Janus-Faced Molecules against Plant Pathogenic Fungi

The high cytotoxicity of the secondary metabolites of mycotoxins is capable of killing microbes and tumour cells alike, similarly to the genotoxic effect characteristic of Janus-faced molecules. The “double-edged sword” effect of several cytotoxins is known, and these agents have, therefore, been utilized only reluctantly against fungal infections. In this review, consideration was given to (a) toxins that could be used against plant and human pathogens, (b) animal models that measure the effect of antifungal agents, (c) known antifungal agents that have been described and efficiently prevent the growth of fungal cells, and (d) the chemical interactions that are characteristic of antifungal agents. The utilization of apoptotic effects against tumour growth by agents that, at the same time, induce mutations may raise ethical issues. Nevertheless, it deserves consideration despite the mutagenic impact of Janus-faced molecules for those patients who suffer from plant pathogenic fungal infections and are older than their fertility age, in the same way that the short-term cytotoxicity of cancer treatment is favoured over the long-term mutagenic effect.

TMOD-08. DEVELOPING DERIVATIVE GBM PDX, IN VIVO, FROM TREATMENT NAÏVE SOURCES

PURPOSE Post-therapy recurrent glioblastoma (GBM) patient-derived xenografts (PDX), developed from corresponding treatment-naïve PDX, could serve as useful resources for identifying therapeutics with activity against recurrent GBM. The goal of this study was to determine whether treatment-naïve intracranial GBM PDX, in mice receiving radiotherapy (RT) and/or temozolomide (TMZ), acquire the same mutations that occur in post-RT+TMZ GBMs from patients. METHODS Luciferase-modified, treatment-naïve GBM PDX were engrafted in the brains of athymic nude mice, followed by treatment with RT only (2 Gy/day x 5), TMZ only (10 mg/kg/day x 5), or RT+TMZ. Bioluminescence imaging was used to monitor intracranial tumor growth, response to treatment, and recurrence from treatment. Some mice with recurrent tumors received additional TMZ treatment. When mice became symptomatic, intracranial tumors were resected and engrafted subcutaneously in a new mouse host, then sequentially propagated subcutaneously into additional host mice. After the third passage, whole-exome sequencing (WES) was done, comparing post-therapy with treatment-naïve PDX sequence variants. RESULTS Analysis of PDX WES showed the following: 1) TMZ consistently caused more genes to incur coding sequence mutations than RT, as much as 13x more; 2) TMZ-treated tumor mutations were mostly G-C to A-T transitions (71-92%), consistent with the known mutagenic effect of TMZ; and 3) post-therapy PDX acquire similar mutations as do recurrent GBMs in patients, for example involving DNA mismatch repair gene MSH6. One of the derivative PDX with MSH6 mutation has been retested for response to RT and TMZ, with results showing its having become TMZ, but not RT resistant. CONCLUSIONS The mutation profiles of RT+TMZ-treated PDX are similar to those reported for GBMs that recur after RT+TMZ in patients. The new PDX resources described here may prove useful for identifying effective treatments against recurrent GBM.

Mutagenic Effect of Proton Beams Characterized by Phenotypic Analysis and Whole Genome Sequencing in Arabidopsis

Protons may have contributed to the evolution of plants as a major component of cosmic-rays and also have been used for mutagenesis in plants. Although the mutagenic effect of protons has been well-characterized in animals, no comprehensive phenotypic and genomic analyses has been reported in plants. Here, we investigated the phenotypes and whole genome sequences of Arabidopsis M2 lines derived by irradiation with proton beams and gamma-rays, to determine unique characteristics of proton beams in mutagenesis. We found that mutation frequency was dependent on the irradiation doses of both proton beams and gamma-rays. On the basis of the relationship between survival and mutation rates, we hypothesized that there may be a mutation rate threshold for survived individuals after irradiation. There were no significant differences between the total mutation rates in groups derived using proton beam or gamma-ray irradiation at doses that had similar impacts on survival rate. However, proton beam irradiation resulted in a broader mutant phenotype spectrum than gamma-ray irradiation, and proton beams generated more DNA structural variations (SVs) than gamma-rays. The most frequent SV was inversion. Most of the inversion junctions contained sequences with microhomology and were associated with the deletion of only a few nucleotides, which implies that preferential use of microhomology in non-homologous end joining was likely to be responsible for the SVs. These results show that protons, as particles with low linear energy transfer (LET), have unique characteristics in mutagenesis that partially overlap with those of low-LET gamma-rays and high-LET heavy ions in different respects.

