BUB1B monoallelic germline variants contribute to prostate cancer predisposition by triggering chromosomal instability.

BACKGROUND: Prostate cancer (PrCa) is the most frequently diagnosed cancer in men. Variants in known moderate- to high-penetrance genes explain less than 5% of the cases arising at early-onset (< 56 years) and/or with familial aggregation of the disease. Considering that BubR1 is an essential component of the mitotic spindle assembly checkpoint, we hypothesized that monoallelic BUB1B variants could be sufficient to fuel chromosomal instability (CIN), potentially triggering (prostate) carcinogenesis. METHODS: To unveil BUB1B as a new PrCa predisposing gene, we performed targeted next-generation sequencing in germline DNA from 462 early-onset/familial PrCa patients and 1,416 cancer patients fulfilling criteria for genetic testing for other hereditary cancer syndromes. To explore the pan-cancer role of BUB1B, we used in silico BubR1 molecular modeling, in vitro gene-editing, and ex vivo patients' tumors and peripheral blood lymphocytes. RESULTS: Rare BUB1B variants were found in ~ 1.9% of the early-onset/familial PrCa cases and in ~ 0.6% of other cancer patients fulfilling criteria for hereditary disease. We further show that BUB1B variants lead to decreased BubR1 expression and/or stability, which promotes increased premature chromatid separation and, consequently, triggers CIN, driving resistance to Taxol-based therapies. CONCLUSIONS: Our study shows that different BUB1B variants may uncover a trigger for CIN-driven carcinogenesis, supporting the role of BUB1B as a (pan)-cancer predisposing gene with potential impact on genetic counseling and treatment decision-making.

Prometaphase.

The establishment of a metaphase plate in which all chromosomes are attached to mitotic spindle microtubules and aligned at the cell equator is required for faithful chromosome segregation in metazoans. The achievement of this configuration relies on the precise coordination between several concurrent mechanisms that start upon nuclear envelope breakdown, mediate chromosome capture at their kinetochores during mitotic spindle assembly and culminate with the congression of all chromosomes to the spindle equator. This period is called 'prometaphase'. Because the nature of chromosome capture by mitotic spindle microtubules is error prone, the cell is provided of error correction mechanisms that sense and correct most erroneous kinetochore-microtubule attachments before committing to separate sister chromatids in anaphase. In this review, aimed for newcomers in the field, more than providing an exhaustive mechanistic coverage of each and every concurrent mechanism taking place during prometaphase, we provide an integrative overview of these processes that ultimately promote the subsequent faithful segregation of chromosomes during mitosis.

SOGA1 and SOGA2/MTCL1 are CLASP-interacting proteins required for faithful chromosome segregation in human cells.

CLASPs are key modulators of microtubule dynamics throughout the cell cycle. During mitosis, CLASPs independently associate with growing microtubule plus-ends and kinetochores and play essential roles in chromosome segregation. In a proteomic survey for human CLASP1-interacting proteins during mitosis, we have previously identified SOGA1 and SOGA2/MTCL1, whose mitotic roles remained uncharacterized. Here we performed an initial functional characterization of human SOGA1 and SOGA2/MTCL1 during mitosis. Using specific polyclonal antibodies raised against SOGA proteins, we confirmed their expression and reciprocal interaction with CLASP1 and CLASP2 during mitosis. In addition, we found that both SOGA1 and SOGA2/MTCL1 are phospho-regulated during mitosis by CDK1. Immunofluorescence analysis revealed that SOGA2/MTCL1 co-localizes with mitotic spindle microtubules and spindle poles throughout mitosis and both SOGA proteins are enriched at the midbody during mitotic exit/cytokinesis. GFP-tagging of SOGA2/MTCL1 further revealed a microtubule-independent localization at kinetochores. Live-cell imaging after siRNA-mediated knockdown of SOGA1 and SOGA2/MTCL1 showed that they are independently required for distinct aspects of chromosome segregation. Thus, SOGA1 and SOGA2/MTCL1 are bona fide CLASP-interacting proteins during mitosis required for faithful chromosome segregation in human cells.

Ecstasy metabolites and monoamine neurotransmitters upshift the Na+/K+ ATPase activity in mouse brain synaptosomes.

