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Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.
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Knowledge of locations and activities of cis -regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our V al i dated S ystematic I ntegrati on (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we showed that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.
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Marek's disease virus (MDV), a naturally oncogenic, highly contagious alpha herpesvirus, induces a T cell lymphoma in chickens that causes severe economic loss. Marek's disease (MD) outcome in an individual is attributed to genetic and environmental factors. Further investigation of the host-virus interaction mechanisms that impact MD resistance is needed to achieve greater MD control. This study analyzed genome-wide DNA methylation patterns in 2 highly inbred parental lines 63 and 72 and 5 recombinant congenic strains (RCS) C, L, M, N, and X strains from those parents. Lines 63 and 72, are MD resistant and susceptible, respectively, whereas the RCS have different combinations of 87.5% Line 63 and 12.5% Line 72. Our DNA methylation cluster showed a strong association with MD incidence. Differentially methylated regions (DMRs) between the parental lines and the 5 RCS were captured. MD-resistant and MD-susceptible markers of DNA methylation were identified as transgenerational epigenetic inheritable. In addition, the growth of v-src DNA tumors and antibody response against sheep red blood cells differed among the 2 parental lines and the RCS. Overall, our results provide very solid evidence that DNA methylation patterns are transgenerational epigenetic inheritance (TEI) in chickens and also play a vital role in MD tumorigenesis and other immune responses; the specific methylated regions may be important modulators of general immunity.
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Herpesvirus Gallináceo 2 , Enfermedad de Marek , Enfermedades de las Ovejas , Animales , Ovinos , Pollos , Resistencia a la Enfermedad/genética , Susceptibilidad a Enfermedades/veterinaria , Epigénesis Genética , Enfermedades de las Ovejas/genéticaRESUMEN
Introduction: Genome-wide association studies (GWAS) have identified genetic markers for cattle production and reproduction traits. Several publications have reported Single Nucleotide Polymorphisms (SNPs) for carcass-related traits in cattle, but these studies were rarely conducted in pasture-finished beef cattle. Hawai'i, however, has a diverse climate, and 100% of its beef cattle are pasture-fed. Methods: Blood samples were collected from 400 cattle raised in Hawai'i islands at the commercial harvest facility. Genomic DNA was isolated, and 352 high-quality samples were genotyped using the Neogen GGP Bovine 100 K BeadChip. SNPs that did not meet the quality control standards were removed using PLINK 1.9, and 85 k high-quality SNPs from 351 cattle were used for association mapping with carcass weight using GAPIT (Version 3.0) in R 4.2. Four models were used for the GWAS analysis: General Linear Model (GLM), the Mixed Linear Model (MLM), the Fixed and Random Model Circulating Probability Unification (FarmCPU), the Bayesian-Information and Linkage-Disequilibrium Iteratively Nested Keyway (BLINK). Results and Discussion: Our results indicated that the two multi-locus models, FarmCPU and BLINK, outperformed single-locus models, GLM and MLM, in beef herds in this study. Specifically, five significant SNPs were identified using FarmCPU, while BLINK and GLM each identified the other three. Also, three of these eleven SNPs ("BTA-40510-no-rs", "BovineHD1400006853", and "BovineHD2100020346") were shared by multiple models. The significant SNPs were mapped to genes such as EIF5, RGS20, TCEA1, LYPLA1, and MRPL15, which were previously reported to be associated with carcass-related traits, growth, and feed intake in several tropical cattle breeds. This confirms that the genes identified in this study could be candidate genes for carcass weight in pasture-fed beef cattle and can be selected for further breeding programs to improve the carcass yield and productivity of pasture-finished beef cattle in Hawai'i and beyond.
