Null cross-modal effects of olfactory training on visual, auditory or olfactory working memory in 6- to 9-year-old children.

Systematic exposure to odours (olfactory training, OT) is a method of smell loss treatment. Due to olfactory system projections to prefrontal brain areas, OT has been hypothesized to enhance cognitive functions, but its effects have been studied predominantly in adults. This study tested OT effects on working memory (WM), i.e., the ability to store and manipulate information for a short time, in healthy children aged 6-9 years. We expected OT to improve olfactory WM and establish cross-modal transfer to visual and auditory WM. Participants performed 12 weeks of bi-daily OT with either 4 odours (lemon, eucalyptus, rose, cloves; OT group) or odourless propylene glycol (placebo group). Pre- and post-training, participants' WM was measured utilizing odours (olfactory WM) or pictures (visual WM) and a word-span task (auditory WM). 84 children (40 girls) completed the study. The analyses revealed no changes in the WM performance following OT. The olfactory WM task was the most difficult for children, highlighting the need to include olfactory-related tasks in educational programmes to improve children's odour knowledge and memory, just as they learn about sounds and pictures. Further neuroimaging research is needed to fully understand the impact of OT on cognitive functions in children.

A pan-cancer atlas of somatic mutations in miRNA biogenesis genes.

It is a well-known and intensively studied phenomenon that the levels of many miRNAs are differentiated in cancer. miRNA biogenesis and functional expression are complex processes orchestrated by many proteins cumulatively called miRNA biogenesis proteins. To characterize cancer somatic mutations in the miRNA biogenesis genes and investigate their potential impact on the levels of miRNAs, we analyzed whole-exome sequencing datasets of over 10 000 cancer/normal sample pairs deposited within the TCGA repository. We identified and characterized over 3600 somatic mutations in 29 miRNA biogenesis genes and showed that some of the genes are overmutated in specific cancers and/or have recurrent hotspot mutations (e.g. SMAD4 in PAAD, COAD and READ; DICER1 in UCEC; PRKRA in OV and LIN28B in SKCM). We identified a list of miRNAs whose level is affected by particular types of mutations in either SMAD4, SMAD2 or DICER1 and showed that hotspot mutations in the RNase domains in DICER1 not only decrease the level of 5p-miRNAs but also increase the level of 3p-miRNAs, including many well-known cancer-related miRNAs. We also showed an association of the mutations with patient survival. Eventually, we created an atlas/compendium of miRNA biogenesis alterations providing a useful resource for different aspects of biomedical research.

Essential thrombocythaemia progression to the fibrotic phase is associated with a decrease in JAK2 and PDL1 levels.

It has been postulated that the changes in the molecular characteristics of the malignant clone(s) and the abnormal activation of JAK-STAT signaling are responsible for myeloproliferative neoplasm progression to more advanced disease phases and the immune escape of the malignant clone. The continuous JAK-STAT pathway activation leads to enhanced activity of the promoter of CD274 coding programmed death-1 receptor ligand (PD-L1), increased PD-L1 level, and the immune escape of MPN cells. The aim of study was to evaluate the PDL1 mRNA and JAK2 mRNA level in molecularly defined essential thrombocythaemia (ET) patients (pts) during disease progression to post-ET- myelofibrosis (post-ET-MF). The study group consisted of 162 ET pts, including 30 pts diagnosed with post-ET-MF. The JAK2V617F, CALR, and MPL mutations were found in 59.3%, 19.1%, and 1.2% of pts, respectively. No copy-number alternations of the JAK2, PDL1, and PDCDL1G2 (PDL2) genes were found. The level of PD-L1 was significantly higher in the JAK2V617F than in the JAK2WT, CALR mutation-positive, and triple-negative pts. The PD-L1 mRNA level was weakly correlated with both the JAK2V617F variant allele frequency (VAF), and with the JAK2V617F allele mRNA level. The total JAK2 level in post-ET-MF pts was lower than in ET pts, despite the lack of differences in the JAK2V617F VAF. In addition, the PD-L1 level was lower in post-ET-MF. A detailed analysis has shown that the decrease in JAK2 and PDL1 mRNA levels depended on the bone marrow fibrosis grade. The PDL1 expression showed no differences in relation to the genotype of the JAK2 haplotypeGGCC_46/1, hemoglobin concentration, hematocrit value, leukocyte, and platelet counts. The observed drop of the total JAK2 and PDL1 levels during the ET progression to the post-ET-MF may reflect the changes in the JAK2V617F positive clone proliferative potential and the PD-L1 level-related immunosuppressive effect. The above-mentioned hypothesis is supported by The Cancer Genome Atlas (TCGA) data, confirming a strong positive association between CD274 (encoding PD-L1), CXCR3 (encoding CXCR3), and CSF1 (encoding M-CSF) expression levels, and recently published results documenting a drop in the CXCR3 level and circulating M-CSF in patients with post-ET-MF.

