BACKGROUND INFORMATION: Cellular prion protein (PrPC ) is infamous for its role in prion diseases. The physiological function of PrPC remains enigmatic, but several studies point to its involvement in cell differentiation processes. To test this possibility, we monitored PrPC changes during the differentiation of prion-susceptible CAD 5 cells, and then we analysed the effect of PrPC ablation on the differentiation process. RESULTS: Neuronal CAD 5 cells differentiate within 5 days of serum withdrawal, with the majority of the cells developing long neurites. This process is accompanied by an up to sixfold increase in PrPC expression and enhanced N-terminal β-cleavage of the protein, which suggests a role for the PrPC in the differentiation process. Moreover, the majority of PrPC in differentiated cells is inside the cell, and a large proportion of the protein does not associate with membrane lipid rafts. In contrast, PrPC in proliferating cells is found mostly on the cytoplasmic membrane and is predominantly associated with lipid rafts. To determine the importance of PrPC in cell differentiation, a CAD 5 PrP-/- cell line with ablated PrPC expression was created using the CRISPR/Cas9 system. We observed no considerable difference in morphology, proliferation rate or expression of molecular markers between CAD 5 and CAD 5 PrP-/- cells during the differentiation initiated by serum withdrawal. CONCLUSIONS: PrPC characteristics, such as cell localisation, level of expression and posttranslational modifications, change during CAD 5 cell differentiation, but PrPC ablation does not change the course of the differentiation process. SIGNIFICANCE: Ablation of PrPC expression does not affect CAD 5 cell differentiation, although we observed many intriguing changes in PrPC features during the process. Our study does not support the concept that PrPC is important for neuronal cell differentiation, at least in simple in vitro conditions.

• Keywords
• cell differentiation, membrane protein, posttranslational modifications,
• MeSH
• Cell Differentiation * MeSH
• Cell Line MeSH
• Membrane Microdomains MeSH
• Mice MeSH
• Neurons cytology   metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Prions metabolism   MeSH
• PrPC Proteins metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Prions MeSH
• PrPC Proteins MeSH

Processing of rRNA in mammalian cells includes a series of cleavages of the primary 47S transcript and results in producing three rRNAs: 18S, 28S and 5.8S. The sequence of the main processing events in human cells has been established, but little is yet known about the dynamics of this process, especially the dynamics of its early stages. In the present study, we used real-time PCR to measure levels of pre-rRNA after inhibition of transcription with actinomycin D. Thus we could estimate the half-life time of rRNA transcripts in two human-derived cell lines, HeLa and LEP (human embryonic fibroblasts), as well as in mouse NIH 3T3 cells. The primary transcripts seemed to be more stable in the human than in the murine cells. Remarkably, the graphs in all cases showed more or less pronounced lag phase, which may reflect preparatory events preceding the first cleavage of the pre-rRNA. Additionally, we followed the dynamics of the decay of the 5'ETS fragment which is degraded only after the formation of 41S rRNA. According to our estimates, the corresponding three (or four) steps of the processing in human cells take five to eight minutes.

• Keywords
• cleavage, half-life time, human, mouse, primary transcript, rRNA processing,
• MeSH
• NIH 3T3 Cells MeSH
• Dactinomycin pharmacology   MeSH
• Transcription, Genetic drug effects   MeSH
• HeLa Cells MeSH
• Humans MeSH
• Mice MeSH
• RNA Processing, Post-Transcriptional genetics   MeSH
• RNA Precursors * genetics   metabolism   MeSH
• RNA, Ribosomal genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Dactinomycin MeSH
• RNA Precursors * MeSH
• RNA, Ribosomal MeSH

We present a simple and sensitive method for the determination of patulin at µg·kg-1 level in apple-based products. Our method relies on the application of an in-line molecularly imprinted polymer solid-phase extraction microcartridge in capillary electrophoresis coupled with mass spectrometry. Capillary zone electrophoresis method has been developed and parameters affecting the in-line process have been carefully optimized. Validation parameters were assessed for patulin, giving LOQ of 1 µg·kg-1 and linearity range 1-100 µg·kg-1 with R2 ≥ 0.997. The LOQ was below the maximum content of patulin requested by the European Union in this type of products. The precision of the peak area and the migration time were less than 14.9 and 1.6%, respectively. Patulin has been analyzed in the presence of 5-hydroxymethylfurfural, which is the main interference in this kind of matrix. The method was applied to assay patulin content in various apple-based products.

