Middle‐Down MS is Ready to Answer Complex Questions in Chromatin Biology

Histones are the most abundant protein family in the cells of complex organisms such as mammals and, together with DNA, they define the backbone of chromatin. Histone PTMs are key players of chromatin biology, as they function as anchors for proteins that bind and modulate chromatin readout, including gene expression. Middle‐down mass spectrometry (MS) has been optimized for about 10 years to study histone N‐terminal tails, but it has been rarely applied to identify the role of coexisting histone marks in biology. In this work, Jiang et al. used middle‐down MS to study the dynamics of coexisting PTMs on histone H4 in two breast cancer cell lines. 1 They found that overall serine 1 phosphorylation (S1ph) is mildly regulated during the cell cycle, but S1ph coexistence frequency with acetylations and methylations on the lysine residues of the N‐terminal tail is remarkably tuned during S phase and G2/M phase. Together, the team placed another benchmark proving that MS analysis of combinatorial histone PTMs provides a more comprehensive view on chromatin state than studying individual marks. We should then constantly question ourselves regarding the limitations of analyzing single PTMs when we attempt to define their effect on protein functions.     

Fertilization,soil and plant community characteristics determine soil microbial activity in managed temperate grasslands

Background and aims

Contaminated soils can impede germination and growth of selected plant species, restricting effective phytoremediation strategies. The purpose of the present study was to enhance the germination and growth of saltgrass [Distichlis spicata (L.) Greene] by evaluating the efficacy of certain seed pretreatments and soil amendments.

Methods

Ten seed pretreatment methods, two amendments, three soil depths and five saline levels were tested under greenhouse conditions.

Results

Saltgrass germination and growth were negatively correlated with increasing salinity levels when NaCl > 85.6 mM. Among ten seed pretreatments (stratification + Proxy 24 h, hot water + Proxy 24 h, stratification, hot water + Proxy 48 h, Proxy 48 h, Proxy 24 h, hot water, scarification, gibberellins, and KMnO₄), the two best methods were stratification + Proxy 24 h and hot water + Proxy 24 h for enhancing saltgrass germination, with the latter pretreatment being especially useful because of its shorter preparation time and high germination rates. Proxy is a commercial ethephon product. Potting soil (5.0 cm depth) was found to be the best amendment for saltgrass germination and growth in hydrocarbon-contaminated soils.

Conclusion

We conclude that direct seeding of saline soils contaminated with petroleum hydrocarbons is a feasible phytoremediation strategy provided that appropriate seed pretreatments and amendments are utilized.

     

p38 MAPK regulates phosphorylation of Bad via PP2A-dependent suppression of the MEK1/2-ERK1/2 survival pathway in TNF-alpha induced endothelial apoptosis

We recently reported that p38 MAPK regulates TNF-induced endothelial apoptosis via phosphorylation and downregulation of Bcl-xL. Here, we describe that such apoptosis includes p38 MAPK-mediated, protein phosphatase 2A (PP2A)-dependent, downregulation of the MEK-ERK pathway. Inhibition of PP2A with fostriecin or calyculin A significantly increased MEK phosphorylation, as did exposure to the p38 MAPK inhibitor SB203580. Inhibition of MEK potentiated TNF-induced caspase-3 activity and cell death, and both those events were suppressed by treatment with fostriecin or calyculin A. Immunoprecipitation experiments revealed an association between p38 MAPK, PP2A and MEK, and the results of a phosphatase assay suggested that PP2A is a downstream target of p38 MAPK. Importantly, phosphorylation of Bad at Ser-112 was found to be regulated by p38 MAPK and PP2A. In summary, the present findings indicate a novel p38 MAPK-mediated apoptosis pathway, involving activation of Bad via PP2A-dependent inhibition of the MEK-ERK pathway.     

Yeast Rev1 is cell cycle regulated, phosphorylated in response to DNA damage and its binding to chromosomes is dependent upon MEC1

Translesion DNA synthesis (TLS) is one of the mechanisms involved in lesion bypass during DNA replication. Three TLS polymerases (Pol) are present in the yeast Saccharomyces cerevisiae: Pol zeta, Pol eta and the product of the REV1 gene. Rev1 is considered a deoxycytidyl transferase because it almost exclusively inserts a C residue in front of the lesion. Even though REV1 is required for most of the UV-induced and spontaneous mutagenesis events, the role of Rev1 is poorly understood since its polymerase activity is often dispensable. Rev1 interacts with several TLS polymerases in mammalian cells and may act as a platform in the switching mechanism required to substitute a replicative polymerase with a TLS polymerase at the sites of DNA lesions. Here we show that yeast Rev1 is a phosphoprotein, and the level of this modification is cell cycle regulated under normal growing conditions. Rev1 is unphosphorylated in G1, starts to be modified while cells are passing S phase and it becomes hyper-phosphorylated in mitosis. Rev1 is also hyper-phosphorylated in response to a variety of DNA damaging agents, including treatment with a radiomimetic drug mostly causing double-strand breaks (DSB). By using the chromosome spreading technique we found the Rev1 is bound to chromosomes throughout the cell cycle, and its binding does not significantly increase in response to genotoxic stress. Therefore, Rev1 phosphorylation does not appear to modulate its binding to chromosomes, suggesting that such modification may influence other aspects of the TLS process. Rev1 binding under damaged and undamaged conditions, is at least partially dependent on MEC1, a gene playing a pivotal role in the DNA damage checkpoint cascade. This genetic dependency may suggest a role for MEC1 in spontaneous mutagenesis events, which require a functional REV1 gene.     

