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The high selectivity and affinity of antibodies toward their antigens have made them a highly valuable tool in disease therapy, diagnosis, and basic research. A plethora of chemical and genetic approaches have been devised to make antibodies accessible to more "undruggable" targets and equipped with new functions of illustrating or regulating biological processes more precisely. In this Review, in addition to introducing how naked antibodies and various antibody conjugates (such as antibody-drug conjugates, antibody-oligonucleotide conjugates, antibody-enzyme conjugates, etc.) work in therapeutic applications, special attention has been paid to how chemistry tools have helped to optimize the therapeutic outcome (i.e., with enhanced efficacy and reduced side effects) or facilitate the multifunctionalization of antibodies, with a focus on emerging fields such as targeted protein degradation, real-time live-cell imaging, catalytic labeling or decaging with spatiotemporal control as well as the engagement of antibodies inside cells. With advances in modern chemistry and biotechnology, well-designed antibodies and their derivatives via size miniaturization or multifunctionalization together with efficient delivery systems have emerged, which have gradually improved our understanding of important biological processes and paved the way to pursue novel targets for potential treatments of various diseases.
Assuntos
Anticorpos , Imunoconjugados , Anticorpos/uso terapêutico , Imunoconjugados/uso terapêutico , Biotecnologia , OligonucleotídeosRESUMO
Domestic ducks are the important host for H5N1 highly pathogenic avian influenza virus (HPAIV) infection and epidemiology, but little is known about the duck T cell response to H5N1 AIV infection. In infection experiments of mallard ducks, we detected significantly increased CD8+ cells and augmented expression of cytotoxicity-associated genes, including granzyme A and IFN-γ, in PBMCs from 5 to 9 d postinfection when the virus shedding was clearly decreased, which suggested the importance of the duck cytotoxic T cell response in eliminating H5N1 infection in vivo. Intriguingly, we found that a CD8high+ population of PBMCs was clearly upregulated in infected ducks from 7 to 9 d postinfection compared with uninfected ducks. Next, we used Smart-Seq2 technology to investigate the heterogeneity and transcriptional differences of the duck CD8+ cells. Thus, CD8high+ cells were likely to be more responsive to H5N1 AIV infection, based on the high level of expression of genes involved in T cell responses, activation, and proliferation, including MALT1, ITK, LCK, CD3E, CD247, CFLAR, IL-18R1, and IL-18RAP. More importantly, we have also successfully cultured H5N1 AIV-specific duck T cells in vitro, to our knowledge, for the first time, and demonstrated that the CD8high+ population was increased with the duck T cell activation and response in vitro, which was consistent with results in vivo. Thus, the duck CD8high+ cells represent a potentially effective immune response to H5N1 AIV infection in vivo and in vitro. These findings provide novel insights and direction for developing effective H5N1 AIV vaccines.
Assuntos
Virus da Influenza A Subtipo H5N1 , Influenza Aviária , Animais , Linfócitos T CD8-Positivos/patologia , Patos , GranzimasRESUMO
Fluorescent molecules are important tools in biological detection, and numerous efforts have been made to develop compounds to meet the desired photophysical properties. For example, tuning the wavelength allows an appropriate penetration depth with minimal interference from the autofluorescence/scattering for a better signal-to-noise contrast. However, there are limited guidelines to rationally design or computationally predict the optical properties from first principles, and factors like the solvent effects will make it more complicated. Herein, we established a database (SMFluo1) of 1181 solvated small-molecule fluorophores covering the ultraviolet-visible-near-infrared absorption window and developed new machine learning models based on deep neural networks for accurately predicting photophysical parameters. The optimal system was applied to 120 out-of-sample compounds, and it exhibited remarkable accuracy with a mean relative error of 1.52%. In this new paradigm, a deep learning algorithm is promising to complement conventional theoretical and experimental studies of fluorophores and to greatly accelerate the discovery of new dyes. Due to its simplicity and efficiency, data from newly developed fluorophores can be easily supplemented to this system to further improve the accuracy across various dye families.
