Marija Ćurlin

A recent evolutionary change affects a regulatory element in the human FOXP2 gene

The FOXP2 gene is required for normal development of speech and language. By isolating and sequencing FOXP2 genomic DNA fragments from a 49,000-year-old Iberian Neandertal and 50 present-day humans, we have identified substitutions in the gene shared by all or nearly all present-day humans but absent or polymorphic in Neandertals. One such substitution is localized in intron 8 and affects a binding site for the transcription factor POU3F2, which is highly conserved among vertebrates. We find that the derived allele of this site is less efficient than the ancestral allele in activating transcription from a reporter construct. The derived allele also binds less POU3F2 dimers than POU3F2 monomers compared with the ancestral allele. Because the substitution in the POU3F2 binding site is likely to alter the regulation of FOXP2 expression, and because it is localized in a region of the gene associated with a previously described signal of positive selection, it is a plausible candidate for having caused a recent selective sweep in the FOXP2 gene.

Comparison of in vitro toxicity of silver ions and silver nanoparticles on human hepatoma cells.

Scientific information on the potential harmful effects of silver nanoparticles (AgNPs) on human health severely lags behind their exponentially growing applications in consumer products. In assessing the toxic risk of AgNP usage, liver, as a detoxifying organ, is particularly important. The aim of this study was to explore the toxicity mechanisms of nano and ionic forms of silver on human hepatoblastoma (HepG2) cells. The results showed that silver ions and citrate‐coated AgNPs reduced cell viability in a dose‐dependent manner. The IC50 values of silver ions and citrate‐coated AgNPs were 0.5 and 50 mg L−1, respectively. The LDH leakage and inhibition of albumin synthesis, along with decreased ALT activity, indicated that treatment with either AgNP or Ag ions resulted in membrane damage and reduced the cell function of human liver cells. Evaluation of oxidative stress markers demonstrating depletion of GSH, increased ROS production, and increased SOD activity, indicated that oxidative stress might contribute to the toxicity effects of nano and ionic forms of silver. The observed toxic effect of AgNP on HepG2 cells was substantially weaker than that caused by ionic silver, while the uptake of nano and ionic forms of silver by HepG2 cells was nearly the same.

Expression analysis of genes involved in TLR2-related signaling pathway: Inflammation and apoptosis after ischemic brain injury

Toll-like receptor 2 (TLR2) is involved in innate immunity in the brain and in the cascade of events after ischemic stroke. The aim of this study was to get an insight into the expression of genes related to TLR2 signaling pathway and associated with inflammation and apoptosis in the later stages of brain response after ischemic injury. Middle cerebral artery occlusion was performed on both wild-type and TLR2(-/-) mice followed by real-time PCR to measure the relative expression of selected genes. In TLR2(-/-) mice expression of genes involved in proinflammatory response was decreased after cerebral ischemia. Tnf was the most prominent cytokine active in the late phase of recovery. Contrary to proinflammatory genes, the expression of Casp8, as a hallmark of apoptosis, was increased in TLR2(-/-) mice, in particular in the late phase of recovery.

Surface coating affects behavior of metallic nanoparticles in a biological environment.

Summary Silver (AgNPs) and maghemite, i.e., superparamagnetic iron oxide nanoparticles (SPIONs) are promising candidates for new medical applications, which implies the need for strict information regarding their physicochemical characteristics and behavior in a biological environment. The currently developed AgNPs and SPIONs encompass a myriad of sizes and surface coatings, which affect NPs properties and may improve their biocompatibility. This study is aimed to evaluate the effects of surface coating on colloidal stability and behavior of AgNPs and SPIONs in modelled biological environments using dynamic and electrophoretic light scattering techniques, as well as transmission electron microscopy to visualize the behavior of the NP. Three dispersion media were investigated: ultrapure water (UW), biological cell culture medium without addition of protein (BM), and BM supplemented with common serum protein (BMP). The obtained results showed that different coating agents on AgNPs and SPIONs produced different stabilities in the same biological media. The combination of negative charge and high adsorption strength of coating agents proved to be important for achieving good stability of metallic NPs in electrolyte-rich fluids. Most importantly, the presence of proteins provided colloidal stabilization to metallic NPs in biological fluids regardless of their chemical composition, surface structure and surface charge. In addition, an assessment of AgNP and SPION behavior in real biological fluids, rat whole blood (WhBl) and blood plasma (BlPl), revealed that the composition of a biological medium is crucial for the colloidal stability and type of metallic NP transformation. Our results highlight the importance of physicochemical characterization and stability evaluation of metallic NPs in a variety of biological systems including as many NP properties as possible.

Does surface coating of metallic nanoparticles modulate their interference with in vitro assays

Screening programs for the evaluation of nanomaterial value and safety rely on in vitro tests. The exceptional physicochemical properties of metallic nanoparticles (NPs), such as large surface area and chemically active surface, may provoke their interference with in vitro methods and analytical techniques used for evaluation of biocompatibility or toxicity of NPs. This study aimed to determine if such interference could be predicted on the basis of the surface characteristics of metallic NPs by investigating the effect of different surface coatings of silver (AgNPs) and maghemite NPs (γ-Fe2O3NPs) on common in vitro assays scoring two of the main cytotoxic endpoints: cell viability and oxidative stress response. We examined optical, adsorptive and chemically reactive types of NP interference with cell viability assays (MTT, MTS, and WST-8) and assays employing fluorescent dyes as markers for production of reactive oxygen species (DCFH-DA and DHE) or glutathione level (MBCl). Each type of tested NPs affected all of the six investigated assays leading to false interpretation of obtained results. The extent and type of interference were dependent on the type and surface coating of NPs as well as on their stability in biological media. The results have shown that interference was concentration-, particle type- and assay type-dependent. This study demonstrated that common in vitro assays, without appropriate cause-and-effect analysis and adaptation or modification, are ineffective in the evaluation of biological effects of metallic NPs due to their interaction with optical readouts and assay components. A comprehensive and feasible experimental setup has been proposed to gain a reproducible and reliable in vitro evaluation as the first step in the health assessment of metallic NPs.

