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'Bacterial-type' ferredoxins host a cubane [4Fe4S]2+/+ cluster that enables these proteins to mediate electron transfer and facilitate a broad range of biological processes. Peptide maquettes based on the conserved cluster-forming motif have previously been reported and used to model the ferredoxins. Herein we explore the integration of a [4Fe4S]-peptide maquette into a H2 -powered electron transport chain. While routinely formed under anaerobic conditions, we illustrate by electron paramagnetic resonance (EPR) analysis that these maquettes can be reconstituted under aerobic conditions by using photoactivated NADH to reduce the cluster at 240â K. Attempts to tune the redox properties of the iron-sulfur cluster by introducing an Fe-coordinating selenocysteine residue were also explored. To demonstrate the integration of these artificial metalloproteins into a semi-synthetic electron transport chain, we utilize a ferredoxin-inspired [4Fe4S]-peptide maquette as the redox partner in the hydrogenase-mediated oxidation of H2 .
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Hidrogenasas , Proteínas Hierro-Azufre , Ferredoxinas/metabolismo , Proteínas Hierro-Azufre/química , Hidrogenasas/metabolismo , Oxidación-Reducción , Péptidos/metabolismo , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
The site-selective modification of peptides and proteins facilitates the preparation of targeted therapeutic agents and tools to interrogate biochemical pathways. Among the numerous bioconjugation techniques developed to install groups of interest, those that generate C(sp3 )-C(sp3 ) bonds are significantly underrepresented despite affording proteolytically stable, biogenic linkages. Herein, a visible-light-mediated reaction is described that enables the site-selective modification of peptides and proteins via desulfurative C(sp3 )-C(sp3 ) bond formation. The reaction is rapid and high yielding in peptide systems, with comparable translation to proteins. Using this chemistry, a range of moieties is installed into model systems and an effective PTM-mimic is successfully integrated into a recombinantly expressed histone.
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Cisteína , Proteínas , Cisteína/química , Proteínas/química , Péptidos/químicaRESUMEN
Herein, we report the synthesis and characterization of a new class of hybrid Wells-Dawson polyoxometalate (POM) containing a diphosphoryl group (P2 O6 X) of the general formula [P2 W17 O57 (P2 O6 X)]6- (X=O, NH, or CR1 R2 ). Modifying the bridging unit X was found to impact the redox potentials of the POM. The ease with which a range of α-functionalized diphosphonic acids (X=CR1 R2 ) can be prepared provides possibilities to access diverse functionalized hybrid POMs. Compared to existing phosphonate hybrid Wells-Dawson POMs, diphosphoryl-substituted POMs offer a wider tunable redox window and enhanced hydrolytic stability. This study provides a basis for the rational design and synthesis of next-generation hybrid Wells-Dawson POMs.
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Interleukin (IL)-11, a multifunctional cytokine, contributes to numerous biological processes, including adipogenesis, hematopoiesis, and inflammation. Asthma, a respiratory disease, is notably characterized by reversible airway obstruction, persistent lung inflammation, and airway hyperresponsiveness (AHR). Nasal insufflation of IL-11 causes AHR in wild-type mice while lung inflammation induced by antigen sensitization and challenge, which mimics features of atopic asthma in humans, is attenuated in mice genetically deficient in IL-11 receptor subunit α-1 (IL-11Rα1-deficient mice), a transmembrane receptor that is required conjointly with glycoprotein 130 to transduce IL-11 signaling. Nevertheless, the contribution of IL-11Rα1 to characteristics of nonatopic asthma is unknown. Thus, based on the aforementioned observations, we hypothesized that genetic deficiency of IL-11Rα1 attenuates lung inflammation and increases airway responsiveness after acute inhalation exposure to ozone (O3), a criteria pollutant and nonatopic asthma stimulus. Accordingly, 4 and/or 24 h after cessation of exposure to filtered room air or O3, we assessed lung inflammation and airway responsiveness in wild-type and IL-11Rα1-deficient mice. With the exception of bronchoalveolar lavage macrophages and adiponectin, which were significantly increased and decreased, respectively, in O3-exposed IL-11Rα1-deficient as compared with O3-exposed wild-type mice, no other genotype-related differences in lung inflammation indices that we quantified were observed in O3-exposed mice. However, airway responsiveness to acetyl-ß-methylcholine chloride (methacholine) was significantly diminished in IL-11Rα1-deficient as compared with wild-type mice after O3 exposure. In conclusion, these results demonstrate that IL-11Rα1 minimally contributes to lung inflammation but is required for maximal airway responsiveness to methacholine in a mouse model of nonatopic asthma.
