A Digital Health Intervention Platform (Active and Independent Management System) to Enhance the Rehabilitation Experience for Orthopedic Joint Replacement Patients: Usability Evaluation Study.

BACKGROUND: Optimal rehabilitation programs for orthopedic joint replacement patients ensure faster return to function, earlier discharge from hospital, and improved patient satisfaction. Digital health interventions show promise as a supporting tool for re-enablement. OBJECTIVE: The main goal of this mixed methods study was to examine the usability of the AIMS platform from the perspectives of both patients and clinicians. The aim of this study was to evaluate a re-enablement platform that we have developed that uses a holistic systems approach to address the de-enablement that occurs in hospitalized inpatients, with the older adult population most at risk. The Active and Independent Management System (AIMS) platform is anticipated to deliver improved patient participation in recovery and self-management through education and the ability to track rehabilitation progression in hospital and after patient discharge. METHODS: Two well-known instruments were used to measure usability: the System Usability Scale (SUS) with 10 items and, for finer granularity, the User Experience Questionnaire (UEQ) with 26 items. In all, 26 physiotherapists and health care professionals evaluated the AIMS clinical portal; and 44 patients in hospital for total knee replacement, total hip replacement, or dynamic hip screw implant evaluated the AIMS app. RESULTS: For the AIMS clinical portal, the mean SUS score obtained was 82.88 (SD 13.07, median 86.25), which would be considered good/excellent according to a validated adjective rating scale. For the UEQ, the means of the normalized scores (range -3 to +3) were as follows: attractiveness=2.683 (SD 0.100), perspicuity=2.775 (SD 0.150), efficiency=2.775 (SD 0.130), dependability=2.300 (SD 0.080), stimulation=1.950 (SD 0.120), and novelty=1.625 (SD 0.090). All dimensions were thus classed as excellent against the benchmarks, confirming the results from the SUS questionnaire. For the AIMS app, the mean SUS score obtained was 74.41 (SD 10.26), with a median of 77.50, which would be considered good according to the aforementioned adjective rating scale. For the UEQ, the means of the normalized scores were as follows: attractiveness=2.733 (SD 0.070), perspicuity=2.900 (SD 0.060), efficiency=2.800 (SD 0.090), dependability=2.425 (SD 0.060), stimulation=2.200 (SD 0.010), and novelty=1.450 (0.260). All dimensions were thus classed as excellent against the benchmarks (with the exception of novelty, which was classed as good), providing slightly better results than the SUS questionnaire. CONCLUSIONS: The study has shown that both the AIMS clinical portal and the AIMS app have good to excellent usability scores, and the platform provides a solid foundation for the next phase of research, which will involve evaluating the effectiveness of the platform in improving patient outcomes after total knee replacement, total hip replacement, or dynamic hip screw.

The characteristics of counterattacks in the Spanish league (La Liga) in 2021-2022.

BACKGROUND: Current studies on counterattacks are limited despite the fact that it is one of the most effective ways to score in soccer. The aim of the study was to investigate the characteristics of counterattacks in the Spanish league (La Liga) for the year 2021-2022. METHODS: The sample used in the study were 2631 counterattacks collected from a total of 380 games. The variables used were: 1) the space which the counterattack had started; 2) the number of players who participated; 3) the number of defending players; 4) the duration of the counterattack; 5) the touches to complete the counterattack; 6) the formation of both teams; 7) the way that counterattack was completed (final action); 8) the minute that the counterattack took place; and 9) which team had a home advantage. The results were analyzed using the Chi-square test. The level of significance was defined at P<0.05. RESULTS: Firstly, it appeared that most counterattacks began from the axis of the defensive zone lasting 6-15 seconds. There were 3-4 attackers participating against 4-6 defenders, using 5-9 touches. In addition, most of them took place in the 46-60 minute of the game and were completed in a shot. CONCLUSIONS: The study can help coaches and analysts to understand the characteristics of the counterattacks and adapt their training in a way to make their teams more effective, both in the attacking and defending phase.

Coronary microvascular dysfunction is associated with impaired cognitive function: the Cerebral-Coronary Connection study (C3 study).

