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Genome editing provides exciting new opportunities to develop transgene-free mutants with desired modifications to meet global food demands. Current developments in genome editing and the diversity of CRISPR-associated proteins with various protospacer adjacent motif regions have redefined our ability to edit plant genomes. Improved Cas nucleases have been developed that could increase editing efficiencies and reduce off-targets as next-generation Cas-nuclease systems for genome-editing crops. Mutant sequencing and integration of data with artificial intelligence approaches could be used to seek any off-target edits and predict possible new protein–protein interactions. Regulatory authorities are increasingly viewing targeted mutagenesis as an extension of conventional plant breeding: the trend is to deregulate edited products if they could have been generated by conventional plant breeding processes. The CRISPR/Cas system comprises RNA-guided nucleases, the target specificity of which is directed by Watson–Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GEd) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GEd crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains. The CRISPR/Cas system comprises RNA-guided nucleases the target specificity of which is directed by Watson–Crick base pairing of target loci with single guide (sg)RNA to induce the desired edits. CRISPR-associated proteins and other engineered nucleases are opening new avenues of research in crops to induce heritable mutations. Here, we review the diversity of CRISPR-associated proteins and strategies to deregulate genome-edited (GE) crops by considering them to be close to natural processes. This technology ensures yield without penalties, advances plant breeding, and guarantees manipulation of the genome for desirable traits. DNA-free and off-target-free GE crops with defined characteristics can help to achieve sustainable global food security under a changing climate, but need alignment of international regulations to operate in existing supply chains. [ABSTRACT FROM AUTHOR]

The prototypic sensors for the induction of innate and adaptive immune responses are the Toll-like receptors (TLRs). Unusually high expression of TLRs in prostate carcinoma (PC), associated with less differentiated, more aggressive and more propagating forms of PC, changed the previous paradigm about the role of TLRs strictly in immune defense system. Our data reveal an entirely novel role of nucleic acids-sensing Toll-like receptors (NA-TLRs) in functional adaptation of malignant cells for supply and digestion of surrounding metabolic substrates from dead cells as specific mechanism of cancer cells survival, by corresponding ligands accelerated degradation and purine/pyrimidine salvage pathway. The spectrophotometric measurement protocols used for the determination of the activity of RNases and DNase II have been optimized in our laboratory as well as the enzyme-linked immunosorbent method for the determination of NF-κB p65 in prostate tissue samples. The protocols used to determine Dicer RNase, AGO2, TARBP2 and PIWIL4 were based on enzyme-linked immunosorbent assay. The amount of pre-existing acid-soluble oligonucleotides was measured and expressed as coefficient of absorbance. The activities of acid DNase II and RNase T2, and the activities of nucleases cleaving TLR3, TLR7/8 and TLR9 ligands (Poly I:C, poly U and unmethylated CpG), increased several times in PC, compared to the corresponding tumor adjacent and control tissue, exerting very high sensitivity and specificity of above 90%. Consequently higher levels of hypoxanthine and NF-κB p65 were reported in PC, whereas the opposite results were observed for miRNA biogenesis enzyme (Dicer RNase), miRNA processing protein (TARB2), miRNA-induced silencing complex protein (Argonaute-AGO) and PIWI-interacting RNAs silence transposon. Considering the crucial role of purine and pyrimidine nucleotides as energy carriers, subunits of nucleic acids and nucleotide cofactors, future explorations will be aimed to design novel anti-cancer immune strategies based on a specific acid endolysosomal nuclease inhibition. [ABSTRACT FROM AUTHOR]

