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Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.
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Acetilação , Cromatina , Lisina , Metilação , Processamento de Proteína Pós-Traducional , Sítio de Iniciação de Transcrição , Animais , Humanos , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Histona Desacetilases/metabolismoRESUMO
Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.
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Dano ao DNA , Histonas , Nucleossomos , Histonas/metabolismo , Histonas/química , Nucleossomos/metabolismo , Humanos , Ligação Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Modelos Moleculares , ATPases Associadas a Diversas Atividades Celulares , DNA HelicasesRESUMO
The terahertz spectrum has the ability to provide high-speed communication and millimeter-level resolution. As a result, terahertz-integrated sensing and communication (ISAC) has been identified as a key enabler for 6G wireless networks. This work discusses a photonics-based D-band communication system for integrated high-resolution localization and high-speed wireless communication. Our empirical results show that a communication rate of 5 Gbps over a distance of 1.5 m and location identification of the target with millimeter-level (<4m m) range resolution can be conducted simultaneously using the same signal. We also show that the error due to the thickness of the beam splitter can be eliminated, while the quantization error and the random drift errors are the limiting factors of the resolution achieved. This experimental demonstration using D-band communication indicates that terahertz ISAC can be realized for 6G networks while considering the underlying system restrictions (e.g., bandwidth limit and lens diameter).
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The occurrence of abnormal phytoplankton blooms is one of the significant changes in coastal ecosystems due to climate change. However, the underlying mechanism of such blooms remains poorly understood due to the complexity of the system. In this study, the data from numerous observations was used to elucidate the unprecedented phytoplankton blooms in the autumn and winter of 2021 in Laizhou Bay, a typical aquaculture bay in the southern Bohai Sea of China. The abundance of phytoplankton cells increased by more than tenfold in the southern waters compared to that in the same period from 2019 to 2020. The phytoplankton bloom was first observed in winter in the Bohai Sea, with the cell abundance in the southern bay exceeding 108 cells L-1 in December 2021. The diversity and evenness of phytoplankton communities decreased in the southern area. Cerataulina pelagica was the dominant algae, comprising 69 % of the total phytoplankton in October and 99 % in December. In autumn 2021, the largest flood of the Yellow River in recent decades occurred. This was attributed to extreme rainfall events within the river basin. The input of substantial riverine nutrients played a significant role in promoting phytoplankton blooms. Correlation analysis indicated the important cumulative impact of the Yellow River on phytoplankton blooms rather than a direct short-term effect. Numerical modeling results indicated that exceptionally high Yellow River discharge in autumn could significantly affect the entire bay from autumn to the following spring. This study may contribute to understanding the abnormal phytoplankton blooms in coastal waters and provide valuable insights for environmental management in river basins and coastal waters.
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Diatomáceas , Fitoplâncton , Ecossistema , Rios , ChinaRESUMO
Chemical proteomics utilizes small-molecule probes to covalently engage with their interacting proteins. Since chemical probes are tagged to the active or binding sites of functional proteins, chemical proteomics can be used to profile protein targets, reveal precise binding sites/mechanisms, and screen inhibitors competing with probes in a biological context. These capabilities of chemical proteomics have great potential to enable discoveries of both drug targets and lead compounds. However, chemical proteomics is limited by the time-consuming bottleneck of sample preparations, which are processed manually. With the advancement of robotics and artificial intelligence, it is now possible to automate workflows to make chemical proteomics sample preparation a high-throughput process. An automated robotic system represents a major technological opportunity to speed up advances in proteomics, open new frontiers in drug target discovery, and broaden the future of chemical biology.