3,4-Methylenedioximethamphetamine (MDMA; "ecstasy") is a psychotropic drug with well-known neurotoxic effects mediated by hitherto not fully understood mechanisms. The Na+- and K+-activated adenosine 5'-triphosphatase (Na+/K+ ATPase), by maintaining the ion gradient across the cell membrane, regulates neuronal excitability. Thus, a perturbation of its function strongly impacts cell homeostasis, ultimately leading to neuronal dysfunction and death. Nevertheless, whether MDMA affects the Na+/K+ ATPase remains unknown. In this study, we used synaptosomes obtained from whole mouse brain to test the effects of MDMA, three of its major metabolites [α-methyldopamine, N-methyl-α-methyldopamine and 5-(glutathion-S-yl)-α-methyldopamine], serotonin (5-HT), dopamine, 3,4-dihydroxy-L-phenylalanine (L-Dopa) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the Na+/K+ ATPase function. A concentration-dependent increase of Na+/K+ ATPase activity was observed in synaptosomes exposed to the tested compounds (concentrations ranging from 0.0625 to 200 µM). These effects were independent of protein kinases A and C activities. Nevertheless, a rescue of the compounds' effects was observed in synaptosomes pre-incubated with the antioxidant N-acetylcysteine (1 mM), suggesting a role for reactive species-regulated pathways on the Na+/K+ ATPase effects. In agreement with this hypothesis, a similar increase in the pump activity was found in synaptosomes exposed to the chemical generator of superoxide radicals, phenazine methosulfate (1-250 µM). This study demonstrates the ability of MDMA metabolites, monoamine neurotransmitters, L-Dopa and DOPAC to alter the Na+/K+ ATPase function. This could represent a yet unknown mechanism of action of MDMA and its metabolites in the brain.

Mechanism of Phase Separation in Aqueous Two-Phase Systems.

Liquid-liquid phase separation underlies the formation of membrane-less organelles inside living cells. The mechanism of this process can be examined using simple aqueous mixtures of two or more solutes, which are able to phase separate at specific concentration thresholds. This work presents the first experimental evidence that mesoscopic changes precede visually detected macroscopic phase separation in aqueous mixtures of two polymers and a single polymer and salt. Dynamic light scattering (DLS) analysis indicates the formation of mesoscopic polymer agglomerates in these systems. These agglomerates increase in size with increasing polymer concentrations prior to visual phase separation. Such mesoscopic changes are paralleled by changes in water structure as evidenced by Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopic analysis of OH-stretch bands. Through OH-stretch band analysis, we obtain quantitative estimates of the relative fractions of four subpopulations of water structures coexisting in aqueous solutions. These estimates indicate that abrupt changes in hydrogen bond arrangement take place at concentrations below the threshold of macroscopic phase separation. We used these experimental observations to develop a model of phase separation in aqueous media.

Arrangement of Hydrogen Bonds in Aqueous Solutions of Different Globular Proteins.

This work presents the first evidence that dissolved globular proteins change the arrangement of hydrogen bonds in water, with different proteins showing quantitatively different effects. Using ATR-FTIR (attenuated total reflection-Fourier transform infrared) spectroscopic analysis of OH-stretch bands, we obtain quantitative estimates of the relative amounts of the previously reported four subpopulations of water structures coexisting in a variety of aqueous solutions. Where solvatochromic dyes can measure the properties of solutions of non-ionic polymers, the results correlate well with ATR-FTIR measurements. In protein solutions to which solvatochromic dye probes cannot be applied, NMR (nuclear magnetic resonance) spectroscopy was used for the first time to estimate the hydrogen bond donor acidity of water. We found strong correlations between the solvent acidity and arrangement of hydrogen bonds in aqueous solutions for several globular proteins. Even quite similar proteins are found to change water properties in dramatically different ways.

DCMC as a Promising Alternative to Bentonite in White Wine Stabilization. Impact on Protein Stability and Wine Aromatic Fraction.