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The paper presents the damage results of thick steel plates subjected to local blast loading using experimental and numerical approaches. Three steel plates with a thickness of 17 mm under the local contact explosion of trinitrotoluene (TNT) explosives were tested, and the damaged parts of the steel plates were scanned using a scanning electron microscope (SEM). ANSYS LS-DYNA software was used to simulate the damage results of the steel plate. By analyzing and comparing the experimental results with the numerical simulation results, the influence law of the TNT acting on the steel plate, the damage mode of the steel plate, the reliability verification of the numerical simulation, and the criterion for judging the damage mode of the steel plate were obtained. Results show that the damage mode of the steel plate changes with the changes in the explosive charge. The diameter of the crater on the surface of the steel plate is mainly related to the diameter of the contact surface between the explosive and the steel plate. The fracture mode of the steel plate in the process of generating cracks is a quasi-cleavage fracture, and the process of generating craters and perforations in the steel plate is a ductile fracture. The damage mode of the steel plates can be divided into three types. The numerical simulation results have minor errors and high reliability, and numerical simulation can be used as an auxiliary tool for experiments. A new criterion is proposed to predict the damage mode of the steel plates under contact explosion.
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Proper knowledge and understanding of climatic variability across different seasons are important in farm management. To learn more about the potential effects of climate change on dairying in Hawaii, we conducted a study on site-specific climate characterization using several variables including rainfall, wind speed (WS), solar radiation, and temperature, at two dairy farms located on Hawai`i Island, Hawai`i, in Ookala named "OK DAIRY" and in Upolu Point named "UP DAIRY." Temperature-humidity index (THI) and WS variations in the hottest four months (June to September) were analyzed to determine when critical thresholds that affect animal health are exceeded. Rainfall data were used to estimate the capacity of forage production in 6-mo wet (November to April) and dry (May to October) seasons. Future projections of temperature and rainfall were assessed using mid- and end-century gridded data products for low (RCP 4.5) and high emissions (RCP 8.5) scenarios. Our results showed that the "OK DAIRY" site received higher rainfall than the "UP DAIRY" site, favoring grass growth and forage availability. In addition, the "UP DAIRY" site was more stressful for animals during the summer (THI 69 to 73) than the "OK DAIRY" site (THI 67 to 70) as the THI exceeded the critical threshold of 68, which is conducive for high-lactating cattle. On the "UP DAIRY" site, the THI did not drop below 68 during the summer nights, which created fewer opportunities for cattle to recover from heat stress. Future projections indicated that air temperature would increase 1.3 to 1.8 °C by mid-century and 1.6 to 3.2 °C by the end-century at both farms, and rainfall will increase at the "OK DAIRY" site and decrease at the "UP DAIRY" site by the end-century. The agriculture and livestock industries, particularly the dairy and beef subsectors in Hawai`i, are vulnerable to climate changes as higher temperatures and less rainfall will have adverse effects on cattle. The findings in this study demonstrated how both observed and projected changes in climate support the development of long-term strategies for breeding and holistic livestock management practices to adapt to changing climate conditions.
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Due to their exceptional catalytic properties for the oxygen reduction reaction (ORR) and other crucial electrochemical reactions, PtCo intermetallic nanoparticle (NP) and single atomic (SA) Pt metal site catalysts have received considerable attention. However, their formation mechanisms at the atomic level during high-temperature annealing processes remain elusive. Here, the thermally driven structure evolution of Pt-Co binary catalyst systems is investigated using advanced in situ electron microscopy, including PtCo intermetallic alloys and single Pt/Co metal sites. The pre-doping of CoN4 sites in carbon supports and the initial Pt NP sizes play essential roles in forming either Pt3 Co intermetallics or single Pt/Co metal sites. Importantly, the initial Pt NP loadings against the carbon support are critical to whether alloying to L12 -ordered Pt3 Co NPs or atomizing to SA Pt sites at high temperatures. High Pt NP loadings (e.g., 20%) tend to lead to the formation of highly ordered Pt3 Co intermetallic NPs with excellent activity and enhanced stability toward the ORR. In contrast, at a relatively low Pt loading (<6 wt%), the formation of single Pt sites in the form of PtC3 N is thermodynamically favorable, in which a synergy between the PtC3 N and the CoN4 sites could enhance the catalytic activity for the ORR, but showing insufficient stability.
