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Dysregulation of IL17A drives numerous inflammatory and autoimmune disorders with inhibition of IL17A using antibodies proven as an effective treatment. Oral anti-IL17 therapies are an attractive alternative option, and several preclinical small molecule IL17 inhibitors have previously been described. Herein, we report the discovery of a novel class of small molecule IL17A inhibitors, identified via a DNA-encoded chemical library screen, and their subsequent optimization to provide in vivo efficacious inhibitors. These new protein-protein interaction (PPI) inhibitors bind in a previously undescribed mode in the IL17A protein with two copies binding symmetrically to the central cavities of the IL17A homodimer.
Assuntos
DNA , Descoberta de Drogas , Interleucina-17 , Bibliotecas de Moléculas Pequenas , Interleucina-17/metabolismo , Interleucina-17/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , DNA/metabolismo , DNA/química , Humanos , Animais , Relação Estrutura-Atividade , Ligação Proteica , CamundongosRESUMO
Marine transgression associated with rising sea levels causes coastal erosion, landscape transitions, and displacement of human populations globally. This process takes two general forms. Along open-ocean coasts, active transgression occurs when sediment-delivery rates are unable to keep pace with accommodation creation, leading to wave-driven erosion and/or landward translation of coastal landforms. It is highly visible, rapid, and limited to narrow portions of the coast. In contrast, passive transgression is subtler and slower, and impacts broader areas. It occurs along low-energy, inland marine margins; follows existing upland contours; and is characterized predominantly by the landward translation of coastal ecosystems. The nature and relative rates of transgression along these competing margins lead to expansion and/or contraction of the coastal zone and-particularly under the influence of anthropogenic interventions-will dictate future coastal-ecosystem response to sea-level rise, as well as attendant, often inequitable, impacts on human populations.
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Efeitos Antropogênicos , Ecossistema , Oceanos e MaresRESUMO
Landward migration of coastal ecosystems in response to sea-level rise is altering coastal carbon dynamics. Although such landscapes rapidly accumulate soil carbon, barrier-island migration jeopardizes long-term storage through burial and exposure of organic-rich backbarrier deposits along the lower beach and shoreface. Here, we quantify the carbon flux associated with the seaside erosion of backbarrier lagoon and peat deposits along the Virginia Atlantic Coast. Barrier transgression leads to the release of approximately 26.1 Gg of organic carbon annually. Recent (1994-2017 C.E.) erosion rates exceed annual soil carbon accumulation rates (1984-2020) in adjacent backbarrier ecosystems by approximately 30%. Additionally, shoreface erosion of thick lagoon sediments accounts for >80% of total carbon losses, despite containing lower carbon densities than overlying salt marsh peat. Together, these results emphasize the impermanence of carbon stored in coastal environments and suggest that existing landscape-scale carbon budgets may overstate the magnitude of the coastal carbon sink.
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Throughout the pandemic, individuals 65 years and older have contributed most COVID-19 related deaths. To best formulate effective vaccination and other prevention policies to protect older adults, large scale observational studies of these higher risk individuals are needed. We conducted a Vaccine Effectiveness (VE) study during the B.1.617.2 Delta variant phase of the pandemic in July and August 2021 in a cohort of 17 million Medicare beneficiaries of which 5.7 million were fully vaccinated. We found that individuals fully vaccinated with the Pfizer-BioNTech BNT162b2 and Moderna mRNA-1273 vaccines in January 2021 had 2.5 times higher breakthrough infections and hospitalizations than those fully vaccinated in March 2021, consistent with waning of vaccine-induced immunity. Measuring VE weekly, we found that VE against hospitalization, and even more so against infection, increased from July 2021 through August 2021, suggesting that in addition to the protective role of vaccination, increased masking or social distancing might have contributed to the unexpected increase in VE. Ongoing monitoring of Medicare beneficiaries should be a priority as new variants continue to emerge, and the VE of the new bivalent vaccines remains to be established. This could be accomplished with a large Medicare claims database and the analytics platform used for this study.
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Recommendations for prioritizing COVID-19 vaccination have focused on the elderly at higher risk for severe disease. Existing models for identifying higher-risk individuals lack the needed integration of socio-demographic and clinical risk factors. Using multivariate logistic regression and random forest modeling, we developed a predictive model of severe COVID-19 using clinical data from Medicare claims for 16 million Medicare beneficiaries and socio-economic data from the CDC Social Vulnerability Index. Predicted individual probabilities of COVID-19 hospitalization were then calculated for population risk stratification and vaccine prioritization and mapping. The leading COVID-19 hospitalization risk factors were non-white ethnicity, end-stage renal disease, advanced age, prior hospitalization, leukemia, morbid obesity, chronic kidney disease, lung cancer, chronic liver disease, pulmonary fibrosis or pulmonary hypertension, and chemotherapy. However, previously reported risk factors such as chronic obstructive pulmonary disease and diabetes conferred modest hospitalization risk. Among all social vulnerability factors, residence in a low-income zip code was the only risk factor independently predicting hospitalization. This multifactor risk model and its population risk dashboard can be used to optimize COVID-19 vaccine allocation in the higher-risk Medicare population.
