Dual Targeted Therapy with p53 siRNA and Epigallocatechingallate in a Triple Negative Breast Cancer Cell Model

Triple-negative breast cancer (TNBC) is a highly aggressive phenotype that is resistant to standard therapy. Thus, the development of alternative therapeutic strategies for TNBC is essential. The purpose of our in vitro study was to evaluate the impact of p53 gene silencing in conjunction with the administration of a natural compound, epigallocatechingallate (EGCG). RT²Profiler PCR Array technology was used to evaluate the impact of dual treatment on the main genes involved in apoptosis in the Hs578T cell culture model of TNBC. Gene expression analysis revealed 28 genes were significantly altered (16 upregulated and 12 downregulated) in response to combined p53 siRNA and EGCG treatment. Further analysis revealed that p53 siRNA and EGCG dual therapy leads to the activation of pro-apoptotic genes and the inhibition of pro-survival genes, autophagy, and cell network formation. These results indicate that this dual therapy targets both the apoptotic and angiogenic pathways, which may improve treatment effectiveness for tumors resistant to conventional treatment.     

Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics

The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL–8 release is detectable within the first 2h and peaks at 4–6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms.     

Synthesis and characterization of platinum(IV) complexes with N,S and S,S heterocyclic ligands

The reactions of [PtMe₃(OAc)(bpy)] (4) with the N,S and S,S containing heterocycles, pyrimidine-2-thione (pymtH), pyridine-2-thione (pytH), thiazoline-2-thione (tztH) and thiophene-2-thiol (tptH), resulted in the formation of the monomeric complexes [PtMe₃(-κS)(bpy)] ( = pymt, 5; pyt, 6; tzt, 7; tpt, 8), where the heterocyclic ligand is coordinated via the exocyclic sulfur atom. In contrast, in the reactions of [PtMe₃(OAc)(Me₂CO)_x] (3, x = 1 or 2) with pymtH, pytH, tztH and tptH dimeric complexes [{PtMe₃(μ-)}₂] (μ- = pymt, 9; pyt, 10; tzt, 11) and the tetrameric complex [{PtMe₃(μ₃-tpt-κS)}₄] (12), respectively, were formed. The complexes were characterized by microanalyses, ¹H and ¹³C NMR spectroscopy and negative ESI-MS (12) measurements. Single-crystal X-ray diffraction analysis of [PtMe₃(pymt-κS)(bpy)] (5) exhibited a conformation where the pymt ligand lies nearly perpendicular to the complex plane above the bpy ligand that was also confirmed by quantum chemical calculations on the DFT level of theory.     

Effect of age on sexually dimorphic selenoprotein expression in mice

Clinical data suggest that selenium (Se) supplementation decreases disease predisposition and severity and accelerates recovery in a variety of pathologies. Pre-supplementation Se levels and sex represent important determinants of these Se-dependent health effects. Accordingly, we previously reported on sexually dimorphic expression patterns of Se-dependent glutathione peroxidase 1, type I deiodinase, and selenoprotein P in young mice. In the present study we investigated whether these differences vary with age. The strong sexual dimorphic expression of hepatic type I deiodinase that was observed in young mice vanished both at the mRNA and enzyme activity level by 1 year of age. In contrast, the strong sex-specific differences in renal type I deiodinase mRNA expression were sustained with age. Accordingly, deiodinase enzymatic activities differed in male and female kidneys, largely independent of age [average of 6.8 vs. 15.7 pmol/(min mg) in males vs. females]. In parallel, hepatic Se concentrations and glutathione peroxidase activities increased in female mice compared to male littermates, establishing a new sexual dimorphism in liver. Thus, age represents another important modifier of the dynamic sex- and tissue-specific selenoprotein expression patterns. These data highlight again the unique physiological regulatory mechanisms that have evolved to control Se metabolism according to the actual needs of the organism.     

