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Background: Marginal seal of a nanohybrid bulk-fill composite compared to a nanohybrid conventional composite, using a universal adhesive (UA) applied in etch-and-rinse (ER) and self-etch (SE) mode was investigated., Material and Methods: Thirty-six intact molars were selected and two standardized cavities in each tooth were prepared and allocated into four groups according to restorative material and etching strategy. All samples were placed in a 1% methylene blue solution for 24 h, then cut in the middle of the restorations obtaining two parts (n=144) and used for microscopic evaluation (50x) for dye penetration measurements., Results: The data were analysed by ANOVA and Tukey post-hoc test ( p <0.05). Marginal seal was influenced by adhesive strategy ( p <0.05) but not from the composite used ( p >0.05)., Conclusions: Simplified restorations with nanohybrid bulk-fill composite showed comparable marginal leakage to incrementally placed nanohybrid composite. The UA used with a ER technique resulted in better marginal seal irrespective of the restorative material used. Key words: Bulk-fill composite, universal adhesive, self-etching, etch-and-rinse, marginal seal., Competing Interests: Conflicts of interest All authors of this manuscript certify that they have no proprietary, financial, or other personal interest of any nature or kind in any product, service, and/or company that is presented in this article., (Copyright: © 2021 Medicina Oral S.L.)

The underlying neurological events accompanying dog domestication remain elusive. To reconstruct the domestication process in an experimental setting, silver foxes (Vulpes vulpes) have been deliberately bred for tame vs aggressive behaviors for more than 50 generations at the Institute for Cytology and Genetics in Novosibirsk, Russia. The hypothalamus is an essential part of the hypothalamic-pituitary-adrenal axis and regulates the fight-or-flight response, and thus, we hypothesized that selective breeding for tameness/aggressiveness has shaped the hypothalamic transcriptomic profile. RNA-seq analysis identified 70 differentially expressed genes (DEGs). Seven of these genes, DKKL1, FBLN7, NPL, PRIMPOL, PTGRN, SHCBP1L and SKIV2L, showed the same direction expression differences in the hypothalamus, basal forebrain and prefrontal cortex. The genes differentially expressed across the three tissues are involved in cell division, differentiation, adhesion and carbohydrate processing, suggesting an association of these processes with selective breeding. Additionally, 159 transcripts from the hypothalamus demonstrated differences in the abundance of alternative spliced forms between the tame and aggressive foxes. Weighted gene coexpression network analyses also suggested that gene modules in hypothalamus were significantly associated with tame vs aggressive behavior. Pathways associated with these modules include signal transduction, interleukin signaling, cytokine-cytokine receptor interaction and peptide ligand-binding receptors (eg, G-protein coupled receptor [GPCR] ligand binding). Current studies show the selection for tameness vs aggressiveness in foxes is associated with unique hypothalamic gene profiles partly shared with other brain regions and highlight DEGs involved in biological processes such as development, differentiation and immunological responses. The role of these processes in fox and dog domestication remains to be determined., (© 2019 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

Animal domestication efforts have led to a shared spectrum of striking behavioral and morphological changes. To recapitulate this process, silver foxes have been selectively bred for tame and aggressive behaviors for more than 50 generations at the Institute for Cytology and Genetics in Novosibirsk, Russia. To understand the genetic basis and molecular mechanisms underlying the phenotypic changes, we profiled gene expression levels and coding SNP allele frequencies in two brain tissue specimens from 12 aggressive foxes and 12 tame foxes. Expression analysis revealed 146 genes in the prefrontal cortex and 33 genes in the basal forebrain that were differentially expressed, with a 5% false discovery rate (FDR). These candidates include genes in key pathways known to be critical to neurologic processing, including the serotonin and glutamate receptor pathways. In addition, 295 of the 31,000 exonic SNPs show significant allele frequency differences between the tame and aggressive populations (1% FDR), including genes with a role in neural crest cell fate determination., Competing Interests: The authors declare no conflict of interest.

