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Abstract A large proportion of colorectal carcinomas (CRC) evolve from colorectal adenomas. However, not all individuals with colonic adenomas have a risk of CRC substantially higher than those of the general population. The aim of the study was to determine the differences or similarities of mutation profile among low- and high-grade adenomas and in situ carcinoma with detailed follow up. We have investigated the mutation spectrum of well-known genes involved in CRC (such as APC, BRAF, EGFR, NRAS, KRAS, PIK3CA, POLE, POLD1, SMAD4, PTEN, and TP53) in a large, well-defined series of 96 adenomas and in situ carcinomas using a high-throughput genotyping technique. Besides, the microsatellite instability and APC and MLH1 promoter methylation were studied as well. We observed a high frequency of pathogenic variants in the studied genes. The APC, KRAS and TP53 mutation frequencies were slightly lower in adenoma samples than in in situ carcinoma samples. Further, when we stratified mutation frequency based on the grade, the frequency distribution was as follows: low-grade adenoma—high-grade adenomas—in situ carcinoma: APC gene 42.9–56.0–54.5%; KRAS gene 32.7–32.0–45.5%; TP53 gene 8.2–20.0–18.2%. The occurrence of KRAS mutation was associated with the presence of villous histology and methylation of the APC promoter was significantly associated with the presence of POLE genetic variations. However, no association was noticed with the presence of any singular mutation and occurrence of subsequent adenoma or CRC. Our data supports the multistep model of gradual accumulation of mutations, especially in the driver genes, such as APC, TP53 and KRAS.

Abstract Background Siderophores are small iron-binding molecules produced by microorganisms to facilitate iron acquisition from the environment. Radiolabelled siderophores offer a promising solution for infection imaging, as they can specifically target the pathophysiological mechanisms of pathogens. Gallium-68 can replace the iron in siderophores, enabling molecular imaging with positron emission tomography (PET). Stereospecific interactions play a crucial role in the recognition of receptors, transporters, and iron utilisation. Furthermore, these interactions have an impact on the host environment, affecting pharmacokinetics and biodistribution. This study examines the influence of siderophore stereoisomerism on imaging properties, with a focus on ferrirubin (FR) and ferrirhodin (FRH), two cis–trans isomeric siderophores of the ferrichrome type. Results Tested siderophores were labelled with gallium-68 with high radiochemical purity. The resulting complexes differed in their in vitro characteristics. [68Ga]Ga-FRH showed less hydrophilic properties and higher protein binding values than [68Ga]Ga-FR. The stability studies confirmed the high radiochemical stability of both [68Ga]Ga-siderophores in all examined media. Both siderophores were found to be taken up by S. aureus, K. pneumoniae and P. aeruginosa with similar efficacy. The biodistribution tested in normal mice showed rapid renal clearance with low blood pool retention and fast clearance from examined organs for [68Ga]Ga-FR, whereas [68Ga]Ga-FRH showed moderate retention in blood, resulting in slower pharmacokinetics. PET/CT imaging of mice injected with [68Ga]Ga-FR and [68Ga]Ga-FRH confirmed findings from ex vivo biodistribution studies. In a mouse model of S. aureus myositis, both radiolabeled siderophores showed radiotracer accumulation at the site of infection. Conclusions The 68Ga-complexes of stereoisomers ferrirubin and ferrirhodin revealed different pharmacokinetic profiles. In vitro uptake was not affected by isomerism. Both compounds had uptake with the same bacterial culture with similar efficacy. PET/CT imaging showed that the [68Ga]Ga-complexes accumulate at the site of S. aureus infection, highlighting the potential of [68Ga]Ga-FR as a promising tool for infection imaging. In contrast, retention of the radioactivity in the blood was observed for [68Ga]Ga-FRH. In conclusion, the stereoisomerism of potential radiotracers should be considered, as even minor structural differences can influence their pharmacokinetics and, consequently, the results of PET imaging.

