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.

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.

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.

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.

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.

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.

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.

Adenylate Kinase 4 Modulates the Resistance of Breast Cancer Cells to Tamoxifen through an m6A-Based Epitranscriptomic Mechanism.

N6-methyladenosine (m6A) is the most abundant internal modification in mRNA and this methylation constitutes an important regulatory mechanism for the stability and translational efficiency of mRNA. In this study, we found that the protein levels of adenylate kinase 4 (AK4) and m6A writer METTL3 are significantly higher in tamoxifen-resistant (TamR) MCF-7 cells than in parental cells. The TamR MCF-7 cells also exhibit increased methylation at multiple m6A consensus motif sites in the 5' untranslated region (5' UTR) of AK4 mRNA, and genetic depletion of METTL3 in TamR MCF-7 cells led to a diminished AK4 protein level and attenuated resistance to tamoxifen. In addition, we observed augmented levels of reactive oxygen species (ROS) and p38 activity in TamR MCF-7 cells, and both are diminished upon genetic depletion of AK4. Reciprocally, overexpression of AK4 in MCF-7 cells stimulates ROS and p38 phosphorylation levels, and it suppresses mitochondrial apoptosis. Moreover, scavenging of intracellular ROS leads to reduced p38 activity and re-sensitizes TamR MCF-7 cells to tamoxifen. Thus, our results uncover a novel m6A-mediated epitranscriptomic mechanism for the regulation of AK4, illustrate the cellular pathways through which increased AK4 expression contributes to tamoxifen resistance, and reveal AK4 as a potential therapeutic target for overcoming tamoxifen resistance.

Elevated Hexokinase II Expression Confers Acquired Resistance to 4-Hydroxytamoxifen in Breast Cancer Cells.

Tamoxifen has been clinically used in treating estrogen receptor (ER)-positive breast cancer for over 30 years. The most challenging aspect associated with tamoxifen therapy is the development of resistance in initially responsive breast tumors. We applied a parallel-reaction monitoring (PRM)-based quantitative proteomic method to examine the differential expression of kinase proteins in MCF-7 and the isogenic tamoxifen-resistant (TamR) cells. We were able to quantify the relative protein expression levels of 315 kinases, among which hexokinase 2 (HK2) and mTOR were up-regulated in TamR MCF-7 cells. We also observed that the TamR MCF-7 cells exhibited elevated rate of glycolysis than the parental MCF-7 cells. In addition, we found that phosphorylation of S6K - a target of mTOR - was much lower in TamR MCF-7 cells, and this phosphorylation level could be restored upon genetic depletion or pharmacological inhibition of HK2. Reciprocally, the level of S6K phosphorylation was diminished upon overexpression of HK2 in MCF-7 cells. Moreover, we observed that HK2 interacts with mTOR, and this interaction inhibits mTOR activity. Lower mTOR activity led to augmented autophagy, which conferred resistance of MCF-7 cells toward tamoxifen. Together, our study demonstrates that elevated expression of HK2 promotes autophagy through inhibiting the mTOR-S6K signaling pathway and results in resistance of MCF-7 breast cancer cells toward tamoxifen; thus, our results uncovered, for the first time, HK2 as a potential therapeutic target for overcoming tamoxifen resistance.

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.

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.

YTHDF2 Binds to 5-Methylcytosine in RNA and Modulates the Maturation of Ribosomal RNA.

5-Methylcytosine is found in both DNA and RNA; although its functions in DNA are well established, the exact role of 5-methylcytidine (m5C) in RNA remains poorly defined. Here we identified, by employing a quantitative proteomics method, multiple candidate recognition proteins of m5C in RNA, including several YTH domain-containing family (YTHDF) proteins. We showed that YTHDF2 could bind directly to m5C in RNA, albeit at a lower affinity than that toward N6-methyladenosine (m6A) in RNA, and this binding involves Trp432, a conserved residue located in the hydrophobic pocket of YTHDF2 that is also required for m6A recognition. RNA bisulfite sequencing results revealed that, after CRISPR-Cas9-mediated knockout of the YTHDF2 gene, the majority of m5C sites in rRNA (rRNA) exhibited substantially augmented levels of methylation. Moreover, we found that YTHDF2 is involved in pre-rRNA processing in cells. Together, our data expanded the functions of the YTHDF2 protein in post-transcriptional regulations of RNA and provided novel insights into the functions of m5C in RNA biology.

