RESUMEN
BACKGROUND: Wenchang chickens are one of the most popular local chicken breeds in the Chinese chicken industry. However, the low feed efficiency is the main shortcoming of this breed. Therefore, there is a need to find a more precise breeding method to improve the feed efficiency of Wenchang chickens. In this study, we explored important candidate genes and variants for feed efficiency and growth traits through genome-wide association study (GWAS) analysis. RESULTS: Estimates of genomic heritability for growth and feed efficiency traits, including residual feed intake (RFI) of 0.05, average daily food intake (ADFI) of 0.21, average daily weight gain (ADG) of 0.24, body weight (BW) at 87, 95, 104, 113 days of age (BW87, BW95, BW104 and BW113) ranged from 0.30 to 0.44. Important candidate genes related to feed efficiency and growth traits were identified, such as PLCE1, LAP3, MED28, QDPR, LDB2 and SEL1L3 genes. CONCLUSION: The results identified important candidate genes for feed efficiency and growth traits in Wenchang chickens and provide a theoretical basis for the development of new molecular breeding technology.
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Pollos , Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple , Animales , Pollos/genética , Pollos/crecimiento & desarrollo , Fenotipo , Alimentación Animal , Sitios de Carácter Cuantitativo , Carácter Cuantitativo HeredableRESUMEN
The benzofuran core inhibitors HCV-796, BMS-929075, MK-8876, compound 2, and compound 9B exhibit good pan-genotypic activity against various genotypes of NS5B polymerase. To elucidate their mechanism of action, multiple molecular simulation methods were used to investigate the complex systems of these inhibitors binding to GT1a, 1b, 2a, and 2b NS5B polymerases. The calculation results indicated that these five inhibitors can not only interact with the residues in the palm II subdomain of NS5B polymerase, but also with the residues in the palm I subdomain or the palm I/III overlap region. Interestingly, the binding of inhibitors with longer substituents at the C5 position (BMS-929075, MK-8876, compound 2, and compound 9B) to the GT1a and 2b NS5B polymerases exhibits different binding patterns compared to the binding to the GT1b and 2a NS5B polymerases. The interactions between the para-fluorophenyl groups at the C2 positions of the inhibitors and the residues at the binding pockets, together with the interactions between the substituents at the C5 positions and the residues at the reverse ß-fold (residues 441-456), play a key role in recognition and the induction of the binding. The relevant studies could provide valuable information for further research and development of novel anti-HCV benzofuran core pan-genotypic inhibitors.
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Antivirales , Benzofuranos , Genotipo , Hepacivirus , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/química , Benzofuranos/química , Benzofuranos/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Hepacivirus/genética , Antivirales/farmacología , Antivirales/química , Simulación de Dinámica Molecular , Simulación del Acoplamiento Molecular , Sitios de Unión , Unión Proteica , Humanos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , ARN Polimerasa Dependiente del ARNRESUMEN
The stimulator of interferon genes (STING) plays a significant role in immune defense and protection against tumor proliferation. Many cyclic dinucleotide (CDN) analogues have been reported to regulate its activity, but the dynamic process involved when the ligands activate STING remains unclear. In this work, all-atom molecular dynamics simulations were performed to explore the binding mode between human STING (hSTING) and four cyclic adenosine-inosine monophosphate analogs (cAIMPs), as well as 2',3'-cGMP-AMP (2',3'-cGAMP). The results indicate that these cAIMPs adopt a U-shaped configuration within the binding pocket, forming extensive non-covalent interaction networks with hSTING. These interactions play a significant role in augmenting the binding, particularly in interactions with Tyr167, Arg238, Thr263, and Thr267. Additionally, the presence of hydrophobic interactions between the ligand and the receptor further contributes to the overall stability of the binding. In this work, the conformational changes in hSTING upon binding these cAIMPs were also studied and a significant tendency for hSTING to shift from open to closed state was observed after binding some of the cAIMP ligands.