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins via their CRISPR-associated Rossmann fold (CARF) domains. Commonly, these are ribonucleases (RNases) that unspecifically degrade both invader and host RNA. To mitigate adverse effects on cell growth, ring nucleases can degrade extant cOAs to switch off ancillary nucleases. Here we show that the model organism Synechocystis sp. PCC 6803 harbors functional CARFdomain effector and ring nuclease proteins. We purified and characterized the two ancillary CARF-domain proteins from the III-D type CRISPR system of this cyanobacterium. The Csx1 homolog, SyCsx1, is a cyclic tetraadenylate(cA4)-dependent RNase with a strict specificity for cytosine nucleotides. The second CARF-domain protein with similarity to Csm6 effectors, SyCsm6, did not show RNase activity in vitro but was able to break down cOAs and attenuate SyCsx1 RNase activity. Our data suggest that the CRISPR systems in Synechocystis confer a multilayered cA4-mediated defense mechanism. [ABSTRACT FROM AUTHOR]

The identification of the protospacer adjacent motif (PAM) sequences of Cas9 nucleases is crucial for their exploitation in genome editing. Here we develop a computational pipeline that was used to interrogate a massively expanded dataset of metagenome and virome assemblies for accurate and comprehensive PAM predictions. This procedure allows the identification and isolation of sequence-tailored Cas9 nucleases by using the target sequence as bait. As proof of concept, starting from the disease-causing mutation P23H in the RHO gene, we find, isolate and experimentally validate a Cas9 which uses the mutated sequence as PAM. Our PAM prediction pipeline will be instrumental to generate a Cas9 nuclease repertoire responding to any PAM requirement. Cas9 proteins require a target-adjacent sequence, the PAM, in order to cleave DNA. Here the authors develop a pipeline to accurately predict PAM sequences in order to facilitate the identification of Cas9s targeting specific sequences, including mutations. [ABSTRACT FROM AUTHOR]

Nucleases used in genome engineering induce hydrolysis of DNA phosphate backbone in a sequence-specific manner. So far CRISPR-Cas, the RNA-guided nucleases, is the most advanced genome engineering system. The CRISPR nucleases allows recognition of a particular genomic sequence with two distinct molecular interactions: first, by direct interaction between the nuclease and the protospacer-adjacent motif, wherein discrete amino acids interact with DNA base pairs; and second, by hybridization of the guide RNA with the target DNA sequence. Here we report the application of the single strand annealing cellular assay to analyze and quantify nuclease activity of wild type and mutant CRISPR-Cpf1. Using this heterologous marker system based on GFP activity, we observed a comparable PAM recognition selectivity with the NGS analysis. The heterologous marker system has revealed that LbCpf1 is a more specific nuclease than AsCpf1 in a cellular context. We controlled the in vitro activity of the Cpf1 nuclease complexes expressed in mammalian cells and demonstrated that they are responsible of the DNA cleavage at the target site. In addition, we generated and tested LbCpf1 variants with several combinations of mutations at the PAM-recognition positions G532, K538 and Y542. Finally, we showed that the results of the in vitro DNA cleavage assay with the wild type and mutants LbCpf1 corroborate with the selection of 6TG resistant cells associated to the genomic disruption of hprt gene. [ABSTRACT FROM AUTHOR]

CRISPR/Cas13a nucleases are currently considered to be the basis for the development of a new generation of biosensors for the ultrasensitive, in-field detection of bacterial and viral pathogens. A recombinant Cas13a nuclease with functional affinity was obtained as a result of heterologous expression in E. coli with a single-step purification process via metal-chelating chromatography with the N-terminal polyhistidine tag. The simplified procedure of Cas13a nuclease purification broadens the possibilities for the development and practical application of diagnostic biosensing systems based on it. Moreover, our results indicate that the currently uncharacterized protein U2PWF1 of Leptotrichia wadei represents Cas13a nuclease. [ABSTRACT FROM AUTHOR]

• Anti-CRISPR proteins (Acrs) allow phages to inhibit CRISPR-Cas immune systems. • Our method uses a cell-free TXTL system to rapidly characterize Acrs. • In the method, Acrs block targeting of a GFP reporter plasmid by a Cas nuclease. • Pre-expressed Acrs, Cas nucleases, and guide RNAs can be frozen and stored. • Diluting the pre-expressed Acr can eliminate non-specific inhibition of the reporter. The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli -derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors. [ABSTRACT FROM AUTHOR]

