RESUMO
BACKGROUND: 4-1BB (CD137) is a co-stimulatory receptor highly expressed on tumor reactive effector T cells and NK cells, which upon stimulation prolongs persistence of tumor reactive effector T and NK cells within the tumor and induces long-lived memory T cells. 4-1BB agonistic antibodies have been shown to induce strong anti-tumor effects that synergize with immune checkpoint inhibitors. The first generation of 4-1BB agonists was, however, hampered by dose-limiting toxicities resulting in suboptimal dose levels or poor agonistic activity. METHODS: ATOR-1017 (evunzekibart), a second-generation Fc-gamma receptor conditional 4-1BB agonist in IgG4 format, was designed to overcome the limitations of the first generation of 4-1BB agonists, providing strong agonistic effect while minimizing systemic immune activation and risk of hepatoxicity. The epitope of ATOR-1017 was determined by X-ray crystallography, and the functional activity was assessed in vitro and in vivo as monotherapy or in combination with anti-PD1. RESULTS: ATOR-1017 binds to a unique epitope on 4-1BB enabling ATOR-1017 to activate T cells, including cells with an exhausted phenotype, and NK cells, in a cross-linking dependent, FcγR-conditional, manner. This translated into a tumor-directed and potent anti-tumor therapeutic effect in vivo, which was further enhanced with anti-PD-1 treatment. CONCLUSIONS: These preclinical data demonstrate a strong safety profile of ATOR-1017, together with its potent therapeutic effect as monotherapy and in combination with anti-PD1, supporting further clinical development of ATOR-1017.
Assuntos
Neoplasias , Linfócitos T , Humanos , Receptores de IgG , Anticorpos Monoclonais/uso terapêutico , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , EpitoposRESUMO
Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Molécula de Adesão da Célula Epitelial/antagonistas & inibidores , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/metabolismo , Humanos , Camundongos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the de novo pyrimidine synthesis pathway, dihydroorotate dehydrogenase (DHODH), and the blockage of uridine transport into cells. These findings hold a 3-fold significance: first, we demonstrate that tenovins, and perhaps other compounds that activate p53, may activate p53 by more than one mechanism; second, that work previously conducted with certain tenovins as SirT1 inhibitors should additionally be viewed through the lens of DHODH inhibition as this is a major contributor to the mechanism of action of the most widely used tenovins; and finally, that small changes in the structure of a small molecule can lead to a dramatic change in the target profile of the molecule even when the phenotypic readout remains static.
Assuntos
Acetanilidas/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Polifarmacologia , Sirtuína 1/antagonistas & inibidores , Tioureia/análogos & derivados , Proteína Supressora de Tumor p53/metabolismo , Autofagia , Proliferação de Células , Di-Hidro-Orotato Desidrogenase , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Tioureia/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d-ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.
Assuntos
Transaminases/metabolismo , Actinobacteria/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Domínio Catalítico , Escherichia coli/metabolismo , Exophiala/enzimologia , Simulação de Acoplamento Molecular , Rhizobiaceae/enzimologia , Alinhamento de Sequência , Estereoisomerismo , Especificidade por Substrato , Transaminases/química , Transaminases/genéticaRESUMO
Over the last decade, serial crystallography, a method to collect complete diffraction datasets from a large number of microcrystals delivered and exposed to an X-ray beam in random orientations at room temperature, has been successfully implemented at X-ray free-electron lasers and synchrotron radiation facility beamlines. This development relies on a growing variety of sample presentation methods, including different fixed target supports, injection methods using gas-dynamic virtual-nozzle injectors and high-viscosity extrusion injectors, and acoustic levitation of droplets, each with unique requirements. In comparison with X-ray free-electron lasers, increased beam time availability makes synchrotron facilities very attractive to perform serial synchrotron X-ray crystallography (SSX) experiments. Within this work, the possibilities to perform SSX at BioMAX, the first macromolecular crystallography beamline at MAX IV Laboratory in Lund, Sweden, are described, together with case studies from the SSX user program: an implementation of a high-viscosity extrusion injector to perform room temperature serial crystallography at BioMAX using two solid supports - silicon nitride membranes (Silson, UK) and XtalTool (Jena Bioscience, Germany). Future perspectives for the dedicated serial crystallography beamline MicroMAX at MAX IV Laboratory, which will provide parallel and intense micrometre-sized X-ray beams, are discussed.
