RESUMO
BACKGROUND AND PURPOSE: In amyotrophic lateral sclerosis (ALS), there is an unmet need for more precise patient characterization through quantitative, ideally operator-independent, assessments of disease extent and severity. Radially sampled averaged magnetization inversion recovery acquisitions (rAMIRA) magnetic resonance imaging enables gray matter (GM) and white matter (WM) area quantitation in the cervical and thoracic spinal cord (SC) with optimized contrast. We aimed to investigate rAMIRA-derived SC GM and SC WM areas and their association with clinical phenotype and disability in ALS. METHODS: A total of 36 patients with ALS (mean [SD] age 61.7 [12.6] years, 14 women) and 36 healthy, age- and sex-matched controls (HCs; mean [SD] age 63.1 [12.1] years, 14 women) underwent two-dimensional axial rAMIRA imaging at the inter-vertebral disc levels C2/3-C5/C6 and the lumbar enlargement level Tmax. ALS Functional Rating Scale-revised (ALSFRS-R) score, muscle strength, and sniff nasal inspiratory pressure (SNIP) were assessed. RESULTS: Compared to HCs, GM and WM areas were reduced in patients at all cervical levels (p < 0.0001). GM area (p = 0.0001), but not WM area, was reduced at Tmax. Patients with King's Stage 3 showed significant GM atrophy at all levels, while patients with King's Stage 1 showed significant GM atrophy selectively at Tmax. SC GM area was significantly associated with muscle force at corresponding myotomes. GM area at C3/C4 was associated with ALSFRS-R (p < 0.001) and SNIP (p = 0.0016). CONCLUSION: Patients with ALS assessed by rAMIRA imaging show significant cervical and thoracic SC GM and SC WM atrophy. SC GM area correlates with muscle strength and clinical disability. GM area reduction at Tmax may be an early disease sign. Longitudinal studies are warranted.
Assuntos
Esclerose Lateral Amiotrófica , Atrofia , Substância Cinzenta , Imageamento por Ressonância Magnética , Humanos , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Esclerose Lateral Amiotrófica/complicações , Feminino , Pessoa de Meia-Idade , Masculino , Substância Cinzenta/diagnóstico por imagem , Substância Cinzenta/patologia , Idoso , Atrofia/patologia , Medula Cervical/diagnóstico por imagem , Medula Cervical/patologia , Vértebras Torácicas/diagnóstico por imagem , Medula Espinal/diagnóstico por imagem , Medula Espinal/patologia , Vértebras Cervicais/diagnóstico por imagem , Substância Branca/diagnóstico por imagem , Substância Branca/patologiaRESUMO
BACKGROUND: CYAD-01 is an autologous chimeric antigen receptor (CAR) T-cell product based on the natural killer (NK) group 2D (NKG2D) receptor, which binds eight ligands that are overexpressed in a wide range of haematological malignancies but are largely absent on non-neoplastic cells. Initial clinical evaluation of a single infusion of CYAD-01 at a low dose in patients with relapsed or refractory acute myeloid leukaemia, myelodysplastic syndromes, and multiple myeloma supported the feasibility of the approach and prompted further evaluation of CYAD-01. The aim of the present study was to determine the safety and recommended phase 2 dosing of CYAD-01 administered without preconditioning or bridging chemotherapy. METHODS: The multicentre THINK study was an open-label, dose-escalation, phase 1 study for patients with relapsed or refractory acute myeloid leukaemia, myelodysplastic syndromes, or multiple myeloma, after at least one previous line of therapy. Patients were recruited from five hospitals in the USA and Belgium. The dose-escalation segment evaluated three dose levels: 3 × 108 (dose level one), 1 × 109 (dose level two), and 3 × 109 (dose level three) cells per infusion with a 3 + 3 Fibonacci study design using a schedule of three infusions at 2-week intervals followed by potential consolidation treatment consisting of three additional infusions. The occurrence of dose-limiting toxicities post-CYAD-01 infusion was assessed as the primary endpoint in the total treated patient population. The trial was registered with ClinicalTrials.gov, NCT03018405, and EudraCT, 2016-003312-12, and has been completed. FINDINGS: Between Feb 6, 2017, and Oct 9, 2018, 25 patients were registered in the haematological dose-escalation segment. Seven patients had manufacturing failure for insufficient yield and two had screening failure. 