Nucleases are enzymes that can degrade genomic DNA and RNA that decrease the accuracy of quantitative measures of those nucleic acids. Here, we study conventional heating, standard microwave irradiation, and Lyse-It, a microwave-based lysing technology, for the potential to fragment and inactivate DNA and RNA endonucleases. Lyse-It employs the use of highly focused microwave irradiation to the sample ultimately fragmenting and inactivating RNase A, RNase B, and DNase I. Nuclease size and fragmentation were determined visually and quantitatively by SDS polyacrylamide gel electrophoresis and the mini-gel Agilent 2100 Bioanalyzer system, with a weighted size calculated to depict the wide range of nuclease fragmentation. Enzyme activity assays were conducted, and the rates were calculated to determine the effect of various lysing conditions on each of the nucleases. The results shown in this paper clearly demonstrate that Lyse-It is a rapid and highly efficient way to degrade and inactivate nucleases so that nucleic acids can be retained for down-stream detection. [ABSTRACT FROM AUTHOR]

Human genome contains a group of more than a dozen similar genes with diverse biological functions including antiviral, antibacterial and angiogenesis activities. The characterized gene products of this group show significant sequence similarity and a common structural fold associated with binding and cleavage of ribonucleic acid (RNA) substrates. Therefore, these proteins have been categorized as members of human pancreatic-type ribonucleases (hRNases). hRNases differ in cell/tissue localization and display distinct substrate binding preferences and a wide range of ribonucleolytic catalytic efficiencies. Limited information is available about structural and dynamical properties that influence this diversity among these homologous RNases. Here, we use computer simulations to characterize substrate interactions, electrostatics and dynamical properties of hRNases 1–7 associated with binding to two nucleotide substrates (ACAC and AUAU). Results indicate that even with complete conservation of active-site catalytic triad associated with ribonucleolytic activity, these enzymes show significant differences in substrate interactions. Detailed characterization suggests that in addition to binding site electrostatic and van der Waals interactions, dynamics of distal regions may also play a role in binding. Another key insight is that a small difference in temperature of 300 K (used in experimental studies) and 310 K (physiological temperature) shows significant changes in enzyme-substrate interactions. [ABSTRACT FROM AUTHOR]

Decellularization of blood vessels is a promising approach to generate native biomaterials for replacement of diseased vessels. The decellularization process affects the mechanical properties of the vascular graft and thus can have a negative impact for in vivo functionality. The aim of this study was to determine how detergents under different fluid dynamics affects decellularization efficacy and mechanical properties of the vascular graft. We applied a protocol utilizing 1% TritonX, 1% Tributyl phosphate (TnBP) and DNase on porcine vena cava. The detergents were applied to the vessels under different conditions; static, agitation and perfusion with 3 different perfusion rates (25, 100 and 400 mL/min). The decellularized grafts were analyzed with histological, immunohistochemical and mechanical tests. We found that decellularization efficacy was equal in all groups, however the luminal ultrastructure of the static group showed remnant cell debris and the 400 mL/min perfusion group showed local damage and tearing of the luminal surface. The mechanical stiffness and maximum tensile strength were not influenced by the detergent application method. In conclusion, our results indicate that agitation or low-velocity perfusion with detergents are preferable methods for blood vessel decellularization. [ABSTRACT FROM AUTHOR]