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Proteínas de la Membrana , Simulación de Dinámica Molecular , Unión Proteica , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Sitios de Unión , Nucleótidos Cíclicos/química , Nucleótidos Cíclicos/metabolismo , Ligandos , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
BACKGROUND: Prior drought stress may change plants response patterns and subsequently increase their tolerance to the same condition, which can be referred to as "drought memory" and proved essential for plants well-being. However, the mechanism of transcriptional drought memory in psammophytes remains unclear. Agriophyllum squarrosum, a pioneer species on mobile dunes, is widely spread in Northern China's vast desert areas with outstanding ability of water use efficiency. Here we conducted dehydration-rehydration treatment on A. squarrosum semi-arid land ecotype AEX and arid land ecotype WW to dissect the drought memory mechanism of A. squarrosum, and to determine the discrepancy in drought memory of two contrasting ecotypes that had long adapted to water heterogeneity. RESULT: Physiological traits monitoring unveiled the stronger ability and longer duration in drought memory of WW than that of AEX. A total of 1,642 and 1,339 drought memory genes (DMGs) were identified in ecotype AEX and WW, respectively. Furthermore, shared DMGs among A. squarrosum and the previously studied species depicted that drought memory commonalities in higher plants embraced pathways like primary and secondary metabolisms; while drought memory characteristics in A. squarrosum were mainly related to response to heat, high light intensity, hydrogen peroxide, and dehydration, which might be due to local adaptation to desert circumstances. Heat shock proteins (HSPs) occupied the center of the protein-protein interaction (PPI) network in drought memory transcription factors (TF), thus playing a key regulatory role in A. squarrosum drought memory. Co-expression analysis of drought memory TFs and DMGs uncovered a novel regulating module, whereby pairs of TFs might function as molecular switches in regulating DMG transforming between high and low expression levels, thus promoting drought memory reset. CONCLUSION: Based on the co-expression analysis, protein-protein interaction prediction, and drought memory metabolic network construction, a novel regulatory module of transcriptional drought memory in A. squarrosum was hypothesized here, whereby recurrent drought signal is activated by primary TF switches, then amplified by secondary amplifiers, and thus regulates downstream complicated metabolic networks. The present research provided valuable molecular resources on plants' stress-resistance basis and shed light on drought memory in A. squarrosum.
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Chenopodiaceae , Ecotipo , Factores de Transcripción/genética , Deshidratación , Sequías , Plantas , Agua , Regulación de la Expresión Génica de las PlantasRESUMEN
Whole-genome genotyping methods are important for breeding. However, it has been challenging to develop a robust method for simultaneous foreground and background genotyping that can easily be adapted to different genes and species. In our study, we accidently discovered that in adapter ligation-mediated PCR, the amplification by primer-template mismatched annealing (PTMA) along the genome could generate thousands of stable PCR products. Based on this observation, we consequently developed a novel method for simultaneous foreground and background integrated genotyping by sequencing (FBI-seq) using one specific primer, in which foreground genotyping is performed by primer-template perfect annealing (PTPA), while background genotyping employs PTMA. Unlike DNA arrays, multiple PCR, or genome target enrichments, FBI-seq requires little preliminary work for primer design and synthesis, and it is easily adaptable to different foreground genes and species. FBI-seq therefore provides a prolific, robust, and accurate method for simultaneous foreground and background genotyping to facilitate breeding in the post-genomics era.
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Genoma , Genotipo , Cartilla de ADN/genética , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary life-cycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships, whereas incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. Our study reveals how bumblebee genes and genomes have evolved across the Bombus phylogeny and identifies variations potentially linked to key ecological and behavioral traits of these important pollinators.
