Degradation Of Chromosomal DNA Research Articles

Quercetin-3-Glucoside Extracted from Apple Pomace Induces Cell Cycle Arrest and Apoptosis by Increasing Intracellular ROS Levels.

Cervical cancer is a life-threatening disease and the fourth most common cancer among women worldwide. Apple pomace is a multifunctional phenolic compound possessing effective biological activity against cervical cancer cells. This study aimed to investigate the anticancer effects of quercetin-3-glucoside (Q3G) extracted from apple pomace in HeLa cell lines and analyze its molecular mechanisms. High-performance liquid chromatography revealed that Q3G, coumaric acid, phloridzin, quercetin, and phloretin are the major polyphenolic compounds constituting apple pomace. Among them, Q3G possessed the greatest antioxidant and anti-inflammatory effects in vitro and exhibited significant cytotoxic effects in HeLa cells in a dose-and time-dependent manner. Flow cytometric analysis indicated that Q3G induced cell cycle arrest at the S phase in a time-dependent manner by altering cyclin-dependent kinase 2. Moreover, it induced apoptosis via chromosomal DNA degradation and increased reactive oxygen species generation. Furthermore, Q3G treatment altered the apoptosis-associated protein expression in the cells by activating caspase-9/-3, downregulating anti-apoptosis protein B-cell lymphoma (Bcl)-2 expressions and up regulating the pro-apoptotic Bcl-2-associated X protein. BH3-interacting domain death agonist cleavage occurred prior to the degradation of an anti-apoptotic Mu-2-related death-inducing gene involved in cell death signaling. Consequently, apple pomace Q3G holds promise as an anti-inflammatory and anticancer agent for treating cervical cancer.

A type III-A CRISPR-Cas system mediates co-transcriptional DNA cleavage at the transcriptional bubbles in close proximity to active effectors.

Many type III CRISPR–Cas systems rely on the cyclic oligoadenylate (cOA) signaling pathway to exert immunization. However, LdCsm, a type III-A lactobacilli immune system mediates efficient plasmid clearance in spite of lacking cOA signaling. Thus, the system provides a good model for detailed characterization of the RNA-activated DNase in vitro and in vivo. We found ATP functions as a ligand to enhance the LdCsm ssDNase, and the ATP enhancement is essential for in vivo plasmid clearance. In vitro assays demonstrated LdCsm cleaved transcriptional bubbles at any positions in non-template strand, suggesting that DNA cleavage may occur for transcribing DNA. Destiny of target plasmid versus nontarget plasmid in Escherichia coli cells was investigated, and this revealed that the LdCsm effectors mediated co-transcriptional DNA cleavage to both target and nontarget plasmids, suggesting LdCsm effectors can mediate DNA cleavage to any transcriptional bubbles in close proximity upon activation. Subcellular locations of active LdCsm effectors were then manipulated by differential expression of LdCsm and CTR, and the data supported the hypothesis. Strikingly, stepwise induction experiments indicated allowing diffusion of LdCsm effector led to massive chromosomal DNA degradation, suggesting this unique IIIA system can facilitate infection abortion to eliminate virus-infected cells.

An improved procedure for the pulsed-field gel electrophoresis analysis of Vibrio vulnificus

Vibrio vulnificus is a marine bacterium that causes septicemia with high mortality via wound infection or seafood ingestion. Subspecies typing of V. vulnificus can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 12% of strains. This study presents a modified PFGE procedure. The outer membrane of V. vulnificus cells was lysed utilizing a sucrose-EDTA method and components in the periplasmic space were removed prior to making plugs. Cytoplasmic digestive enzymes were inactivated by an extended proteinase K reaction. Experimental results indicated that five out of eight strains exhibiting smeared DNA in a previous study (Appl. Environ. Microbiol. 70: 5153-5158. 2004) achieved clear banding patterns. This modified PFGE procedure can be applied to improve PFGE typing of untypeable strains of V. Vulnificus.