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Artificial lipid bilayers have been used for several decades to study channel-forming pores and ion channels in membranes. Until recently, the classical two-chamber setups have been primarily used for studying the biophysical properties of pore forming proteins. Within the last 10 years, instruments for automated lipid bilayer measurements have been developed and are now commercially available. This chapter focuses on protein purification and reconstitution of channel-forming proteins into lipid bilayers using a classic setup and on the commercially available systems, the Orbit mini and Orbit 16.
Assuntos
Eletrofisiologia/instrumentação , Canais Iônicos/metabolismo , Bicamadas Lipídicas/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fenômenos Eletrofisiológicos , Desenho de Equipamento , Escherichia coli/genética , Expressão Gênica , Humanos , Canais Iônicos/genética , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/química , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mutação Puntual , Porinas/genética , Porinas/metabolismo , Transformação GenéticaRESUMO
The storage of organic carbon in the terrestrial biosphere directly affects atmospheric concentrations of carbon dioxide over a wide range of timescales. Within the terrestrial biosphere, the magnitude of carbon storage can vary in response to environmental perturbations such as changing temperature or hydroclimate1, potentially generating feedback on the atmospheric inventory of carbon dioxide. Although temperature controls the storage of soil organic carbon at mid and high latitudes2,3, hydroclimate may be the dominant driver of soil carbon persistence in the tropics4,5; however, the sensitivity of tropical soil carbon turnover to large-scale hydroclimate variability remains poorly understood. Here we show that changes in Indian Summer Monsoon rainfall have controlled the residence time of soil carbon in the Ganges-Brahmaputra basin over the past 18,000 years. Comparison of radiocarbon ages of bulk organic carbon and terrestrial higher-plant biomarkers with co-located palaeohydrological records6 reveals a negative relationship between monsoon rainfall and soil organic carbon stocks on a millennial timescale. Across the deglaciation period, a depletion of basin-wide soil carbon stocks was triggered by increasing rainfall and associated enhanced soil respiration rates. Our results suggest that future hydroclimate changes in tropical regions are likely to accelerate soil carbon destabilization, further increasing atmospheric carbon dioxide concentrations.
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Dióxido de Carbono/análise , Dióxido de Carbono/história , Sequestro de Carbono , Solo/química , Clima Tropical , Atmosfera/química , Ciclo do Carbono , Planeta Terra , História Antiga , Chuva , Estações do Ano , Temperatura , Fatores de TempoRESUMO
Combinatorial selective isotope labeling is a valuable tool to facilitate polypeptide backbone resonance assignment in cases of low sensitivity or extensive chemical shift degeneracy. It involves recording of 15N-HSQC and 2D HN-projections of triple-resonance spectra on a limited set of samples containing different combinations of labeled and unlabeled amino acid types. Using labeling schemes in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in 15N or 13C isotopes - individually as well as simultaneously - usually yields abundant amino-acid type information of consecutive residues i and iâ¯-â¯1. Although this results in a large number of anchor points that can be used in the sequential assignment process, for most amide signals the exact positioning of the corresponding residue the polypeptide sequence still relies on matching intra- and interresidual 13C chemical shifts obtained from 3D spectra. An obvious way to obtain more sequence-specific assignments directly with combinatorial labeling would be to increase the number of samples. This is, however, undesirable because of increased sample preparation efforts and costs. Irrespective of the number of samples, unambiguous assignments cannot be accomplished for iâ¯-â¯1/i pairs that are not unique in the sequence. Here we show that the ambiguity for non-unique pairs can be resolved by including information about the labeling state of residues iâ¯+â¯1 and iâ¯-â¯2. Application to a 35-residue peptide resulted in complete assignments of all detectable signals in the 15N HSQC which, due to its repetitive sequence and 13C chemical shift degeneracies, was difficult to achieve by other means. For a medium-sized protein (165 residues, rotational correlation time 8.2â¯ns) the improved protocol allowed the extent of backbone amide assignment to be expanded to 88% solely using a suite of 2D 1H-15N correlated spectra.
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Técnicas de Química Combinatória , Nitrogênio/química , Peptídeos/química , Amidas/química , Aminoácidos/química , Isótopos de Carbono , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Dobramento de Proteína , Espécies Reativas de NitrogênioRESUMO
The abundance of organic carbon (OC) in vegetation and soils (~2,600 PgC) compared to carbon in the atmosphere (~830 PgC) highlights the importance of terrestrial OC in global carbon budgets. The residence time of OC in continental reservoirs, which sets the rates of carbon exchange between land and atmosphere, represents a key uncertainty in global carbon cycle dynamics. Retention of terrestrial OC can also distort bulk OC- and biomarker-based paleorecords, yet continental storage timescales remain poorly quantified. Using "bomb" radiocarbon (14C) from thermonuclear weapons testing as a tracer, we model leaf-wax fatty acid and bulk OC 14C signatures in a river-proximal marine sediment core from the Bay of Bengal in order to constrain OC storage timescales within the Ganges-Brahmaputra (G-B) watershed. Our model shows that 79-83% of the leaf-waxes in this core were stored in continental reservoirs for an average of 1,000-1,200 calendar years, while the remainder was stored for an average of 15 years. This age structure distorts high-resolution organic paleorecords across geologically rapid events, highlighting that compound-specific proxy approaches must consider storage timescales. Furthermore, these results show that future environmental change could destabilize large stores of old - yet reactive - OC currently stored in tropical basins.
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Cotranslational insertion of membrane proteins into defined nanoparticle membranes has been developed as an efficient process to produce highly soluble samples in native-like environments and to study lipid-dependent effects on protein structure and function. Numerous examples of the structural and functional characterization of transporters, ion channels, or G-protein-coupled receptors in cotranslationally formed nanodisc complexes demonstrate the versatility of this approach, although the basic underlying mechanisms of membrane insertion are mainly unknown. We have revealed the first aspects of the insertion of proteins into nanodiscs by combining cell-free expression, noncovalent mass spectrometry, and NMR spectroscopy. We provide evidence of cooperative insertion of homo-oligomeric complexes and demonstrate the possibility to modulate their stoichiometry by modifying reaction conditions. Additionally, we show that significant amounts of lipid are released from the nanodiscs upon insertion of larger protein complexes.
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Lasers , Proteínas de Membrana/análise , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Building on insights gained from the discovery of the antimalarial ozonide arterolane (OZ277), we now describe the structure-activity relationship (SAR) of the antimalarial ozonide artefenomel (OZ439). Primary and secondary amino ozonides had higher metabolic stabilities than tertiary amino ozonides, consistent with their higher pKa and lower log D7.4 values. For primary amino ozonides, addition of polar functional groups decreased in vivo antimalarial efficacy. For secondary amino ozonides, additional functional groups had variable effects on metabolic stability and efficacy, but the most effective members of this series also had the highest log D7.4 values. For tertiary amino ozonides, addition of polar functional groups with H-bond donors increased metabolic stability but decreased in vivo antimalarial efficacy. Primary and tertiary amino ozonides with cycloalkyl and heterocycle substructures were superior to their acyclic counterparts. The high curative efficacy of these ozonides was most often associated with high and prolonged plasma exposure, but exposure on its own did not explain the presence or absence of either curative efficacy or in vivo toxicity.
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Adamantano/análogos & derivados , Antimaláricos/uso terapêutico , Malária/tratamento farmacológico , Peróxidos/uso terapêutico , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Adamantano/administração & dosagem , Adamantano/sangue , Adamantano/farmacologia , Adamantano/uso terapêutico , Animais , Antimaláricos/administração & dosagem , Antimaláricos/sangue , Antimaláricos/farmacologia , Feminino , Masculino , Camundongos , Peróxidos/administração & dosagem , Peróxidos/sangue , Peróxidos/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.
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Proteínas de Membrana/metabolismo , Multimerização Proteica , Espectrometria de Massas , Ligação Proteica , Dobramento de Proteína , Biologia SintéticaRESUMO
OBJECTIVES: Cortical bone drilling for preparation of screw placement is common in multiple surgical fields. The heat generated while drilling may reach thresholds high enough to cause osteonecrosis. This can compromise implant stability. Orthopaedic drill bits are several orders more expensive than their similarly sized, publicly available industrial counterparts. We hypothesize that an industrial bit will generate less heat during drilling, and the bits will not generate more heat after multiple cortical passes. METHODS: We compared 4 4.0 mm orthopaedic and 1 3.97 mm industrial drill bits. Three types of each bit were drilled into porcine femoral cortices 20 times. The temperature of the bone was measured with thermocouple transducers. The heat generated during the first 5 drill cycles for each bit was compared to the last 5 cycles. These data were analyzed with analysis of covariance. RESULTS: The industrial drill bit generated the smallest mean increase in temperature (2.8 ± 0.29°C) P < 0.0001. No significant difference was identified comparing the first 5 cortices drilled to the last 5 cortices drilled for each bit. The P-values are as follows: Bosch (P = 0.73), Emerge (P = 0.09), Smith & Nephew (P = 0.08), Stryker (P = 0.086), and Synthes (P = 0.16). The industrial bit generated less heat during drilling than its orthopaedic counterparts. The bits maintained their performance after 20 drill cycles. CONCLUSIONS: Consideration should be given by manufacturers to design differences that may contribute to a more efficient cutting bit. Further investigation into the reuse of these drill bits may be warranted, as our data suggest their efficiency is maintained after multiple uses.
