The ß-catenin C terminus links Wnt and sphingosine-1-phosphate signaling pathways to promote vascular remodeling and atherosclerosis.

Canonical Wnt and sphingosine-1-phosphate (S1P) signaling pathways are highly conserved systems that contribute to normal vertebrate development, with key consequences for immune, nervous, and cardiovascular system function; despite these functional overlaps, little is known about Wnt/ß-catenin-S1P cross-talk. In the vascular system, both Wnt/ß-catenin and S1P signals affect vessel maturation, stability, and barrier function, but information regarding their potential coordination is scant. We report an instance of functional interaction between the two pathways, including evidence that S1P receptor 1 (S1PR1) is a transcriptional target of ß-catenin. By studying vascular smooth muscle cells and arterial injury response, we find a specific requirement for the ß-catenin carboxyl terminus, which acts to induce S1PR1, and show that this interaction is essential for vascular remodeling. We also report that pharmacological inhibition of the ß-catenin carboxyl terminus reduces S1PR1 expression, neointima formation, and atherosclerosis. These findings provide mechanistic understanding of how Wnt/ß-catenin and S1P systems collaborate during vascular remodeling and inform strategies for therapeutic manipulation.

Hemp-Derived CBD Used in Food and Food Supplements.

Cannabis sativa L., a plant historically utilized for textile fibers, oil, and animal feed, is progressively being recognized as a potential food source. This review elucidates the nutritional and functional attributes of hemp and cannabidiol (CBD) within the context of food science. Hemp is characterized by the presence of approximately 545 secondary metabolites, among which around 144 are bioactive cannabinoids of primary importance. The study looks in detail at the nutritional components of cannabis and the potential health benefits of CBD, encompassing anti-inflammatory, anxiolytic, and antipsychotic effects. The review deals with the legislation and potential applications of hemp in the food industry and with the future directions of cannabis applications as well. The paper emphasizes the need for more scientific investigation to validate the safety and efficacy of hemp components in food products, as current research suggests that CBD may have great benefits for a wide range of consumers.

Effect of Extraction Methods on Aroma Profile, Antioxidant Activity and Sensory Acceptability of Specialty Coffee Brews.

Specialty coffees from various geographical origins were processed using different extraction methods. Four extraction techniques were employed: cold brew (CB), espresso (ES), French press (FR), and aeropress (AE). The potential health benefits of coffee brews were linked to their antioxidant activity, as determined by the DPPH assay, and total polyphenol content (TPC) measured through the Folin-Ciocalteu reducing-capacity assay. The Columbia (C) espresso coffee type (omni-roasting) exhibited the highest antioxidant activity (86.31 ± 0.70) µmol/100 mL, with a TPC value of (44.41 ± 0.35) mg GAE/g. Quantitative analyses of caffeine and chlorogenic acid were conducted using high-performance liquid chromatography (HPLC). The evaluation of coffee aroma profiles involved the application of headspace solid-phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS) and was complemented by sensory analysis following the Specialty Coffee Association (SCA) standard protocol. The predominant volatile compounds found in all samples included furans, phenols, pyrazines, and terpenes. The EY espresso type (medium dark roasting) had the highest levels of most coffee volatiles. The C cold brew type (omni-roasting) was rated as the preferred coffee in terms of its sensory characteristics and flavour. In summary, ES and CB were found to be more effective extraction methods for the parameters assessed.

Inactivation of the tumor suppressor gene Apc synergizes with H. pylori to induce DNA damage in murine gastric stem and progenitor cells.

Helicobacter pylori infection is a major risk factor for the development of gastric cancer. The bacteria reside in close proximity to gastric surface mucous as well as stem and progenitor cells. Here, we take advantage of wild-type and genetically engineered murine gastric organoids and organoid-derived monolayers to study the cellular targets of H. pylori-induced DNA damage and replication stress and to explore possible interactions with preexisting gastric cancer driver mutations. We find using alkaline comet assay, single-molecule DNA fiber assays, and immunofluorescence microscopy of DNA repair foci that H. pylori induces transcription-dependent DNA damage in actively replicating, Leucine-rich-repeat containing G-Protein-Coupled Receptor 5 (Lgr5)-positive antral stem and progenitor cells and their Troy-positive corpus counterparts, but not in other gastric epithelial lineages. Infection-dependent DNA damage is aggravated by Apc inactivation, but not by Trp53 or Smad4 loss, or Erbb2 overexpression. Our data suggest that H. pylori induces DNA damage in stem and progenitor cells, especially in settings of hyperproliferation due to constitutively active Wnt signaling.