• Keywords
• Capillary electrophoresis, In-line preconcentrator, Mass spectrometry, Molecularly imprinted polymer, Mycotoxin, Patulin,
• MeSH
• Furaldehyde analogs & derivatives   chemistry   MeSH
• Food Analysis methods   MeSH
• Electrophoresis, Capillary methods   MeSH
• Solid Phase Extraction methods   MeSH
• Food Contamination analysis   MeSH
• Limit of Detection MeSH
• Malus chemistry   MeSH
• Molecular Imprinting MeSH
• Patulin analysis   MeSH
• Polymers chemistry   MeSH
• Food-Processing Industry MeSH
• Tandem Mass Spectrometry methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Furaldehyde MeSH
• 5-hydroxymethylfurfural MeSH Browser
• Patulin MeSH
• Polymers MeSH

We report here the presence of numerous processed pseudogenes derived from the W family of endogenous retroviruses in the human genome. These pseudogenes are structurally colinear with the retroviral mRNA followed by a poly(A) tail. Our analysis of insertion sites of HERV-W processed pseudogenes shows a strong preference for the insertion motif of long interspersed nuclear element (LINE) retrotransposons. The genomic distribution, stability during evolution, and frequent truncations at the 5' end resemble those of the pseudogenes generated by LINEs. We therefore suggest that HERV-W processed pseudogenes arose by multiple and independent LINE-mediated retrotransposition of retroviral mRNA. These data document that the majority of HERV-W copies are actually nontranscribed promoterless pseudogenes. The current search for HERV-Ws associated with several human diseases should concentrate on a small subset of transcriptionally competent elements.

• MeSH
• Long Interspersed Nucleotide Elements genetics   MeSH
• Endogenous Retroviruses genetics   MeSH
• Phylogeny MeSH
• GC Rich Sequence genetics   MeSH
• Genome, Human MeSH
• Virus Integration genetics   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• RNA Processing, Post-Transcriptional genetics   MeSH
• Pseudogenes genetics   MeSH
• RNA, Viral genetics   MeSH
• Base Sequence genetics   MeSH
• RNA Stability genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• RNA, Viral MeSH

We designed and evaluated an innovative computer-aided-learning environment based on the on-line integration of computer controlled medical diagnostic devices and a medical information system for use in the preclinical medical physics education of medical students. Our learning system simulates the actual clinical environment in a hospital or primary care unit. It uses a commercial medical information system for on-line storage and processing of clinical type data acquired during physics laboratory classes. Every student adopts two roles, the role of 'patient' and the role of 'physician'. As a 'physician' the student operates the medical devices to clinically assess 'patient' colleagues and records all results in an electronic 'patient' record. We also introduced an innovative approach to the use of supportive education materials, based on the methods of adaptive e-learning. A survey of student feedback is included and statistically evaluated. The results from the student feedback confirm the positive response of the latter to this novel implementation of medical physics and informatics in preclinical education. This approach not only significantly improves learning of medical physics and informatics skills but has the added advantage that it facilitates students' transition from preclinical to clinical subjects.

• MeSH
• Biophysics education   MeSH
• Electrocardiography instrumentation   MeSH
• Electronic Health Records * MeSH
• Physics education   MeSH
• Blood Pressure MeSH
• Physicians * MeSH
• Microscopy instrumentation   MeSH
• Online Systems MeSH
• Tomography, X-Ray Computed instrumentation   MeSH
• Hearing Tests instrumentation   MeSH
• Stents MeSH
• Education, Medical, Undergraduate methods   MeSH
• Systems Integration * MeSH
• Ultrasonography instrumentation   MeSH
• Equipment and Supplies * MeSH
• Vision Tests instrumentation   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The review presents an evaluation of the development of on-line, at-line and in-line sample treatment coupled with capillary and microchip electrophoresis over the last 10 years. In the first part, it describes different types of flow-gating interfaces (FGI) such as cross-FGI, coaxial-FGI, sheet-flow-FGI, and air-assisted-FGI and their fabrication using molding into polydimethylsiloxane and commercially available fittings. The second part deals with the coupling of capillary and microchip electrophoresis with microdialysis, solid-phase, liquid-phase, and membrane based extraction techniques. It mainly focuses on modern techniques such as extraction across supported liquid membrane, electroextraction, single drop microextraction, head space microextraction, and microdialysis with high spatial and temporal resolution. Finally, the design of sequential electrophoretic analysers and fabrication of SPE microcartridges with monolithic and molecularly imprinted polymeric sorbents are discussed. Applications include the monitoring of metabolites, neurotransmitters, peptides and proteins in body fluids and tissues to study processes in living organisms, as well as the monitoring of nutrients, minerals and waste compounds in food, natural and wastewater.

• Keywords
• Capillary electrophoresis, Electromembrane extraction, Flow-gating interface, Liquid-phase extraction, Microchip electrophoresis, Microdialysis, On-line coupling, Sequential analysis, Solid-phase extraction,
• MeSH
• Electrophoresis, Capillary methods   MeSH
• Electrophoresis, Microchip * methods   MeSH
• Microdialysis MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

A new HPLC column-switching method using large volume sample injection and fused-core columns for on-line solid phase extraction have been developed for the determination of the following carbamates and pyrethroids: aldicarb, carbaryl, pirimicarb, carbofuran, kadethrin, flumethrin, fenpropathrin, fenoxycarb, tau-fluvalinate and fenvalerate, in surface water samples. Sudan I was used as internal standard. The proposed method was performed using 100 µl sample injection followed by an on-line solid phase extraction procedure and finally the compounds were identified and quantified by liquid chromatography with ultraviolet detection. The separation was carried out on C-18 reversed phase column based on fused-core particle technology. The influence of the injected sample volume, the variables affecting to SPE process and the conditions for the separation on an analytical column, were studied and optimized. The limits of detection ranged from 5.5 to 8.9 µg L(-1), and limits of quantification from 18.4 to 29.7 µg L(-1), while inter- and intra-day variability was under 15%. This new analytical procedure was satisfactorily applied for the determination of these organic pollutants in surface water samples located in Czech Republic. Concentration levels were found for some of these pollutants up to 26.11 µg L(-1) in the river Elbe and up to 34.53 µg L(-1) in the closed lakes samples.