16 patients were treated with CYAD-01 (three with multiple myeloma and three with acute myeloid leukaemia at dose level one; three with acute myeloid leukaemia at dose level two; and six with acute myeloid leukaemia and one with myelodysplastic syndromes at dose level three). Median follow-up was 118 days (IQR 46-180). Seven patients (44%) had grade 3 or 4 treatment-related adverse events. In total, five patients (31%) had grade 3 or 4 cytokine release syndrome across all dose levels. One dose-limiting toxicity of cytokine release syndrome was reported at dose level three. No treatment-related deaths occurred, and the maximum tolerated dose was not reached. Three (25%) of 12 evaluable patients with relapsed or refractory acute myeloid leukaemia or myelodysplastic syndromes had an objective response. Among responders, two patients with acute myeloid leukaemia proceeded to allogeneic haematopoietic stem-cell transplantation (HSCT) after CYAD-01 treatment, with durable ongoing remissions (5 and 61 months). INTERPRETATION: Treatment with a multiple CYAD-01 infusion schedule without preconditioning is well tolerated and shows anti-leukaemic activity, although without durability outside of patients bridged to allogeneic HSCT. These phase 1 data support the proof-of-concept of targeting NKG2D ligands by CAR T-cell therapy. Further clinical studies with NKG2D-based CAR T-cells are warranted, potentially via combinatorial antigen targeted approaches, to improve anti-tumour activity. FUNDING: Celyad Oncology.
Assuntos
Leucemia Mieloide Aguda , Mieloma Múltiplo , Síndromes Mielodisplásicas , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/uso terapêutico , Imunoterapia Adotiva , Síndrome da Liberação de Citocina , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológicoRESUMO
Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus-the conjugative type IV secretion system (T4SS)-produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament-the conjugative pilus-that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein-protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Sistemas de Secreção Tipo IV , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Conjugação Genética , DNA/genética , Evolução Molecular , Fímbrias Bacterianas/metabolismo , Plasmídeos/genética , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/metabolismo , Sistemas de Secreção Tipo IV/ultraestruturaRESUMO
BACKGROUND: The Gold Coast criteria (GCC) have been proposed as a means of selecting patients for amyotrophic lateral sclerosis (ALS) clinical trials. We aimed to characterise disease progression according to the GCC. METHODS: Data from population-based ALS registries from the Netherlands and Belgium were analysed. The GCC additionally define ALS as lower motor neuron (LMN) dysfunction in ≥2 body regions without upper motor neuron dysfunction. Therefore, the revised El Escorial criteria (rEEC) were supplemented with a 'Gold Coast ALS' category for patients with only LMN dysfunction in ≥2 body regions. We assessed survival time, ALS Functional Rating Scale (ALSFRS-R) progression rates and between-patient variability per diagnostic category. RESULTS: We included 5957 ALS patients, of whom 600 (10.1%) fulfilled the GCC but not the rEEC, and 95 (1.6%) fulfilled only the rEEC. ALSFRS-R progression rates were similar for the rEEC (0.84 points/month) and GCC (0.81 points/month) with similar variability (standard deviation of 0.59 vs. 0.60) and median survival time (17.8 vs.18.7 months). Survival time and average progression rates varied (p<0.001) between categories. Per category, however, there was considerable between-patient variability with progression rates ranging from: -2.10 to -0.14 (definite), -1.94 to -0.06 (probable), -2.10 to -0.02 (probable laboratory supported), -1.79 to -0.02 (possible) and -1.31 to 0.08 (Gold Coast). CONCLUSIONS: The GCC broaden the definition of ALS, allowing more patients to participate in trials, while minimally impacting population heterogeneity. Given the large variability per diagnostic category, selecting only specific categories for trials may not result in a more homogeneous study population.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Bélgica/epidemiologia , Progressão da Doença , Países BaixosRESUMO
Parkinson's disease (PD) is a fatal neurodegenerative disorder that is primarily caused by the degeneration and loss of dopaminergic neurons of the substantia nigra in the ventral midbrain. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset PD identified to date, with G2019S being the most frequent LRRK2 mutation, which is responsible for up to 1-2% of sporadic PD and up to 6% of familial PD cases. As no treatment is available for this devastating disease, developing new therapeutic strategies is of foremost importance. Cellular models are commonly used for testing novel potential neuroprotective compounds. However, current cellular PD models either lack physiological relevance to dopaminergic neurons or are too complex and costly for scaling up the production process and for screening purposes. In order to combine biological relevance and throughput, we have developed a PD model in Lund human mesencephalic (LUHMES) cell-derived dopaminergic neurons by overexpressing wild-type (WT) and G2019S LRRK2 proteins. We show that these cells can differentiate into dopaminergic-like neurons and that expression of mutant LRRK2 causes a range of different phenotypes, including reduced nuclear eccentricity, altered mitochondrial and lysosomal morphologies, and increased dopaminergic cell death. This model could be used to elucidate G2019S LRRK2-mediated dopaminergic neural dysfunction and to identify novel molecular targets for disease intervention. In addition, our model could be applied to high-throughput and phenotypic screenings for the identification of novel PD therapeutics.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/fisiologia , Modelos Biológicos , Doença de Parkinson/metabolismo , Códon , Neurônios Dopaminérgicos/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismoRESUMO
Over the past two decades, the El Escorial criteria (EEC) have been used as eligibility criteria in major randomized controlled trials. One of the goals of the revised EEC was to allow earlier diagnosis and, thus earlier trial inclusion by introducing a new category, namely "clinically probable laboratory supported" ALS. This category allowed EMG findings to be taken into account assuming that EMG is more sensitive than the clinical examination in detecting lower motor neuron signs. Recently, Edaravone has been licensed in several countries for the treatment of ALS based on a randomized controlled trial in a selected group of ALS patients excluding the EEC category "clinically probable laboratory supported". The major reason was that in a post hoc analysis of the first Edaravone trial this group comprised many slow progressors. As it is unclear whether this bias towardslow progressors was a study-specific problem or related to the category itself, we performed an analysis in the PRO-ACT dataset. In the PRO-ACT dataset, progression in ALS patients included at baseline into the "clinically probable laboratory supported" category was significantly slower (-0.53 in ALSFRS/month) compared to the other EEC categories (-0.68 in ALSFRS/month; p < 0.001) and exhibited a significantly longer diagnostic delay (13.5 months vs. 11.7 months, p < 0.001). This suggests that the bias toward slow progressors in the "clinically probable laboratory supported" category is an inherent problem of the category and thus does not fulfill the previous goal of earlier diagnosis, raising several questions concerning the application of this category.
Assuntos
Esclerose Lateral Amiotrófica/diagnóstico , Viés , Diagnóstico Tardio , Diagnóstico Precoce , Adulto , Progressão da Doença , Eletromiografia , Feminino , Humanos , Laboratórios , Masculino , Pessoa de Meia-IdadeRESUMO
The limited regenerative capacity of the heart after a myocardial infarct results in remodeling processes that can progress to congestive heart failure (CHF). Several strategies including mechanical stabilization of the weakened myocardium and regenerative approaches (specifically stem cell technologies) have evolved which aim to prevent CHF. However, their final performance remains limited motivating the need for an advanced strategy with enhanced efficacy and reduced deleterious effects. An epicardial carrier device enabling a targeted application of a biomaterial-based therapy to the infarcted ventricle wall could potentially overcome the therapy and application related issues. Such a device could play a synergistic role in heart regeneration, including the provision of mechanical support to the remodeling heart wall, as well as providing a suitable environment for in situ stem cell delivery potentially promoting heart regeneration. In this study, we have developed a novel, single-stage concept to support the weakened myocardial region post-MI by applying an elastic, biodegradable patch (SPREADS) via a minimal-invasive, closed chest intervention to the epicardial heart surface. We show a significant increase in %LVEF 14â¯days post-treatment when GS (clinical gold standard treatment) was compared to GSâ¯+â¯SPREADS + Gel with and without cells (pâ¯≤â¯0.001). Furthermore, we did not find a significant difference in infarct quality or blood vessel density between any of the groups which suggests that neither infarct quality nor vascularization is the mechanism of action of SPREADS. The SPREADS device could potentially be used to deliver a range of new or previously developed biomaterial hydrogels, a remarkable potential to overcome the translational hurdles associated with hydrogel delivery to the heart.