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Asma , Ozono , Neumonía , Humanos , Ratones , Animales , Cloruro de Metacolina/efectos adversos , Ozono/toxicidad , Interleucina-11/efectos adversos , Asma/genética , Neumonía/inducido químicamente , Neumonía/genética , Neumonía/complicaciones , Receptores de Interleucina-11 , Líquido del Lavado BronquioalveolarRESUMEN
Post-translational modifications (PTMs) enhance the repertoire of protein function and mediate or influence the activity of many cellular processes. The preparation of site-specifically and homogeneously modified proteins, to apply as tools to understand the biological role of PTMs, is a challenging task. Herein, we describe a visible-light-mediated desulfurative C(sp3 )-C(sp3 ) bond forming reaction that enables the site-selective installation of Nϵ -modified sidechains into peptides and proteins of interest. Rapid, operationally simple, and tolerant to ambient atmosphere, we demonstrate the installation of a range of lysine (Lys) PTMs into model peptide systems and showcase the potential of this technology by site-selectively installing an Nϵ Ac sidechain into recombinantly expressed ubiquitin (Ub).
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Péptidos , ProteínasRESUMEN
OBJECTIVE: The Maternal Mental Health in Canada, 2018/2019, survey reported that 18% of 7,085 mothers who recently gave birth reported "feelings consistent with postpartum depression" based on scores ≥7 on a 5-item version of the Edinburgh Postpartum Depression Scale (EPDS-5). The EPDS-5 was designed as a screening questionnaire, not to classify disorders or estimate prevalence; the extent to which EPDS-5 results reflect depression prevalence is unknown. We investigated EPDS-5 ≥7 performance relative to major depression prevalence based on a validated diagnostic interview, the Structured Clinical Interview for DSM (SCID). METHODS: We searched Medline, Medline In-Process & Other Non-Indexed Citations, PsycINFO, and the Web of Science Core Collection through June 2016 for studies with data sets with item response data to calculate EPDS-5 scores and that used the SCID to ascertain depression status. We conducted an individual participant data meta-analysis to estimate pooled percentage of EPDS-5 ≥7, pooled SCID major depression prevalence, and the pooled difference in prevalence. RESULTS: A total of 3,958 participants from 19 primary studies were included. Pooled prevalence of SCID major depression was 9.2% (95% confidence interval [CI] 6.0% to 13.7%), pooled percentage of participants with EPDS-5 ≥7 was 16.2% (95% CI 10.7% to 23.8%), and pooled difference was 8.0% (95% CI 2.9% to 13.2%). In the 19 included studies, mean and median ratios of EPDS-5 to SCID prevalence were 2.1 and 1.4 times. CONCLUSIONS: Prevalence estimated based on EPDS-5 ≥7 appears to be substantially higher than the prevalence of major depression. Validated diagnostic interviews should be used to establish prevalence.