Quantification of aniline and N-methylaniline in indigo

Aniline and N-methylaniline are common contaminants in commercially produced indigo. It is known, that commercially produced indigo contains up to 0.6% aniline and 0.4% N-methylaniline by weight and indigo dye shows a small mutagenic effect, most probably due to the presence of these contaminants. The present work describes a new and powerful analytical method to determine the concentration of these contaminants in indigo. This method is based on the transformation of water insoluble indigo into soluble leucoindigo and allows therefore the acidic extraction of the aromatic contaminants. This transformation step is essential, because the main part of these contaminants are strongly included in the indigo crystals. The amount of extracted aniline and N-methylaniline from the leucoindigo solution was quantified with high performance liquid chromatography (HPLC, combined with a photo diode array detector). A possible accumulation of the aromatic amines at the indigo crystal surface was investigated using FTIR and by adsorption studies. Therefore this method allows an accurate monitoring of these toxic by-products in the indigo dye, which is important for an economic and environmental assessment of the denim production.

In Vitro Effects of Particulate Matter Associated with a Wildland Fire in the North-West of Italy

Wildland fires, increasing in recent decades in the Mediterranean region due to climate change, can contribute to PM levels and composition. This study aimed to investigate biological effects of PM2.5 (Ø < 2.5 µm) and PM10 (Ø < 10 µm) collected near a fire occurred in the North-West of Italy in 2017 and in three other areas (urban and rural areas). Organic extracts were assessed for mutagenicity using Ames test (TA98 and TA100 strains), cell viability (WST-1 and LDH assays) and genotoxicity (Comet assay) with human bronchial cells (BEAS-2B) and estrogenic activity using a gene reporter assay (MELN cells). In all sites, high levels of PM10 and PM2.5 were measured during the fire suggesting that near and distant sites were influenced by fire pollutants. The PM10 and PM2.5 extracts induced a significant mutagenicity in all sites and the mutagenic effect was increased with respect to historical data. All extracts induced a slight increase of the estrogenic activity but a possible antagonistic activity of PM samples collected near fire was observed. No cytotoxicity or DNA damage was detected. Results confirm that fires could be relevant for human health, since they can worsen the air quality increasing PM concentrations, mutagenic and estrogenic effects.

The mutagenic effect of tobacco smoke on male fertility

Despite the association between tobacco use and the harmful effects on general health as well as male fertility parameters, smoking remains globally prevalent. The main content of tobacco smoke is nicotine and its metabolite cotinine. These compounds can pass the blood-testis barrier, which subsequently causes harm of diverse degree to the germ cells. Although controversial, smoking has been shown to cause not only a decrease in sperm motility, sperm concentration, and an increase in abnormal sperm morphology, but also genetic and epigenetic aberrations in spermatozoa. Both animal and human studies have highlighted the occurrence of sperm DNA-strand breaks (fragmentation), genome instability, genetic mutations, and the presence of aneuploids in the germline of animals and men exposed to tobacco smoke. The question to be asked at this point is, if smoking has the potential to cause all these genetic aberrations, what is the extent of damage? Hence, this review aimed to provide evidence that smoking has a mutagenic effect on sperm and how this subsequently affects male fertility. Additionally, the role of tobacco smoke as an aneugen will be explored. We furthermore aim to incorporate the epidemiological aspects of the aforementioned and provide a holistic approach to the topic.

