General Gel-sol Method to Synthesize Various Highly Fluorescent Nanoclusters and Assay of Nuclease with the Near Infrared-emitting Gold Nanoclusters.

A general egg white gel-sol strategy for fabrication of highly fluorescent Au, Ag, Cu, and Pt nanoclusters (NCs) and the first example of using Au NCs for assay of nuclease activity and inhibition were described. The Au NCs enabled bright red fluorescence, and the other Ag, Cu, and Pt NCs have highly blue emission. The red-emitting Au NCs were further applied in assay of S1 nuclease activity and inhibition. Free hemin efficiently quenches the emission of Au NCs by photoinduced electron transfer due to the formation of Au NCs-hemin conjugates. However, G-quadruplex/hemin exerts negligible effect on its fluorescence due to no Au NCs-hemin conjugate formed. There are stronger electrostatic repulsion effects between both negatively charged G-quadruplex and Au NCs. Therefore, a novel G-quadruplex/hemin-based Au NCs fluorescent sensor for S1 nuclease was designed. A known G-rich oligonucleotide (ODN) serves as not only substrate for S1 nuclease but also for the construction of G-quadruplex/hemin. The G-rich ODN is hydrolyzed into fragments by S1 nuclease resulting in no G-quadruplex/hemin formation. Therefore, the free hemin quenches Au NCs fluorescence remarkably and the assay of S1 nuclease activity and inhibition has accomplished. Both the fluorescent NCs syntheses and the detection of S1 nuclease are facile and efficient.

Perfect Complementarity Mechanism for Aphid Control: Oligonucleotide Insecticide Macsan-11 Selectively Causes High Mortality Rate for Macrosiphoniella sanborni Gillette.

Macrosiphoniella sanborni is a widespread pest of Chrysanthemum morifolium that causes significant damage to world floriculture. Chemical insecticides and biological methods of control have a number of disadvantages that can be improved by using oligonucleotide insecticides. In this article, we present, for the first time, the results of using oligonucleotide insecticides, for which the target sequence is an internal transcribed spacer (ITS) in a polycistronic rRNA transcript. The mortality of wingless aphid individuals after a Macsan-11 treatment was recorded at a level of 67.15 ± 3.32% 7 days after a single treatment with a solution at a concentration of 100 ng/µL and 97.38 ± 2.49% 7 days after a double treatment with a solution of the same concentration and a daily interval. The contact use of the control oligonucleotide (ACTG)2ACT-11. as well as the oligonucleotide insecticides Macsan-11(3') and Macsan-11(5') was not accompanied by insect mortality. Given the high variability in the internal transcribed spacer, which has proven to be a promising target for the action of oligonucleotide insecticides, it is possible to create selective preparations. This study showed the prospects of ribosomal insect pest genes as targets for the action of olinscides, and also demonstrated the high specificity of such insecticidal agents.

Targeted Knockout of the Vegfa Gene in the Retina by Subretinal Injection of RNP Complexes Containing Cas9 Protein and Modified sgRNAs.

The therapeutic effect of retinal gene therapy using CRISPR/Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of single guide RNAs (sgRNAs) and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro. To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs). In K562 cells, msgRNAs significantly increased the insertion/deletion (indel) frequency (25%) compared with unmodified counterparts leading to robust knockout of the VEGFA gene encoding vascular endothelial growth factor A (96% indels). Likewise, in HEK293 cells, lipoplexes containing varying amounts of RNP and EGFP mRNA showed efficient VEGFA knockout (43% indels) and strong EGFP expression, indicative of efficacious functional knockout using small amounts of RNP. In mice, subretinal injections of equivalent lipoplexes yielded 6% indels in Vegfa of isolated EGFP-positive RPE cells. However, signs of toxicity following delivery of lipoplexes containing high amounts of RNP were observed. Although the mechanism resulting in the varying efficacy remains to be elucidated, our data suggest that a single subretinal injection of RNPs carrying msgRNAs and SpCas9 induces targeted retinal indel formation, thus providing a clinically relevant strategy relying on nonviral delivery of short-lived nuclease activity.

The Antimicrobial Peptide Gad-1 Clears Pseudomonas aeruginosa Biofilms under Cystic Fibrosis Conditions.