Assuntos
Corantes Fluorescentes , Redes Neurais de Computação , Humanos , Aprendizado de Máquina , SolventesRESUMO
PURPOSE: Nasopharyngeal carcinoma (NPC) is a malignant tumor endangering human health. Gemcitabine or cisplatin chemotherapy has been regarded as effective treatment for patients with locoregionally advanced NPC. However, the effect of gemcitabine plus cisplatin concurrent chemoradiotherapy (CCRT) remained controversial among the studies. Therefore, we conducted this meta-analysis to assess the efficacy and safety of induction chemotherapy by gemcitabine and cisplatin (GP regimen) in patients with locoregionally advanced NPC. METHODS: A systematic literature search was performed using PubMed, Web of Science, and Embase to evaluate the survival benefit and toxicity profiles of patients with locoregionally advanced NPC who were treated with CCRT. A random-effects model or a fixed-effects model was used to pool the data according to the heterogeneity among the included studies. RESULTS: A total of five studies with 1286 patients met the inclusion criteria and were included in this meta-analysis. Pooled estimate showed that GP regimen was associated with significant improvements in OS (HR = 0.57, 95% CI 0.45, 0.73; P < 0.001), DFS (HR = 0.56, 95% CI 0.47, 0.66; P < 0.001), and DRFS (HR = 0.51, 95% CI 0.36, 0.73; P < 0.001), but not in LRFS (HR = 0.54, 95% CI 0.25, 1.19; P = 0.126) and ORR (RR = 1.30, 95% CI 0.54, 3.09; P = 0.556). Moreover, the incidence of adverse events of all grades (RR = 1.15, 95%CI 0.11, 1.38; P = 0.063) or grade 3-4 (RR = 0.96, 95%CI 0.57, 1.29; P = 0.385), was comparable between GP regimen and control treatments. CONCLUSION: Our meta-analysis indicated that the patients with locoregionally advanced NPC could benefit from the regimen of gemcitabine plus cisplatin induction chemotherapy.
Assuntos
Cisplatino , Quimioterapia de Indução , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas , Protocolos de Quimioterapia Combinada Antineoplásica , Desoxicitidina/análogos & derivados , Humanos , Quimioterapia de Indução/efeitos adversos , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , GencitabinaRESUMO
The Corona Virus Disease reported in 2019 (COVID-19) poses a significant threat to human and public health. Its early and accurate detection can reduce the spread and recurrence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Real-time reverse transcription fluorescent quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for detecting the nucleic acid of SARS-CoV-2. This study developed and tested a dual-target (ORF1ab and N gene) one-step nested RT-qPCR (DTO-N-PCR) to detect SARS-CoV-2. Ten-fold serial dilutions of mixed synthetic DNA from SARS-CoV-2 ORF1ab and N gene were used as templates to test the sensitivity of DTO-N-PCR. Its specificity was subsequently tested using throat swab specimens from 10 COVID-19 patients and 35 healthy participants. DTO-N-PCR was more sensitive and specific than conventional RT-qPCR. It has unique features, including a dual-target (ORF1ab and N gene), rapid one-step operation of reverse transcription and PCR, four pairs of inner and outer primers, and specific probes. These features aid in its rapid, accurate, and efficient detection of SARS-CoV-2 RNA.
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COVID-19 , Ácidos Nucleicos , COVID-19/diagnóstico , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
Catalytic asymmetric dynamic kinetic resolution of configurationally labile bridged biaryls is emerging as a powerful strategy for atropisomer synthesis. However, the reported examples suffer from an inherent challenge as the reactivity is highly dependent on the torsional strain of the biaryl substrates, which significantly narrows down the scope and hampers the application. Herein, we report our discovery and development of a torsional strain-independent reaction between biaryl thionolactones and activated isocyanides. By employing auto-tandem silver catalysis, a universal synthesis of both tri- and tetra-ortho-substituted thiazole-containing biaryls was realized in high yields with high enantioselectivities. In addition, these products could be facilely converted to a novel type of bridged biaryls bearing an eight-membered lactone. Mechanistic studies were carried out to elucidate the cause of this unusual torsional strain-independent reactivity.
Assuntos
Cianetos , Lactonas , Tiazóis , Catálise , Cianetos/química , Lactonas/química , Prata/química , Estereoisomerismo , Tiazóis/químicaRESUMO
In contrast to the well-established [3+2] cycloaddition reactions, the catalytic enantioselective [3+n] (n≥3) cycloaddition reaction of activated isocyanides for the preparation of six-membered or larger ring systems has remained underdeveloped. Herein, we report the first example of highly diastereo- and enantioselective [3+3] cycloaddition of activated isocyanides with azomethine imines. By employing silver catalysis, a wide range of biologically important bicyclic 1,2,4-triazines were obtained in high yields (up to 99 %) with good to excellent stereoselectivities (up to >20 : 1 dr, 99 % ee). In addition, the same catalytic system could be applied to both the late-stage functionalization of complex bioactive molecules and the kinetic resolution of racemic azomethine imines, further highlighting its versatility and synthetic utility.
