Multifunctional activity-based chemical probes for sirtuins.

The sirtuin family of NAD+-dependent protein deacylases has gained significant attention during the last two decades, owing to their unique enzymatic activities as well as their critical roles in a broad array of cellular events. Innovative chemical probes are heavily pursued for the functional annotation and pharmacological perturbation of this group of "eraser" enzymes. We have developed several series of activity-based chemical probes (ABPs) to interrogate the functional state of active sirtuins in complex biological samples. They feature a simple Ala-Ala-Lys tripeptide backbone with a thioacyl "warhead", a photoaffinity group (benzophenone or diazirine), and a bioorthogonal group (terminal alkyne or azido) for conjugation to reporters. When applied in a comparative fashion, these probes reveal the changes of active sirtuin contents under different physiological conditions. Additionally, they can also be utilized in a competitive manner for inhibitor discovery. The Nobel-winning "click" conjugation to a fluorophore allows the visualization of the active enzymes, while the covalent adduct to a biotin leads to the affinity capture of the protein of interest. Furthermore, the "clickable" tag enables the easy access to proteolysis targeting chimeras (PROTACs) that effectively degrade human SIRT2 in HEK293 cells, albeit at micromolar concentrations. These small molecule probes offer unprecedented opportunities to investigate the biological functions and physiological relevance of the sirtuin family.

Development of Second Generation Activity-Based Chemical Probes for Sirtuins.

NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacylases, namely, the sirtuins, are important cell adaptor proteins that alter cell physiology in response to low calorie conditions. They are thought to mediate the beneficial effects of calorie restriction to extend longevity and improve health profiles. Novel chemical probes are highly desired for a better understanding of sirtuin's roles in various biological processes. We developed a group of remarkably simple activity-based chemical probes for the investigation of active sirtuin content in complex native proteomes. These probes harbor a thioacyllysine warhead, a diazirine photoaffinity tag, as well as a terminal alkyne bioorthogonal functional group. Compared to their benzophenone-containing counterparts, these new probes demonstrated improved labeling efficiency and sensitivity, shortened irradiation time, and reduced background signal. They were applied to the labeling of individual recombinant proteins, protein mixtures, and whole cell lysate. These cell permeable small molecule probes also enabled the cellular imaging of sirtuin activity change. Taken together, our study provides new chemical biology tools and future drug discovery strategies for perturbing the activity of different sirtuin isoforms.

Tomato Yellow Leaf Curl Virus V2 Protein Plays a Critical Role in the Nuclear Export of V1 Protein and Viral Systemic Infection.

Geminiviruses are an important group of circular, single-stranded DNA viruses that cause devastating diseases in crops. Geminiviruses replicate their genomic DNA in the nucleus and the newly synthesized viral DNA is subsequently transported to the cytoplasm for further cell-to-cell and long-distance movement to establish systemic infection. Thus, nucleocytoplasmic transportation is crucial for successful infection by geminiviruses. For Tomato yellow leaf curl virus (TYLCV), the V1 protein is known to bind and shuttle viral genomic DNA, however, the role of the V2 protein in this process is still unclear. Here, we report that the V1 protein is primarily localized in the nucleus when expressed but the nucleus-localized V1 protein dramatically decreases when co-expressed with V2 protein. Moreover, the V2-facilitated nuclear export of V1 protein depends on host exportin-α and a specific V1-V2 interaction. Chemical inhibition of exportin-α or a substitution at cysteine 85 of the V2 protein, which abolishes the V1-V2 interaction, blocks redistribution of the V1 protein to the perinuclear region and the cytoplasm. When the V2C85S mutation is incorporated into a TYLCV infectious clone, the TYLCV-C85S causes delayed onset of very mild symptoms compared to wild-type TYLCV, suggesting that the V1-V2 interaction and, thus, the V2-mediated nuclear export of the V1 protein is crucial for viral spread and systemic infection. Our data point to a critical role of the V2 protein in promoting the nuclear export of the V1 protein and viral systemic infection, likely by promoting V1 protein-mediated nucleocytoplasmic transportation of TYLCV genomic DNA.

South Carolina 20 Years of Diabetes--A Public Health Concern.

