Erratum - Tri-Lineage Differentiation of NTERA2 Clone D1 Cells towards Neural, Hepatic and Osteogenic Lineages in Vitro.

The images in Fig. 4 were not presented correctly. The correct version of Fig. 4: see the last page of pdf. The original article was published in Folia Biologica (Praha) Volume 67, No. 5-6 (2021), 174-182. https://doi.org/10.14712/fb2021067050174.

The relationship between cognitive control and lexical conflict resolution in developmental dyslexia.

Previous studies have revealed that cognitive control functions contribute to the resolution of lexical interference. Both cognitive control (CC) deficits and reduced speed of lexical retrieval in Rapid Automatized Naming (RAN) tasks are characteristics of Developmental Dyslexia (DD), but it is still not fully understood how these deficits relate to each other and to reading problems. To examine this question, we tested adolescents with DD (n = 38), poor readers (PR; n = 25) and typical readers (TR; n = 33) matched on age and IQ, on CC functions with Stroop, Stop Signal, Simon, Backward Digit Span and n-back tasks and on lexical retrieval and lexical conflict resolution with RAN of pictures in semantically homogeneous vs. mixed trials. As expected, in the blocked RAN Task DD individuals showed longer naming times and a greater effect of lexical conflict resolution (indexed by difference scores of naming times in the homogeneous and mixed conditions) than TR participants. We also found significant group differences (TR = PR > DD) in CC measures. Naming time was associated with CC, while the lexical interference effect did not show any association with this set of abilities. These findings suggest that DD individuals show impairments in multiple cognitive functions, such as cognitive control, lexical retrieval and lexical conflict resolution. Our results also suggest that CC functions are involved in lexical retrieval, but we have not found evidence for their involvement in lexical conflict resolution processes.

Controlling the Structure of Carbon Deposit by Nitrogen Doping Catalytic Chemical Vapor Deposition Synthesis.

Nitrogen doped multi-walled carbon nanotubes and other carbon nanoparticles were synthesized by catalytic chemical vapor deposition of tripropylamine and acetylene on CaCO3-supported cobalt catalyst (5 wt%), prepared by impregnation, and various precursors. Each synthesis was performed by using either the pure nitrogenous organic compound or its mixture with acetone. Transmission electron microscopy studies revealed a significant difference both in the yield and the diversity of the carbon deposits. Every synthesis resulted in bamboo-like nanotubes, and nearly all of them also in onion-like structures. Electron energy loss spectroscopy studies of the samples indicated the presence of nitrogen and calcium (caused by the catalyst support). High-resolution transmission electron microscopy and X-ray diffraction measurements were also performed to characterize the samples.

Adhesive Management of Anterior Tooth Wear in Combination with the Dahl Concept-A 27-Month Observational Case Series.

Localized anterior maxillary tooth wear caused by erosion and attrition with loss of interocclusal space is difficult to manage. This observational case-series study reports six cases with worn anterior dentition treated with labial ceramic and palatal direct resin composite veneers at an increased vertical dimension of occlusion without restoration of unaffected posterior teeth. Thirty-six palatal direct veneers were made in six patients from a nanohybrid resin composite with the help of a wax-up-based template at an increased vertical dimension. After the complete re-establishment of posterior occlusion, 40 labial lithium-disilicate ceramic veneers were fabricated with a mock-up-guided method. The sandwich veneers were evaluated according to the United States Public Health Service (USPHS) criteria after a mean service time of 22.7 months. Re-establishment of posterior contacts as well as subjective patient satisfaction and function were evaluated. The overall success of the labial ceramic veneers was excellent. The quality of the palatal resin composite restorations was found to be good with predominantly "Alpha" scores. The marginal quality (11.1% and 33.3% of integrity and discoloration, respectively) and surface roughness (16.7%) showed small deteriorations indicated by "Beta" scores. The resin composite showed, in general, signs of wear facets which resulted in "Beta" scores in 44.4% of the cases. Posterior contacts re-established firmly within 4 weeks in all cases. Patient satisfaction with esthetics and function was high. The short-term outcome of this non-invasive treatment option is favorable and promising.