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Adaptación Biológica/genética , Abejas/genética , Evolución Biológica , Genoma de los Insectos , Animales , Uso de Codones , Elementos Transponibles de ADN , Dieta , Conducta Alimentaria , Componentes del Gen , Tamaño del Genoma , Selección GenéticaRESUMEN
Lysine-ε-acetylation (Kac) is a reversible post-translational modification that plays important roles during plant-pathogen interactions. Some pathogens can deliver secreted effectors encoding acetyltransferases or deacetylases into host cell to directly modify acetylation of host proteins. However, the function of these acetylated host proteins in plant-pathogen defense remains to be determined. Employing high-resolution tandem mass spectrometry, we analyzed protein abundance and lysine acetylation changes in maize infected with Puccinia polysora (P. polysora) at 0 h, 12 h, 24 h, 48 h and 72 h. A total of 7412 Kac sites from 4697 proteins were identified, and 1732 Kac sites from 1006 proteins were quantified. Analyzed the features of lysine acetylation, we found that Kac is ubiquitous in cellular compartments and preferentially targets lysine residues in the -F/W/Y-X-X-K (ac)-N/S/T/P/Y/G- motif of the protein, this Kac motif contained proteins enriched in basic metabolism and defense-associated pathways during fungal infection. Further analysis of acetylproteomics data indicated that maize regulates cellular processes in response to P. polysora infection by altering Kac levels of histones and non-histones. In addition, acetylation of pathogen defense-related proteins presented converse patterns in signaling transduction, defense response, cell wall fortification, ROS scavenging, redox reaction and proteostasis. Our results provide informative resources for studying protein acetylation in plant-pathogen interactions, not only greatly extending the understanding on the roles of acetylation in vivo, but also providing a comprehensive dynamic pattern of Kac modifications in the process of plant immune response.
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Lisina , Zea mays , Lisina/metabolismo , Zea mays/metabolismo , Procesamiento Proteico-Postraduccional , Puccinia , Acetilación , Proteoma/metabolismoRESUMEN
KEY MESSAGE: Morphological, genetic and transcriptomic characterizations of an EMS-induced wheat paired spikelets (PS) mutant were performed. A novel qualitative locus WPS1 on chromosome 1D was identified. Grain yield of wheat is significantly associated with inflorescence or spike architecture. However, few genes related to wheat spike development have been identified and their underlying mechanisms are largely unknown. In this study, we characterized an ethyl methanesulfonate (EMS)-induced wheat mutant, wheat paired spikelets 1 (wps1). Unlike a single spikelet that usually develops at each node of rachis, a secondary spikelet appeared below the primary spikelet at most of the rachis nodes of wps1. The microscope observation showed that the secondary spikelet initiated later than the primary spikelet. Genetic analysis suggested that the PS of wps1 is controlled by a single dominant nuclear gene, designated WHEAT PAIRED SPIKELETS 1 (WPS1). Further RNA-seq based bulked segregant analysis and molecular marker mapping localized WPS1 in an interval of 208.18-220.92 Mb on the chromosome arm 1DL, which is different to known genes related to spike development in wheat. By using wheat omics data, TraesCS1D02G155200 encoding a HD-ZIP III transcription factor was considered as a strong candidate gene for WPS1. Transcriptomic analysis indicated that PS formation in wps1 is associated with auxin-related pathways and may be regulated by networks involving TB1, Ppd1, FT1, VRN1, etc. This study laid the solid foundation for further validation of the causal gene of WPS1 and explored its regulatory mechanism in PS formation and inflorescence development, which may benefit to kernel yield improvement of wheat based on optimization or design of spike architecture in the future.
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Transcriptoma , Triticum , Grano Comestible/genética , Perfilación de la Expresión Génica , Inflorescencia/genética , Triticum/genéticaRESUMEN
A novel obligate anaerobic organism, designated DONG20-135T, was isolated from human faeces collected in Beijing, PR China. Cells were Gram-stain-negative, rod-shaped, non-motile and non-spore-forming. Growth occurred at 25â45 °C (optimum, 30â35 °C), a pH range of 6-9 (optimum, pH 8) and in the presence of 0â3.5â% (w/v) NaCl (optimum, 0.5â1.5â%). The major fatty acids were C16â:â0, C18â:â1 ω9c and C10â:â0, the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, four glycolipids, six aminolipids, three aminophospholipids and four unidentified lipids. No respiratory quinones were detected. The cell-wall peptidoglycan of the strain was A1γ type, containing meso-diaminopimelic acid. The 16S rRNA gene sequences shared a lower identity (<92.7â% similarity) with the described species. The phylogenetic tree based on 16S rRNA gene sequences and the protein-concatamer tree showed that strain DONG20-135T formed a distinct lineage within the family Erysipelotrichaceae. The genomic DNA G + C content was 42.2âmol%. Based on the results of phenotypic, chemotaxonomic and genomic analyses, strain DONG20-135T represents a novel genus of the family Erysipelotrichaceae, for which the name Copranaerobaculum intestinale gen. nov., sp. nov. is proposed (=KCTC 15868T=CGMCC 1.17357T).