Bacterial infections in cystic fibrosis (CF) patients are an emerging health issue and lead to a premature death. CF is a hereditary disease that creates a thick mucus in the lungs that is prone to bacterial biofilm formation, specifically Pseudomonas aeruginosa biofilms. These biofilms are very difficult to treat because many of them have antibiotic resistance that is worsened by the presence of extracellular DNA (eDNA). eDNA helps to stabilize biofilms and can bind antimicrobial compounds to lessen their effects. The metallo-antimicrobial peptide Gaduscidin-1 (Gad-1) eradicates established P. aeruginosa biofilms through a combination of modes of action that includes nuclease activity that can cleave eDNA in biofilms. In addition, Gad-1 exhibits synergistic activity when used with the antibiotics kanamycin and ciprofloxacin, thus making Gad-1 a new lead compound for the potential treatment of bacterial biofilms in CF patients.

Copper(II) Complexes Containing Natural Flavonoid Pomiferin Show Considerable In Vitro Cytotoxicity and Anti-inflammatory Effects.

A series of new heteroleptic copper(II) complexes of the composition [Cu(L)(bpy)]NO3·2MeOH (1), [Cu(L)(dimebpy)]NO3·2H2O (2), [Cu(L)(phen)]NO3·2MeOH (3), [Cu(L)(bphen)]NO3·MeOH (4), [Cu(L)(dppz)]NO3·MeOH (5) was prepared, where HL = 3-(3,4-dihydroxyphenyl)-5-hydroxy-8,8-dimethyl-6-(3-methylbut-2-ene-1-yl)-4H,8H-benzo[1,2-b:3,4-b']dipyran-4-one, (pomiferin) and bpy = 2,2'-bipyridine, dimebpy = 4,4'-dimethyl-2,2'-bipyridine, phen = 1,10-phenanthroline, bphen = 4,7-diphenyl-1,10-phenanthroline, and dppz = dipyrido[3,2-a:2',3'-c]phenazine. The complexes were characterized using elemental analysis, infrared and UV/Vis spectroscopies, mass spectrometry, thermal analysis and conductivity measurements. The in vitro cytotoxicity, screened against eight human cancer cell lines (breast adenocarcinoma (MCF-7), osteosarcoma (HOS), lung adenocarcinoma (A549), prostate adenocarcinoma (PC-3), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), colorectal adenocarcinoma (Caco-2) and monocytic leukemia (THP-1), revealed the complexes as effective antiproliferative agents, with the IC50 values of 2.2-13.0 µM for the best performing complexes 3 and 5. All the complexes 1-5 showed the best activity against the A2780R cells (IC50 = 2.2-6.6 µM), and moreover, the complexes demonstrated relatively low toxicity on healthy human hepatocytes, with IC50 > 100 µM. The complexes were evaluated by the Annexin V/propidium iodide apoptosis assay, induction of cell cycle modifications in A2780 cells, production of reactive oxygen species (ROS), perturbation of mitochondrial membrane potential, inhibition of apoptosis and inflammation-related signaling pathways (NF-κB/AP-1 activity, NF-κB translocation, TNF-α secretion), and tested for nuclease mimicking activity. The obtained results revealed the corresponding complexes to be effective antiproliferative and anti-inflammatory agents.

Neutrophil extracellular trap formation and nuclease activity in septic patients.

BACKGROUND: There is little knowledge, whether in patients with sepsis neutrophil extracellular trap (NET) formation and NET degrading nuclease activity are altered. Thus, we tested the hypotheses that 1) NET formation from neutrophils of septic patients is increased compared to healthy volunteers, both without stimulation and following incubation with mitochondrial DNA (mtDNA), a damage-associated molecular pattern, or phorbol 12-myristate 13-acetate (PMA; positive control) and 2) that serum nuclease activities are increased as well. METHODS: Following ethic committee approval, we included 18 septic patients and 27 volunteers in this prospective observational trial. Blood was withdrawn and NET formation from neutrophils was analyzed in vitro without stimulation and following incubation with mtDNA (10 µg/well) or PMA (25 nmol). Furthermore, serum nuclease activity was assessed using gel electrophoresis. RESULTS: In contrast to our hypothesis, in septic patients, unstimulated NET release from neutrophils was decreased by 46.3% (4.3% ± 1.8 SD vs. 8.2% ± 2.9, p ≤ 0.0001) and 48.1% (4.9% ± 2.5 vs. 9.4% ± 5.2, p = 0.002) after 2 and 4 h compared to volunteers. mtDNA further decreased NET formation in neutrophils from septic patients (4.7% ± 1.2 to 2.8% ± 0,8; p = 0.03), but did not alter NET formation in neutrophils from volunteers. Of note, using PMA, as positive control, we ensured that neutrophils were still able to form NETs, with NET formation increasing to 73.2% (±29.6) in septic patients and 91.7% (±7.1) in volunteers (p = 0.22). Additionally, we show that serum nuclease activity (range: 0-6) was decreased in septic patients by 39.6% (3 ± 2 vs 5 ± 0, median and ICR, p = 0.0001) compared to volunteers. CONCLUSIONS: Unstimulated NET formation and nuclease activity are decreased in septic patients. mtDNA can further reduce NET formation in sepsis. Thus, neutrophils from septic patients show decreased NET formation in vitro despite diminished nuclease activity in vivo. TRIAL REGISTRATION: DRKS00007694, german clinical trials database (DRKS). Retrospectively registered 06.02.2015.

