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Borrelia spp. are transmitted to humans by the bite of an infected tick. In Europe, Borrelia afzelii and Borrelia garinii are the main causative agents of Lyme borreliosis, one of the most prevalent tick-borne diseases in the northern hemisphere. In bacteria such as Borrelia spp., a restriction-modification system (RMS) protects against the harmful introduction of foreign DNA. The RMS comprises two activities: methyltransferase and endonuclease. This study is aimed to characterize the RMS of B. afzelii and B. garinii. First, we identified potential RMS genes. The predicted genes were cloned into a methylase-deficient Escherichia coli strain and digested with methylation-sensitive restriction enzymes to verify methyltransferase activity. Additionally, the RMS proteins were purified to evaluate endonuclease activity. Subsequently, methylated and unmethylated plasmids were used to investigate the effect of methylation on endonuclease activity and transformation efficiency. We identified four possible RMS genes in B. afzelii and four RMS genes in B. garinii. We analyzed the presence of these genes in patient isolates and observed a high degree of heterogeneity. The restriction pattern of DNA methylated by each of the four recombinantly expressed genes provided strong evidence that all encode adenine-specific methyltransferases. After 24 h of incubation with purified RMS proteins, we observed complete digestion of unmethylated plasmid DNA, demonstrating endonuclease activity. Finally, we proved that methylation protects against endonuclease activity and increases transformation efficiency. [ABSTRACT FROM AUTHOR]

Mesophilic Argonautes (Agos) from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics. This study characterizes a novel Ago from Pseudobutyrivibrio ruminis (PrAgo), which can cleave single-stranded DNA using guide DNA (gDNA). PrAgo, functioning as a multi-turnover enzyme, effectively cleaves DNA using 5′-phosphate gDNA, 14–30 nucleotides in length, in the presence of both Mn2+ and Mg2+ ions. PrAgo demonstrates DNA cleavage activity over a broad pH range (pH 4–12), with optimal activity at pH 11. As a mesophilic enzyme, PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65 °C, particularly at 65 °C. PrAgo does not show strong preferences for the 5′-nucleotide in gDNA. It shows high tolerance for single-base mismatches, except at positions 13 and 15 of gDNA. Continuous double-nucleotide mismatches at positions 10–16 of gDNA significantly reduce cleavage activity. Furthermore, PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65 °C. Additionally, molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency. These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology. [ABSTRACT FROM AUTHOR]

The food enzyme endonuclease (Aspergillus nuclease S1; EC 3.1.30.1) is produced with the non‐genetically modified Penicillium citrinum strain NP 11–15 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.006 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1010 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 168,333. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially for individuals allergic to Penicillium. However, the likelihood of such reactions will not exceed the likelihood of allergic reactions to Penicillium. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use. [ABSTRACT FROM AUTHOR]

Cervical cancer is the fourth most common malignancy in women globally. Chemotherapies, targeted therapies, and immunotherapies in the treatment of cervical cancer are usually accompanied by effective and adverse effects. Therefore, finding other efficient and accurate molecular targets remains essential to improve the treatment benefits of cervical cancer patients. MCPIP1 (monocyte chemoattractant protein-induced protein 1) is a kind of endonuclease with a CCCH zinc finger domain and a PilT-N-terminal (PIN) domain, and its function in cervical cancer is unknown. We found that MCPIP1 inhibits cell proliferation and promotes cell apoptosis of cervical cancer. Additionally, MCPIP1 suppresses mRNA and protein expression of the apoptotic inhibitor XIAP by decreasing its mRNA stability. Mechanically, MCPIP1 binds to the XIAP mRNA via its CCCH zinc finger domain and degrades the XIAP mRNA via the endonuclease activity coming from its PIN domain. Our study clarifies that MCPIP1 promotes cervical cancer cell apoptosis by suppressing the expression of XIAP, thereby impeding cervical cancer progression. Moreover, targeted delivery of MCPIP1 with engineered Salmonella typhimurium leads to tumor growth retardation in the HeLa xenograft tumor model in mice. Therefore, our study may provide a theoretical basis for formulating clinical treatment strategies for cervical cancer. [ABSTRACT FROM AUTHOR]

Abstract Mesophilic Argonautes (Agos) from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics. This study characterizes a novel Ago from Pseudobutyrivibrio ruminis (PrAgo), which can cleave single-stranded DNA using guide DNA (gDNA). PrAgo, functioning as a multi-turnover enzyme, effectively cleaves DNA using 5′-phosphate gDNA, 14–30 nucleotides in length, in the presence of both Mn2+ and Mg2+ ions. PrAgo demonstrates DNA cleavage activity over a broad pH range (pH 4–12), with optimal activity at pH 11. As a mesophilic enzyme, PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65 °C, particularly at 65 °C. PrAgo does not show strong preferences for the 5′-nucleotide in gDNA. It shows high tolerance for single-base mismatches, except at positions 13 and 15 of gDNA. Continuous double-nucleotide mismatches at positions 10–16 of gDNA significantly reduce cleavage activity. Furthermore, PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65 °C. Additionally, molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency. These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology. Graphical Abstract

Abstract Background Endonucleases play a crucial role in plant growth and stress response by breaking down nuclear DNA. However, the specific members and biological functions of the endonuclease encoding genes in wheat remain to be determined. Results In this study, we identified a total of 26 TaENDO family genes at the wheat genome-wide level. These genes were located on chromosomes 2 A, 2B, 2D, 3 A, 3B, and 3D and classified into four groups, each sharing similar gene structures and conserved motifs. Furthermore, we identified diverse stress-response and growth-related cis-elements in the promoter of TaENDO genes, which were broadly expressed in different organs, and several TaENDO genes were significantly induced under drought and salt stresses. We further examined the biological function of TaENDO23 gene since it was rapidly induced under drought stress and exhibited high expression in spikes and grains. Subcellular localization analysis revealed that TaENDO23 was localized in the cytoplasm of wheat protoplasts. qRT-PCR results indicated that the expression of TaENDO23 increased under PEG6000 and abscisic acid treatments, but decreased under NaCl treatment. TaENDO23 mainly expressed in leaves and spikes. A kompetitive allele-specific PCR (KASP) marker was developed to identify single nucleotide polymorphisms in TaENDO23 gene in 256 wheat accessions. The alleles with TaENDO23-HapI haplotypes had higher grain weight and size compared to TaENDO23-HapII. The geographical and annual frequency distributions of the two TaENDO23 haplotypes revealed that the elite haplotype TaENDO23-HapI was positively selected in the wheat breeding process. Conclusion We systematically analyzed the evolutionary relationships, gene structure characteristics, and expression patterns of TaENDO genes in wheat. The expression of TaENDO23, in particular, was induced under drought stress, mainly expressed in the leaves and grains. The KASP marker of TaENDO23 gene successfully distinguished between the wheat accessions, revealing TaENDO23-HapI as the elite haplotype associated with improved grain weight and size. These findings provide insights into the evolution and characteristics of TaENDO genes at the genome-wide level in wheat, laying the foundation for further biological analysis of TaENDO23 gene, especially in response to drought stress and grain development.