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Inteligência Artificial , Proteômica , Automação , Proteínas/química , Descoberta de DrogasRESUMO
Glycogen is a highly branched biomacromolecule that functions as a glucose buffer. It is involved in multiple diseases such as glycogen storage disorders, diabetes, and even liver cancer, where the imbalance between biosynthetic and catabolic enzymes results in structural alterations and abnormal accumulation of glycogen that can be toxic to cells. Accurate and sensitive glycogen quantification and structural determination are prerequisites for understanding the phenotypes and biological functions of glycogen under these conditions. In this research, we furthered cell glycogen characterization by presenting a highly sensitive method to measure the glycogen content and degree of branching. The method employed a novel fructose density gradient as an alternative to the traditional sucrose gradient to fractionate glycogen from cell mixtures using ultracentrifugation. Fructose was used to avoid the large glucose background, allowing the method to be highly quantitative. The glycogen content was determined by quantifying 1-phenyl-3-methyl-5-pyrazolone (PMP)-derivatized glucose residues obtained from acid-hydrolyzed glycogen using ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry (UHPLC/QqQ-MS). The degree of branching was determined through linkage analysis where the glycogen underwent permethylation, hydrolysis, PMP derivatization, and UHPLC/QqQ-MS analysis. The new approach was used to study the effect of insulin on the glycogen phenotypes of human hepatocellular carcinoma (Hep G2) cells. We observed that cells produced greater amounts of glycogen with less branching under increasing insulin levels before reaching the cell's insulin-resistant state, where the trend reversed and the cells produced less but higher-branched glycogen. The advantage of this method lies in its high sensitivity in characterizing both the glycogen level and the structure of biological samples.
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Glicogênio , Insulinas , Humanos , Espectrometria de Massas/métodos , Cromatografia Líquida , Cromatografia Líquida de Alta Pressão/métodos , Glucose/análise , EdaravoneRESUMO
Post-transcriptional modifications of RNA strongly influence the RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in the fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before MS identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA MS increases both coverage and throughput, while EMLC LC-MS orthogonally separates RNA molecules of different lengths and charges. The combination of the two methods offers a broadly applicable platform to improve the length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.
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Espectrometria de Mobilidade Iônica , RNA , Sequência de Bases , Espectrometria de Massas/métodos , Cromatografia Líquida , Espectrometria de Mobilidade Iônica/métodosRESUMO
Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.
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Ribonucleosídeos , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Camundongos , RNA/química , Processamento Pós-Transcricional do RNA , Ribonucleosídeos/análise , Ribonucleosídeos/química , Ribonucleosídeos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Extensive multi-species harmful algal blooms (HABs) were triggered by Super Typhoon Lekima in Laizhou Bay (Bohai Sea) in August 2019. After conducting two field cruises before and after the typhoon passage, we employed both high-performance liquid chromatography (HPLC)-pigment and microscopic methods to study the changes in the phytoplankton community and biomass. Following the passage of Lekima, the average surface salinity decreased, while dissolved inorganic nitrogen and dissolved silicate concentrations increased in the study area. The phytoplankton abundance and Chl a significantly increased after the typhoon event. Post-typhoon, the highest abundance values of Pseudo-nitzschia spp., Noctiluca scintillans, and Coscinodiscus spp. reached 106 cells/L and those of Bacillaria paxillifera, Ceratium spp., and Gymnodinium catenatum were in the order of 105 cells/L. HPLC-pigment CHEMTAX analysis showed that the biomass (Chl a) of dinoflagellates, diatoms, cryptophytes, chlorophytes, and haptophytes increased significantly after the typhoon. The increase in Chl a concentration was mainly attributable to large-sized phytoplankton, which are mostly diatoms and dinoflagellates. This study highlights that typhoons may cause HABs by introducing large amounts of freshwater and nutrients and change the phytoplankton community in a temperate and inner bay.
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Tempestades Ciclônicas , Diatomáceas , Dinoflagellida , China , Proliferação Nociva de Algas , FitoplânctonRESUMO
We have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the site-specific glycan alterations associated with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Glycopeptides associated with disease severity were also identified. Multinomial regression modelling was employed to construct and validate multi-analyte diagnostic models capable of accurately distinguishing PBC, PSC, and healthy controls from one another (AUC = 0.93 ± 0.03). Finally, to investigate how disease-relevant environmental factors can influence glycosylation, we characterized the ability of bile acids known to be differentially expressed in PBC to alter glycosylation. We hypothesize that this could be a mechanism by which altered self-antigens are generated and become targets for immune attack. This work demonstrates the utility of the MRM method to identify diagnostic site-specific glycan classifiers capable of distinguishing even related autoimmune diseases from one another.