Assuntos
Cristalografia por Raios X/instrumentação , Síncrotrons , Desenho de Equipamento , Laboratórios , Compostos de Silício , Suécia , TemperaturaRESUMO
Peptidoglycan hydrolytic enzymes are considered to be a promising alternative to conventional antibiotics in combating bacterial infections. To identify novel hydrolytic enzymes, we performed a database search with the sequences of two thermostable endolysins with high bactericidal activity, studied earlier in our laboratory. Both these enzymes originate from Thermus scotoductus bacteriophages MAT2119 and vB_Tsc2631. A lytic enzyme LysC from Clostridium intestinale URNW was found to have the highest amino acid sequence similarity to the bacteriophage proteins and was chosen for further analysis. The recombinant enzyme showed strong activity against its host bacteria C. intestinale, as well as against C. sporogenes, Bacillus cereus, Micrococcus luteus, and Staphylococcus aureus, on average causing a 5.12 ± 0.14 log reduction of viable S. aureus ATCC 25923 cells in a bactericidal assay. Crystallographic studies of the protein showed that the catalytic site of LysC contained a zinc atom coordinated by amino acid residues His50, His147, and Cys155, a feature characteristic for type 2 amidases. Surprisingly, neither of these residues, nor any other of the four conserved residues in the vicinity of the active site, His51, Thr52, Tyr76, and Thr153, were essential to maintain the antibacterial activity of LysC. Therefore, our attention was attracted to the intrinsically disordered and highly positively charged N-terminal region of the enzyme. Potential antibacterial activity of this part of the sequence, predicted by the Antimicrobial Sequence Scanning System, AMPA, was confirmed in our experimental studies; the truncated version of LysC (LysCΔ2-23) completely lacked antibacterial activity. Moreover, a synthetic peptide, which we termed Intestinalin, with a sequence identical to the first thirty amino acids of LysC, displayed substantial anti-staphylococcal activity with IC50 of 6 µg/mL (1.5 µM). This peptide was shown to have α-helical conformation in solution in the presence of detergents which is a common feature of amphipathic α-helical antimicrobial peptides.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Clostridium/enzimologia , Endopeptidases/isolamento & purificação , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Bacteriófagos/enzimologia , Domínio Catalítico , Simulação por Computador , Sequência Conservada , Cristalografia por Raios X , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/efeitos dos fármacos , Proteínas Virais/químicaRESUMO
The galectins are a family of galactose-binding proteins playing key roles in inflammatory processes and cancer. However, they are structurally very closely related, and discovery of highly selective inhibitors is challenging. In this work, we report the design of novel inhibitors binding to a subsite unique to galectin-3, which confers both high selectivity and affinity towards galectin-3. Olefin cross metathesis between allyl ß-C-galactopyranosyl and 1-vinylnaphthalenes or acylation of aminomethyl ß-C-galactopyranosyl with 1-naphthoic acid derivatives gave C-galactopyranosyls carrying 1-naphthamide structural elements that interacted favorably with a galectin-3 unique subsite according to molecular modeling and X-ray structural analysis of two inhibitor-galectin-3 complexes. Affinities were down to sub-µM and selectivities over galectin-1, 2, 4 N-terminal domain, 4 C-terminal domain, 7, 8 N-terminal domain, 9 N-terminal domain, and 9 C-terminal domain were high. These results show that high affinity and selectivity for a single galectin can be achieved by targeting unique subsites, which holds promise for further development of small and selective galectin inhibitors.
Assuntos
Galactose , Galectina 3/química , Acilação , Proteínas Sanguíneas , Cristalografia por Raios X , Galactose/análogos & derivados , Galactose/síntese química , Galactose/química , Galectinas , Humanos , Domínios ProteicosRESUMO
We report the synthesis of linear and branched (1â4)-d-galactans. Four tetrasaccharides and one pentasaccharide were accessed by adopting a procedure of regioselective ring opening of a 4,6-O-naphthylidene protecting group followed by glycosylation using phenyl thioglycoside donors. The binding of the linear pentasaccharide with galectin-3 is also investigated by the determination of a co-crystal structure. The binding of the (1â4)-linked galactan to Gal-3 highlights the oligosaccharides of pectic galactan, which is abundant in the human diet, as putative Gal-3 ligands.
RESUMO
The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30-40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Šresolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.
