Human RNA-binding protein HNRNPD interacts with and regulates the repair of deoxyribouridine in DNA.

Deoxyribouridine (dU) is an abnormal nucleoside in DNA and plays vital roles in multiple biological and physiological processes. Here, we conducted a mass spectrometry-based screen for dU-binding proteins and found that the heterogeneous nuclear ribonucleoprotein D (HNRNPD) could preferentially bind to dU-containing DNA. We also discovered that HNRNPD engages in the 5-Fluorouracil (5FU)-induced DNA damage response and can modulate the repair of dU in DNA in vitro and in human cells. Moreover, using a shuttle vector- and next-generation sequencing-based method, we unveiled the crucial role of HNRNPD in promoting the replicative bypass of dU in human cells. Taken together, these findings suggested that HNRNPD is a novel dU-bearing DNA-binding protein capable of regulating the removal of dU in DNA, and provided new insights into the molecular mechanisms of dU-associated diseases.

m1A in CAG repeat RNA binds to TDP-43 and induces neurodegeneration.

Microsatellite repeat expansions within genes contribute to a number of neurological diseases1,2. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology3-9. Here we reveal that the adenosine in CAG repeat RNA can be methylated to N1-methyladenosine (m1A) by TRMT61A, and that m1A can be demethylated by ALKBH3. We also observed that the m1A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m1A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m1A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, our study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m1A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion.

Single-step genome-wide association analyses of claw horn lesions in Holstein cattle using linear and threshold models.

BACKGROUND: Lameness in dairy cattle is primarily caused by foot lesions including the claw horn lesions (CHL) of sole haemorrhage (SH), sole ulcers (SU), and white line disease (WL). This study investigated the genetic architecture of the three CHL based on detailed animal phenotypes of CHL susceptibility and severity. Estimation of genetic parameters and breeding values, single-step genome-wide association analyses, and functional enrichment analyses were performed. RESULTS: The studied traits were under genetic control with a low to moderate heritability. Heritability estimates of SH and SU susceptibility on the liability scale were 0.29 and 0.35, respectively. Heritability of SH and SU severity were 0.12 and 0.07, respectively. Heritability of WL was relatively lower, indicating stronger environmental influence on the presence and development of WL than the other two CHL. Genetic correlations between SH and SU were high (0.98 for lesion susceptibility and 0.59 for lesion severity), whereas genetic correlations of SH and SU with WL also tended to be positive. Candidate quantitative trait loci (QTL) were identified for all CHL, including some on Bos taurus chromosome (BTA) 3 and 18 with potential pleiotropic effects associated with multiple foot lesion traits. A genomic window of 0.65 Mb on BTA3 explained 0.41, 0.50, 0.38, and 0.49% of the genetic variance for SH susceptibility, SH severity, WL susceptibility, and WL severity, respectively. Another window on BTA18 explained 0.66, 0.41, and 0.70% of the genetic variance for SH susceptibility, SU susceptibility, and SU severity, respectively. The candidate genomic regions associated with CHL harbour annotated genes that are linked to immune system function and inflammation responses, lipid metabolism, calcium ion activities, and neuronal excitability. CONCLUSIONS: The studied CHL are complex traits with a polygenic mode of inheritance. Most traits exhibited genetic variation suggesting that animal resistance to CHL can be improved with breeding. The CHL traits were positively correlated, which will facilitate genetic improvement for resistance to CHL as a whole. Candidate genomic regions associated with lesion susceptibility and severity of SH, SU, and WL provide insights into a global profile of the genetic background underlying CHL and inform genetic improvement programmes aiming at enhancing foot health in dairy cattle.

Human Mitochondrial Protein HSPD1 Binds to and Regulates the Repair of Deoxyinosine in DNA.

The generation of deoxyinosine (dI) in DNA is one of the most important sources of genetic mutations, which may lead to cancer and other human diseases. A further understanding of the biological consequences of dI necessitates the identification and functional characterizations of dI-binding proteins. Herein, we employed a mass spectrometry-based proteomics approach to detect the cellular proteins that may sense the presence of dI in DNA. Our results demonstrated that human mitochondrial heat shock protein 60 (HSPD1) can interact with dI-bearing DNA. We further demonstrated the involvement of HSPD1 in the sodium nitrite-induced DNA damage response and in the modulation of dI levels in vitro and in human cells. Together, these findings revealed HSPD1 as a novel dI-binding protein that may play an important role in the mitochondrial DNA damage control in human cells.

Next-Generation Sequencing-Based Analysis of the Effects of N1- and N6-Methyldeoxyadenosine Adducts on DNA Transcription.