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Ácidos Grasos , Fosfolípidos , Anaerobiosis , Técnicas de Tipificación Bacteriana , Composición de Base , China , ADN Bacteriano/genética , Ácidos Grasos/química , Heces , Humanos , Fosfolípidos/química , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Agriophyllum squarrosum (L.) Moq., a pioneer plant endemic to the temperate deserts of Asia, could be domesticated into an ideal crop with outstanding ecological and medicinal characteristics. A previous study showed differential organic acid accumulation between two in situ altitudinal ecotypes. To verify whether this accumulation was determined by environmental or genetic factors, we conducted organic acid targeted metabolic profiling among 14 populations of A. squarrosum collected from regions with different altitudes based on a common garden experiment. Results showed that the most abundant organic acid in A. squarrosum was citric acid (96.03%, 2322.90 µg g-1). Association analysis with in situ environmental variables showed that salicylic acid content was positively correlated with altitudinal gradient. Considering the enrichment of salicylic acid and protocatechualdehyde in high-altitude populations based on the common garden experiment, and the high expression of their biosynthesis relative genes (i.e., PAL and C4H) in the in situ high-altitude ecotype, we propose that organic acid accumulation could be involved in local adaptation to high altitudes. This study not only addresses the molecular basis of local adaptation involving the accumulation of organic acids in the desert plant A. squarrosum but also provides a method to screen wild germplasms to mitigate the impact of global climate change.
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Chenopodiaceae , Plantas Medicinales , Aclimatación , Adaptación Fisiológica , Altitud , Cambio ClimáticoRESUMEN
External environmental factors can cause an imbalance in intestinal flora. For people living in the extremes of a plateau climate, lack of oxygen is a primary health challenge that leads to a series of reactions. We wondered how intestinal microorganisms might change in a simulated plateau environment and what changes might occur in the host organism and intestinal microorganisms in the absence of hypoxia-related factors. In this study, mice carrying a knockout of hypoxia-inducible factor 1ß (Hif-1ß) in myeloid cells and wild-type mice were raised in a composite hypoxic chamber to simulate a plateau environment at 5,000 m of elevation for 14 days. The mice carrying the myeloid Hif-1ß deletion displayed aggravated hypoxic phenotypes in comparison to and significantly greater weight loss and significantly higher cardiac index values than the wild-type group. The levels of some cytokines increased in the hypoxic environment. Analysis of 16S rRNA sequencing results showed that hypoxia had a significant effect on the gut microbiota in both wild-type and Hif-1ß-deficient mice, especially on the first day. The levels of members of the Bacteroidaceae family increased continuously from day 1 to day 14 in Hif-1ß deletion mice, and they represented an obviously different group of bacteria at day 14 compared with the wild-type mice. Butyrate-producing bacteria, such as Butyricicoccus, were found in wild-type mice only after 14 days in the hypoxic environment. In conclusion, hypoxia caused heart enlargement, greater weight loss, and obvious microbial imbalance in myeloid Hif-1ß-deficient mice. This study revealed genetic and microecological pathways for research on mechanisms of hypoxia.
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Translocador Nuclear del Receptor de Aril Hidrocarburo/deficiencia , Microbioma Gastrointestinal , Eliminación de Gen , Hipoxia/genética , Células Mieloides/metabolismo , Animales , Biodiversidad , Femenino , Hipoxia/metabolismo , Ratones , Ratones Noqueados , Células Mieloides/inmunología , FenotipoRESUMEN
Flowering time is one of the most critical traits for plants' life cycles, which is influenced by various environment changes, such as global warming. Previous studies have suggested that to guarantee reproductive success, plants have shifted flowering times to adapt to global warming. Although many studies focused on the molecular mechanisms of early flowering, little was supported by the repeated sampling at different time points through the changing climate. To fully dissect the temporal and spatial evolutionary genetics of flowering time, we investigated nucleotide variation in ten flowering time candidate genes and nine reference genes for the same ten wild-barley populations sampled 28 years apart (1980-2008). The overall genetic differentiation was significantly greater in the descendant populations (2008) compared with the ancestral populations (1980); however, local adaptation tests failed to detect any single-nucleotide polymorphism (SNP)/indel under spatial-diversifying selection at either time point. By contrast, the WFABC (Wright-Fisher ABC-based approach) that detected 54 SNPs/indels was under strong selection during the past 28 generations. Moreover, all these 54 alleles were segregated in the ancestral populations, but fixed in the descendent populations. Among the top ten SNPs/indels, seven were located in genes of FT1 (FLOWERING TIME LOCUS T 1), CO1 (CONSTANS-LIKE PROTEIN 1), and VRN-H2 (VERNALIZATION-H2), which have been documented to be associated with flowering time regulation in barley cultivars. This study might suggest that all ten populations have undergone parallel evolution over the past few decades in response to global warming, and even an overwhelming local adaptation and ecological differentiation.