Selenoproteins of the thyroid gland: expression, localization and possible function of glutathione peroxidase 3

The thyroid gland has an exceptionally high selenium content, even during selenium deficiency. At least 11 selenoproteins are expressed, which may be involved in the protection of the gland against the high amounts of H2O2 produced during thyroid hormone biosynthesis. As determined here by in situ hybridization and Northern blotting experiments, glutathione peroxidases (GPx) 1 and 4 and selenoprotein P were moderately expressed, occurring selectively in the follicular cells and in leukocytes of germinal follicles of thyroids affected by Hashimoto's thyroiditis. Selenoprotein 15 was only marginally expressed and distributed over all cell types. GPx3 mRNA was exclusively localized to the thyrocytes, showed the highest expression levels and was down-regulated in 5 of 6 thyroid cancer samples as compared to matched normal controls. GPx3 could be extracted from thyroidal colloid by incubation with 0.5% sodium dodecyl sulfate indicating that this enzyme is (i) secreted into the follicular lumen and (ii) loosely attached to the colloidal thyroglobulin. These findings are consistent with a role of selenoproteins in the protection of the thyroid from possible damage by H2O2. Particularly, GPx3 might use excess H2O2 and catalyze the polymerization of thyroglobulin to the highly cross-linked storage form present in the colloid.     

Comparative analysis of peanut NBS-LRR gene clusters suggests evolutionary innovation among duplicated domains and erosion of gene microsynteny

? Plant genomes contain numerous disease resistance genes (R genes) that play roles in defense against pathogens. Scarcity of genetic polymorphism makes peanut (Arachis hypogaea) especially vulnerable to a wide variety of pathogens. ? Here, we isolated and characterized peanut bacterial artificial chromosomes (BACs) containing a high density of R genes. Analysis of two genomic regions identified several TIR-NBS-LRR (Toll-interleukin-1 receptor, nucleotide-binding site, leucine-rich repeat) resistance gene analogs or gene fragments. We reconstructed their evolutionary history characterized by tandem duplications, possibly facilitated by transposon activities. We found evidence of both intergenic and intragenic gene conversions and unequal crossing-over, which may be driving forces underlying the functional evolution of resistance. ? Analysis of the sequence mutations, protein secondary structure and three-dimensional structures, all suggest that LRR domains are the primary contributor to the evolution of resistance genes. The central part of LRR regions, assumed to serve as the active core, may play a key role in the resistance function by having higher rates of duplication and DNA conversion than neighboring regions. The assumed active core is characterized by significantly enriched leucine residue composition, accumulation of positively selected sites, and shorter beta sheets. ? Homologous resistance gene analog (RGA)-containing regions in peanut, soybean, Medicago, Arabidopsis and grape have only limited gene synteny and microcollinearity.     

The role of osteopontin (OPN/SPP1) haplotypes in the susceptibility to Crohn's disease

Background

Osteopontin represents a multifunctional molecule playing a pivotal role in chronic inflammatory and autoimmune diseases. Its expression is increased in inflammatory bowel disease (IBD). The aim of our study was to analyze the association of osteopontin (OPN/SPP1) gene variants in a large cohort of IBD patients.

Methodology/Principal Findings

Genomic DNA from 2819 Caucasian individuals (n = 841 patients with Crohn''s disease (CD), n = 473 patients with ulcerative colitis (UC), and n = 1505 healthy unrelated controls) was analyzed for nine OPN SNPs (rs2728127, rs2853744, rs11730582, rs11739060, rs28357094, rs4754 = p.Asp80Asp, rs1126616 = p.Ala236Ala, rs1126772 and rs9138). Considering the important role of osteopontin in Th17-mediated diseases, we performed analysis for epistasis with IBD-associated IL23R variants and analyzed serum levels of the Th17 cytokine IL-22. For four OPN SNPs (rs4754, rs1126616, rs1126772 and rs9138), we observed significantly different distributions between male and female CD patients. rs4754 was protective in male CD patients (p = 0.0004, OR = 0.69). None of the other investigated OPN SNPs was associated with CD or UC susceptibility. However, several OPN haplotypes showed significant associations with CD susceptibility. The strongest association was found for a haplotype consisting of the 8 OPN SNPs rs2728127-rs2853744-rs11730582-rs11439060-rs28357094-rs112661-rs1126772-rs9138 (omnibus p-value = 2.07×10⁻⁸). Overall, the mean IL-22 secretion in the combined group of OPN minor allele carriers with CD was significantly lower than that of CD patients with OPN wildtype alleles (p = 3.66×10⁻⁵). There was evidence for weak epistasis between the OPN SNP rs28357094 with the IL23R SNP rs10489629 (p = 4.18×10⁻²) and between OPN SNP rs1126616 and IL23R SNP rs2201841 (p = 4.18×10⁻²) but none of these associations remained significant after Bonferroni correction.

