CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination

Shigella flexneri Regulation of ARF6 Activation during Bacterial Entry via an IpgD-Mediated Positive Feedback Loop

ABSTRACT Entry into cells is critical for virulence of the human bacterial pathogens Shigella spp. Shigella spp. induce membrane ruffle formation and macropinocytic uptake, but the events instigating this process are incompletely understood. The host small GTPase ADP-ribosylation factor 6 (ARF6) functions in membrane trafficking at the plasma membrane and activates membrane ruffle formation. We demonstrate that ARF6 is required for efficient Shigella flexneri entry, is activated by S. flexneri dependent on the phosphatase activity of the type III secreted effector IpgD, and depends on cytohesin guanine nucleotide exchange factors (GEFs) for recruitment to entry sites. The cytohesin GEF ARF nucleotide binding site opener (ARNO) is recruited to these sites, also dependent on IpgD phosphatase activity. ARNO recruitment is independent of ARF6, indicating that, in addition to the described recruitment of ARNO by ARF6, ARNO is recruited upstream of ARF6. Our data provide evidence that ARF6, IpgD, phosphoinositide species, and ARNO constitute a previously undescribed positive feedback loop that amplifies ARF6 activation at bacterial entry sites, thereby promoting efficient S. flexneri uptake

Shedding of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus from adult and pediatric bathers in marine waters

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>including methicillin resistant <it>S. aureus</it>, MRSA, are human colonizing bacteria that commonly cause opportunistic infections primarily involving the skin in otherwise healthy individuals. These infections have been linked to close contact and sharing of common facilities such as locker rooms, schools and prisons Waterborne exposure and transmission routes have not been traditionally associated with <it>S. aureus </it>infections. Coastal marine waters and beaches used for recreation are potential locations for the combination of high numbers of people with close contact and therefore could contribute to the exposure to and infection by these organisms. The primary aim of this study was to evaluate the amount and characteristics of the shedding of methicillin sensitive <it>S. aureus</it>, MSSA and MRSA by human bathers in marine waters.</p> <p>Results</p> <p>Nasal cultures were collected from bathers, and water samples were collected from two sets of pools designed to isolate and quantify MSSA and MRSA shed by adults and toddlers during exposure to marine water. A combination of selective growth media and biochemical and polymerase chain reaction analysis was used to identify and perform limited characterization of the <it>S. aureus </it>isolated from the water and the participants. Twelve of 15 MRSA isolates collected from the water had identical genetic characteristics as the organisms isolated from the participants exposed to that water while the remaining 3 MRSA were without matching nasal isolates from participants. The amount of <it>S. aureus </it>shed per person corresponded to 10⁵to 10⁶CFU per person per 15-minute bathing period, with 15 to 20% of this quantity testing positive for MRSA.</p> <p>Conclusions</p> <p>This is the first report of a comparison of human colonizing organisms with bacteria from human exposed marine water attempting to confirm that participants shed their own colonizing MSSA and MRSA into their bathing milieu. These findings clearly demonstrate that adults and toddlers shed their colonizing organisms into marine waters and therefore can be sources of potentially pathogenic <it>S. aureus </it>and MRSA in recreational marine waters. Additional research is needed to evaluate recreational beaches and marine waters as potential exposure and transmission pathways for MRSA.</p

Urinary Nitric Oxide Levels Are Associated with Blood Pressure, Fruit and Vegetable Intake and Total Polyphenol Excretion in Adolescents from the SI! Program.