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Adaptación Fisiológica/genética , Flores/fisiología , Hordeum/genética , Selección Genética , Alelos , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Genética de Población , Hordeum/fisiología , Mutación INDEL , Israel , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Culturomics can ascertain traces of microorganisms to be cultivated using different strategies and identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry or 16S rDNA sequencing. However, to cater to all requirements of microorganisms and isolate as many species as possible, multiple culture conditions must be used, imposing a heavy workload. In addition, the fast-growing bacteria (e.g., Escherichia) surpass the slow-growing bacteria in culture by occupying space and using up nutrients. Besides, some bacteria (e.g., Pseudomonas) suppress others by secreting antibacterial metabolites, making it difficult to isolate bacteria with lower competence. Applying inhibitors to restrain fast-growing bacteria is one method to cultivate more bacterial species from human feces. RESULTS: We applied CHIR-090, an LpxC enzyme inhibitor that has antibacterial activity against most Gram-negative bacteria, to culturomics of human fresh feces. The antibacterial activity of CHIR-090 was first assessed on five Gram-negative species of bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, and Bacteroides vulgatus), all of which are commonly isolated from the human gut. Then, we assessed suitable concentrations of the inhibitor. Finally, CHIR-090 was applied in blood culture bottles for bacterial cultivation. In total, 102 species from five samples were identified. Of these, we found one new species, two species not reported previously in the human gut, and 11 species not previously isolated from humans. CONCLUSIONS: CHIR-090 can suppress E. coli, P. aeruginosa, K. pneumoniae, Pro. vulgaris, but not B. vulgatus. Compared with the non-inhibitor group, CHIR-090 increased bacteria isolation by 23.50%, including four species not reported in humans and one new species. Application of LpxC enzyme inhibitor in culturomics increased the number of species isolated from the human gut.
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Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Técnicas Bacteriológicas/métodos , Inhibidores Enzimáticos/farmacología , Microbioma Gastrointestinal , Adulto , Bacterias/aislamiento & purificación , Cultivo de Sangre/métodos , ADN Bacteriano/genética , Heces/microbiología , Voluntarios Sanos , Humanos , Ácidos Hidroxámicos/farmacología , Análisis de Secuencia de ADN , Treonina/análogos & derivados , Treonina/farmacologíaRESUMEN
PKA (protein lysine acetylation) is a key post-translational modification involved in the regulation of various biological processes in rice. So far, rice acetylome data is very limited due to the highly-dynamic pattern of protein expression and PKA modification. In this study, we performed a comprehensive quantitative acetylome profile on four typical rice tissues, i.e., the callus, root, leaf, and panicle, by using a mass spectrometry (MS)-based, label-free approach. The identification of 1536 acetylsites on 1454 acetylpeptides from 890 acetylproteins represented one of the largest acetylome datasets on rice. A total of 1445 peptides on 887 proteins were differentially acetylated, and are extensively involved in protein translation, chloroplast development, and photosynthesis, flowering and pollen fertility, and root meristem activity, indicating the important roles of PKA in rice tissue development and functions. The current study provides an overall view of the acetylation events in rice tissues, as well as clues to reveal the function of PKA proteins in physiologically-relevant tissues.