Tri-Lineage Differentiation of NTERA2 Clone D1 Cells towards Neural, Hepatic and Osteogenic Lineages in Vitro.

Over the past decades, the in vitro use of pluripotent cell lines gained a crucial role in toxicology, preclinical drug testing and developmental biology. NTERA2 clone D1 cells were identified as pluripotent cells with high potential for neural differentiation. Although they are commonly used cellular sources in neuropharmacology and neurodevelopmental studies, their endodermal and mesodermal differentiation potential awaits further characterization. Here, we devised improved protocols for hepatogenic and osteogenic differentiation of NTERA2 clone D1 cells. Our in vitro differentiation assays showed significant up-regulation of multiple hepatogenic markers. We also observed robust mineralization and osteogenic marker expression of NTERA2 clone D1 cells upon in vitro osteogenic induction. These results suggest that NTERA2 clone D1 cells may be utilized as an in vitro model system to study various aspects of liver biology and osteogenesis. In addition, tri-lineage differentiation of NTERA2 clone D1 cells may serve as a simple experimental control system when validating pluripotency of other cell types.

Next-generation sequencing identifies novel mitochondrial variants in pituitary adenomas.

PURPOSE: Disrupted mitochondrial functions and genetic variants of mitochondrial DNA (mtDNA) have been observed in different human neoplasms. Next-generation sequencing (NGS) can be used to detect even low heteroplasmy-level mtDNA variants. We aimed to investigate the mitochondrial genome in pituitary adenomas by NGS. METHODS: We analysed 11 growth hormone producing and 33 non-functioning [22 gonadotroph and 11 hormone immunonegative] pituitary adenomas using VariantPro™ Mitochondrion Panel on Illumina MiSeq instrument. Revised Cambridge Reference Sequence (rCRS) of the mtDNA was used as reference. Heteroplasmy was determined using a 3% cutoff. RESULTS: 496 variants were identified in pituitary adenomas with overall low level of heteroplasmy (7.22%). On average, 35 variants were detected per sample. Samples harbouring the highest number of variants had the highest Ki-67 indices independently of histological subtypes. We identified eight variants (A11251G, T4216C, T16126C, C15452A, T14798C, A188G, G185A, and T16093C) with different prevalences among different histological groups. T16189C was found in 40% of non-recurrent adenomas, while it was not present in the recurrent ones. T14798C and T4216C were confirmed by Sanger sequencing in all 44 samples. 100% concordance was found between NGS and Sanger method. CONCLUSIONS: NGS is a reliable method for investigating mitochondrial genome and heteroplasmy in pituitary adenomas. Out of the 496 detected variants, 414 have not been previously reported in pituitary adenoma. The high number of mtDNA variants may contribute to adenoma genesis, and some variants (i.e., T16189C) might associate with benign behaviour.

Limitations of high throughput methods for miRNA expression profiles in non-functioning pituitary adenomas.

Microarray, RT-qPCR based arrays and next-generation-sequencing (NGS) are available high-throughput methods for miRNA profiling (miRNome). Analytical and biological performance of these methods were tested in identification of biologically relevant miRNAs in non-functioning pituitary adenomas (NFPA). miRNome of 4 normal pituitary (NP) and 8 NFPA samples was determined by these platforms and expression of 21 individual miRNAs was measured on 30 (20 NFPA and 10 NP) independent samples. Complex bioinformatics was used. 132 and 137 miRNAs were detected by all three platforms in NP and NFPA, respectively, of which 25 were differentially expressed (fold change > 2). The strongest correlation was observed between microarray and TaqMan-array, while the data obtained by NGS were the most discordant despite of various bioinformatics settings. As a technical validation we measured the expression of 21 selected miRNAs by individual RT-qPCR and we were able to validate 35.1%, 76.2% and 71.4% of the miRNAs revealed by SOLiD, TLDA and microarray result, respectively. We performed biological validation using an extended number of samples (20 NFPAs and 8 NPs). Technical and biological validation showed high correlation (p < 0.001; R = 0.96). Pathway and network analysis revealed several common pathways but no pathway showed the same activation score. Using the 25 platform-independent miRNAs developmental pathways were the top functional categories relevant for NFPA genesis. The difference among high-throughput platforms is of great importance and selection of screening method can influence experimental results. Validation by another platform is essential in order to avoid or to minimalize the platform specific errors.