Nitric oxide (NO) is important to cardiovascular health (CVH), and its bioavailability could be regulated by the antioxidant effect of polyphenols, improving endothelial function and consequently blood pressure (BP). However, scant research has been carried out on NO and CVH correlates in adolescent populations. Therefore, our aim was to investigate the association between NO and the CVH status and other health factors in adolescents. NO, total polyphenol excretion (TPE), anthropometric measurements, BP, blood lipid profile, blood glucose, diet, physical activity, and smoking status were recorded, while CVH score was classified as ideal, intermediate, and poor. Negative associations were observed between NO and body mass index, body fat percentage, BP, and triglycerides; and positive associations between NO and skeletal muscle percentage, HDL-cholesterol, fruit and vegetable intake, and TPE was observed. To capture more complex interactions among different factors, multiple linear regression was performed, obtaining a significant association between NO and fruit and vegetable intake (β = 0.175), TPE (β = 0.225), and systolic BP (β = -0.235). We conclude that urinary NO levels are positively associated with the consumption of fruits and vegetables rich in antioxidants such as polyphenols and negatively associated with systolic BP.The SI! Program for Secondary Schools trial was supported by the SHE Foundation, the la Caixa Foundation (LCF/PR/CE16/10700001), the Fundació la Marató de TV3 (grant number 369/C/2016). Support was also provided by the Ministerio de Ciencia, Innovación y Universidades (PID2020-114022RB-I00), CIBEROBN from the Instituto de Salud Carlos III, ISCIII from the Ministerio de Ciencia, Innovación y Universidades (AEI/FEDER, UE), and Generalitat de Catalunya. RF-J is a recipient of grant PI19/01704 funded by the Fondo de Investigación Sanitaria- Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund/European Social Fund a way to make Europe/Investing in your future. The CNIC is supported by the ISCIII, the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). GS-B was the recipient of grant LCF/PR/MS19/12220001 funded by la Caixa Foundation (ID 100010434). AT-R is a Serra Húnter fellow. EL-S was a FI-SDUR (EMC/3345/2020) fellowship from the Generalitat de Catalunya. JM-G was a postgraduate fellow of the Ministerio de Ciencia e Innovación of Spain at the Residencia de Estudiantes (2020–ongoing).S

Naturally Occurring Osmolyte, Trehalose Induces Functional Conformation in an Intrinsically Disordered Activation Domain of Glucocorticoid Receptor

Intrinsically disordered (ID) regions are frequently found in the activation domains of many transcription factors including nuclear hormone receptors. It is believed that these ID regions promote molecular recognition by creating large surfaces suitable for interactions with their specific protein binding partners, which is a critical component of gene regulation by transcription factors. It has been hypothesized that conditional folding of these activation domains may be a prerequisite for their efficient interaction with specific coregulatory proteins, and subsequent transcriptional activity leading to the regulation of target gene(s). In this study, we tested whether a naturally occurring osmolyte, trehalose can promote functionally ordered conformation in glucocorticoid receptor's major activation function domain, AF1, which is found to exist as an ID protein, and requires an efficient interaction with coregulatory proteins for optimal activity. Our data show that trehalose induces an ordered conformation in AF1 such that its interaction with steroid receptor coactivator-1 (SRC-1), a critical coregulator of glucocorticoid receptor's activity, is greatly enhanced

Characteristic mTOR activity in Hodgkin-lymphomas offers a potential therapeutic target in high risk disease – a combined tissue microarray, in vitro and in vivo study

BACKGROUND: Targeting signaling pathways is an attractive approach in many malignancies. The PI3K/Akt/mTOR pathway is activated in a number of human neoplasms, accompanied by lower overall and/or disease free survival. mTOR kinase inhibitors have been introduced in the therapy of renal cell carcinoma and mantle cell lymphoma, and several trials are currently underway. However, the pathological characterization of mTOR activity in lymphomas is still incomplete. METHODS: mTOR activity and the elements of mTOR complexes were investigated by immunohistochemistry on tissue microarrays representing different human non-Hodgkin-lymphomas (81 cases) and Hodgkin-lymphomas (87 cases). The expression of phospho-mTOR, phospho-4EBP1, phospho-p70S6K, phospho-S6, Rictor, Raptor and Bcl-2, Bcl-xL, Survivin and NF-kappaB-p50 were evaluated, and mTOR activity was statistically analyzed along with 5-year survival data. The in vitro and in vivo effect of the mTOR inhibitor rapamycin was also examined in human Hodgkin-lymphoma cell lines. RESULTS: The majority (>50%) of mantle cell lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, anaplastic large-cell lymphoma and Hodgkin-lymphoma cases showed higher mTOR activity compared to normal lymphoid tissues. Hodgkin-lymphoma was characterized by high mTOR activity in 93% of the cases, and Bcl-xL and NF-kappaB expression correlated with this mTOR activity. High mTOR activity was observed in the case of both favorable and unfavorable clinical response. Low mTOR activity was accompanied by complete remission and at least 5-year disease free survival in Hodgkin-lymphoma patients. However, statistical analysis did not identify correlation beetween mTOR activity and different clinical data of HL patients, such as survival. We also found that Rictor (mTORC2) was not overexpressed in Hodgkin-lymphoma biopsies and cell lines. Rapamycin inhibited proliferation and induced apoptosis in Hodgkin-lymphoma cells both in vitro and in vivo, moreover, it increased the apoptotic effect of chemotherapeutic agents. CONCLUSIONS: Targeting mTOR activity may be a potential therapeutic tool in lymphomas. The presence of mTOR activity probably indicates that the inclusion of mTOR inhibition in the therapy of Hodgkin-lymphomas may be feasible and beneficial, especially when standard protocols are ineffective, and it may also allow dose reduction in order to decrease late treatment toxicity. Most likely, the combination of mTOR inhibitors with other agents will offer the highest efficiency for achieving the best clinical response