ABSTRACT Canine parvovirus (CPV) can cause high morbidity and mortality rates in puppies, posing a significant threat to both pet dogs and the breeding industry. Rapid, accurate, and convenient detection methods are important for the early intervention and treatment of canine parvovirus. In this study, we propose a visual CPV detection system called nucleic acid mismatch enzyme digestion (NMED). This system combines loop-mediated isothermal amplification (LAMP), endonuclease for gene mismatch detection, and colloidal gold lateral chromatography. We demonstrated that NMED can induce the binding of the amplicon from the sample to the specific labeling probe, which in turn triggers digestion by the endonuclease. The sensitivity and visual visibility of LAMP were increased by combining endonuclease and colloidal gold lateral chromatography assisted by a simple temperature-controlled device. The sensitivity of the NMED assay was 1 copy/μL, which was consistent with quantitative PCR (qPCR). The method was validated with 20 clinical samples that potentially had CPV infection; 15 positive samples and 5 negative samples were evaluated; and the detection accuracy was consistent with that of qPCR. As a rapid, accurate, and convenient molecular diagnostic method, NMED has great potential for application in the field of pathogenic microorganism detection.IMPORTANCEThe NMED method has been established in the laboratory and used for CPV detection. The method has several advantages, including simple sampling, high sensitivity, intuitive results, and no requirement for expensive equipment. The establishment of this method has commercial potential and offers a novel approach and concept for the future development of clinical detection of pathogenic microorganisms.

Abstract The food enzyme endonuclease (Aspergillus nuclease S1; EC 3.1.30.1) is produced with the non‐genetically modified Penicillium citrinum strain NP 11–15 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in the processing of yeast and yeast products. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.006 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1010 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 168,333. A search for homology of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, especially for individuals allergic to Penicillium. However, the likelihood of such reactions will not exceed the likelihood of allergic reactions to Penicillium. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Chili (Capsicum annuum L.) is a highly valued vegetable, renowned for its unique taste and aroma. However, chili production faces challenges in meeting the high demand due to infections caused by pathogens such as ChiVMV (potyvirus). Previous studies have suggested that chili eIF4E1 plays a crucial role in potyvirus gene transcription. Therefore, this study explores the potential of CRISPR‐Cas9‐based genome editing to enhance chili resistance by introducing premature stop codons or truncated proteins. Two sgRNAs were designed, targeting the first and second intron of the eIF4E1 gene. The production of Cas9 protein was assessed with varying IPTG concentrations in Escherichia coli BL21(DE3), carrying 4xNLS‐pMJ915v2‐sfGFP plasmid with a TEV protease cut‐site at the N terminal. The findings indicate that the optimal IPTG concentration is 500 µM. Purification using an IMAC column confirmed the presence of Cas9 in the initial 2 mL of the eluted fractions, as indicated by numerous background proteins. Nevertheless, successful formation of Cas9‐RNP complexes was achieved for both sgRNAs. The nucleolytic activity of Tag‐Cas9 (carrying the MBP‐tag) and Cas9 was confirmed through in vitro endonuclease activity assays. The next step involved transfecting chili protoplasts with these RNP complexes to edit the chili eIF4E1 gene.

Nucleotide excision repair (NER) is a multistep biochemical process that maintains the integrity of the genome. Unlike other mechanisms that maintain genomic integrity, NER is distinguished by two irreversible nucleolytic events that are executed by the xeroderma pigmentosum group G (XPG) and xeroderma pigmentosum group F (XPF) structure-specific endonucleases. Beyond nucleolysis, XPG and XPF regulate the overall efficiency of NER through various protein–protein interactions. The current experiments evaluated whether an environmental stressor could negatively affect the expression of Xpg (Ercc5: excision repair cross-complementing 5) or Xpf (Ercc4: excision repair cross-complementing 4) in the mammalian cochlea. Ubiquitous background noise was used as an environmental stressor. Gene expression levels for Xpg and Xpf were quantified from the cochlear neurosensory epithelium after noise exposure. Further, nonlinear cochlear signal processing was investigated as a functional consequence of changes in endonuclease expression levels. Exposure to stressful background noise abrogated the expression of both Xpg and Xpf, and these effects were associated with pathological nonlinear signal processing from receptor cells within the mammalian inner ear. Given that exposure to environmental sounds (noise, music, etc.) is ubiquitous in daily life, sound-induced limitations to structure-specific endonucleases might represent an overlooked genomic threat. [ABSTRACT FROM AUTHOR]

An electrochemically reduced graphene oxide (ERGO) electrode-based electrochemical assay was developed for rapid, sensitive, and straightforward analysis of both activity and inhibition of the endonuclease EcoRV. The procedure uses a DNA substrate designed for EcoRV, featuring a double-stranded DNA (dsDNA) region labeled with methylene blue (MB) and a single-stranded DNA (ssDNA) region immobilized on the ERGO surface. The ERGO electrode, immobilized with the DNA substrate, was subsequently exposed to a sample containing EcoRV. Upon enzymatic hydrolysis, the cleaved dsDNA fragments were detached from the ERGO surface, leading to a decrease in the MB concentration near the electrode. This diminished the electron transfer efficiency for MB reduction, resulting in a decreased reduction current. This assay demonstrates excellent specificity and high sensitivity, with a limit of detection (LOD) of 9.5 × 10−3 U mL−1. Importantly, it can also measure EcoRV activity in the presence of aurintricarboxylic acid, a known inhibitor, highlighting its potential for drug discovery and clinical diagnostic applications. [ABSTRACT FROM AUTHOR]

A manuscript published in the scientific journal "Nature" in 1996 reported on the cloning of sheep by somatic cell nuclear transfer (SCNT) into enucleated oocytes. The donor nuclei came from an established cell line, which in turn were derived from a sheep fetus. This method provided fundamental insights into understanding the molecular basis of cellular totipotency, pluripotency and the mechanisms underlying nuclear genome reprogramming. However, it was not until the birth of the cloned sheep "Dolly" that cloning came to the attention of the general public. However, new technologies often only unfold their impact in combination with other findings. In the case of cloning by somatic nuclear transfer, this is the combination with genome editing, as the combination of these two techniques enables the efficient generation of animals with edited genomes. This summary therefore aims to provide a comprehensive overview of the animals that have already been cloned in the past and the current status of edited farm animals in the context of commercial and pre-commercial applications. This overview will explicitly cover applications in the agricultural and food sector and does not claim to be exhaustive with regard to applications that serve biomedical purposes. In terms of content, the opportunities and risks associated with these technologies are addressed, with particular reference to the current legal framework in various countries around the world. [ABSTRACT FROM AUTHOR]

Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses encode for a Replicase (Rep) that is essential for viral replication. Rep is a helicase with three domains: an endonuclease, an oligomeric, and an ATPase domain (ED, OD, and AD). Our recent cryo-EM structure of the porcine circovirus 2 (PCV2) Rep provided the first structure of a CRESS-DNA Rep. The structure visualized the ED to be highly mobile, Rep to form a homo-hexamer, bound ssDNA and nucleotides, and the AD to adopt a staircase arrangement around the ssDNA. We proposed a hand-over-hand mechanism by the ADs for ssDNA translocation. The hand-over-hand mechanism requires extensive movement of the AD. Here, we scrutinize this mechanism using all-atom Molecular Dynamics (MD) simulation of Rep in three states: (1) Rep bound to ssDNA and ADP, (2) Rep bound to ssDNA, and (3) Rep by itself. Each of the 700 nsec simulations converges within 200 nsec and provides important insight into the dynamics of Rep, the dynamics of Rep in the presence of these biomolecules, and the importance of ssDNA and ADP in driving the AD to adopt the staircase arrangement around the ssDNA. To the best of our knowledge, this is the first example of an all-atom MD simulation of a CRESS-DNA Rep. This study sets the basis of further MD studies aimed at obtaining a chemical understanding of how Rep uses nucleotide binding and hydrolysis to translocate ssDNA. [ABSTRACT FROM AUTHOR]

The Oropouche virus (OROV) is a member of the family Peribunyaviridae (order Bunyavirales) and the cause of a dengue-like febrile illness transmitted mainly by biting midges and mosquitoes. In this study, we aimed to explore acylphloroglucinols and xanthohumol from hops (Humulus lupulus L.) as a promising alternative for antiviral therapies. The evaluation of the inhibitory potential of hops compounds on the viral cycle of OROV was performed through two complementary approaches. The first approach applies cell-based assay post-inoculation experiments to explore the inhibitory potential on the latest steps of the viral cycle, such as genome translation, replication, virion assembly, and virion release from the cells. The second part covers in silico methods evaluating the ability of those compounds to inhibit the activity of the endonuclease domain, which is essential for transcription, binding, and cleaving RNA. In conclusion, the beta acids showed strongest inhibitory potential in post-treatment assay (EC50 = 26.7 µg/mL). Xanthohumol had the highest affinity for OROV endonuclease followed by colupulone and cohumulone. This result contrasts with that observed for docking and MM/PBSA analysis, where cohumulone was found to have a higher affinity. Finally, among the three tested ligands, Lys92 and Arg33 exhibited the highest affinity with the protein. [ABSTRACT FROM AUTHOR]

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.

Restriction modification (RM) systems are one of the ubiquitous yet primitive defense responses employed by bacteria and archaea with the primary role of safeguarding themselves against invading bacteriophages. Protection of the host occurs by the cleavage of the invading foreign DNA via restriction endonucleases with concomitant methylation of host DNA with the aid of a methyltransferase counterpart. RM systems have been extensively studied in bacteria, however, in the case of archaea there are limited reports of RM enzymes that are investigated to date owing to their inhospitable growth demands. This review aims to broaden the knowledge about what is known about the diversity of RM systems in archaea and encapsulate the current knowledge on restriction and modification enzymes characterized in archaea so far and the role of RM systems in the milieu of archaeal biology.

Degradation of lignin, a natural macromolecule, by laccase via substrate radicals has been intensively studied. However, the interactions between laccase and DNA have not been thoroughly elucidated to date. In this report, we demonstrate that laccase has the endonuclease-mimetic activity with the sequence preference. The decay of plasmid and cDNA was observed with atomic force microscopy (AFM), electron paramagnetic resonance (EPR) and DNA sequencing. Concomitantly, Image 1 appeared, which produced the DNA lesion. The sequencing results showed that A and AA were the preferred nucleotides flanking the cleaved sites. The hydrogen bonds between complementary base pairs and the base redox potential were responsible for the long-lived charge transfer state within the A-repetitive sequence governing the sequence preference. This discovery implies alternative strategies of surviving the phosphorus stress and resistance to alien DNA in certain species, in which laccase is abundant. These results enrich our understanding of the mechanisms of DNA damage and are important for the interpretation of disease treatment.

Abstract Background Mycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly induces leprosy by causing permanent damage to the skin, nerves, limbs and eyes as well as deformities and disabilities. Moreover, ML grows slowly and is nonculturable in vitro. Given the prevalence of leprosy, a highly sensitive and rapid method for the early diagnosis of leprosy is urgently needed. Results In this study, we devised a novel tool for the diagnosis of leprosy by combining restriction endonuclease, real-time fluorescence analysis and multiple cross displacement amplification (E-RT-MCDA). To establish the system, primers for the target gene RLEP were designed, and the optimal conditions for E-RT-MCDA at 67 °C for 36 min were determined. Genomic DNA from ML, various pathogens and clinical samples was used to evaluate and optimize the E-RT-MCDA assay. The limit of detection (LoD) was 48.6 fg per vessel for pure ML genomic DNA, and the specificity of detection was as high as 100%. In addition, the detection process could be completed in 36 min by using a real-time monitor. Conclusion The E-RT-MCDA method devised in the current study is a reliable, sensitive and rapid technique for leprosy diagnosis and could be used as a potential tool in clinical settings.

It is every biochemist's dream to reconstitute a biological process in vitro using defined components, because doing so not only reduces a biological phenomenon to one or a series of biochemical reactions, but also defines the minimal list of essential components. In this issue of Genes & Development, Boreikaite and colleagues (pp. 210-224) and Schmidt and colleagues (pp. 195-209) report their independent reconstitution of human pre-mRNA 3' end processing.

Structures provide a critical breakthrough step for biological analyses, and small angle X-ray scattering (SAXS) is a powerful structural technique to study dynamic DNA repair proteins. As toxic and mutagenic repair intermediates need to be prevented from inadvertently harming the cell, DNA repair proteins often chaperone these intermediates through dynamic conformations, coordinated assemblies, and allosteric regulation. By measuring structural conformations in solution for both proteins, DNA, RNA, and their complexes, SAXS provides insight into initial DNA damage recognition, mechanisms for validation of their substrate, and pathway regulation. Here, we describe exemplary SAXS analyses of a DNA damage response protein spanning from what can be derived directly from the data to obtaining super resolution through the use of SAXS selection of atomic models. We outline strategies and tactics for practical SAXS data collection and analysis. Making these structural experiments in reach of any basic and clinical researchers who have protein, SAXS data can readily be collected at government-funded synchrotrons, typically at no cost for academic researchers. In addition to discussing how SAXS complements and enhances cryo-electron microscopy, X-ray crystallography, NMR, and computational modeling, we furthermore discuss taking advantage of recent advances in protein structure prediction in combination with SAXS analysis.

