Base Pair Substitutions Research Articles

Molecular identification and studies on genetic diversity and structure-related GC heterogeneity of Spatholobus Suberectus based on ITS2

To determine the role of internal transcribed spacer 2 (ITS2) in the identification of Spatholobus suberectus and explore the genetic diversity of S. suberectus. A total of 292 ITS2s from S. suberectus and 17 other plant species were analysed. S. suberectus was clustered separately in the phylogenetic tree. The genetic distance between species was greater than that within S. suberectus. Synonymous substitution rate (Ks) analysis revealed that ITS2 diverged the most recently within S. suberectus (Ks = 0.0022). These findings suggested that ITS2 is suitable for the identification of S. suberectus. The ITS2s were divided into 8 haplotypes and 4 evolutionary branches on the basis of secondary structure, indicating that there was variation within S. suberectus. Evolutionary analysis revealed that the GC content of paired regions (pGC) was greater than that of unpaired regions (upGC), and the pGC showed a decreasing trend, whereas the upGC remained unchanged. Single-base mutation was the main cause of base pair substitution. In both the initial state and the equilibrium state, the substitution rate of GC was higher than that of AU. The increase in the GC content was partly attributed to GC-biased gene conversion (gBGC). High GC content reflected the high recombination and mutation rates of ITS2, which is the basis for species identification and genetic diversity. We characterized the sequence and structural characteristics of S. suberectus ITS2 in detail, providing a reference and basis for the identification of S. suberectus and its products, as well as the protection and utilization of wild resources.

Chemical composition, mutagenicity, and cytotoxicity of urban submicron particulate matter (PM1) in Agra, India

This study, conducted in Agra, India, examined the mass concentrations, chemical compositions, and seasonal variations of submicron particles (PM1). The concentrations of metals, water-soluble inorganic ions (WSIs) including anions (F−, Cl−, NO₃−, SO₄2−) and cations (Ca2+, K+, Mg2+, NH₄+, Na+), organic carbon (OC) and elemental carbon (EC) and polycyclic aromatic hydrocarbons (PAHs) in PM1 extract were determined using Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES), Ion Chromatography, Thermogravimetric Analysis and Gas Chromatography-Mass Spectrometry (GC–MS) respectively. For morphological observation of PM1 particles, Field Emission Scanning Electron Microscopy-Energy Dispersive X-ray spectrometry (FESEM-EDS) was used. The annual average concentration of PM1 was 82.9 ± 33.4 μg/m3, which exceeds the World Health Organisation's (WHO) safe limit for PM2.5 of 5 μg/m3 by a factor of 17. The PM1 mass composition included metals (31 %), WSIs (28 %), OC and EC (9.8 %), and PAHs (0.4 %). Winter recorded the highest PM1 concentration (96.1 ± 25.8 μg/m3), followed by post-monsoon, summer, and monsoon seasons. The average concentration of PAHs was 364.6 ± 226.6 ng/m3. Positive Matrix Factorization (PMF) identified traffic, emissions from biomass/coal and wood combustion, industrial/stationary sources, and secondary aerosols as potential contributors. The Ames test revealed the presence of frameshift mutations and base pair substitutions, especially in winter and post-monsoon. Additionally, PM1 exhibited cytotoxic effects on V-79 cells, with heightened toxicity during winter and prolonged exposure in other seasons. This study underscores the urgent need to address local emission sources and establish regulatory standards for PM1 in urban areas.

Prediction of Deleterious Single Amino Acid Polymorphisms with a Consensus Holdout Sampler.