Sources of thymidine and analogs fueling futile damage-repair cycles and ss-gap accumulation during thymine starvation in Escherichia coli

Thymine deprivation in thyA mutant E. coli causes thymineless death (TLD) and is the mode of action of popular antibacterial and anticancer drugs, yet the mechanisms of TLD are still unclear. TLD comprises three defined phases: resistance, rapid exponential death (RED) and survival, with the nature of the resistance phase and of the transition to the RED phase holding key to TLD pathology. We propose that a limited source of endogenous thymine maintains replication forks through the resistance phase. When this source ends, forks undergo futile break-repair cycle during the RED phase, eventually rendering the chromosome non-functional. Two obvious sources of the endogenous thymine are degradation of broken chromosomal DNA and recruitment of thymine from stable RNA. However, mutants that cannot degrade broken chromosomal DNA or lack ribo-thymine, instead of shortening the resistance phase, deepen the RED phase, meaning that only a small fraction of T-starved cells tap into these sources. Interestingly, the substantial chromosomal DNA accumulation during the resistance phase is negated during the RED phase, suggesting futile cycle of incorporation and excision of wrong nucleotides. We tested incorporation of dU or rU, finding some evidence for both, but DNA-dU incorporation accelerates TLD only when intracellular [dUTP] is increased by the dut mutation. In the dut ung mutant, with increased DNA-dU incorporation and no DNA-dU excision, replication is in fact rescued even without dT, but TLD still occurs, suggesting different mechanisms. Finally, we found that continuous DNA synthesis during thymine starvation makes chromosomal DNA increasingly single-stranded, and even the dut ung defect does not completely block this ss-gap accumulation. We propose that instability of single-strand gaps underlies the pathology of thymine starvation.

Stable maintenance of the rudivirus SIRV3 in a carrier state in Sulfolobus islandicus despite activation of the CRISPR-Cas immune response by a second virus SMV1

ABSTRACTCarrier state viral infection constitutes an equilibrium state in which a limited fraction of a cellular population is infected while the remaining cells are transiently resistant to infection. This type of infection has been characterized for several bacteriophages but not, to date, for archaeal viruses. Here we demonstrate that the rudivirus SIRV3 can produce a host-dependent carrier state infection in the model crenarchaeon Sulfolobus. SIRV3 only infected a fraction of a Sulfolobus islandicus REY15A culture over several days during which host growth was unimpaired and no chromosomal DNA degradation was observed. CRISPR spacer acquisition from SIRV3 DNA was induced by coinfecting with the monocaudavirus SMV1 and it was coincident with increased transcript levels from subtype I-A adaptation and interference cas genes. However, this response did not significantly affect the carrier state infection of SIRV3 and both viruses were maintained in the culture over 12 days during which SIRV3 anti-CRISPR genes were shown to be expressed. Transcriptome and proteome analyses demonstrated that most SIRV3 genes were expressed at varying levels over time whereas SMV1 gene expression was generally low. The study yields insights into the basis for the stable infection of SIRV3 and the resistance to the different host CRISPR-Cas interference mechanisms. It also provides a rationale for the commonly observed coinfection of archaeal cells by different viruses in natural environments.

New N-benzhydrylpiperazine/1,3,4-oxadiazoles conjugates inhibit the proliferation, migration, and induce apoptosis in HeLa cancer cells via oxidative stress-mediated mitochondrial pathway.

N-benzhydrylpiperazine and 1,3,4-oxadiazoles are pharmacologically active scaffolds which exhibits significant inhibitory growth effects against various cancer cells, however, antiproliferation effects and the underlying mechanism for inducing apoptosis for aforementioned scaffolds addressing HeLa cancer cells remains uncertain. In this study, N-benzhydrylpiperazine clubbed with 1,3,4-oxadiazoles (4a-4h) were synthesized, subsequently characterized using high resolution spectroscopic techniques and eventually evaluated for their antiproliferation potential by inducing apoptosis in HeLa cancer cells. The MTT assay screening results revealed that among all, compound 4d ( N-benzhydryl-4-((5-(4-aminophenyl)-1,3,4-oxadiazol-2-yl)methyl)piperazine) in particular, exhibited IC 50 value of 28.13 ± 0.21 μg/mL and significantly inhibited the proliferation of HeLa cancer cells in concentration-dependent manner. The in vitro anticancer assays for treated HeLa cells resulted in alterations in the cell morphology, reduction in colony formation, and inhibition of cell migration in concentration-dependent treatment. Furthermore, G2/M phase arrest, variations in the nuclear morphology, degradation of chromosomal DNA confirmed the ongoing apoptosis in treated HeLa cells. Increase in the expression of cytochrome C and caspase-3 confirmed the involvement of intrinsic mitochondrial pathway regulating the cell death. Also, elevation in reactive oxygen species level and loss of mitochondrial membrane potential signified that compound 4d induced apoptosis in HeLa cells by generating the oxidative stress. Therefore, compound 4d may act as a potent chemotherapeutic agent against human cervical cancer.