Transfer RNAs require essential maturation steps to become functional. Among them, RNase P removes 5′ leader sequences of pre‐tRNAs. Although RNase P was long thought to occur universally as ribonucleoproteins, different types of protein‐only RNase P enzymes were discovered in both eukaryotes and prokaryotes. Interestingly, all these enzymes belong to the super‐group of PilT N‐terminal‐like nucleases (PIN)‐like ribonucleases. This wide family of enzymes can be subdivided into major subgroups. Here, we review recent studies at both functional and mechanistic levels on three PIN‐like ribonucleases groups containing enzymes connected to tRNA maturation and/or translation regulation. The evolutive distribution of these proteins containing PIN‐like domains as well as their organization and fusion with various functional domains is discussed and put in perspective with the diversity of functions they acquired during evolution, for the maturation and homeostasis of tRNA and a wider array of RNA substrates. © 2019 IUBMB Life, 2019 © 2019 IUBMB Life, 71(8):1117–1125, 2019 [ABSTRACT FROM AUTHOR]

The epithelial-mesenchymal transition (EMT) plays a key role in tumor progression, drug resistance and metastasis. Recently, numerous microRNA (miRNA) have been described to regulate EMT in tumor progression. In this study, we found that conditioned medium from the LC212 non-small-cell lung cancer (NSCLC) cell line (LC212-CM) induces morphological changes and overexpression of Vimentin, CD90, SMAD 2/3, SLUG and TWIST in A549 NSCLC cells, consistent with a mesenchymal phenotype. To identify the soluble mediators in LC212-CM involved in this phenomenon, we performed miRNA profiling and TGF-β1 quantification. We found that LC212-CM contains high levels of TGF-β1 as well as different secreted miRNAs. We focused our attention on Homo sapiens-microRNA21 (hsa-miR21), one of most relevant miRNA associated with lung cancer progression, metastasis and EMT. An hsa-miR21 antagomiR was able to prevent the LC212-CM-induced EMT phenotype in A549 cells. Furthermore, we found that TGF-β1 and hsa-miR21 cooperate in the induction of EMT in A549 cells. Intriguingly, TGF-β1 was found to induce hsa-miR21 expression in A549 cell, thus suggesting that the hsa-miR21 mediates at least in part the pro-EMT effects of TGF-β1. In conclusion, hsa-miR21 and TGF-β1 are involved in autocrine and paracrine circuits that regulate the EMT status of lung cancer cells. [ABSTRACT FROM AUTHOR]

Two long-term potentially oil exposed Baltic Sea coastal sites near old oil refineries and harbours were compared to nearby less exposed sites in terms of bacterial, archaeal and fungal microbiomes and oil degradation potential. The bacterial, archaeal and fungal diversities were similar in oil exposed and less exposed sampling sites based on bacterial and archaeal 16S rRNA gene and fungal 5.8S rRNA gene amplicon sequencing from both DNA and RNA fractions. The number of genes participating in alkane degradation (alkB) or PAH-ring hydroxylation (PAH–RHDα) were detected by qPCR in all water and sediment samples. These numbers correlated with the number of bacterial 16S rRNA gene copies in sediment samples but not with the concentration of petroleum hydrocarbons or PAHs. This indicates that both the clean and the more polluted sites at the Baltic Sea coastal areas have a potential for petroleum hydrocarbon degradation. The active community (based on RNA) of the coastal Baltic Sea water differed largely from the total community (based on DNA). The most noticeable difference was seen in the bacterial community in the water samples were the active community was dominated by Cyanobacteria and Proteobacteria whereas in total bacterial community Actinobacteria was the most abundant phylum. The abundance, richness and diversity of Fungi present in water and sediment samples was in general lower than that of Bacteria and Archaea. Furthermore, the sampling location influenced the fungal community composition, whereas the bacterial and archaeal communities were not influenced. This may indicate that the fungal species that are adapted to the Baltic Sea environments are few and that Fungi are potentially more vulnerable to or affected by the Baltic Sea conditions than Bacteria and Archaea. [ABSTRACT FROM AUTHOR]