Gender Differences in Sleep Deprivation Effects on Risk and Inequality Aversion: Evidence from an Economic Experiment

Excessive working hours—even at night—are becoming increasingly common in our modern 24/7 society. The prefrontal cortex (PFC) is particularly vulnerable to the effects of sleep loss and, consequently, the specific behaviors subserved by the functional integrity of the PFC, such as risk-taking and pro-social behavior, may be affected significantly. This paper seeks to assess the effects of one night of sleep deprivation on subjects’ risk and social preferences, which are probably the most explored behavioral domains in the tradition of Experimental Economics. This novel cross-over study employs thirty-two university students (gender-balanced) participating to 2 counterbalanced laboratory sessions in which they perform standard risk and social preference elicitation protocols. One session was after one night of undisturbed sleep at home, and the other was after one night of sleep deprivation in the laboratory. Sleep deprivation causes increased sleepiness and decreased alertness in all subjects. After sleep loss males make riskier decisions compared to the rested condition, while females do the opposite. Females likewise show decreased inequity aversion after sleep deprivation. As for the relationship between cognitive ability and economic decisions, sleep deprived individuals with higher cognitive reflection show lower risk aversion and more altruistic behavior. These results show that one night of sleep deprivation alters economic behavior in a gender-sensitive way. Females’ reaction to sleep deprivation, characterized by reduced risky choices and increased egoism compared to males, may be related to intrinsic psychological gender differences, such as in the way men and women weigh up probabilities in their decision-making, and/or to the different neurofunctional substrate of their decision-making.The authors acknowledge financial support from the Spanish Ministry of Economic Competititveness (ECO2012-34928), Italian Ministry of University and Research MIUR (PRIN 20103S5RN3_002), Generalitat Valenciana (Research Projects Gruposo3/086), the Instituto Valenciano de Investigaciones Económicas (IVIE), and the Ministero della Salute (RF-2009-1528677)

Filovirus RefSeq Entries: Evaluation and Selection of Filovirus Type Variants, Type Sequences, and Names

Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences

Expert range maps of global mammal distributions harmonised to three taxonomic authorities