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Autoimunidade , Colangite Esclerosante/imunologia , Cirrose Hepática Biliar/imunologia , Polissacarídeos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Ácidos e Sais Biliares/sangue , Ácidos e Sais Biliares/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Colangite Esclerosante/sangue , Colangite Esclerosante/diagnóstico , Diagnóstico Diferencial , Glicômica/métodos , Glicopeptídeos/sangue , Glicopeptídeos/imunologia , Glicosilação , Humanos , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/diagnóstico , Polissacarídeos/sangue , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
BACKGROUND: Glycoproteomics deals with glycoproteins that are formed by post-translational modification when sugars (like fucose and sialic acid) are attached to protein. Glycosylation of proteins influences function, but whether glycosylation is altered by diet is unknown. OBJECTIVE: To evaluate the effect of consuming a diet based on the Dietary Guidelines for Americans on circulating glycoproteins that have previously been associated with cardiometabolic diseases. DESIGN: Forty-four women, with one or more metabolic syndrome characteristics, completed an 8-week randomized controlled feeding intervention (n = 22) consuming a diet based on the Dietary Guidelines for Americans (DGA 2010); the remaining consumed a 'typical American diet' (TAD, n = 22). Fasting serum samples were obtained at week0 (baseline) and week8 (post-intervention); 17 serum proteins were chosen for targeted analyses. Protein standards and serum samples were analyzed in a UHPLC-MS protocol to determine peptide concentration and their glycan (fucosylation or sialylation) profiles. Data at baseline were used in correlational analyses; change in proteins and glycans following intervention were used in non-parametric analyses. RESULTS: At baseline, women with more metabolic syndrome characteristics had more fucosylation (total di-fucosylated proteins: p = 0.045) compared to women with a lesser number of metabolic syndrome characteristics. Dietary refined grain intake was associated with increased total fucosylation (ρ = - 0.530, p < 0.001) and reduced total sialylation (ρ = 0.311, p = 0.042). After the 8-week intervention, there was higher sialylation following the DGA diet (Total di-sialylated protein p = 0.018, poly-sialylated orosomucoid p = 0.012) compared to the TAD diet. CONCLUSIONS: Based on this study, glycosylation of proteins is likely affected by dietary patterns; higher sialylation was associated with a healthier diet pattern. Altered glycosylation is associated with several diseases, particularly cancer and type 2 diabetes, and this study raises the possibility that diet may influence disease state by altering glycosylation. CLINICAL TRIAL REGISTRATION: NCT02298725 at clinicaltrials.gov; https://clinicaltrials.gov/ct2/show/NCT02298725 .
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Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Proteínas Sanguíneas/metabolismo , Doenças Cardiovasculares/prevenção & controle , Dieta , Feminino , Glicosilação , HumanosRESUMO
C-dot-based composites with phosphorescence have been widely reported due to their attractive potential in various applications. But easy quenching of phosphorescence induced by oxygen or instability of matrices remained a tricky problem. Herein, we reported a Si-doped-CD (Si-CD)-based RTP materials with long lifetime by embedding Si-CDs in sulfate crystalline matrices. The resultant Si-CD@sulfate composites exhibited a long lifetime up to 1.07 s, and outstanding stability under various ambient conditions. The intriguing RTP phenomenon was attributed to the C = O bond and the doping of Si element due to the fact that sulfates could effectively stabilize the triplet states of Si-CDs, thus enabling the intersystem crossing (ISC). Meanwhile, we confirmed that the ISC process and phosphorescence emission could be effectively regulated based on the heavy atom effect. This research introduced a new perspective to develop materials with regulated RTP performance and high stability.
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One natural p-terphenyl glycoside, gliocladinin C, and two furano-polyene derivatives, chaetominins A and B, were isolated from potato endophytic fungus Chaetomium subaffine. The absolute configurations of these compounds were elucidated by HR-ESI-MS, NMR, the DP4+ probabilities and electronic circular dichroism (ECD) spectra. Furthermore, gliocladinin C and chaetominin A showed cytotoxic activity against two selected human tumor cell lines (Hep-2 and HepG-2).
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Antineoplásicos/química , Chaetomium/metabolismo , Compostos de Terfenil/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Chaetomium/química , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Compostos de Terfenil/farmacologiaRESUMO
In this study, we use an anaerobic-aerobic integrated denitrification (Fe/C-ZACID) device with an iron-carbon-activated carbon and zeolite composite filter to remove nitrogen from simulated low carbon-nitrogen ratio (C/N) sewage. The impacts of dissolved oxygen (DO) level, hydraulic retention time (HRT), C/N and nitrate recirculation ratio on denitrification performance were studied. The results show that when HRT was 6 h, DO was 3 ± 0.1 mg/L, influent C/N was 3, and nitrate recirculation ratio was 100%, and removal rates of 95% for ammonia and 85% for total nitrogen (TN) were achieved. A beaker comparison test demonstrated that this synergistic denitrification system included heterotrophic denitrification, physicochemical denitrification, iron autotrophic denitrification and hydrogen autotrophic denitrification, etc. The Fe/C-ZACID device has a high-efficiency nitrogen removal effect for low C/N ratio sewage and strong shock resistance, which provides technical support and a theoretical basis for advanced denitrification of rural domestic sewage.
