Aberrant DNA N6 -methyladenine incorporation via adenylate kinase 1 is suppressed by ADAL deaminase-dependent 2'-deoxynucleotide pool sanitation.

Intracellular decay of N6 -methyladenine (m6A)-containing RNA potentially induces aberrant N6 -methyl-2'-adenine (6mdA) misincorporation into DNA. Biophysically, misincorporated 6mdA may destabilize the DNA duplex in a manner similar to bona fide methylated 6mdA DNA, thereby affecting DNA replication and transcription. Utilizing heavy stable isotope labeling and ultrasensitive UHPLC-MS/MS assay, we demonstrate that intracellular m6A-RNA decay does not generate free 6mdA species, nor lead to any misincorporated DNA 6mdA in most mammalian cell lines tested, unveiling the existence of a sanitation mechanism that prevents 6mdA misincorporation. Depletion of deaminase ADAL increases the levels of free 6mdA species, concomitant with the presence of DNA-misincorporated 6mdA resulting from intracellular RNA m6A decay, suggesting that ADAL catabolizes 6mdAMP in vivo. Furthermore, we show that the overexpression of adenylate kinase 1 (AK1) promotes 6mdA misincorporation, while AK1 knockdown diminishes 6mdA incorporation, in ADAL-deficient cells. We conclude that ADAL together with other factors (such as MTH1) contributes to 2'-deoxynucleotide pool sanitation in most cells but compromised sanitation (e.g., in NIH3T3 cells) and increased AK1 expression may facilitate aberrant 6mdA incorporation. This sanitation mechanism may provide a framework for the maintenance of the epigenetic 6mdA landscape.

Stella safeguards the oocyte methylome by preventing de novo methylation mediated by DNMT1.

Postnatal growth of mammalian oocytes is accompanied by a progressive gain of DNA methylation, which is predominantly mediated by DNMT3A, a de novo DNA methyltransferase1,2. Unlike the genome of sperm and most somatic cells, the oocyte genome is hypomethylated in transcriptionally inert regions2-4. However, how such a unique feature of the oocyte methylome is determined and its contribution to the developmental competence of the early embryo remains largely unknown. Here we demonstrate the importance of Stella, a factor essential for female fertility5-7, in shaping the oocyte methylome in mice. Oocytes that lack Stella acquire excessive DNA methylation at the genome-wide level, including in the promoters of inactive genes. Such aberrant hypermethylation is partially inherited by two-cell-stage embryos and impairs zygotic genome activation. Mechanistically, the loss of Stella leads to ectopic nuclear accumulation of the DNA methylation regulator UHRF18,9, which results in the mislocalization of maintenance DNA methyltransferase DNMT1 in the nucleus. Genetic analysis confirmed the primary role of UHRF1 and DNMT1 in generating the aberrant DNA methylome in Stella-deficient oocytes. Stella therefore safeguards the unique oocyte epigenome by preventing aberrant de novo DNA methylation mediated by DNMT1 and UHRF1.

Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing.

The regulatory role of N(6)-methyladenosine (m(6)A) and its nuclear binding protein YTHDC1 in pre-mRNA splicing remains an enigma. Here we show that YTHDC1 promotes exon inclusion in targeted mRNAs through recruiting pre-mRNA splicing factor SRSF3 (SRp20) while blocking SRSF10 (SRp38) mRNA binding. Transcriptome assay with PAR-CLIP-seq analysis revealed that YTHDC1-regulated exon-inclusion patterns were similar to those of SRSF3 but opposite of SRSF10. In vitro pull-down assay illustrated a competitive binding of SRSF3 and SRSF10 to YTHDC1. Moreover, YTHDC1 facilitates SRSF3 but represses SRSF10 in their nuclear speckle localization, RNA-binding affinity, and associated splicing events, dysregulation of which, as the result of YTHDC1 depletion, can be restored by reconstitution with wild-type, but not m(6)A-binding-defective, YTHDC1. Our findings provide the direct evidence that m(6)A reader YTHDC1 regulates mRNA splicing through recruiting and modulating pre-mRNA splicing factors for their access to the binding regions of targeted mRNAs.

Artificially Evolved Superbinder for Specific Recognition of N6-Methyladenine Base Modification in DNA and RNA.

N6-Methyladenine (m6A/6mA) is a functional epigenetic base modification found in RNA and DNA. By selecting one RNA m6A reader as a template, we created a series of libraries of 3 × 108 RNA m6A reader mutants and developed a yeast surface-display-based evolution approach. Using high-throughput fluorescence-activated cell sorting, we ultimately obtained three evolved 6mA-binding proteins (e6mABPs), which displayed increased affinity for 6mA-containing DNA and reduced affinity for 6mA-free DNA. These e6mABPs are applicable for m6A/6mA enrichment and are potentially applied for modulating cell behavior.