Analysis of thermophilic fungal populations during phase II of composting for the cultivation of Agaricus subrufescens

The composition and genetic diversity of fungal populations during phase II of compost production for the cultivation of Agaricus subrufescens was determined using culture-dependent and -independent methods on days 3, 6, 10, 12, and 14 of phase II composting. The isolates were morphologically characterized and subsequently analyzed using repetitive extragenic palindromic sequences (rep-PCR), and the intergenic region was sequenced to genetically identify the isolates. Changes on in the filamentous fungi population were analyzed using denaturing gradient gel electrophoresis (DGGE), and the resulting bands were sequenced. The population did not significantly change from day 3 to 10 (2.55 x 10⁵ –6 x 10⁵ CFU g^?1), and maximum counts on day 14 of phase II composting (6.92 log CFU g^?1). In the morphological characterization, Scytalidium thermophilum, Thermomyces lanuginosus, and Thermomyces ibadanensis were the most abundant identified species. The 26 most abundant isolates identified by morphological analysis were characterized using rep-PCR. A significant amount of genetic diversity was detected among the isolates of all three studied species. Based on the DGGE analysis, the diversity of the fungi was reduced during phase II composting, and S. thermophilum was the predominant species identified throughout the entire process. Thus, this study presents the first report of the involvement of T. ibadanensis in the production of compost for Agaricus mushroom cultivation.     

Micromere-specific cell surface proteins of 16-cell stage sea urchin embryos

Evidence is presented of cell-type specificity of surface proteins from the 16-cell stage sea urchin embryo. The protein composition of the micromere cell surface has been examined by 125I labelling of intact cells followed by SDS-PAGE. In Arbacia punctulata, four high molecular weight (HMW) proteins are detected on the surface of isolated micromeres--but not on mesomere-macromere fractions. In Strongylocentrotus droebachiensis, a micromere-specific protein of 133 K molecular weight (MW) was identified. This 133 K protein binds to wheat germ agglutinin (WGA) but not to concanavalin A (conA). Lectin binding was studied using a new technique. The procedure involves the separation, by SDS-PAGE, of iodinated cell-surface proteins followed by their electrophoretic transfer to lectin-coated nitrocellulose membranes. Using this procedure, cell-type-specific surface proteins which are also lectin-binding-specific, were detected.     

Biomimesis and biomorphic transformations: New concepts applied to bone regeneration

In the last decades the activity of material scientists was more and more directed to the development of biomimetic scaffolds, able to drive and address cell activity towards proper differentiation and the repair of diseased human tissues. In case of bone, this requires the synthesis of three-dimensional constructs able to exchange chemical signals promoting osteogenesis and to progressively be resorbed during the formation and remodelling of new bone. Besides, particularly for the regeneration of extensive portions of bone, a morphological and mechanical biomimesis is also required, to allow cell colonization and formation of a proper vascularization tree. The healing of load-bearing bones also requires scaffolds with a hierarchically organized morphology, to provide improved biomechanical behaviour and allow a proper mechano-transduction of the mechanical stimuli down to the cell level. The present paper is an overview of the current technologies and devices developed in the last decade for the regeneration of bone tissue. In particular, novel biomimetic and biomorphic scaffolds, obtained by the controlled transformation of native ligneous structures, promise to adequately face the problem of obtaining complex hierarchical structures, not achievable otherwise by any currently existing manufacturing techniques.     

Anti-Trichomonas vaginalis activity of Hypericum polyanthemum extract obtained by supercritical fluid extraction and isolated compounds

The anti-Trichomonas vaginalis activity of Hypericum polyanthemum extract obtained by supercritical fluid extraction (50 °C, 150 bar) and the chemical compounds isolated and purified from this extract (benzopyrans HP1, HP2, HP3, and phloroglucinol derivative uliginosin B) were assessed. All samples had anti-T. vaginalis activity; however, HP1 demonstrated the best selectivity against this protozoan (metronidazole-resistant and susceptible isolates), with no cytotoxicity on mammalian cells (selectivity index of 73.97). Moreover, HP1 had activity against a metronidazole-resistant isolate (52% of viable trophozoites), and this effect was higher when tested with a low concentration of metronidazole (23% of viable trophozoites). Experiments demonstrated that all isolated compounds caused damage to the parasites' membrane (> 90% of LDH release) and do not present a notable hemolytic effect, although HP2 and uliginosin B exhibited cytotoxicity against mammalian cells. Therefore, the analyzed molecules are promising prototypes for new antiprotozoal drugs, especially HP1, which seems to improve metronidazole's effect on a resistant T. vaginalis isolate.