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Depresión Posparto/epidemiología , Depresión Posparto/psicología , Tamizaje Masivo/métodos , Madres/psicología , Canadá/epidemiología , Depresión Posparto/diagnóstico , Trastorno Depresivo Mayor , Medicina Basada en la Evidencia , Femenino , Humanos , Embarazo , Prevalencia , Escalas de Valoración PsiquiátricaRESUMEN
Establishing and maintaining protected areas (PAs) are key tools for biodiversity conservation. However, this approach is insufficient for many species, particularly those that are wide-ranging and sparse. The cheetah Acinonyx jubatus exemplifies such a species and faces extreme challenges to its survival. Here, we show that the global population is estimated at â¼7,100 individuals and confined to 9% of its historical distributional range. However, the majority of current range (77%) occurs outside of PAs, where the species faces multiple threats. Scenario modeling shows that, where growth rates are suppressed outside PAs, extinction rates increase rapidly as the proportion of population protected declines. Sensitivity analysis shows that growth rates within PAs have to be high if they are to compensate for declines outside. Susceptibility of cheetah to rapid decline is evidenced by recent rapid contraction in range, supporting an uplisting of the International Union for the Conservation of Nature (IUCN) Red List threat assessment to endangered. Our results are applicable to other protection-reliant species, which may be subject to systematic underestimation of threat when there is insufficient information outside PAs. Ultimately, conserving many of these species necessitates a paradigm shift in conservation toward a holistic approach that incentivizes protection and promotes sustainable human-wildlife coexistence across large multiple-use landscapes.
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Acinonyx , Conservación de los Recursos Naturales , África , Animales , Asia , Biodiversidad , Simulación por Computador , Extinción Biológica , Modelos Biológicos , Dinámica Poblacional/tendencias , Factores de RiesgoRESUMEN
The development of site-selective chemistry targeting the canonical amino acids enables the controlled installation of desired functionalities into native peptides and proteins. Such techniques facilitate the development of polypeptide conjugates to advance therapeutics, diagnostics, and fundamental science. We report a versatile and selective method to functionalize peptides and proteins through free-radical-mediated dechalcogenation. By exploiting phosphine-induced homolysis of the C-Se and C-S bonds of selenocysteine and cysteine, respectively, we demonstrate the site-selective installation of groups appended to a persistent radical trap. The reaction is rapid, operationally simple, and chemoselective. The resulting aminooxy linker is stable under a variety of conditions and selectively cleavable in the presence of a low-oxidation-state transition metal. We have explored the full scope of this reaction using complex peptide systems and a recombinantly expressed protein.
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People with lived experience are individuals who have first-hand experience of the medical condition(s) being considered. The value of including the viewpoints of people with lived experience in health policy, health care, and health care and systems research has been recognized at many levels, including by funding agencies. However, there is little guidance or established best practices on how to include non-academic reviewers in the grant review process. Here we describe our approach to the inclusion of people with lived experience in every stage of the grant review process. After a budget was created for a specific call, a steering committee was created. This group included researchers, people with lived experience, and health systems administrators. This group developed and issued the call. After receiving proposals, stage one was scientific review by researchers. Grants were ranked by this score and a short list then reviewed by people with lived experience as stage two. Finally, for stage three, the Steering Committee convened and achieved consensus based on information drawn from stages one and two. Our approach to engage people with lived experience in the grant review process was positively reviewed by everyone involved, as it allowed for patient perspectives to be truly integrated. However, it does lengthen the review process. The proposed model offers further practical insight into including people with lived experience in the review process.
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Organización de la Financiación , Participación del Paciente , Comités Consultivos , Política de SaludRESUMEN
Polyproline sequences are highly abundant in prokaryotic and eukaryotic proteins, where they serve as key components of secondary structure. To date, construction of the proline-proline motif has not been possible owing to steric congestion at the ligation junction, together with an n â π* electronic interaction that reduces the reactivity of acylated proline residues at the C-terminus of peptides. Here, we harness the enhanced reactivity of prolyl selenoesters and a trans-γ-selenoproline moiety to access the elusive proline-proline junction for the first time through a diselenide-selenoester ligation-deselenization manifold. The efficient nature of this chemistry is highlighted in the high-yielding one-pot assembly of two proline-rich polypeptide targets, submaxillary gland androgen regulated protein 3B and lumbricin-1. This method provides access to the most challenging of ligation junctions, thus enabling the construction of previously intractable peptide and protein targets of increasing structural complexity.