Protein haze in white wine is one of the most common non-microbial defects of commercial wines, with bentonite being the main solution utilized by the winemaking industry to tackle this problem. Bentonite presents some serious disadvantages, and several alternatives have been proposed. Here, an alternative based on a new cellulose derivative (dicarboxymethyl cellulose, DCMC) is proposed. To determine the efficiency of DCMC as a bentonite alternative, three monovarietal wines were characterized, and their protein instability and content determined by a heat stability test (HST) and the Bradford method, respectively. The wines were treated with DCMC to achieve stable wines, as shown by the HST, and the efficacy of the treatments was assessed by determining, before and after treatment, the wine content in protein, phenolic compounds, sodium, calcium, and volatile organic compounds (VOCs) as well as the wine pH. DCMC applied at dosages such as those commonly employed for bentonite was able to reduce the protein content in all tested wines and to stabilize all but the Moscatel de Setúbal varietal wine. In general, DCMC was shown to induce lower changes in the wine pH and phenolic content than bentonite, reducing the wine calcium content. Regarding which VOCs are concerned, DCMC produced a general impact similar to that of bentonite, with differences depending on wine variety. The results obtained suggest that DCMC can be a sustainable alternative to bentonite in protein white wine stabilization.

Interfacial tension and mechanism of liquid-liquid phase separation in aqueous media.

The organization of multiple subcellular compartments is controlled by liquid-liquid phase separation. Phase separation of this type occurs with the emergence of interfacial tension. Aqueous two-phase systems formed by two non-ionic polymers can be used to separate and analyze biological macromolecules, cells and viruses. Phase separation in these systems may serve as the simple model of phase separation in cells also occurring in aqueous media. To better understand liquid-liquid phase separation mechanisms, interfacial tension was measured in aqueous two-phase systems formed by dextran and polyethylene glycol and by polyethylene glycol and sodium sulfate in the presence of different additives. Interfacial tension values depend on differences between the solvent properties of the coexisting phases, estimated experimentally by parameters representing dipole-dipole, ion-dipole, ion-ion, and hydrogen bonding interactions. Based on both current and literature data, we propose a mechanism for phase separation in aqueous two-phase systems. This mechanism is based on the fundamental role of intermolecular forces. Although it remains to be confirmed, it is possible that these may underlie all liquid-liquid phase separation processes in biology.

Synthesis of new hetero-arylidene-9(10H)-anthrone derivatives and their biological evaluation.

New hetero-arylidene-9(10H)-anthrone derivatives (1) were synthesized from reaction of 1,2-dimethyl-3-alkyl imidazolium salts (2) and 9-anthracenecarboxaldehyde. Ion exchange of the anion with dioctyl sulfosuccinate and lithium bis(trifluoromethanesulfonyl)imide led to the preparation of other derivatives. The antiproliferative effect of the compounds was evaluated in human ovarian (A2780) and colorectal (HCT116) carcinoma cell lines and in normal primary human fibroblasts. Compound 1 presented an antiproliferative effect related to the imidazolium pattern of substitution with compounds having a decyl group at the R-position (1c and 3c) showing the highest cytotoxic activities in all cell lines independently of the counter ion. Compounds 1b and 1c internalize A2780 cancer cells via a passive or an active transport, respectively, inducing A2780 cell death via an extrinsic apoptosis (1b) or intrinsic apoptosis and oncosis (1c). The localization of both compounds in the cytoplasm coupled to the absence of reactive oxygen species (ROS) induction suggest that the mechanisms of toxicity might be different than those of other anthracyclines currently used in chemotherapy.

Invasive Plants: Turning Enemies into Value.

In this review, a brief description of the invasive phenomena associated with plants and its consequences to the ecosystem is presented. Five worldwide invasive plants that are a threat to Portugal were selected as an example, and a brief description of each is presented. A full description of their secondary metabolites and biological activity is given, and a resume of the biological activity of extracts is also included. The chemical and pharmaceutical potential of invasive species sensu lato is thus acknowledged. With this paper, we hope to demonstrate that invasive species have potential positive attributes even though at the same time they might need to be controlled or eradicated. Positive attributes include chemical and pharmaceutical properties and developing these could help mitigate the costs of management and eradication.

Synthetic Approaches to A Challenging and Unusual Structure-An Amino-Pyrrolidine Guanine Core.