The dynamics of the cellular proportion of mutant mtDNA molecules is crucial for mitochondrial diseases. Cellular populations of mitochondria are under homeostatic control, but the details of the control mechanisms involved remain elusive. Here, we use stochastic modelling to derive general results for the impact of cellular control on mtDNA populations, the cost to the cell of different mtDNA states, and the optimisation of therapeutic control of mtDNA populations. This formalism yields a wealth of biological results, including that an increasing mtDNA variance can increase the energetic cost of maintaining a tissue, that intermediate levels of heteroplasmy can be more detrimental than homoplasmy even for a dysfunctional mutant, that heteroplasmy distribution (not mean alone) is crucial for the success of gene therapies, and that long-term rather than short intense gene therapies are more likely to beneficially impact mtDNA populations. [ABSTRACT FROM AUTHOR]

Numerous Gram-negative pathogens use a Type III Secretion System (T3SS) to promote virulence by injecting effector proteins into targeted host cells, which subvert host cell processes. Expression of T3SS and the effectors is triggered upon host cell contact, but the underlying mechanism is poorly understood. Here, we report a novel strategy of Yersinia pseudotuberculosis in which this pathogen uses a secreted T3SS translocator protein (YopD) to control global RNA regulators. Secretion of the YopD translocator upon host cell contact increases the ratio of post-transcriptional regulator CsrA to its antagonistic small RNAs CsrB and CsrC and reduces the degradosome components PNPase and RNase E levels. This substantially elevates the amount of the common transcriptional activator (LcrF) of T3SS/Yop effector genes and triggers the synthesis of associated virulence-relevant traits. The observed hijacking of global riboregulators allows the pathogen to coordinate virulence factor expression and also readjusts its physiological response upon host cell contact. [ABSTRACT FROM AUTHOR]

A review of the article "A comprehensive overview and evaluation of circular RNA detection tools" which appeared in a previous issue of the periodical "PLOS Computational Biology" is presented.

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity. [ABSTRACT FROM AUTHOR]

Proteins of the Fanconi Anemia (FA) complementation group are required for crosslink (CL) repair in humans and their loss leads to severe pathological phenotypes. Here we characterize a homolog of the Fe-S cluster helicase FANCJ in the model plant Arabidopsis, AtFANCJB, and show that it is involved in interstrand CL repair. It acts at a presumably early step in concert with the nuclease FAN1 but independently of the nuclease AtMUS81, and is epistatic to both error-prone and error-free post-replicative repair in Arabidopsis. The simultaneous knock out of FANCJB and the Fe-S cluster helicase RTEL1 leads to induced cell death in root meristems, indicating an important role of the enzymes in replicative DNA repair. Surprisingly, we found that AtFANCJB is involved in safeguarding rDNA stability in plants. In the absence of AtRTEL1 and AtFANCJB, we detected a synergetic reduction to about one third of the original number of 45S rDNA copies. It is tempting to speculate that the detected rDNA instability might be due to deficiencies in G-quadruplex structure resolution and might thus contribute to pathological phenotypes of certain human genetic diseases. [ABSTRACT FROM AUTHOR]

In their comment, Chen and Chuang [] pointed out several weak points of our recent paper []. Here we respond in detail to clarify the dataset we used in our work. We also discuss the three confounding factors they listed in their comment. [ABSTRACT FROM AUTHOR]

We have developed a new method for selectively sorting droplets containing growing bacteria using a fluorescence resonance energy transfer (FRET)-based RNA probe. Bacteria and the FRET-based RNA probe are encapsulated into nanoliter-scale droplets, which are incubated to allow for cell growth. The FRET-based RNA probe is cleaved by RNase derived from the bacteria propagated in the droplets, resulting in an increase in fluorescence intensity. The fluorescent droplets containing growing bacteria are distinguishable from quenching droplets, which contain no cells. We named this method FNAP-sort based on the use of a fluorescent nucleic acid probe in droplets for bacterial sorting. Droplets containing the FRET-based RNA probe and four species of pure cultures, which grew in the droplets, were selectively enriched on the basis of fluorescence emission. Furthermore, fluorescent droplets were sorted from more than 500,000 droplets generated using environmental soil bacteria and the FRET-based RNA probe on days 1, 3, and 7 with repeated incubation and sorting. The bacterial compositions of sorted droplets differed on days 1, 3, and 7; moreover, on day 7, the bacterial composition of the fluorescent droplets was drastically different from that of the quenching droplets. We believe that FNAP-sort is useful for high-throughput cultivation and sorting of environmental samples containing bacteria with various growth rates, including slow-growing microbes that require long incubation times. [ABSTRACT FROM AUTHOR]