Seasonal influenza A and B viruses represent a global concern. Antiviral drugs are crucial to treat severe influenza in high-risk patients and prevent virus spread in case of a pandemic. The emergence of viruses showing drug resistance, in particular for the recently licensed polymerase inhibitor baloxavir marboxil, drives the need for developing alternative antivirals. The endonuclease activity residing in the N-terminal domain of the polymerase acidic protein (PAN) is crucial for viral RNA synthesis and a validated target for drug design. Its function can be impaired by molecules bearing a metal-binding pharmacophore (MBP) able to coordinate the two divalent metal ions in the active site. In the present work, the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold is explored for the inhibition of influenza virus PA endonuclease. The structure-activity relationship was analysed by modifying the substituents on the lipophilic moiety linked to the MBP. The new compounds exhibited nanomolar inhibitory activity in a FRET-based enzymatic assay, and a few compounds (15-17, 21) offered inhibition in the micromolar range, in a cell-based influenza virus polymerase assay. When investigated against a panel of PA-mutant forms, compound 17 was shown to retain full activity against the baloxavir-resistant I38T mutant. This was corroborated by docking studies providing insight into the binding mode of this novel class of PA inhibitors.

Oropouche virus (OROV) is an emerging vector-borne arbovirus found in South America that causes Oropouche fever, a febrile infection similar to dengue fever. It has a high epidemic potential, causing illness in over 500,000 cases diagnosed since the virus was first discovered in 1955. Currently, the prevention of human viral infection depends on vaccination, but availability for many viruses is limited, and they are classified as neglected viruses. At present, there are no vaccines or antiviral treatments available. An alternative approach to limiting the spread of the virus is to selectively disrupt viral replication mechanisms. Here, we demonstrate the inhibitory effect of acridones, which efficiently inhibited viral replication by 99.9 % in vitro. To evaluate possible mechanisms of action, we conducted tests with dsRNA, an intermediate in virus replication, as well as MD simulations, docking, and binding free energy analysis. The results showed a strong interaction between FAC21 and the OROV endonuclease, which possibly limits the interaction of viral RNA with other proteins. Therefore, our results suggest a dual mechanism of antiviral action, possibly caused by ds-RNA intercalation. In summary, our findings demonstrate that a new generation of antiviral drugs could be developed based on the selective optimization of molecules.

The cap-snatching mechanism mediated by cap-dependent endonuclease, which is common among the negative-stranded, segmented RNA viruses in Orthomyxoviridae, Bunyaviridae, and Arenaviridae, is crucial for viral transcription and replication and is thus an attractive target for antiviral drug development. Herein, tanshinone I and its analog tanshinone IIA were identified as candidate compounds with broad-spectrum antiviral activities against bandaviruses, including severe fever with thrombocytopenia syndrome virus, Heartland virus, and Guertu virus. Additionally, the broad-spectrum antiviral activity was observed in influenza A virus and arenavirus. Further study demonstrated that tanshinone I exhibited potent antiviral activity in vitro and significantly reduced the viral loads in vivo. The underlying mechanism was speculated to involve tanshinone I binding to the active pocket of the L protein endonuclease domain to inhibit cap cleavage. This study reports candidate broad-spectrum antiviral compounds against negative-stranded, segmented RNA viruses, highlighting the endonuclease involved in the cap-snatching process as a reliable antiviral target for discovering broad-spectrum antivirals. IMPORTANCE The spread of avian-borne, tick-borne, and rodent-borne pathogens has the potential to pose a serious threat to human health, and candidate vaccines as well as therapeutics for these pathogens are urgently needed. Tanshinones, especially tanshinone I, were identified as a cap-dependent endonuclease inhibitor with broad-spectrum antiviral effects on negative-stranded, segmented RNA viruses including bandavirus, orthomyxovirus, and arenavirus from natural products, implying an important resource of candidate antivirals from the traditional Chinese medicines. This study supplies novel candidate antivirals for the negative-stranded, segmented RNA virus and highlights the endonuclease involved in the cap-snatching process as a reliable broad-spectrum antiviral target. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly induces leprosy by causing permanent damage to the skin, nerves, limbs and eyes as well as deformities and disabilities. Moreover, ML grows slowly and is nonculturable in vitro. Given the prevalence of leprosy, a highly sensitive and rapid method for the early diagnosis of leprosy is urgently needed. Results: In this study, we devised a novel tool for the diagnosis of leprosy by combining restriction endonuclease, real-time fluorescence analysis and multiple cross displacement amplification (E-RT-MCDA). To establish the system, primers for the target gene RLEP were designed, and the optimal conditions for E-RT-MCDA at 67 °C for 36 min were determined. Genomic DNA from ML, various pathogens and clinical samples was used to evaluate and optimize the E-RT-MCDA assay. The limit of detection (LoD) was 48.6 fg per vessel for pure ML genomic DNA, and the specificity of detection was as high as 100%. In addition, the detection process could be completed in 36 min by using a real-time monitor. Conclusion: The E-RT-MCDA method devised in the current study is a reliable, sensitive and rapid technique for leprosy diagnosis and could be used as a potential tool in clinical settings. [ABSTRACT FROM AUTHOR]

Abstract Introduction During evolution, blood-feeding arthropods developed a complex salivary mixture that can interfere with host haemostatic and immune response, favoring blood acquisition and pathogen transmission. Therefore, a survey of the salivary gland contents can lead to the identification of molecules with potent pharmacological activity in addition to increase our understanding of the molecular mechanisms underlying the hematophagic behaviour of arthropods. The southern house mosquito, Culex quinquefasciatus, is a vector of several pathogenic agents, including viruses and filarial parasites that can affect humans and wild animals. Results Previously, a Sanger-based transcriptome of the salivary glands (sialome) of adult C. quinquefasciatus females was published based on the sequencing of 503 clones organized into 281 clusters. Here, we revisited the southern mosquito sialome using an Illumina-based RNA-sequencing approach of both male and female salivary glands. Our analysis resulted in the identification of 7,539 coding DNA sequences (CDS) that were functionally annotated into 25 classes, in addition to 159 long non-coding RNA (LncRNA). Additionally, comparison of male and female libraries allowed the identification of female-enriched transcripts that are potentially related to blood acquisition and/or pathogen transmission. Conclusion Together, these findings represent an extended reference for the identification and characterization of the proteins containing relevant pharmacological activity in the salivary glands of C. quinquefasciatus mosquitoes.