The synthesis of an unreported 2-aminopyrrolidine-1-carboxamidine unit is here described for the first time. This unusual and promising structure was attained through the oxidative decarboxylation of amino acids using the pair of reagents, silver(I)/peroxydisulfate (Ag(I)/S2O82-) followed by intermolecular (in the case of l-proline derivative) and intramolecular trapping (in the case of acyl l-arginine) by N-nucleophiles. The l-proline approach has a broader scope for the synthesis of 2-aminopyrrolidine-1-carboxamidine derivatives, whereas the intramolecular cyclization afforded by the l-acylarginines, when applied, results in higher yields. The former allowed the first synthesis of cernumidine, a natural alkaloid isolated in 2011 from Solanum cernuum Vell, as its racemic form.

Coherent-hybrid STED: high contrast sub-diffraction imaging using a bi-vortex depletion beam.

Stimulated emission depletion (STED) fluorescence microscopy squeezes an excited spot well below the wavelength scale using a doughnut-shaped depletion beam. To generate a doughnut, a scale-free vortex phase modulation (2D-STED) is often used because it provides maximal transverse confinement and radial-aberration immunity (RAI) to the central dip. However, RAI also means blindness to a defocus term, making the axial origin of fluorescence photons uncertain within the wavelength scale provided by the confocal detection pinhole. Here, to reduce the uncertainty, we perturb the 2D-STED phase mask so as to change the sign of the axial concavity near focus, creating a dilated dip. By providing laser depletion power, the dip can be compressed back in three dimensions to retrieve lateral resolution, now at a significantly higher contrast. We test this coherent-hybrid STED (CH-STED) mode in x-y imaging of complex biological structures, such as the dividing cell. The proposed strategy creates an orthogonal direction in the STED parametric space that uniquely allows independent tuning of resolution and contrast using a single depletion beam in a conventional (circular polarization-based) STED setup.

N-Heterocyclic Olefin Catalysis for the Ring Opening of Cyclic Amidine Compounds: A Pathway to the Synthesis of ε-Caprolactam- and γ-Lactam-Derived Amines.

For the first time, 1,2-dimethyl-3-ethylimidazolium iodide (1a) catalyzes the ring opening of the bicyclic amidine system of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) or DBN (1,5-diazabicyclo[4.3.0]non-5-ene) on reaction with aldehydes. The mechanism here proposed involves an N-heterocyclic olefin (NHO) catalytic species that acts as a nucleophile to promote the cyclic amidine ring opening. The resulting ε-caprolactam- and γ-lactam-derived imines were obtained in moderate to excellent yields (28-99%) and reduced to the corresponding amines by sodium borohydride. Confirmation of the imine product was achieved via single-crystal X-ray diffraction studies.

Effect of an Intrinsically Disordered Plant Stress Protein on the Properties of Water.

Dehydrins are plant proteins that are able to protect plants from various forms of dehydrative stress such as drought, cold, and high salinity. Dehydrins can prevent enzymes from losing activity after freeze/thaw treatments. Previous studies had suggested that the dehydrins function by a molecular shield effect, essentially preventing a denatured enzyme from aggregating with another enzyme. Therefore, the larger the dehydrin, the larger the shield and theoretically the more effective the protection. Although this relationship holds for smaller dehydrins, it fails to explain why larger dehydrins are less efficient than would be predicted from their size. Using solvatochromic dyes to probe the solvent features of water, we first confirm that the dehydrins do not bind the dyes, which would interfere with interpretation of the data. We then show that the dehydrins have an effect on three solvent properties of water (dipolarity/polarizability, hydrogen-bond donor acidity and hydrogen-bond acceptor basicity), which can contribute to the protective mechanism of these proteins. Interpretation of these data suggests that although polyethylene glycol and dehydrins have similar protective effects, dehydrins may more efficiently modify the hydrogen-bonding ability of bulk water to prevent enzyme denaturation. This possibly explains why dehydrins recover slightly more enzyme activity than polyethylene glycol.

Secondary Metabolites and Biological Activity of Invasive Macroalgae of Southern Europe.