Assuntos
Proteínas de Bactérias/química , Antígeno CD11b/química , Proteínas Hemolisinas/química , Leucocidinas/química , Staphylococcus aureus/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Antígeno CD11b/metabolismo , Cristalografia por Raios X , Células HL-60 , Proteínas Hemolisinas/metabolismo , Humanos , Leucocidinas/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/metabolismo , Relação Estrutura-AtividadeRESUMO
Carbohydrate-binding modules (CBMs) are key components of many carbohydrate-modifying enzymes. CBMs affect the activity of these enzymes by modulating bonding and catalysis. To further characterize and study CBM-ligand binding interactions, neutron crystallographic studies of an engineered family 4-type CBM in complex with a branched xyloglucan ligand were conducted. The first neutron crystal structure of a CBM-ligand complex reported here shows numerous atomic details of hydrogen bonding and water-mediated interactions and reveals the charged state of key binding cleft amino acid side chains.
Assuntos
Metabolismo dos Carboidratos , Carboidratos/química , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Enzimas/química , Enzimas/metabolismo , Glucanos/química , Glucanos/metabolismo , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Nêutrons , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Rhodothermus/enzimologia , Água , Xilanos/química , Xilanos/metabolismoRESUMO
This article presents results from an electronic survey completed by intermittent catheterization users who were registered members of a global web-based database. The results provide data on complication rates, health care utilization, and benefits from using hydrophilic-coated urinary catheters isotonic to urine.
Assuntos
Aceitação pelo Paciente de Cuidados de Saúde , Cateterismo Urinário/efeitos adversos , Cateteres Urinários/efeitos adversos , Desenho de Equipamento , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
Engineering of novel carbohydrate-binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein-carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi-specific carbohydrate-binding module (CBM) X-2 L110F bound to a branched oligomer of xyloglucan (XXXG). X-2 L110F is an engineered CBM that can recognize xyloglucan, xylans and ß-glucans. The structural observations of the present study compared with previously reported structures of X-2 L110F in complex with linear oligomers, show that the π-surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X-2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH-π interactions can influence binding specificity and that flexibility is a key feature for the multi-specificity displayed by X-2 L110F, allowing for the accommodation of branched ligands.
Assuntos
Glucanos/química , Lectinas/química , Modelos Moleculares , Oligossacarídeos de Cadeias Ramificadas/química , Xilanos/química , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Configuração de Carboidratos , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Glucanos/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Ligação de Hidrogênio , Lectinas/genética , Lectinas/metabolismo , Ligantes , Mutação , Oligossacarídeos de Cadeias Ramificadas/metabolismo , Fenilalanina/química , Conformação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rhodothermus/enzimologia , Xilanos/metabolismoRESUMO
Galectin-8 contains two different carbohydrate recognition domains (CRDs). Selective inhibitors for at least one CRD are desirable for galectin-8 biology studies and potentially for pharmacological purposes. Structure-guided design led to the discovery of potent and selective glycomimetic-heterocycle hybrid ligands, with a 4-(p-bromophenyl)phthalazinone derivative displaying a 34 µM K d for galectin-8N (N-terminal CRD), no binding to galectin-8C (C-terminal CRD), -1, -3, -4N, -7, -9C, or -9N, and >40-fold selectivity over galectin-4C. Selectivity was achieved with the halogenated 4-phenylphthalazinone moiety occupying a galectin-8N-specific sub-pocket. A 1.30 Å resolution X-ray structure revealed the phthalazinone moiety stacking with Arg45 and the 4-bromophenyl moiety stacking both Arg59 and Tyr141 of galectin-8N. Physicochemical and in vitro ADME studies revealed a desirable LogD, which also translated to good passive permeability. The chemical, microsome, and plasma stability support these compounds as promising tool compounds and candidates for hit-to-lead optimization.
RESUMO
We have previously described a new series of selective and orally available galectin-1 inhibitors resulting in the thiazole-containing glycomimetic GB1490. Here, we show that the introduction of polar substituents to the thiazole ring results in galectin-1-specific compounds with low nM affinities. X-ray structural analysis of a new ligand-galectin-1 complex shows changes in the binding mode and ligand-protein hydrogen bond interactions compared to the GB1490-galectin-1 complex. These new high affinity ligands were further optimized with respect to affinity and ADME properties resulting in the galectin-1-selective GB1908 (Kd galectin-1/3 0.057/6.0 µM). In vitro GB1908 inhibited galectin-1-induced apoptosis in Jurkat cells (IC50 = 850 nM). Pharmacokinetic experiments in mice revealed that a dose of 30 mg/kg b.i.d. results in free levels of GB1908 in plasma over galectin-1 Kd for 24 h. GB1908 dosed with this regimen reduced the growth of primary lung tumor LL/2 in a syngeneic mouse model.