Strains of red fox (Vulpes vulpes) with markedly different behavioural phenotypes have been developed in the famous long-term selective breeding programme known as the Russian farm-fox experiment. Here we sequenced and assembled the red fox genome and re-sequenced a subset of foxes from the tame, aggressive and conventional farm-bred populations to identify genomic regions associated with the response to selection for behaviour. Analysis of the re-sequenced genomes identified 103 regions with either significantly decreased heterozygosity in one of the three populations or increased divergence between the populations. A strong positional candidate gene for tame behaviour was highlighted: SorCS1, which encodes the main trafficking protein for AMPA glutamate receptors and neurexins and suggests a role for synaptic plasticity in fox domestication. Other regions identified as likely to have been under selection in foxes include genes implicated in human neurological disorders, mouse behaviour and dog domestication. The fox represents a powerful model for the genetic analysis of affiliative and aggressive behaviours that can benefit genetic studies of behaviour in dogs and other mammals, including humans.

In the version of this Article originally published, there were some errors in the affiliations: Stephen J. O'Brien's affiliations were incorrectly listed as 8,9; they should have been 7,9. Affiliation 3 was incorrectly named the Institute of Cytology and Genetics of the Russian Academy of Sciences; it should have read Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences. Affiliation 4 was incorrectly named the Institute of Molecular and Cell Biology of the Russian Academy of Sciences; it should have read Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences. These have now been corrected.

Domesticated species exhibit a suite of behavioral, endocrinological, and morphological changes referred to as "domestication syndrome." These changes may include a reduction in reactivity of the hypothalamic-pituitary-adrenal (HPA) axis and specifically reduced adrenocorticotropic hormone release from the anterior pituitary. To investigate the biological mechanisms targeted during domestication, we investigated gene expression in the pituitaries of experimentally domesticated foxes ( Vulpes vulpes ). RNA was sequenced from the anterior pituitary of six foxes selectively bred for tameness ("tame foxes") and six foxes selectively bred for aggression ("aggressive foxes"). Expression, splicing, and network differences identified between the two lines indicated the importance of genes related to regulation of exocytosis, specifically mediated by cAMP, organization of pseudopodia, and cell motility. These findings provide new insights into biological mechanisms that may have been targeted when these lines of foxes were selected for behavior and suggest new directions for research into HPA axis regulation and the biological underpinnings of domestication., (Copyright © 2018 Hekman et al.)

Individuals involved in a social interaction exhibit different behavioral traits that, in combination, form the individual's behavioral responses. Selectively bred strains of silver foxes (Vulpes vulpes) demonstrate markedly different behaviors in their response to humans. To identify the genetic basis of these behavioral differences we constructed a large F 2 population including 537 individuals by cross-breeding tame and aggressive fox strains. 98 fox behavioral traits were recorded during social interaction with a human experimenter in a standard four-step test. Patterns of fox behaviors during the test were evaluated using principal component (PC) analysis. Genetic mapping identified eight unique significant and suggestive QTL. Mapping results for the PC phenotypes from different test steps showed little overlap suggesting that different QTL are involved in regulation of behaviors exhibited in different behavioral contexts. Many individual behavioral traits mapped to the same genomic regions as PC phenotypes. This provides additional information about specific behaviors regulated by these loci. Further, three pairs of epistatic loci were also identified for PC phenotypes suggesting more complex genetic architecture of the behavioral differences between the two strains than what has previously been observed.

There were performed researches on morphometric (the nuclear area, perimeter, the maximum and minimum diameters) and densitometric (optical density, integrated optical density) parameters of nuclei of cells on the cytologic smears received from 28 patients with atypical hyperplasia and an initial breast cancer. Significant reduction of measured nuclei parameters was noted among: moderately - and high-grade ductal carcinoma in situ, atypical ductal hyperplasia, lobular carcinoma in situ, an invasive papillary cancer, intraductal papilloma. The lowest value of the measured parameters was noted at love-grade ductal carcinoma in situ and intraductal papillary cancer. It was established that morphometric and densitometric indicators at atypical hyperplasia of an epithelium and an initial breast cancer in some cases overlap each other that is at the bottom of difficulties of differential cytomorphological diagnostics and quite often forces to formulate the cytologic conclusion in the presumable form.

