that Uri1p may coordinate translation initiation and cotranslational quality control.

Recent research has begun to elucidate the global network of cytosolic and membrane protein interactions. The resulting interactome map facilitates numerous biological studies, including those for cell signalling, protein trafficking and protein regulation. Due to the hydrophobic nature of membrane proteins such as tyrosine kinases, G-protein coupled receptors, membrane bound phosphatases and transporters it is notoriously difficult to study their relationship to signaling molecules, the cytoskeleton, or any other interacting partners. Although conventional yeast-two hybrid is a simple and robust technique that is effective in the identification of specific protein-protein interactions, it is limited in its use for membrane proteins. However, the split-ubiquitin membrane based yeast two-hybrid assay (MYTH) has been described as a tool that allows for the identification of membrane protein interactions. In the MYTH system, ubiquitin has been split into two halves, each of which is fused to a protein, at least one of which is membrane bound. Upon interaction of these two proteins, the two halves of ubiquitin are reconstituted and a transcription factor that is fused to the membrane protein is released. The transcription factor then enters the nucleus and activates transcription of reporter genes. Currently, large-scale MYTH screens using cDNA or gDNA libraries are performed to identify and map the binding partners of various membrane proteins. Thus, the MYTH system is proving to be a powerful tool for the elucidation of specific protein-protein interactions, contributing greatly to the mapping of the membrane protein interactome.

All previous experimental and theoretical studies of molecular interactions at metal surfaces show that electronically nonadiabatic influences increase with molecular velocity. We report the observation of a nonadiabatic electronic effect that follows the opposite trend: The probability of electron emission from a low-work function surface--Au(111) capped by half a monolayer of Cs--increases as the velocity of the incident NO molecule decreases during collisions with highly vibrationally excited NO(X(2)pi((1/2)), V = 18; V is the vibrational quantum number of NO), reaching 0.1 at the lowest velocity studied. We show that these results are consistent with a vibrational autodetachment mechanism, whereby electron emission is possible only beyond a certain critical distance from the surface. This outcome implies that important energy-dissipation pathways involving nonadiabatic electronic excitations and, furthermore, not captured by present theoretical methods may influence reaction rates at surfaces.

Using visible and x-ray photoelectron spectroscopy, we measured the work function of a Au(111) surface at a well-defined submonolayer coverage of Cs. For a Cs coverage producing a photoemission maximum with a He-Ne laser, the work function is 1.61+/-0.08 eV, consistent with previous assumptions used to analyze vibrationally promoted electron emission. A discussion of possible Cs layer structures is also presented.