BACKGROUND: It remains unknown whether the presence of coronary microcirculatory dysfunction (CMD) correlates with its equivalent condition in the brain, cerebral small vessel disease (CSVD). The cerebral-coronary connection (C3), a prospective blinded study, investigated the prevalence of CMD in patients with coronary artery disease (CAD) and its association with CSVD and cognitive function. METHODS AND RESULTS: Patients with documented CAD fulfilling inclusion criteria underwent physiological assessment of epicardial vessels and the microcirculation using intracoronary pressure and Doppler. Coronary microcirculation-related indices included coronary flow reserve (CFR) and hyperaemic microvascular resistance. Brain magnetic resonance imaging, transcranial Doppler (TCD), and neurocognitive examination were performed. Overall, 67 patients were included in the study (mean age 66 years, 73% female). Patients with abnormal CFR (<2.0) (55.2%) showed higher burden of white-matter hyperintensities: 43.2 vs. 20.0% (P = 0.044). After statistical adjustment, low CFR was associated with lower grey matter volume (P = 0.024) and with parameters of white-matter microstructural damage in diffusion-tensor imaging (lower fractional anisotropy and higher mean diffusivity, P = 0.029 and P = 0.032, respectively). Low CFR was associated with higher resistive (P = 0.027) and pulsatility (P = 0.043) values on TCD, and worse neurocognitive test scores (lower mini mental state examination, P = 0.025, and slower Trail Making Test A, P = 0.034). CONCLUSIONS: Coronary microcirculatory dysfunction is frequent in patients with CAD and correlates with CSVD, abnormal cerebral flow haemodynamics, and significant cognitive impairment. These findings support the hypothesis that microvascular dysfunction in the heart and the brain are part of a single pathological process affecting microcirculation in patients with CAD. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT04131075.

A multisource database tracking the impact of the COVID-19 pandemic on the communities of Boston, MA, USA.

A pandemic, like other disasters, changes how systems work. In order to support research on how the COVID-19 pandemic impacted the dynamics of a single metropolitan area and the communities therein, we developed and made publicly available a "data-support system" for the city of Boston. We actively gathered data from multiple administrative (e.g., 911 and 311 dispatches, building permits) and internet sources (e.g., Yelp, Craigslist), capturing aspects of housing and land use, crime and disorder, and commercial activity and institutions. All the data were linked spatially through BARI's Geographical Infrastructure, enabling conjoint analysis. We curated the base records and aggregated them to construct ecometric measures (i.e., descriptors of a place) at various geographic scales, all of which were also published as part of the database. The datasets were published in an open repository, each accompanied by a detailed documentation of methods and variables. We anticipate updating the database annually to maintain the tracking of the records and associated measures.

Among classic myeloproliferative neoplasms, essential thrombocythemia is associated with the greatest risk of venous thromboembolism during COVID-19.

In a multicenter European retrospective study including 162 patients with COVID-19 occurring in essential thrombocythemia (ET, n = 48), polycythemia vera (PV, n = 42), myelofibrosis (MF, n = 56), and prefibrotic myelofibrosis (pre-PMF, n = 16), 15 major thromboses (3 arterial and 12 venous) were registered in 14 patients, of whom all, but one, were receiving LMW-heparin prophylaxis. After adjustment for the competing risk of death, the cumulative incidence of arterial and venous thromboembolic events (VTE) reached 8.5% after 60 days follow-up. Of note, 8 of 12 VTE were seen in ET. Interestingly, at COVID-19 diagnosis, MPN patients had significantly lower platelet count (p < 0.0001) than in the pre-COVID last follow-up.This decline was remarkably higher in ET (-23.3%, p < 0.0001) than in PV (-16.4%, p = 0.1730) and was associated with higher mortality rate (p = 0.0010) for pneumonia. The effects of possible predictors of thrombosis, selected from those clinically relevant and statistically significant in univariate analysis, were examined in a multivariate model. Independent risk factors were transfer to ICU (SHR = 3.73, p = 0.029), neutrophil/lymphocyte ratio (SHR = 1.1, p = 0.001) and ET phenotype (SHR = 4.37, p = 0.006). The enhanced susceptibility to ET-associated VTE and the associated higher mortality for pneumonia may recognize a common biological plausibility and deserve to be delved to tailor new antithrombotic regimens including antiplatelet drugs.

Platelet releasate normalises the compromised muscle regeneration in a mouse model of hyperlipidaemia.