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Desnitrificação , Eliminação de Resíduos Líquidos/métodos , Zeolitas , Anaerobiose , Reatores Biológicos , Carvão Vegetal , Filtração , Ferro , Nitrogênio , EsgotosRESUMO
Inorganic hydrated salt phase change materials (PCMs) hold promise for improving the energy conversion efficiency of thermal systems and facilitating the exploration of renewable thermal energy. Hydrated salts, however, often suffer from low thermal conductivity, supercooling, phase separation, leakage and poor solar absorptance. In recent years, compounding hydrated salts with functional carbon materials has emerged as a promising way to overcome these shortcomings and meet the application demands. This work reviews the recent progress in preparing carbon-enhanced hydrated salt phase change composites for thermal management applications. The intrinsic properties of hydrated salts and their shortcomings are firstly introduced. Then, the advantages of various carbon materials and general approaches for preparing carbon-enhanced hydrated salt PCM composites are briefly described. By introducing representative PCM composites loaded with carbon nanotubes, carbon fibers, graphene oxide, graphene, expanded graphite, biochar, activated carbon and multifunctional carbon, the ways that one-dimensional, two-dimensional, three-dimensional and hybrid carbon materials enhance the comprehensive thermophysical properties of hydrated salts and affect their phase change behavior is systematically discussed. Through analyzing the enhancement effects of different carbon fillers, the rationale for achieving the optimal performance of the PCM composites, including both thermal conductivity and phase change stability, is summarized. Regarding the applications of carbon-enhanced hydrate salt composites, their use for the thermal management of electronic devices, buildings and the human body is highlighted. Finally, research challenges for further improving the overall thermophysical properties of carbon-enhanced hydrated salt PCMs and pushing towards practical applications and potential research directions are discussed. It is expected that this timely review could provide valuable guidelines for the further development of carbon-enhanced hydrated salt composites and stimulate concerted research efforts from diverse communities to promote the widespread applications of high-performance PCM composites.
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To identify the relationship between leadership and work readiness in a cohort of new head nurses in China. This cross-sectional study enrolled 225 newly appointed head nurses in public tertiary hospitals in China, which were selected using convenience sampling. Data were collected using online questionnaires that included a sociodemographic characteristics form, the Nursing Managers Leadership Scale (NMLS), and the New Nurse Leaders' Job Readiness Scale (NNLJRS). IBM SPSS v.25 was used for statistical analysis. The overall mean scores of NMLS (100.50â ±â 17.64) and NNLJRS (111.90â ±â 15.84) of the 225 new nurse leaders were at moderate levels. The results of the Pearson correlation analysis and the hierarchical regression analysis further indicated that there was a significant positive correlation between leadership and work readiness of new head nurses (râ =â 0.85, Pâ <â .001), as well as charisma (ßâ =â 0.19, Pâ <â .01), affinity (ßâ =â 0.18, Pâ <â .01), coordination ability (ßâ =â 0.32, Pâ <â .01), and motivational ability (ßâ =â 0.21, Pâ <â .01) in leadership were found to be positively associated with work readiness. This study found that the leadership and work readiness of the new head nurses still needed improvement. A significant relationship was found between these 2 variables, and charisma, affinity, coordination ability, and motivational ability in the leadership ability of the new head nurses facilitated the level of work readiness. Nursing administration should create a leadership development series program focusing on the development of charisma, affinity, coordination ability, and motivational ability to support the work readiness of new nurse managers and help them with role transition.