OBJECTIVE: To assess and enumerate the trends in diabetes prevalence, morbidity and mortality rates in South Carolina (SC) within the past 2 decades. MATERIALS AND METHODS: We analyzed state-level data from vital records, Behavioral Risk Factor Surveillance System, Children's Health Assessment Survey and Administrative Claim Files. RESULTS: Over the past 20 years, there has been an average 2.5% annual increase in diabetes prevalence among adults in SC (P < 0.01). Although a typical reduction in mortality rate of 2.2% has been observed during the same period, the increased number of people living with diabetes (from 5.0% in 1995 to 12.0% in 2014) has brought more need for diabetes care, particularly for severe in-hospital cases and cases with crisis at the emergency department, totaling $404 million in annual costs. CONCLUSIONS: SC has experienced an epidemic of diabetes. Coupled with declining trends in mortality and increased hospitalization and emergency department visits, the state is experiencing historical morbidity and complications due to diabetes. The shift in complexity of the disease onset and management has resulted in more individuals living with cardiovascular disease and other comorbidities. The cost of care for all South Carolinians with diabetes is estimated to exceed 2.8 billion dollars in 2014 and projected to be more than 4 billion dollars by 2020. If the diabetes prevalence trend of increasing rates continues over the next 20 years, the number of individuals living with diabetes and its complications would rise to 1.3 million in SC.

C-terminal methylation of truncated neuropeptides: an enzyme-assisted extraction artifact involving methanol.

Neuropeptides are the largest class of signaling molecules used by nervous systems. Today, neuropeptide discovery commonly involves chemical extraction from a tissue source followed by mass spectrometric characterization. Ideally, the extraction procedure accurately preserves the sequence and any inherent modifications of the native peptides. Here, we present data showing that this is not always true. Specifically, we present evidence showing that, in the lobster Homarus americanus, the orcokinin family members, NFDEIDRSGFG-OMe and SSEDMDRLGFG-OMe, are non-native peptides generated from full-length orcokinin precursors as the result of a highly selective peptide modification (peptide truncation with C-terminal methylation) that occurs during extraction. These peptides were observed by MALDI-FTMS and LC-Q-TOFMS analyses when eyestalk ganglia were extracted in a methanolic solvent, but not when tissues were dissected, co-crystallized with matrix, and analyzed directly with methanol excluded from the sample preparation. The identity of NFDEIDRSGFG-OMe was established using MALDI-FTMS/SORI-CID, LC-Q-TOFMS/MS, and comparison with a peptide standard. Extraction substituting deuterated methanol for methanol confirmed that the latter is the source of the C-terminal methyl group, and MS/MS confirmed the C-terminal localization of the added CD3. Surprisingly, NFDEIDRSGFG-OMe is not produced via a chemical acid-catalyzed esterification. Instead, the methylated peptide appears to result from proteolytic truncation in the presence of methanol, as evidenced by a reduction in conversion with the addition of a protease-inhibitor cocktail; heat effectively eliminated the conversion. This unusual and highly specific extraction-derived peptide conversion exemplifies the need to consider both chemical and biochemical processes that may modify the structure of endogenous neuropeptides.

Superluminous supernovae at redshifts of 2.05 and 3.90.

A rare class of 'superluminous' supernovae that are about ten or more times more luminous at their peaks than other types of luminous supernova has recently been found at low to intermediate redshifts. A small subset of these events have luminosities that evolve slowly and result in radiated energies of up to about 10(51) ergs. Therefore, they are probably examples of 'pair-instability' or 'pulsational pair-instability' supernovae with estimated progenitor masses of 100 to 250 times that of the Sun. These events are exceedingly rare at low redshift, but are expected to be more common at high redshift because the mass distribution of the earliest stars was probably skewed to high values. Here we report the detection of two superluminous supernovae, at redshifts of 2.05 and 3.90, that have slowly evolving light curves. We estimate the rate of events at redshifts of 2 and 4 to be approximately ten times higher than the rate at low redshift. The extreme luminosities of superluminous supernovae extend the redshift limit for supernova detection using present technology, previously 2.36 (ref. 8), and provide a way of investigating the deaths of the first generation of stars to form after the Big Bang.