Apoptotic Cells Induced Signaling for Immune Homeostasis in Macrophages and Dendritic Cells.

Inefficient and abnormal clearance of apoptotic cells (efferocytosis) contributes to systemic autoimmune disease in humans and mice, and inefficient chromosomal DNA degradation by DNAse II leads to systemic polyarthritis and a cytokine storm. By contrast, efficient clearance allows immune homeostasis, generally leads to a non-inflammatory state for both macrophages and dendritic cells (DCs), and contributes to maintenance of peripheral tolerance. As many as 3 × 108 cells undergo apoptosis every hour in our bodies, and one of the primary “eat me” signals expressed by apoptotic cells is phosphatidylserine (PtdSer). Apoptotic cells themselves are major contributors to the “anti-inflammatory” nature of the engulfment process, some by secreting thrombospondin-1 (TSP-1) or adenosine monophosphate and possibly other immune modulating “calm-down” signals that interact with macrophages and DCs. Apoptotic cells also produce “find me” and “tolerate me” signals to attract and immune modulate macrophages and DCs that express specific receptors for some of these signals. Neither macrophages nor DCs are uniform, and each cell type may variably express membrane proteins that function as receptors for PtdSer or for opsonins like complement or opsonins that bind to PtdSer, such as protein S and growth arrest-specific 6. Macrophages and DCs also express scavenger receptors, CD36, and integrins that function via bridging molecules such as TSP-1 or milk fat globule-EGF factor 8 protein and that differentially engage in various multi-ligand interactions between apoptotic cells and phagocytes. In this review, we describe the anti-inflammatory and pro-homeostatic nature of apoptotic cell interaction with the immune system. We do not review some forms of immunogenic cell death. We summarize the known apoptotic cell signaling events in macrophages and DCs that are related to toll-like receptors, nuclear factor kappa B, inflammasome, the lipid-activated nuclear receptors, Tyro3, Axl, and Mertk receptors, as well as induction of signal transducer and activator of transcription 1 and suppressor of cytokine signaling that lead to immune system silencing and DC tolerance. These properties of apoptotic cells are the mechanisms that enable their successful use as therapeutic modalities in mice and humans in various autoimmune diseases, organ transplantation, graft-versus-host disease, and sepsis.

Expression of a Vibrio parahaemolyticus toxin in Escherichia coli results in chromosomal DNA degradation.

A toxin-antitoxin system, vp1842/vp1843, locates within a superintegron on the Vibrio parahaemolyticus genome chromosome I whose toxin gene vp1843 encodes a DNA nicking endonuclease. We found that the vp1843 expression in Escherichia coli cells strongly induced chromosomal DNA degradation. On the basis of these observations, we discuss a possible physiological role of vp1842/vp1843 in V. parahaemolyticus.

NQO-Induced DNA-Less Cell Formation Is Associated with Chromatin Protein Degradation and Dependent on A0A1-ATPase in Sulfolobus.