Single Amino Acid Polymorphisms (SAPs) or nonsynonymous Single Nucleotide Variants (nsSNVs) are the most common genetic variations. They result from missense mutations where a single base pair substitution changes the genetic code in such a way that the triplet of bases (codon) at a given position is coding a different amino acid. Since genetic mutations sometimes cause genetic diseases, it is important to comprehend and foresee which variations are harmful and which ones are neutral (not causing changes in the phenotype). This can be posed as a classification problem. Computational methods using machine intelligence are gradually replacing repetitive and exceedingly overpriced mutagenic tests. By and large, uneven quality, deficiencies, and irregularities of nsSNVs datasets debase the convenience of artificial intelligence-based methods. Subsequently, strong and more exact approaches are needed to address these problems. In the present work paper, we show a consensus classifier built on the holdout sampler, which appears strong and precise and outflanks all other popular methods. We produced 100 holdouts to test the structures and diverse classification variables of diverse classifiers during the training phase. The finest performing holdouts were chosen to develop a consensus classifier and tested using a k-fold (1 ≤ k ≤5) cross-validation method. We also examined which protein properties have the biggest impact on the precise prediction of the effects of nsSNVs. Our Consensus Holdout Sampler outflanks other popular algorithms, and gives excellent results, highly accurate with low standard deviation. The advantage of our method emerges from using a tree of holdouts, where diverse LM/AI-based programs are sampled in diverse ways.

Kataegis in clinical and molecular subgroups of primary breast cancer

Kataegis is a hypermutation phenomenon characterized by localized clusters of single base pair substitution (SBS) reported in multiple cancer types. Despite a high frequency in breast cancer, large-scale analyses of kataegis patterns and associations with clinicopathological and molecular variables in established breast cancer subgroups are lacking. Therefore, WGS profiled primary breast cancers (n = 791) with associated clinical and molecular data layers, like RNA-sequencing data, were analyzed for kataegis frequency, recurrence, and associations with genomic contexts and functional elements, transcriptional patterns, driver alterations, homologous recombination deficiency (HRD), and prognosis in tumor subgroups defined by ER, PR, and HER2/ERBB2 status. Kataegis frequency was highest in the HER2-positive(p) subgroups, including both ER-negative(n)/positive(p) tumors (ERnHER2p/ERpHER2p). In TNBC, kataegis was neither associated with PAM50 nor TNBC mRNA subtypes nor with distant relapse in chemotherapy-treated patients. In ERpHER2n tumors, kataegis was associated with aggressive characteristics, including PR-negativity, molecular Luminal B subtype, higher mutational burden, higher grade, and expression of proliferation-associated genes. Recurrent kataegis loci frequently targeted regions commonly amplified in ER-positive tumors, while few recurrent loci were observed in TNBC. SBSs in kataegis loci appeared enriched in regions of open chromatin. Kataegis status was not associated with HRD in any subgroup or with distinct transcriptional patterns in unsupervised or supervised analysis. In summary, kataegis is a common hypermutation phenomenon in established breast cancer subgroups, particularly in HER2p subgroups, coinciding with an aggressive tumor phenotype in ERpHER2n disease. In TNBC, the molecular implications and associations of kataegis are less clear, including its prognostic value.

A single base pair substitution in zebrafish distinguishes between innate and acute startle behavior regulation.

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animal's life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line (escapist) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of the synaptotagmin 7a (syt7a) gene that is tightly linked to the escapist acoustic hypersensitivity phenotype. By generating animals in which we deleted the syt7a open reading frame, and subsequent complementation testing with the escapist line, we demonstrate that loss of syt7a function is not the cause of the escapist behavioral phenotype. Nonetheless, escapist mutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that in escapist mutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss of syt7a as causative for the escapist phenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds in escapist larvae can operate independently from those regulating acute threshold regulation.

EVALUATION OF THE CYTOTOXICITY AND GENOTOXICOLOGICAL SAFETY PROFILE OF BIOACTIVE SILVER(I) COMPLEXES WITH AMINOADAMANTANE LIGANDS

Silver(I) complexes of amantadine (atdH), memantine (mtnH) and rimantadine (rtdH), named Ag-atd, Ag-mtn and Ag-rtd, respectively, were recently synthesized and described in the literature as promising antibacterial agents. In the present study, the cytotoxicity of such complexes was evaluated against cultures of primary epidermal keratinocytes (HaCaT) and murine melanoma tumor cells (B16-F10), and mutagenicity was studied by the Ames test to investigate their abilities to induce gene mutations. The Ames test was performed using Salmonella Typhimurium strains (TA98, TA100, TA102 and TA97a) capable of detecting frameshift and base pair substitution gene mutations, in experiments with and without metabolic activation (microsomal fraction S9). This study revealed significant cytotoxic activity against tumor cells and selectivity of Ag-atd and Ag-rtd complexes when compared to non-tumor human keratinocyte cells. Moreover, the Ag(I) complexes did not induce a significant growth in the number of revertant colonies when comparing with the negative control, both in the experiments without and with metabolic activation, indicating absence of mutagenic activity. The results are encouraging and collaborate in the genotoxicological investigations necessary for understanding the interaction and ability of the silver complexes to induce mutations and contribute to ensure their uses as future antibacterial or antitumor agents.