The collapsin response mediator protein (CRMP) family proteins are intracellular mediators of neurotrophic factors regulating neurite structure/spine formation and are essential for dendrite patterning and directional axonal pathfinding during brain developmental processes. Among this family, CRMP5/DPYSL5 plays a significant role in neuronal migration, axonal guidance, dendrite outgrowth, and synapse formation by interacting with microtubules. Here, we report the identification of missense mutations in DPYSL5 in nine individuals with brain malformations, including corpus callosum agenesis and/or posterior fossa abnormalities, associated with variable degrees of intellectual disability. A recurrent de novo p.Glu41Lys variant was found in eight unrelated patients, and a p.Gly47Arg variant was identified in one individual from the first family reported with Ritscher-Schinzel syndrome. Functional analyses of the two missense mutations revealed impaired dendritic outgrowth processes in young developing hippocampal primary neuronal cultures. We further demonstrated that these mutations, both located in the same loop on the surface of DPYSL5 monomers and oligomers, reduced the interaction of DPYSL5 with neuronal cytoskeleton-associated proteins MAP2 and βIII-tubulin. Our findings collectively indicate that the p.Glu41Lys and p.Gly47Arg variants impair DPYSL5 function on dendritic outgrowth regulation by preventing the formation of the ternary complex with MAP2 and βIII-tubulin, ultimately leading to abnormal brain development. This study adds DPYSL5 to the list of genes implicated in brain malformation and in neurodevelopmental disorders.

ABSTRACTThe need for integrated passive devices (IPDs) emerges from the increasing consumer demand for electronic product miniaturization. Metal-insulator-metal (MIM) capacitors are vital components of IPD systems. Developing new materials and technologies is essential for advancing capacitor characteristics and co-integrating with other electronic passives. Here we present an innovative electrochemical technology joined with the sputter-deposition of Al and Zr layers to synthesize novel planar nanocomposite metal-oxide dielectrics consisting of ZrO2 nanorods self-embedded into the nanoporous Al2O3 matrix such that its pores are entirely filled with zirconium oxide. The technology is utilized in MIM capacitors characterized by modern surface and interface analysis techniques and electrical measurements. In the 95–480 nm thickness range, the best-achieved MIM device characteristics are the one-layer capacitance density of 112 nF·cm−2, the loss tangent of 4·10−3 at frequencies up to 1 MHz, the leakage current density of 40 pA·cm−2, the breakdown field strength of up to 10 MV·cm−1, the energy density of 100 J·cm−3, the quadratic voltage coefficient of capacitance of 4 ppm·V−2, and the temperature coefficient of capacitance of 480 ppm·K−1 at 293–423 K at 1 MHz. The outstanding performance, stability, and tunable capacitors’ characteristics allow for their application in low-pass filters, coupling/decoupling/bypass circuits, RC oscillators, energy-storage devices, ultrafast charge/discharge units, or high-precision analog-to-digital converters. The capacitor technology based on the non-porous planar anodic-oxide dielectrics complements the electrochemical conception of IPDs that combined, until now, the anodized aluminum interconnection, microresistors, and microinductors, all co-related in one system for use in portable electronic devices.

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Most genes associated with neurodevelopmental disorders (NDDs) were identified with an excess of de novo mutations (DNMs) but the significance in case-control mutation burden analysis is unestablished. Here, we sequence 63 genes in 16,294 NDD cases and an additional 62 genes in 6,211 NDD cases. By combining these with published data, we assess a total of 125 genes in over 16,000 NDD cases and compare the mutation burden to nonpsychiatric controls from ExAC. We identify 48 genes (25 newly reported) showing significant burden of ultra-rare (MAF

Abstract Background Congenital myasthenic syndromes (CMSs) are characterized by hypotonia, episodic apnea, muscle weakness, ptosis and generalized fatigability. CMS type 20 (CMS20) is a rare disorder caused by variants in SLC5A7. In contrast to most other CMSs, CMS20 is also associated with neurodevelopmental disorders (NDDs). Only 19 patients from 14 families have been reported so far. Methods We studied a 12‐year‐old boy with symptoms manifested at six weeks of age. Later, he also showed speech delay, moderate intellectual disability and autism. Analysis of CMS genes known at the time of clinical diagnosis yielded no results. Trio exome sequencing (ES) was performed. Results ES revealed compound heterozygosity for two SLC5A7 variants, p.(Asn431Lys) and p.(Ile291Thr). While the first variant was absent from all databases, the second variant has already been described in one patient. In silico analysis of known pathogenic SLC5A7 variants showed that variants with a higher predicted deleteriousness may be associated with earlier onset and increased severity of neuromuscular manifestations. Conclusion Our patient confirms that CMS20 can be associated with NDDs. The study illustrates the strength of ES in deciphering the genetic basis of rare diseases, contributes to characterization of CMS20 and suggests trends in genotype–phenotype correlation in CMS20.