Results are presented of preoperative and intraoperative cytological diagnosis of breast cancer metastases in regional lymph nodes in 84 patients. Of them in 42 there was studied material of fine-needle aspiration biopsy, in 42 - smears of sentinel lymph nodes. In all cases cytohistological comparisons were carried out. The effectiveness of cytological examination of material of fine-needle aspiration biopsy was 90.5%. Urgent cytology of smears of sentinel lymph nodes allowed revealing metastases in 5 of 10 patients. The use of immunohistochemical examination increases the likelihood of detection of micrometastases and isolated tumor cell clusters.

The silver fox (Vulpes vulpes) offers a novel model for studying the genetics of social behavior and animal domestication. Selection of foxes, separately, for tame and for aggressive behavior has yielded two strains with markedly different, genetically determined, behavioral phenotypes. Tame strain foxes are eager to establish human contact while foxes from the aggressive strain are aggressive and difficult to handle. These strains have been maintained as separate outbred lines for over 40 generations but their genetic structure has not been previously investigated. We applied a genotyping-by-sequencing (GBS) approach to provide insights into the genetic composition of these fox populations. Sequence analysis of EcoT22I genomic libraries of tame and aggressive foxes identified 48,294 high quality SNPs. Population structure analysis revealed genetic divergence between the two strains and more diversity in the aggressive strain than in the tame one. Significant differences in allele frequency between the strains were identified for 68 SNPs. Three of these SNPs were located on fox chromosome 14 within an interval of a previously identified behavioral QTL, further supporting the importance of this region for behavior. The GBS SNP data confirmed that significant genetic diversity has been preserved in both fox populations despite many years of selective breeding. Analysis of SNP allele frequencies in the two populations identified several regions of genetic divergence between the tame and aggressive foxes, some of which may represent targets of selection for behavior. The GBS protocol used in this study significantly expanded genomic resources for the fox, and can be adapted for SNP discovery and genotyping in other canid species.

The red fox (Vulpes vulpes) demonstrates a variety of coat colors including platinum, a common phenotype maintained in farm-bred fox populations. Foxes heterozygous for the platinum allele have a light silver coat and extensive white spotting, whereas homozygosity is embryonic lethal. Two KIT transcripts were identified in skin cDNA from platinum foxes. The long transcript was identical to the KIT transcript of silver foxes, whereas the short transcript, which lacks exon 17, was specific to platinum. The KIT gene has several copies in the fox genome: an autosomal copy on chromosome 2 and additional copies on the B chromosomes. To identify the platinum-specific KIT sequence, the genomes of one platinum and one silver fox were sequenced. A single nucleotide polymorphism (SNP) was identified at the first nucleotide of KIT intron 17 in the platinum fox. In platinum foxes, the A allele of the SNP disrupts the donor splice site and causes exon 17, which is part of a segment that encodes a conserved tyrosine kinase domain, to be skipped. Complete cosegregation of the A allele with the platinum phenotype was confirmed by linkage mapping (LOD 25.59). All genotyped farm-bred platinum foxes from Russia and the US were heterozygous for the SNP (A/G), whereas foxes with different coat colors were homozygous for the G allele. Identification of the platinum mutation suggests that other fox white-spotting phenotypes, which are allelic to platinum, would also be caused by mutations in the KIT gene., (© 2015 Stichting International Foundation for Animal Genetics.)

This paper presents an analysis of the results of cytological diagnostics of breast tumors in 3415 patients who underwent tests in the Laboratory of Cytology for 2009-2013. In 1434 patients there were performed cytohistological correlations. Sensitivity of cytology was 99.1%, specificity--93.5%, predictive value of the positive test--98.5%, predictive value of the negative test--95.9%, accuracy--98%. Non-informative material was obtained in 13.5% of cases. The main factors that reduced effectiveness of cytological method were non-informative and little informative material and also difficulties in conducting of differential cytomorphological diagnostics.