Prolonged carriage of OXA-244-carbapenemase-producing Escherichia coli complicates epidemiological investigations

The rapid increase of OXA-244-producing Escherichia coli, predominantly driven by genetically clustered isolates of sequence type (ST)38, has been observed in at least nine European countries, including Germany. However, the reasons for the spread of OXA-244-producing E. coli remain unclear. Here, we aim to evaluate the possibility of prolonged carriage. We identified a total of six different patients with repeated detection of OXA-244-producing E. coli isolates, which were subjected to both short and long-read whole-genome sequencing (WGS). Besides allelic differences using core genome multilocus sequence typing (cgMLST) analyses, we obtained numbers of single-nucleotide polymorphisms (SNPs) to calculate individual base-pair substitution (BPS) rates. To assess possible re-exposure and risk factors for prolonged carriage, case interviews were conducted. The time between detections ranged from eleven months to more than three years. Initial isolates originated in three+ out of six cases from clinical samples, whereas remaining samples were from screening, mostly in the inpatient setting. As expected, cgMLST analyses showed low numbers of allelic differences between isolates of each case ranging from 1 to 4, whereas numbers of SNPs were between 2 and 99 (mean = 36), thus clearly highlighting the discrepancy between these different bacterial typing approaches. For five out of six cases, observed BPS rates suggest that patients can be colonized with OXA-244-producing E. coli, including ST38 cluster isolates, for extensively long times. Thus, we may have previously missed the epidemiological link between cases because exposure to OXA-244-producing E. coli could have occurred in a time frame, which has not been evaluated in previous investigations. Our results may help to guide future epidemiological investigations as well as to support the interpretation of genetic diversity of OXA-244-producing E. coli, particularly among ST38 cluster isolates.

Mutagenicity Assessment of Homologous Series of Methyl Ester Sulphonates (MES) Using the Bacterial Reverse Mutation (Ames) Test.

Methyl ester sulphonate (MES) is an anionic surfactant that is suitable to be used as an active ingredient in household products. Four palm-based MES compounds with various carbon chains, namely C12, C14, C16 and C16/18 MES, were assayed by the in vitro bacterial reverse mutation (Ames) test in the Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and the Escherichia coli strain WP2 uvrA, with the aim of establishing the safety data of the compounds, specifically their mutagenicity. The test was also carried out on linear alkylbenzene sulphonate (LAS) for comparison. The plate incorporation method was conducted according to the Organization for Economic Cooperation and Development (OECD) Test Guideline 471. All compounds were tested at five analysable non-cytotoxic concentrations, varying from .001mg/plate to 5mg/plate, with and without S-9 metabolic activation. All tested concentrations showed no significant increase in the number of revertant colonies compared to revertant colonies of the negative control. The Ames test indicated that each concentration of C12, C14, C16, C16/18 MES, and LAS used in this study induced neither base-pair substitutions nor frame-shift mutations in the S. typhimurium strains TA98, TA100, TA1535, and TA1537 and the E. coli strain WP2 uvrA. The results showed that C12, C14, C16 and C16/18 MES have no potential mutagenic properties in the presence and absence of S-9 metabolic activation, similarly to LAS. Therefore, the MES is safe to be used as an alternative to petroleum-based surfactants for household cleaning products.

Genotoxic and Mutagenic Effects of the Alternaria Mycotoxin Alternariol in Combination with the Process Contaminant Acrylamide.