To investigate DNA damage response in the model crenarchaeon Sulfolobus islandicus, four different DNA damage agents were tested for their effects on cell death of this archaeon, including UV irradiation, methyl methanesulfonate, cisplatin, and 4-nitroquinoline 1-oxide (NQO). Cell death featured with DNA-less cell formation was revealed in DNA damage treatment with each agent. Cellular responses upon NQO treatment were characterized in details, and following sequential events were revealed, including: a modest accumulation of G1/S phase cells, membrane depolarization, proteolytic degradation of chromatin proteins, and chromosomal DNA degradation. Further insights into the process were gained from studying drugs that affect the archaeal ATP synthase, including a proton gradient uncoupler and an ATP synthase inhibitor. Whereas the proton uncoupler-mediated excess proton influx yielded cell death as observed for the NQO treatment, inhibition of ATP synthase attenuated NQO-induced membrane depolarization and DNA-less cell formation. In conclusion, the NQO-induced cell death in S. islandicus is characterized by proteolytic degradation of chromatin protein, and chromosomal DNA degradation, which probably represents a common feature for the cell death induced by different DNA damage agents.

Reverse Gyrase Functions in Genome Integrity Maintenance by Protecting DNA Breaks In Vivo.

Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophilic life remain to be demonstrated. Here, we investigated the roles of TopR1 in genome stability maintenance in S. islandicus in response to the treatment of methyl methanesulfonate (MMS), a DNA alkylation agent. Lethal MMS treatment induced two successive events: massive chromosomal DNA backbone breakage and subsequent DNA degradation. The former occurred immediately after drug treatment, leading to chromosomal DNA degradation that concurred with TopR1 degradation, followed by chromatin protein degradation and DNA-less cell formation. To gain a further insight into TopR1 function, the expression of the enzyme was reduced in S. islandicus cells using a CRISPR-mediated mRNA interference approach (CRISPRi) in which topR1 mRNAs were targeted for degradation by endogenous III-B CRISPR-Cas systems. We found that the TopR1 level was reduced in the S. islandicus CRISPRi cells and that the cells underwent accelerated genomic DNA degradation during MMS treatment, accompanied by a higher rate of cell death. Taken together, these results indicate that TopR1 probably facilitates genome integrity maintenance by protecting DNA breaks from thermo-degradation in vivo.

Induction of Cell Death by Betulinic Acid through Induction of Apoptosis and Inhibition of Autophagic Flux in Microglia BV-2 Cells.

Betulinic acid (BA), a natural pentacyclic triterpene found in many medicinal plants is known to have various biological activity including tumor suppression and anti-inflammatory effects. In this study, the cell-death induction effect of BA was investigated in BV-2 microglia cells. BA was cytotoxic to BV-2 cells with IC50 of approximately 2.0 μM. Treatment of BA resulted in a dose-dependent chromosomal DNA degradation, suggesting that these cells underwent apoptosis. Flow cytometric analysis further confirmed that BA-treated BV-2 cells showed hypodiploid DNA content. BA treatment triggered apoptosis by decreasing Bcl-2 levels, activation of capase-3 protease and cleavage of PARP. In addition, BA treatment induced the accumulation of p62 and the increase in conversion of LC3-I to LC3-II, which are important autophagic flux monitoring markers. The increase in LC3-II indicates that BA treatment induced autophagosome formation, however, accumulation of p62 represents that the downstream autophagy pathway is blocked. It is demonstrated that BA induced cell death of BV-2 cells by inducing apoptosis and inhibiting autophagic flux. These data may provide important new information towards understanding the mechanisms by which BA induce cell death in microglia BV-2 cells.

Mechanisms of Antibacterial Action of Quinoxaline 1,4-di-N-oxides against Clostridium perfringens and Brachyspira hyodysenteriae.

Quinoxaline 1,4-di-N-oxides (QdNOs) are a class of bioreductive compounds, however, their antibacterial mechanisms are still unclarified. The aim of this study was to assess the ability of two representative QdNO drugs, cyadox (CYA) and olaquindox (OLA), to produce reactive oxide species (ROS) in Gram-positive anaerobe Clostridium perfringens CVCC1125 and Gram-negative anaerobe Brachyspira hyodysenteriae B204. In addition, the effects of QdNOs on the integrity of bacterial cell walls and membranes as well as the morphological alterations and DNA oxidative damage in C. perfringens and B. hyodysenteriae were analyzed. It was demonstrated that under anaerobic conditions, QdNOs were metabolized into the reduced products which did not show any antibacterial activity. A significant dose-related increase of intracellular ROS level and intracellular hydroxyl radicals were evident in bacteria exposed to QdNOs. The result of biochemical assay showed that the cell walls and membranes of the bacteria treated with QdNOs were damaged. After exposure to 1/2MIC to 4MIC of CYA and OLA, C. perfringens and B. hyodysenteriae became elongated and filamentous. Morphological observation with scanning and transmission electron microscopes revealed rupture, loss of cytoplasmic material and cell lysis in QdNO-treated bacteria, indicating serious damage of cells. There was an increase of 8-OHdG in the two strains treated by QdNOs, but it was lower in C. perfringens CVCC1125 than in B. hyodysenteriae B204. Agarose gel electrophoresis showed the degradation of chromosomal DNA in both of the two anaerobes treated by QdNOs. The results suggest that QdNOs may kill C. perfringens and B. hyodysenteriae via the generation of ROS and hydroxyl radicals from the bacterial metabolism of QdNOs, which cause oxidative damage in bacteria under anaerobic conditions.