Hyperactive DNA Cutting for Unbiased UHPLC-MS/MS Quantification of Epigenetic DNA Marks by Engineering DNase I Mutants.

Epigenetic DNA modifications, such as 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine, are associated with a variety of diseases and potential biomarkers for cancer diagnosis and therapy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays are considered to be the gold standard for qualitative and quantitative detection of DNA modifications. DNA digestion for converting long DNA polymer into 2'-deoxynucleosides is an important preprocessing step to achieve sensitive and accurate LC-MS/MS quantification. Here, we showed that, as stimulated by divalent metal ions, Mg2+ and Mn2+, the engineered human DNase I Q9R:E13R:N74K mutant can efficiently digest DNA in the presence of monovalent metal ions at a high concentration (e.g., 1 M NaCl), showing hyperactivity on DNA cutting. We also found that the engineered DNase I mutants display exceptional DNA-cutting activity over a wider pH range (5.5-9.5). Due to their hyperactivity and high salt tolerance, the engineered DNase I mutants cut DNA 5mC and dC efficiently. Benefitting from this DNA-cutting hyperactivity, we demonstrated an LC-MS/MS assay for unbiased and accurate quantification of DNA 5mC.

Necrosulfonamide Selectively Induces DNA Double-Strand Breaks in Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy that causes endless pain for patients and accounts for thousands of deaths worldwide. The development of an effective AML treatment is a topic of ongoing interest. Here, we demonstrated that a pyroptosis inhibitor necrosulfonamide (NSA) can selectively induce highly toxic double-strand breaks and kill AML cells. Mechanistically, reactive oxygen species (ROS) were the key effectors mediating the toxicity of NSA. These results probably indicate that NSA is a novel candidate for the treatment of AML.

Nanoparticles-mediated CRISPR-Cas9 gene therapy in inherited retinal diseases: applications, challenges, and emerging opportunities.

Inherited Retinal Diseases (IRDs) are considered one of the leading causes of blindness worldwide. However, the majority of them still lack a safe and effective treatment due to their complexity and genetic heterogeneity. Recently, gene therapy is gaining importance as an efficient strategy to address IRDs which were previously considered incurable. The development of the clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has strongly empowered the field of gene therapy. However, successful gene modifications rely on the efficient delivery of CRISPR-Cas9 components into the complex three-dimensional (3D) architecture of the human retinal tissue. Intriguing findings in the field of nanoparticles (NPs) meet all the criteria required for CRISPR-Cas9 delivery and have made a great contribution toward its therapeutic applications. In addition, exploiting induced pluripotent stem cell (iPSC) technology and in vitro 3D retinal organoids paved the way for prospective clinical trials of the CRISPR-Cas9 system in treating IRDs. This review highlights important advances in NP-based gene therapy, the CRISPR-Cas9 system, and iPSC-derived retinal organoids with a focus on IRDs. Collectively, these studies establish a multidisciplinary approach by integrating nanomedicine and stem cell technologies and demonstrate the utility of retina organoids in developing effective therapies for IRDs.

Quantification of Epigenetic DNA Modifications in the Subchromatin Structure Matrix Attachment Regions by Stable Isotope Dilution UHPLC-MS/MS Analysis.

To date, subchromatin structure-based quantification of epigenetic DNA modifications is limited. Matrix attachment regions (MARs), an important subchromatin structure, contain DNA elements that specifically bind chromatin to the nuclear matrix in eukaryotes and are involved in a number of diseases. Here, we exploited a high-salt extraction-based subchromatin fractionation approach for the isolation of MAR DNA and other fractions and further developed heavy stable isotope-diluted ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) for the specific quantification of epigenetic DNA modifications in the subchromatin structures. By this approach, we showed for the first time that the content of a DNA demethylation intermediate, 5-hydroxymethylcytosine (5hmdC), in MARs decreased significantly in four tested cell lines compared to the contents in genomic DNA. In particular, the content of DNA 5hmdC in the MARs of 293T cell lines decreased the most at approximately 41.09%. Together, our findings implicate that MAR DNA is less sensitive than genomic DNA to DNA demethylation.

METTL3-mediated m6A modification is required for cerebellar development.

N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout (cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of newborn cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 depletion-induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.

Reversal of the Inflammatory Responses in Fabry Patient iPSC-Derived Cardiovascular Endothelial Cells by CRISPR/Cas9-Corrected Mutation.