Self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) are carefully controlled by extrinsic and intrinsic factors, to ensure the lifelong process of hematopoiesis. Apurinic/apyrimidinic endonuclease 1 (APEX1) is a multifunctional protein implicated in DNA repair and transcriptional regulation. Although previous studies have emphasized the necessity of studying APEX1 in a lineage-specific context and its role in progenitor differentiation, no studies have assessed the role of APEX1, nor its two enzymatic domains, in supporting adult HSPC function. In this study, we demonstrated that complete loss of APEX1 from murine bone marrow HSPCs (induced by CRISPR/Cas9) caused severe hematopoietic failure following transplantation, as well as a HSPC expansion defect in culture conditions maintaining in vivo HSC functionality. Using specific inhibitors against either the nuclease or redox domains of APEX1 in combination with single cell transcriptomics (CITE-seq), we found that both APEX1 nuclease and redox domains are regulating mouse HSPCs, but through distinct underlying transcriptional changes. Inhibition of the APEX1 nuclease function resulted in loss of HSPCs accompanied by early activation of differentiation programs and enhanced lineage commitment. By contrast, inhibition of the APEX1 redox function significantly downregulated interferon-stimulated genes and regulons in expanding HSPCs and their progeny, resulting in dysfunctional megakaryocyte-biased HSPCs, as well as loss of monocytes and lymphoid progenitor cells. In conclusion, we demonstrate that APEX1 is a key regulator for adult regenerative hematopoiesis, and that the APEX1 nuclease and redox domains differently impact proliferating HSPCs. [ABSTRACT FROM AUTHOR]

Formation of the 3′ end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3′-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field. [ABSTRACT FROM AUTHOR]

Xeroderma pigmentosum group G (XPG) protein is both a functional partner in multiple DNA damage responses (DDR) and a pathway coordinator and structure-specific endonuclease in nucleotide excision repair (NER). Different mutations in the XPG gene ERCC5 lead to either of two distinct human diseases: Cancer-prone xeroderma pigmentosum (XP-G) or the fatal neurodevelopmental disorder Cockayne syndrome (XP-G/CS). To address the enigmatic structural mechanism for these differing disease phenotypes and for XPG's role in multiple DDRs, here we determined the crystal structure of human XPG catalytic domain (XPGcat), revealing XPG-specific features for its activities and regulation. Furthermore, XPG DNA binding elements conserved with FEN1 superfamily members enable insights on DNA interactions. Notably, all but one of the known pathogenic point mutations map to XPGcat, and both XP-G and XP-G/CS mutations destabilize XPG and reduce its cellular protein levels. Mapping the distinct mutation classes provides structure-based predictions for disease phenotypes: Residues mutated in XP-G are positioned to reduce local stability and NER activity, whereas residues mutated in XP-G/CS have implied long-range structural defects that would likely disrupt stability of the whole protein, and thus interfere with its functional interactions. Combined data from crystallography, biochemistry, small angle X-ray scattering, and electron microscopy unveil an XPG homodimer that binds, unstacks, and sculpts duplex DNA at internal unpaired regions (bubbles) into strongly bent structures, and suggest how XPG complexes may bind both NER bubble junctions and replication forks. Collective results support XPG scaffolding and DNA sculpting functions in multiple DDR processes to maintain genome stability.

Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.

Toxin-antitoxin (TA) systems are widespread in bacteria and archaea plasmids and genomes to regulate DNA replication, gene transcription, or protein translation. Higher eukaryotic and prokaryotic nucleotide-binding (HEPN) and minimal nucleotidyltransferase (MNT) domains are prevalent in prokaryotic genomes and constitute TA pairs. However, three gene pairs (MTH304/305, 408/409, and 463/464) of Methanothermobacter thermautotropicus ΔH HEPN-MNT family have not been studied as TA systems. Among these candidates, our study characterizes the MTH463/MTH464 TA system. MTH463 expression inhibited Escherichia coli growth, whereas MTH464 did not and blocked MTH463 instead. Using site-directed MTH463 mutagenesis, we determined that amino acids R99G, H104A, and Y106A from the R[ɸX]4-6H motif are involved with MTH463 cell toxicity. Furthermore, we established that purified MTH463 could degrade MS2 phage RNA, whereas purified MTH464 neutralized MTH463 activity in vitro. Our results indicate that the endonuclease toxin MTH463 (encoding a HEPN domain) and its cognate antitoxin MTH464 (encoding the MNT domain) may act as a type II TA system in M. thermautotropicus ΔH. This study provides initial and essential information studying TA system functions, primarily archaea HEPN-MNT family. [ABSTRACT FROM AUTHOR]

Introduction: During evolution, blood-feeding arthropods developed a complex salivary mixture that can interfere with host haemostatic and immune response, favoring blood acquisition and pathogen transmission. Therefore, a survey of the salivary gland contents can lead to the identification of molecules with potent pharmacological activity in addition to increase our understanding of the molecular mechanisms underlying the hematophagic behaviour of arthropods. The southern house mosquito, Culex quinquefasciatus, is a vector of several pathogenic agents, including viruses and filarial parasites that can affect humans and wild animals. Results: Previously, a Sanger-based transcriptome of the salivary glands (sialome) of adult C. quinquefasciatus females was published based on the sequencing of 503 clones organized into 281 clusters. Here, we revisited the southern mosquito sialome using an Illumina-based RNA-sequencing approach of both male and female salivary glands. Our analysis resulted in the identification of 7,539 coding DNA sequences (CDS) that were functionally annotated into 25 classes, in addition to 159 long non-coding RNA (LncRNA). Additionally, comparison of male and female libraries allowed the identification of female-enriched transcripts that are potentially related to blood acquisition and/or pathogen transmission. Conclusion: Together, these findings represent an extended reference for the identification and characterization of the proteins containing relevant pharmacological activity in the salivary glands of C. quinquefasciatus mosquitoes. [ABSTRACT FROM AUTHOR]

An electrochemically reduced graphene oxide (ERGO) electrode-based electrochemical assay was developed for rapid, sensitive, and straightforward analysis of both activity and inhibition of the endonuclease EcoRV. The procedure uses a DNA substrate designed for EcoRV, featuring a double-stranded DNA (dsDNA) region labeled with methylene blue (MB) and a single-stranded DNA (ssDNA) region immobilized on the ERGO surface. The ERGO electrode, immobilized with the DNA substrate, was subsequently exposed to a sample containing EcoRV. Upon enzymatic hydrolysis, the cleaved dsDNA fragments were detached from the ERGO surface, leading to a decrease in the MB concentration near the electrode. This diminished the electron transfer efficiency for MB reduction, resulting in a decreased reduction current. This assay demonstrates excellent specificity and high sensitivity, with a limit of detection (LOD) of 9.5 × 10−3 U mL−1. Importantly, it can also measure EcoRV activity in the presence of aurintricarboxylic acid, a known inhibitor, highlighting its potential for drug discovery and clinical diagnostic applications.