Synthesis, Characterization and Photocatalytic Efficiency of ZnO/MWCNT Nanocomposites Prepared Under Different Solvent Conditions.

Here we report the application of zinc oxide (ZnO) coated multi-walled carbon nanotube (MWCNT) composites in the photocatalytic decomposition of acetaldehyde (AA). Zinc oxide nanoparticles were successfully coated on the multi-walled carbon nanotube via impregnation process using zinc acetate (Zn(CH3COO)2×2H2O) as precursor and sodium dodecyl sulfate (SDS) treated multiwalled carbon nanotube as raw material under different solvent conditions. The applied solvents during preparation were ethanol (EtOH) and water (H2O). As-prepared materials were characterized by thermal analysis (TG), X-ray diffraction (XRD), specific surface area measurement (BET) and transmission electron microscopy (TEM) techniques. Photocatalytic efficiencies of as-prepared composites were investigated in a stationary reactor equipped with UV lamp. Decomposition of acetaldehyde was followed by using gas chromatography (GC). Observations revealed that using impregnation method and different solvents the preparation of ZnO/MWCNT nanocomposites can be controlled easily. The highest degradation rate was achieved with the nanocomposite was synthetized using ethanol as solvent. The photocatalytic experiments revealed that the composite has higher photocatalytic activity than that of both the zinc oxide nanoparticles and the mechanical mixture of multi-walled carbon nanotube and zinc oxide.

Tracking down protein-protein interactions via a FRET-system using site-specific thiol-labeling.

Förster resonance energy transfer is among the most popular tools to follow protein-protein interactions. Although limited to certain cases, site-specific fluorescent labeling of proteins via natural functions by means of chemical manipulations can redeem laborious protein engineering techniques. Herein we report on the synthesis of a heterobifunctional tag and its use in site-specific protein labeling studies aiming at exploring protein-protein interactions. The oxadiazole-methylsulfonyl functionality serves as a thiol specific warhead that enables easy and selective installation of fluorescent labels through a bioorthogonal motif. Mitogen activated protein kinase (MAPK14) and its substrate mitogen activated protein kinase activated kinase (MAPKAP2) or its docking motif, a 22 amino acid-long peptide fragment, were labeled with a donor and an acceptor, respectively. Evolution of strong FRET signals upon protein-protein interactions supported the specific communication between the partners. Using an efficient FRET pair allowed the estimation of dissociation constants for protein-protein and peptide-protein interactions (145 nM and 240 nM, respectively).

Altered neurite morphology and cholinergic function of induced pluripotent stem cell-derived neurons from a patient with Kleefstra syndrome and autism.

The aim of the present study was to establish an in vitro Kleefstra syndrome (KS) disease model using the human induced pluripotent stem cell (hiPSC) technology. Previously, an autism spectrum disorder (ASD) patient with Kleefstra syndrome (KS-ASD) carrying a deleterious premature termination codon mutation in the EHMT1 gene was identified. Patient specific hiPSCs generated from peripheral blood mononuclear cells of the KS-ASD patient were differentiated into post-mitotic cortical neurons. Lower levels of EHMT1 mRNA as well as protein expression were confirmed in these cells. Morphological analysis on neuronal cells differentiated from the KS-ASD patient-derived hiPSC clones showed significantly shorter neurites and reduced arborization compared to cells generated from healthy controls. Moreover, density of dendritic protrusions of neuronal cells derived from KS-ASD hiPSCs was lower than that of control cells. Synaptic connections and spontaneous neuronal activity measured by live cell calcium imaging could be detected after 5 weeks of differentiation, when KS-ASD cells exhibited higher sensitivity of calcium responses to acetylcholine stimulation indicating a lower nicotinic cholinergic tone at baseline condition in KS-ASD cells. In addition, gene expression profiling of differentiated neuronal cells from the KS-ASD patient revealed higher expression of proliferation-related genes and lower mRNA levels of genes involved in neuronal maturation and migration. Our data demonstrate anomalous neuronal morphology, functional activity and gene expression in KS-ASD patient-specific hiPSC-derived neuronal cultures, which offers an in vitro system that contributes to a better understanding of KS and potentially other neurodevelopmental disorders including ASD.