NEW FINDINGS: What is the central question of this study? What is the impact of obesity-independent hyperlipidaemia on skeletal muscle stem cell function of ApoE-deficient (ApoE-/- ) mice? What is the main finding and its importance? Compromised muscle stem cell function accounts for the impaired muscle regeneration in hyperlipidaemic ApoE-/- mice. Importantly, impaired muscle regeneration is normalised by administration of platelet releasate. ABSTRACT: Muscle satellite cells are important stem cells for skeletal muscle regeneration and repair after injury. ApoE-deficient mice, an established mouse model of hyperlipidaemia and atherosclerosis, show evidence of oxidative stress-induced lesions and fat infiltration in skeletal muscle followed by impaired repair after injury. However, the mechanisms underpinning attenuated muscle regeneration remain to be fully defined. Key to addressing the latter is to understand the properties of muscle stem cells from ApoE-deficient mice and their myogenic potential. Muscle stem cells from ApoE-deficient mice were cultured both ex vivo (on single fibres) and in vitro (primary myoblasts) and their myogenic capacity was determined. Skeletal muscle regeneration was studied on days 5 and 10 after cardiotoxin injury. ApoE-deficient muscle stem cells showed delayed activation and differentiation on single muscle fibres ex vivo. Impaired proliferation and differentiation profiles were also evident on isolated primary muscle stem cells in culture. ApoE-deficient mice displayed impaired skeletal muscle regeneration after acute injury in vivo. Administration of platelet releasate in ApoE-deficient mice reversed the deficits of muscle regeneration after acute injury to wild-type levels. These findings indicate that muscle stem cell myogenic potential is perturbed in skeletal muscle of a mouse model of hyperlipidaemia. We propose that platelet releasate could be a therapeutic intervention for conditions with associated myopathy such as peripheral arterial disease.

High mortality rate in COVID-19 patients with myeloproliferative neoplasms after abrupt withdrawal of ruxolitinib.

We report the clinical presentation and risk factors for survival in 175 patients with myeloproliferative neoplasms (MPN) and COVID-19, diagnosed between February and June 2020. After a median follow-up of 50 days, mortality was higher than in the general population and reached 48% in myelofibrosis (MF). Univariate analysis, showed a significant relationship between death and age, male gender, decreased lymphocyte counts, need for respiratory support, comorbidities and diagnosis of MF, while no association with essential thrombocythemia (ET), polycythemia vera (PV), and prefibrotic-PMF (pre-PMF) was found. Regarding MPN-directed therapy ongoing at the time of COVID-19 diagnosis, Ruxolitinib (Ruxo) was significantly more frequent in patients who died in comparison with survivors (p = 0.006). Conversely, multivariable analysis found no effect of Ruxo alone on mortality, but highlighted an increased risk of death in the 11 out of 45 patients who discontinued treatment. These findings were also confirmed in a propensity score matching analysis. In conclusion, we found a high risk of mortality during COVID-19 infection among MPN patients, especially in MF patients and/or discontinuing Ruxo at COVID-19 diagnosis. These findings call for deeper investigation on the role of Ruxo treatment and its interruption, in affecting mortality in MPN patients with COVID-19.

Mild dyserythropoiesis and ß-like globin gene expression imbalance due to the loss of histone chaperone ASF1B.

The expression of the human ß-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to ß). The γ- to ß-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (ß-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to ß-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to ß-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching.

A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology.

Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to Mirasol PRT or PSL, and proves to be a methodology suitable to phenotype platelets in an unbiased manner, in various physiological contexts.

Platelet releasate promotes skeletal myogenesis by increasing muscle stem cell commitment to differentiation and accelerates muscle regeneration following acute injury.