Terminal differentiation of villus tip enterocytes is governed by distinct Tgfß superfamily members.

The protective and absorptive functions of the intestinal epithelium rely on differentiated enterocytes in the villi. The differentiation of enterocytes is orchestrated by sub-epithelial mesenchymal cells producing distinct ligands along the villus axis, in particular Bmps and Tgfß. Here, we show that individual Bmp ligands and Tgfß drive distinct enterocytic programs specific to villus zonation. Bmp4 is expressed from the centre to the upper part of the villus and activates preferentially genes connected to lipid uptake and metabolism. In contrast, Bmp2 is produced by villus tip mesenchymal cells and it influences the adhesive properties of villus tip epithelial cells and the expression of immunomodulators. Additionally, Tgfß induces epithelial gene expression programs similar to those triggered by Bmp2. Bmp2-driven villus tip program is activated by a canonical Bmp receptor type I/Smad-dependent mechanism. Finally, we establish an organoid cultivation system that enriches villus tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Our data suggest that not only a Bmp gradient but also the activity of individual Bmp drives specific enterocytic programs.

A Physicochemical Study of the Antioxidant Activity of Corn Silk Extracts.

Corn silk (CS) extracts are reported to contain flavonoids (appx. 59.65 mg quercetin/g), polysaccharides (appx. 58.75 w.%), steroids (appx. 38.3 × 10-3 to 368.9 × 10-3 mg/mL), polyphenols (appx. 77.89 mg/GAE/g) and other functional biological substances. This study investigated the antioxidant activity of corn silk extracts related to their functional compounds. The radical scavenging effect of corn silk extracts was evaluated by the spin-trapping electron paramagnetic resonance (EPR) technique, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonate) (ABTS•+) free radical measurement, ferric ion-reducing antioxidant power, and copper ion reductive capacity. It was found that the maturity stage of CS plant materials and the applied extraction procedure of their bioactive compounds have a profound effect on the radical scavenging capacity. Differences in the antioxidant activity of the studied corn silk samples based on their maturity were also confirmed. The strongest DPPH radical scavenging effect was observed for the corn silk mature stage (CS-M)stage (CS-MS) (65.20 ± 0.90)%, followed by the silky stage (CS-S) (59.33 ± 0.61)% and the milky stage (CS-M) (59.20 ± 0.92)%, respectively. In general, the final maturity stage (CS-MS) provided the most potent antioxidant effect, followed by the earliest maturity stage (CS-S) and the second maturity stage (CS-M).

Physico-Chemical Study of Curcumin and Its Application in O/W/O Multiple Emulsion.

Curcuma is a world-renowned herb known for its immense health benefits. In this study, physicochemical analyses were performed on the curcumin standard sample and curcumin multiple emulsions. The emulsions were analysed for thermal and structural stability for 21 days. Confocal laser microscopy (CLSM) was performed in order to observe the emulsion encapsulation. Modulated differential scanning calorimetry (MDSC) and HPLC methods revealed a variety of curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin, and cyclocurcumin) in the investigated curcumin standard. In addition, the MDSC method was found to be suitable and comparable to HPLC for determining the curcuminoid substances. The analysis of the curcumin release revealed a value of 0.18 w.% after 14 days as the equilibrium value. Furthermore, an increase in the sizes of the emulsions was observed at the end of the 21-day study. The emulsion stability index (ESI) was used to measure the stability of multiple emulsions. The ESI reached 55.8% between 7 and 21 days later. Nano droplets of the oil phase loaded with dispersed curcumin particles captured inside the water-based carboxymethylcellulose micelles were clearly observed by CLSM.

Glycoprotein (GP)96 Is Essential for Maintaining Intestinal Epithelial Architecture by Supporting Its Self-Renewal Capacity.