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells.

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factorsinduce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatmentwasrequired for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 10(10) cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

The Antimicrobial Properties of Silver Nanoparticles in Bacillus subtilis Are Mediated by Released Ag+ Ions.

The superior antimicrobial properties of silver nanoparticles (Ag NPs) are well-documented, but the exact mechanisms underlying Ag-NP microbial toxicity remain the subject of intense debate. Here, we show that Ag-NP concentrations as low as 10 ppm exert significant toxicity against Bacillus subtilis, a beneficial bacterium ubiquitous in the soil. Growth arrest and chromosomal DNA degradation were observed, and flow cytometric quantification of propidium iodide (PI) staining also revealed that Ag-NP concentrations of 25 ppm and above increased membrane permeability. RedoxSensor content analysis and Phag-GFP expression analysis further indicated that reductase activity and cytosolic protein expression decreased in B. subtilis cells treated with 10–50 ppm of Ag NPs. We conducted X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses to directly clarify the valence and fine structure of Ag atoms in B. subtilis cells placed in contact with Ag NPs. The results confirmed the Ag species in Ag NP-treated B. subtilis cells as Ag2O, indicating that Ag-NP toxicity is likely mediated by released Ag+ ions from Ag NPs, which penetrate bacterial cells and are subsequently oxidized intracellularly to Ag2O. These findings provide conclusive evidence for the role of Ag+ ions in Ag-NP microbial toxicity, and suggest that the impact of inappropriately disposed Ag NPs to soil and water ecosystems may warrant further investigation.

Harnessing Type I and Type III CRISPR-Cas systems for genome editing.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR array and a donor DNA containing a non-target sequence. Transformation of a pGE plasmid would yield two alternative fates to transformed cells: wild-type cells are to be targeted for chromosomal DNA degradation, leading to cell death, whereas those carrying the mutant gene would survive the cell killing and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided the protospacer adjacent motif (PAM) of an uncharacterized PAM-dependent CRISPR-Cas system can be predicted by bioinformatic analysis.

Tea Extract Prevents Arsenic-mediated DNA Damage and Death of Murine Thymocytes in vitro

Groundwater contamination by arsenic has created a major health hazard in Bangladesh by affecting millions of people. In this study, we report cytotoxic effects of arsenic in primary culture of murine thymocytes and the counteractive actions of tea extract to reduce this cytotoxic effects. When murine thymocytes were incubated for shorter period (1 h) with higher concentrations (50 and 100 ?M) of sodium arsenite (NaAsO2), cell viability was decreased to 79.06 ± 0.52% and 62.53 ± 0.23%, respectively. In case of longer incubation (16 hrs) with a wide range of NaAsO2 concentration (1-100 ?M), cell viability was reduced from 89.30 ± 0.84% to 79.0 ± 0.52% by 1 ?M NaAsO2, and this reduction was continued with increasing concentration reaching to 29.60 ± 0.72% by 100 ?M. Tea is known to possess antioxidant property and we found that this tea extract reduced NaAsO2-mediated death of the cells in culture. After 16 h of incubation, the chromosomal DNA of 5 ?M NaAsO2-exposed cells was found degraded suggesting apoptotic death of the cells. Interestingly, this degradation of chromosomal DNA was blocked by tea extract. All of these results together suggest a future therapeutic application of tea extract to reduce or block arsenic toxicity.Dhaka Univ. J. Pharm. Sci. 14(1): 79-85, 2015 (June)

Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis.