The Oropouche virus (OROV) is a member of the family Peribunyaviridae (order Bunyavirales) and the cause of a dengue-like febrile illness transmitted mainly by biting midges and mosquitoes. In this study, we aimed to explore acylphloroglucinols and xanthohumol from hops (Humulus lupulus L.) as a promising alternative for antiviral therapies. The evaluation of the inhibitory potential of hops compounds on the viral cycle of OROV was performed through two complementary approaches. The first approach applies cell-based assay post-inoculation experiments to explore the inhibitory potential on the latest steps of the viral cycle, such as genome translation, replication, virion assembly, and virion release from the cells. The second part covers in silico methods evaluating the ability of those compounds to inhibit the activity of the endonuclease domain, which is essential for transcription, binding, and cleaving RNA. In conclusion, the beta acids showed strongest inhibitory potential in post-treatment assay (EC50 = 26.7 µg/mL). Xanthohumol had the highest affinity for OROV endonuclease followed by colupulone and cohumulone. This result contrasts with that observed for docking and MM/PBSA analysis, where cohumulone was found to have a higher affinity. Finally, among the three tested ligands, Lys92 and Arg33 exhibited the highest affinity with the protein.

Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses encode for a Replicase (Rep) that is essential for viral replication. Rep is a helicase with three domains: an endonuclease, an oligomeric, and an ATPase domain (ED, OD, and AD). Our recent cryo-EM structure of the porcine circovirus 2 (PCV2) Rep provided the first structure of a CRESS-DNA Rep. The structure visualized the ED to be highly mobile, Rep to form a homo-hexamer, bound ssDNA and nucleotides, and the AD to adopt a staircase arrangement around the ssDNA. We proposed a hand-over-hand mechanism by the ADs for ssDNA translocation. The hand-over-hand mechanism requires extensive movement of the AD. Here, we scrutinize this mechanism using all-atom Molecular Dynamics (MD) simulation of Rep in three states: (1) Rep bound to ssDNA and ADP, (2) Rep bound to ssDNA, and (3) Rep by itself. Each of the 700 nsec simulations converges within 200 nsec and provides important insight into the dynamics of Rep, the dynamics of Rep in the presence of these biomolecules, and the importance of ssDNA and ADP in driving the AD to adopt the staircase arrangement around the ssDNA. To the best of our knowledge, this is the first example of an all-atom MD simulation of a CRESS-DNA Rep. This study sets the basis of further MD studies aimed at obtaining a chemical understanding of how Rep uses nucleotide binding and hydrolysis to translocate ssDNA.

Transfer RNAs (tRNAs) represent the most abundant class of RNA molecules in the cell and are key players during protein synthesis and cellular homeostasis. Aberrations in the extensive tRNA biogenesis pathways lead to severe neurological disorders in humans. Mutations in the tRNA splicing endonuclease (TSEN) and its associated RNA kinase cleavage factor polyribonucleotide kinase subunit 1 (CLP1) cause pontocerebellar hypoplasia (PCH), a heterogeneous group of neurodegenerative disorders, that manifest as underdevelopment of specific brain regions typically accompanied by microcephaly, profound motor impairments, and child mortality. Recently, we demonstrated that mutations leading to specific PCH subtypes destabilize TSEN in vitro and cause imbalances of immature to mature tRNA ratios in patient‐derived cells. However, how tRNA processing defects translate to disease on a systems level has not been understood. Recent findings suggested that other cellular processes may be affected by mutations in TSEN/CLP1 and obscure the molecular mechanisms of PCH emergence. Here, we review PCH disease models linked to the TSEN/CLP1 machinery and discuss future directions to study neuropathogenesis. [ABSTRACT FROM AUTHOR]

The eukaryotic Argonaute proteins (eAgos) play an important role in the RNA interference pathway. The function and mechanism of prokaryotic Argonaute proteins (pAgos) in vivo are still unclear although the structure of pAgos and eAgos are highly homologous. Most of the reported pAgos have a preference for 5′P-gDNA, but MpAgo originated from bacteria Marinitoga piezophila preferentially uses 5′OH-gRNA to target DNA and RNA. To enrich our knowledge of this type of Argonaute proteins, here we report an Argonaute protein derived from Tepiditoga spiralis (TsAgo). Like MpAgo, TsAgo has a preference for 5′OH-gRNA. Meanwhile, TsAgo has DNA and RNA cleavage activity in presence of Mn2+ and Mg2+, and TsAgo has catalytic activity at 37–70 °C. In addition, TsAgo can tolerate mismatches in the 5′-end and 3′-tail regions of guides but is sensitive to mismatches in the 5′-seed and central regions of guides, especially the central region. Furthermore, the EMSA assay reveals that TsAgo exhibits a stronger binding affinity for 5′OH-gRNA than 5′P-gRNA which is consistent with its cleavage activity. Moreover, the structural modeling analysis demonstrates that like MpAgo, TsAgo has an ordered α5 at the C terminus of the PIWI domain which may hinder to binding of 5′ phosphate. Importantly, we find that TsAgo can target and cut plasmid DNA in vitro at 60 °C under the direction of RNA guides. These studies broaden our understanding of pAgos, and demonstrate that TsAgo can be regarded as an RNA-guided programmable nuclease for cleaving plasmids. • TsAgo has a preference for 5′OH-gRNA. • TsAgo bears high specificity in the central region of guides. • TsAgo can use 5′OH-gRNA to recognize and cleave plasmids. [ABSTRACT FROM AUTHOR]

Ribosomal RNA (rRNA) processing and maturation are fundamentally important for ribosome biogenesis, but the mechanisms in archaea, the third form of life, remains largely elusive. This study aimed to investigate the rRNA maturation process in Methanococcus maripaludis, a representative archaeon lacking known 3′-5′ exonucleases. Through cleavage site identification and enzymatic assays, the splicing endonuclease EndA was determined to process the bulge-helix-bulge (BHB) motifs in 16S and 23S rRNA precursors. After splicing, the circular processing intermediates were formed and this was confirmed by quantitative RT-PCR and Northern blot. Ribonuclease assay revealed a specific cleavage at a 10-nt A/U-rich motif at the mature 5′ end of pre-16S rRNA, which linearized circular pre-16S rRNA intermediate. Further 3′-RACE and ribonuclease assays determined that the endonuclease Nob1 cleaved the 3′ extension of pre-16S rRNA, and so generated the mature 3′ end. Circularized RT-PCR (cRT-PCR) and 5′-RACE identified two cleavage sites near helix 1 at the 5′ end of 23S rRNA, indicating that an RNA structure-based endonucleolytic processing linearized the circular pre-23S rRNA intermediate. In the maturation of pre-5S rRNA, multiple endonucleolytic processing sites were determined at the 10-nt A/U-rich motif in the leader and trailer sequence. This study demonstrates that endonucleolytic processing, particularly at the 10-nt A/U-rich motifs play an essential role in the pre-rRNA maturation of M. maripaludis, indicating diverse pathways of rRNA maturation in archaeal species. [ABSTRACT FROM AUTHOR]

Prokaryotic Argonaute (pAgo) nucleases with precise DNA cleavage activity show great potential for gene manipulation. Extensive biochemical studies have revealed that recognition of guides with different 5′ groups by Ago is important for biocatalysis. Here, we identified an Ago from the thermophilic Thermus parvatiensis (TpsAgo) and analyzed the regulatory effect of 5′-modified guides on TpsAgo cleavage activity. Recombinant TpsAgo cleaves single-stranded DNA and RNA targets at 65–90°C, which is mediated by a 5′ hydroxyl or phosphate DNA guide. Notably, TpsAgo can utilize various 5′-modified DNA guides for catalysis, including 5′-NH2C6, 5′-Biotin, 5′-FAM and 5′-SHC6 guides. Moreover, TpsAgo performs programmable cleavage of double-stranded DNA at temperatures over 80°C and strongly tolerates NaCl concentrations up to 3.2 M. These results provide insight into the catalytic performance of Agos by guide regulation, which may facilitate their biotechnological applications.