Angiostrongylus vasorum and Crenosoma vulpis are parasites in the respiratory tract of domestic and wild carnivores. Recently, an increasing number of lungworm infections in dogs has been recognized in Europe. In this report, the results of the first copro-epidemiological study of A. vasorum and C. vulpis in dogs from Slovakia are presented. The Baermann technique and the modified flotation method with zinc sulphate solution (specific gravity 1.2) was used for lungworm detection. In addition, conventional PCR was performed for species confirmation. The majority of lungworm infections were found in the eastern part of the country. A relatively high prevalence (4.13%) of A. vasorum was detected in different parts of Slovakia. Infection was detected most frequently incidentally in asymptomatic dogs. Within this study, crenosomosis was detected for the first time in dogs from Slovakia. Since one infected dog had no travel history, the case is considered autochthonous. An autochthonous case report of angiostrongylosis in a Hanoverian hound puppy from central Slovakia is also described.

Abstract Background Intellectual disability (ID) is a feature of many rare diseases caused by thousands of genes. This genetic heterogeneity implies that pathogenic variants in a specific gene are found only in a small number of patients, and difficulties arise in the definition of prevailing genotype and characteristic phenotype associated with that gene. One of such very rare disorders is autosomal recessive ID type 66 (OMIM #618221) caused by defects in C12orf4. Up to now, six families have been reported with mostly truncating variants. The spectrum of the clinical phenotype was not emphasized in previous reports, and detailed phenotype was not always available from previous patients, especially from large cohort studies. Methods Exome sequencing was performed in a consanguineous Armenian family with two affected adult brothers. Results The patients carry a novel homozygous nonsense C12orf4 variant. The integration of previous data and phenotyping of the brothers indicate that the clinical picture of C12orf4 defects involves hypotonia in infancy, rather severe ID, speech impairment, and behavioral problems such as aggressiveness, unstable mood, and autistic features. Several other symptoms are more variable and less consistent. Conclusion This rather nonsyndromic and nonspecific clinical picture implies that additional patients with C12orf4 defects will likely continue to be identified using the “genotype‐first” approach, rather than based on clinical assessment. The phenotype needs further delineation in future reports.

The circadian rhythms in physiological and behavioral functions are driven by a pacemaker located in the suprachiasmatic nucleus (SCN). The rhythms continue in constant darkness and depend on cell-cell communication between neurons and glia. The SCN astrocytes generate also a circadian rhythm in extracellular adenosine 5′-triphosphate (ATP) accumulation, but molecular mechanisms that regulate ATP release are poorly understood. Here, we tested the hypothesis that ATP is released via the plasma membrane purinergic P2X7 receptors (P2X7Rs) and P2Y receptors (P2YRs) which have been previously shown to be expressed in the SCN tissue at transcriptional level. We have investigated this hypothesis using SCN organotypic cultures, primary cultures of SCN astrocytes, ATP bioluminescent assays, immunohistochemistry, patch-clamping, and calcium imaging. We found that extracellular ATP accumulation in organotypic cultures followed a circadian rhythm, with a peak between 24:00 and 04:00 h, and the trough at ~12:00 h. ATP rhythm was inhibited by application of AZ10606120, A438079, and BBG, specific blockers of P2X7R, and potentiated by GW791343, a positive allosteric modulator of this receptor. Double-immunohistochemical staining revealed high expression of the P2X7R protein in astrocytes of SCN slices. PPADS, a non-specific P2 antagonist, and MRS2179, specific P2Y1R antagonist, also abolished ATP rhythm, whereas the specific P2X4R blocker 5-BDBD was not effective. The pannexin-1 hemichannel blocker carbenoxolone displayed a partial inhibitory effect. The P2Y1R agonist MRS2365, and the P2Y2R agonist MRS2768 potentiated ATP release in organotypic cultures and increase intracellular Ca2+ level in cultured astrocytes. Thus, SCN utilizes multiple purinergic receptor systems and pannexin-1 hemichannels to release ATP.