Background: Two strains of the silver fox (Vulpes vulpes), with markedly different behavioral phenotypes, have been developed by long-term selection for behavior. Foxes from the tame strain exhibit friendly behavior towards humans, paralleling the sociability of canine puppies, whereas foxes from the aggressive strain are defensive and exhibit aggression to humans. To understand the genetic differences underlying these behavioral phenotypes fox-specific genomic resources are needed., Results: cDNA from mRNA from pre-frontal cortex of a tame and an aggressive fox was sequenced using the Roche 454 FLX Titanium platform (> 2.5 million reads & 0.9 Gbase of tame fox sequence; >3.3 million reads & 1.2 Gbase of aggressive fox sequence). Over 80% of the fox reads were assembled into contigs. Mapping fox reads against the fox transcriptome assembly and the dog genome identified over 30,000 high confidence fox-specific SNPs. Fox transcripts for approximately 14,000 genes were identified using SwissProt and the dog RefSeq databases. An at least 2-fold expression difference between the two samples (p < 0.05) was observed for 335 genes, fewer than 3% of the total number of genes identified in the fox transcriptome., Conclusions: Transcriptome sequencing significantly expanded genomic resources available for the fox, a species without a sequenced genome. In a very cost efficient manner this yielded a large number of fox-specific SNP markers for genetic studies and provided significant insights into the gene expression profile of the fox pre-frontal cortex; expression differences between the two fox samples; and a catalogue of potentially important gene-specific sequence variants. This result demonstrates the utility of this approach for developing genomic resources in species with limited genomic information.

During the second part of the twentieth century, Belyaev selected tame and aggressive foxes (Vulpes vulpes), in an effort known as the "farm-fox experiment", to recapitulate the process of animal domestication. Using these tame and aggressive foxes as founders of segregant backcross and intercross populations we have employed interval mapping to identify a locus for tame behavior on fox chromosome VVU12. This locus is orthologous to, and therefore validates, a genomic region recently implicated in canine domestication. The tame versus aggressive behavioral phenotype was characterized as the first principal component (PC) of a PC matrix made up of many distinct behavioral traits (e.g. wags tail; comes to the front of the cage; allows head to be touched; holds observer's hand with its mouth; etc.). Mean values of this PC for F1, backcross and intercross populations defined a linear gradient of heritable behavior ranging from tame to aggressive. The second PC did not follow such a gradient, but also mapped to VVU12, and distinguished between active and passive behaviors. These data suggest that (1) there are at least two VVU12 loci associated with behavior; (2) expression of these loci is dependent on interactions with other parts of the genome (the genome context) and therefore varies from one crossbred population to another depending on the individual parents that participated in the cross.

Strains of silver foxes, selectively bred at the Institute of Cytology and Genetics of the Russian Academy of Sciences, are a well established, novel model for studying the genetic basis of behavior, and the processes involved in canine domestication. Here we describe a method to measure fox behavior as quantitative phenotypes which distinguish populations and resegregate in experimental pedigrees. We defined 50 binary observations that nonredundantly and accurately distinguished behaviors in reference populations and cross-bred pedigrees. Principal-component analysis dissected out the independent elements underlying these behaviors. PC1 accounted for >44% of the total variance in measured traits. This system clearly distinguished tame foxes from aggressive and wildtype foxes. F1 foxes yield intermediate values that extend into the ranges of both the tame and aggressive foxes, while the scores of the backcross generation resegregate. These measures can thus be used for QTL mapping to explore the genetic basis of tame and aggressive behavior in foxes, which should provide new insights into the mechanisms of mammalian behavior and canine domestication.

Small interfering RNAs (siRNA) provide a powerful approach for sequence-specific silencing of gene expression. In the present study we investigated inhibition of c-myc gene expression by siRNAs targeted to the sequence 1452-1470 b. in third exon of c-myc mRNA and to homologous regions in second exons of c-myc (697-715 b.) and N-myc (302-320 b.) mRNAs. siRNAs were prepared enzymatically according to the scheme, including dsDNA-templates preparation using Klenow fragment, separate in vitro transcription of each RNA strand with subsequent hybridization and removal of leader sequences by T1 RNase. Investigation of c-myc gene silencing by siRNAs revealed that enzymatically prepared siRNAs induce stronger inhibition of c-myc expression, than siRNA with the same sequence prepared by chemical synthesis. It was found that down-regulation of c-myc gene expression by investigated siRNAs results in efficient inhibition and even complete arrest of carcinoma cell proliferation, moreover, the extend of growth inhibition correlates with the level of siRNA-mediated reduction of c-myc mRNA.