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Compuestos de Organoselenio/química , Péptidos/síntesis química , Prolina/análogos & derivados , Proteínas y Péptidos Salivales/síntesis química , Secuencias de Aminoácidos , Antibacterianos/síntesis química , Humanos , Compuestos de Organoselenio/síntesis química , Prolina/síntesis química , Staphylococcus aureus/efectos de los fármacos , EstereoisomerismoRESUMEN
Entry into mitosis is driven by the phosphorylation of thousands of substrates, under the master control of Cdk1. During entry into mitosis, Cdk1, in collaboration with MASTL kinase, represses the activity of the major mitotic protein phosphatases, PP1 and PP2A, thereby ensuring mitotic substrates remain phosphorylated. For cells to complete and exit mitosis, these phosphorylation events must be removed, and hence, phosphatase activity must be reactivated. This reactivation of phosphatase activity presumably requires the inhibition of MASTL; however, it is not currently understood what deactivates MASTL and how this is achieved. In this study, we identified that PP1 is associated with, and capable of partially dephosphorylating and deactivating, MASTL during mitotic exit. Using mathematical modelling, we were able to confirm that deactivation of MASTL is essential for mitotic exit. Furthermore, small decreases in Cdk1 activity during metaphase are sufficient to initiate the reactivation of PP1, which in turn partially deactivates MASTL to release inhibition of PP2A and, hence, create a feedback loop. This feedback loop drives complete deactivation of MASTL, ensuring a strong switch-like activation of phosphatase activity during mitotic exit.
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Quinasas Ciclina-Dependientes/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/fisiología , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Quinasa CDC2 , Línea Celular Tumoral , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Modelos Teóricos , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
The design, synthesis and formulation of non-viral gene delivery vectors is an area of renewed research interest. Amongst the most efficient non-viral gene delivery systems are lipopolyplexes, in which cationic peptides are co-formulated with plasmid DNA and lipids. One advantage of lipopolyplex vectors is that they have the potential to be targeted to specific cell types by attaching peptide targeting ligands on the surface, thus increasing both the transfection efficiency and selectivity for disease targets such as cancer cells. In this paper, we have investigated two different modes of displaying cell-specific peptide targeting ligands at the surface of lipopolyplexes. Lipopolyplexes formulated with bimodal peptides, with both receptor binding and DNA condensing sequences, were compared with lipopolyplexes with the peptide targeting ligand directly conjugated to one of the lipids. Three EGFR targeting peptide sequences were studied, together with a range of lipid formulations and maleimide lipid structures. The biophysical properties of the lipopolyplexes and their transfection efficiencies in a basal-like breast cancer cell line were investigated using plasmid DNA bearing genes for the expression of firefly luciferase and green fluorescent protein. Fluorescence quenching experiments were also used to probe the macromolecular organisation of the peptide and pDNA components of the lipopolyplexes. We demonstrated that both approaches to lipopolyplex targeting give reasonable transfection efficiencies, and the transfection efficiency of each lipopolyplex formulation is highly dependent on the sequence of the targeting peptide. To achieve maximum therapeutic efficiency, different peptide targeting sequences and lipopolyplex architectures should be investigated for each target cell type.
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Péptidos Catiónicos Antimicrobianos/química , Neoplasias de la Mama/terapia , ADN/química , Técnicas de Transferencia de Gen , Lípidos/química , Péptidos Catiónicos Antimicrobianos/síntesis química , Neoplasias de la Mama/metabolismo , ADN/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Vectores Genéticos/química , Vectores Genéticos/genética , Humanos , Ligandos , Plásmidos/química , Conformación Proteica , Propiedades de Superficie , TransfecciónRESUMEN
The use of native chemical ligation at selenocysteine (Sec) residues with peptide thioesters and additive-free selenocystine ligation with peptides bearing phenyl selenoesters, in concert with one-pot oxidative deselenization chemistry, is described. These approaches provide a simple and rapid method for accessing native peptides with serine in place of Sec at the ligation junction. The efficiency of both variants of the one-pot ligation-oxidative deselenization chemistry is probed through the synthesis of a MUC5AC-derived glycopeptide.