Conclusions/Significance

Our study identified OPN haplotypes as modifiers of CD susceptibility, while the combined effects of certain OPN variants may modulate IL-22 secretion.     

Social competition among gynes in halictine bees: The influence of bee size and pheromones on behavior

Solitary gynes of two species of social bees, Lasioglossum (Evylaeus) malachurumand L. (E.) pauxillum(Hymenoptera: Halictidae), were observed in the field and in the laboratory during the solitary (spring) phase of their life cycles. Fighting over nests among gynes of the former species is common when nests are being provisioned and can result in serious injury or even death to one or both interactants. The payoff is occasional acquisition of a nest. In contrast, fighting was never observed in the field among gynes of L. pauxillum.Several factors determine the outcome of such fights in L. malachuram,these include relative sizes of the two opponents as well as nest ownership. Dyadic interactions in the laboratory reveal that size influences the behavioral strategies of the gynes of both species, with the larger of two individuals in a dyadic interaction being on average more aggressive. Furthermore, in a second experimental series with L. malachurum,application to the smaller gyne of synthetically derived macrocyclic lactones found in the species' Dufour's gland pheromone mixture significantly decreased the aggressive tendencies of the larger such that its behavior was no longer significantly different from that for the smaller gyne. Therefore, among gynes, aggressive pheromonal signaling, coupled with other possible signal modalities, is probably an integral part in the communication system.     

A truncated RHAMM protein for discovering novel therapeutic peptides

The receptor for hyaluronan mediated motility (RHAMM, gene name HMMR) belongs to a group of proteins that bind to hyaluronan (HA), a high-molecular weight anionic polysaccharide that has pro-angiogenic and inflammatory properties when fragmented. We propose to use a chemically synthesized, truncated version of the protein (706–767), 7?kDa RHAMM, as a target receptor in the screening of novel peptide-based therapeutic agents. Chemical synthesis by Fmoc-based solid-phase peptide synthesis, and optimization using pseudoprolines, results in RHAMM protein of higher purity and yield than synthesis by recombinant protein production. 7?kDa RHAMM was evaluated for its secondary structure, ability to bind the native ligand, HA, and its bioactivity. This 62-amino acid polypeptide replicates the HA binding properties of both native and recombinant RHAMM protein. Furthermore, tubulin-derived HA peptide analogues that bind to recombinant RHAMM and were previously reported to compete with HA for interactions with RHAMM, bind with a similar affinity and specificity to the 7?kDa RHAMM. Therefore, in terms of its key binding properties, the 7?kDa RHAMM mini-protein is a suitable replacement for the full-length recombinant protein.     

First record of the non-indigenous jellyfish <Emphasis Type="Italic">Blackfordia virginica</Emphasis> (Mayer, 1910) in the Baltic Sea

Marine invasions are of increasing concern for biodiversity conservation worldwide. Gelatinous macrozooplankton contain members, which have become globally invasive, for example the ctenophore Mnemiopsis leidyi or the hydromedusae Blackfordia virginica. B. virginica is characterised by a large salinity tolerance, with a brackish-water habitat preference, and by a metagenic life history strategy with an alternation between sexually reproducing planktonic medusae and asexually reproducing benthic polyps to complete the life cycle. In this study we analysed 8 years of ichthyoplankton survey data (2010–2017) from the Kiel Canal and 14 ichthyoplankton summer surveys in the central Baltic Sea (2008–2017). We report the first presence of B. virginica in northern Europe, namely from the southwestern Baltic Sea and the Kiel Canal. In the Kiel Canal, B. virginica was first sporadically sighted in 2014 and 2015 and has developed persistent populations since summer 2016. Changes in size-frequency distributions during summer 2016 indicate active recruitment in the Kiel Canal at salinities between 7 and 13 and temperatures?>?14 °C. Close vicinity to and direct connection with the southwestern Baltic Sea, where B. virginica was observed during 2017, indicate that the Baltic Sea and other brackish-water habitats of Northern Europe are at risk for colonisation of this non-indigenous species. Our results highlight that monitoring activities should consider gelatinous macrozooplankton for standard assessments to allow for the detection of non-indigenous species at an early stage of their colonisation.