Assuntos
Antineoplásicos , Galectina 1 , Neoplasias Pulmonares , Galectina 1/antagonistas & inibidores , Galectina 1/metabolismo , Humanos , Animais , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Administração Oral , Apoptose/efeitos dos fármacos , Relação Estrutura-Atividade , Células Jurkat , Descoberta de Drogas , Cristalografia por Raios X , Tiazóis/farmacocinética , Tiazóis/farmacologia , Tiazóis/químicaRESUMO
De novo drug design aims to generate molecules from scratch that possess specific chemical and pharmacological properties. We present a computational approach utilizing interactome-based deep learning for ligand- and structure-based generation of drug-like molecules. This method capitalizes on the unique strengths of both graph neural networks and chemical language models, offering an alternative to the need for application-specific reinforcement, transfer, or few-shot learning. It enables the "zero-shot" construction of compound libraries tailored to possess specific bioactivity, synthesizability, and structural novelty. In order to proactively evaluate the deep interactome learning framework for protein structure-based drug design, potential new ligands targeting the binding site of the human peroxisome proliferator-activated receptor (PPAR) subtype gamma are generated. The top-ranking designs are chemically synthesized and computationally, biophysically, and biochemically characterized. Potent PPAR partial agonists are identified, demonstrating favorable activity and the desired selectivity profiles for both nuclear receptors and off-target interactions. Crystal structure determination of the ligand-receptor complex confirms the anticipated binding mode. This successful outcome positively advocates interactome-based de novo design for application in bioorganic and medicinal chemistry, enabling the creation of innovative bioactive molecules.
Assuntos
Aprendizado Profundo , Desenho de Fármacos , PPAR gama , Humanos , Ligantes , PPAR gama/metabolismo , PPAR gama/agonistas , PPAR gama/química , Sítios de Ligação , Ligação ProteicaRESUMO
Glypicans are a family of cell-surface proteoglycans that regulate Wnt, hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling. Loss-of-function mutations in glypican core proteins and in glycosaminoglycan-synthesizing enzymes have revealed that glypican core proteins and their glycosaminoglycan chains are important in shaping animal development. Glypican core proteins consist of a stable α-helical domain containing 14 conserved Cys residues followed by a glycosaminoglycan attachment domain that becomes exclusively substituted with heparan sulfate (HS) and presumably adopts a random coil conformation. Removal of the α-helical domain results in almost exclusive addition of the glycosaminoglycan chondroitin sulfate, suggesting that factors in the α-helical domain promote assembly of HS. Glypican-1 is involved in brain development and is one of six members of the vertebrate family of glypicans. We expressed and crystallized N-glycosylated human glypican-1 lacking HS and N-glycosylated glypican-1 lacking the HS attachment domain. The crystal structure of glypican-1 was solved using crystals of selenomethionine-labeled glypican-1 core protein lacking the HS domain. No additional electron density was observed for crystals of glypican-1 containing the HS attachment domain, and CD spectra of the two protein species were highly similar. The crystal structure of N-glycosylated human glypican-1 core protein at 2.5 Å, the first crystal structure of a vertebrate glypican, reveals the complete disulfide bond arrangement of the conserved Cys residues, and it also extends the structural knowledge of glypicans for one α-helix and two long loops. Importantly, the loops are evolutionarily conserved in vertebrate glypican-1, and one of them is involved in glycosaminoglycan class determination.
Assuntos
Cristalografia por Raios X/métodos , Glipicanas/química , Animais , Membrana Celular/metabolismo , Dicroísmo Circular , Clonagem Molecular , Sequência Conservada , Dissulfetos/química , Glicoproteínas/química , Glicosilação , Células HEK293 , Heparitina Sulfato/química , Humanos , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteoglicanas/químicaRESUMO
Thylakoids are chlorophyll-containing membranes in chloroplasts that have been isolated from green leaves. It has been previously shown that thylakoids supplemented with a high-fat meal can affect cholecystokinin (CCK), ghrelin, insulin and blood lipids in humans, and can act to suppress food intake and prevent body weight gain in rodents. This study investigates the addition of thylakoids to a high carbohydrate meal and its effects upon hunger motivation and fullness, and the levels of glucose, insulin, CCK, ghrelin and tumour necrosis factor (TNF)-alpha in overweight women. Twenty moderately overweight female subjects received test meals on three different occasions; two thylakoid enriched and one control, separated by 1 week. The test meals consisted of a high carbohydrate Swedish breakfast, with or without addition of thylakoids. Blood samples and VAS-questionnaires were evaluated over a 4-h period. Addition of thylakoids suppressed hunger motivation and increased secretion of CCK from 180 min, and prevented postprandial hypoglycaemia from 90 min following food intake. These effects indicate that thylakoids may intensify signals of satiety. This study therefore suggests that the dietary addition of thylakoids could aid efforts to reduce food intake and prevent compensational eating later in the day, which may help to reduce body weight over time.