Though it is an essential process, transcription can be a source of genomic instability. For instance, it may generate RNA:DNA hybrids as the nascent transcript hybridizes with the complementary DNA template. These hybrids, called R-loops, act as a major cause of replication fork stalling and DNA breaks. In this study, we show that lowering transcription and R-loop levels in plastids of Arabidopsis thaliana reduces DNA rearrangements and mitigates plastid genome instability phenotypes. This effect can be observed on a genome-wide scale, as the loss of the plastid sigma transcription factor SIG6 prevents DNA rearrangements by favoring conservative repair in the presence of ciprofloxacin-induced DNA damage or in the absence of plastid genome maintenance actors such as WHY1/WHY3, RECA1 and POLIB. Additionally, resolving R-loops by the expression of a plastid-targeted exogenous RNAse H1 produces similar results. We also show that highly-transcribed genes are more susceptible to DNA rearrangements, as increased transcription of the psbD operon by SIG5 correlates with more locus-specific rearrangements. The effect of transcription is not specific to Sigma factors, as decreased global transcription levels by mutation of heat-stress-induced factor HSP21, mutation of nuclear-encoded polymerase RPOTp, or treatment with transcription-inhibitor rifampicin all prevent the formation of plastid genome rearrangements, especially under induced DNA damage conditions. [ABSTRACT FROM AUTHOR]

Fragmentation of DNA is the very important first step in preparing nucleic acids for next-generation sequencing. Here we report a novel Fragmentation Through Polymerization (FTP) technique, which is a simple, robust, and low-cost enzymatic method of fragmentation. This method generates double-stranded DNA fragments that are suitable for direct use in NGS library construction and allows the elimination of the additional step of reparation of DNA ends. [ABSTRACT FROM AUTHOR]

Sputum often contains large amounts of contaminating bacterial DNA that, if not eliminated during RNA isolation, may interfere with gene expression studies. During RNA isolation only repeated DNase treatment can effectively remove contaminating bacterial DNA from samples, but this compromises RNA quality. In this study we tested alternative methods to facilitate the removal of DNA and improve the quality of RNA obtained. Sputum samples obtained from patients with chronic obstructive pulmonary disease were processed with dithiothreitol and subjected to various RNA isolation methods, yet with modified protocols. Modifications included prolonged DNase treatment or vortexing of sputum cells in the presence of beads prior to RNA isolation. Bacterial DNA contamination was tested by PCR using universal bacterial primers, while RNA quality was assessed by real-time PCR using GAPDH primers for amplicons of different length. We found that the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column was able to completely eliminate bacterial DNA, if sputum cells were lysed in the presence of bashing beads. Notably, compared with the standard protocol, the modified procedure yielded better quality RNA as well, as indicated by improved threshold profiles of qPCR. Bead vortexing of cells was less effective when combined with other RNA isolation methods, and the repeated DNase treatment needed to completely remove contaminating DNA from the samples reduced the quality of RNA markedly. Bead vortexing in combination with certain RNA extraction methods greatly facilitates the isolation of sputum RNA that is free of contaminating bacterial DNA, and is suitable for downstream applications. [ABSTRACT FROM AUTHOR]

The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, dependent on several key residues. Yet some differences between various CdtB subunit activities, and discrepancies between biochemical and cellular data, have been observed. To better characterise the role of CdtB in the induction of DNA damage, we affinity-purified wild-type and mutants of CdtB, issued from E. coli and H. ducreyi, under native and denaturing conditions. We then compared their nuclease activity by a classic in vitro assay using plasmid DNA, and two different eukaryotic assays–the first assay where host cells were transfected with a plasmid encoding CdtB, the second assay where host cells were directly transfected with purified CdtB. We show here that in vitro nuclease activities are difficult to quantify, whereas CdtB activities in host cells can be easily interpreted and confirmed the loss of function of the catalytic mutant. Our results highlight the importance of performing multiple assays while studying the effects of bacterial genotoxins, and indicate that the classic in vitro assay should be complemented with cellular assays. [ABSTRACT FROM AUTHOR]

The apolipoprotein E-C1-C4-C2 gene cluster at 19q13.32 encodes four amphipathic apolipoproteins. The influence of APOE common polymorphisms on plasma lipid/lipoprotein profile, especially on LDL-related traits, is well recognized; however, little is known about the role of other genes/variants in this gene cluster. In this study, we evaluated the role of common and uncommon/rare genetic variation in this gene region on inter-individual variation in plasma lipoprotein levels in non-Hispanic Whites (NHWs) and African blacks (ABs). In the variant discovery step, the APOE, APOC1, APOC4, APOC2 genes were sequenced along with their flanking and hepatic control regions (HCR1 and HCR2) in 190 subjects with extreme HDL-C/TG levels. The next step involved the genotyping of 623 NHWs and 788 ABs for the identified uncommon/rare variants and common tagSNPs along with additional relevant SNPs selected from public resources, followed by association analyses with lipid traits. A total of 230 sequence variants, including 15 indels were identified, of which 65 were novel. A total of 70 QC-passed variants in NHWs and 108 QC-passed variants in ABs were included in the final association analyses. Single-site association analysis of SNPs with MAF>1% revealed 20 variants in NHWs and 24 variants in ABs showing evidence of association with at least one lipid trait, including several variants exhibiting independent associations from the established APOE polymorphism even after multiple-testing correction. Overall, our study has confirmed known associations and also identified novel associations in this genomic region with various lipid traits. Our data also support the contribution of both common and uncommon/rare variation in this gene region in affecting plasma lipid profile in the general population. [ABSTRACT FROM AUTHOR]

DNA-RNA hybrids arise in all cell types, and are removed by multiple enzymes, including the trimeric ribonuclease, RNase H2. Mutations in human RNase H2 result in Aicardi–Goutières syndrome (AGS), an inflammatory brain disorder notable for being a Mendelian mimic of congenital viral infection. Previous studies have shown that several AGS-associated mutations of the RNase H2B subunit do not affect trimer stability or catalytic activity and are clustered on the surface of the complex, leading us to speculate that these mutations might impair important interactions of RNase H2 with so far unidentified proteins. In this study, we show that AGS mutations in this cluster impair the interaction of RNase H2 with several members of the CoREST chromatin-silencing complex that include the histone deacetylase HDAC2 and the demethylase KDM1A, the transcriptional regulators RCOR1 and GTFII-I as well as ZMYM3, an MYM-type zinc finger protein. We also show that the interaction is mediated by the zinc finger protein ZMYM3, suggesting that ZMYM3 acts as a novel type of scaffold protein coordinating interactions between deacetylase, demethylase and RNase H type enzymes, raising the question of whether coordination between histone modifications and the degradation of RNA-DNA hybrids may be required to prevent inflammation in humans. [ABSTRACT FROM AUTHOR]

The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm. [ABSTRACT FROM AUTHOR]

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus. [ABSTRACT FROM AUTHOR]

Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called 'off-targets' by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content of the target correlates with efficiency and that Cas9 from Staphylococcus aureus (SaCas9) is comparatively most efficient at inducing mutations. We also demonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use. [ABSTRACT FROM AUTHOR]