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An effective and universal strategy is developed to enhance the stability of the non-noble-metal M-Nx /C catalyst in proton exchange membrane fuel cells (PEMFCs) by improving the bonding strength between metal ions and chelating polymers, i.e., poly(acrylic acid) (PAA) homopolymer and poly(acrylic acid-maleic acid) (P(AA-MA)) copolymer with different AA/MA ratios. Mössbauer spectroscopy and X-ray absorption spectroscopy (XAS) reveal that the optimal P(AA-MA)-Fe-N catalyst with a higher Fe3+ -polymer binding constant possesses longer FeN bonds and exclusive Fe-N4 /C moiety compared to PAA-Fe-N, which consists of ≈15% low-coordinated Fe-N2 /N3 structures. The optimized P(AA-MA)-Fe-N catalyst exhibits outstanding ORR activity and stability in both half-cell and PEMFC cathodes, with the retention rate of current density approaching 100% for the first 37 h at 0.55 V in an H2 -air fuel cell. Density functional theory (DFT) calculations suggest that the Fe-N4 /C site could optimize the difference between the adsorption energy of the Fe atoms on the support (Ead ) and the bulk cohesive energy (Ecoh ) relative to Fe-N2 /N3 moieties, thereby strongly stabilizing Fe centers against demetalation.
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Beef cattle raised under grass-fed and grain-fed have many differences, including metabolic efficiency and meat quality. To investigate these two regimens' intrinsic influence on beef cattle, we used high-throughput sequencing and metabolomics analyses to explore differentially expressed genes (DEGs) and metabolimic networks in the liver. A total of 200 DEGs, 76 differentially expressed miRNAs (DEmiRNAs), and two differentially expressed lncRNAs (DElncRNAs) were detected between regimen groups. Metabolic processes and pathways enriched functional genes including target genes of miRNAs and lncRNAs. We found that many genes were involved in energy, retinol and cholesterol metabolism, and bile acid synthesis. Combined with metabolites such as low glucose concentration, high cholesterol concentration, and increased primary bile acid concentration, these genes were mainly responsible for lowering intramuscular fat, low cholesterol, and yellow meat in grass-fed cattle. Additionally, we identified two lncRNAs and eight DEGs as potential competing endogenous RNAs (ceRNAs) to bind miRNAs by the interaction network analysis. These results revealed that the effects of two feeding regimens on beef cattle were mainly induced by gene expression changes in metabolic pathways mediated via lncRNAs, miRNAs, and ceRNAs, and contents of metabolites in the liver. It may provide a clue on feeding regimens inducing the metabolic regulations.
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We elucidate the structural evolution of CoN4 sites during thermal activation by developing a zeolitic imidazolate framework (ZIF)-8-derived carbon host as an ideal model for Co2+ ion adsorption. Subsequent in situ X-ray absorption spectroscopy analysis can dynamically track the conversion from inactive Co-OH and Co-O species into active CoN4 sites. The critical transition occurs at 700 °C and becomes optimal at 900 °C, generating the highest intrinsic activity and four-electron selectivity for the oxygen reduction reaction (ORR). DFT calculations elucidate that the ORR is kinetically favored by the thermal-induced compressive strain of Co-N bonds in CoN4 active sites formed at 900 °C. Further, we developed a two-step (i.e., Co ion doping and adsorption) Co-N-C catalyst with increased CoN4 site density and optimized porosity for mass transport, and demonstrated its outstanding fuel cell performance and durability.