Novel fluorescent-based reporter cell line engineered for monitoring homologous recombination events.

Homologous recombination (HR) faithfully restores DNA double-strand breaks. Defects in this HR repair pathway are associated with cancer predisposition. In genetic engineering, HR has been used extensively to study gene function and it represents an ideal method of gene therapy for single gene disorders. Here, we present a novel assay to measure HR in living cells. The HR substrate consisted of a non-fluorescent 3' truncated form of the eGFP gene and was integrated into the AAVS1 locus, known as a safe harbor. The donor DNA template comprised a 5' truncated eGFP copy and was delivered via AAV particles. HR mediated repair restored full-length eGFP coding sequence, resulting in eGFP+ cells. The utility of our assay in quantifying HR events was validated by exploring the impact of the overexpression of HR promoters and the siRNA-mediated silencing of genes known to play a role in DNA repair on the frequency of HR. We conclude that this novel assay represents a useful tool to further investigate the mechanisms that control HR and test continually emerging tools for HR-mediated genome editing.

Profile of Basal Cell Carcinoma Mutations and Copy Number Alterations - Focus on Gene-Associated Noncoding Variants.

Basal cell carcinoma (BCC) of the skin is the most common cancer in humans, characterized by the highest mutation rate among cancers, and is mostly driven by mutations in genes involved in the hedgehog pathway. To date, almost all BCC genetic studies have focused exclusively on protein-coding sequences; therefore, the impact of noncoding variants on the BCC genome is unrecognized. In this study, with the use of whole-exome sequencing of 27 tumor/normal pairs of BCC samples, we performed an analysis of somatic mutations in both protein-coding sequences and gene-associated noncoding regions, including 5'UTRs, 3'UTRs, and exon-adjacent intron sequences. Separately, in each region, we performed hotspot identification, mutation enrichment analysis, and cancer driver identification with OncodriveFML. Additionally, we performed a whole-genome copy number alteration analysis with GISTIC2. Of the >80,000 identified mutations, ~50% were localized in noncoding regions. The results of the analysis generally corroborated the previous findings regarding genes mutated in coding sequences, including PTCH1, TP53, and MYCN, but more importantly showed that mutations were also clustered in specific noncoding regions, including hotspots. Some of the genes specifically mutated in noncoding regions were identified as highly potent cancer drivers, of which BAD had a mutation hotspot in the 3'UTR, DHODH had a mutation hotspot in the Kozak sequence in the 5'UTR, and CHCHD2 frequently showed mutations in the 5'UTR. All of these genes are functionally implicated in cancer-related processes (e.g., apoptosis, mitochondrial metabolism, and de novo pyrimidine synthesis) or the pathogenesis of UV radiation-induced cancers. We also found that the identified BAD and CHCHD2 mutations frequently occur in melanoma but not in other cancers via The Cancer Genome Atlas analysis. Finally, we identified a frequent deletion of chr9q, encompassing PTCH1, and unreported frequent copy number gain of chr9p, encompassing the genes encoding the immune checkpoint ligands PD-L1 and PD-L2. In conclusion, this study is the first systematic analysis of coding and noncoding mutations in BCC and provides a strong basis for further analyses of the variants in BCC and cancer in general.

Pan-cancer analysis of somatic mutations in miRNA genes.