Sex, Age, and Bodyweight as Determinants of Extracellular DNA in the Plasma of Mice: A Cross-Sectional Study.

Extracellular DNA (ecDNA) is studied as a possible biomarker, but also as a trigger of the immune responses important for the pathogenesis of several diseases. Extracellular deoxyribonuclease (DNase) activity cleaves ecDNA. The aim of our study was to describe the interindividual variability of ecDNA and DNase activity in the plasma of healthy mice, and to analyze the potential determinants of the variability, including sex, age, and bodyweight. In this experiment, 58 adult CD1 mice (41 females and 31 males) of a variable age (3 to 16 months old) and bodyweight (females 25.7 to 52.1 g, males 24.6 to 49.6 g) were used. The plasma ecDNA was measured using a fluorometric method. The nuclear ecDNA and mitochondrial ecDNA were quantified using real-time PCR. The deoxyribonuclease activity was assessed using the single radial enzyme diffusion method. The coefficient of variance for plasma ecDNA was 139%, and for DNase 48%. Sex differences were not found in the plasma ecDNA (52.7 ± 73.0 ηg/mL), but in the DNase activity (74.5 ± 33.5 K.u./mL for males, and 47.0 ± 15.4 K.u./mL for females). There were no associations between plasma ecDNA and bodyweight or the age of mice. Our study shows that the variability of plasma ecDNA and DNase in adult healthy mice is very high. Sex, age, and bodyweight seem not to be major determinants of ecDNA variability in healthy mice. As ecDNA gains importance in the research of several diseases, it is of importance to understand its production and cleavage. Further studies should, thus, test other potential determinants, taking into account cleavage mechanisms other than DNase.

Design, Synthesis, and Biological Evaluation of Benzimidazole-Derived Biocompatible Copper(II) and Zinc(II) Complexes as Anticancer Chemotherapeutics.

Herein, we have synthesized and characterized a new benzimidazole-derived "BnI" ligand and its copper(II) complex, [Cu(BnI)2], 1, and zinc(II) complex, [Zn(BnI)2], 2, using elemental analysis and various spectroscopic techniques. Interaction of complexes 1 and 2 with the biomolecules viz. HSA (human serum albumin) and DNA were studied using absorption titration, fluorescence techniques, and in silico molecular docking studies. The results exhibited the significant binding propensity of both complexes 1 and 2, but complex 1 showed more avid binding to HSA and DNA. Also, the nuclease activity of 1 and 2 was analyzed for pBR322 DNA, and the results obtained confirmed the potential of the complexes to cleave DNA. Moreover, the mechanistic pathway was studied in the presence of various radical scavengers, which revealed that ROS (reactive oxygen species) are responsible for the nuclease activity in complex 1, whereas in complex 2, the possibility of hydrolytic cleavage also exists. Furthermore, the cytotoxicity of the ligand and complexes 1 and 2 were studied on a panel of five different human cancer cells, namely: HepG2, SK-MEL-1, HT018, HeLa, and MDA-MB 231, and compared with the standard drug, cisplatin. The results are quite promising against MDA-MB 231 (breast cancer cell line of 1), with an IC50 value that is nearly the same as the standard drug. Apoptosis was induced by complex 1 on MDA-MB 231 cells predominantly as studied by flow cytometry (FACS). The adhesion and migration of cancer cells were also examined upon treatment of complexes 1 and 2. Furthermore, the in vivo chronic toxicity profile of complexes 1 and 2 was also studied on all of the major organs of the mice, and found them to be less toxic. Thus, the results warrant further investigations of complex 1.

Synthesis, characterization, DNA interactions, DNA cleavage, radical scavenging activity, antibacterial, anti-proliferative and docking studies of new transition metal complexes.