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While constitutive CCCTC-binding factor (CTCF)-binding sites are needed to maintain relatively invariant chromatin structures, such as topologically associating domains, the precise roles of CTCF to control cell-type-specific transcriptional regulation remain poorly explored. We examined CTCF occupancy in different types of primary blood cells derived from the same donor to elucidate a new role for CTCF in gene regulation during blood cell development. We identified dynamic, cell-type-specific binding sites for CTCF that colocalize with lineage-specific transcription factors. These dynamic sites are enriched for single-nucleotide polymorphisms that are associated with blood cell traits in different linages, and they coincide with the key regulatory elements governing hematopoiesis. CRISPR-Cas9-based perturbation experiments demonstrated that these dynamic CTCF-binding sites play a critical role in red blood cell development. Furthermore, precise deletion of CTCF-binding motifs in dynamic sites abolished interactions of erythroid genes, such as RBM38, with their associated enhancers and led to abnormal erythropoiesis. These results suggest a novel, cell-type-specific function for CTCF in which it may serve to facilitate interaction of distal regulatory emblements with target promoters. Our study of the dynamic, cell-type-specific binding and function of CTCF provides new insights into transcriptional regulation during hematopoiesis.
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Factor de Unión a CCCTC/metabolismo , Eritropoyesis , Elementos Reguladores de la Transcripción , Sitios de Unión , Línea Celular , Células Cultivadas , Elementos de Facilitación Genéticos , Células Eritroides/citología , Células Eritroides/metabolismo , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Activación TranscripcionalRESUMEN
Lung cancer is the most common cancer worldwide. Epigenetic modifications like DNA methylation play fundamental roles in the dynamic process of lung cancer. The objective of this study was to use methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) to identify novel and high-confidence DNA methylation in lung tumor. We first compared the whole-genome DNA methylation of three lung cancer cell lines, including A549, H1299, and SK-MES-1, against BEAS-2B, a lung/bronchial normal epithelial cell line. We then used pyrosequencing and OneStep qMethyl kit methods to verify the results in the cell line specimens. MBD-Seq identified 279, 8046, and 22 887 differentially methylated regions (DMRs), respectively, with 120 common DMRs among three comparison groups. Three DMRs were consistent with the MBD-Seq results by both pyrosequencing and OneStep qMethyl validations. Furthermore, OneStep qMethyl kit was also performed for functional validation of these three potential DMRs in sputum DNA from clinical participants. We successfully identified one new DMR adjacent to ATG16L2. The novel DMR might have an important function in lung carcinogenesis. Further validation of the finding in clinical specimens of lung cancer patients and functional analysis of this novel DMR in the development of lung cancer through transcriptional silencing of ATG16L2 are warranted.
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Proteínas Relacionadas con la Autofagia/genética , Metilación de ADN , Técnicas Genéticas , Neoplasias Pulmonares/genética , Proteínas Relacionadas con la Autofagia/química , Carcinogénesis/genética , Línea Celular Tumoral , Epigénesis Genética , Genoma Humano , Humanos , Dominios ProteicosRESUMEN
Electrocatalysts with single metal atoms as active sites have received increasing attention owing to their high atomic utilization efficiency and exotic catalytic activity and selectivity. This review aims to provide a comprehensive summary on the recent development of such single-atom electrocatalysts (SAECs) for various energy-conversion reactions. The discussion starts with an introduction of the different types of SAECs, followed by an overview of the synthetic methodologies to control the atomic dispersion of metal sites and atomically resolved characterization using state-of-the-art microscopic and spectroscopic techniques. In recognition of the extensive applications of SAECs, the electrocatalytic studies are dissected in terms of various important electrochemical reactions, including hydrogen evolution reaction (HER), oxygen evolution reaction (OER), oxygen reduction reaction (ORR), carbon dioxide reduction reaction (CO2RR), and nitrogen reduction reaction (NRR). Examples of SAECs are deliberated in each case in terms of their catalytic performance, structure-property relationships, and catalytic enhancement mechanisms. A perspective is provided at the end of each section about remaining challenges and opportunities for the development of SAECs for the targeted reaction.