BACKGROUND: miRNAs are considered important players in oncogenesis, serving either as oncomiRs or suppressormiRs. Although the accumulation of somatic alterations is an intrinsic aspect of cancer development and many important cancer-driving mutations have been identified in protein-coding genes, the area of functional somatic mutations in miRNA genes is heavily understudied. METHODS: Here, based on the analysis of large genomic datasets, mostly the whole-exome sequencing of over 10,000 cancer/normal sample pairs deposited within the TCGA repository, we undertook an analysis of somatic mutations in miRNA genes. FINDINGS: We identified and characterized over 10,000 somatic mutations and showed that some of the miRNA genes are overmutated in Pan-Cancer and/or specific cancers. Nonrandom occurrence of the identified mutations was confirmed by a strong association of overmutated miRNA genes with KEGG pathways, most of which were related to specific cancer types or cancer-related processes. Additionally, we showed that mutations in some of the overmutated genes correlate with miRNA expression, cancer staging, and patient survival. INTERPRETATION: Our study is the first comprehensive Pan-Cancer study of cancer somatic mutations in miRNA genes. It may help to understand the consequences of mutations in miRNA genes and the identification of miRNA functional mutations. The results may also be the first step (form the basis and provide the resources) in the development of computational and/or statistical approaches/tools dedicated to the identification of cancer-driver miRNA genes. FUNDING: This work was supported by research grants from the Polish National Science Centre 2016/22/A/NZ2/00184 and 2015/17/N/NZ3/03629.

Somatic Mutations in miRNA Genes in Lung Cancer-Potential Functional Consequences of Non-Coding Sequence Variants.

A growing body of evidence indicates that miRNAs may either drive or suppress oncogenesis. However, little is known about somatic mutations in miRNA genes. To determine the frequency and potential consequences of miRNA gene mutations, we analyzed whole exome sequencing datasets of 569 lung adenocarcinoma (LUAD) and 597 lung squamous cell carcinoma (LUSC) samples generated in The Cancer Genome Atlas (TCGA) project. Altogether, we identified 1091 somatic sequence variants affecting 522 different miRNA genes and showed that half of all cancers had at least one such somatic variant/mutation. These sequence variants occurred in most crucial parts of miRNA precursors, including mature miRNA and seed sequences. Due to our findings, we hypothesize that seed mutations may affect miRNA:target interactions, drastically changing the pool of predicted targets. Mutations may also affect miRNA biogenesis by changing the structure of miRNA precursors, DROSHA and DICER cleavage sites, and regulatory sequence/structure motifs. We identified 10 significantly overmutated hotspot miRNA genes, including the miR-379 gene in LUAD enriched in mutations in the mature miRNA and regulatory sequences. The occurrence of mutations in the hotspot miRNA genes was also shown experimentally. We present a comprehensive analysis of somatic variants in miRNA genes and show that some of these genes are mutational hotspots, suggesting their potential role in cancer.

Effect of exogenous butyrate on the gastrointestinal tract of sheep. II. Hydrolytic activity in the rumen and structure and function of the small intestine.

The aim of this study was to determine the effect of exogenous butyrate on the activity of carbohydrate-digesting enzymes in the reticuloruminal digesta and structure and selected functions of the small intestine in sheep. Eighteen rams (30.8 ± 2.1 kg; 12 to 15 mo of age) were fed for 14 d a diet without (CTRL) or with sodium butyrate (BUT; 36 g/kg of offered DM). Butyrate concentration in the reticuloruminal fluid and proximal small intestinal digesta was greater for BUT compared with CTRL (P ≤ 0.05). Amylolytic activity was greater, whereas cellulolytic and xylanolytic activity in the reticuloruminal digesta was less for BUT compared with CTRL (P ≤ 0.04). Relative to BW, small intestinal tissue mass and small intestine length did not differ between treatments (P ≥ 0.15); however, absolute length of the small intestine was greater for BUT compared with CTRL (P = 0.04). In the duodenum, crypt depth tended (P = 0.10) to be greater, whereas in the ileum, crypt depth and muscularis thickness tended (P = 0.10) to be less for BUT compared with CTRL. Mitosis-to-apoptosis ratio in the proximal jejunum was greater for CTRL compared with BUT (P = 0.02). Expression of G-protein-coupled receptor 43 mRNA in the duodenal epithelium was greater for BUT compared with CTRL (P < 0.01). On the other hand, peptide transporter 1 mRNA expression in the distal sections of the small intestine, as well as activity of aminopeptidase A and dipeptidylpeptidase IV, were greater for CTRL (P ≤ 0.05). In summary, exogenous butyrate supplementation in feed affects hydrolytic activity in the rumen, and increased butyrate flow out of the reticulorumen affects both proximal and distal sections of the small intestine in sheep.