Interaction of oligonucleotides condensed into long concatemeric complexes with cancer cells was investigated. Pairs of 24- and 25-mer oligodeoxyribonucleotides were designed so that they could hybridize and form concatemeric structures. Pre-assembling of the oligonucleotides into concatemers considerably enhanced their ability to bind to human embryo kidney 293 cells and neuroblastoma IMR-32 cells as compared to free oligonucleotides. Efficiency of concatemers binding to the cells is improved with increase of the length and concentration of concatemeric complexes. The obtained results suggest incorporation of pharmacologically active oligonucleotides into concatemeric complexes as an approach to improvement of their cellular interaction.

Suppression of c-myc proto-oncogene expression by small interfering RNA (siRNA) in human epidermoid carcinoma KB-3-1 and neuroblastoma SK-N-MC cell lines was investigated. The siRNA duplex targeted to the exon 3 of c-myc mRNA (siRNA-I) was prepared by in vitro transcription using T7 RNA polymerase and short double-stranded DNA (dsDNA) templates. siRNA-I was shown to efficiently decrease c-myc mRNA expression in both tumor cell lines and to arrest their proliferation. Incubation of KB-3-1 cells with 150 nM siRNA-I results in a 92% decrease in the c-myc mRNA level and an 83% decrease in the protein level. In SK-N-MC cells, 150 nM siRNA-I causes a 60% decrease in the c-myc mRNA level and a 55% decrease in the protein level. The reduction of the c-myc mRNA level correlates with the inhibition of cell proliferation; 150 nM siRNA-I causes a 2.5-fold reduction in the SK-N-MC proliferation rate and a 15-fold decrease in the proliferation rate and complete arrest of cell division in KB-3-1 cells. siRNA-I has little effect on proliferation of the IMR-32 cells that overexpress the N-myc but not the c-myc gene, demonstrating that siRNA-I antiproliferation activity is mediated by specific block of c-myc expression.

Inhibition of p-glycoprotein (PGP) expression and reverse of multidrug resistance (MDR) phenotype in KB-8-5 cells by synthetic 21-bp double-stranded oligoribonucleotides were investigated. siRNA constructs for the efficient down regulation of MDR1 that are active in nanomolar concentrations and cause reversal of MDR phenotype in cells were developed.

Suppression of c-myc protooncogene expression in KB-3-1 cells by siRNA was investigated. The siRNA duplex targeted to the exon 3 of c-myc mRNA was prepared by in vitro transcription with T7 RNA polymerase on short dsDNA-templates. It was found that incubation of KB-3-1 cells in the presence of 75 nM siRNA results in decrease of the c-myc mRNA level down to 5% of the level in the control cells and significant decline of KB-3-1 cell proliferation rate. Using 200 nM siRNA four-fold decrease of KB-3-1 cells proliferation rate was observed and this effect was stable at least 96 h after transfection.

Interaction of transcription factors with functional regions of gene mdr1 promoter has been studied. Binding of transcription factors to double-stranded (ds) oligonucleotides, mimicking four important regulatory regions before and after treatment of KB and K-562 cell lines with doxorubicin, cytarabine, and vinblastine was analysed by gel-shift assay. Gene induction resulted in enhancement of the main complex A(A1) proteins formation on the all four regions in both the cell lines. Inhibition of each complex formation by an excess of non-radiolabelled ds oligonucleotides suggests the possibility of regions interaction during promoter activation. Another indication of interaction of regulatory regions during activation is overlapping minor complexes sets. The results suggest that the four studied regulatory regions are important for mdr1 gene activation. Corresponding oligonucleotides mimicking these regions can be employed as inhibitors of gene transcription that bind specific transcriptional factors.