DNA methylation can occur naturally or be induced by various environmental and chemotherapeutic agents. The regioisomeric N1- and N6-methyldeoxyadenosine (1mdA and 6mdA, respectively) represent an important class of methylated DNA adducts. In this study, we developed a shuttle vector- and next-generation sequencing-based assay to quantitatively assess the effects of 1mdA and 6mdA on the accuracy and efficiency of DNA transcription. Our results revealed that 1mdA can induce multiple types of mutant transcripts and strongly inhibit DNA transcription, whereas 6mdA is a nonmutagenic DNA adduct that can exhibit a subtle but significant inhibitory effect on DNA transcription in vitro and in human cells. Moreover, our results demonstrated that the transcription-coupled nucleotide excision repair pathway is dispensable for the removal of 1mdA and 6mdA from the template DNA strand in human cells. These findings provided new important insights into the functional interplay between DNA methylation modifications and transcription in mammalian cells.

Development of an Endonuclease V-Assisted Analytical Method for Sequencing Analysis of Deoxyinosine in DNA.

Deoxyinosine (dI) is a highly mutagenic lesion that preferentially pairs with deoxycytidine during replication, which may induce A to G transition and ultimately contribute to carcinogenesis. Therefore, finding the site of dI modification in DNA is of great value for both basic research and clinical applications. Herein, we developed a novel method to sequence the dI modification site in DNA, which utilizes endonuclease V (EndoV)-dependent deamination repair to specifically label the modification site with biotin-14-dATP that allows the affinity enrichment of dI-bearing DNA for sequencing. We have achieved efficient determination of the location of the modified nucleotide in dI-bearing plasmid DNA with the assistance of EndoV-dependent deamination repair. We have also successfully applied this approach to locate the dI modification sites in the mitochondrial DNA of human cells. Our method should be generally applicable for genome-wide sequencing analysis of dI modifications in living organisms.

Next-Generation Sequencing-Based Analysis of the Roles of DNA Polymerases ν and θ in the Replicative Bypass of 8-Oxo-7,8-dihydroguanine in Human Cells.

DNA polymerase (Pol) ν and Pol θ are two specialized A-family DNA polymerases that function in the translesion synthesis of certain DNA lesions. However, the biological functions of human Pols ν and θ in cellular replicative bypass of 8-oxo-7,8-dihydroguanine (8-oxoG), an important carcinogenesis-related biomarker of oxidative DNA damage, remain unclear. Herein, we showed that depletion of Pols ν and θ in human cells could cause an elevated hypersensitivity to oxidative stress induced by hydrogen peroxide. Using next-generation sequencing-based lesion bypass and mutagenesis assay, we further demonstrated that Pols ν and θ had important roles in promoting translesion synthesis of 8-oxoG in human cells. We also found that the depletion of Pol ν, but not Pol θ, caused a substantial reduction in G → T mutation frequency for 8-oxoG. These findings provided novel insights into the involvement of A-family DNA polymerases in oxidative DNA damage response.

Mechanism associated with the positive effect of nanocellulose on nitrogen retention in a manure composting system.

Additives can play important roles in effectively inhibiting nitrogen losses during livestock manure composting due to the activities of microbes. This study investigated the effects of adding nanocellulose at 300 mg/kg, 600 mg/kg, and 900 mg/kg (NC900) on nitrogen conversion, nitrogen conversion functional genes, and related microorganisms during composting. The results showed that compared with the control, nanocellulose hindered the ammoniation reaction. In addition, NC900 promoted nitrification, interfered with the denitrification process, and reduced the abundance of the nirK gene, thereby increasing the nitrate nitrogen content and decreasing ammonia spillover. NC900 promoted nitrogen fixation by increasing the abundance of members of Rhizobiales, which play important roles in nitrogen fixation. In general, compared with the control, NC900 improved the retention of nitrogen by controlling ammonia emissions. The results obtained in this study demonstrate that nanocellulose can be applied in the treatment of organic solid waste and agricultural production.

Weighted Gene Co-expression Network Analysis Identifies Specific Modules and Hub Genes Related to Subacute Ruminal Acidosis.