Assuntos
Iminas , Prata , Compostos Azo , Catálise , Cianetos , Reação de Cicloadição , Estereoisomerismo , TiossemicarbazonasRESUMO
A novel mid-infrared (MIR) laser crystal Co/Er:PbF2 was successfully grown. The use of Er3+ ion co-doping to sensitize a Co2+ ion and enhance the 2.1-4.2 µm broadband MIR emission of the Co2+ ion in a PbF2 crystal was studied for the first time, to the best of our knowledge. The Er3+ ion was demonstrated to be an effective sensitizer of the Co2+ ion, making the Co/Er:PbF2 crystal propitious to be pumped by commercialized laser diodes. Furthermore, with Er3+ ion co-doping, the local symmetry of Co2+ and Er3+ ions was seriously distorted, thereby enhancing the 2.1-4.2 µm MIR emissions. This study provides a path for designing MIR laser materials with optimal performance.
RESUMO
Protein-protein interactions play pivotal roles in life, and the protein interaction affinity confers specific protein interaction events in physiology or pathology. Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research to detect molecular interactions in vitro and in vivo. The FRET assay provides very high sensitivity and efficiency. Several attempts have been made to develop the FRET assay into a quantitative measurement for protein-protein interaction affinity in the past. However, the progress has been slow due to complicated procedures or because of challenges in differentiating the FRET signal from other direct emission signals from donor and receptor. This review focuses on recent developments of the quantitative FRET analysis and its application in the determination of protein-protein interaction affinity (KD), either through FRET acceptor emission or donor quenching methods. This paper mainly reviews novel theatrical developments and experimental procedures rather than specific experimental results. The FRET-based approach for protein interaction affinity determination provides several advantages, including high sensitivity, high accuracy, low cost, and high-throughput assay. The FRET-based methodology holds excellent potential for those difficult-to-be expressed proteins and for protein interactions in living cells.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Algoritmos , Cinética , Modelos Teóricos , Ligação Proteica , Proteínas/metabolismo , SoluçõesRESUMO
SUMOylation is one of the posttranslational modifications that mediate cellular activities such as transcription, DNA repair, and signal transduction and is involved in the cell cycle. However, only a limited number of small molecule inhibitors have been identified to study its role in cellular processes. Here, we report a Förster resonance energy transfer (FRET) high-throughput screening assay based on the interaction between E2 Ubc9 and E3 PIAS1. Of the 3200 compounds screened, 34 (1.1%) showed higher than 50% inhibition and 4 displayed dose-response inhibitory effects. By combining this method with a label-free surface plasmon resonance (SPR) assay, false positives were excluded leading to discovering WNN0605-F008 and WNN1062-D002 that bound to Ubc9 with KD values of 1.93 ± 0.62 and 5.24 ± 3.73 µM, respectively. We examined the effect of the two compounds on SUMO2-mediated SUMOylation of RanGAP1, only WNN0605-F008 significantly inhibited RanGAP1 SUMOylation, whereas WNN1062-D002 did not show any inhibition. These compounds, with novel chemical scaffolds, may serve as the initial material for developing new SUMOylation inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Ativadoras de GTPase/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sumoilação/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Animais , Inibidores Enzimáticos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Células MCF-7 , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas Inibidoras de STAT Ativados/metabolismo , Multimerização Proteica/efeitos dos fármacos , Células Sf9 , Bibliotecas de Moléculas Pequenas/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Spodoptera , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The firefly luciferin analog thioluciferin (S-luc) was synthesised as a key element of bioluminescent reporters for oxidation state and thiol/disulfide equilibria. It shows blue-shifts in absorption and fluorescence compared to luciferin, and is a modest luciferase substrate. These features are attributed to a π-system that is less conjugated than luciferin.