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Cistina/análogos & derivados , Compuestos de Organoselenio/química , Selenocisteína/química , Cromatografía Líquida de Alta Presión , Cistina/química , Glicopéptidos/síntesis química , Glicopéptidos/química , Humanos , Espectrometría de Masas , Mucina 5AC/química , Oxidación-ReducciónRESUMEN
We describe an unprecedented reaction between peptide selenoesters and peptide dimers bearing N-terminal selenocystine that proceeds in aqueous buffer to afford native amide bonds without the use of additives. The selenocystine-selenoester ligations are complete in minutes, even at sterically hindered junctions, and can be used in concert with one-pot deselenization chemistry. Various pathways for the transformation are proposed and probed through a combination of experimental and computational studies. Our new reaction manifold is also showcased in the total synthesis of two proteins.
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Proteínas Bacterianas/síntesis química , Corismato Mutasa/síntesis química , Cistina/análogos & derivados , Compuestos de Organoselenio/química , Péptidos/química , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Corismato Mutasa/química , Corismato Mutasa/metabolismo , Cistina/química , Ésteres/química , Conformación Molecular , Mycobacterium tuberculosis/enzimología , Compuestos de Selenio/químicaRESUMEN
Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under-utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high-yielding step. When coupled with ligation at Sec, this transformation provides a new approach to programmed ligations at Ser residues. This new reaction is compatible with a wide range of functionality, including the presence of unprotected amino acid side chains and appended glycans. The utility of the methodology is demonstrated in the rapid synthesis of complex glycopeptide fragments of the epithelial glycoproteins MUC5AC and MUC4 and through the total synthesis of the structured, cysteine (Cys)-free protein eglinâ C.
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Cisteína/química , Glicopéptidos/síntesis química , Selenocisteína/química , Secuencia de Aminoácidos , Animales , Glicopéptidos/química , Hirudo medicinalis/química , Humanos , Datos de Secuencia Molecular , Mucina 5AC/síntesis química , Mucina 5AC/química , Mucina 4/síntesis química , Mucina 4/química , Oxidación-Reducción , Proteínas/síntesis química , Proteínas/químicaRESUMEN
Sonic hedgehog (Shh) signaling is essential to the patterning of the embryonic neural tube, but its presence and function in the postmitotic differentiated neurons in the brain remain largely uncharacterized. We recently showed that Shh and its signaling components, Patched and Smoothened, are expressed in postnatal and adult hippocampal neurons. We have now examined whether Shh signaling has a function in these neurons. Using cultured hippocampal neurons as a model system, we found that presynaptic terminals become significantly larger in response to the application of Shh. Ultrastructural examination confirmed the enlarged presynaptic profiles and also revealed variable increases in the size of synaptic vesicles, with a resulting loss of uniformity. Furthermore, electrophysiological analyses showed significant increases in the frequency, but not the amplitude, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in response to Shh, providing functional evidence of the selective role of Shh in presynaptic terminals. Thus, we conclude that Shh signaling regulates the structure and functional properties of presynaptic terminals of hippocampal neurons.