Weighted gene co-expression network analysis (WGCNA) was used to understand the pathogenesis of subacute ruminal acidosis (SARA) and identify potential genes related to the disease. Microarray data from dataset GSE143765, which included 22 cows with and nine cows without SARA, were downloaded from the NCBI Gene Expression Omnibus (GEO). Results of WGCNA identified highly correlated modules of sample genes, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses allowed further biological insights into SARA-related modules. The protein-protein interaction (PPI) network, modules from the PPI network, and cistron annotation enrichment of modules were also analyzed. A total of 14,590 DEGs were used for the WGCNA. Construction of a protein-protein network identified DCXR, MMP15, and MMP17 as hub genes. Functional annotation showed that DCXR mainly exhibited L-xylulose reductase (NADP+) activity, glucose metabolic process, xylulose metabolic process, and carbonyl reductase (NADPH) activity, which are involved in the pentose and glucuronate interconversion pathways. MMP15 and MMP17 mainly have a collagen catabolic process. Overall, the results of this study aid the clarification of the biological and metabolic processes associated with SARA at the molecular level. The data highlight potential mechanisms for the future development of intervention strategies to reduce or alleviate the risk of SARA.

Comprehensive expression profiles of mRNAs, lncRNAs and miRNAs in Kashin-Beck disease identified by RNA-sequencing.

Kashin-Beck disease (KBD) is a chronic, endemic and deforming osteochondropathy, whose basic pathological alterations include apoptosis and necrosis of chondrocytes in articular cartilage and growth plates and imbalanced extracellular matrix metabolism. Numerous studies have reported that long noncoding RNAs (lncRNAs) and microRNA (miRNAs) are aberrantly expressed in KBD. Our study was comprised of 5 KBD patients and 5 healthy individuals and we compared the expression profiles of mRNAs, lncRNAs and miRNAs through RNA-sequencing (RNA-seq). Bioinformatic analysis of GO and KEGG was employed to conduct functional annotation and pathway enriched analysis. In total, 3194 mRNAs, 4103 lncRNAs and 1550 miRNAs were detected to be differentially expressed by RNA-seq (P < 0.05; |log2FC| ≥1). The lysosome pathway, Wnt signaling pathway, TNF signaling pathway, endocytosis and mTOR signaling pathway were identified to be involved in the KBD development according to the result of the KEGG analysis. In addition, a ceRNA network based on lncRNA-miRNA-mRNA was constructed to probe the intricate regulatory mechanism and interaction between transcripts, which was visualized using the Cytoscape software. The ce-lncRNAs of four aberrantly expressed genes, FOSB, EGR3, BCAM and SOX6, were determined through the network. Among the identified DElncRNAs, we selected 8 differentially expressed lncRNAs to confirm the reliability of RNA-seq data by qRT-PCR in 11 KBD patients and 11 healthy individuals. We aimed to provide a comprehensive understanding ofmRNA, lncRNA and miRNA alterations between KBD patients and healthy individuals, and meanwhile reveal several potential causative molecular and signaling pathways involved in KBD.

Insights into the associations of copper and zinc with nitrogen metabolism during manure composting with shrimp shell powder.

The application of shrimp shell powder (SSP) in manure composting can promote the maturation of compost and reduce the associated environmental risk. This study investigated the response of adding SSP at different levels (CK: 0, L: 5%, M: 10%, and H: 15%) on heavy metal resistance genes (MRGs), nitrogen functional genes, enzymes, and microorganisms. SSP inhibited nitrification and denitrification via decreasing the abundances of functional genes and key enzymes related to Cu, Zn, and MRGs. The nitrate reductase and nitrous-oxide reductase in the denitrification pathway were lower under H. Phylogenetic trees indicated that Burkholderiales sp. had strong relationships with OTU396 and OTU333, with important roles in the nitrogen cycle and plant growth. Redundancy analysis and structural equation modeling showed the complex response between heavy metal and nitrogen that bio-Cu and bio-Zn had positive significantly relationships with nirK-type and amoA-type bacteria, and amoA-type bacteria might be hotspot of cueO.

Responses of bacterial communities and antibiotic resistance genes to nano-cellulose addition during pig manure composting.

Treatment with exogenous additives during composting can help to alleviate the accumulation of antibiotic resistance genes (ARGs) caused by the direct application of pig manure to farmland. In addition, nano-cellulose has an excellent capacity for adsorbing pollutants. Thus, the effects of adding 300, 600, and 900 mg/kg nano-cellulose to compost on the bacterial communities, mobile genetic elements (MGEs), and ARGs were determined in this study. After composting, treatment with nano-cellulose significantly reduced the relative abundance of ARGs, which was lowest in the compost product with 600 mg/kg added nano-cellulose. Nano-cellulose inhibited the rebound in ARGs from the cooling period to the maturity period, and weakened the selective pressure of heavy metals on microorganisms by passivating bio-Cu. The results also showed that MGEs explained most of the changes in the abundances of ARGs, and MGEs had direct effects on ARGs. The addition of 600 mg/kg nano-cellulose reduced the abundances of bacterial genera associated with ermQ, tetG, and other genes, and the number of links (16) between ARGs and MGEs was lowest in the treatment with 600 mg/kg added nano-cellulose. Therefore, adding 600 mg/kg nano-cellulose reduced the abundances of ARGs by affecting host bacteria and MGEs. The results obtained in this study demonstrate the positive effect of nano-cellulose on ARG pollution in poultry manure, where adding 600 mg/kg nano-cellulose was most effective at reducing the abundances of ARGs.

