Identification and characterization of the first fragment hits for SETDB1 Tudor domain

SET domain bifurcated protein 1 (SETDB1) is a human histone-lysine methyltransferase which is amplified in human cancers and was shown to be crucial in the growth of non-small and small cell lung carcinoma. In addition to its catalytic domain, SETDB1 harbors a unique tandem tudor domain which recognizes histone sequences containing both methylated and acetylated lysines, and likely contributes to its localization on chromatin. Using X-ray crystallography and NMR spectroscopy fragment screening approaches, we have identified the first small molecule fragment hits that bind to histone peptide binding groove of the Tandem Tudor Domain (TTD) of SETDB1. Herein, we describe the binding modes of these fragments and analogues and the biophysical characterization of key compounds. These confirmed small molecule fragments will inform the development of potent antagonists of SETDB1 interaction with histones.     

Dispersal limitation: Evolutionary origins and consequences in arthropods

Niche and dispersal ability are key traits for explaining the geographical structuring of species into discrete populations, and its evolutionary significance. Beyond their individual effects, the interplay between species niche and its geographic limits, together with the evolutionary lability of dispersal ability, can underpin trait diversification and speciation when exposed to gradients of selection. In this issue of Molecular Ecology, two complementary papers demonstrate how evolutionary lability for dispersal ability linked to niche shift can drive such a model in a context that includes selection. Both papers investigate the evolution of dispersal limitation in arthropods across altitudinal gradients, but using taxa with contrasting ecologies. McCulloch et al. (2019) investigate the evolution of wing loss at higher altitudes in stoneflies, a taxon inhabiting freshwater systems. Suzuki et al. (2019) report a similar phenomenon, but involving wing reduction at higher altitudes in scorpionflies, a taxon associated with moist terrestrial habitats. Here, we compare and contrast the results of both studies to explore their broader implications for understanding diversification and speciation within arthropods.     

Towards alien plant prioritization in Italy: methodological issues and first results

In recent decades, multiple actions have been taken to counteract the relentless expansion of invasive alien species as well as to gain a better understanding of their effects on ecosystems. Here, we describe the approach designed by the Italian Botanical Society that is aimed at selecting a list of candidate alien plants to be subjected to a prioritization procedure. We selected a total of 96 species on the basis of data related to their occurrence on both a national and regional scale, their invasiveness and their potential to invade plant communities and/or habitats of community concern. This list represents the first result obtained by applying this standardized workflow and is a first step towards the identification of those alien species that should be included in the national list according to Regulation (EU) n. 1143/2014.     

Characterization of chromate-resistant and -reducing bacteria by traditional means and by a high-throughput phenomic technique for bioremediation purposes

To select strains for the bioremediation of Cr(VI)-polluted environments, four highly Cr(VI)-resistant bacterial isolates were identified and characterized using both traditional techniques and a novel approach called phenotype microarrays. The isolates were identified as members of Pseudomonas mendocina (strains 34 and 56) and members of Pseudomonas corrugata (strains 22 and 28). Results showed that it was possible, by varying the carbon/energy source, to decouple bacterial growth and Cr(VI) reduction, inasmuch as some carbon/energy sources were more effective electron donors for chromate reduction, whereas other sources supported growth but not an effective chromate reduction. The isolates were characterized by a novel high-throughput technique, phenotype microarrays (PM)-Biolog, which can test up to 2000 cellular phenotypes simultaneously. The isolates belonging to P. corrugata had PM profiles different from those of the isolates belonging to P. mendocina. Such differences were related to the capacity of the isolates to resist various chemicals, pH values, and osmolytic substances. With the PM technique a very large amount of information about the fitness of isolates in the presence of different stressors could be obtained.     

Expression and activity of cyclooxygenase isoforms in skeletal muscles and myocardium of humans and rodents.