BACKGROUND & AIMS: Glycoprotein (GP)96 is an endoplasmic reticulum-resident master chaperone for cell surface receptors including the Wnt co-receptors low-density lipoprotein-receptor-related protein 5/6. Intestinal epithelial cell (IEC)-specific deletion of Gp96 is embryonically lethal. However, the role of GP96 in adult intestinal tissue and especially within the intestinal stem cell (ISC) niche is unknown. Here, we investigated how GP96 loss interferes with intestinal homeostasis by compromising viability, proliferation, and differentiation of IECs. METHODS: Tamoxifen was used to induce Cre-mediated deletion of Gp96 in GP96-VillincreERT2 (Cre recombinase-Estrogen-Receptor Transgene 2) mice and intestinal organoids. With H&E and immunofluorescence staining we assessed alterations in intestinal morphology and the presence and localization of IEC types. Real-time polymerase chain reaction and Western blot analysis were performed to explore the molecular mechanisms underlying the severe phenotype of Gp96 KO mice and organoids. RESULTS: IEC-specific deletion of Gp96 in adult mice resulted in a rapid degeneration of the stem cell niche, followed by complete eradication of the epithelial layer and death within a few days. These effects were owing to severe defects in ISC renewal and premature ISC differentiation, which resulted from defective Wnt and Notch signaling. Furthermore, depletion of GP96 led to massive induction of endoplasmic reticulum stress. Although effects on ISC renewal and adequate differentiation were partly reversed upon activation of Wnt/Notch signaling, viability could not be restored, indicating that reduced viability was mediated by other mechanisms. CONCLUSIONS: Our work shows that GP96 plays a fundamental role in regulating ISC fate and epithelial regeneration and therefore is indispensable for maintaining intestinal epithelial homeostasis.

Active eosinophils regulate host defence and immune responses in colitis.

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.

Functional and Quality Profile Evaluation of Butters, Spreadable Fats, and Shortenings Available from Czech Market.

The aim of this study was to assess the functional properties of butters, spreadable fats, and shortenings, collected from the Czech market, in correlation with their nutritional values declared by the producers. Various methods were applied to determine relevant parameters of the products. Using penetration tests, samples were characterized by specific textural attributes according to their composition and processing type, particularly for the presence of milk/vegetable fats. Using differential scanning calorimetry (DSC), thermal peaks corresponding to medium- and high-melting triacylglycerol fractions were detected in the ranges 15-16 °C and 31.5-34.5 °C, respectively. Rheological analysis revealed that the viscoelasticity of samples was related to frequency behavior of the fat structure, characterized by the dominance of elastic modulus (G') over viscous modulus (G″) up to the frequency of 10 Hz. This indicated good emulsion stability of the products in the region of linear viscoelasticity. For spreadable fats, the structure was resistant to phase separation in the whole frequency range under study (0.1-100 Hz). The results showed that the applied techniques can be successfully used to characterize the processing and compositional quality of butters and vegetable fats.

GATA3 mediates nonclassical ß-catenin signaling in skeletal cell fate determination and ectopic chondrogenesis.

Skeletal precursors are mesenchymal in origin and can give rise to distinct sublineages. Their lineage commitment is modulated by various signaling pathways. The importance of Wnt signaling in skeletal lineage commitment has been implicated by the study of ß-catenin-deficient mouse models. Ectopic chondrogenesis caused by the loss of ß-catenin leads to a long-standing belief in canonical Wnt signaling that determines skeletal cell fate. As ß-catenin has other functions, it remains unclear whether skeletogenic lineage commitment is solely orchestrated by canonical Wnt signaling. The study of the Wnt secretion regulator Gpr177/Wntless also raises concerns about current knowledge. Here, we show that skeletal cell fate is determined by ß-catenin but independent of LEF/TCF transcription. Genomic and bioinformatic analyses further identify GATA3 as a mediator for the alternative signaling effects. GATA3 alone is sufficient to promote ectopic cartilage formation, demonstrating its essential role in mediating nonclassical ß-catenin signaling in skeletogenic lineage specification.

The Relationship between Bone Conduction Hearing Threshold Shifts after Surgery for Chronic Otitis Media with Cholesteatoma According to STAM, EAONO/JOS, and SAMEO-ATO Classifications.

