Individual Sweet Taste Perception Influences Salivary Characteristics After Orosensory Stimulation With Sucrose and Noncaloric Sweeteners.

Emerging evidence points to a major role of salivary flow and viscoelastic properties in taste perception and mouthfeel. It has been proposed that sweet-tasting compounds influence salivary characteristics. However, whether perceived differences in the sensory properties of structurally diverse sweet-tasting compounds contribute to salivary flow and saliva viscoelasticity as part of mouthfeel and overall sweet taste perception remains to be clarified. In this study, we hypothesized that the sensory diversity of sweeteners would differentially change salivary characteristics in response to oral sweet taste stimulation. Therefore, we investigated salivary flow and saliva viscoelasticity from 21 healthy test subjects after orosensory stimulation with sucrose, rebaudioside M (RebM), sucralose, and neohesperidin dihydrochalcone (NHDC) in a crossover design and considered the basal level of selected influencing factors, including the basal oral microbiome. All test compounds enhanced the salivary flow rate by up to 1.51 ± 0.12 g/min for RebM compared to 1.10 ± 0.09 g/min for water within the 1st min after stimulation. The increase in flow rate was moderately correlated with the individually perceived sweet taste (r = 0.3, p < 0.01) but did not differ between the test compounds. The complex viscosity of saliva was not affected by the test compounds, but the analysis of covariance showed that it was associated (p < 0.05) with mucin 5B (Muc5B) concentration. The oral microbiome was of typical composition and diversity but was strongly individual-dependent (permutational analysis of variance (PERMANOVA): R 2 = 0.76, p < 0.001) and was not associated with changes in salivary characteristics. In conclusion, this study indicates an impact of individual sweet taste impressions on the flow rate without measurable changes in the complex viscosity of saliva, which may contribute to the overall taste perception and mouthfeel of sweet-tasting compounds.

DNA methylation landscapes of 1538 breast cancers reveal a replication-linked clock, epigenomic instability and cis-regulation.

DNA methylation is aberrant in cancer, but the dynamics, regulatory role and clinical implications of such epigenetic changes are still poorly understood. Here, reduced representation bisulfite sequencing (RRBS) profiles of 1538 breast tumors and 244 normal breast tissues from the METABRIC cohort are reported, facilitating detailed analysis of DNA methylation within a rich context of genomic, transcriptional, and clinical data. Tumor methylation from immune and stromal signatures are deconvoluted leading to the discovery of a tumor replication-linked clock with genome-wide methylation loss in non-CpG island sites. Unexpectedly, methylation in most tumor CpG islands follows two replication-independent processes of gain (MG) or loss (ML) that we term epigenomic instability. Epigenomic instability is correlated with tumor grade and stage, TP53 mutations and poorer prognosis. After controlling for these global trans-acting trends, as well as for X-linked dosage compensation effects, cis-specific methylation and expression correlations are uncovered at hundreds of promoters and over a thousand distal elements. Some of these targeted known tumor suppressors and oncogenes. In conclusion, this study demonstrates that global epigenetic instability can erode cancer methylomes and expose them to localized methylation aberrations in-cis resulting in transcriptional changes seen in tumors.

CDK13 cooperates with CDK12 to control global RNA polymerase II processivity.

The RNA polymerase II (POLII)-driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3' polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.

Regulation of the RNAPII Pool Is Integral to the DNA Damage Response.

In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.

Application of encapsulated natural bioactive compounds from red pepper waste in yogurt.

Aim: The aim of this study was to encapsulate red pepper waste (RPW) bioactives and monitor their stability in yogurt.Methods: RPW extract was encapsulated in whey protein using spray and freeze-drying techniques. Physicochemical characteristics of encapsulates were evaluated, and better encapsulates were used to develop functional yogurt. Retention of bioactives was followed over 21 days of storage, and sensory analyses were assessed.Results: Freeze-dried encapsulates (FDE) showed better characteristics like water activity, moisture content, solubility, flowing and colour properties, and, therefore, incorporated in yogurt. Yogurt with FDE successfully retained carotenoids (71.43%) and caused increasing of polyphenol retention (up to 123.73%). This yogurt exhibited higher sensory and general acceptability scores compared to control sample. The fortification of yogurts had a positive influence on maintaining the initial number of lactic acid bacteria during storage.Conclusion: Freeze drying and utilisation of pepper waste are efficient for functional food development, with improved nutritional, colour and bioactive properties.

Analysis of RNA polymerase II ubiquitylation and proteasomal degradation.

Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RNAPII and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well.

Global transcriptional analysis identifies a novel role for SOX4 in tumor-induced angiogenesis.

The expression of the transcription factor SOX4 is increased in many human cancers, however, the pro-oncogenic capacity of SOX4 can vary greatly depending on the type of tumor. Both the contextual nature and the mechanisms underlying the pro-oncogenic SOX4 response remain unexplored. Here, we demonstrate that in mammary tumorigenesis, the SOX4 transcriptional network is dictated by the epigenome and is enriched for pro-angiogenic processes. We show that SOX4 directly regulates endothelin-1 (ET-1) expression and can thereby promote tumor-induced angiogenesis both in vitro and in vivo. Furthermore, in breast tumors, SOX4 expression correlates with blood vessel density and size, and predicts poor-prognosis in patients with breast cancer. Our data provide novel mechanistic insights into context-dependent SOX4 target gene selection, and uncover a novel pro-oncogenic role for this transcription factor in promoting tumor-induced angiogenesis. These findings establish a key role for SOX4 in promoting metastasis through exploiting diverse pro-tumorigenic pathways.