ATF3 Modulates the Resistance of Breast Cancer Cells to Tamoxifen through an N6-Methyladenosine-Based Epitranscriptomic Mechanism.

Tamoxifen has been used for years for treating estrogen receptor-positive breast cancer; drug resistance, however, constitutes one of the main challenges for this therapy. We found that the protein expression level of ATF3 is significantly higher in tamoxifen-resistant (TamR) MCF-7 cells than the corresponding parental cancer cells. In addition, ATF3 protein expression is positively correlated with the resistance of TamR MCF-7 cells to 4-hydroxytamoxifen (4-OHT). Mechanistically, elevated ATF3 protein expression in TamR MCF-7 cells results from a lower level of expression of YTHDF2, an m6A reader protein, and the ensuing stabilization and increased translational efficiency of ATF3 mRNA. Additionally, TamR MCF-7 cells exhibited decreased methylation at A131, a consensus motif site for m6A, in the 5'-untranslated region (5'-UTR) of ATF3 mRNA. Moreover, augmented ATF3 stimulates the expression of ABCB1, an efflux pump that confers drug resistance in breast cancer cells, and ATF3 itself is also positively regulated by adenylate kinase 4. Together, our results uncovered a novel molecular target for m6A modification (i.e., ATF3 mRNA) and the epitranscriptomic regulator for this target (i.e., YTHDF2). We also illustrated the role of ATF3 in drug resistance, revealed its downstream target (i.e., ABCB1), and suggested ATF3 as a candidate therapeutic target for overcoming drug resistance in cancer cells.

The genome of the warm-season turfgrass African bermudagrass (Cynodon transvaalensis).

Cynodon species can be used for multiple purposes and have high economic and ecological significance. However, the genetic basis of the favorable agronomic traits of Cynodon species is poorly understood, partially due to the limited availability of genomic resources. In this study, we report a chromosome-scale genome assembly of a diploid Cynodon species, C. transvaalensis, obtained by combining Illumina and Nanopore sequencing, BioNano, and Hi-C. The assembly contains 282 scaffolds (~423.42 Mb, N50 = 5.37 Mb), which cover ~93.2% of the estimated genome of C. transvaalensis (~454.4 Mb). Furthermore, 90.48% of the scaffolds (~383.08 Mb) were anchored to nine pseudomolecules, of which the largest was 60.78 Mb in length. Evolutionary analysis along with transcriptome comparison provided a preliminary genomic basis for the adaptation of this species to tropical and/or subtropical climates, typically with dry summers. The genomic resources generated in this study will not only facilitate evolutionary studies of the Chloridoideae subfamily, in particular, the Cynodonteae tribe, but also facilitate functional genomic research and genetic breeding in Cynodon species for new leading turfgrass cultivars in the future.

Chemical proteomic profiling of UTP-binding proteins in human cells.

Nucleotide-binding proteins play important roles in a variety of biological processes. While ATP- and GTP-binding proteins have been well studied, the systematical identification of UTP-interacting proteins remains under investigated. Here, we developed a chemical proteomic strategy using a biotinylated UTP affinity probe coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) method to enrich, identify and quantify UTP-binding proteins at the entire proteome scale. By performing labeling reactions with high vs low concentrations of UTP probe (100 and 10 µM) or with the UTP probe in the presence of free UTP in stable isotope labeling by amino acids in cell culture (SILAC) experiments, we identified more than 70 potential UTP-binding proteins which are involved in multiple cellular processes, such as translational elongation and protein folding. We also validated the UTP-binding capability of the cytoskeletal protein ACTB by using cellular thermal shift assay (CETSA). Together, we performed a high-throughput chemical proteomics-based analysis to identify, for the first time, UTP-binding proteins in human proteome, which should be applicable for the identification and quantification of UTP-binding proteins in other organisms.

Enhanced removal of antibiotic resistance genes and mobile genetic elements during swine manure composting inoculated with mature compost.