Programmed DNA double-strand breaks (DSBs) occur during antigen receptor gene recombination, namely V(D)J recombination in developing B lymphocytes and class switch recombination (CSR) in mature B cells. Repair of these DSBs by classical end-joining (c-NHEJ) enables the generation of diverse BCR repertoires for efficient humoral immunity. Deletion of or mutation in c-NHEJ genes in mice and humans confer various degrees of primary immune deficiency and predisposition to lymphoid malignancies that often harbor oncogenic chromosomal translocations. In the absence of c-NHEJ, alternative end-joining (A-EJ) catalyzes robust CSR and to a much lesser extent, V(D)J recombination, but the mechanisms of A-EJ are only poorly defined. In this review, we introduce recent advances in the understanding of A-EJ in the context of V(D)J recombination and CSR with emphases on DSB end processing, DNA polymerases and ligases, and discuss the implications of A-EJ to lymphoid development and chromosomal translocations.

Abstract Background Prokaryotic Argonaute (pAgo) proteins are well-known oligonucleotide-directed endonucleases, which contain a conserved PIWI domain required for endonuclease activity. Distantly related to pAgos, PIWI-RE family, which is defined as PIWI with conserved R and E residues, has been suggested to exhibit divergent activities. The distinctive biochemical properties and physiological functions of PIWI-RE family members need to be elucidated to explore their applications in gene editing. Results Here, we describe the catalytic performance and cellular functions of a PIWI-RE family protein from Pseudomonas stutzeri (PsPIWI-RE). Structural modelling suggests that the protein possesses a PIWI structure similar to that of pAgo, but with different PAZ-like and N-terminal domains. Unlike previously reported pAgos, recombinant PsPIWI-RE acts as an RNA-guided DNA nuclease, as well as a DNA-guided RNA nuclease. It cleaves single-stranded DNA at temperatures ranging from 20 to 65 °C, with an optimum temperature of 45 °C. Mutation at D525 or D610 significantly reduced its endonuclease activity, confirming that both residues are key for catalysis. Comparing with wild-type, mutant with PIWI-RE knockout is more sensitive to ciprofloxacin as DNA replication inhibitor, suggesting PIWI-RE may potentially be involved in DNA replication. Conclusion Our study provides the first insights into the programmable nuclease activity and biological function of the unknown PIWI-RE family of proteins, emphasizing their important role in vivo and potential application in genomic DNA modification. Graphical Abstract

Metal-organic framework play an important role in developing novel detection techniques. Herein, an ultrathin two-dimensional (2D) metal-organic framework nanosheet (Cu-TCPP) assembled microflower was successfully synthesized and applied for ultrasensitive detection. The high detection performance of Cu-TCPP microflower was attributed to high-capacity aptamer adsorption and specific desorption resulting from numerous approachable active sites, and its ultrathin nanosheets microflower-like structure. Moreover, the Cu-TCPP microflower had overcame the disadvantages of 2D MOF nanosheets which tended to agglomerate. Moreover, it exhibited excellent fluorescence quenching performance as a donor for fluorescence resonance energy transfer. Acetamiprid, a new chloronicotinic neurotoxic insecticide was used as the model analyte. The ultrasensitive sensor was developed by combining the Cu-TCPP microflower with deoxyribonuclease Ⅰ signal enhanced fluorescence. When acetamiprid was present, the fluorescent labeled aptamer probe was selectively bound to acetamiprid and desorbed from the Cu-TCPP microflower, resulting in fluorescence recovery. Subsequently, DNase Ⅰ was used to cleave the DNA strands of the aptamer probe, and acetamiprid was released to participate in a new cycle. The detection limit of the assay was as low as 5.56 pg/mL with a linear range 50.00 pg/mL∼5.00 μg/mL, and it was a universal platform to detect other analytes. [Display omitted] • The Cu-TCPP microflower was synthetized with superior active sites and FRET performance. • The Cu-TCPP microflower and DNase I assisted amplification was combined for ultrasensitive aptasensor. • The aptasensor for sensitive and selective detection of acetamiprid with LOD 5.56 pg/mL and linear range 50 ∼ 5,000,000 pg/mL. [ABSTRACT FROM AUTHOR]

Here, we report a novel endonuclease and N 6-adenine DNA methyltransferase (m6A methyltransferase) in the Ureaplasma parvum SV3F4 strain. Our previous study found that the SV3F4 strain carries 17 unique genes, which are not encoded in the two previously reported U. parvum serovar 3 strain, OMC-P162 and ATCC 700970. Of these 17 unique genes, UP3_ c0261 and UP3_ c0262, were originally annotated as encoding hypothetical proteins. Comparative genomics analyses more recently indicated they encode a Type II restriction endonuclease and an m6A methyltransferase, respectively. The UP3_c0261 and UP3_c0262 genes were individually expressed and purified in Escherichia coli. The UP3_c0261 recombinant protein showed endonuclease activity on the pT7Blue vector, recognizing and cleaving a GTNAC motif, resulting in a 5 base 5' extension. The UP3_c0261 protein digested a polymerase chain reaction (PCR) product harboring the GTNAC motif. The endonuclease UP3_c0261 was designated as UpaF4I. Treatment of the PCR product with the recombinant protein UP3_c0262 completely blocked the restriction enzyme activity of UpaF4I. Analysis of the treated PCR product harboring a modified nucleotide by UP3_c0262 with HPLC-MS/MS and MS/MS showed that UP3_c0262 was an m6A methyltransferase containing a methylated A residue in both DNA strands of the GTNAC motif. Whole genome methylation analysis of SV3F4 showed that 99.9 % of the GTNAC motif was m6A modified. These results suggest the UP3_c0261 and UP3_c0262 genes may act as a novel Type II restriction-modification system in the Ureaplasma SV3F4 strain. • UP3_c0261 protein is a Type II endonuclease, designated as UpaF4I. • UpaF4I recognized the ↓GTNAC↑ motif, resulting in a 5 base 5' extension. • UP3_c0262 completely blocked UpaF4I restriction enzyme activity. • UP3_c0262 protein (M.UpaF4I) is an m6A DNA methyltransferase. [ABSTRACT FROM AUTHOR]