Humans are constantly exposed to mixtures of different xenobiotics through their diet. One emerging concern is the Alternaria mycotoxin alternariol (AOH), which can occur in foods typically contaminated by the process contaminant acrylamide (AA). AA is a byproduct of the Maillard reaction produced in carbohydrate-rich foods during thermal processing. Given the genotoxic properties of AOH and AA as single compounds, as well as their potential co-occurrence in food, this study aimed to assess the cytotoxic, genotoxic, and mutagenic effects of these compounds in combination. Genotoxicity was assessed in HepG2 cells by quantifying the phosphorylation of the histone γ-H2AX, induced as a response to DNA double-strand breaks (DSBs). Mutagenicity was tested in Salmonella typhimurium strains TA98 and TA100 by applying the Ames microplate format test. Our results showed the ability of AOH and AA to induce DSBs and increase revertant numbers in S. typhimurium TA100, with AOH being more potent than AA. However, no synergistic effects were observed during the combined treatments. Notably, the results of the study suggest that the compounds exert mutagenic effects primarily through base pair substitutions. In summary, the data indicate no immediate cause for concern regarding synergistic health risks associated with the consumption of foods co-contaminated with AOH and AA.

Lowering the pH leads to the disaggregation of NiO and ZnO nanoparticles and modifies the mutagenic response.

In a changing environmental scenario, acid rain can have a significant impact on aquatic ecosystems. Acidification is known to produce corrosion in metals, hence increasing their harmful effects on the environment, organisms and human health. The prevalent use of metallic nanoparticles (NPs) in everyday products raises concerns regarding exposure and nanotoxicity even in these acidified conditions. We thus report on the cytotoxic and genotoxic potential of nickel oxide (NiO-NP) and zinc oxide (ZnO-NP) NPs when suspended in aqueous media in light of pH variations (7.5 and 5). A modified microsuspension method of the Salmonella/microsome assay was adopted, and strains (TA97a, TA98, TA100, TA102) were exposed to NPs (10-1280 μg/plate) with and without a metabolization fraction. The acidic condition favored disaggregation and caused a decrease in NPs size. Mutagenicity was observed in all samples and different strains, with greater DNA base pair substitution damage (TA100 and TA102), but extrinsic conditions (pH) suggest different action mechanisms of NiO-NP and ZnO-NP on genetic content. Mutagenic activity was found to increase upon metabolic activation (TA98, TA100, and TA102) demonstrating the bioactivity of NiO-NP and ZnO-NP in relation to metabolites generated by the mammalian p450 system in vitro. Modifications in the Salmonella assay methodology increased cell exposure time. The observed responses recommend this modified assay as one of the methodologies of choice for nanoecotoxicological evaluation. These findings emphasize the significance of incorporating the environmental context when evaluating the toxicity of metal-based NPs.

Contribution of the SOS response and the DNA repair systems to norfloxacin induced mutations in E. coli

Antibiotic-resistant bacteria severely threaten human health. Besides spontaneous mutations generated by endogenous factors, the resistance might also originate from mutations induced by certain antibiotics, such as the fluoroquinolones. Such antibiotics increase the genome-wide mutation rate by introducing replication errors from the SOS response pathway or decreasing the efficiency of the DNA repair systems. However, the relative contributions of these molecular mechanisms remain unclear, hindering understanding of the generation of resistant pathogens. Here, using newly-accumulated mutations of wild-type and SOS-uninducible Escherichia coli strains, as well as those of the strains deficient for the mismatch repair (MMR) and the oxidative damage repair pathways, we find that the SOS response is the major mutagenesis contributor in mutation elevation, responsible for ~ 30–50% of the total base-pair substitution (BPS) mutation-rate elevation upon treatment with sublethal levels of norfloxacin (0 ~ 50 ng/mL). We further estimate the significance of the effects on other mutational features of these mechanisms (i.e., transversions, structural variations, and mutation spectrum) in E. coli using linear models. The SOS response plays a positive role in all three mutational features (mutation rates of BPSs, transversions, structural variations) and affects the mutational spectrum. The repair systems significantly reduce the BPS mutation rate and the transversion rate, regardless of whether antibiotics are present, while significantly increasing the structural variation rate in E. coli. Our results quantitatively disentangle the contributions of the SOS response and DNA repair systems in antibiotic-induced mutagenesis.

Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos

Human germline gene correction by targeted nucleases holds great promise for reducing mutation transmission. However, recent studies have reported concerning observations in CRISPR-Cas9-targeted human embryos, including mosaicism and loss of heterozygosity (LOH). The latter has been associated with either gene conversion or (partial) chromosome loss events. In this study, we aimed to correct a heterozygous basepair substitution in PLCZ1, related to infertility. In 36% of the targeted embryos that originated from mutant sperm, only wild-type alleles were observed. By performing genome-wide double-digest restriction site-associated DNA sequencing, integrity of the targeted chromosome (i.e., no deletions larger than 3 Mb or chromosome loss) was confirmed in all seven targetedGENType-analyzed embryos (mutant editing and absence of mutation), while short-range LOH events (shorter than 10 Mb) were clearly observed by single-nucleotide polymorphism assessment in two of these embryos. These results fuel the currently ongoing discussion on double-strand break repair in early human embryos, making a case for the occurrence of gene conversion events or partial template-based homology-directed repair.

Incidence, Seroprevalence, and In-shelter Transmission of SARS-CoV-2 in Cats

Cats are susceptible to infection with SARS-CoV-2 and can transmit the virus to humans. Experimentally infected cats shed virus for 5-10 days and generate antibodies within 7 days, while naive cats cohoused with these cats shed virus orally within 24 hours. Shelters intake cats from SARS-CoV-2 endemic communities and house them in close proximity, creating the opportunity for shelter-acquired disease. Shelter cat populations could serve as a reservoir for the virus, where it might mutate and spill back into human populations. This study aimed to determine the incidence and seroprevalence of SARS-CoV-2 in cats housed in a shelter. Cats over 1 kg undergoing anesthesia between April and December 2020 in a large shelter were enrolled in the study using convenience sampling. Pharyngeal, conjunctival, and serum samples were collected. The pharyngeal/conjunctival swabs were analyzed by Real-Time PCR using N1 and N2 probes derived from the virus's nucleocapsid protein (N) portion. One sample positive for N1 within the 40 cycles specified by CDC protocol but only positive for N2 after 41 cycles (late rise) was sequenced. The seroprevalence of SARS-CoV-2 antibodies was assessed using the cPass SARS-CoV-2 Neutralization Antibody Detection Kit. During the study period, 7,179 cats entered the shelter, and 350 were sampled. Most sampled cats were owner surrender (45%) or stray (43%), which was different (P<0.0001) from the overall population (26% owner surrender and 64% stray). Seven (2%) samples were PCR positive (95%CI 0.8-4.0%), 199 (57%) were negative, and 144 (41%) were inconclusive. Of the inconclusive samples, 85 (59%) were positive for N1 but negative for N2. The median length of stay before sampling (LOSBS) was 4 days (IQR 1-10) for all sampled cats and 13 days (IQR 5-22) for PCR-positive cats. Of the 131 serum samples tested to date, 11 (8%) were positive (95%CI 4.3-14.5). The median LOSBS for antibody-positive cats was 5 (IQR 1-5; range 1-8). One positive cat with a LOSBS of 5 days was PCR and antibody positive. The cat with a sequenced sample had a LOSBS of 13 days, was antibody negative, and had both base pair deletions and substitutions in the N2 target probe region. Consistent with in-shelter transmission, the median LOSBS for cats positive by PCR was greater than the documented SARS-CoV-2 infectious period. All but one of the seropositive cats had a LOSBS of 5 days or less, suggesting the previous infection was community-acquired. The sequenced sample was credibly from a shelter-acquired infection, and the base pair deletions and substitutions were consistent with a feline-specific strain circulating in the shelter.

In vitroandin silicoevaluation of the design of nano-phyto-drug candidate for oral use againstStaphylococcus aureus