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Increasing catalytic activity and durability of atomically dispersed metal-nitrogen-carbon (M-N-C) catalysts for the oxygen reduction reaction (ORR) cathode in proton-exchange-membrane fuel cells remains a grand challenge. Here, a high-power and durable Co-N-C nanofiber catalyst synthesized through electrospinning cobalt-doped zeolitic imidazolate frameworks into selected polyacrylonitrile and poly(vinylpyrrolidone) polymers is reported. The distinct porous fibrous morphology and hierarchical structures play a vital role in boosting electrode performance by exposing more accessible active sites, providing facile electron conductivity, and facilitating the mass transport of reactant. The enhanced intrinsic activity is attributed to the extra graphitic N dopants surrounding the CoN4 moieties. The highly graphitized carbon matrix in the catalyst is beneficial for enhancing the carbon corrosion resistance, thereby promoting catalyst stability. The unique nanoscale X-ray computed tomography verifies the well-distributed ionomer coverage throughout the fibrous carbon network in the catalyst. The membrane electrode assembly achieves a power density of 0.40 W cm-2 in a practical H2 /air cell (1.0 bar) and demonstrates significantly enhanced durability under accelerated stability tests. The combination of the intrinsic activity and stability of single Co sites, along with unique catalyst architecture, provide new insight into designing efficient PGM-free electrodes with improved performance and durability.
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[This corrects the article DOI: 10.1186/s40104-020-00482-x.].
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BACKGROUND: Grass-fed and grain-fed Angus cattle differ in the diet regimes. However, the intricate mechanisms of different beef quality and other phenotypes induced by diet differences are still unclear. Diet affects mitochondrial function and dynamic behavior in response to changes in energy demand and supply. In this study, we examined the mtDNA copy number, mitochondria-related genes expression, and metabolic biomarkers in grass-fed and grain-fed Angus cattle. RESULTS: We found that the grass-fed group had a higher mtDNA copy number than the grain-fed group. Among different tissues, the mtDNA copy number was the highest in the liver than muscle, rumen, and spleen. Based on the transcriptome of the four tissues, a lower expression of mtDNA-encoded genes in the grass-fed group compared to the grain-fed group was discovered. For the mitochondria-related nuclear genes, however, most of them were significantly down-regulated in the muscle of the grass-fed group and up-regulated in the other three tissues. In which, COX6A2, POLG2, PPIF, DCN, and NDUFA12, involving in ATP synthesis, mitochondrial replication, transcription, and maintenance, might contribute to the alterations of mtDNA copy number and gene expression. Meanwhile, 40 and 23 metabolic biomarkers were identified in the blood and muscle of the grain-fed group compared to a grass-fed group, respectively. Integrated analysis of the altered metabolites and gene expression revealed the high expression level of MDH1 in the grain-fed group might contribute to the mitochondrial NADH oxidation and spermidine metabolism for adapting the deletion mtDNA copy number. CONCLUSIONS: Overall, the study may provide further deep insight into the adaptive and regulatory modulations of the mitochondrial function in response to different feeding systems in Angus cattle.
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BACKGROUND: Marek's disease (MD) is a highly neoplastic disease primarily affecting chickens, and remains as a chronic infectious disease that threatens the poultry industry. Copy number variation (CNV) has been examined in many species and is recognized as a major source of genetic variation that directly contributes to phenotypic variation such as resistance to infectious diseases. Two highly inbred chicken lines, 63 (MD-resistant) and 72 (MD-susceptible), as well as their F1 generation and six recombinant congenic strains (RCSs) with varied susceptibility to MD, are considered as ideal models to identify the complex mechanisms of genetic and molecular resistance to MD. RESULTS: In the present study, to unravel the potential genetic mechanisms underlying resistance to MD, we performed a genome-wide CNV detection using next generation sequencing on the inbred chicken lines with the assistance of CNVnator. As a result, a total of 1649 CNV regions (CNVRs) were successfully identified after merging all the nine datasets, of which 90 CNVRs were overlapped across all the chicken lines. Within these shared regions, 1360 harbored genes were identified. In addition, 55 and 44 CNVRs with 62 and 57 harbored genes were specifically identified in line 63 and 72, respectively. Bioinformatics analysis showed that the nearby genes were significantly enriched in 36 GO terms and 6 KEGG pathways including JAK/STAT signaling pathway. Ten CNVRs (nine deletions and one duplication) involved in 10 disease-related genes were selected for validation by using quantitative real-time PCR (qPCR), all of which were successfully confirmed. Finally, qPCR was also used to validate two deletion events in line 72 that were definitely normal in line 63. One high-confidence gene, IRF2 was identified as the most promising candidate gene underlying resistance and susceptibility to MD in view of its function and overlaps with data from previous study. CONCLUSIONS: Our findings provide valuable insights for understanding the genetic mechanism of resistance to MD and the identified gene and pathway could be considered as the subject of further functional characterization.