Assuntos
Dissulfetos/química , Luciferina de Vaga-Lumes/química , Luciferases de Vaga-Lume/metabolismo , Luminescência , Compostos de Sulfidrila/química , Animais , Medições LuminescentesRESUMO
BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. Early detection of individuals at risk of lung cancer is critical to reduce the mortality rate. OBJECTIVE: The aim of this study was to develop and validate a prospective risk prediction model to identify patients at risk of new incident lung cancer within the next 1 year in the general population. METHODS: Data from individual patient electronic health records (EHRs) were extracted from the Maine Health Information Exchange network. The study population consisted of patients with at least one EHR between April 1, 2016, and March 31, 2018, who had no history of lung cancer. A retrospective cohort (N=873,598) and a prospective cohort (N=836,659) were formed for model construction and validation. An Extreme Gradient Boosting (XGBoost) algorithm was adopted to build the model. It assigned a score to each individual to quantify the probability of a new incident lung cancer diagnosis from October 1, 2016, to September 31, 2017. The model was trained with the clinical profile in the retrospective cohort from the preceding 6 months and validated with the prospective cohort to predict the risk of incident lung cancer from April 1, 2017, to March 31, 2018. RESULTS: The model had an area under the curve (AUC) of 0.881 (95% CI 0.873-0.889) in the prospective cohort. Two thresholds of 0.0045 and 0.01 were applied to the predictive scores to stratify the population into low-, medium-, and high-risk categories. The incidence of lung cancer in the high-risk category (579/53,922, 1.07%) was 7.7 times higher than that in the overall cohort (1167/836,659, 0.14%). Age, a history of pulmonary diseases and other chronic diseases, medications for mental disorders, and social disparities were found to be associated with new incident lung cancer. CONCLUSIONS: We retrospectively developed and prospectively validated an accurate risk prediction model of new incident lung cancer occurring in the next 1 year. Through statistical learning from the statewide EHR data in the preceding 6 months, our model was able to identify statewide high-risk patients, which will benefit the population health through establishment of preventive interventions or more intensive surveillance.
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Registros Eletrônicos de Saúde/tendências , Neoplasias Pulmonares/epidemiologia , Estudos de Coortes , Detecção Precoce de Câncer , Feminino , Humanos , Incidência , Maine , Masculino , Estudos Prospectivos , Estudos RetrospectivosRESUMO
The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain-binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders.
Assuntos
Peptídeo 1 Semelhante ao Glucagon , Receptores Acoplados a Proteínas G , Animais , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMO
An asymmetric cobalt-catalyzed hydroboration/cyclization of 1,7-enynes to synthesize chiral six-membered N-heterocyclic compounds was developed. A variety of aniline-tethered 1,7-enynes react with pinacolborane to afford the corresponding chiral boryl-functionalized quinoline derivatives in high yields with high enantioselectivity. This cobalt-catalyzed asymmetric cyclization of 1,7-enyens provides a general approach to access a series of chiral quinoline derivatives containing quaternary stereocenters.
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Acute liver failure (ALF) is associated with high mortality, and a poor understanding of the underlying pathophysiology has resulted in a lack of effective treatments so far. Here, using an amatoxin-induced rhesus monkey model of ALF, we panoramically revealed the cellular and molecular events that lead to the development of ALF. The challenged monkeys with toxins underwent a typical course of ALF including severe hepatic injury, systemic inflammation and eventual death. Adaptive immune was not noticeably disturbed throughout the progress of ALF. A systematic examination of serum factors and cytokines revealed that IL-6 increase was the most rapid and drastic. Interestingly, we found that IL-6 was mainly produced by circulating monocytes. Furthermore, ablation of monocyte-derived IL-6 in mice decreased liver injury and systemic inflammation following chemical injection. Our findings reveal a critical role of circulating monocytes in initiating and accelerating ALF, indicating a potential therapeutic target in clinical treatment for ALF.
Assuntos
Amanitinas/toxicidade , Encefalopatia Hepática/imunologia , Interleucina-6/imunologia , Lipopolissacarídeos/toxicidade , Falência Hepática Aguda/imunologia , Monócitos/imunologia , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Citocinas/genética , Citocinas/imunologia , Progressão da Doença , Expressão Gênica , Encefalopatia Hepática/induzido quimicamente , Encefalopatia Hepática/genética , Encefalopatia Hepática/patologia , Interleucina-6/deficiência , Interleucina-6/genética , L-Lactato Desidrogenase/sangue , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/genética , Falência Hepática Aguda/patologia , Testes de Função Hepática , Macaca mulatta , Camundongos , Monócitos/patologiaRESUMO
The divergent, stereoselective formal [3+2] cycloadditions of allenoates with activated isocyanides catalyzed by silver or phosphine-based catalysts were investigated. Silver catalysis is capable of delivering a range of 3H-pyrroles in high stereoselectivities. These enantioenriched heterocycles can either undergo sequential cyclisation with isocyanoacetates to deliver unprecedented bicyclic imidazolines with excellent yields and stereoselectivity or undergo unusual aromatization pathways leading to polysubstituted pyrroles. On the other hand, a simple mix-and-go procedure using an amino acid-derived phosphine as the catalyst produces pyrroles bearing a benzylic stereocenter with good enantioselectivity.