Assuntos
Colecistocinina/sangue , Carboidratos da Dieta/administração & dosagem , Fome/efeitos dos fármacos , Hipoglicemia/prevenção & controle , Sobrepeso/sangue , Tilacoides , Adulto , Idoso , Glicemia/efeitos dos fármacos , Colecistocinina/efeitos dos fármacos , Dieta/métodos , Carboidratos da Dieta/sangue , Suplementos Nutricionais , Feminino , Grelina/sangue , Grelina/efeitos dos fármacos , Humanos , Fome/fisiologia , Hipoglicemia/sangue , Hipoglicemia/complicações , Insulina/sangue , Pessoa de Meia-Idade , Sobrepeso/complicações , Período Pós-Prandial/efeitos dos fármacos , Período Pós-Prandial/fisiologia , Saciação/efeitos dos fármacos , Saciação/fisiologia , Resposta de Saciedade/efeitos dos fármacos , Resposta de Saciedade/fisiologia , Método Simples-Cego , Inquéritos e Questionários , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
Climate change is increasing the risk for extreme weather events such as heatwaves, including in northern countries like Sweden, which until recent years has had limited experiences of coping with extreme heat. Based on predictions that Sweden will be more frequently exposed to heatwaves in the future, it is imperative to increase the societal resilience and adaptation measures. This paper presents a qualitative interview study involving 19 participants and their experiences of caring for vulnerable people during the heatwave in 2018. The participants represent four different organizations (working directly or indirectly with vulnerable people) in two municipalities in Sweden, including preschools, homes for the elderly, homecare services, and care homes for people with functional impairments, which were all impacted during the heatwave. This study contributes new empirical insights about the heatwave in 2018 and, in particular, similarities and differences in both experiences and adaptation measures across the four organizations. The findings show how both staff and vulnerable people suffered from the consequences of heat which increased vulnerability, how some organizations lacked enough (qualified) staff to secure routines, and that few evaluations and formal changes were done after the heatwave.
Assuntos
Mudança Climática , Temperatura Alta , Pré-Escolar , Humanos , Idoso , Suécia , CidadesRESUMO
Purpose: To explore the clinical practice development of different surgical techniques when installing bone-anchored hearing implants and their associated trends in outcomes. Design: Retrospective study of 228 bone-anchored hearing implants in 200 patients, performed over a 10-year period between 2012 and 2022 in a referral hospital. Method: Real-world data of demography, etiology, surgical setup, complications, and audiological outcomes were collected. Eligibility criteria from clinical practice were applied. Results: The minimally invasive technique is associated with shorter surgery duration, 20 vs. 44â min as compared to a linear incision technique. The minimally invasive technique was also associated with a lower occurrence of complications when compared to linear incision techniques (intraoperative; 1.8% vs. 4.9%, postoperative; 49% vs. 66%). Most differences were seen in complications relating to skin and wound healing. Conclusion: Adoption of a minimally invasive surgical technique for the installations of bone-anchored hearing implants can reduce surgical complexity without compromising safety aspects or clinical benefits.
RESUMO
A new series of orally available α-d-galactopyranosides with high affinity and specificity toward galectin-1 have been discovered. High affinity and specificity were achieved by changing six-membered aryl-triazolyl substituents in a series of recently published galectin-3-selective α-d-thiogalactosides (e.g., GB1107 Kd galectin-1/3 3.7/0.037 µM) for five-membered heterocycles such as thiazoles. The in vitro pharmacokinetic properties were optimized, resulting in several galectin-1 inhibitors with favorable properties. One compound, GB1490 (Kd galectin-1/3 0.4/2.7 µM), was selected for further characterization toward a panel of galectins showing a selectivity of 6- to 320-fold dependent on galectin. The X-ray structure of GB1490 bound to galectin-1 reveals the compound bound in a single conformation in the carbohydrate binding site. GB1490 was shown to reverse galectin-1-induced apoptosis of Jurkat cells at low µM concentrations. No cell cytotoxicity was observed for GB1490 up to 90 µM in the A549 cells. In pharmacokinetic studies in mice, GB1490 showed high oral bioavailability (F% > 99%).