The late-onset type of Fabry disease (FD) with GLA IVS4 + 919G > A mutation has been shown to lead to cardiovascular dysfunctions. In order to eliminate variations in other aspects of the genetic background, we established the isogenic control of induced pluripotent stem cells (iPSCs) for the identification of the pathogenetic factors for FD phenotypes through CRISPR/Cas9 genomic editing. We adopted droplet digital PCR (ddPCR) to efficiently capture mutational events, thus enabling isolation of the corrected FD from FD-iPSCs. Both of these exhibited the characteristics of pluripotency and phenotypic plasticity, and they can be differentiated into endothelial cells (ECs). We demonstrated the phenotypic abnormalities in FD iPSC-derived ECs (FD-ECs), including intracellular Gb3 accumulation, autophagic flux impairment, and reactive oxygen species (ROS) production, and these abnormalities were rescued in isogenic control iPSC-derived ECs (corrected FD-ECs). Microarray profiling revealed that corrected FD-derived endothelial cells reversed the enrichment of genes in the pro-inflammatory pathway and validated the downregulation of NF-κB and the MAPK signaling pathway. Our findings highlighted the critical role of ECs in FD-associated vascular dysfunctions by establishing a reliable isogenic control and providing information on potential cellular targets to reduce the morbidity and mortality of FD patients with vascular complications.

Care for Patients with Stroke During the COVID-19 Pandemic: Physical Therapy and Rehabilitation Suggestions for Preventing Secondary Stroke.

Infection with the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the development of the novel 2019 coronavirus disease (COVID-19) and associated clinical symptoms, which typically presents as an upper respiratory syndrome such as pneumonia. Growing evidence indicates an increased prevalence of neurological involvement (e.g., in the form of stroke) during virus infection. COVID-19 has been suggested to be more than a lung infection because it affects the vasculature of the lungs and other organs and increases the risk of thrombosis. Patients with stroke are vulnerable to secondary events as a result not only of their poor vascular condition but also of their lack of access to rehabilitation resources. Herein, we review current knowledge regarding the pathophysiology of COVID-19, its possible association with neurological involvement, and current drug therapies. Suggestions are also offered regarding the potential for current neurorehabilitation therapies to be taught and practiced at home.

A Review of SARS-CoV-2 and the Ongoing Clinical Trials.

The sudden outbreak of 2019 novel coronavirus (2019-nCoV, later named SARS-CoV-2) in Wuhan, China, which rapidly grew into a global pandemic, marked the third introduction of a virulent coronavirus into the human society, affecting not only the healthcare system, but also the global economy. Although our understanding of coronaviruses has undergone a huge leap after two precedents, the effective approaches to treatment and epidemiological control are still lacking. In this article, we present a succinct overview of the epidemiology, clinical features, and molecular characteristics of SARS-CoV-2. We summarize the current epidemiological and clinical data from the initial Wuhan studies, and emphasize several features of SARS-CoV-2, which differentiate it from SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), such as high variability of disease presentation. We systematize the current clinical trials that have been rapidly initiated after the outbreak of COVID-19 pandemic. Whereas the trials on SARS-CoV-2 genome-based specific vaccines and therapeutic antibodies are currently being tested, this solution is more long-term, as they require thorough testing of their safety. On the other hand, the repurposing of the existing therapeutic agents previously designed for other virus infections and pathologies happens to be the only practical approach as a rapid response measure to the emergent pandemic, as most of these agents have already been tested for their safety. These agents can be divided into two broad categories, those that can directly target the virus replication cycle, and those based on immunotherapy approaches either aimed to boost innate antiviral immune responses or alleviate damage induced by dysregulated inflammatory responses. The initial clinical studies revealed the promising therapeutic potential of several of such drugs, including favipiravir, a broad-spectrum antiviral drug that interferes with the viral replication, and hydroxychloroquine, the repurposed antimalarial drug that interferes with the virus endosomal entry pathway. We speculate that the current pandemic emergency will be a trigger for more systematic drug repurposing design approaches based on big data analysis.

Vertical Ultrafiltration-Facilitated DNA Digestion for Rapid and Sensitive UHPLC-MS/MS Detection of DNA Modifications.