Lens induction is a classical developmental model allowing investigation of cell specification, spatiotemporal control of gene expression, as well as how transcription factors are integrated into highly complex gene regulatory networks (GRNs). Pax6 represents a key node in the gene regulatory network governing mammalian lens induction. Meis1 and Meis2 homeoproteins are considered as essential upstream regulators of Pax6 during lens morphogenesis based on their interaction with the ectoderm enhancer (EE) located upstream of Pax6 transcription start site. Despite this generally accepted regulatory pathway, Meis1-, Meis2- and EE-deficient mice have surprisingly mild eye phenotypes at placodal stage of lens development. Here, we show that simultaneous deletion of Meis1 and Meis2 in presumptive lens ectoderm results in arrested lens development in the pre-placodal stage, and neither lens placode nor lens is formed. We found that in the presumptive lens ectoderm of Meis1/Meis2 deficient embryos Pax6 expression is absent. We demonstrate using chromatin immunoprecipitation (ChIP) that in addition to EE, Meis homeoproteins bind to a remote, ultraconserved SIMO enhancer of Pax6. We further show, using in vivo gene reporter analyses, that the lens-specific activity of SIMO enhancer is dependent on the presence of three Meis binding sites, phylogenetically conserved from man to zebrafish. Genetic ablation of EE and SIMO enhancers demostrates their requirement for lens induction and uncovers an apparent redundancy at early stages of lens development. These findings identify a genetic requirement for Meis1 and Meis2 during the early steps of mammalian eye development. Moreover, they reveal an apparent robustness in the gene regulatory mechanism whereby two independent "shadow enhancers" maintain critical levels of a dosage-sensitive gene, Pax6, during lens induction.

The aim of our study was to determine the prevalence of hepatitis E virus (HEV) in domestic pigs and to investigate the genetic divergence of swine HEV in the Czech Republic. To this end, a one-step real time RT-PCR assay was introduced as a screening method while nested RT-PCR was used as an additional method to obtain specific sequences from the HEV genome and thus to perform sequence analysis. A total of 63 piglets originating from 14 farms were examined. Bile and intestinal contents were collected from each animal. At least one HEV RNA positive piglet was found in ten (71.4%) of the monitored farms. HEV RNA was most frequently detected in bile samples (34.9%) compared to intestinal content samples (22.2%). In nine piglets (14.3%), both biological samples were HEV RNA-positive. Based on these results sequence and phylogenetic analysis of one randomly selected HEV isolate originating from each HEV RNA-positive farm was performed. Analysis of 287 bp PCR products of the ORF1 gene showed that all the studied HEV isolates could be classified into genotype 3 and subgenotypes 3f and 3g. The failure to find any 100% homology between our isolates and HEV isolates deposited in the GenBank confirms the significant variability within the HEV genome.

Effects of sodium humate (HuNa), containing 61.9% of humic acids in dry matter, on utilisation and retention of dietary iodine were investigated in balance experiments carried out in pigs. A control group was fed a commercial diet containing 0.1 mg of iodine per kg. The daily intake of iodine was 129.2 µg per animal or 4.08 µg per kg live weight. The recommended intake of dietary iodine is 0.25 to 0.30 mg per kg at 88% dry matter. The experimental group (HuNa) was fed the same diet supplemented with 3% of sodium humate which contained 0.5 mg of iodine per kg at 90.04% dry matter. The supplementation increased the daily intake to 141.4 µg per animal, or 4.45 µg per kg live weight. The supplementation of HuNa increased significantly the amount of iodine excreted in faeces (53.2 ± 11.0 vs. 43.0 ± 6.47 µg per animal per day; P < 0.05) and nonsignificantly the urinary iodine excretion (31.8 ± 8.93 vs. 29.0 ± 11.3 µg per animal per day). Differences between the HuNa and the control groups in blood serum iodine concentrations (18.8 vs. 18.1 µg per litre) and urinary iodine concentrations (32.5 vs. 34.4 µg per litre) were nonsignificant. It is evident from the concentrations that the pigs suffered from a serious iodine deficiency. Compared with controls, the HuNa group showed significantly higher triiodothyronine concentration (P < 0.05) and nonsignificantly lower thyroxine and iodine concentrations in blood serum. Dietary iodine utilisation was lower in the HuNa than in the control group (62.3 vs. 66.6%). The results show that utilisation of dietary iodine was limited and that sodium humate (humic acids) reduced iodine utilisation and retention and affected also other parameters indicating their goitrogenic effects.