Livestock manure is a major source of antibiotic resistance genes (ARGs) that enter the environment. This study assessed the effects of inoculation with mature compost (MC) on the fates of ARGs and the bacterial community during swine manure composting. The results showed that MC prolonged the thermophilic period and promoted the decomposition of organic matter, which was due to the rapid growth and reproduction of thermophilic bacteria (Bacillus, Thermobifida, and Thermobacillus). MC significantly reduced the relative abundances of ARGs (1.02 logs) and mobile genetic elements (MGEs) (1.70 logs) after composting, especially sulfanilamide resistance genes. The total ARGs removal rate was 1.11 times higher in MC than the control. Redundancy analysis and structural equation modeling showed that horizontal gene transfer mediated by MGEs (ISCR1 and intI1) was the main direct factor related to the changes in ARGs during composting, whereas the C/N ratio and pH were the two most important indirect factors. Network analysis showed that members of Firmicutes comprising Romboutsia, Clostridisensu_stricto_1, and Terrisporobacter were the main bacterial hosts of ARGs and MGEs. MC reduced the risk of ARGs transmission by decreasing the abundances of bacterial hosts. Thus, MC is a promising strategy for reducing the proliferation risk of ARGs.

Modulation of N-terminal methyltransferase 1 by an N6-methyladenosine-based epitranscriptomic mechanism.

Protein α-N-methylation is an evolutionarily conserved type of post-translational modification; however, little is known about the regulatory mechanisms for this modification. Methylation at the N6 position of adenosine in mRNAs is dynamic and modulates their stability, splicing, and translational efficiency. Here, we found that the expression of N-terminal methyltransferase 1 (NTMT1) protein is altered by depletion of those genes encoding the reader/writer/eraser proteins of N6-methyladenosine (m6A). We also observed that MRG15 is N-terminally methylated by NTMT1, and this methylation could also be modulated by reader/writer/eraser proteins of m6A. Together, these results revealed a novel m6A-based epitranscriptomic mechanism in regulating protein N-terminal methylation.

Microbial succession and molecular ecological networks response to the addition of superphosphate and phosphogypsum during swine manure composting.

This study assessed the effects of superphosphate (SPP) and phosphogypsum (PPG) on the bacterial and fungal community succession and molecular ecological networks during composting. Adding SPP and PPG had positive effects on the bacterial richness and diversity, negative effects on the fungal richness and diversity. The microbial diversity and richness were higher in PPG than SPP. Non-metric multidimensional scaling analysis clearly separated SPP and PPG from the control treatment with no additives. The dominant genera comprised Turicibacter, Bacillus, norank_o_SBR1031, Thermobifida, norank_f_Limnochordaceae, Truepera, Thermopolyspora, Mycothermus, Dipodascus, Thermomyces, and unclassified_p_Ascomycota. In all treatments, the major bacterial species differed clearly in the later thermophilic, cooling, and maturation composting stages, whereas the main fungal species varied significantly in the thermophilic stage. The changes in the dominant microorganisms in SPP and PPG may have inhibited or promoted the degradation of organic matter during various composting stages. Adding SPP and PPG led to more complex bacterial networks and less complex fungal networks, where SPP had more adverse effects on the fungal networks than PPG. SPP and PPG could potentially alter the co-occurrence patterns of the bacterial and fungal communities by changing the most influential species. SPP and PPG changed the composition and succession of the microbial community by influencing different physiochemical properties during various composting stages where the pH was the main explanatory factor. Overall, this study provides new insights into the effects of SPP and PPG on the microbial community and its interactions during composting.

Effects of phosphogypsum and medical stone on nitrogen transformation, nitrogen functional genes, and bacterial community during aerobic composting.

This study explored the effects of adding phosphogypsum (PPG), medical stone (MS), and both (PPM) during composting on nitrogen transformation, nitrogen functional genes, the bacterial community, and their relationships with NH3 and N2O emissions. Adding MS and PPM reduced NH3 emissions by 25.78-68.37% and N2O emissions by 19.00-42.86%. PPG reduced NH3 emissions by 59.74% but slightly increased N2O emissions by 8.15%. MS was strongly correlated with the amoA-dominated nitrification process. PPG and PPM had strong correlations with nirS- and nirK-dominated, and nosZ-dominated denitrification processes, respectively. PPM promoted nitrification and denitrification processes more than PPG and MS. Different functional bacteria had key roles in nitrification and denitrification during different composting stages. Firmicutes probably contributed to the conversion and release of nitrogen in the thermophilic period, whereas Proteobacteria, Chloroflexi, Bacteroidetes, and other phyla might have played important roles in the cooling and maturation periods. PPM obtained the greatest reductions in NH3 and N2O release via the regulation of environmental variables, nitrogen functional genes, and the bacterial community. Overall, these results provide insights at a molecular level into the effects of PPG and MS on nitrogen transformation and NH3 and N2O emissions during composting.