RESUMO
The cyclization of allenoates with activated isocyanides was reported for the first time. While Ag catalysis led to an unprecedented enantioselective synthesis of 3H pyrroles, a simple procedure using PPh3 produced a wide range of polysubstituted 1H pyrroles with high efficiency.
Assuntos
Alcenos/química , Cianetos/química , Pirróis/química , Pirróis/síntese química , Catálise , Técnicas de Química Sintética , Ciclização , Estereoisomerismo , Especificidade por SubstratoRESUMO
SUMOylation plays important roles in many key physiological and pathological processes. The SUMOylation cascade involves a heterodimer of activating enzyme, E1 (Aos1/Uba2); a conjugating enzyme, E2 (Ubc9); and many ligase enzymes, E3. Focusing on the activation step of the SUMOylation process, we examined the interaction of E1 with its substrates. Previous studies reported the Km of E1 enzymes in ubiquitin and other ubiquitin-like pathways, but the Km of the SUMO paralogs (SUMO2 and SUMO3) is unknown. Here, by using quantitative FRET to measure the SUMO E1 enzyme kinetics of SUMO1, 2, and 3 and ATP under steady state conditions, we found that the enzyme kinetics from the quantitative FRET method are comparable to those from conventional radioactive assays. Additionally, the kinetic constants, Km , of SUMO2 (3.418 ± 0.9131 µM) and SUMO3 (2.764 ± 0.75 µM) [FW1] are approximately four to five times higher than that of SUMO1 Km (0.7458 ± 0.1105 µM). These results demonstrate the advantages of FRET technology for determining Km , including the ability to monitor reaction progress in real-time with high-throughput and high-sensitivity in an environmentally friendly manner. The processes discussed here extend the utility of quantitative FRET in characterizing protein-protein interactions and enzyme kinetics.
Assuntos
Trifosfato de Adenosina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Enzimas Ativadoras de Ubiquitina/metabolismo , Cinética , Ligação ProteicaRESUMO
Förster resonance energy transfer (FRET) has been widely used in biological and biomedical research because it can determine molecule or particle interactions within a range of 1-10 nm. The sensitivity and efficiency of FRET strongly depend on the distance between the FRET donor and acceptor. Historically, FRET assays have been used to quantitatively deduce molecular distances. However, another major potential application of the FRET assay has not been fully exploited, that is, the use of FRET signals to quantitatively describe molecular interactive events. In this review, we discuss the use of quantitative FRET assays for the determination of biochemical parameters, such as the protein interaction dissociation constant (K(d)), enzymatic velocity (k(cat)) and K(m). We also describe fluorescent microscopy-based quantitative FRET assays for protein interaction affinity determination in cells as well as fluorimeter-based quantitative FRET assays for protein interaction and enzymatic parameter determination in solution.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Animais , Ensaios Enzimáticos/métodos , Humanos , Cinética , Microscopia de Fluorescência/métodos , Peptídeo Hidrolases/metabolismo , Mapeamento de Interação de Proteínas/métodosRESUMO
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a valuable tool for elucidating molecular interactions in vitro and in vivo. Quantitative FRET analysis is a powerful method for determining biochemical parameters and molecular distances at nanometer levels. Recently, we reported theoretical developments and experimental procedures for determining the dissociation constant, Kd and enzymatic kinetics parameters, Kcat and KM, of protein interactions with the engineered FRET pair, CyPet and YPet. The strong FRET signal from this pair made these developments possible. However, the direct link of fluorescent proteins with proteins of interests may interfere with the folding of some fusion proteins. Here, we report a new protein engineering strategy for improving FRET signals by adding a linker between the fluorescent protein and the targeted protein. This improvement allowed us to follow the covalent conjugation of NEDD8 to its E2 ligase in the presence of E1 and ATP, which was difficult to determine without linker. Three linkers, LAEAAAKEAA, TSGSPGLQEFGT, and LAAALAAA, which are alpha helix or random coil, all significantly improved the FRET signals. Our results show a general methodology for improving trans-FRET signals to effectively determine biochemical reaction intermediates.