Assuntos
Implantes Absorvíveis , Terapia Baseada em Transplante de Células e Tecidos/instrumentação , Hidrogéis/administração & dosagem , Células-Tronco Mesenquimais , Infarto do Miocárdio/terapia , Tecido Adiposo/citologia , Animais , Materiais Biocompatíveis , Movimento Celular/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Desenho de Equipamento , Feminino , Humanos , Ácido Hialurônico , Hidrogéis/química , Hidrogéis/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/efeitos dos fármacos , Infarto do Miocárdio/fisiopatologia , Pericárdio , Suínos , ViscosidadeRESUMO
OBJECTIVE: There is still insufficient knowledge about natural history in adult spinal muscular atrophy, thus valid markers for treatment and disease monitoring are urgently needed. METHODS: We studied hand muscle innervation pattern of 38 adult genetically confirmed 5q spinal muscular atrophy (SMA) patients by the motor unit number index (MUNIX) method. Data were compared to healthy controls and amyotrophic lateral sclerosis (ALS) patients and systematically correlated to typical disease-relevant scores and other clinical as well as demographic characteristics. RESULTS: Denervation of hand muscles in adult SMA was not evenly distributed. By calculation of the MUNIX ratios, we identified a specific hand muscle wasting pattern for SMA which is different to the split hand in ALS. Furthermore, MUNIX parameters strongly correlated with established disease course parameters independent of disease stages. CONCLUSION: We found a pathophysiological remarkable denervation pattern of hand muscles, a 'reversed split hand'. MUNIX of single hand muscles correlated well with disease severity and thus represents an easily available biomarker for adult SMA. SIGNIFICANCE: Our data show the power of the MUNIX method as a biomarker for upcoming questions in adult SMA.
Assuntos
Mãos/inervação , Mãos/fisiologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Atrofia Muscular Espinal/fisiopatologia , Recrutamento Neurofisiológico/fisiologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Atrofia Muscular Espinal/diagnóstico , Adulto JovemRESUMO
Injectable hydrogels that aim to mechanically stabilise the weakened left ventricle wall to restore cardiac function or to deliver stem cells in cardiac regenerative therapy have shown promising data. However, the clinical translation of hydrogel-based therapies has been limited due to difficulties injecting them through catheters. We have engineered a novel catheter, Advanced Materials Catheter (AMCath), that overcomes translational hurdles associated with delivering fast-gelling covalently cross-linked hyaluronic acid hydrogels to the myocardium. We developed an experimental technique to measure the force required to inject such hydrogels and determined the mechanical/viscoelastic properties of the resulting hydrogels. The preliminary in vivo feasibility of delivering fast-gelling hydrogels through AMCath was demonstrated by accessing the porcine left ventricle and showing that the hydrogel was retained in the myocardium post-injection (three 200 µL injections delivered, 192, 204 and 183 µL measured). However, the mechanical properties of the hydrogels were reduced by passage through AMCath (≤20.62% reduction). We have also shown AMCath can be used to deliver cardiopoietic adipose-derived stem cell-loaded hydrogels without compromising the viability (80% viability) of the cells in vitro. Therefore, we show that hydrogel/catheter compatibility issues can be overcome as we have demonstrated the minimally invasive delivery of a fast-gelling covalently cross-linked hydrogel to the beating myocardium.
Assuntos
Materiais Biocompatíveis/administração & dosagem , Cateteres Cardíacos , Sistemas de Liberação de Medicamentos/instrumentação , Ácido Hialurônico/administração & dosagem , Hidrogéis/administração & dosagem , Animais , Linhagem Celular , Células Imobilizadas/citologia , Células Imobilizadas/transplante , Reagentes de Ligações Cruzadas/administração & dosagem , Desenho de Equipamento , Humanos , Injeções , Infarto do Miocárdio/terapia , Transplante de Células-Tronco , Células-Tronco/citologia , SuínosRESUMO
OBJECTIVE: Motor Unit Number Index (MUNIX) is a quantitative neurophysiological method that reflects loss of motor neurons in Amyotrophic Lateral Sclerosis (ALS) in longitudinal studies. It has been utilized in one natural history ALS study and one drug trial (Biogen USA) after training and qualification of raters. METHODS: Prior to testing patients, evaluators had to submit test-retest data of 4 healthy volunteers. Twenty-seven centres with 36 raters measured MUNIX in 4 sets of 6 different muscles twice. Coefficient of variation of all measurements had to be <20% to pass the qualification process. MUNIX COV of the first attempt, number of repeated measurements and muscle specific COV were evaluated. RESULTS: COV varied considerably between raters. Mean COV of all raters at the first measurements was 12.9%⯱â¯13.5 (median 8.7%). Need of repetitions ranged from 0 to 43 (mean 10.7⯱â¯9.1, median 8). Biceps and first dorsal interosseus muscles showed highest repetition rates. MUNIX variability correlated considerably with variability of compound muscle action potential. CONCLUSION: MUNIX revealed generally good reliability, but was rater dependent and ongoing support for raters was needed. SIGNIFICANCE: MUNIX can be implemented in large clinical trials as an outcome measure after training and a qualification process.
