Mutations to azaguanine resistance induced in cultured diploid human fibroblasts by the carcinogen, N-acetoxy-2-Acetylaminofluorene

The ability of the carcinogen, N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), to induce mutations to azaguanine resistance in diploid human cells was quantitatively investigated and shown to be dose-dependent. The 8-azaguanine (AG) resistance was shown to be heritable in the absence of mutagen or selective agent and the cells of the mutant clones were shown to retain normal sensitivity to N-AcO-AAF.     

Assessing DNA for fish identifications from reference collections: the good,bad and ugly shed light on formalin fixation and sequencing approaches

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.     

Benefiting others and self: Production of vitamins in plants

Vitamins maintain growth and development in humans, animals, and plants. Because plants serve as essential producers of vitamins, increasing the vitamin contents in plants has become a goal of crop breeding worldwide. Here, we begin with a summary of the functions of vitamins. We then review the achievements to date in elucidating the molecular mechanisms underlying how vitamins are synthesized, transported, and regulated in plants. We also stress the exploration of variation in vitamins by the use of forward genetic approaches, such as quantitative trait locus mapping and genome-wide association studies. Overall, we conclude that exploring the diversity of vitamins could provide new insights into plant metabolism and crop breeding.     

Chemotherapy regimens induce inhibitory immune checkpoint protein expression on stem-like and senescent-like oesophageal adenocarcinoma cells

Use of immune checkpoint inhibitors (ICIs) with chemotherapy to enhance responses in oesophageal adenocarcinoma (OAC) is an attractive approach. We identified subpopulations of OAC cells expressing inhibitory immune checkpoint (IC) ligands (PD-L1, PD-L2 and CD160) and receptors (PD-1, TIGIT, TIM-3, LAG-3 and A2aR) in vitro and in ex vivo biopsies. Combination chemotherapy regimens FLOT and CROSS promote a more immune-resistant phenotype through upregulation of IC ligands and receptors on OAC cells in vitro. Importantly, this study investigated if OAC cells, enriched for ICs exhibited a more stem-like and senescent-like phentoype. FLOT preferentially upregulates PD-L1 on a stem-like OAC cell phenotype, defined by ALDH activity. Expression of senescence-associated β-galactosidase is induced in a subpopulation of OAC cells following FLOT and CROSS chemotherapy treatment, along with enhanced expression of TIM-3 and A2aR ICs. Blockade of PD-1 signalling in OAC cells induced apoptosis and enhanced FLOT and CROSS chemotherapy toxicity in vitro. Upregulation of ICs on OAC cells following chemotherapy may represent potential mechanisms of chemo-immune resistance. Combination ICIs may be required to enhance the efficacy of chemotherapy and immunotherapy in OAC patients.     

Redox status in workers occupationally exposed to long-term low levels of ionizing radiation: A pilot study

Objectives: Reactive oxygen species (ROS), including superoxide (O₂^??), play an important role in the biological effects of ionizing radiation. The human body has developed different antioxidant systems to defend against excessive levels of ROS. The aim of the present study is to investigate the redox status changes in the blood of radiologic technologists and compare these changes to control individuals.

Methods: We enrolled 60 medical workers: 20 occupationally exposed to ionizing radiation (all radiologic technologists), divided in three subgroups: conventional radiography (CR), computerized tomography (CT), and interventional radiography (IR) and 40 age- and gender-matched unexposed controls. Levels of O₂^?? and malondialdehyde (MDA) in blood were measured as an index of redox status, as were the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase. Redox status was also assessed by measuring levels of reduced and oxidized glutathione (GSH, GSSG, respectively).

Results: Levels of O₂^?? and MDA, and SOD activity in the blood of IR and CT-exposed subjects were significantly higher than both the CR-exposed subjects and control individuals. However, there were no statistically significant differences in the levels of catalase, GSH and ratio of GSH/GSSG between exposed workers and control individuals.

Discussion: This study suggests that healthcare workers in CT and IR occupationally exposed to radiation have an elevated circulating redox status as compared to unexposed individuals.     

Native Prey and Invasive Predator Patterns of Foraging Activity: The Case of the Yellow-Legged Hornet Predation at European Honeybee Hives

Contrary to native predators, which have co-evolved with their prey, alien predators often benefit from native prey naïveté. Vespa velutina, a honeybee predator originating from Eastern China, was introduced into France just before 2004. The present study, based on video recordings of two beehives at an early stage of the invasion process, intends to analyse the alien hornet hunting behaviour on the native prey, Apis mellifera, and to understand the interaction between the activity of the predator and the prey during the day and the season. Chasing hornets spent most of their time hovering facing the hive, to catch flying honeybees returning to the hive. The predation pressure increased during the season confirming previous study based on predator trapping. The number of honeybee captures showed a maximum peak for an intermediate number of V. velutina, unrelated to honeybee activity, suggesting the occurrence of competition between hornets. The number of honeybees caught increased during midday hours while the number of hornets did not vary, suggesting an increase in their efficacy. These results suggest that the impact of V. velutina on honeybees is limited by its own biology and behaviour and did not match the pattern of activity of its prey. Also, it could have been advantageous during the invasion, limiting resource depletion and thus favouring colonisation. This lack of synchronization may also be beneficial for honeybee colonies by giving them an opportunity to increase their activity when the hornets are less effective.     

Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase

Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination, UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an inactive form prior to ssDNA generation.     

Genetic variation in DNMT3B and increased global DNA methylation is associated with suicide attempts in psychiatric patients

Recently, a significant epigenetic component in the pathology of suicide has been realized. Here we investigate candidate functional SNPs in epigenetic‐regulatory genes, DNMT1 and DNMT3B, for association with suicide attempt (SA) among patients with co‐existing psychiatric illness. In addition, global DNA methylation levels [5‐methyl cytosine (5‐mC%)] between SA and psychiatric controls were quantified using the Methylflash Methylated DNA Quantification Kit. DNA was obtained from blood of 79 suicide attempters and 80 non‐attempters, assessed for DSM‐IV Axis I disorders. Functional SNPs were selected for each gene (DNMT1; n = 7, DNMT3B; n = 10), and genotyped. A SNP (rs2424932) residing in the 3′ UTR of the DNMT3B gene was associated with SA compared with a non‐attempter control group (P = 0.001; Chi‐squared test, Bonferroni adjusted P value = 0.02). Moreover, haplotype analysis identified a DNMT3B haplotype which differed between cases and controls, however this association did not hold after Bonferroni correction (P = 0.01, Bonferroni adjusted P value = 0.56). Global methylation analysis showed that psychiatric patients with a history of SA had significantly higher levels of global DNA methylation compared with controls (P = 0.018, Student's t‐test). In conclusion, this is the first report investigating polymorphisms in DNMT genes and global DNA methylation quantification in SA risk. Preliminary findings suggest that allelic variability in DNMT3B may be relevant to the underlying diathesis for suicidal acts and our findings support the hypothesis that aberrant DNA methylation profiles may contribute to the biology of suicidal acts. Thus, analysis of global DNA hypermethylation in blood may represent a biomarker for increased SA risk in psychiatric patients.