Lipopolysaccharide Stimulates the Growth of Bacteria That Contribute to Ruminal Acidosis.

Lipopolysaccharide (LPS) has been reported to contribute to a ruminal acidosis of cattle by affecting ruminal bacteria. The goal of this study was to determine how LPS affects the growth of pure cultures of ruminal bacteria, including those that contribute to ruminal acidosis. We found that dosing LPS (200,000 EU) increased the maximum specific growth rates of four ruminal bacterial species (Streptococcus bovis JB1, Succinivibrio dextrinosolvens 24, Lactobacillus ruminis RF1, and Selenomonas ruminantium HD4). Interestingly, all the species ferment sugars and produce lactate, contributing to acidosis. Species that consume lactate or ferment fiber were not affected by LPS. We found that S. bovis JB1 failed to grow in LPS as the carbon source in the media; growth of S. bovis JB1 was increased by LPS when glucose was present. Growth of Megasphaera elsdenii T81, which consumes lactate, was not different between the detoxified (lipid A delipidated) and regular LPS. However, the maximum specific growth rate of S. bovis JB1 was greater in regular LPS than detoxified LPS. Mixed bacteria from a dual-flow continuous culture system were collected to determine changes of metabolic capabilities of bacteria by LPS, and genes associated with LPS biosynthesis were increased by LPS. In summary, LPS was not toxic to bacteria, and lipid A of LPS stimulated the growth of lactate-producing bacteria. Our results indicate that LPS not only is increased during acidosis but also may contribute to ruminal acidosis development by increasing the growth of lactic acid-producing bacteria.IMPORTANCE Gram-negative bacteria contain lipopolysaccharide (LPS) coating their thin peptidoglycan cell wall. The presence of LPS has been suggested to be associated with a metabolic disorder of cattle-ruminal acidosis-through affecting ruminal bacteria. Ruminal acidosis could reduce feed intake and milk production and increase the incidence of diarrhea, milk fat depression, liver abscesses, and laminitis. However, how LPS affects bacteria associated with ruminal acidosis has not been studied. In this study, we investigated how LPS affects the growth of ruminal bacteria by pure cultures, including those that contribute to acidosis, and the functional genes of ruminal bacteria. Thus, this work serves to further our understanding of the roles of LPS in the pathogenesis of ruminal acidosis, as well as providing information that may be useful for the prevention of ruminal acidosis and reducetion of economic losses for farmers.

Budesonide/Fomoterol in combination with Montelukast in the treatment of Bronchial Asthma.

OBJECTIVE: To analyze the clinical effect of budesonide/fomoterol combined with montelukast in the treatment of chronic persistent asthma. METHODS: Ninety-four patients with asthma who came to our hospital for treatment from April 2017 to April 2019 were randomly divided into control group and observation group, with 47 patients in each group. The control group was treated with budesonide/formoterol, and the observation group was treated with montelukast on the basis of the control group. The treatment effect of the two groups was observed and compared. RESULTS: The total efficacy rate of the observation group was significantly higher than that of the control group (P<0.05); the daytime symptom score and nighttime symptom score of the observation group were significantly higher than those of the control group (P<0.05). The pulmonary function indexes of the two groups after treatment were significantly higher than that before treatment, and the improvement of the observation group was more significant (P<0.05); the FeNO and EO levels of the observation group after treatment were superior to those of the control group, and the difference was statistically significant (P<0.05). CONCLUSION: Budesonide/formoterol powder inhalation combined with montelukast can effectively improve the lung function, reduce the level of inflammatory factors, and accelerate the regression of symptoms in the treatment of chronic persistent asthma. It is worth clinical application.

Characterization of the L genome segment of an orthohantavirus isolated from Niviventer confucianus.