In this review a brief description of the invasive phenomena associated with algae and its consequences on the ecosystem are presented. Three examples of invasive algae of Southern Europe, belonging to Rodophyta, Chlorophyta, and Phaeophyta, were selected, and a brief description of each genus is presented. A full description of their secondary metabolites and biological activity is given and a summary of the biological activity of extracts is also included. In Asparagopsis we encounter mainly halogenated compounds. From Caulerpa, several terpenoids and alkaloids were isolated, while in Sargassum, meroterpenoids prevail.

Developments in the Reactivity of 2-Methylimidazolium Salts.

Unexpected and unusual reactivity of 2-methylimidazolium salts toward aryl-N-sulfonylimines and aryl aldehydes is here reported. Upon reaction with aryl-N-sulfonylimines, the addition product, arylethyl-2-imidazolium-1-tosylamide (3), is formed with moderate to good yields, while upon reaction with aldehydes, the initial addition product (6) observed in NMR and HPLC-MS experimental analysis is postulated by us as an intermediate to the final conversion to carboxylic acids. Studies in the presence and absence of molecular oxygen allow us to conclude that the imidazolium salts is crucial for the oxidation. A detailed mechanistic study was carried out to provide insights regarding this unexpected reactivity.

The Role of Spongia sp. in the Discovery of Marine Lead Compounds.

A comprehensive review on the chemistry of Spongia sp. is here presented, together with the biological activity of the isolated compounds. The compounds are grouped in sesquiterpene quinones, diterpenes, C21 and other linear furanoterpenes, sesterterpenes, sterols (including secosterols), macrolides and miscellaneous compounds. Among other reports we include studies on the intraspecific diversity of a Mediterranean species, compounds isolated from associated sponge and nudibranch and compounds isolated from S. zimocca and the red seaweed Laurentia microcladia. Under biological activity a table of the reported biological activities of the various compounds and the biological screening of extracts are described. The present review covers the literature from 1971 to 2015.

Structural features important for differences in protein partitioning in aqueous dextran-polyethylene glycol two-phase systems of different ionic compositions.

Partitioning of 15 proteins in dextran-70-polyethylene glycol (PEG)-8000 aqueous two-phase systems (ATPSs) in the presence of 0.01M sodium phosphate buffer, pH7.4 was studied. The effect of salt additives (NaCl, CsCl, Na2SO4, NaClO4 and NaSCN) at different concentrations on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the protein partition coefficients in ATPSs with and without salt additives. The results obtained show that the presence and concentration of salt additives affect the protein partition behavior. Analysis of ATPSs in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning could not be explained by the protein size. The structural signatures for the proteins were constructed from partition coefficient values in four ATPSs with different salt additives, and the structural distances were calculated using cytochrome c as the reference structure. The structural distances for all the examined proteins (except lysozyme) were found to be interrelated. Analysis of about 50 different descriptors of the protein structures revealed that the partition behavior of proteins is determined by the peculiarities of their surfaces (e.g., the number of water-filled cavities and the averaged hydrophobicity of the surface residues) and by the intrinsic flexibility of the protein structure measured in terms of the B-factor (or temperature factor).

Effects of osmolytes on protein-solvent interactions in crowded environment: Analyzing the effect of TMAO on proteins in crowded solutions.

We analyzed the effect of a natural osmolyte, trimethylamine N-oxide (TMAO), on structural properties and conformational stabilities of several proteins under macromolecular crowding conditions by a set of biophysical techniques. We also used the solvent interaction analysis method to look at the peculiarities of the TMAO-protein interactions under crowded conditions. To this end, we analyzed the partitioning of these proteins in TMAO-free and TMAO-containing aqueous two-phase systems (ATPSs). These ATPSs had the same polymer composition of 6.0 wt.% PEG-8000 and 12.0 wt.% dextran-75, and same ionic composition of 0.01 M K/NaPB, pH 7.4. These analyses revealed that there is no direct interaction of TMAO with proteins, suggesting that the TMAO effects on the protein structure in crowded solutions occur via the effects of this osmolyte on solvent properties of aqueous media. The effects of TMAO on protein structure in the presence of polymers were rather complex and protein-specific. Curiously, our study revealed that in highly concentrated polymer solutions, TMAO does not always act to promote further protein folding.