Identification of inhibitors regulating cell proliferation and FUS-DDIT3 expression in myxoid liposarcoma using combined DNA, mRNA, and protein analyses.

FUS-DDIT3 belongs to the FET (FUS, EWSR1, and TAF15) family of fusion oncogenes, which collectively are considered to be key players in tumor development. Even though over 90% of all myxoid liposarcomas (MLS) have a FUS-DDIT3 gene fusion, there is limited understanding of the signaling pathways that regulate its expression. In order to study cell proliferation and FUS-DDIT3 regulation at mRNA and protein levels, we first developed a direct cell lysis approach that allows DNA, mRNA, and protein to be analyzed in the same sample using quantitative PCR, reverse transcription quantitative qPCR and proximity ligation assay, respectively.

Multifunctional adaptor protein Daxx interacts with chromatin-remodelling ATPase Brg1.

Multifunctional adapter and chaperone protein Daxx participates in the regulation of a number of mainly transcription-related processes. Most notably in a complex with chromatin-remodelling ATPase ATRX, Daxx serves as a histone H3.3 chaperone at telomeric regions and certain genes.

Target antigens for Hs-14 monoclonal antibody and their various expression in normozoospermic and asthenozoospermic men.

Background: Poor semen quality is one of the main causes of infertility. We have generated a set of monoclonal antibodies to human sperm and used them to investigate sperm quality. Some of these antibodies found differences in the expression of proteins between normal sperm and pathological sperm displaying severe defects.

Kinetic analysis of decreased sperm fertilizing ability by fluorides and fluoroaluminates: a tool for analyzing the effect of environmental substances on biological events.

Fluorides and fluoroaluminates decrease mouse sperm fertilizing potential by modifying the process of sperm preparation for fertilization, so-called capacitation, followed by acrosome reaction (AR). Capacitation was monitored by protein tyrosine phosphorylation (pTyr), and AR was induced consequently. The aim of this study was to apply kinetic analysis to the previously obtained dependences of pTyr and AR at capacitation times, and propose a mathematical theory for a mechanism when sperm maturation ability is amended by external stimuli.

Gene expression profiling of changes induced by maternal diabetes in the embryonic heart.

Cardiovascular defects are one of the most common congenital defects associated with maternal diabetes. Based on whole embryo gene expression microarray analysis, 11 genes were chosen for temporal expression analysis of diabetes-exposed hearts. The majority of the selected genes were deregulated in diabetes-exposed hearts compared to our controls at E13.

Exposure to endocrine disruptor induces transgenerational epigenetic deregulation of microRNAs in primordial germ cells.

In mammals, germ cell differentiation is initiated in the Primordial Germ Cells (PGCs) during fetal development. Prenatal exposure to environmental toxicants such as endocrine disruptors may alter PGC differentiation, development of the male germline and induce transgenerational epigenetic disorders. The anti-androgenic compound vinclozolin represents a paradigmatic example of molecule causing transgenerational effects on germ cells.

Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

Background: Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay.

Methylation analysis of histone H4K12ac-associated promoters in sperm of healthy donors and subfertile patients.

Background: Histone to protamine exchange and the hyperacetylation of the remaining histones are hallmarks of spermiogenesis. Acetylation of histone H4 at lysine 12 (H4K12ac) was observed prior to full decondensation of sperm chromatin after fertilization suggesting an important role for the regulation of gene expression in early embryogenesis. Similarly, DNA methylation may contribute to gene silencing of several developmentally important genes.

Local dynamics of proteins and DNA evaluated from crystallographic B factors.

The dynamics of protein and nucleic acid structures is as important as their average static picture. The local molecular dynamics concealed in diffraction images is expressed as so-called B factors. To find out how the crystal-derived B factors represent the dynamic behaviour of atoms and residues of proteins and DNA in their complexes, the distributions of scaled B factors from a carefully curated data set of over 700 protein-DNA crystal structures were analyzed [Schneider et al.

Rheological study of chitosan acetate solutions containing chitin nanofibrils.

Rheological properties of chitosan acetate solutions containing chitin nanofibrils (n-chitin) and biocompatible plasticizers intended for preparation of biodegradable films are reported in the steady, oscillatory and transient shear flow. The experiments were performed on slurries with an optimum proportion of 65/35 wt.% between chitosan and n-chitin in the films which was determined from our results of mechanical properties and absorption of water vapor.

Comparison of reverse transcription quantitative real-time PCR, flow cytometry, and immunohistochemistry for detection of monoclonality in lymphomas.

In healthy humans, 60-70% of the B lymphocytes produce kappa light chains, while the remaining cells produce lambda light chains. Malignant transformation and clonal expansion of B lymphocytes lead to an altered kappa : lambda expression ratio, which is an important diagnostic criteria of lymphomas. Here, we compared three methods for clonality determination of suspected B cell lymphomas.

Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells.

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin-23 receptor (IL-23R), which is a key element of proinflammatory IL-23-mediated signaling. A library of variants derived from the three-helix bundle scaffold of the albumin-binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high-affinity binders of recombinant extracellular IL-23R.

Partial deficiency of HIF-1α stimulates pathological cardiac changes in streptozotocin-induced diabetic mice.

Background: Diabetic cardiomyopathy is associated with a number of functional and structural pathological changes such as left ventricular dysfunction, cardiac remodeling, and apoptosis. The primary cause of diabetic cardiomyopathy is hyperglycemia, the metabolic hallmark of diabetes. Recent studies have shown that a diabetic environment suppresses hypoxia-inducible factor (HIF)-1α protein stability and function.

Bioinformatic analysis of the protein/DNA interface.

To investigate the principles driving recognition between proteins and DNA, we analyzed more than thousand crystal structures of protein/DNA complexes. We classified protein and DNA conformations by structural alphabets, protein blocks [de Brevern, Etchebest and Hazout (2000) (Bayesian probabilistic approach for predicting backbone structures in terms of protein blocks. Prots.

Direct cell lysis for single-cell gene expression profiling.

The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here, we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR.

Increasing affinity of interferon-γ receptor 1 to interferon-γ by computer-aided design.

We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ.

Expression analysis of MND1/GAJ, SPATA22, GAPDHS and ACR genes in testicular biopsies from non-obstructive azoospermia (NOA) patients.

Background: High-throughput studies provide a wide spectrum of genes for use as predictive markers during testicular sperm extraction (TESE) in combination with ICSI. In this work, we used the specimens from testicular biopsies of men with non-obstructive azoospermia who underwent TESE to investigate the expression of spermatogenesis-related genes MND1, SPATA22, GAPDHS and ACR.

Methods: Testicular biopsy specimens were subdivided into three groups: hypospermatogenesis (HS); maturation arrest (MA); and Sertoli cell-only syndrome (SCO).

Increased susceptibility of HIF-1α heterozygous-null mice to cardiovascular malformations associated with maternal diabetes.

Cardiovascular malformations are the most common manifestation of diabetic embryopathy. The molecular mechanisms underlying the teratogenic effect of maternal diabetes have not been fully elucidated. Using genome-wide expression profiling, we previously demonstrated that exposure to maternal diabetes resulted in dysregulation of the hypoxia-inducible factor 1 (HIF-1) pathway in the developing embryo.

Lipopolysaccharide treatment suppresses spontaneously developing ankylosing enthesopathy in B10.BR male mice: the potential role of interleukin-10.

Background: Ankylosing enthesopathy (ANKENT) is an animal model of human ankylosing spondylitis. ANKENT is an inflammatory disease affecting the ankle and tarsal joints of the hind limbs in susceptible mouse strains. In the disease, the participation of intestinal microbiota components was suggested.

Glutamate carboxypeptidase II: an overview of structural studies and their importance for structure-based drug design and deciphering the reaction mechanism of the enzyme.

Recent years witnessed rapid expansion of our knowledge about structural features of human glutamate carboxypeptidase II (GCPII). There are over thirty X-ray structures of human GCPII (and of its close ortholog GCPIII) publicly available at present. They include structures of ligand-free wild-type enzymes, complexes of wild-type GCPII/GCPIII with structurally diversified inhibitors as well as complexes of the GCPII(E424A) inactive mutant with several substrates.

Assessing oestrogenic effects of brominated flame retardants hexabromocyclododecane and tetrabromobisphenol A on MCF-7 cells.

Tetrabromobisphenol A (TBBPA) is the main flame retardant used in printed circuit boards and laminates. The human population is highly exposed to TBBPA as it is used in consumer electronics as well as office and communication equipment. The main use of hexabromocyclododecane (HBCD) is in insulation foam boards, which are widely used in the construction sector.

Gene expression profiling of sex differences in HIF1-dependent adaptive cardiac responses to chronic hypoxia.

Although physiological responses to chronic hypoxia, including pulmonary hypertension and right ventricular hypertrophy, have been well described, the molecular mechanisms involved in cardiopulmonary adaptations are still not fully understood. We hypothesize that adaptive responses to chronic hypoxia are the result of altered transcriptional regulations in the right and left ventricles. Here we report results from the gene expression profiling of adaptive responses in a chronically hypoxic heart.

The optimization of the protocol for immunofluorescence on fish spermatozoa.

In comparison with mammals, the fertilization of fish occurs predominantly outside the organism in a water environment, where fish spermatozoa require specific conditions to interact with oocytes. It is evident that optimal conditions for fish and mammalian spermatozoa are quite different. This paper describes a special approach to handling fish (common carp and Siberian sturgeon) spermatozoa in comparison with the samples originating from mammals (boar).