• Keywords
• Carbamates, Chromatography, Large volume sample injection, On-line solid phase extraction (on-line SPE), Pyrethroids, Surface water,
• MeSH
• Water Pollutants, Chemical analysis   MeSH
• Chromatography, Liquid MeSH
• Solid Phase Extraction methods   MeSH
• Insecticides analysis   MeSH
• Lakes MeSH
• Carbamates analysis   MeSH
• Environmental Monitoring methods   MeSH
• Online Systems MeSH
• Organic Chemicals analysis   MeSH
• Pyrethrins analysis   MeSH
• Rivers MeSH
• Reproducibility of Results MeSH
• Spectrophotometry, Ultraviolet MeSH
• Water chemistry   MeSH
• Chromatography, High Pressure Liquid methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• Water Pollutants, Chemical MeSH
• Insecticides MeSH
• Carbamates MeSH
• Organic Chemicals MeSH
• Pyrethrins MeSH
• Water MeSH

We describe the molecular etiology of β(+)-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the β-globin gene (HBB). The transcript level of the affected β-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed β-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the β-globin(L1) allele also resulted from a reduced rate of transcription and decreased production of full-length β-globin primary transcripts. The promoter and enhancer sequences of the β-globin(L1) allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the β-globin(L1) transcription despite permanent β-globin(L1) promoter CpG methylation. This result indicates that the decreased rate of transcription from the β-globin(L1) allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the β-globin gene represents a novel etiology of β-thalassemia.

• Keywords
• HBB, LINE-1, epigenetic repression, β-globin, β-thalassemia,
• MeSH
• Alleles MeSH
• Alternative Splicing MeSH
• beta-Globins genetics   MeSH
• beta-Thalassemia genetics   MeSH
• CpG Islands MeSH
• Long Interspersed Nucleotide Elements * MeSH
• Adult MeSH
• Transcription, Genetic MeSH
• Introns * MeSH
• Mutagenesis, Insertional * MeSH
• Humans MeSH
• DNA Methylation MeSH
• Gene Order MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation MeSH
• RNA Stability MeSH
• Gene Silencing MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Case Reports MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta-Globins MeSH

Transmitted light holographic microscopy is particularly used for quantitative phase imaging of transparent microscopic objects such as living cells. The study of the cell is based on extraction of the dynamic data on cell behaviour from the time-lapse sequence of the phase images. However, the phase images are affected by the phase aberrations that make the analysis particularly difficult. This is because the phase deformation is prone to change during long-term experiments. Here, we present a novel algorithm for sequential processing of living cells phase images in a time-lapse sequence. The algorithm compensates for the deformation of a phase image using weighted least-squares surface fitting. Moreover, it identifies and segments the individual cells in the phase image. All these procedures are performed automatically and applied immediately after obtaining every single phase image. This property of the algorithm is important for real-time cell quantitative phase imaging and instantaneous control of the course of the experiment by playback of the recorded sequence up to actual time. Such operator's intervention is a forerunner of process automation derived from image analysis. The efficiency of the propounded algorithm is demonstrated on images of rat fibrosarcoma cells using an off-axis holographic microscope.

• Keywords
• Aberration compensation, cell tracking, digital image processing, quantitative phase imaging, real-time holography,
• MeSH
• Algorithms MeSH
• Fibrosarcoma pathology   MeSH
• Holography methods   MeSH
• Image Interpretation, Computer-Assisted methods   MeSH
• Rats MeSH
• Microscopy, Phase-Contrast methods   MeSH
• Cell Line, Tumor MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional processing of mRNA expressed from extrachromosomal DNA.

• MeSH
• Cell Line MeSH
• Cell Cycle drug effects   MeSH
• Chlorocebus aethiops MeSH
• Cyclin-Dependent Kinases antagonists & inhibitors   MeSH
• DNA, Viral MeSH
• Phosphorylation drug effects   MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• Kidney drug effects   metabolism   MeSH
• Humans MeSH
• RNA Processing, Post-Transcriptional drug effects   MeSH
• Promoter Regions, Genetic drug effects   MeSH
• Purines pharmacology   MeSH
• RNA Polymerase II genetics   metabolism   MeSH
• Roscovitine MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cyclin-Dependent Kinases MeSH
• DNA, Viral MeSH
• Protein Kinase Inhibitors MeSH
• olomoucine II MeSH Browser
• Purines MeSH
• RNA Polymerase II MeSH
• Roscovitine MeSH