The compound N-(2-hydroxybenzylidene)-1-ethyl-1, 4-dihydro-7-methyl-4-oxo-1, 8 naphthyridine-3-carbohydrazide (LH) and its Cu (II), Co (II) and Zn (II) complexes were synthesized and characterized. The absorption spectral titrations and competitive DNA binding studies depicted those complexes of title compound bind to CT-DNA through intercalation. Interestingly [Cu (II)-(L2)] showed relatively high binding constant value (6.61 x 10(5) M(-1)) compared to [Co (II)-(L2)] (4.378× 10(5) M(-1)) and [Zn (II)-(L2)] (3.1x10(5) M(-1)). Ligand and its complexes were also examined for DNA nuclease activity against pBR-322 plasmid DNA, which showed that [Cu (II)-(L2)] had the best hydrolytic cleavage property displaying prominent double-strand DNA cleavage. In addition, antioxidant activities of the ligand and its metal complexes investigated through scavenging effects for DPPH radical in- vitro, indicated their potentiality as good antioxidants. The in vitro anti-bacterial study inferred the better anti-bacterial activity of [Cu (II)-(L2)] and this was also correlated theoretically by employing docking studies wherein [Cu (II)-(L2)] displayed good Gold score and Chem score. Finally the in vitro anti- proliferative activity of studied compounds was tested against HeLa and MCF-7 cell lines. Interestingly [Cu (II)-(L2)] displayed lower IC50 value and lower percentage of viability in both HeLa and MCF-7 cell lines.

Nuclear degraded sperm subpopulation is affected by poor chromatin compaction and nuclease activity.

There is an interest in the nuclear degraded sperm subpopulation because, although it is present in a low percentage in all semen samples, patient groups such as varicocele and rearranged genome carriers show high levels of these degraded spermatozoa. This study is designed with two objectives in mind: first, incubations of H2 O2 and nuclease on DTT-treated and untreated samples to show the aetiology of this subpopulation and second, assessment of the correlation between the protamine ratio and nuclear degraded spermatozoa. A very high increase in the nuclear degraded subpopulation has been found with nuclease incubation, and it is even higher when it has been merged with nuclear decompaction using DTT. Alternatively, incubation with H2 O2 with and without DTT did not show such a significant increase in nuclear degraded spermatozoa. The protamine ratio correlated with this subpopulation, showing, in patients, that poor nuclear compaction would turn the sperm susceptible to degradation. Then, the assessment of nuclear degraded spermatozoa might not be only a measure of DNA degradation but also an indicator of chromatin compaction in the spermatozoa. Different patient groups would fit this model for sperm nuclear degradation, such as varicocele patients, who show a high percentage of immature spermatozoa and nuclear degraded spermatozoa, and reorganised genome carriers, where reorganisation might also cause poor chromatin compaction on the sperm nucleus.

DNA/HSA interaction and nuclease activity of an iron(III) amphiphilic sulfonated corrole.

The DNA binding of amphiphilic iron(III) 2,17-bis(sulfonato)-5,10,15-tris(pentafluorophenyl)corrole complex (Fe-SC) was studied using spectroscopic methods and viscosity measurements. Its nuclease-like activity was examined by using pBR322 DNA as a target. The interaction of Fe-SC with human serum albumin (HSA) in vitro was also examined using multispectroscopic techniques. Experimental results revealed that Fe-SC binds to ct-DNA via an outside binding mode with a binding constant of 1.25 × 10(4) M(-1). This iron corrole also displays good activity during oxidative DNA cleavage by hydrogen peroxide or tert-butyl hydroperoxide oxidants, and high-valent (oxo)iron(V,VI) corrole intermediates may play an important role in DNA cleavage. Fe-SC exhibits much stronger binding affinity to site II than site I of HSA, indicating a selective binding tendency to HSA site II. The HSA conformational change induced by Fe-SC was confirmed by UV/Vis and CD spectroscopy.

Purification, characterization and structure of nucleoside diphosphate kinase from Drosophila melanogaster.

Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme found in all organisms and cell types, which catalyzes the transfer of the phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. The gene encoding for NDPK from Drosophila melanogaster was amplified from the genomic DNA. The recombinant NDPK (rNDPK) was overexpressed in Escherichia coli and purified to homogeneity by Ni-NTA agarose affinity chromatography, HiTrap SP HP cation exchange chromatography and HiLoad 16/60 Superdex 200 gel filtration chromatography. The gel filtration chromatography and analytical ultracentrifugation showed that rNDPK was a trimer in solution. The binding affinity of NDPs with rNDPK, measured by isothermal titration calorimetry, indicated that the purines nucleotides show higher binding affinity compared with pyrimidines. The rNDPK had a definite nuclease activity in vitro, which could cleave supercoiled plasmid DNA, but had no effect on dsDNA and ssDNA. Furthermore, the structure for NDPK was determined by using the sitting drop vapor diffusion method. In the final model, the asymmetric unit is made of three molecules, each of which consists of a four-stranded anti-parallel ß-sheets and seven α-helices. Sequence alignment and structure comparison illustrated that the simulated nucleotide-binding active site are conserved.

Duck plague virus pUL15 performs a nonspecial cleavage activity through its C terminal nuclease domain in vitro.

Duck plague virus (DPV), also known as anatid herpesvirus, is a double-stranded DNA virus and a member of α herpesvirus. DPV pUL15 is a homolog of herpes simplex virus 1 (HSV-1) pUL15, a terminase large subunit, and plays a key role in the cleavage and packaging of the viral concatemeric genome. However, the sequence similarity between DPV pUL15 and its homologs is low, and it is not sure if DPV pUL15 has the potential to cleave the concatemeric genome as same as its homologs. Here, we expressed the C terminal domain of DPV pUL15 to explore the nuclease function of DPV pUL15. The main results showed that (Ⅰ) DPV pUL15 C-terminal domain possesses nonspecific nuclease activity and lacks the DNA binding ability. (Ⅱ) DPV pUL15 nuclease activity needs to coordinate with divalent metal ions and tends to be more active at high temperatures. (Ⅲ) Even though the structure of DPV pUL15 nuclease domain is relatively conserved, the mutations of conserved amino acids on the nuclease domain do not significantly inhibit the nuclease activity.

Beyond a platform protein for the degradosome assembly: The Apoptosis-Inducing Factor as an efficient nuclease involved in chromatinolysis.

The Apoptosis-Inducing Factor (AIF) is a moonlighting flavoenzyme involved in the assembly of mitochondrial respiratory complexes in healthy cells, but also able to trigger DNA cleavage and parthanatos. Upon apoptotic-stimuli, AIF redistributes from the mitochondria to the nucleus, where upon association with other proteins such as endonuclease CypA and histone H2AX, it is proposed to organize a DNA-degradosome complex. In this work, we provide evidence for the molecular assembly of this complex as well as for the cooperative effects among its protein components to degrade genomic DNA into large fragments. We have also uncovered that AIF has nuclease activity that is stimulated in the presence of either Mg2+ or Ca2+. Such activity allows AIF by itself and in cooperation with CypA to efficiently degrade genomic DNA. Finally, we have identified TopIB and DEK motifs in AIF as responsible for its nuclease activity. These new findings point, for the first time, to AIF as a nuclease able to digest nuclear dsDNA in dying cells, improving our understanding of its role in promoting apoptosis and opening paths for the development of new therapeutic strategies.

An RNA-based catalytic hairpin assembly circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs.

CRISPR-Cas nuclease-based nucleic acid detection has exhibited extraordinary value in the field of molecular diagnostics, but it usually involves two separate reaction steps of nucleic acid amplification and Cas-based endpoint detection, resulting in the use of multiple enzymes, inconvenient operation, and potential carry-over contamination. Here, we propose an RNA-based catalytic hairpin assembly (CHA) circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs (miRNAs) at an isothermal condition. This method relies on the rational design of a spacer-blocking crRNA as a bridge between the two systems. The target miRNA can specifically trigger RNA-based CHA and induce a configurational change of the blocked crRNAs into precursor crRNAs (pre-crRNAs), which can be processed into mature crRNAs to function by leveraging the inherent RNase activities of Cas12a. In this way, the developed circuit achieves a femtomolar detection limit and shows an accurate detection of miRNA levels in different cell lines. Therefore, our method would provide a new paradigm to develop miRNA detection methods based on the CRISPR/Cas system.

Nuclease activity: an exploitable biomarker in bacterial infections.