LC-MS/MS technologies provide important and powerful analytical tools for chemical structure-dependent identification and quantification of epigenetically crucial DNA modifications. To perform LC-MS/MS analysis, it is better to convert DNA to 2'-deoxynucleosides through enzymatic digestion. Here, we observed that inorganic cations Na+ and K+ and phosphate buffers, which were often found in various DNA solutions, significantly inhibited DNA digestion as catalyzed by typical set of DNase I, snake venom phosphodiesterase, and calf intestine alkaline phosphatase, leading to poor or varying performance on UHPLC-MS/MS analysis. We then developed an efficient and unique vertical-ultrafiltration approach, enabling us to remove these inorganic salts without DNA loss. Consequently, the removal of inorganic salts by ultrafiltration facilitated the followed DNA digestion and thus enhanced the final UHPLC-MS/MS detection. Benefiting from the developed vertical-ultrafiltration approach, it is also feasible to integrate the desalting step with the other two steps of DNA digestion and protein removal. By investigating the time course of DNA digestion, we observed a differential release rate of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine causing a measurement bias on the methylation frequency. We further exploited Mg2+ to eliminate this bias by stimulating DNase set-based DNA digestion. These innovative approaches enable us to perform rapid, sensitive, and robust UHPLC-MS/MS analysis of methylated DNA 2'-deoxycytidine, demethylation intermediates, and probably other DNA modifications.

Predominance of N6-Methyladenine-Specific DNA Fragments Enriched by Multiple Immunoprecipitation.

N6-methyladenine (6mA) is a rediscovered DNA modification in eukaryotic genomes. To explore the distribution and functions of 6mA, it is of paramount option to use immunoprecipitation to select 6mA-containing DNA fragments for genome-wide sequencing. Presumably, most of the 6mA-free fragments are removed, and the copulling down of the residual is stochastic and sequence-independent and thus they should not be called as peaks by computation. Surprisingly, here we show the predominance of 6mA-free fragments in the pulled-down fractions. By taking advantage of the submicromolar affinity of the antibodies, we further develop an elegant, multiple-round immunoprecipitation (MrIP) approach and show that 6mA-containing fragments can be enriched over 9100-fold and dominate in the final pulled-down fractions. This biochemical approach would greatly reduce the peak calling bias, which is caused by handling of dominated 6mA-free DNA fragments with an assumption-based algorithm computation and facilitates 6mA-pertinent data mining. The MrIP concept is extendable for the genome-wide sequencing of diverse DNA modifications.

Metabolically Generated Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N6-Methyladenine in Human Cells.

DNA N6-methyl-2'-deoxyadenosine (6mdA) is an epigenetic modification in both eukaryotes and bacteria. Here we exploited stable isotope-labeled deoxynucleoside [15N5]-2'-deoxyadenosine ([15N5]-dA) as an initiation tracer and for the first time developed a metabolically differential tracing code for monitoring DNA 6mdA in human cells. We demonstrate that the initiation tracer [15N5]-dA undergoes a specific and efficient adenine deamination reaction leading to the loss the exocyclic amine 15N, and further utilizes the purine salvage pathway to generate mainly both [15N4]-dA and [15N4]-2'-deoxyguanosine ([15N4]-dG) in mammalian genomes. However, [15N5]-dA is largely retained in the genomes of mycoplasmas, which are often found in cultured cells and experimental animals. Consequently, the methylation of dA generates 6mdA with a consistent coding pattern, with a predominance of [15N4]-6mdA. Therefore, mammalian DNA 6mdA can be potentially discriminated from that generated by infecting mycoplasmas. Collectively, we show a promising approach for identification of authentic DNA 6mdA in human cells and determine if the human cells are contaminated with mycoplasmas.

Proteomics analysis and proteogenomic characterization of different physiopathological human lenses.

BACKGROUND: The aim of the present study was to identify the proteomic differences among human lenses in different physiopathological states and to screen for susceptibility genes/proteins via proteogenomic characterization. METHODS: The total proteomes identified across the regenerative lens with secondary cataract (RLSC), congenital cataract (CC) and age-related cataract (ARC) groups were compared to those of normal lenses using isobaric tagging for relative and absolute protein quantification (iTRAQ). The up-regulated proteins between the groups were subjected to biological analysis. Whole exome sequencing (WES) was performed to detect genetic variations. RESULTS: The most complete human lens proteome to date, which consisted of 1251 proteins, including 55.2% previously unreported proteins, was identified across the experimental groups. Bioinformatics functional annotation revealed the common involvement of cellular metabolic processes, immune responses and protein folding disturbances among the groups. RLSC-over-expressed proteins were characteristically enriched in the intracellular immunological signal transduction pathways. The CC groups featured biological processes relating to gene expression and vascular endothelial growth factor (VEGF) signaling transduction, whereas the molecular functions corresponding to external stress were specific to the ARC groups. Combined with WES, the proteogenomic characterization narrowed the list to 16 candidate causal molecules. CONCLUSIONS: These findings revealed common final pathways with diverse upstream regulation of cataractogenesis in different physiopathological states. This proteogenomic characterization shows translational potential for detecting susceptibility genes/proteins in precision medicine.