AIM: The use of platelets as biomaterials has gained intense research interest. However, the mechanisms regarding platelet-mediated skeletal myogenesis remain to be established. The aim of this study was to determine the role of platelet releasate in skeletal myogenesis and muscle stem cell fate in vitro and ex vivo respectively. METHODS: We analysed the effect of platelet releasate on proliferation and differentiation of C2C12 myoblasts by means of cell proliferation assays, immunohistochemistry, gene expression and cell bioenergetics. We expanded in vitro findings on single muscle fibres by determining the effect of platelet releasate on murine skeletal muscle stem cells using protein expression profiles for key myogenic regulatory factors. RESULTS: TRAP6 and collagen used for releasate preparation had a more pronounced effect on myoblast proliferation vs thrombin and sonicated platelets (P < 0.05). In addition, platelet concentration positively correlated with myoblast proliferation. Platelet releasate increased myoblast and muscle stem cell proliferation in a dose-dependent manner, which was mitigated by VEGFR and PDGFR inhibition. Inhibition of VEGFR and PDGFR ablated MyoD expression on proliferating muscle stem cells, compromising their commitment to differentiation in muscle fibres (P < 0.001). Platelet releasate was detrimental to myoblast fusion and affected differentiation of myoblasts in a temporal manner. Most importantly, we show that platelet releasate promotes skeletal myogenesis through the PDGF/VEGF-Cyclin D1-MyoD-Scrib-Myogenin axis and accelerates skeletal muscle regeneration after acute injury. CONCLUSION: This study provides novel mechanistic insights on the role of platelet releasate in skeletal myogenesis and set the physiological basis for exploiting platelets as biomaterials in regenerative medicine.

Identification of underlying and transfusion-related platelet qualitative alterations in the hemato-oncologic patient.

Hemato-oncologic patients with chemotherapy-induced thrombocytopenia are one of the populations receiving platelet transfusions. The general practice with these patients is to give prophylactic platelet transfusions when platelet counts fall below 10×109PLT/L. However, in more than 40% of these patients, platelet transfusion does not prevent bleeding. The reason of the low efficacy of platelet transfusion in the context of chemotherapy patients is not entirely understood. We therefore aimed at immunophenotyping the expression of platelet surface and activation markers and thrombopoietin levels from hemato-oncologic patients before and after transfusion. A more detailed follow-up was performed in three patients that underwent autologous bone marrow transplantation. As previously reported, basal platelet activation was observed in hemato-oncologic patients. Based on flow cytometry parameters, i.e. the percentage of positivity and mean fluorescence intensity (MFI) distribution, our data provide an additional interpretation of platelet acquired qualitative changes in the hemato-oncologic patient. From our results we propose: first, the underlying activation of platelets in the hemato-oncologic patient is accompanied by loss of expression of the platelet receptors that are susceptible to protease-mediated shedding; second, soon after transfusion, the newly circulating donor platelets show additional activation, which may result in subsequent platelet receptor recycling and potential accelerated clearance of these activated platelets. In conclusion, the immunophenotype of circulating platelets changes after prophylactic platelet transfusion. Next to platelet count increment, exploration of this immunophenotype might help to explain transfusion refractory bleeding in hemato-oncologic patients. Eventually this may lead to personalization and improvement of the present platelet transfusion support regime.

Calreticulin in Essential Thrombocythemia: StressINg OUT the Megakaryocyte Nucleus.

Calreticulin (CALR) is a multifaceted protein primarily involved in intracellular protein control processes. The identification of CALR mutations in essential thrombocythemia (ET) and primary myelofibrosis that are mutually exclusive with the JAK2 V617F mutation has stirred an intensive research interest about the molecular functions of CALR and its mutants in myeloproliferative neoplasms (MPNs) and its diagnostic/prognostic value. The recently characterized protein-protein interaction of CALR mutants and MPL receptor has advanced our knowledge on the functional role of CALR mutants in thrombocythemia but it has also uncovered limitations of the current established research models. Human cell lines and mouse models provide useful information but they lack the advantages provided by ex vivo primary cultures of physiologically relevant to the disease cell types [i.e., megakaryocytes (MKs), platelets]. The results from gene expression and chromatin occupancy analysis have focused on the JAK-STAT pathway activated in both JAK2 V617F- and CALR-mutated MPN patient groups, although a more complete analysis is needed to be performed in MKs. Stress related processes seem to be affected in CALR mutant ET-MKs, but the precise mechanism is not known yet. Herein, we describe a culture method for human MKs from peripheral blood progenitors, which could help further toward an unbiased characterization of the role of CALR in ET and MK differentiation.

Repercussion of Megakaryocyte-Specific Gata1 Loss on Megakaryopoiesis and the Hematopoietic Precursor Compartment.

During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cKOMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches.

Analysis of phenotype and outcome in essential thrombocythemia with CALR or JAK2 mutations.