Conflicting data have been reported on cyclooxygenase (COX)-1 and COX-2 expression and activity in striated muscles, including skeletal muscles and myocardium, in particular it is still unclear whether muscle cells are able to produce prostaglandins (PGs). We characterized the expression and enzymatic activity of COX-1 and COX-2 in the skeletal muscles and in the myocardium of mice, rats and humans. By RT-PCR, COX-1 and COX-2 mRNAs were observed in homogenates of mouse and rat hearts, and in different types of skeletal muscles from all different species. By Western blotting, COX-1 and -2 proteins were detected in skeletal muscles and hearts from rodents, as well as in skeletal muscles from humans. Immunoperoxidase stains showed that COX-1 and -2 were diffusely expressed in the myocytes of different muscles and in the myocardiocytes from all different species. In the presence of arachidonic acid, which is the COX enzymatic substrate, isolated skeletal muscle and heart samples from rodents released predominantly PGE(2). The biosynthesis of PGE(2) was reduced between 50 and 80% (P < 0.05 vs. vehicle) in the presence of either COX-1- or COX-2-selective blockers, demonstrating that both isoforms are enzymatically active. Exogenous PGE(2) added to isolated skeletal muscle preparations from rodents did not affect contraction, whereas it significantly fastened relaxation of a slow type muscle, such as soleus. In conclusion, COX-1 and COX-2 are expressed and enzymatically active in myocytes of skeletal muscles and hearts of rodents and humans. PGE(2) appears to be the main product of COX activity in striated muscles.     

Oxidized transthyretin in amniotic fluid as an early marker of preeclampsia

Preeclampsia is a pregnancy-specific hypertensive syndrome and a major cause of maternal and fetal morbidity and mortality. At the present time, no reliable screening tests to identify women at risk are available. We have compared the amniotic fluids (AF) proteomic maps of five preeclamptic patients with those of five controls. The analysis was carried out by two-dimensional electrophoresis followed by peptide mapping and tandem mass spectrometric analysis. Besides the implementation of the previously published AF proteomic maps, our results show that transthyretin (TTR), the protein responsible for transporting both the thyroid hormone tyroxine and the retinol binding protein, is present in the AF of both preeclamptic and control women as a mixture of dimeric and post-translationally modified monomeric forms. Although the nature of these forms is similar in both groups, the preeclamptic women showed a significant increase in the amount of monomeric proteins with respect to the control group. Since the TTR monomeric forms are the results of different oxidizing reactions, we hypothesize that the higher oxidative stress in preeclampsia is the major destabilizing factor of the TTR functional dimeric form in the preeclamptic women.     

Mouse fibroblasts are reprogrammed to Oct-4 and Rex-1 gene expression and alkaline phosphatase activity by embryonic stem cell extracts

A recent remarkable study has shown that when mouse NIH-3T3 fibroblasts are exposed to an embryonic stem cell (ESC) extract, the majority of them expresses the Oct-4 gene, form ESC-like colonies, and embryoid-like bodies that differentiate into cells of the three germ layers. The use of cell extracts for inducing cell dedifferentiation could be a powerful system to obtain large quantities of pluripotent cells. It is thus of crucial importance that the robustness of this method of cell transdifferentiation is tested by other laboratories before it is advanced to a more ambitious use in cell therapy programs. We report here our experimental observations using the same reprogramming protocol on STO and NIH-3T3 mouse fibroblasts. Three are the main results: first, we confirmed an enduring reprogramming activity of the ESC extract, although on a much smaller number of cells that varies from approximately 0.003 to 0.04% of the total population of fibroblasts and with an effect limited to the induction of Oct-4 and Rex-1 gene expression and alkaline phosphatase activity. Second, the expression of OCT-4, SSEA-1, and Forssman antigen proteins was never detected. Third, our work has clearly demonstrated that ESCs may survive the procedure of extract preparation, may be source of contamination that is expanded in culture and give false positive results.     

Proteomic analysis of activity-dependent synaptic plasticity in hippocampal neurons

Following long-term treatment with bicuculline and tetrodotoxin (TTX) aimed at modifying synaptic activity in cultured neurons, we used a proteomic approach to identify the associated changes in protein expression. The neurons were left untreated, or treated with bicuculline or TTX, and fractionated by means of differential detergent extraction, after which the proteins in each fraction were separated by means of two-dimensional (2D) gel electrophoresis, and 57 proteins of interest were identified by mass spectrometry. The proteins that showed altered expression and/or post-translational modifications include proteins or enzymes involved in regulating cell and protein metabolism, the cytoskeleton, or mitochondrial activity. These results suggest that extensive alterations in neuronal protein expression take place as a result of increased or decreased synaptic activity.