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Procesamiento Proteico-Postraduccional , Proteoma , Proteómica , Acetilación , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Biología Computacional/métodos , Lisina/metabolismo , Especificidad de Órganos , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteómica/métodosRESUMEN
Replication protein A (RPA) is a conserved heterotrimeric protein complex comprising RPA1, RPA2, and RPA3 subunits involved in multiple DNA metabolism pathways attributable to its single-stranded DNA binding property. Unlike other species possessing a single RPA2 gene, rice (Oryza sativa) possesses three RPA2 paralogs, but their functions remain unclear. In this study, we identified RPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (rpa2c) exhibited reduced bivalent formation, leading to chromosome nondisjunction. In rpa2c, chiasma frequency is reduced by ~78% compared with the wild type and is accompanied by loss of the obligate chiasma. The residual ~22% chiasmata fit a Poisson distribution, suggesting loss of crossover control. RPA2c colocalized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of RPA2c to the chromosomes. In addition, RPA2c partially colocalized with MER3 during late leptotene, thus indicating that RPA2c is required for class I crossover formation at a late stage of homologous recombination. Furthermore, we identified RPA1c, an RPA1 subunit with nearly overlapping distribution to RPA2c, required for ~79% of chiasmata formation. Our results demonstrate that an RPA complex comprising RPA2c and RPA1c is required to promote meiotic crossovers in rice.
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Intercambio Genético , Meiosis , Oryza/genética , Proteínas de Plantas/metabolismo , Proteína de Replicación A/metabolismo , ADN Bacteriano , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutagénesis Insercional , No Disyunción Genética , Oryza/citología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Proteína de Replicación A/genéticaRESUMEN
Whole-genome genotyping (WGG) stands as a pivotal element in genomic-assisted plant breeding. Nevertheless, sequencing-based approaches for WGG continue to be costly, primarily owing to the high expenses associated with library preparation and the laborious protocol. During prior development of foreground and background integrated genotyping by sequencing (FBI-seq), we discovered that any sequence-specific primer (SP) inherently possesses the capability to amplify a massive array of stable and reproducible non-specific PCR products across the genome. Here, we further improved FBI-seq by replacing the adapter ligated by Tn5 transposase with an arbitrary degenerate (AD) primer. The protocol for the enhanced FBI-seq unexpectedly mirrors a simplified thermal asymmetric interlaced (TAIL)-PCR, a technique that is widely used for isolation of flanking sequences. However, the improved TAIL-PCR maximizes the primer-template mismatched annealing capabilities of both SP and AD primers. In addition, leveraging of next-generation sequencing enhances the ability of this technique to assay tens of thousands of genome-wide loci for any species. This cost-effective, user-friendly, and powerful WGG tool, which we have named TAIL-PCR by sequencing (TAIL-peq), holds great potential for widespread application in breeding programs, thereby facilitating genome-assisted crop improvement.
RESUMEN
BACKGROUND: Colorectal cancer (CRC) is the third most common cancer in the world, and a strong relationship exists between CRC and gut microbiota, which affects the occurrence, development, and metastasis of cancer. Bioinformatics-based analyses revealed that the abundance of Parvimonas micra (P. micra) in the feces of patients with cancer is significantly higher than that in healthy people. Therefore, an important relationship may exist between P. micra and CRC. METHODS: We first confirmed that P. micra can promote the proliferation of cell lines through cell experiments and mouse models. Then we selected the signaling pathways and content of exosomes to promote the development of CRC by transcriptomics and microRNA sequencing. Finally, we confirmed that P. micra promoted CRC development through miR-218-5p/Ras/ERK/c-Fos pathway through the in vivo and in vitro experiments. RESULTS: First, it was confirmed by in vitro and in vivo experiments that P. micra can promote the development of CRC. Transcriptome analysis after the coincubation of bacteria and cells revealed that P. micra promoted cell proliferation by activating the Ras/ERK/c-Fos pathway. Furthermore, microRNA sequencing analysis of the cells and exosomes showed that miR-218-5p and protein tyrosine phosphatase receptor R (PTPRR) were the key factors involved in activating the Ras/ERK/c-Fos pathway, and the miR-218-5p inhibitor was used to confirm the role of microRNA in xenograft mice. CONCLUSION: This experiment confirmed that P. micra promoted the development of CRC by upregulating miR-218-5p expression in cells and exosomes, inhibiting PTPRR expression, and ultimately activating the Ras/ERK/c-Fos signaling pathway.