Teneurins are type 2 transmembrane proteins expressed by developing neurons during periods of synaptogenesis and apoptosis. Neurons expressing teneurin-1 synapse with other teneurin-1-expressing neurons, and neurons expressing teneurin-2 synapse with other teneurin-2-expressing neurons. Knockdowns and mutations of teneurins lead to abnormal neuronal connections, but the mechanisms underlying teneurin action remain unknown. Teneurins appear to have evolved via horizontal gene transfer from prokaryotic proteins involved in bacterial self-recognition. The bacterial teneurin-like proteins contain a cytotoxic C-terminal domain that is encapsulated in a tyrosine-aspartic acid repeat barrel. Teneurins are likely to be organized in the same way, but it is unclear if the C-terminal domains of teneurins have cytotoxic properties. Here we show that expression of teneurin C-terminal domains or the addition of purified teneurin C-terminal domains leads to an increase in apoptosis in vitro The C-terminal domains of teneurins are most similar to bacterial nucleases, and purified C-terminal domains of teneurins linearize pcDNA3 and hydrolyze mitochondrial DNA. We hypothesize that yet to be identified stimuli lead to the release of the encapsulated teneurin C-terminal domain into the intersynaptic region, resulting in programmed cell death or the disruption of mitochondrial DNA and the subsequent pruning of inappropriate contacts.

Abstract Thermophilic Argonaute proteins (Agos) have been shown to utilize small DNA guides for cleaving complementary DNA in vitro, which shows great potential for nucleic acid detection. In this study, we explored mesophilic Agos for the detection of small molecule by cooperating with allosteric transcription factors (aTFs). Two Agos from mesophilic bacteria, Paenibacillus borealis (PbAgo) and Brevibacillus laterosporus (BlAgo), showed nuclease activity for single-stranded DNA at moderate temperatures (37 °C) by using 5′-phosphorylated and 5′-hydroxylated DNA guides. Both Agos perform programmable cleavage of double-stranded DNA, especially in AT-rich regions of plasmid. Furthermore, we developed a simple and low-cost p-hydroxybenzoic acid detection method based on DNA-guided DNA cleavage of Agos and the allosteric effect of HosA, which expands the potential application of small molecule detection by Agos.

Plant gene editing has become an important molecular tool to revolutionize modern breeding of crops. Over the past years, remarkable advancement has been made in developing robust and efficient editing methods for plants. Despite a variety of available genome editing methods, the discovery of most recent system of clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins (CRISPR-Cas) has been one of the biggest advancement in this path, with being the most efficient approach for genome manipulation. Until recently, genetic manipulations were confined to methods, like Agrobacterium-mediated transformations, zinc-finger nucleases, and TAL effector nucleases. However this technology supersedes all other methods for genetic modification. This RNA-guided CRISPR-Cas system is being rapidly developed with enhanced functionalities for better use and greater possibilities in biological research. In this review, we discuss and sum up the application of this simple yet powerful tool of CRISPR-Cas system for crop improvement with recent advancement in this technology. [ABSTRACT FROM AUTHOR]

Riemerella anatipestifer is an important bacterial pathogen in the global duck industry and causes heavy economic losses. In our previous study, we demonstrated that R. anatipestifer type IX secretion system components GldK and GldM, and the secretion protein metallophosphoesterase, acted as virulence factors. In this study, R. anatipestifer AS87_RS02955 was investigated for virulence and enzymatic activity properties. We constructed AS87_RS02955 mutation and complementation strains to assess bacterial virulence. In vivo bacterial loads showed a significantly reduced bacterial loads in the blood of ducks infected with mutant strain Yb2D02955, which was recovered in the blood of ducks infected with the complementation strain cYb2D02955, demonstrating that AS87_RS02955 was associated with virulence. Further studies showed AS87_RS02955 was a novel nonspecific endonuclease with no functionally conserved domain, but enzymatic activity toward DNA and RNA was indicated. DNase activity was activated by Zn21, Cu21, Mg21, Ca21, and Mn21 ions but inhibited by ethylenediaminetetraacetic acid. RNase activity was independent of metal cations, but stimulated by Mg21, Ca21, and Mn21. RAS87_RS02955 enzymatic activity was active across a broad pH and temperature range. Moreover, we identified four sites in rAS87_RS02955, F39, F92, I134, and F145, which were critical for enzymatic activity. In summary, we showed that R. anatipestifer AS87_RS02955 encoded a novel endonuclease with important roles in bacterial virulence. IMPORTANCE R. anatipestifer AS87_RS02955 was identified as a novel T9SS effector and displayed a nonspecific endonuclease activity in this study. The protein did not contain a conserved His-Asn-His motif structure, which is similar to the endonuclease from Prevotella sp. Its mutant strain Yb2D02955 demonstrated significantly attenuated virulence, suggesting AS87_RS02955 is an important virulence factor. Moreover, AS87_RS02955 displayed nonspecific endonuclease activity to cleave l DNA and MS2 RNA, while four protein sites were critical for endonuclease activity. In conclusion, R. anatipestifer AS87_RS02955 plays important roles in bacterial virulence. [ABSTRACT FROM AUTHOR]

Canine parvovirus (CPV) can cause high morbidity and mortality rates in puppies, posing a significant threat to both pet dogs and the breeding industry. Rapid, accurate, and convenient detection methods are important for the early intervention and treatment of canine parvovirus. In this study, we propose a visual CPV detection system called nucleic acid mismatch enzyme digestion (NMED). This system combines loop-mediated isothermal amplification (LAMP), endonuclease for gene mismatch detection, and colloidal gold lateral chromatography. We demonstrated that NMED can induce the binding of the amplicon from the sample to the specific labeling probe, which in turn triggers digestion by the endonuclease. The sensitivity and visual visibility of LAMP were increased by combining endonuclease and colloidal gold lateral chromatography assisted by a simple temperature-controlled device. The sensitivity of the NMED assay was 1 copy/μL, which was consistent with quantitative PCR (qPCR). The method was validated with 20 clinical samples that potentially had CPV infection; 15 positive samples and 5 negative samples were evaluated; and the detection accuracy was consistent with that of qPCR. As a rapid, accurate, and convenient molecular diagnostic method, NMED has great potential for application in the field of pathogenic microorganism detection., Importance: The NMED method has been established in the laboratory and used for CPV detection. The method has several advantages, including simple sampling, high sensitivity, intuitive results, and no requirement for expensive equipment. The establishment of this method has commercial potential and offers a novel approach and concept for the future development of clinical detection of pathogenic microorganisms., Competing Interests: The authors declare no conflict of interest.