Assuntos
Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/fisiopatologia , Recrutamento Neurofisiológico/fisiologia , Esclerose Lateral Amiotrófica/epidemiologia , Feminino , Humanos , Estudos Longitudinais , MasculinoRESUMO
Celyad recently initiated several clinical trials with the CYAD-01 product, a natural killer group 2D (NKG2D)-based chimeric antigen receptor (CAR), in both solid and hematologic tumor types. This review discusses the unique properties of CYAD-01, expecting to provide a new paradigm to fight against solid tumors.
Assuntos
Imunoterapia Adotiva , Neoplasias/terapia , Receptores de Antígenos Quiméricos/uso terapêutico , Linfócitos T/fisiologia , Terapias em Estudo , Ensaios Clínicos como Assunto/métodos , Aprovação de Drogas , Indústria Farmacêutica , Humanos , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/tendências , Neoplasias/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Projetos de Pesquisa , Linfócitos T/transplante , Terapias em Estudo/métodos , Terapias em Estudo/tendências , Estados Unidos , United States Food and Drug AdministrationRESUMO
Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells. Given the complex choreography of the substrate through the secretion apparatus, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems.
Assuntos
Sistemas de Secreção Bacterianos , Escherichia coli/química , Escherichia coli/ultraestrutura , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Sistemas de Secreção Bacterianos/genética , Membrana Celular/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestruturaRESUMO
The small heat shock protein αB-crystallin is an oligomeric molecular chaperone that binds aggregation-prone proteins. As a component of the proteostasis system, it is associated with cataract, neurodegenerative diseases, and myopathies. The structural determinants for the regulation of its chaperone function are still largely elusive. Combining different experimental approaches, we show that phosphorylation-induced destabilization of intersubunit interactions mediated by the N-terminal domain (NTD) results in the remodeling of the oligomer ensemble with an increase in smaller, activated species, predominantly 12-mers and 6-mers. Their 3D structures determined by cryo-electron microscopy and biochemical analyses reveal that the NTD in these species gains flexibility and solvent accessibility. These modulated properties are accompanied by an increase in chaperone activity in vivo and in vitro and a more efficient cooperation with the heat shock protein 70 system in client folding. Thus, the modulation of the structural flexibility of the NTD, as described here for phosphorylation, appears to regulate the chaperone activity of αB-crystallin rendering the NTD a conformational sensor for nonnative proteins.
Assuntos
Modelos Moleculares , Chaperonas Moleculares/química , Conformação Proteica , Cadeia B de alfa-Cristalina/química , Cromatografia em Gel , Clonagem Molecular , Microscopia Crioeletrônica , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Chaperonas Moleculares/metabolismo , Fosforilação , Corantes de Rosanilina , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.
