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Radical cure of Plasmodium vivax malaria must include elimination of quiescent "hypnozoite" forms in the liver; however, the only FDA-approved treatments are contraindicated in many vulnerable populations. To identify new drugs and drug targets, we screened the Repurposing, Focused Rescue, and Accelerated Medchem library against P. vivax liver stages and identified the DNA methyltransferase inhibitors hydralazine and cadralazine as active against hypnozoites. We then used bisulfite sequencing and immunostaining to identify cytosine modifications in the infectious stage (sporozoites) and liver stages, respectively. A subsequent screen of epigenetic inhibitors revealed hypnozoites are broadly sensitive to histone acetyltransferase and methyltransferase inhibitors, indicating that several epigenetic mechanisms are likely modulating hypnozoite persistence. Our data present an avenue for the discovery and development of improved radical cure antimalarials.

The 2nd CASMS conference was held virtually through Gather. Town platform from October 17 to 21, 2022, with a total of 363 registrants including an outstanding and diverse group of scientists at the forefront of their research fields from both academia and industry worldwide, especially in the United States and China. The conference offered a 5-day agenda with an exciting scientific program consisting of two plenary lectures, 14 parallel symposia, and 4 special sessions in which a total of 97 invited speakers presented technological innovations and their applications in proteomics & biological mass spectrometry and metabo-lipidomics & pharmaceutical mass spectrometry. In addition, 18 invited speakers/panelists presented at 3 research-focused and 2 career development workshops. Moreover, 144 posters, 54 lightning talks, 5 sponsored workshops, and 14 exhibitions were presented, from which 20 posters and 8 lightning talks received presentation awards. Furthermore, the conference featured 1 MCP lectureship and 5 young investigator awardees for the first time to highlight outstanding mid-career and early-career rising stars in mass spectrometry from our society. The conference provided a unique scientific platform for young scientists (i.e., graduate students, postdocs and junior faculty/investigators) to present their research, meet with prominent scientists, and learn about career development and job opportunities (http://casms.org).

With the rapid development of industrial control modernization, traditional manufacturing is closely integrated with the Internet. The networking of industrial control systems not only improves the manufacturing capacity of enterprises, but also facilitates the control and management of remote production sites, but at the same time it also brings a series of network security issues. This paper proposes a frequency feature vector construction method based on Modbus TCP message sequence. This method uses a sliding window to sequentially extract the combined sequence of the function code and the register address, obtains the pattern subsequence through the extraction, and then converts the frequency of the pattern subsequence in the feature library into a frequency feature vector.

Affected by the inherent ideas like "Focus on Function Realization, Despise Security Protection", there are lots of hidden threats in the industrial control system of wind farms (ICS-WF), such as unreasonable IP configuration, failure in virus detection and killing, which are prone to illegal invasion and attack from the cyberspace. Those unexpected unauthorized accesses are quite harmful for the stable operation of the wind farms and regional power grid. Therefore, by investigating the current security situation and needs of ICS-WF, analyzing the characteristics of ICS-WF’s architecture and internal communication, and integrating the ideas of the classified protection of cybersecurity, this paper proposes a new customized cybersecurity strategy for ICS-WF based on the barrel theory. We also introduce an new anomalous intrusion detection technology for ICS-WF, which is developed based on statistical models of wind farm network characteristics. Finally, combined all these work with the network security offense and defense drill in the industrial control safety simulation laboratory of wind farms, this research formulates a three-dimensional comprehensive protection solution for ICS-WF, which significantly improves the cybersecurity level of ICS-WF.

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.

DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms.

Tumor cells, including cancer stem cells (CSCs) resistant to radio- and chemotherapy, must enhance metabolism to meet the extra energy demands to repair and survive such genotoxic conditions. However, such stress-induced adaptive metabolic alterations, especially in cancer cells that survive radiotherapy, remain unresolved. In this study, we found that CPT1 (Carnitine palmitoyl transferase I) and CPT2 (Carnitine palmitoyl transferase II), a pair of rate-limiting enzymes for mitochondrial fatty acid transportation, play a critical role in increasing fatty acid oxidation (FAO) required for the cellular fuel demands in radioresistant breast cancer cells (RBCs) and radiation-derived breast cancer stem cells (RD-BCSCs). Enhanced CPT1A/CPT2 expression was detected in the recurrent human breast cancers and associated with a worse prognosis in breast cancer patients. Blocking FAO via a FAO inhibitor or by CRISPR-mediated CPT1A/CPT2 gene deficiency inhibited radiation-induced ERK activation and aggressive growth and radioresistance of RBCs and RD-BCSCs. These results revealed that switching to FAO contributes to radiation-induced mitochondrial energy metabolism, and CPT1A/CPT2 is a potential metabolic target in cancer radiotherapy.

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) inthe regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically thehighly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modificationas a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric O2-, N3-, and O4-ethylthymidine (O2-, N3-, and O4-EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at O2-EtdT and N3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove O2-EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove O4-EtdT lesion. For the N3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove-sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.

DNMT1 (DNA methyltransferase 1) is responsible for propagating the DNA methylation patterns during DNA replication. DNMT1 contains, in addition to a C-terminal methyltransferase domain, a large N-terminal regulatory region that is composed of an RFTS (replication foci targeting sequence) domain, a CXXC zinc finger domain and a pair of BAH (bromo adjacent homology) domains. The regulatory domains of DNMT1 mediate a network of protein-protein and protein-DNA interactions to control the recruitment and enzymatic activity of DNMT1. Here we report the crystal structure of human DNMT1 with all the structural domains (hDNMT1, residues 351-1600) in complex with S-adenosyl-l-homocysteine at 2.62Å resolution. The RFTS domain directly associates with the methyltransferase domain, thereby inhibiting the substrate binding of hDNMT1. Through structural analysis, mutational, biochemical and enzymatic studies, we further identify that a linker sequence between the CXXC and BAH1 domains, aside from its role in the CXXC domain-mediated DNMT1 autoinhibition, serves as an important regulatory element in the RFTS domain-mediated autoinhibition. In comparison with the previously determined structure of mouse DNMT1, this study also reveals a number of distinct structural features that may underlie subtle functional diversity observed for the two orthologues. In addition, this structure provides a framework for understanding the functional consequence of disease-related hDNMT1 mutations.

Thiopurines are effective immunosuppressants and anticancer agents. However, the long-term use of thiopurines was found to be associated with a significantly increased risk of various types of cancer. To date, the specific mechanism(s) underlying the carcinogenicity associated with thiopurine treatment remain(s) unclear. Herein, we constructed duplex pTGFP-Hha10 shuttle vectors carrying a 6-thioguanine ((S)G) or S⁶-methylthioguanine (S⁶mG) at a unique site and allowed the vectors to propagate in three different human cell lines. Analysis of the replication products revealed that although neither thionucleoside blocked considerably DNA replication in any of the human cell lines, both (S)G and S⁶mG were mutagenic, resulting in G→A mutation at frequencies of ~8% and ~39%, respectively. Consistent with what was found from our previous study in E. coli cells, our data demonstrated that the mutagenic properties of (S)G and S⁶mG provided significant evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline. Keywords: Reactive oxygen species, Free radicals, Genotoxic stress, Oxidative lesions, Endogenous DNA damage, Cellular senescence, Aging