Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.     

Volatile exposure within the honeybee hive and its effect on olfactory discrimination

Honeybees of different ages and reproductive castes cohabit in the hive where they are exposed to many odors that might affect associative learning. Our aim was to analyze the role of odors pre-exposed as volatiles on appetitive learning in honeybees of different ages and search for their long-term effect both under natural and laboratory conditions. By evaluating memory acquisition and retention through a differential proboscis extension response conditioning, we found that hive-exposed odors offered as a reinforced conditioned stimulus during training promoted a learning-reduced effect [latent inhibition (LI)]. On the other hand, no effect was found when the non-reinforced conditioned stimulus was pre-exposed. The LI effect varied with the odor identity. However, only slight differences were found with the age of the bees. Exposure-conditioning intervals longer than 24 h did not show an LI effect unless the odor concentration was increased or exposure was prolonged. Our results show that pre-exposed volatiles could either reduce learning performance, if this odor is later associated with food, or be irrelevant in the case that alternative scented resources circulate within the colony. The differential effects found according to the olfactory exposure characteristics could strongly influence the propagation of chemosensory information within the hive.     

Post-translational modifications in MeHg-induced neurotoxicity

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.     

Epidermal growth factor modulation of prostaglandins and nitrite biosynthesis in rat fetal membranes

The production of prostaglandins (PGs) and nitric oxide (NO) by amnion tissue may play a significant role in parturition. It is thought that epidermal growth factor (EGF) may be one of the fetal signals that governs the initiation of labor. The aim of the present study was to investigate the effect of EGF in vivo on the PGs and nitrite production of rat fetal membranes. We have evaluated the regulation of PGs and nitrite production in rat fetal membranes ex vivo. The intra-uterine administration of EGF 500 ng in day 21 of pregnancy induced increases in PGE(2) (P<0.001) and PGF(2alpha) (P<0.01) compared to the control fetal membranes from pregnant rats on day 22. Also, this dose of EGF diminished nitrate production significantly (P<0.01). We found that fetal membranes at term (days 18-22 of gestation) expressed EGF-R. The NO donor, nitroprussiate 300 and 600 microM, elicited an inhibitory effect on the PGE(2) and PGF(2alpha) stimulated synthesis. On the other hand, indomethacin 10(-6) and 10(-7)M, a non-selective cyclooxygenase inhibitor, reverted the inhibitory effect exerted by EGF. Hence, rat fetal membranes were found to express epidermal growth factor receptors and, under the effect of EGF, PGs and nitrites production pathways interact probably to prevent a toxic effect caused by an exacerbated synthesis of these mediators.     

Analysis of genotypic diversity inCercospora beticola Sacc. field isolates

Genetic variability and population structure ofCercospora beticola, the causal agent of Cercospora leaf spot in sugarbeet, from four sugarbeet-growing regions of Greece were investigated using growth rate, pathogenicity, and mini- and microsatellite DNA fingerprinting. Mycelial growth and pathogenicity were very diverse within and between groups, and no correlation was found between these features and the geographic origin of the isolates. High diversity was found by micro- and minisatellite fingerprinting, with an average gene diversity of 0.21, and no significant differences among populations. Among the 46 isolates, 45 different genotypes were identified, showing a high degree of genotype diversity. Analysis of the genetic profiles provided no evidence for regional patterns of variation (ΦF_ST=0.01, P=0.261) and the analysis of molecular variation (AMOVA) revealed that genetic variability was due mainly to variations within (99%) rather than between (1%) populations. Such a low level of genetic differentiation is reflected by a migration rate value Nm of 4.7. The high migration rate cannot be referred to splash dispersed conidia. To justify the absence of a regional structure in these C.beticola populations, we must suppose the existence of a long-distance means of dispersal, such as seed transmission and/or man mediated transmission.     

Viridans Group Streptococci Clinical Isolates: MALDI-TOF Mass Spectrometry versus Gene Sequence-Based Identification

Viridans Group Streptococci (VGS) species-level identification is fundamental for patients management. Matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI-TOF MS) has been used for VGS identification but discrimination within the Mitis group resulted difficult. In this study, VGS identifications with two MALDI-TOF instruments, the Biotyper (Bruker) and the VITEK MS (bioMérieux) have been compared to those derived from tuf, soda and rpoB genes sequencing. VGS isolates were clustered and a dendrogram constructed using the Biotyper 3.0 software (Bruker). RpoB gene sequencing resulted the most sensitive and specific molecular method for S. pneumonia identification and was used as reference method. The sensitivity and the specificity of the VITEK MS in S. pneumonia identification were 100%, while the Biotyper resulted less specific (92.4%). In non pneumococcal VGS strains, the group-level correlation between rpoB and the Biotyper was 100%, while the species-level correlation was 61% after database upgrading (than 37% before upgrading). The group-level correlation between rpoB and the VITEK MS was 100%, while the species-level correlation was 36% and increases at 69% if isolates identified as S. mitis/S. oralis are included. The less accurate performance of the VITEK MS in VGS identification within the Mitis group was due to the inability to discriminate between S. mitis and S. oralis. Conversely, the Biotyper, after the release of the upgraded database, was able to discriminate between the two species. In the dendrogram, VGS strains from the same group were grouped into the same cluster and had a good correspondence with the gene-based clustering reported by other authors, thus confirming the validity of the upgraded version of the database. Data from this study demonstrated that MALDI-TOF technique can represent a rapid and cost saving method for VGS identification even within the Mitis group but improvements of spectra database are still recommended.     

On the expression of cytokeratins and their distribution in some rabbit gland tissues.

The composition and distribution of cytokeratins were studied in a series of exocrine and endocrine glands in the rabbit, by means of anti-human monoclonal antibodies. Rabbit cytokeratins are known to be homologous to those in man, and their in situ distribution was also found to be similar. In acinic cells of the exocrine glands, only cytokeratins 8 and 18 were detected, whereas in ductal cells 1, 5, 10, 11 and 13, 19 were also present. The myoepithelial cells surrounding acini and basal duct cells were stained with the antibodies directed against cytokeratins 13 and 1, 5, 10, 11. Among the endocrine glands, a higher reactivity was observed in the thyroid gland and pancreatic islets, while pituitary and adrenal gland often remained unstained. Although the expression of cytokeratins was found to be almost homogeneous in each glandular cell type, a certain variability was observed, depending on the organ examined and the treatment carried out.     

Male gonads morphology,spermatogenesis and sperm ultrastructure of the seahorse Hippocampus guttulatus (Syngnathidae)

Testes morphology, spermatogenetic process and mature sperm ultrastructure were analysed in Hippocampus guttulatus, using both light and transmission electron microscopy. Both testes were organized in a single large germinal compartment, with a central lumen. Spermatocysts only contained spermatogonia and primary spermatocytes. Inside the testis lumen, together with mature sperm, two types of large mono‐nucleate cells, flagellate and aflagellate, were present. Both types of cells were interpreted as developing germ cells precociously released inside the testis lumen, where their maturation was completed. According to the different morphological features of the nuclei, such as chromatin condensation degree, aspect of the nuclear fossa and others, the flagellate cells were unquestionably developing spermatids. On the contrary, the developmental stage of the aflagellate was more difficult to interpreted. They could be secondary spermatocytes or young spermatids. No dimorphic sperm were recognizable, the only sperm type observed have features typical of the intro‐sperm reports in other syngnathids species. They had a cylindrical head, a short midpiece, characterized by two mitochondrial rings housed inside a cytoplasmic collar, and a long flagellum. These and previous data about the same topic reported on other syngnathids species were compared and discussed.