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Pollos/genética , Variaciones en el Número de Copia de ADN , Resistencia a la Enfermedad/genética , Enfermedad de Marek/genética , Animales , Pollos/virología , Ontología de Genes , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
Current methods can illuminate the genome-wide activity of CRISPR-Cas9 nucleases, but are not easily scalable to the throughput needed to fully understand the principles that govern Cas9 specificity. Here we describe 'circularization for high-throughput analysis of nuclease genome-wide effects by sequencing' (CHANGE-seq), a scalable, automatable tagmentation-based method for measuring the genome-wide activity of Cas9 in vitro. We applied CHANGE-seq to 110 single guide RNA targets across 13 therapeutically relevant loci in human primary T cells and identified 201,934 off-target sites, enabling the training of a machine learning model to predict off-target activity. Comparing matched genome-wide off-target, chromatin modification and accessibility, and transcriptional data, we found that cellular off-target activity was two to four times more likely to occur near active promoters, enhancers and transcribed regions. Finally, CHANGE-seq analysis of six targets across eight individual genomes revealed that human single-nucleotide variation had significant effects on activity at ~15.2% of off-target sites analyzed. CHANGE-seq is a simplified, sensitive and scalable approach to understanding the specificity of genome editors.
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Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Epigénesis Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Secuencia de Bases , Línea Celular , Cromatina/genética , Edición Génica , Variación Genética , Genoma Humano , Humanos , Aprendizaje AutomáticoRESUMEN
The urgent need to address the high-cost issue of proton-exchange membrane fuel cell (PEMFC) technologies, particularly for transportation applications, drives the development of simultaneously highly active and durable platinum group metal-free (PGM-free) catalysts and electrodes. The past decade has witnessed remarkable progress in exploring PGM-free cathode catalysts for the oxygen reduction reaction (ORR) to overcome sluggish kinetics and catalyst instability in acids. Among others, scientists have identified the newly emerging atomically dispersed transition metal (M: Fe, Co, or/and Mn) and nitrogen co-doped carbon (M-N-C) catalysts as the most promising alternative to PGM catalysts. Here, we provide a comprehensive review of significant breakthroughs, remaining challenges, and perspectives regarding the M-N-C catalysts in terms of catalyst activity, stability, and membrane electrode assembly (MEA) performance. A variety of novel synthetic strategies demonstrated effectiveness in improving intrinsic activity, increasing active site density, and attaining optimal porous structures of catalysts. Rationally designing and engineering the coordination environment of single metal MNx sites and their local structures are crucial for enhancing intrinsic activity. Increasing the site density relies on the innovative strategies of restricting the migration and agglomeration of single metal sites into metallic clusters. Relevant understandings provide the correlations among the nature of active sites, nanostructures, and catalytic activity of M-N-C catalysts at the atomic scale through a combination of experimentation and theory. Current knowledge of the transferring catalytic properties of M-N-C catalysts to MEA performance is limited. Rationally designing morphologic features of M-N-C catalysts play a vital role in boosting electrode performance through exposing more accessible active sites, realizing uniform ionomer distribution, and facilitating mass/proton transports. We outline future research directions concerning the comprehensive evaluation of M-N-C catalysts in MEAs. The most considerable challenge of current M-N-C catalysts is the unsatisfied stability and rapid performance degradation in MEAs. Therefore, we further discuss practical methods and strategies to mitigate catalyst and electrode degradation, which is fundamentally essential to make M-N-C catalysts viable in PEMFC technologies.