Background and Aims. A very common statistical method used in statistical analysis of medical data is the Cox proportional hazard model (Cox model). The aim of this paper is to describe statistical data analysis as a process beginning with data management, followed by statistical coding and statistical analysis, including survival analysis and the Cox model, and finishing with the interpretation of results. The methods are applied to the data from stomatology - the influence of selected variables on the fracture of endodontic equipment. Statistical analyses were performed in R. Methods. Exploratory analysis and visualization methods are firstly presented. The survival analysis methods of Kaplan- Meier's product limit estimate, estimate of the cumulative hazard function, and Nair's 95% confidence bands are then calculated. Finally, the conditional hazard is modelled with the Cox model where regression parameters are tested with the Wald test and the proportionality of hazard with the Schoenfeld test. Results. In the Cox model, covariates influencing the fracture of endodontic equipment, the radius of the dental canal, the size and brand of the endodontic equipment (Mtwo, ProTaper, Revo-S, and Wizard Navigator) were used. The regression parameters of the radius of the dental canal and endodontic equipment size proved to be statistically significant. Compared to the reference brand Mtwo endodontic equipment, the regression parameters of the ProTaper and Revo-S drills proved to be statistically significant. Conclusions. Fracturing endodontic equipment during a root canal treatment often leads to undesirable resting of the tool in the root canal of the patient's tooth, which can lead to less successful endodontic treatment and thus less satisfactory prognosis. The dentist's efforts should allow the use of endodontic equipment for sufficient root canal treatment with minimum risk of fracturing the equipment inside the root system. Removal of the equipment is often very difficult and sometimes fails, resulting in subsequent hermetic closure of the root canal by the root filling not being possible and thereby greatly reducing satisfactory prognosis for the tooth. [ABSTRACT FROM AUTHOR]

Arrays of self-organized WO 3 -based semiconductor nanorods are prepared from a thin W layer, W/Ti bilayer (tungsten-on-titanium), and W-10at.%Ti alloy layer via the porous-anodic-alumina (PAA)-assisted anodization at various conditions to address the radius/length ratio of~13/130 and ~70/700 nm (respectively ‘small’ and ‘big’ nanorods). Doping the WO 3 nanorods with TiO 2 was achieved, for the first time, simply by anodizing the W/Ti and W-10at.%Ti layers through the alumina nanopores. The post-anodizing treatments combined PAA dissolution with annealing in air and vacuum at 500–550 °C to alter the film composition, crystal structure, and electrical properties. The air-annealed big nanorods comprising monoclinic and triclinic WO 3 crystal phases reveal their superior performance in photoelectrochemical (PEC) water splitting, showing a low onset potential (0.5 V RHE ) and a competitive value of photocurrent (15.5 mA cm −2 ) in 0.1 mol dm −3 Na 2 SO 4 solution (pH 5.0) under chopped illumination at a single wavelength of 405 nm, 1 W cm −2 , with no sign of photocorrosion. Paradoxically, the presence of monoclinic WO 2.9 phase in the vacuum-annealed nanorods worsens the PEC behavior and stimulates the peroxo-assisted dissolution. Unexpectedly, electrochemically doping both the WO 3 and WO 2.9 big nanorods with TiO 2 causes the photocurrent to decrease dramatically. An advanced approach developed for modeling charge transport processes in the PAA-assisted WO x nanorods predicts a 7-fold further rise in the solar current should the big nanorods grow longer (1.5 µm) and wider (300 nm) to absorb a bigger portion of light and support a thicker depletion layer, without, however, getting fully depleted, which is the case of the small nanorods. [ABSTRACT FROM AUTHOR]

Here we synthesize a 3-D metal/oxide/metal nanostructured film that merges the benefits of advanced nanocomposite inorganic materials with the flexibility of nonlithographic electrochemical technologies based on so-called porous-anodic-alumina-assisted anodizing of a refractory metal and the point electrodeposition of noble metals. The film is composed of a thin niobium oxide layer with spatially-ordered upright-standing niobium oxide nanocolumns, assembled between the two parallel electrodes, which work as long aspect ratio semiconducting nanochannels whose resistivity is greatly impacted by chemisorption reactions when a gas interacts with the film. A laboratory gas sensor employing the film, assembled on a standard TO-8 Metal Can Package, shows superior characteristics for H 2 and especially C 2 H 5 OH detection. [ABSTRACT FROM AUTHOR]