Background: This study focuses on the hearing threshold for bone conduction (BC) after middle-ear surgery. Methods: A total of 92 patients (120 ears) were treated for newly diagnosed chronic otitis media with cholesteatoma (2013−2018). BC was examined at frequencies of 0.5, 1, 2, and 4 kHz prior to and 1 year after surgery. STAM classification for cholesteatoma location, EAONO/JOS for stage, and surgery according to SAMEO-ATO classification were applied. The bone conduction threshold was compared for individual frequencies in patients with occurrence/absence of cholesteatoma in different locations. Results: For the occurrence of cholesteatoma in the attic (A), a statistically significant difference was found at 4 kHz (p < 0.001), in the supratubal recess (S1) at 4 kHz (p = 0.003), and for the mastoid (M) at 0.5 kHz (p = 0.024), at 1 kHz (p = 0.032), and at 2 kHz (p = 0.039). Conclusions: Cholesteatoma location can influence the post-operative hearing threshold for bone conduction.

Research Note: Impact of applied thermal treatment on textural, and sensory properties and cooking loss of selected chicken and turkey cuts as affected by cooking technique.

The effect of various cooking methods (roasting, broiling, grilling, frying, and stewing) on cooking loss (CL) and textural and sensory properties of selected chicken (breast fillet, thigh, and thigh fillet) and turkey (breast fillet, thigh) cuts in relation to the applied apparatus was evaluated. Diverse results were recorded according to the method, the type of poultry meat, and the cut of poultry meat. Additionally, CL and shear force (SF) values in all examined samples were influenced by the culinary technique, the type of poultry meat, and the poultry meat cut. The lowest CL and shear SF values were reported when the samples were treated using a method with higher heating rates and/or temperatures and shorter cooking times. Additionally, lower values of CL and SF were obtained for chicken meat compared to turkey meat (thighs). In general, the applied culinary technique affected the sensory properties of the samples tested. High sensory scores were recorded for grilled chicken breast fillets and fried turkey breast fillets (irrespective of the applied apparatus). On the whole, it could be stated that culinary techniques at high temperature requiring shorter times (such as frying, grilling, and roasting) were evaluated to be more effective (in terms of CL and SF).

Epithelial Wnt secretion drives the progression of inflammation-induced colon carcinoma in murine model.

Colon cancer is initiated by stem cells that escape the strict control. This process is often driven through aberrant activation of Wnt signaling by mutations in components acting downstream of the receptor complex that unfetter tumor cells from the need for Wnts. Here we describe a class of colon cancer that does not depend on mutated core components of the Wnt pathway. Genetically blocking Wnt secretion from epithelial cells of such tumors results in apoptosis, reduced expression of colon cancer markers, followed by enhanced tumor differentiation. In contrast to the normal colonic epithelium, such tumor cells autosecrete Wnts to maintain their uncontrolled proliferative behavior. In humans, we determined certain cases of colon cancers in which the Wnt pathway is hyperactive, but not through mutations in its core components. Our findings illuminate the path in therapy to find further subtypes of Wnt-dependent colon cancer that might be responsive to Wnt secretion inhibitors.

The interactions of Bcl9/Bcl9L with ß-catenin and Pygopus promote breast cancer growth, invasion, and metastasis.

Canonical Wnt/ß-catenin signaling is an established regulator of cellular state and its critical contributions to tumor initiation, malignant tumor progression and metastasis formation have been demonstrated in various cancer types. Here, we investigated how the binding of ß-catenin to the transcriptional coactivators B-cell CLL/lymphoma 9 (Bcl9) and Bcl9-Like (Bcl9L) affected mammary gland carcinogenesis in the MMTV-PyMT transgenic mouse model of metastatic breast cancer. Conditional knockout of both Bcl9 and Bcl9L resulted into tumor cell death. In contrast, disrupting the interaction of Bcl9/Bcl9L with ß-catenin, either by deletion of their HD2 domains or by a point mutation in the N-terminal domain of ß-catenin (D164A), diminished primary tumor growth and tumor cell proliferation and reduced tumor cell invasion and lung metastasis. In comparison, the disruption of HD1 domain-mediated binding of Bcl9/Bcl9L to Pygopus had only moderate effects. Interestingly, interfering with the ß-catenin-Bcl9/Bcl9L-Pygo chain of adapters only partially impaired the transcriptional response of mammary tumor cells to Wnt3a and TGFß treatments. Together, the results indicate that Bcl9/Bcl9L modulate but are not critically required for canonical Wnt signaling in its contribution to breast cancer growth and malignant progression, a notion consistent with the "just-right" hypothesis of Wnt-driven tumor progression.