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Proteínas Hedgehog/metabolismo , Hipocampo/citología , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Animales , Células HEK293 , Humanos , Neurotransmisores/metabolismo , Tamaño de los Órganos , Terminales Presinápticos/metabolismo , Ratas , Transducción de Señal , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructuraRESUMEN
The convergent assembly of peptide fragments by native chemical ligation has revolutionized the way in which proteins can be accessed by chemical synthesis. A variation of native chemical ligation involves the reaction of peptides bearing an N-terminal selenocysteine residue with peptide thioesters, which proceeds through the same mechanism as the parent reaction. This transformation was first investigated in 2001 for the installation of selenocysteine into peptides and proteins via ligation chemistry. The recent discovery that selenocysteine residues within peptides can be chemoselectively deselenized without the concomitant desulfurization of cysteine residues has led to renewed interest in ligation chemistry at selenocysteine. This review outlines the use of selenocysteine in ligation chemistry as well as recent investigations of chemoselective ligation-deselenization chemistry at other selenol-derived amino acids that have the potential to greatly expand the number of targets that can be accessed by chemical synthesis.
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Aminoácidos/metabolismo , Compuestos de Selenio/química , Aminoácidos/química , Estructura Molecular , Péptidos/química , Péptidos/metabolismo , Selenocisteína/química , Selenocisteína/metabolismoRESUMEN
Synthetic methods that enable the macrocyclisation of peptides facilitate the development of effective therapeutic and diagnostic tools. Herein we report a peptide cyclisation strategy based on intramolecular interception of visible-light-mediated cysteine desulfurisation. This method allows cyclisation of unprotected peptides in an aqueous solution via the installation of a hydrocarbon linkage. We explore the limits of this chemistry using a range of model peptides of increasing length and complexity, including peptides of biological/therapeutic relevance. The method is applied to replace the native disulfide of the peptide hormone, oxytocin, with a proteolytically/redox-stable hydrocarbon, and internal macrocyclisation of an MCL-1-binding peptide.
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Across eukaryotic proteomes, tryptophan is the least abundant of the 20 canonical amino acids, which makes it an ideal chemical handle for the late-stage functionalization of peptide and protein scaffolds with minimal production of undesired isoforms. Herein, we report the photocatalytic C2-alkylation of tryptophan using bromodifluoroacetate/acetamide-derived radical precursors. This rapid visible-light-mediated reaction is additive-free, operationally simple, and tolerates diverse functionality. We demonstrate the late-stage modification of a variety of complex peptides, including examples of biological significance.
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Péptidos , Triptófano , Triptófano/química , Péptidos/química , Proteínas/química , Aminoácidos/química , AlquilaciónRESUMEN
The seven-item Hospital Anxiety and Depression Scale Depression subscale (HADS-D) and the total score of the 14-item HADS (HADS-T) are both used for major depression screening. Compared to the HADS-D, the HADS-T includes anxiety items and requires more time to complete. We compared the screening accuracy of the HADS-D and HADS-T for major depression detection. We conducted an individual participant data meta-analysis and fit bivariate random effects models to assess diagnostic accuracy among participants with both HADS-D and HADS-T scores. We identified optimal cutoffs, estimated sensitivity and specificity with 95% confidence intervals, and compared screening accuracy across paired cutoffs via two-stage and individual-level models. We used a 0.05 equivalence margin to assess equivalency in sensitivity and specificity. 20,700 participants (2,285 major depression cases) from 98 studies were included. Cutoffs of ≥7 for the HADS-D (sensitivity 0.79 [0.75, 0.83], specificity 0.78 [0.75, 0.80]) and ≥15 for the HADS-T (sensitivity 0.79 [0.76, 0.82], specificity 0.81 [0.78, 0.83]) minimized the distance to the top-left corner of the receiver operating characteristic curve. Across all sets of paired cutoffs evaluated, differences of sensitivity between HADS-T and HADS-D ranged from -0.05 to 0.01 (0.00 at paired optimal cutoffs), and differences of specificity were within 0.03 for all cutoffs (0.02-0.03). The pattern was similar among outpatients, although the HADS-T was slightly (not nonequivalently) more specific among inpatients. The accuracy of HADS-T was equivalent to the HADS-D for detecting major depression. In most settings, the shorter HADS-D would be preferred. (PsycInfo Database Record (c) 2023 APA, all rights reserved).