Expression of G protein-coupled oestrogen receptor in melanoma and in pregnancy-associated melanoma.

BACKGROUND: The hormone sensitivity of melanoma and the role of 'classical' oestrogen receptor (ER) α and ß in tumour progression have been intensively studied with rather contradictory results. The presence of 'non-classical' G protein-coupled oestrogen receptor (GPER) has not been investigated on human melanoma tissues. OBJECTIVE: To analyse the expression of GPER, ERα and ERß in pregnancy-associated (PAM) and in non-pregnancy-associated (NPAM) melanomas in correlation with traditional prognostic markers and disease-free survival (DFS). METHODS: Receptor protein levels were tested using immunohistochemistry in 81 formalin-fixed paraffin-embedded melanoma tissues. PAMs (n = 38) were compared with age- and Breslow thickness-matched cases (n = 43) including non-pregnant women (NPAM-W) (n = 22) and men (NPAM-M) (n = 21). The association between receptor expression and DFS was analysed by uni- and multivariate Cox proportional hazards regression. RESULTS: G protein-coupled oestrogen receptor was detected both in PAMs and NPAMs. In 39 of the 41 (95.1%) GPER-positive melanomas, GPER and ERß were co-expressed. GPER/ERß-positive melanomas were significantly more common in PAM compared to NPAM (P = 0.0001) with no significant difference between genders (P = 0.4383). In PAMs, the distribution of GPER and ERß was similar (78.4% vs. 81.6%; P = 0.8504), while in NPAM, ERß was the representative ER (60.5% vs. 27.9%; P = 0.0010) without gender difference (59.1% vs. 61.9%). GPER-/ERß-positive melanomas were associated with lower Breslow thickness, lower mitotic rate and higher presence of peritumoral lymphocyte infiltration (PLI) compared to GPER-/ERß-negative cases (P = 0.0156, P = 0.0036 and P = 0.0001) predicting a better DFS (HR = 0.785, 95% CI 0.582-1.058). Despite the significantly higher frequency of GPER and ERß expression in PAM, no significant difference was found in DFS between PAM and NPAM. All but one case failed to show ERα expression. CONCLUSIONS: The presence of GPER and its simultaneous expression with ERß can serve as a new prognostic indicator in a significant subpopulation of melanoma patients.

Correction: A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags.

Correction for 'A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags' by B. Söveges, et al., Org. Biomol. Chem., 2016, 14, 6071-6078.

A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags.

Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.

Digestibility and metabolism of dietary guanidino acetic acid fed to broilers.

In two feeding experiments the retention of supplemental guanidine acetic acid (GAA) in broilers was investigated. In both experiments, the same three treatments were used; the basal feed was supplemented with 0, 0.6, or 6.0 g GAA per kg of feed. While in a growth study (experiment 1) day-old, male Ross 308 broilers were fed diets for 35 days, these diets were fed for only 8 days to fistulated broilers 34 days of age in a balance study (experiment 2). Feeding 0.6 g/kg GAA did not improve growth performance whereas 6.0 g/kg GAA resulted in a reduction of feed consumption and consequently of weight gain (P ≤ 0.05). Feed conversion was not affected and was 1.48 to 1.49 in all treatments. Increasing levels of dietary GAA gradually increased the creatine concentration in breast muscle and liver tissues (P ≤ 0.05) indicating a transformation and retention of dietary GAA as creatine. In experiment 2 the non-supplemented basal diet allowed us to determine the endogenous GAA, creatine, and creatinine excretions. Accordingly, only small amounts of these metabolites were recovered in feces while they were much higher in urine. Increasing dietary GAA intake increased fecal and renal GAA, creatine, and creatinine excretion and was significant (P ≤ 0.05) at 6.0 g/kg dietary GAA compared to no or 0.6 g/kg GAA supplementation. The mean true fecal digestibility of GAA (99%) was unaffected by the level of supplemental GAA. Considering renal GAA excretions, true availability of supplemental GAA was reduced with increasing dose (83% vs. 71%; P ≤ 0.05). Taking into account creatine and creatinine excretions above those of the basal diet, as they are a consequence of increasing dietary supply, true availability of supplemental GAA shrank from 76% (0.6 g/kg GAA) to 46% (6.0 g/kg GAA; P ≤ 0.05). Changes in blood creatine and creatinine levels reflected the changes observed in the liver and muscle tissues and may suggest increased transport to excretion organs. Data from these experiment were used to estimate the creatine requirement.