Tracing colonic embryonic transcriptional profiles and their reactivation upon intestinal damage.

We lack a holistic understanding of the genetic programs orchestrating embryonic colon morphogenesis and governing damage response in the adult. A window into these programs is the transcriptomes of the epithelial and mesenchymal cell populations in the colon. Performing unbiased single-cell transcriptomic analyses of the developing mouse colon at different embryonic stages (embryonic day 14.5 [E14.5], E15.5, and E18.5), we capture cellular and molecular profiles of the stages before, during, and after the appearance of crypt structures, as well as in a model of adult colitis. The data suggest most adult lineages are established by E18.5. We find embryonic-specific gene expression profiles and cell populations that reappear in response to tissue damage. Comparison of the datasets from mice and human colitis suggests the processes are conserved. In this study, we provide a comprehensive single-cell atlas of the developing mouse colon and evidence for the reactivation of embryonic genes in disease.

Parsing ß-catenin's cell adhesion and Wnt signaling functions in malignant mammary tumor progression.

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, ß-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of ß-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of ß-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of ß-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of ß-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by ß-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-ß-induced EMT. Our results uncouple the signaling from the adhesion function of ß-catenin and underline the importance of Wnt/ß-catenin-dependent transcription in malignant tumor progression of breast cancer.

Differential regulation of ß-catenin-mediated transcription via N- and C-terminal co-factors governs identity of murine intestinal epithelial stem cells.

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/ß-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic ß-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of ß-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with ß-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune ß-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/ß-catenin output requires selective modulation of gene expression by transcriptional co-factors.

Distinct populations of crypt-associated fibroblasts act as signaling hubs to control colon homeostasis.

Despite recent progress in recognizing the importance of mesenchymal cells for the homeostasis of the intestinal system, the current picture of how these cells communicate with the associated epithelial layer remains unclear. To describe the relevant cell populations in an unbiased manner, we carried out a single-cell transcriptome analysis of the adult murine colon, producing a high-quality atlas of matched colonic epithelium and mesenchyme. We identify two crypt-associated colonic fibroblast populations that are demarcated by different strengths of platelet-derived growth factor receptor A (Pdgfra) expression. Crypt-bottom fibroblasts (CBFs), close to the intestinal stem cells, express low levels of Pdgfra and secrete canonical Wnt ligands, Wnt potentiators, and bone morphogenetic protein (Bmp) inhibitors. Crypt-top fibroblasts (CTFs) exhibit high Pdgfra levels and secrete noncanonical Wnts and Bmp ligands. While the Pdgfralow cells maintain intestinal stem cell proliferation, the Pdgfrahigh cells induce differentiation of the epithelial cells. Our findings enhance our understanding of the crosstalk between various colonic epithelial cells and their associated mesenchymal signaling hubs along the crypt axis-placing differential Pdgfra expression levels in the spotlight of intestinal fibroblast identity.

Antimicrobial-resistance in Escherichia coli isolatedfrom wild pheasants (Phasianus colchicus).

The purpose of this study was to examine the prevalence of antimicrobial resistance among 180 Escherichia coli strains isolated from 200 wild pheasants caught in rural areas of the Czech Republic (Eastern Moravia) and Slovakia (Western Region). The isolates were also classified into phylogenetic groups by the multiplex PCR method. Our findings demonstrated that 130 strains were resistant to ampicillin (72%), 160 strains to cephalothin (89%), and 40 strains to tetracycline (22%). Ten strains were found to be resistant to chloramphenicol and sulfamethoxazole/trimethoprim (5.6%). In turn, all strains were sensitive to cefoperazone/sulbactam, ciprofloxacin, colistin, gentamicin, and piperacillin/tazobactam. Ten of the 180 isolates (5.6%) exhibited multi-resistant phenotypes, including resistances against beta-lactams, aminoglycosides, tetracyclines, sulphonamides, and chloramphenicol. As far as we know, this is the first report describing antimicrobial resistance in E. coli from pheasants.