Retroviruses have evolved multiple means to counteract host restriction factors such as single-stranded DNA-specific deoxycytidine deaminases (APOBEC3s, A3s). These include exclusion of A3s from virions by an A3-unreactive nucleocapsid or expression of an A3-neutralizing protein (Vif, Bet). However, a number of retroviruses package A3s and do not encode apparent vif- or bet-like genes, yet they replicate in the presence of A3s. The mode by which they overcome deleterious restriction remains largely unknown. Here we show that the prototypic betaretrovirus, mouse mammary tumor virus (MMTV), packages similar amounts of A3s as HIV-1ΔVif, yet its proviruses carry a significantly lower level of A3-mediated deamination events than the lentivirus. The G-to-A mutation rate increases when the kinetics of reverse transcription is reduced by introducing a mutation (F120L) to the DNA polymerase domain of the MMTV reverse transcriptase (RT). A similar A3-sensitizing effect was observed when the exposure time of single-stranded DNA intermediates to A3s during reverse transcription was lengthened by reducing the dNTP concentration or by adding suboptimal concentrations of an RT inhibitor to infected cells. Thus, the MMTV RT has evolved to impede access of A3s to transiently exposed minus DNA strands during reverse transcription, thereby alleviating inhibition by A3 family members. A similar mechanism may be used by other retroviruses and retrotransposons to reduce deleterious effects of A3 proteins. [ABSTRACT FROM AUTHOR]

Putrescine belongs to the large group of polyamines, an essential class of metabolites that exists throughout all kingdoms of life. The Salmonella speF gene encodes an inducible ornithine decarboxylase that produces putrescine from ornithine. Putrescine can be also synthesized from arginine in a parallel metabolic pathway. Here, we show that speF expression is controlled at multiple levels through regulatory elements contained in a long leader sequence. At the heart of this regulation is a short open reading frame, orf34, which is required for speF production. Translation of orf34 interferes with Rho-dependent transcription termination and helps to unfold an inhibitory RNA structure sequestering speF ribosome-binding site. Two consecutive arginine codons in the conserved domain of orf34 provide a third level of speF regulation. Uninterrupted translation of orf34 under conditions of high arginine allows the formation of a speF mRNA structure that is degraded by RNase G, whereas ribosome pausing at the consecutive arginine codons in the absence of arginine enables the formation of an alternative structure that is resistant to RNase G. Thus, the rate of ribosome progression during translation of the upstream ORF influences the dynamics of speF mRNA folding and putrescine production. The identification of orf34 and its regulatory functions provides evidence for the evolutionary conservation of ornithine decarboxylase regulatory elements and putrescine production. [ABSTRACT FROM AUTHOR]

Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor β-induced (TGFBI) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI-targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy. [ABSTRACT FROM AUTHOR]

Staphylococcus epidermidis is a bacterium frequently isolated from contaminated platelet concentrates (PCs), a blood product used to treat bleeding disorders in transfusion patients. PCs offer an accidental niche for colonization of S. epidermidis by forming biofilms and thus avoiding clearance by immune factors present in this milieu. Using biochemical and microscopy techniques, we investigated the structural changes of the peptidoglycan (PG) and the biofilm matrix of S. epidermidis biofilms formed in whole-blood derived PCs compared to biofilms grown in glucose-supplemented trypticase soy broth (TSBg). Both, the PG and the biofilm matrix are primary mechanisms of defense against environmental stress. Here we show that in PCs, the S. epidermidis biofilm matrix is mainly of a proteinaceous nature with extracellular DNA, in contrast to the predominant polysaccharide nature of the biofilm matrix formed in TSBg cultures. PG profile studies demonstrated that the PG of biofilm cells remodels during PC storage displaying fewer muropeptides variants than those observed in TSBg. The PG muropeptides contain two chemical modifications (amidation and O-acetylation) previously associated with resistance to antimicrobial agents by other staphylococci. Our study highlights two key structural features of S. epidermidis that are remodeled when exposed to human platelets and could be used as targets to reduce septic transfusions events. [ABSTRACT FROM AUTHOR]