Assuntos
Membrana Celular/metabolismo , Lipídeos/química , Lipopeptídeos/química , Peptídeos Cíclicos/química , 1,2-Dipalmitoilfosfatidilcolina/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Fenômenos Fisiológicos Bacterianos , Calorimetria/métodos , Difenilexatrieno/química , Fluoresceínas/química , Lauratos/química , Luz , Bicamadas Lipídicas/química , Lipossomos/química , Micelas , Microscopia de Força Atômica/métodos , Microscopia de Fluorescência/métodos , Fosfatidilcolinas/química , Fosfolipídeos/química , Rodaminas/química , Espalhamento de Radiação , Espectrometria de Fluorescência/métodosRESUMO
Small heat shock proteins (sHsps) are molecular chaperones that prevent the aggregation of nonnative proteins. The sHsps investigated to date mostly form large, oligomeric complexes. The typical bacterial scenario seemed to be a two-component sHsps system of two homologous sHsps, such as the Escherichia coli sHsps IbpA and IbpB. With a view to expand our knowledge on bacterial sHsps, we analyzed the sHsp system of the bacterium Deinococcus radiodurans, which is resistant against various stress conditions. D. radiodurans encodes two sHsps, termed Hsp17.7 and Hsp20.2. Surprisingly, Hsp17.7 forms only chaperone active dimers, although its crystal structure reveals the typical α-crystallin fold. In contrast, Hsp20.2 is predominantly a 36mer that dissociates into smaller oligomeric assemblies that bind substrate proteins stably. Whereas Hsp20.2 cooperates with the ATP-dependent bacterial chaperones in their refolding, Hsp17.7 keeps substrates in a refolding-competent state by transient interactions. In summary, we show that these two sHsps are strikingly different in their quaternary structures and chaperone properties, defining a second type of bacterial two-component sHsp system.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , Deinococcus/genética , Deinococcus/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico Pequenas/ultraestrutura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Dobramento de Proteína , Multimerização Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Estresse FisiológicoRESUMO
OBJECTIVES: Therapy for relapsing-remitting multiple sclerosis with natalizumab (Tysabri; Biogen Idec) has been shown to be effective in the reduction of the clinical relapse rate and disability progression. However, real-life longitudinal data, including years before baseline, are rare. METHODS: An observational single-center study was carried out. We analyzed data from 64 consecutive patients with multiple sclerosis. RESULTS: After 1 year of treatment (n = 64), score on the Expanded Disability Status Scale (EDSS) decreased by 0.47 points (P = 0.047) and the annualized relapse rate (ARR) decreased by 82% (P < 0.001). After 2 years (n = 41), EDSS score was still reduced by 0.28 (not significant) and ARR was reduced by 69% (P < 0.001). After 3 years (n = 23), EDSS score was reduced by 0.26 (not significant), and ARR was reduced by 77% (P < 0.001). Reduction of EDSS score and ARR did not depend on baseline ARR (1-2 vs >2) or EDSS score and was not biased by exceptional high disease activity or relapses around baseline. CONCLUSIONS: These real-life data reinforce that natalizumab is effective over years, reduces ARR, and stabilizes EDSS score independent of baseline ARR, baseline EDSS score, or baseline treatment.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Avaliação da Deficiência , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/fisiopatologia , Adulto , Idoso , Estudos de Coortes , Bases de Dados Factuais/tendências , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/epidemiologia , Natalizumab , Suíça/epidemiologia , Resultado do Tratamento , Adulto JovemRESUMO
Hypochlorite is a powerful oxidant produced by neutrophils to kill invading microorganisms. Despite this important physiological role of HOCl in fighting bacterial infections, no hypochlorite-specific stress response has been identified yet. Here, we identified a hypochlorite-responsive transcription factor, YjiE, which is conserved in proteobacteria and eukaryotes. YjiE forms unusual dodecameric ring-like structures in vitro that undergo large DNA-induced conformational changes to form dimers and tetramers as shown by transmission electron microscopy and analytical ultracentrifugation. Such smaller oligomers are predominant in hypochlorite-stressed cells and are the active species as shown by fluorescence anisotropy and analytical ultracentrifugation. YjiE regulates a large number of genes upon hypochlorite stress. Among them are genes involved in cysteine, methionine biosynthesis, and sulfur metabolism (up-regulated) and genes involved in iron acquisition and homeostasis (down-regulated), thus supposedly replenishing oxidized metabolites and decreasing the hypochlorite-mediated amplification of intracellular reactive oxygen species. As a result, YjiE specifically confers hypochlorite resistance to E. coli cells. Thus, to our knowledge, YjiE is the first described hypochlorite-specific transcription factor.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Ácido Hipocloroso/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desinfetantes/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genéticaRESUMO
The molecular chaperone αB-crystallin, the major player in maintaining the transparency of the eye lens, prevents stress-damaged and aging lens proteins from aggregation. In nonlenticular cells, it is involved in various neurological diseases, diabetes, and cancer. Given its structural plasticity and dynamics, structure analysis of αB-crystallin presented hitherto a formidable challenge. Here we present a pseudoatomic model of a 24-meric αB-crystallin assembly obtained by a triple hybrid approach combining data from cryoelectron microscopy, NMR spectroscopy, and structural modeling. The model, confirmed by cross-linking and mass spectrometry, shows that the subunits interact within the oligomer in different, defined conformations. We further present the molecular architectures of additional well-defined αB-crystallin assemblies with larger or smaller numbers of subunits, provide the mechanism how "heterogeneity" is achieved by a small set of defined structural variations, and analyze the factors modulating the oligomer equilibrium of αB-crystallin and thus its chaperone activity.