SOX4 can redirect TGF-ß-mediated SMAD3-transcriptional output in a context-dependent manner to promote tumorigenesis.

Expression of the transcription factor SOX4 is often elevated in human cancers, where it generally correlates with tumor-progression and poor-disease outcome. Reduction of SOX4 expression results in both diminished tumor-incidence and metastasis. In breast cancer, TGF-ß-mediated induction of SOX4 has been shown to contribute to epithelial-to-mesenchymal transition (EMT), which controls pro-metastatic events. Here, we identify SMAD3 as a novel, functionally relevant SOX4 interaction partner. Genome-wide analysis showed that SOX4 and SMAD3 co-occupy a large number of genomic loci in a cell-type specific manner. Moreover, SOX4 expression was required for TGF-ß-mediated induction of a subset of SMAD3/SOX4-co-bound genes regulating migration and extracellular matrix-associated processes, and correlating with poor-prognosis. These findings identify SOX4 as an important SMAD3 co-factor controlling transcription of pro-metastatic genes and context-dependent shaping of the cellular response to TGF-ß. Targeted disruption of the interaction between these factors may have the potential to disrupt pro-oncogenic TGF-ß signaling, thereby impairing tumorigenesis.

A Biobank of Breast Cancer Explants with Preserved Intra-tumor Heterogeneity to Screen Anticancer Compounds.

The inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts (PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.

Context-Specific Effects of TGF-ß/SMAD3 in Cancer Are Modulated by the Epigenome.

The transforming growth factor beta (TGF-ß) signaling pathway exerts opposing effects on cancer cells, acting as either a tumor promoter or a tumor suppressor. Here, we show that these opposing effects are a result of the synergy between SMAD3, a downstream effector of TGF-ß signaling, and the distinct epigenomes of breast-tumor-initiating cells (BTICs). These effects of TGF-ß are associated with distinct gene expression programs, but genomic SMAD3 binding patterns are highly similar in the BTIC-promoting and BTIC-suppressing contexts. Our data show cell-type-specific patterns of DNA and histone modifications provide a modulatory layer by determining accessibility of genes to regulation by TGF-ß/SMAD3. LBH, one such context-specific target gene, is regulated according to its DNA methylation status and is crucial for TGF-ß-dependent promotion of BTICs. Overall, these results reveal that the epigenome plays a central and previously overlooked role in shaping the context-specific effects of TGF-ß in cancer.

Antifungal efficiency assessment of the TiO2 coating on façade paints.

The work studies the photocatalytic activity and the antifungal efficiency of the TiO2/Zn-Al coatings placed on the target commercial façade paints. The photocatalytic active nanocomposite based on TiO2 and Zn-Al-layered double hydroxides (ZnAl-LDHs) was synthesized by a wet impregnation technique with 3 % w/w TiO2. The freshly prepared suspension was applied by spray technique on the surfaces of the white façade paints. The goal of the work was to develop a method that quickly quantifies the antifungal activity of the commercial façade paints with and without biocidal components covered with a photocatalytic coating. The essence of the proposed method is the monitoring of the fungal growth (artificial ageing conditions) and the quantification of its development (UV-A 0.13 mWcm(-2)) on the façade paint surfaces. A special fungus nutrient (potato dextrose agar (PDA)) was inoculated with the spores of the Aspergillus niger ATCC 6275, and the test samples (façade paints with and without photocatalytic coating) were placed on the inoculated nutrient in the petri dishes. The images of the fungal growth on the samples of the facade paints, during a period of 5 days, were imported into Matlab R2012a where they were converted to binary images (BW), based on the adequate threshold. The percentage of the surface coverage was calculated by applying the specifically written program code which determines the ratio of the black and white pixels. The black pixels correspond to the surface covered with hyphae and mycelia of the fungus.

TGFß induces the formation of tumour-initiating cells in claudinlow breast cancer.

The role of transforming growth factor-beta (TGFß) in the progression of different molecular subtypes of breast cancer has not been clarified. Here we show that TGFß increases breast tumour-initiating cell (BTIC) numbers but only in claudin(low) breast cancer cell lines by orchestrating a specific gene signature enriched in stem cell processes that predicts worse clinical outcome in breast cancer patients. NEDD9, a member of the Cas family of integrin scaffold proteins, is necessary to mediate these TGFß-specific effects through a positive feedback loop that integrates TGFß/Smad and Rho-actin-SRF-dependent signals. In normal human mammary epithelium, TGFß induces progenitor activity only in the basal/stem cell compartment, where claudin(low) cancers are presumed to arise. These data show opposing responses to TGFß in both breast malignant cell subtypes and normal mammary epithelial cell subpopulations and suggest therapeutic strategies for a subset of human breast cancers.