We sequenced and analyzed the L segment of the RNA genome of Hantaan virus (HTNV) strain NC167. This segment is 6,533 nucleotides in length and contains a single open reading frame (ORF) of 6,456 nucleotides in the antigenome sense that encodes the viral RNA-dependent RNA polymerase, which is 2,153 amino acids long with a predicted molecular mass of 246 kDa. The 5' terminus of the viral RNA was found to contain the expected sequences that are conserved in orthohantaviruses. According to the phylogenetics and levels of sequence similarity, the L segment of HTNV NC167 is similar to but clearly distinct from the L segments of other orthohantaviruses.

Efficient Elimination of Chlorinated Organics on a Phosphoric Acid Modified CeO2 Catalyst: A Hydrolytic Destruction Route.

The development of efficient technologies to prevent the emission of hazardous chlorinated organics from industrial sources without forming harmful byproducts, such as dioxins, is a major challenge in environmental chemistry. Herein, we report a new hydrolytic destruction route for efficient chlorinated organics elimination and demonstrate that phosphoric acid-modified CeO2 (HP-CeO2) can decompose chlorobenzene (CB) without forming polychlorinated congeners under the industry-relevant reaction conditions. The active site and reaction pathway were investigated, and it was found that surface phosphate groups initially react with CB and water to form phenol and HCl, followed by deep oxidation. The high on-stream stability of the catalyst was due to the efficient generation of HCl, which removes Cl from the catalyst surface and ensures O2 activation and therefore deep oxidation of the hydrocarbons. Subsequent density functional theory calculations revealed a distinctly decreased formation energy of an oxygen vacancy at nearest (VO-1) and next-nearest (VO-2) surface sites to the bonded phosphate groups, which likely contributes to the high rate of oxidation observed over the catalyst. Significantly, no dioxins, which are frequently formed in the conventional oxidation route, were observed. This work not only reports an efficient route and corresponding phosphate active site for chlorinated organics elimination but also illustrates that the rational design of the reaction route can solve some of the most important challenges in environmental catalysis.

Position-dependent effects of regioisomeric methylated adenine and guanine ribonucleosides on translation.

Reversible methylation of the N6 or N1 position of adenine in RNA has recently been shown to play significant roles in regulating the functions of RNA. RNA can also be alkylated upon exposure to endogenous and exogenous alkylating agents. Here we examined how regio-specific methylation at the hydrogen bonding edge of adenine and guanine in mRNA affects translation. When situated at the third codon position, the methylated nucleosides did not compromise the speed or accuracy of translation under most circumstances. When located at the first or second codon position, N1-methyladenosine (m1A) and m1G constituted robust blocks to both Escherichia coli and wheat germ extract translation systems, whereas N2-methylguanosine (m2G) moderately impeded translation. While m1A, m2G and N6-methyladenosine (m6A) did not perturb translational fidelity, O6-methylguanosine (m6G) at the first and second codon positions was strongly and moderately miscoding, respectively, and it was decoded as an adenosine in both systems. The effects of methylated ribonucleosides on translation could be attributed to the methylation-elicited alterations in base pairing properties of the nucleobases, and the mechanisms of ribosomal decoding contributed to the position-dependent effects. Together, our study afforded important new knowledge about the modulation of translation by methylation of purine nucleobases in mRNA.

Identification of YTH Domain-Containing Proteins as the Readers for N1-Methyladenosine in RNA.

N1-methyladenosine (m1A) is an important post-transcriptional modification in RNA; however, the exact biological role of m1A remains to be determined. By employing a quantitative proteomics method, we identified multiple putative protein readers of m1A in RNA, including several YTH domain family proteins. We showed that YTHDF1-3 and YTHDC1, but not YTHDC2, could bind directly to m1A in RNA. We also found that Trp432 in YTHDF2, a conserved residue in the hydrophobic pocket of the YTH domain that is necessary for its binding to N6-methyladenosine (m6A), is required for its recognition of m1A. An analysis of previously published data revealed transcriptome-wide colocalization of YTH domain-containing proteins and m1A sites in HeLa cells, suggesting that YTH domain-containing proteins can bind to m1A in cells. Together, our results uncovered YTH domain-containing proteins as readers for m1A in RNA and provided new insight into the functions of m1A in RNA biology.