The JAK2 V617F mutation, the thrombopoietin receptor MPL W515K/L mutation and calreticulin (CALR) mutations are mutually exclusive in essential thrombocythemia and support a novel molecular categorization of essential thrombocythemia. CALR mutations account for approximately 30% of cases of essential thrombocythemia. In a retrospective study, we examined the frequency of MPL and CALR mutations in JAK2 V617F-negative cases of essential thrombocythemia (n=103). In addition, we compared the clinical phenotype and outcome of CALR mutant cases of essential thrombocythemia with a cohort of JAK2 V617F-positive essential thrombocythemia (n=57). CALR-positive cases represented 63.7% of double-negative cases of essential thrombocythemia, and most carried CALR type 1 or type 2 indels. However, we also identified one patient who was positive for both the JAK2 V617F and the CALR mutations. This study revealed that CALR mutant essential thrombocythemia is associated with younger age, higher platelet counts, lower erythrocyte counts, leukocyte counts, hemoglobin, and hematocrit, and increased risk of progression to myelofibrosis in comparison with JAK2 V617F-positive essential thrombocythemia. Analysis of the CALR mutant group according to indel type showed that CALR type 1 deletion is strongly associated with male gender. CALR mutant patients had a better overall survival than JAK2 V617F-positive patients, in particular patients of age 60 years or younger. In conclusion, this study in a Belgian cohort of patients supports and extends the growing body of evidence that CALR mutant cases of essential thrombocythemia are phenotypically distinct from JAK2 V617F-positive cases, with regards to clinical and hematologic presentation as well as overall survival.

TAF10 Interacts with the GATA1 Transcription Factor and Controls Mouse Erythropoiesis.

The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cells in vivo, we observed a differentiation block accompanied by deregulated GATA1 target genes, including Gata1 itself, suggesting functional cross talk between GATA1 and TAF10. Additionally, we analyzed by mass spectrometry the composition of TFIID and SAGA complexes in mouse and human cells and found that their global integrity is maintained, with minor changes, during erythroid cell differentiation and development. In agreement with our functional data, we show that TAF10 interacts directly with GATA1 and that TAF10 is enriched on the GATA1 locus in human fetal erythroid cells. Thus, our findings demonstrate a cross talk between canonical TFIID and SAGA complexes and cell-specific transcription activators during development and differentiation.

Developments in FINDbase worldwide database for clinically relevant genomic variation allele frequencies.

FINDbase (http://www.findbase.org) aims to document frequencies of clinically relevant genomic variations, namely causative mutations and pharmacogenomic markers, worldwide. Each database record includes the population, ethnic group or geographical region, the disorder name and the related gene, accompanied by links to any related databases and the genetic variation together with its frequency in that population. Here, we report, in addition to the regular data content updates, significant developments in FINDbase, related to data visualization and querying, data submission, interrelation with other resources and a new module for genetic disease summaries. In particular, (i) we have developed new data visualization tools that facilitate data querying and comparison among different populations, (ii) we have generated a new FINDbase module, built around Microsoft's PivotViewer (http://www.getpivot.com) software, based on Microsoft Silverlight technology (http://www.silverlight.net), that includes 259 genetic disease summaries from five populations, systematically collected from the literature representing the documented genetic makeup of these populations and (iii) the implementation of a generic data submission tool for every module currently available in FINDbase.

The architecture of human general transcription factor TFIID core complex.

The initiation of gene transcription by RNA polymerase II is regulated by a plethora of proteins in human cells. The first general transcription factor to bind gene promoters is transcription factor IID (TFIID). TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks. TFIID is a megadalton-sized multiprotein complex composed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs). Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. Histone fold domains are prevalent in TAFs, and histone-like tetramer and octamer structures have been proposed in TFIID. A functional core-TFIID subcomplex was revealed in Drosophila nuclei, consisting of a subset of TAFs (TAF4, TAF5, TAF6, TAF9 and TAF12). These core subunits are thought to be present in two copies in holo-TFIID, in contrast to TBP and other TAFs that are present in a single copy, conveying a transition from symmetry to asymmetry in the TFIID assembly pathway. Here we present the structure of human core-TFIID determined by cryo-electron microscopy at 11.6 Å resolution. Our structure reveals a two-fold symmetric, interlaced architecture, with pronounced protrusions, that accommodates all conserved structural features of the TAFs including the histone folds. We further demonstrate that binding of one TAF8-TAF10 complex breaks the original symmetry of core-TFIID. We propose that the resulting asymmetric structure serves as a functional scaffold to nucleate holo-TFIID assembly, by accreting one copy each of the remaining TAFs and TBP.