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Liderança , Humanos , Estudos Transversais , China , Feminino , Masculino , Adulto , Inquéritos e Questionários , Enfermeiros Administradores/psicologia , Recursos Humanos de Enfermagem Hospitalar/psicologia , Recursos Humanos de Enfermagem Hospitalar/organização & administração , Adulto JovemRESUMO
OBJECTIVES: This study aimed to investigate the current status of innovative behaviours among nurses in traditional Chinese medicine (TCM) hospitals using latent profile analysis, identify potential subgroups and their population characteristics and explore factors associated with different categories. DESIGN: Cross-sectional study. SETTING: Six TCM hospitals in Anhui, China. PARTICIPANTS: From 1 April 2023 to 31 July 2023, a total of 642 registered nurses with more than 1 year of work experience were recruited from the clinical departments of six TCM hospitals using a stratified cluster sampling method. 529 valid questionnaires were recovered, presenting a validity rate of 82.40%. PRIMARY AND SECONDARY OUTCOME MEASURES: Data were collected through online surveys containing a sociodemographic questionnaire, the Nurse Innovative Behaviour Scale, the Nurse Adversity Quotient Self-Evaluation Scale and the Conditions for Work Effectiveness Questionnaire-II. Latent profile analysis was performed to identify categorisation features of nurses' innovative behaviour in TCM hospitals. Multiple logistic regression analyses were used to investigate associated factors with profile membership. RESULTS: TCM hospital nurses' innovative behaviours were mainly classified into three types of latent profiles: low innovative behaviour (35.3%), moderate innovative behaviour (48.4%) and high innovative behaviour (16.3%). The results of multiple logistic regression analyses indicated that gender, monthly income, department, hospital level, position, nurse competency level, any training attended related to TCM knowledge and skills, adversity quotient level and structural empowerment level were the influencing factors for the potential profiles. CONCLUSIONS: The innovative behaviour of nurses in TCM hospitals can be classified into three categories. Studying the heterogeneity of the innovative behaviour of nurses in TCM hospitals and its associated factors provides evidence for nursing administrators and educators to develop individualised interventions based on each latent characteristic to improve the innovative behaviour of nurses in TCM hospitals. It is of great significance to the heritage and innovative development of TCM nursing.
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Recursos Humanos de Enfermagem Hospitalar , Humanos , Estudos Transversais , China , Feminino , Adulto , Masculino , Inquéritos e Questionários , Recursos Humanos de Enfermagem Hospitalar/psicologia , Medicina Tradicional Chinesa , Atitude do Pessoal de Saúde , Pessoa de Meia-Idade , Enfermeiras e Enfermeiros/psicologiaRESUMO
Cell membrane glycoproteins are generally highly fucosylated and sialylated, and post-translational modifications play important roles in the proteins' functions of signaling, binding and cellular processing. For these reasons, methods for measuring sialic acid-mediated protein-protein interactions have been developed. However, determining the role of fucose in these interactions has been limited by technological barriers that have thus far hindered the ability to characterize and observe fucose-mediated protein-protein interactions. Herein, we describe a method to metabolically label mammalian cells with modified fucose, which incorporates a bioorthogonal group into cell membrane glycoproteins thereby enabling the characterization of cell-surface fucose interactome. Copper-catalyzed click chemistry was used to conjugate a proximity labeling probe, azido-FeBABE. Following the addition of hydrogen peroxide (H2O2), the fucose-azido-FeBABE catalyzed the formation of hydroxyl radicals, which in turn oxidized the amino acids in the proximity of the labeled fucose residue. The oxidized peptides were identified using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Variations in degree of protein oxidation were obtained with different H2O2 reaction times yielding the acquisition of spatial information of the fucose-interacting proteins. In addition, specific glycoprotein-protein interactions were constructed for Galectin-3 (LEG3) and Galectin-3-binding protein (LG3BP) illustrating the further utility of the method. This method identifies new fucose binding partners thereby enhancing our understanding of the cell glycocalyx.
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The STRICTA checklist is the guideline for reporting clinical trials undertaken using acupuncture intervention. As an extension of the CONSORT checklist, the STRICTA checklist facilitates the reporting quality of acupuncture clinical trials. The clinical research paradigm changes along with the development of science and technology. It is crucial to ensure whether or not the existing STRICTA checklist guides the reporting clinical trials of acupuncture now and in the future as well. This paper introduces the development and the updating procedure of the STRICTA checklist, analyzes the characteristics of utility and the limitation, and proposes several suggestions on the difficulties and challenges encountered in the implementation of the STRICTA checklist of current version so as to advance the further update and improvement.
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Terapia por Acupuntura , Lista de Checagem , Humanos , Terapia por Acupuntura/normas , Ensaios Clínicos como Assunto/normas , Projetos de Pesquisa/normasRESUMO
In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx6K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes.