RESUMO
Translocation into the endoplasmic reticulum (ER) is an initial and crucial biogenesis step for all secreted and endomembrane proteins in eukaryotes. ER insertion can take place through the well-characterized signal recognition particle (SRP)-dependent pathway or the less-studied route of SRP-independent translocation. To better understand the prevalence of the SRP-independent pathway, we systematically defined the translocational dependence of the yeast secretome. By combining hydropathy-based analysis and microscopy, we uncovered that a previously unappreciated fraction of the yeast secretome translocates without the aid of the SRP. Furthermore, we validated a family of SRP-independent substrates-the glycosylphosphatidylinositol (GPI)-anchored proteins. Studying this family, we identified a determinant for ER targeting and uncovered a network of cytosolic proteins that facilitate SRP-independent targeting and translocation. These findings highlight the underappreciated complexity of SRP-independent translocation, which enables this pathway to efficiently cope with its extensive substrate flux.
Assuntos
Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Redes e Vias Metabólicas , Saccharomyces cerevisiae/citologia , Partícula de Reconhecimento de Sinal/metabolismoRESUMO
Nonradiative exciton relaxation processes are critical for energy transduction and transport in optoelectronic materials, but how these processes are connected to the underlying crystal structure and the associated electron, exciton, and phonon band structures, as well as the interactions of all these particles, is challenging to understand. Here, we present a first-principles study of exciton-phonon relaxation pathways in pentacene, a paradigmatic molecular crystal and optoelectronic semiconductor. We compute the momentum- and band-resolved exciton-phonon interactions, and use them to analyze key scattering channels. We find that both exciton intraband scattering and interband scattering to parity-forbidden dark states occur on the same â¼100 fs timescale as a direct consequence of the longitudinal-transverse splitting of the bright exciton band. Consequently, exciton-phonon scattering exists as a dominant nonradiative relaxation channel in pentacene. We further show how the propagation of an exciton wave packet is connected with crystal anisotropy, which gives rise to the longitudinal-transverse exciton splitting and concomitant anisotropic exciton and phonon dispersions. Our results provide a framework for understanding the role of exciton-phonon interactions in exciton nonradiative lifetimes in molecular crystals and beyond.
RESUMO
Pentacene is an important model organic semiconductor in both the singlet exciton fission (SF) and organic electronics communities. We have investigated the effect of changing crystal structure on the SF process, generating multiple triplet excitons from an initial singlet exciton, and subsequent triplet recombination. Unlike for similar organic semiconductors that have strong SF sensitive to polymorphism, we find almost no quantitative difference between the kinetics of triplet pair (TT) formation in the two dominant polymorphs of pentacene. Both pairwise dimer coupling and momentum-space crystal models predict much faster TT formation from the bright singlet excited state of the Bulk vs ThinFilm polymorph, contrasting with the experiment. GW and Bethe-Salpeter equation calculations, including exciton-phonon coupling, reveal that ultrafast phonon-driven transitions in the ThinFilm polymorph compensate the intrinsically slower purely Coulomb-mediated TT formation channel, rationalizing the similarity in observed rates. Taking into account the influence of subtle structural distinctions on both the direct and phonon-mediated SF pathways reveals a predictive capability to these methods, expected to be applicable to a wide variety of molecular crystals.
RESUMO
Exciton dynamics, lifetimes, and scattering are directly related to the exciton dispersion or band structure. Here, we present a general theory for exciton band structure within both ab initio and model Hamiltonian approaches. We show that contrary to common assumption, the exciton band structure contains nonanalytical discontinuities-a feature which is impossible to obtain from the electronic band structure alone. These discontinuities are purely quantum phenomena, arising from the exchange scattering of electron-hole pairs. We show that the degree of these discontinuities depends on materials' symmetry and dimensionality, with jump discontinuities occurring in 3D and different orders of removable discontinuities in 2D and 1D, whose details depend on the exciton degeneracy and material thickness. We connect these features to the early stages of exciton dynamics, radiative lifetimes, and diffusion constants, in good correspondence with recent experimental observations, revealing that the discontinuities in the band structure lead to ultrafast ballistic transport and suggesting that measured exciton diffusion and dynamics are influenced by the underlying exciton dispersion.
Assuntos
Elétrons , DifusãoRESUMO
Advances in single-cell genomics enable commensurate improvements in methods for uncovering lineage relations among individual cells. Current sequencing-based methods for cell lineage analysis depend on low-resolution bulk analysis or rely on extensive single-cell sequencing, which is not scalable and could be biased by functional dependencies. Here we show an integrated biochemical-computational platform for generic single-cell lineage analysis that is retrospective, cost-effective, and scalable. It consists of a biochemical-computational pipeline that inputs individual cells, produces targeted single-cell sequencing data, and uses it to generate a lineage tree of the input cells. We validated the platform by applying it to cells sampled from an ex vivo grown tree and analyzed its feasibility landscape by computer simulations. We conclude that the platform may serve as a generic tool for lineage analysis and thus pave the way toward large-scale human cell lineage discovery.
Assuntos
Linhagem da Célula , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Algoritmos , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Masculino , Microfluídica/métodos , Pessoa de Meia-Idade , Análise de Sequência de DNA/economia , Análise de Sequência de DNA/normas , Análise de Célula Única/economia , Análise de Célula Única/normasRESUMO
OBJECTIVE: We aimed to identify unique patterns of eye-movements measures reflecting inattentive reading among adults with and without ADHD. METHOD & RESULTS: We recorded eye-movements during uninterrupted text reading of typically developed (TD) and ADHD adults. First, we found significantly longer reading time for the ADHD group than the TD group. Further, we detected cases in which words were reread more than twice and found that such occasions were much more frequent in participants with ADHD than in TD participants. Moreover, we discovered that the first reading pass of these words was less sensitive to the length of the word than the first pass of words read only once, indicating a less meaningful reading. CONCLUSION: We propose that high rate of words that were reread is a correlate of inattentive reading which is more pronounced among ADHD readers. Implications of the findings in the context of reading comprehension are discussed.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Movimentos Oculares , Adulto , Humanos , CompreensãoRESUMO
OBJECTIVE: The present study evaluated the near (attention) and far (reading, ADHD symptoms, learning, and quality of life) transfer effects of a Computerized Progressive Attention Training (CPAT) versus Mindfulness Based Stress Reduction (MBSR) practice among adults with ADHD compared to a passive group. METHOD: Fifty-four adults participated in a non-fully randomized controlled trial. Participants in the intervention groups completed eight 2-hr weekly training sessions. Outcomes were assessed before, immediately after, and 4 months post-intervention, using objective tools: attention tests, eye-tracker, and subjective questionnaires. RESULTS: Both interventions showed near-transfer to various attention functions. The CPAT produced far-transfer effects to reading, ADHD symptoms, and learning while the MBSR improved the self-perceived quality of life. At follow-up, all improvements except for ADHD symptoms were preserved in the CPAT group. The MBSR group showed mixed preservations. CONCLUSION: Both interventions have beneficial effects, however only the CPAT group exhibited improvements compared to the passive group.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Atenção Plena , Humanos , Adulto , Qualidade de Vida , Transtorno do Deficit de Atenção com Hiperatividade/terapia , Atenção , Estresse Psicológico/terapiaRESUMO
We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.
Assuntos
Tratamento Farmacológico da COVID-19 , Proteases 3C de Coronavírus , Inibidores de Protease de Coronavírus , Descoberta de Drogas , SARS-CoV-2 , Humanos , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/química , Simulação de Acoplamento Molecular , Inibidores de Protease de Coronavírus/síntese química , Inibidores de Protease de Coronavírus/química , Inibidores de Protease de Coronavírus/farmacologia , Relação Estrutura-Atividade , Cristalografia por Raios XRESUMO
High-throughput nanomole-scale synthesis allows for late-stage functionalization (LSF) of compounds in an efficient and economical manner. Here, we demonstrated that copper-catalyzed azide-alkyne cycloaddition could be used for the LSF of covalent kinase inhibitors at the nanoscale, enabling the synthesis of hundreds of compounds that did not require purification for biological assay screening, thus reducing experimental time drastically. We generated crude libraries of inhibitors for the kinase MKK7, derived from two different parental precursors, and analyzed them via the high-throughput In-Cell Western assay. Select inhibitors were resynthesized, validated via conventional biological and biochemical methods such as western blots and liquid chromatography-mass spectrometry (LC-MS) labeling, and successfully co-crystallized. Two of these compounds showed over 20-fold increased inhibitory activity compared to the parental compound. This study demonstrates that high-throughput LSF of covalent inhibitors at the nanomole-scale level can be an auspicious approach in improving the properties of lead chemical matter.
Assuntos
Alcinos , Azidas , Alcinos/química , Azidas/química , Reação de Cicloadição , Ensaios de Triagem em Larga Escala , Espectrometria de Massas/métodosRESUMO
Government-sanctioned use of nerve agents (NA) has escalated dramatically in recent years. Oxime reactivators of organophosphate (OP)-inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) serve as antidotes toward poisoning by OPNAs. The oximes used as therapeutics are quaternary compounds that cannot penetrate the blood-brain barrier (BBB). There remains an urgent need for the development of next generation OPNA therapeutics. We have developed two high-throughput screening (HTS) assays using a fluorogenic NA surrogate, O-ethyl methylphosphonyl O-4-methyl-3-cyano-coumarin (EMP-MeCyC). EMP-MeCyC detoxification and EMP-BChE reactivation screening campaigns of ~155,000 small molecules resulted in the identification of 33 nucleophile candidates, including non-quaternary oximes. Four of the oximes were reactivators of both Sarin- and VX-inhibited BChE and directly detoxified Sarin. One oxime also detoxified VX. The novel reactivators included a non-quaternary pyridine amidoxime, benzamidoxime, benzaldoxime and a piperidyl-ketoxime. The VX-inhibited BChE reactivation reaction rates by these novel molecules were similar to those observed with known bis-quaternary reactivators and faster than mono-quaternary pyridinium oximes. Notably, we discovered the first ketoxime reactivator of OP-ChEs and detoxifier of OPNAs. Preliminary toxicological studies demonstrated that the newly discovered non-quaternary oximes were relatively non-toxic in mice. The discovery of unique non-quaternary oximes opens the door to the design of novel therapeutics and decontamination agents following OPNA exposure.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Agentes Neurotóxicos/toxicidade , Oximas/farmacologia , Animais , Ativação Enzimática , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
Designing covalent inhibitors is increasingly important, although it remains challenging. Here, we present covalentizer, a computational pipeline for identifying irreversible inhibitors based on structures of targets with non-covalent binders. Through covalent docking of tailored focused libraries, we identify candidates that can bind covalently to a nearby cysteine while preserving the interactions of the original molecule. We found â¼11,000 cysteines proximal to a ligand across 8,386 complexes in the PDB. Of these, the protocol identified 1,553 structures with covalent predictions. In a prospective evaluation, five out of nine predicted covalent kinase inhibitors showed half-maximal inhibitory concentration (IC50) values between 155 nM and 4.5 µM. Application against an existing SARS-CoV Mpro reversible inhibitor led to an acrylamide inhibitor series with low micromolar IC50 values against SARS-CoV-2 Mpro. The docking was validated by 12 co-crystal structures. Together these examples hint at the vast number of covalent inhibitors accessible through our protocol.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/química , SARS-CoV-2/enzimologia , Proteínas da Matriz Viral/antagonistas & inibidores , Acrilamida/química , Acrilamida/metabolismo , Sítios de Ligação , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico , Biologia Computacional/métodos , Bases de Dados de Proteínas , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/metabolismo , SARS-CoV-2/isolamento & purificação , Proteínas da Matriz Viral/metabolismoRESUMO
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is threatening public health as it spreads worldwide across diverse environments. Its genetic hallmark, the mecA gene, confers resistance to many ß-lactam antibiotics. Here, we show that, in addition, mecA provides a broad selective advantage across diverse chemical environments. Competing fluorescently labelled wild-type and mecA-deleted CA-MRSA USA400 strains across ~57,000 compounds supplemented with subinhibitory levels of the ß-lactam drug cefoxitin, we find that mecA provides a widespread advantage across ß-lactam and non ß-lactam antibiotics, non-antibiotic drugs and even diverse natural and synthetic compounds. This advantage depends on the presence of cefoxitin and is strongly associated with the compounds' physicochemical properties, suggesting that it may be mediated by differential compounds permeability into the cell. Indeed, mecA protects the bacteria against increased cell-envelope permeability under subinhibitory cefoxitin treatment. Our findings suggest that CA-MRSA success might be driven by a cell-envelope mediated selective advantage across diverse chemical compounds.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Staphylococcus aureus Resistente à Meticilina/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Cefoxitina/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Modelos Logísticos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Análise Multivariada , PermeabilidadeRESUMO
Differentiation therapy has been recently revisited as a prospective approach in cancer therapy by targeting the aberrant growth, and repairing the differentiation and cell death programs of cancer cells. However, differentiation therapy of solid tumors is a challenging issue and progress in this field is limited. We performed High Throughput Screening (HTS) using a novel dual multiplex assay to discover compounds, which induce differentiation of human colon cancer cells. Here we show that the protein arginine methyl transferase (PRMT) type 1 inhibitor, MS023, is a potent inducer of colon cancer cell differentiation with a large therapeutic window. Differentiation changes in the highly aggressive human colon cancer cell line (HT-29) were proved by proteomic and genomic approaches. Growth of HT-29 xenograft in nude mice was significantly delayed upon MS023 treatment and immunohistochemistry of tumor indicated differentiation changes. These findings may lead to development of clinically effective anti-cancer drugs based on the mechanism of cancer cell differentiation.
Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Diferenciação Celular , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Humanos , Camundongos , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Intraoral local anesthesia is essential for delivering dental care. However, it is often perceived by some patients as the most painful and in some instances as the only painful part of the treatment, leading in extreme cases to avoidance of dental care. The present study measured the variables of pain, pressure, and discomfort caused by 4 commonly used local anesthesia injections: local infiltration, mental nerve block, inferior alveolar nerve block, and periodontal ligament injections. Patients were asked to grade pain, discomfort, and pressure on a visual analog scale as associated with needle insertion, operator finger position in the mouth, and pressure at injection. The inferior alveolar injection was graded to be the most painful followed by periodontal ligament and then mental nerve block injections. The periodontal ligament injections yielded the highest pressure scores. The inferior alveolar block injection yielded significantly more discomfort than local infiltration and mental nerve block injections when comparing finger and needle position. Local infiltration in the anterior maxillary region yielded the highest needle insertion and finger position discomfort scores. The present study suggests that the dental operator should be aware of local anesthesia injection pain, pressure, and discomfort together with efficacy of technique.
Assuntos
Anestesia Dentária/efeitos adversos , Anestésicos Locais/administração & dosagem , Dor/etiologia , Adolescente , Adulto , Idoso , Anestésicos Locais/efeitos adversos , Queixo/inervação , Feminino , Dedos/fisiologia , Humanos , Injeções/efeitos adversos , Injeções/instrumentação , Masculino , Nervo Mandibular , Nervo Maxilar , Pessoa de Meia-Idade , Agulhas/efeitos adversos , Bloqueio Nervoso/efeitos adversos , Medição da Dor , Ligamento Periodontal , PressãoRESUMO
BACKGROUND: The topoisomerases Top1, Top2alpha and Top2beta are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2beta are subject to proteasomal degradation, this phenomena was not demonstrated for Top2alpha. METHODOLOGY/PRINCIPAL FINDINGS: We show here that Top2alpha is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2beta. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2alpha degradation, increases the persistence of Top2alpha-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2alpha in-vitro and cellular overexpression of Bmi1 increases drug induced Top2alpha ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2alpha ubiquitination and drug-induced Top2alpha degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner. CONCLUSIONS/SIGNIFICANCE: The discovery that poisoned Top2alpha is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.