Aim: Comprehensive, global information on species' occurrences is an essential biodiversity variable and central to a range of applications in ecology, evolution, biogeography and conservation. Expert range maps often represent a species' only available distributional information and play an increasing role in conservation assessments and macroecology. We provide global range maps for the native ranges of all extant mammal species harmonised to the taxonomy of the Mammal Diversity Database (MDD) mobilised from two sources, the Handbook of the Mammals of the World (HMW) and the Illustrated Checklist of the Mammals of the World (CMW). Location: Global. Taxon: All extant mammal species. Methods: Range maps were digitally interpreted, georeferenced, error-checked and subsequently taxonomically aligned between the HMW (6253 species), the CMW (6431 species) and the MDD taxonomies (6362 species). Results: Range maps can be evaluated and visualised in an online map browser at Map of Life (mol.org) and accessed for individual or batch download for non-commercial use. Main conclusion: Expert maps of species' global distributions are limited in their spatial detail and temporal specificity, but form a useful basis for broad-scale characterizations and model-based integration with other data. We provide georeferenced range maps for the native ranges of all extant mammal species as shapefiles, with species-level metadata and source information packaged together in geodatabase format. Across the three taxonomic sources our maps entail, there are 1784 taxonomic name differences compared to the maps currently available on the IUCN Red List website. The expert maps provided here are harmonised to the MDD taxonomic authority and linked to a community of online tools that will enable transparent future updates and version control.Fil: Marsh, Charles J.. Yale University; Estados UnidosFil: Sica, Yanina. Yale University; Estados UnidosFil: Burguin, Connor. University of New Mexico; Estados UnidosFil: Dorman, Wendy A.. University of Yale; Estados UnidosFil: Anderson, Robert C.. University of Yale; Estados UnidosFil: del Toro Mijares, Isabel. University of Yale; Estados UnidosFil: Vigneron, Jessica G.. University of Yale; Estados UnidosFil: Barve, Vijay. University Of Florida. Florida Museum Of History; Estados UnidosFil: Dombrowik, Victoria L.. University of Yale; Estados UnidosFil: Duong, Michelle. University of Yale; Estados UnidosFil: Guralnick, Robert. University Of Florida. Florida Museum Of History; Estados UnidosFil: Hart, Julie A.. University of Yale; Estados UnidosFil: Maypole, J. Krish. University of Yale; Estados UnidosFil: McCall, Kira. University of Yale; Estados UnidosFil: Ranipeta, Ajay. University of Yale; Estados UnidosFil: Schuerkmann, Anna. University of Yale; Estados UnidosFil: Torselli, Michael A.. University of Yale; Estados UnidosFil: Lacher, Thomas. Texas A&M University; Estados UnidosFil: Wilson, Don E.. National Museum of Natural History; Estados UnidosFil: Abba, Agustin Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Estudios Parasitológicos y de Vectores. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Centro de Estudios Parasitológicos y de Vectores; ArgentinaFil: Aguirre, Luis F.. Universidad Mayor de San Simón; BoliviaFil: Arroyo Cabrales, Joaquín. Instituto Nacional de Antropología E Historia, Mexico; MéxicoFil: Astúa, Diego. Universidade Federal de Pernambuco; BrasilFil: Baker, Andrew M.. Queensland University of Technology; Australia. Queensland Museum; AustraliaFil: Braulik, Gill. University of St. Andrews; Reino UnidoFil: Braun, Janet K.. Oklahoma State University; Estados UnidosFil: Brito, Jorge. Instituto Nacional de Biodiversidad; EcuadorFil: Busher, Peter E.. Boston University; Estados UnidosFil: Burneo, Santiago F.. Pontificia Universidad Católica del Ecuador; EcuadorFil: Camacho, M. Alejandra. Pontificia Universidad Católica del Ecuador; EcuadorFil: de Almeida Chiquito, Elisandra. Universidade Federal do Espírito Santo; BrasilFil: Cook, Joseph A.. University of New Mexico; Estados UnidosFil: Cuéllar Soto, Erika. Sultan Qaboos University; OmánFil: Davenport, Tim R. B.. Wildlife Conservation Society; TanzaniaFil: Denys, Christiane. Muséum National d'Histoire Naturelle; FranciaFil: Dickman, Christopher R.. The University Of Sydney; AustraliaFil: Eldridge, Mark D. B.. Australian Museum; AustraliaFil: Fernandez Duque, Eduardo. University of Yale; Estados UnidosFil: Francis, Charles M.. Environment And Climate Change Canada; CanadáFil: Frankham, Greta. Australian Museum; AustraliaFil: Freitas, Thales. Universidade Federal do Rio Grande do Sul; BrasilFil: Friend, J. Anthony. Conservation And Attractions; AustraliaFil: Giannini, Norberto Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - Tucumán. Unidad Ejecutora Lillo; ArgentinaFil: Gursky-Doyen, Sharon. Texas A&M University; Estados UnidosFil: Hackländer, Klaus. Universitat Fur Bodenkultur Wien; AustriaFil: Hawkins, Melissa. National Museum of Natural History; Estados UnidosFil: Helgen, Kristofer M.. Australian Museum; AustraliaFil: Heritage, Steven. University of Duke; Estados UnidosFil: Hinckley, Arlo. Consejo Superior de Investigaciones Científicas. Estación Biológica de Doñana; EspañaFil: Holden, Mary. American Museum of Natural History; Estados UnidosFil: Holekamp, Kay E.. Michigan State University; Estados UnidosFil: Humle, Tatyana. University Of Kent; Reino UnidoFil: Ibáñez Ulargui, Carlos. Consejo Superior de Investigaciones Científicas. Estación Biológica de Doñana; EspañaFil: Jackson, Stephen M.. Australian Museum; AustraliaFil: Janecka, Mary. University of Pittsburgh at Johnstown; Estados Unidos. University of Pittsburgh; Estados UnidosFil: Jenkins, Paula. Natural History Museum; Reino UnidoFil: Juste, Javier. Consejo Superior de Investigaciones Científicas. Estación Biológica de Doñana; EspañaFil: Leite, Yuri L. R.. Universidade Federal do Espírito Santo; BrasilFil: Novaes, Roberto Leonan M.. Universidade Federal do Rio de Janeiro; BrasilFil: Lim, Burton K.. Royal Ontario Museum; CanadáFil: Maisels, Fiona G.. Wildlife Conservation Society; Estados UnidosFil: Mares, Michael A.. Oklahoma State University; Estados UnidosFil: Marsh, Helene. James Cook University; AustraliaFil: Mattioli, Stefano. Università degli Studi di Siena; ItaliaFil: Morton, F. Blake. University of Hull; Reino UnidoFil: Ojeda, Agustina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Provincia de Mendoza. Instituto Argentino de Investigaciones de las Zonas Áridas. Universidad Nacional de Cuyo. Instituto Argentino de Investigaciones de las Zonas Áridas; ArgentinaFil: Ordóñez Garza, Nicté. Instituto Nacional de Biodiversidad; EcuadorFil: Pardiñas, Ulises Francisco J.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Diversidad y Evolución Austral; ArgentinaFil: Pavan, Mariana. Universidade de Sao Paulo; BrasilFil: Riley, Erin P.. San Diego State University; Estados UnidosFil: Rubenstein, Daniel I.. University of Princeton; Estados UnidosFil: Ruelas, Dennisse. Museo de Historia Natural, Lima; PerúFil: Schai-Braun, Stéphanie. Universitat Fur Bodenkultur Wien; AustriaFil: Schank, Cody J.. University of Texas at Austin; Estados UnidosFil: Shenbrot, Georgy. Ben Gurion University of the Negev; IsraelFil: Solari, Sergio. Universidad de Antioquia; ColombiaFil: Superina, Mariella. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; ArgentinaFil: Tsang, Susan. American Museum of Natural History; Estados UnidosFil: Van Cakenberghe, Victor. Universiteit Antwerp; BélgicaFil: Veron, Geraldine. Université Pierre et Marie Curie; FranciaFil: Wallis, Janette. Kasokwa-kityedo Forest Project; UgandaFil: Whittaker, Danielle. Michigan State University; Estados UnidosFil: Wells, Rod. Flinders University.; AustraliaFil: Wittemyer, George. State University of Colorado - Fort Collins; Estados UnidosFil: Woinarski, John. Charles Darwin University; AustraliaFil: Upham, Nathan S.. University of Yale; Estados UnidosFil: Jetz, Walter. University of Yale; Estados Unido