Type IIn supernovae at redshift z approximately 2 from archival data.

Supernovae have been confirmed to redshift z approximately 1.7 (refs 1, 2) for type Ia (thermonuclear detonation of a white dwarf) and to z approximately 0.7 (refs 1, 3-5) for type II (collapse of the core of the star). The subclass type IIn (ref. 6) supernovae are luminous core-collapse explosions of massive stars and, unlike other types, are very bright in the ultraviolet, which should enable them to be found optically at redshifts z approximately 2 and higher. In addition, the interaction of the ejecta with circumstellar material creates strong, long-lived emission lines that allow spectroscopic confirmation of many events of this type at z approximately 2 for 3-5 years after explosion (ref. 14). Here we report three spectroscopically confirmed type IIn supernovae, at redshifts z = 0.808, 2.013 and 2.357, detected in archival data using a method designed to exploit these properties at z approximately 2. Type IIn supernovae directly probe the formation of massive stars at high redshift. The number found to date is consistent with the expectations of a locally measured stellar initial mass function, but not with an evolving initial mass function proposed to explain independent observations at low and high redshift.

Molecular, mass spectral, and physiological analyses of orcokinins and orcokinin precursor-related peptides in the lobster Homarus americanus and the crayfish Procambarus clarkii.

Recently, cDNAs encoding prepro-orcokinins were cloned from the crayfish Procambarus clarkii; these cDNAs encode multiple copies of four orcokinin isoforms as well as several other peptides. Using the translated open reading frames of the P. clarkii transcripts as queries, five ESTs encoding American lobster Homarus americanus orthologs were identified via BLAST analysis. From these clones, three cDNAs, each encoding one of two distinct prepro-hormones, were characterized. Predicted processing of the deduced prepro-hormones would generate 13 peptides, 12 of which are conserved between the 2 precursors: the orcokinins NFDEIDRSGFGFN (3 copies), NFDEIDRSGFGFH (2 copies) and NFDEIDRSGFGFV (2 copies), FDAFTTGFGHN (an orcomyotropin-related peptide), SSEDMDRLGFGFN, GDY((SO3))DVYPE, VYGPRDIANLY and SAE. Additionally, one of two longer peptides (GPIKVRFLSAIFIPIAAPARSSPQQDAAAGYTDGAPV or APARSSPQQDAAAGYTDGAPV) is predicted from each prepro-hormone. MALDI-FTMS analyses confirmed the presence of all predicted orcokinins, the orcomyotropin-related peptide, and three precursor-related peptides, SSEDMDRLGFGFN, GDYDVYPE (unsulfated) and VYGPRDIANLY, in H. americanus neural tissues. SAE and the longer, unshared peptides were not detected. Similar complements of peptides are predicted from P. clarkii transcripts; the majority of these were detected in its neural tissues with mass spectrometry. Truncated orcokinins not predicted from any precursor were also detected in both species. Consistent with previous studies in the crayfish Orconectes limosus, NFDEIDRSGFGFN increased mid-/hindgut motility in P. clarkii. Surprisingly, the same peptide, although native to H. americanus, did not affect gut motility in this species. Together, our results provide the framework for future investigations of the regulation and physiological function of orcokinins/orcokinin precursor-related peptides in astacideans.

Specificity of the MMPI-2 Fake Bad Scale as a marker for personal injury malingering.

Psychologists who evaluate patients in medicolegal contexts should utilize objective assessment data with empirically established sensitivity and specificity for identifying negative response bias. The purpose of this study was to investigate the specificity of the Fake Bad Scale for identifying negative response bias in personal injury claimants. The cutoff scores proposed by Lees-Haley and colleagues were applied a federal prison, medical outpatients, and patients from to inmate volunteers from substance abuse unit. Half of the inmates were given instructions to malinger psychopathology to affect the adjudication process, and the remaining inmates and all of the hospital patients were given standard instructions. The original cutoff scores correctly identified the majority of inmates instructed to malinger psychopathology, but these scores resulted in unacceptably high rates of false positive classifications. The revised cutoff scores resulted in fewer false positives, i.e., 8%-24%.

