Combination of TRAIL with Bortezomib Shifted Apoptotic Signaling from DR4 to DR5 Death Receptor by Selective Internalization and Degradation of DR4

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) mediates apoptosis in cancer cells through death receptors DR4 and DR5 preferring often one receptor over another in the cells expressing both receptors. Receptor selective mutant variants of TRAIL and agonistic antibodies against DR4 and DR5 are highly promising anticancer agents. Here using DR5 specific mutant variant of TRAIL - DR5-B we have demonstrated for the first time that the sensitivity of cancer cells can be shifted from one TRAIL death receptor to another during co-treatment with anticancer drugs. First we have studied the contribution of DR4 and DR5 in HCT116 p53+/+ and HCT116 p53−/− cells and demonstrated that in HCT116 p53+/+ cells the both death receptors are involved in TRAIL-induced cell death while in HCT116 p53−/− cells prevailed DR4 signaling. The expression of death (DR4 and DR5) as well as decoy (DcR1 and DcR2) receptors was upregulated in the both cell lines either by TRAIL or by bortezomib. However, combined treatment of cells with two drugs induced strong time-dependent and p53-independent internalization and further lysosomal degradation of DR4 receptor. Interestingly DR5-B variant of TRAIL which do not bind with DR4 receptor also induced elimination of DR4 from cell surface in combination with bortezomib indicating the ligand-independent mechanism of the receptor internalization. Eliminatory internalization of DR4 resulted in activation of DR5 receptor thus DR4-dependent HCT116 p53−/− cells became highly sensitive to DR5-B in time-dependent manner. Internalization and degradation of DR4 receptor depended on activation of caspases as well as of lysosomal activity as it was completely inhibited by Z-VAD-FMK, E-64 and Baf-A1. In light of our findings, it is important to explore carefully which of the death receptors is active, when sensitizing drugs are combined with agonistic antibodies to the death receptors or receptor selective variants of TRAIL to enhance cancer treatment efficiency.     

Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes

The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.     

First record of the littoral family Isotogastruridae (Collembola) in Asia

The new species Isotogastrura trichaetosasp. n. isdescribed from a sand beach of Hainan, South China. It differs from all its congeners by 3+3 axial setae on Abd. IV (vs. 2+2) and by the presence of a pair of tubercles on Abd.VI. The geography of this strictly littoral genus is discussed.     

A survey of East Palaearctic Lycosidae (Araneae). 7. A new species of Acantholycosa Dahl, 1908 from the Russian Far East

Acantholycosa azarkinaesp. n. is described from the Maritime Province of Russia on the basis of both sexes. Acantholycosa norvegica (Thorell, 1872) is reported from the Maritime Province for the first time. A key and illustrations to all six species that occur in Far East Asia are provided.     

Learning probabilistic models of hydrogen bond stability from molecular dynamics simulation trajectories

Background

The estimation of individual ancestry from genetic data has become essential to applied population genetics and genetic epidemiology. Software programs for calculating ancestry estimates have become essential tools in the geneticist's analytic arsenal.

Results

Here we describe four enhancements to ADMIXTURE, a high-performance tool for estimating individual ancestries and population allele frequencies from SNP (single nucleotide polymorphism) data. First, ADMIXTURE can be used to estimate the number of underlying populations through cross-validation. Second, individuals of known ancestry can be exploited in supervised learning to yield more precise ancestry estimates. Third, by penalizing small admixture coefficients for each individual, one can encourage model parsimony, often yielding more interpretable results for small datasets or datasets with large numbers of ancestral populations. Finally, by exploiting multiple processors, large datasets can be analyzed even more rapidly.

Conclusions

The enhancements we have described make ADMIXTURE a more accurate, efficient, and versatile tool for ancestry estimation.     

Confident phosphorylation site localization using the Mascot Delta Score

Large scale phosphorylation analysis is more and more getting into focus of proteomic research. Although it is now possible to identify thousands of phosphorylated peptides in a biological system, confident site localization remains challenging. Here we validate the Mascot Delta Score (MD-score) as a simple method that achieves similar sensitivity and specificity for phosphosite localization as the published Ascore, which is mainly used in conjunction with Sequest. The MD-score was evaluated using liquid chromatography-tandem MS data of 180 individually synthesized phosphopeptides with precisely known phosphorylation sites. We tested the MD-score for a wide range of commonly available fragmentation methods and found it to be applicable throughout with high statistical significance. However, the different fragmentation techniques differ strongly in their ability to localize phosphorylation sites. At 1% false localization rate, the highest number of correctly assigned phosphopeptides was achieved by higher energy collision induced dissociation in combination with an Orbitrap mass analyzer followed very closely by low resolution ion trap spectra obtained after electron transfer dissociation. Both these methods are significantly better than low resolution spectra acquired after collision induced dissociation and multi stage activation. Score thresholds determined from simple calibration functions for each fragmentation method were stable over replicate analyses of the phosphopeptide set. The MD-score outperforms the Ascore for tyrosine phosphorylated peptides and we further show that the ability to call sites correctly increases with increasing distance of two candidate sites within a peptide sequence. The MD-score does not require complex computational steps which makes it attractive in terms of practical utility. We provide all mass spectra and the synthetic peptides to the community so that the development of present and future localization software can be benchmarked and any laboratory can determine MD-scores and localization probabilities for their individual analytical set up.     

Two-photon absorption properties of fluorescent proteins

Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.     

In vivo multispectral photoacoustic and photothermal flow cytometry with multicolor dyes: a potential for real-time assessment of circulation, dye-cell interaction, and blood volume

Recently, photoacoustic (PA) flow cytometry (PAFC) has been developed for in vivo detection of circulating tumor cells and bacteria targeted by nanoparticles. Here, we propose multispectral PAFC with multiple dyes having distinctive absorption spectra as multicolor PA contrast agents. As a first step of our proof-of-concept, we characterized high-speed PAFC capability to monitor the clearance of three dyes (Indocyanine Green [ICG], Methylene Blue [MB], and Trypan Blue [TB]) in an animal model in vivo and in real time. We observed strong dynamic PA signal fluctuations, which can be associated with interactions of dyes with circulating blood cells and plasma proteins. PAFC demonstrated enumeration of circulating red and white blood cells labeled with ICG and MB, respectively, and detection of rare dead cells uptaking TB directly in bloodstream. The possibility for accurate measurements of various dye concentrations including Crystal Violet and Brilliant Green were verified in vitro using complementary to PAFC photothermal (PT) technique and spectrophotometry under batch and flow conditions. We further analyze the potential of integrated PAFC/PT spectroscopy with multiple dyes for rapid and accurate measurements of circulating blood volume without a priori information on hemoglobin content, which is impossible with existing optical techniques. This is important in many medical conditions including surgery and trauma with extensive blood loss, rapid fluid administration, and transfusion of red blood cells. The potential for developing a robust clinical PAFC prototype that is safe for human, and its applications for studying the liver function are further highlighted.