Cervical mucus: from biochemical structure to clinical implications.

Structure of human cervical mucus plays a pivotal role in female fertility and protection of reproductive health. Investigation of biochemical and biophysical structure of cervical mucus remains a challenge due to complex structural proteins, high content of oligosaccharides and cyclic variability of its structure. We present the current knowledge on chemical and biophysical features of cervical mucus and regulation of its secretion, relevant clinical observations and underexplored elements. The latter relates to biochemical background of physical properties and antimicrobial activity of cervical mucus, and regulation of its production.

STAM2, a member of the endosome-associated complex ESCRT-0 is highly expressed in neurons.

STAM2 (signal transducing adaptor molecule 2), a subunit of the ESCRT-0 complex, is an endosomal protein acting as a regulator of receptor signaling and trafficking. To analyze STAM2 in the nervous system, its gene expression and protein localization in the mouse brain were identified using three methods: mRNA in situ hybridization, immunohistochemistry, and via lacZ reporter in frame with Stam2 gene using the gene trap mouse line Stam2(Gt1Gaj). STAM2 intracellular localization was analyzed by subcellular fractionation and co-immunofluorescence using confocal microscopy. Stam2 was strongly expressed in the cerebral and cerebellar cortex, hippocampal formation, olfactory bulb, and medial habenula. The majority of STAM2-positive cells co-stained with the neuronal markers. In neurons STAM2 was found in the early endosomes and also in the nucleus. The other members of the ESCRT-0 complex co-localized with STAM2 in the cytoplasm, but they were not present in the nucleus. The newly identified neuron-specific nuclear localization of STAM2, together with its high expression in the brain indicated that STAM2 might have a specific function in the mouse nervous system.

Krüppel-like transcription factor 8 (Klf8) is expressed and active in the neurons of the mouse brain.

Krüppel-like transcription factor 8 (KLF8) is a transcription factor suggested to be involved in various cellular events, including malignant cell transformation, still its expression in the adult rodent brain remained unknown. To analyze Klf8 in the mouse brain and to identify cell types expressing it, a specific transgenic Klf8(Gt1Gaj) mouse was used. The resulting Klf8 gene-driven β-galactosidase activity was visualized by X-gal histochemical staining of the brain sections. The obtained results were complemented by in situ RNA hybridization and immunohistochemistry. Klf8 was highly expressed throughout the adult mouse brain gray matter including the cerebral cortex, hippocampus, olfactory bulb, hypothalamus, pallidum, and striatum, but not in the cerebellum. Immunofluorescent double-labeling revealed that KLF8-immunoreactive cells were neurons, and the staining was located in their nucleus. This was the first study showing that Klf8 was highly expressed in various regions of the mouse brain and in particular in the neurons, where it was localized in the cell nuclei.

Impact of cationic surfactant on the self-assembly of sodium caseinate.

The impact of a cationic surfactant, dodecylammonium chloride (DDACl), on the self-assembly of sodium caseinate (SC) has been investigated by light scattering, zeta potential, and rheological measurements as well as by microscopy (transmission electron and confocal laser scanning microscopy). In SC dilute solutions concentration-dependent self-assembly proceeds through the formation of spherical associates and their aggregation into elongated structures composed of connected spheres. DDACl interacts with SC via its hydrophilic and hydrophobic groups, inducing changes in SC self-assembled structures. These changes strongly depend on the surfactant aggregation states (monomeric or micellar) as well as concentration ratio of both components, leading to the formation of soluble and insoluble complexes of nano- to microdimensions. DDACl monomers interact with SC self-assembled entities in a different way compared to their micelles. Surfactant monomers form soluble complexes (similar to surfactant mixed micelles) at lower SC concentration but insoluble gelatinous complexes at higher SC concentration. At surfactant micellar concentration soluble complexes with casein chains wrapped around surfactant micelles are formed. This study suggests that the use of proper cationic surfactant concentration will allow modification and control of structural changes of SC self-assembled entities.

Neurons and a subset of interstitial cells of Cajal in the enteric nervous system highly express Stam2 gene.

Signal transducing adaptor molecule 2 (STAM2) is a phosphotyrosine protein, which is a member of the endosomal sorting complex required for transport (ESCRT‐0) and is involved in the sorting process of the mono‐ubiquitinated endosomal cargo for degradation in the lysosome. Analysis of gene trap mice carrying lacZ in frame with Stam2 revealed beta‐galactosidase activity in the enteric nervous system (both in the myenteric and submucosal plexus) throughout the digestive tract. STAM2 immunostaining confirmed that the observed beta‐galactosidase activity coincided with high Stam2 expression. To identify cell types with high Stam2 expression, STAM2 immunostaining was colocalized with the neuronal markers microtubule‐associated protein 2 and protein gene product 9.5 and with c‐kit as a marker for interstitial cells of Cajal (ICCs). STAM2 and c‐kit positive cells comprised a subset of ICCs in the enteric nervous system. Qualitative and quantitative analysis of the morphology of the enteric nervous system in the homozygous mice carrying gene trap insertion in the Stam2 gene did not reveal phenotype changes; therefore, STAM2 function in the digestive tube remains elusive. Anat Rec, 2012.