Association between convalescent plasma treatment and mortality in COVID-19: a collaborative systematic review and meta-analysis of randomized clinical trials.

Funder: laura and john arnold foundationBACKGROUND: Convalescent plasma has been widely used to treat COVID-19 and is under investigation in numerous randomized clinical trials, but results are publicly available only for a small number of trials. The objective of this study was to assess the benefits of convalescent plasma treatment compared to placebo or no treatment and all-cause mortality in patients with COVID-19, using data from all available randomized clinical trials, including unpublished and ongoing trials (Open Science Framework, https://doi.org/10.17605/OSF.IO/GEHFX ). METHODS: In this collaborative systematic review and meta-analysis, clinical trial registries (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform), the Cochrane COVID-19 register, the LOVE database, and PubMed were searched until April 8, 2021. Investigators of trials registered by March 1, 2021, without published results were contacted via email. Eligible were ongoing, discontinued and completed randomized clinical trials that compared convalescent plasma with placebo or no treatment in COVID-19 patients, regardless of setting or treatment schedule. Aggregated mortality data were extracted from publications or provided by investigators of unpublished trials and combined using the Hartung-Knapp-Sidik-Jonkman random effects model. We investigated the contribution of unpublished trials to the overall evidence. RESULTS: A total of 16,477 patients were included in 33 trials (20 unpublished with 3190 patients, 13 published with 13,287 patients). 32 trials enrolled only hospitalized patients (including 3 with only intensive care unit patients). Risk of bias was low for 29/33 trials. Of 8495 patients who received convalescent plasma, 1997 died (23%), and of 7982 control patients, 1952 died (24%). The combined risk ratio for all-cause mortality was 0.97 (95% confidence interval: 0.92; 1.02) with between-study heterogeneity not beyond chance (I2 = 0%). The RECOVERY trial had 69.8% and the unpublished evidence 25.3% of the weight in the meta-analysis. CONCLUSIONS: Convalescent plasma treatment of patients with COVID-19 did not reduce all-cause mortality. These results provide strong evidence that convalescent plasma treatment for patients with COVID-19 should not be used outside of randomized trials. Evidence synthesis from collaborations among trial investigators can inform both evidence generation and evidence application in patient care