Rescuing eGFP-Tagged Canine Distemper Virus for 40 Serial Passages Separately in Ribavirin- and Non-Treated Cells: Comparative Analysis of Viral Mutation Profiles

Due to lacking a proofreading mechanism in their RNA-dependent RNA polymerases (RdRp), RNA viruses generally possess high mutation frequencies, making them evolve rapidly to form viral quasispecies during serial passages in cells, especially treated with mutagens, like ribavirin. Canine distemper virus (CDV) belongs to the genus Morbillivirus. Its L protein functions as an RdRp during viral replication. In this study, a recombinant enhanced green fluorescence protein-tagged CDV (rCDV-eGFP) was rescued from its cDNA clone, followed by viral identification and characterization at passage-7 (P7). This recombinant was independently subjected to extra 40 serial passages (P8 to 47) in ribavirin- and non-treated cells. Two viral progenies, undergoing passages in ribavirin- and non-treated VDS cells, were named rCDV-eGFP-R and -N, respectively. Both progenies were simultaneously subjected to next-generation sequencing (NGS) at P47 for comparing their quasispecies diversities with each other. The rCDV-eGFP-R and -N showed 62 and 23 single-nucleotide mutations (SNMs) in individual antigenomes, respectively, suggesting that the ribavirin conferred a mutagenic effect on the rCDV-eGFP-R. The spectrum of 62 SNMs contained 26 missense and 36 silent mutations, and that of 23 SNMs was composed of 17 missense and 6 silent mutations. Neither the rCDV-eGFP-R nor -N exhibited nonsense mutation in individual antigenomes. We speculate that the rCDV-eGFP-R may contain at least one P47 sub-progeny characterized by high-fidelity replication in cells. If such a sub-progeny can be purified from the mutant swarm, its L protein would elucidate a molecular mechanism of CDV high-fidelity replication.

Evolutionary dynamics of pseudoautosomal region 1 in humans and great apes

The pseudoautosomal region 1 (PAR1) is a 2.7 Mb telomeric region of human sex chromosomes. As the largest point of contact between the X and Y, PAR1 has a crucial role in ensuring proper segregation of sex chromosomes during male meiosis, exposing it to extreme recombination and associated mutational processes. We investigate PAR1 evolution using population genomic datasets of extant humans, eight populations of great apes and two archaic human genome sequences. We find that the PAR1 sequence is closer to nucleotide equilibrium than autosomal telomeric sequences. We detect a difference between long-term substitution patterns and extant diversity in PAR1 that is mainly driven by the conflict between strong mutation and recombination-associated fixation bias at CpG sites. Additionally, we detect excess C→G mutations in PAR1 of all great ape species, specific to the mutagenic effect of male recombination. Analysis of differences between frequencies of alleles segregating in females and males provided no evidence for sexually antagonistic selection in this region. Furthermore, despite recent evidence for Y chromosome introgression from humans into Neanderthals, we find that the Neanderthal PAR1 retained similarity to the Denisovan sequence, as is the case for the X chromosome and the autosomes. Lastly, we study repeat content and double-strand break hotspot regions in PAR1 and find that they may play roles in ensuring the obligate X-Y recombination event during male meiosis. Our study provides an unprecedented quantification of population genetic forces and insight into evolutionary processes governing PAR1 biology.

Population sequencing data reveal a compendium of mutational processes in the human germ line

Biological mechanisms underlying human germline mutations remain largely unknown. We statistically decompose variation in the rate and spectra of mutations along the genome using volume-regularized nonnegative matrix factorization. The analysis of a sequencing dataset (TOPMed) reveals nine processes that explain the variation in mutation properties between loci. We provide a biological interpretation for seven of these processes. We associate one process with bulky DNA lesions that resolve asymmetrically with respect to transcription and replication. Two processes track direction of replication fork and replication timing, respectively. We identify a mutagenic effect of active demethylation primarily acting in regulatory regions and a mutagenic effect of LINE repeats. We localize a mutagenic process specific to oocytes from population sequencing data. This process appears transcriptionally asymmetric.