Until recently, transcriptome analyses of single cells have been confined to eukaryotes. The information obtained from single-cell transcripts can provide detailed insight into spatiotemporal gene expression, and it could be even more valuable if expanded to prokaryotic cells. Transcriptome analysis of single prokaryotic cells is a recently developed and powerful tool. Here we describe a procedure that allows amplification of the total transcript of a single prokaryotic cell for in-depth analysis. This is performed by using a laser-capture microdissection instrument for single-cell isolation, followed by reverse transcription via Moloney murine leukemia virus, degradation of chromosomal DNA with McrBC and DpnI restriction enzymes, single-stranded cDNA (ss-cDNA) ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to double-stranded cDNA (ds-cDNA) by Φ29 polymerase. This procedure takes ∼5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.

A simple, fast and cost-effective method of synthesis of cupric oxide nanoparticle with promising antibacterial potency: Unraveling the biological and chemical modes of action

BackgroundGradual attainment of bacterial resistance to antibiotics led us to develop a robust method of synthesis of stable, colloidal cupric oxide nanoparticle of physiological pH with potential antibacterial action. MethodsCu(II) oxide NP was synthesized by reduction–oxidation of CuCl2, using polyvinyl alcohol as stabilizer. Characteristics and antibacterial activity of the particles were investigated by techniques like UV–Vis spectrophotometry, DLS, AFM, TEM, EDS, FTIR, AAS, agar plating, FACS, gel electrophoresis and XPS. ResultsThe NPs were about 50nm in size and cubic in shape with two surface plasmon peaks at 266 and 370nm and had semi-conducting behavior with a band gap of 3.40 and 3.96eV. About 80% of precursor CuCl2 was converted to NP. The minimum inhibitory and the minimum bactericidal concentrations of CuO-NP were respectively 120 and 160μg/mL for Escherichia coli and 180 and 195μg/mL for Staphylococcus aureus in Luria–Bertani medium. In growth media, the NPs got modified by media organics with displacement of the stabilizer PVA molecules. This modified NP (around 240nm) killed cells by generating ROS, which finally caused membrane lipid per-oxidation and chromosomal DNA degradation in NP-treated cells. ConclusionReports indicate that we are among the few who had prepared CuO-NP in colloidal form. The antibacterial potency of our particle in growth media was much promising than other reports. Our findings demonstrated that ‘particle-specific’ effect, not ‘ion-specific’ one, was responsible for the NP action. General significanceThe NP may be used as a sterilizing agent in various bioprocesses and as substituent of antibiotics, after thorough toxicological study.

The Deletion of rnhB in Mycobacterium smegmatis Does Not Affect the Level of RNase HII Substrates or Influence Genome Stability

RNase HII removes RNA from RNA/DNA hybrids, such as single ribonucleotides and RNA primers generated during DNA synthesis. Both, RNase HII substrates and RNase HII deficiency have been associated with genome instability in several organisms, and genome instability is a major force leading to the acquisition of drug resistance in bacteria. Understanding the mechanisms that underlie this phenomenon is one of the challenges in identifying efficient methods to combat bacterial pathogens. The aim of the present study was set to investigate the role of rnhB, presumably encoding RNase HII, in maintaining genome stability in the M. tuberculosis model organism Mycobacterium smegmatis. We performed gene replacement through homologous recombination to obtain mutant strains of Mycobacterium smegmatis lacking the rnhB gene. The mutants did not present an altered phenotype, according to the growth rate in liquid culture or susceptibility to hydroxyurea, and did not show an increase in the spontaneous mutation rate, determined using the Luria-Delbrück fluctuation test for streptomycin resistance in bacteria. The mutants also did not present an increase in the level of RNase HII substrates, measured as the level of alkaline degradation of chromosomal DNA or determined through immunodetection. We conclude that proteins other than RnhB proteins efficiently remove RNase HII substrates in M. smegmatis. These results highlight differences in the basic biology between Mycobacteria and eukaryotes and between different species of bacteria.