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Neoplasias Colorrectales , Firmicutes , MicroARNs , Animales , Humanos , Ratones , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/microbiología , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Transducción de Señal , Firmicutes/patogenicidadRESUMEN
BACKGROUND: The elevated circulating toxins secondary to the impairment of intestinal barrier integrity commonly elicit a chronic inflammatory response and finally contribute to multiple diseases. These toxins, including bacterial by-products and heavy metals, are the potent risk factors for the development of recurrent spontaneous abortion (RSA). Preclinical evidence suggests that several dietary fibers can restore intestinal barrier function and decrease the accumulation of heavy metals. However, it is uncertain whether treatment with a newly developed blend of dietary fibers product (Holofood) benefits patients with RSA. METHODS: In this trial, we enrolled 70 adult women with RSA, who were randomly assigned into the experiment group and the control group in a 2:1 ratio. Upon the basis of conventional therapy, subjects in the experiment group (n = 48) received 8 weeks oral administration with Holofood three times daily at a dose of 10 g each time. Subjects without Holofood consumption were set as the control (n = 22). Blood samples were collected for the determinations of metabolic parameters, heavy mental lead, and the indices related to intestinal barrier integrity (D-lactate, bacterial endotoxin, and diamine oxidase activity). RESULTS: The reduction amplitude in blood lead from baseline to week 8 was 40.50 ± 54.28 (µg/L) in the experiment group as compared with 13.35 ± 36.81 (µg/L) in the control group (P = 0.037). The decreased level of serum D-lactate from baseline to week 8 was 5.58 ± 6.09 (mg/L) in the experiment group as compared with - 2.38 ± 8.90 (mg/L, P < 0.0001) in the control group. The change in serum DAO activity from baseline to week 8 was 3.26 ± 2.23 (U/L) in the experiment group as compared with - 1.24 ± 2.22 (U/L, P < 0.0001) in the control group. Participants who received Holofood had a greater decline in blood endotoxin from baseline to week 8 than those in the control group. Moreover, by comparing with the self-baseline, Holofood consumption significantly decreased the blood levels of lead, D-lactate, bacterial endotoxin, and DAO activity. CONCLUSION: Our results suggest that Holofood affords a clinically relevant improvements in blood lead level and intestinal barrier dysfunction in patients with RSA.
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Aborto Espontáneo , Plomo , Humanos , Adulto , Femenino , Embarazo , Plomo/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Aborto Espontáneo/metabolismo , Endotoxinas/metabolismo , Fibras de la Dieta/uso terapéutico , Fibras de la Dieta/metabolismo , Ácido Láctico/metabolismoRESUMEN
Culturomics employs various cultivating conditions to obtain different types of bacteria and new species. However, current culturomics lacks a highly efficient method for isolating specific pathobionts. Immunomagnetic bead technology, which uses magnetic beads conjugated with antibodies for capturing the antigen to realize enrichment of the targets, has been employed as an alternative method. In this study, we developed a novel method, immunomagnetic bead-enriched culturomics (IMBEC), in which magnetic bead-conjugated antibodies purified from the fecal samples of patients with colorectal cancer (CRC) were used to enrich and isolate potential pathobionts. A protocol for enriching potential pathobionts via immunomagnetic capture was developed by optimizing the concentrations of coupling reagents, NaCl, and detergent. The efficacy of pathobiont enrichment was compared between antibody-coated magnetic beads (antibody group) and nonconjugated blank magnetic beads (blank group). To determine the proinflammatory potential of isolates from both groups, we investigated their ability to induce cytokine production in THP-1 macrophages. This protocol was employed for isolating bacteria from 10 fecal samples of patients with CRC, which were simultaneously compared with those isolated from the blank group. A total of 209 bacterial species were isolated from both groups, including 173 from the antibody group, 160 from the blank group, and 124 from both groups. Bacteria isolated from the antibody group produced more proinflammatory cytokines than those isolated from the blank group. IMBEC is a promising method for relatively specific isolation of potential pathobionts for a particular disease of interest.