Si vous êtes chargé de ... : guide de gestion à l' usage des responsables des soins de santé primaires / Rosemary McMahon, Elizabeth Barton et Maurice Piot ; en collaboration avec Naomi Gelina et Felton Ross / On being in charge : a guide to management in primary health care / Guía para la gestión de la atención primaria de salud

Ce livre est la deuxième édition révisée d'un guide de formation très populaire, traduit en 16 langues, conçu pour aider les agents de santé, et notamment les infirmières, les sages-femmes et les auxiliaires médicaux, à améliorer leurs compétences gestionnaires. Constatant le lien étroit qui existe entre une bonne gestion et des soins de santé de qualité, ce manuel explique comment utiliser un large éventail d'outils gestionnaires simples pour tirer le meilleur profit de maigres ressources, par une meilleure gestion du temps ou par un gaspillage moindre des médicaments. L'ouvrage propose de nombreux exercices et présente des exemples pratiques, des études de cas, des illustrations, des tableaux et des échantillons de formulaires qui aideront le lecteur à adopter une attitude orientée vers la solution des problèmes et lui donneront des conseils et des suggestions pour ses problèmes de tous les jours. Le guide est divisé en 14 chapitres divisés en quatre grande parties, chacune d'entre elles pouvant être étudiée séparément ou comme partie d'un tout, en fonction des besoins d'apprentissage individuels. L'exercice préliminaire, qui a rencontré beaucoup de succès dans la première édition, aide le lecteur à diagnostiquer ses carences gestionnaires et le guide vers les chapitres qui l'aideront à combler ses lacunes. Chaque chapitre commence avec une description des objectifs d'apprentissage et chaque partie se termine avec des exercices destinés à faciliter l'apprentissage individuel et la solution de problèmes en équipes. Cette deuxième édition offre 70 nouvelles pages de solutions proposées aux exercices

On being in charge : a guide to management in primary health care / by Rosemary McMahon, Elizabeth Barton & Maurice Piot ; in collaboration with Naomi Gelina & Felton Ross / Si vous êtes chargé de ... : guide de gestion à l' usage des responsables des soins de santé primaires / Guía para la gestión de la atención primaria de salud

A practical, instructive training guide that has proven its capacity to improve the managerial skills of middle-level health workers responsible for the management or supervision of health services. Presented as an activity-centered working guide, the book uses numerous questionnaires, exercises, practical examples, illustrations, charts, and sample forms to help readers relate advice and suggestions to their own daily problems. Emphasis is placed on ways to improve the management of health services provided by full-time, qualified staff. The guide features 18 chapters presented in four main parts, any one of which can be studied separately or as part of the whole, according to individual learning needs. The first provides a straightforward explanation of the general principles and functions of management. Part two is concerned with personal relations, offering advice on how to encourage and coordinate a health team, communicate with groups of people, and supervise in a supportive way. The third and most extensive part presents problem-solving methods for the management of common problems involving equipment, drug supply, money, time, space in the office or clinic as well as in the community, and paperwork. The guide concludes with chapters addressing the management of primary health care services, including advice on how to plan, implement, and evaluate health activities. Each chapter begins with a statement of learning objectives and each part concludes with a number of exercises to facilitate self-learning. Widely used and highly praised, published in 1980 and reprinted many times since, the book has demonstrated its enduring value as both a problem-solving reference work and a training guide for the testing and development of managerial skills

On being in charge : A guide to management in primary health care / On being in charge : A guide to management in primary health care

Primary health crae has long been accepted as the best route to health for all, but its success in achieving that goal depends largely on effective management. Identifying health problems, assigning priorities to their solution, planning and implementing programmes, supervising staff, and evaluating results are among the major demands made of health service managers, particularly at district level. Administrative weakness in these areas, however, combined with an almost inevitable scarcity of human and material resources, is frequently a casue of failutre to meet community needs$This instructive, practical guide aims to improve the managerial skills of middle-level health workers, on whom these responsibilities generally devolve. It is designed for use both in formal training courses and by the individual engaged in private study. The book is devided into four essentially self-contained parts, each with practical examples, questionnaires and illustrations that help relate the information to health workers' own experiences. Clear learning objectives are presented at the beginning of each chapter, and the book includes a large number of exercises designed to evaluate progress towards these objetives