INTRODUCTION: In the increasingly challenging field of clinical microbiology, diagnosis is a cornerstone whose accuracy and timing are crucial for the successful management, therapy, and outcome of infectious diseases. Currently employed biomarkers of infectious diseases define the scope and limitations of diagnostic techniques. As such, expanding the biomarker catalog is crucial to address unmet needs and bring about novel diagnostic functionalities and applications. AREAS COVERED: This review describes the extracellular nucleases of 15 relevant bacterial pathogens and discusses the potential use of nuclease activity as a diagnostic biomarker. Articles were searched for in PubMed using the terms: 'nuclease,' 'bacteria,' 'nuclease activity' or 'biomarker.' For overview sections, original and review articles between 2000 and 2019 were searched for using the terms: 'infections,' 'diagnosis,' 'bacterial,' 'burden,' 'challenges.' Informative articles were selected. EXPERT OPINION: Using the catalytic activity of nucleases offers new possibilities compared to established biomarkers. Nucleic acid activatable reporters in combination with different transduction platforms and delivery methods can be used to detect disease-associated nuclease activity patterns in vitro and in vivo for prognostic and diagnostic applications. Even when these patterns are not obvious or of unknown etiology, screening platforms could be used to identify new disease reporters.

Dual targeting of Saccharomyces cerevisiae Pso2 to mitochondria and the nucleus, and its functional relevance in the repair of DNA interstrand crosslinks.

Repair of DNA interstrand crosslinks involves a functional interplay among different DNA surveillance and repair pathways. Previous work has shown that interstrand crosslink-inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA, and its pso2/snm1 mutants exhibit a petite phenotype followed by loss of mitochondrial DNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. Here, by combining a wide range of approaches with in vitro and in vivo analyses, we interrogated the subcellular localization and function of Pso2. We found evidence that the nuclear-encoded Pso2 contains 1 mitochondrial targeting sequence and 2 nuclear localization signals (NLS1 and NLS2), although NLS1 resides within the mitochondrial targeting sequence. Further analysis revealed that Pso2 is a dual-localized interstrand crosslink repair protein; it can be imported into both nucleus and mitochondria and that genotoxic agents enhance its abundance in the latter. While mitochondrial targeting sequence is essential for mitochondrial Pso2 import, either NLS1 or NLS2 is sufficient for its nuclear import; this implies that the 2 nuclear localization signal motifs are functionally redundant. Ablation of mitochondrial targeting sequence abrogated mitochondrial Pso2 import, and concomitantly, raised its levels in the nucleus. Strikingly, mutational disruption of both nuclear localization signal motifs blocked the nuclear Pso2 import; at the same time, they enhanced its translocation into the mitochondria, consistent with the notion that the relationship between mitochondrial targeting sequence and nuclear localization signal motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual targeting of Pso2 into 2 DNA-bearing organelles is discussed.

Ultra-Sensitive and Specific Detection of S. aureus Bacterial Cultures Using an Oligonucleotide Probe Integrated in a Lateral Flow-Based Device.

The identification of pathogens causing infectious diseases is still based on laborious and time-consuming techniques. Therefore, there is an urgent need for the development of novel methods and devices that can considerably reduce detection times, allowing the health professionals to administer the right treatment at the right time. Lateral flow-based systems provide fast, cheap and easy to use alternatives for diagnosis. Herein, we report on a lateral flow approach for specifically detecting S. aureus bacteria within 6 h.

When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer.

DNA polymerases catalyze nucleotidyl transfer, the central reaction in synthesis of DNA polynucleotide chains. They function not only in DNA replication, but also in diverse aspects of DNA repair and recombination. Some DNA polymerases can perform translesion DNA synthesis, facilitating damage tolerance and leading to mutagenesis. In addition to these functions, many DNA polymerases conduct biochemically distinct reactions. This review presents examples of DNA polymerases that carry out nuclease (3'-5' exonuclease, 5' nuclease, or end-trimming nuclease) or lyase (5' dRP lyase) extracurricular activities. The discussion underscores how DNA polymerases have a remarkable ability to manipulate DNA strands, sometimes involving relatively large intramolecular movement.

The integrity and assay performance of tissue mitochondrial DNA is considerably affected by choice of isolation method.

The integrity of mitochondrial DNA (mtDNA) isolated from solid tissues is critical for analyses such as long-range PCR, but is typically assessed under conditions that fail to provide information on the individual mtDNA strands. Using denaturing gel electrophoresis, we show that commonly-used isolation procedures generate mtDNA containing several single-strand breaks per strand. Through systematic comparison of DNA isolation methods, we identify a procedure yielding the highest integrity of mtDNA that we demonstrate displays improved performance in downstream assays. Our results highlight the importance of isolation method choice, and serve as a resource to researchers requiring high-quality mtDNA from solid tissues.