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Transferência de Energia , Fêmur/fisiologia , Fêmur/cirurgia , Temperatura Alta , Equipamentos Ortopédicos , Osteotomia/instrumentação , Animais , Temperatura Corporal/fisiologia , Desenho de Equipamento , Análise de Falha de Equipamento , Fricção , Indústrias/instrumentação , SuínosRESUMO
Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial 15 N-, 13 Cα -, and 13 C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves 15 N, 1-13 C, 2-13 C, fully 15 N/13 C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide 1 H and 15 N resonances need to be obtained, even in cases where sensitivity is the limiting factor.
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Ciclofilinas/metabolismo , Aminoácidos/metabolismo , Isótopos de Carbono/química , Sistema Livre de Células , Peptidil-Prolil Isomerase F , Ciclofilinas/química , Ciclofilinas/genética , Humanos , Ligantes , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear BiomolecularRESUMO
Primary sarcomas of the thumb metacarpal are rare malignant lesions. Surgical treatment involves amputation versus tumor resection with thumb reconstruction. If complete tumor resection is possible, thumb preservation may be considered, as the thumb is vital to hand function. Following tumor resection, previous reports have described graft reconstruction with fusion to the trapezium or scaphoid. We present two cases of sarcoma necessitating resection of the thumb metacarpal that were reconstructed with an arthrodesis of the proximal phalanx to the second metacarpal shaft. Arthrodesis to the second metacarpal allows robust bony contact for fusion as well as improved resting position of the thumb. At 2- and 4-year follow-up, both patients have a stable, pain-free thumb without evidence of local recurrence.
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Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.
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Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Canais de Potássio/biossíntese , Canais de Potássio/química , Proteínas de Bactérias/genética , Sistema Livre de Células , Cristalografia por Raios X , Técnicas In Vitro , Bicamadas Lipídicas/química , Proteínas de Membrana/genética , Modelos Moleculares , Canais de Potássio/genética , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Cell-free protein production has become a core technology in the rapidly spreading field of synthetic biology. In particular the synthesis of membrane proteins, highly problematic proteins in conventional cellular production systems, is an ideal application for cell-free expression. A large variety of artificial as well as natural environments for the optimal co-translational folding and stabilization of membrane proteins can rationally be designed. The high success rate of cell-free membrane protein production allows to focus on individually selected targets and to modulate their functional and structural properties with appropriate supplements. The efficiency and robustness of lysates from Escherichia coli strains allow a wide diversity of applications and we summarize current strategies for the successful production of high quality membrane protein samples.
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Escherichia coli/genética , Proteínas de Membrana/biossíntese , Sistema Livre de Células , Proteínas de Membrana/química , Proteínas de Membrana/genética , Biossíntese de Proteínas , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The crystal structure of a nucleotide exchange factor in white blood cells reveals an autoinhibitory mechanism that reinforces the switch-like behaviour of the signalling protein Ras.
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Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , HumanosRESUMO
A platform that incorporates computational library design, parallel solution-phase synthesis, continuous flow hydrogenation, and automated high throughput purification and reformatting technologies was applied to the production of a 120-member library of 1-aryl-4-aminopiperidine analogues for drug discovery screening. The application described herein demonstrates the advantages of computational library design coupled with a flexible, modular approach to library synthesis. The enabling technologies described can be readily adopted by the traditional medicinal chemist without extensive training and lengthy process development times.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Simulação de Dinâmica Molecular , Piperidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Algoritmos , Animais , Linhagem Celular , Permeabilidade da Membrana Celular/fisiologia , Humanos , Microssomos/química , Microssomos/metabolismo , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Ratos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Solubilidade , SuínosRESUMO
We demonstrate the utility of a microfluidic platform in which water-in-oil droplet compartments serve to miniaturize cell lysate assays by a million-fold for directed enzyme evolution. Screening hydrolytic activities of a promiscuous sulfatase demonstrates that this extreme miniaturization to the single-cell level does not come at a high price in signal quality. Moreover, the quantitative readout delivers a level of precision previously limited to screening methodologies with restricted throughput. The sorting of 3 × 10(7) monodisperse droplets per round of evolution leads to the enrichment of clones with improvements in activity (6-fold) and expression (6-fold). The detection of subtle differences in a larger number of screened clones provides the combination of high sensitivity and high-throughput needed to rescue a stalled directed evolution experiment and make it viable.