The interface between HOPG and 1-butyl-3-methyl-imidazolium hexafluorophosphate.

The interface between highly oriented pyrolytic graphite (HOPG) and 1-butyl-3-metyl-imidazolium hexafluorophosphate (BMIPF6) has been studied using cyclic voltammetry, electrochemical impedance spectroscopy, immersion charge measurements and in situ scanning tunneling microscopy (in situ STM). The results are compared with those obtained with Au(100) in BMIPF6 (Phys. Chem. Chem. Phys., 2011, 13, 11627). The main result is that the high frequency capacitance spectra on the two systems are similar to each other, however at low frequencies some slow interfacial processes cause the appearance of a second capacitance arc on Au(100), which is absent for HOPG. The slow processes are attributed to the rearrangement of the Au surface structure and to the formation of ionic liquid adlayers--these are visualized by in situ STM.

Post-ischemic treatment with L-kynurenine sulfate exacerbates neuronal damage after transient middle cerebral artery occlusion.

Since brain ischemia is one of the leading causes of adult disability and death, neuroprotection of the ischemic brain is of particular importance. Acute neuroprotective strategies usually have the aim of suppressing glutamate excitotoxicity and an excessive N-methyl-d-aspartate (NMDA) receptor function. Clinically tolerated antagonists should antagonize an excessive NMDA receptor function without compromising the normal synaptic function. Kynurenic acid (KYNA) an endogenous metabolite of the tryptophan metabolism, may be an attractive neuroprotectant in this regard. The manipulation of brain KYNA levels was earlier found to effectively enhance the histopathological outcome of experimental ischemic/hypoxic states. The present investigation of the neuroprotective capacity of L-kynurenine sulfate (L-KYNs) administered systemically after reperfusion in a novel distal middle cerebral artery occlusion (dMCAO) model of focal ischemia/reperfusion revealed that in contrast with earlier results, treatment with L-KYNs worsened the histopathological outcome of dMCAO. This contradictory result indicates that post-ischemic treatment with L-KYNs may be harmful.

Effects of tampons and menses on the composition and diversity of vaginal microbial communities over time.

OBJECTIVE: To investigate the influence of menses on the vaginal microbiota and determine whether tampons that differ in material composition influence these bacterial communities in different ways. DESIGN: A single-centre trial with randomised, complete block design. SETTING: Procter & Gamble facility. SAMPLE: Seven self-declared healthy, female volunteers of reproductive age. METHODS: Volunteers used a pad and two types of tampons during the study, one product exclusively each month for three sequential menstrual cycles. During menses and once each mid-cycle, vaginal bacterial community composition was characterised by cultivation-independent methods based on pyrosequencing of V1-V2 variable regions of 16S ribosomal RNA genes. MAIN OUTCOME MEASURES: Changes in the species composition, abundance and diversity in vaginal bacterial communities over time and between treatments. RESULTS: The vaginal microbiotas of all seven women were dominated by Lactobacillus spp. at mid-cycle, and the compositions of those communities were largely consistent between cycles. Community dynamic patterns during menses varied considerably and were more or less individualised. In three of the seven women the community diversity during pad use was significantly different from at least one tampon cycle. CONCLUSIONS: Changes in the composition of the vaginal microbiota during menses were common, but the magnitude of change varied between women. Despite these changes, most communities were capable of resuming a composition similar to previous mid-cycle sampling times following menstruation. Overall we conclude that the two tampons tested do not significantly impact the vaginal microbiota in different ways; however, larger studies should be performed to confirm these findings.