Recent advances in high-performance liquid chromatography tandem mass spectrometry techniques for analysis of DNA damage and epigenetic modifications.

Environmental exposure would cause DNA damage and epigenetic modification changes, potentially resulting in physiological dysfunction, thereby triggering diseases and even cancer. DNA damage and epigenetic modifications are thus promising biomarkers for environmental exposures and disease states. Benefiting from its high sensitivity and accuracy, high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) is considered the "gold standard technique" for investigating epigenetic DNA modifications. This review summarizes the recent advancements of UHPLC-MS/MS-based technologies for DNA damage and epigenetic modifications analysis, mainly focusing on the innovative methods developed for UHPLC-MS/MS-related pretreatment technologies containing efficient genomic DNA digestion and effective removal of the inorganic salt matrix, and the new strategies for improving detection sensitivity of liquid chromatography-mass spectrometry. Moreover, we also summarized the novel hyphenated techniques of the advanced UHPLC-MS/MS coupled with other separation and analysis methods for the measurement of DNA damage and epigenetic modification changes in special regions and fragments of chromosomes.

Adenosine Deaminase-Like Gene-Carried Lentivirus Toolkit for Identification of DNA N6-Methyladenine Origins.

Post-replicative DNA N6-methyladenine (pr6mdA) can form via bona fide methylase-catalyzed adenine methylation, playing a pivotal role in embryonic development and other biological processes. Surprisingly, pre-methylated adenine can be erroneously incorporated into DNA as misincorporated N6-methyladenine (i6mdA) via DNA polymerase-mediated replication. Despite pr6mdA and i6mdA sharing identical chemical structures, their biological functions diverge significantly, presenting a substantial challenge in distinguishing between the two. Here, for the first-time, it is exploited that the adenosine deaminase-like (Adal) protein and a corresponding activity-null mutant to construct an Adal lentivirus toolkit. With this newly designed toolkit, both pr6mdA and i6mdA can be identified and quantified simultaneously. The presence of 6mdA in the bone marrow cells of mice is shown, with its levels serving as indicators for growth with age, probably reflecting the cellular stress-caused changes in RNA decay, nucleotide pool sanitation, and transcription. Collectively, a powerful toolkit to advance understanding of both pr6mdA and i6mdA is demonstrated.

n-3 polyunsaturated fatty acids delay intervertebral disc degeneration by inhibiting nuclear receptor coactivator 4-mediated iron overload.

n-3 polyunsaturated fatty acids (PUFAs) are closely related to the progression of numerous chronic inflammatory diseases, but the role of n-3 PUFAs in the intervertebral disc degeneration (IVDD) remains unclear. In this study, male C57BL/6 wildtype mice (WT group, n = 30) and fat-1 transgenic mice (TG group, n = 30) were randomly selected to construct the IVDD model. The results demonstrated that the optimized composition of PUFAs in the TG mice had a significant impact on delaying IVDD and cellular senescence of intervertebral disc (IVD). Mechanismly, n-3 PUFAs inhibited IVD senescence by alleviating NCOA4-mediated iron overload. NCOA4 overexpression promoted iron overload and weakened the pro-proliferation and anti-senescence effect of DHA on the IVD cells. Furthermore, this study futher revealed n-3 PUFAs downregulated NCOA4 expression by inactiviting the LGR5/ß-catenin signaling pathway. This study provides an important theoretical basis for preventing and treating IVDD and low back pain.

Using a static magnetic field to attenuate the severity in COVID-19-invaded lungs.

Two important factors affecting the progress of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the S-protein binding function of ACE2 receptors and the membrane fluidity of host cells. This study aimed to evaluate the effect of static magnetic field (SMF) on S-protein/ACE2 binding and cellular membrane fluidity of lung cells, and was performed in vitro using a Calu-3 cell model and in vivo using an animal model. The ability of ACE2 receptors to bind to SARS-CoV-2 spike protein on host cell surfaces under SMF stimulation was evaluated using fluorescence images. Host lung cell membrane fluidity was tested using fluorescence polarization to determine the effects of SMF. Our results indicate that 0.4 T SMF can affect binding between S-protein and ACE2 receptors and increase Calu-3 cell membrane fluidity, and that SMF exposure attenuates LPS-induced alveolar wall thickening in mice. These results may be of value for developing future non-contact, non-invasive, and low side-effect treatments to reduce disease severity in COVID-19-invaded lungs.