Onopordum acanthiumis a medicinal plant with many important properties, such as antibacterial, anticancer, and anti-hypotensive properties. Although various studies reported the biological activities ofO. acanthium, there is no study on its nano-phyto-drug formulation. The aim of this study is to develop a candidate nano-drug based on phytotherapeutic constituents and evaluate its efficiencyin vitroandin silico. In this context, poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) ofO. acanthiumextract (OAE) were synthesized and characterized. It was determined that the average particle size of OAE-PLGA-NPs was 214.9 ± 6.77 nm, and the zeta potential was −8.03 ± 0.85 mV, and PdI value was 0.064 ± 0.013. The encapsulation efficiency of OAE-PLGA-NPs was calculated as 91%, and the loading capacity as 75.83%. Thein vitrodrug release study showed that OAE was released from the PLGA NPs with 99.39% over the 6 days. Furthermore, the mutagenic and cytotoxic activity of free OAE and OAE-PLGA-NPs were evaluated by the Ames test and MTT test, respectively. Although 0.75 and 0.37 mg/mL free OAE concentrations caused both frameshift mutation and base pair substitution (p< 0.05), the administered OAE–PLGA NP concentrations were not mutagenic. It was determined with the MTT analysis that the doses of 0.75 and 1.5 mg/mL of free OAE had a cytotoxic effect on the L929 fibroblast cell line (p< 0.05), and OAE-PLGA-NPs had no cytotoxic effect. Moreover, the interaction between the OAE andS. aureuswas also investigated using the molecular docking analysis method. The molecular docking and molecular dynamics (MD) results were implemented to elucidate theS. aureusMurE inhibition potential of OAE. It was shown that quercetin in the OAE content interacted significantly with the substantial residues in the catalytic pocket of theS. aureusMurE enzyme, and quercetin performed four hydrogen bond interactions corresponding to a low binding energy of −6.77 kcal/mol with catalytic pocket binding residues, which are crucial for the inhibition mechanism ofS. aureusMurE. Finally, the bacterial inhibition values of free OAE and OAE–PLGA NPs were determined againstS. aureususing a microdilution method. The antibacterial results showed that the inhibition value of the OAE–PLGA NPs was 69%. In conclusion, from thein vitroandin silicoresults of the nano-sized OAE-PLGA NP formulation produced in this study, it was evaluated that the formulation may be recommended as a safe and effective nano-phyto-drug candidate againstS. aureus.

De Novo Structural Variations of Escherichia coli Detected by Nanopore Long-Read Sequencing.

Spontaneous mutations power evolution, whereas large-scale structural variations (SVs) remain poorly studied, primarily because of the lack of long-read sequencing techniques and powerful analytical tools. Here, we explore the SVs of Escherichia coli by running 67 wild-type (WT) and 37 mismatch repair (MMR)-deficient (ΔmutS) mutation accumulation lines, each experiencing more than 4,000 cell divisions, by applying Nanopore long-read sequencing and Illumina PE150 sequencing and verifying the results by Sanger sequencing. In addition to precisely repeating previous mutation rates of base-pair substitutions and insertion and deletion (indel) mutation rates, we do find significant improvement in insertion and deletion detection using long-read sequencing. The long-read sequencing and corresponding software can particularly detect bacterial SVs in both simulated and real data sets with high accuracy. These lead to SV rates of 2.77 × 10-4 (WT) and 5.26 × 10-4 (MMR-deficient) per cell division per genome, which is comparable with previous reports. This study provides the SV rates of E. coli by applying long-read sequencing and SV detection programs, revealing a broader and more accurate picture of spontaneous mutations in bacteria.

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

Assessment of DNA mutagenicity induced by He-Ne laser using Salmonella typhimurium strains.

Helium-neon (He-Ne) laser mutagenesis is widely used in microbiology and plant breeding. In this study, two frameshift mutant representative strains of Salmonella typhimurium TA97a and TA98 and two base pair substitution types TA100 and TA102 were employed as model microorganisms to assess DNA mutagenicity induced by He-Ne laser (3J·cm-2·s-1, 632.8nm) for 10, 20, and 30min. The results revealed that the optimal laser application was 6h in the mid-logarithmic growth stage. Low-power He-Ne laser for short treatment inhibited cell growth, and continued treatment stimulated the metabolism. The effects of the laser on TA98 and TA100 were the most prominent. Sequencing results from 1500 TA98 revertants showed that there were 88 insertion and deletion (InDel) types in the hisD3052 gene, of which the InDels unique to laser were 21 more than that of the control. Sequencing results from 760 TA100 revertants indicated that laser treatment created Pro (CCC) in the product of the hisG46 gene more likely to be replaced by His (CAC) or Ser (TCC) than by Leu (CTC). Two unique non-classical base substitutions, CCC → TAC and CCC → CAA, also appeared in the laser group. These findings will provide a theoretical basis for further exploration of laser mutagenesis breeding. KEY POINTS: • Salmonella typhimurium served as model organism for laser mutagenesis study. • Laser promoted the occurrence of InDels in the hisD3052 gene of TA98. • Laser promoted the occurrence of base substitution in the hisG46 gene of TA100.

Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays

Propranolol is a widely used β-blocker that can generate a nitrosated derivative, N-nitroso propranolol (NNP). NNP has been reported to be negative in the bacterial reverse mutation test (the Ames test) but genotoxic in other in vitro assays. In the current study, we systematically examined the in vitro mutagenicity and genotoxicity of NNP using several modifications of the Ames test known to affect the mutagenicity of nitrosamines, as well as a battery of genotoxicity tests using human cells. We found that NNP induced concentration-dependent mutations in the Ames test, both in two tester strains that detect base pair substitutions, TA1535 and TA100, as well as in the TA98 frameshift-detector strain. Although positive results were seen with rat liver S9, the hamster liver S9 fraction was more effective in bio-transforming NNP into a reactive mutagen. NNP also induced micronuclei and gene mutations in human lymphoblastoid TK6 cells in the presence of hamster liver S9. Using a panel of TK6 cell lines that each expresses a different human cytochrome P450 (CYP), CYP2C19 was identified as the most active enzyme in the bioactivation of NNP to a genotoxicant among those tested. NNP also induced concentration-dependent DNA strand breakage in metabolically competent 2-dimensional (2D) and 3D cultures of human HepaRG cells. This study indicates that NNP is genotoxic in a variety of bacterial and mammalian systems. Thus, NNP is a mutagenic and genotoxic nitrosamine and a potential human carcinogen.

Role of KatG in Francisella tularensis Susceptibility to Resazurin.

The CDC classifies Francisella tularensis as a Category A bioterrorism agent. Due to the risk of potential release of antibiotic-resistant F. tularensis strains, new therapeutics against F. tularensis must be developed. Resazomycins are resazurin (Rz)-based compounds that exhibit antimicrobial activity against F. tularensis and other gram-negative bacteria. The mode of action of resazomycins is not understood, but potential targets of the antibiotic were identified in a high throughput screen for Rz-resistant isolates. The FTL_1504 gene was identified as being mutated in all of the Rz-resistant (Rzr) strains sequenced. FTL_1504 encodes for the catalase KatG which catalyzes the decomposition of hydrogen peroxide to water and oxygen. Moreover, KatG has also been shown to play an essential role in the activity of a different antibiotic, isonazid. Four different coding mutations were characterized in katG that included single base pair substitutions and a deletion with all resulting in the introduction of a premature stop codon. All Rzr mutants tested had reduced catalase activity compared to wild-type F. tularensis LVS with three of the four mutations resulting in loss of KatG expression. Based on this data, we hypothesize KatG plays a role in the susceptibility of F. tularensis to resazurin. To address this, we are generating a katG deletion mutant in F. tularensis LVS and measuring its Rz susceptibility via agar dilution assays. Investigation of the contribution of KatG may unveil a unique role for the oxidative stress response in the mode of action of this family of antibiotics

DNA-free genome editing for ZmPLA1 gene via targeting immature embryos in tropical maize

ABSTRACT Doubled haploid (DH) production accelerates the development of homozygous lines in a single generation. In maize, haploids are widely produced by the use of haploid inducer Stock 6, earlier reported in 1959. Three independent studies reported haploid induction in maize which is triggered due to a 4 bp frame-shift mutation in matrilineal (ZmPLA1) gene. The present study was focused on the generation of mutants for ZmPLA1 gene in maize inbred line LM13 through site-directed mutagenesis via CRISPR/Cas9-mediated ribonucleoprotein (RNP) complex method to increase the haploid induction rate. Three single guide RNAs (sgRNAs) for the ZmPLA1 gene locus were used for transforming the 14 days old immature embryos via bombardment. 373 regenerated plants were subjected to mutation detection followed by Sanger’s sequencing. Out of three putative mutants identified, one mutant depicted one base pair substitution and one base pair deletion at the target site.