Speciation, phytoavailability, and accumulation of toxic elements and sulfur by humic acid-fertilized lemongrass and common sage in a sandy soil treated with heavy oil fly ash: A trial for management of power stations wastes.

Globally, power stations generate huge amounts of the hazardous waste heavy oil fly ash (HOFA), which is rich in Ni, V, Fe, S, and dumped into landfills. Thus, exploring new approaches for a safe recycling and sustainable management of HOFA is needed and of great environmental interest. The potential application of HOFA as an amendment to sandy soils has not been studied yet. This is the first research investigating the potentiality of using HOFA as a soil conditioner. To this end, we conducted a greenhouse experiment in order to investigate the impacts of HOFA addition (1.2, 2.4, 3.6 t ha-1) to sandy soil on the total and available content of nutrients (e.g., S, Fe, Mn, Cu, Zn) and toxic elements (TEs; e.g., Cd, Co, Cr, Ni, Pb, V) in the soil and their phytoextraction and translocation by lemongrass (Cymbopogon citratus) and common sage (Salvia officinalis). We also assessed the impact of humic acid (HA) foliar application (50 and 100 l ha-1) on the growth and elements accumulation by the two plants. The studied HOFA was acidic and highly enriched in S (43,268.0), V (3,527.0), Ni (1774.0), and Fe (15,159.0) (units in mg kg-1). The X-ray absorption near edge structure (XANES) data showed that V in HOFA was composed primarily of V(IV) sorbed onto goethite, V(V) sorbed onto humic substances, in the forms of V2O3, and VCl4. Addition of the lower doses of HOFA (1.2 and 2.4 t ha-1) did not change significantly soil pH, salinity, and the total and available elements content compared to the unamended soil. Although the elements content in the 3.6 t ha-1 HOFA-treated soil was significantly higher than the untreated, the total content of all elements (except for Ni) was lower than the maximum allowable concentrations in soils. HOFA addition, particularly in the highest dose (3.6 t ha-1), decreased significantly the growth and biomass of both plants. Common sage accumulated more elements than lemongrass; however, the elements content in the plants was lower than the critical concentrations for sensitive plants. The foliar application of humic acid enhanced significantly the plant growth and increased their tolerance to the HOFA-induced stress. We conclude that the addition of HOFA up to 2.4 t ha-1 in a single application as amendment to sandy soils is not likely to create any TE toxicity problems to plants, particularly if combined with a foliar application of humic acid; however, repeated additions of HOFA may induce toxicity. These findings should be verified under field conditions.

SOX17 enables immune evasion of early colorectal adenomas and cancers.

A hallmark of cancer is the avoidance of immune destruction. This process has been primarily investigated in locally advanced or metastatic cancer1-3; however, much less is known about how pre-malignant or early invasive tumours evade immune detection. Here, to understand this process in early colorectal cancers (CRCs), we investigated how naive colon cancer organoids that were engineered in vitro to harbour Apc-null, KrasG12D and Trp53-null (AKP) mutations adapted to the in vivo native colonic environment. Comprehensive transcriptomic and chromatin analyses revealed that the endoderm-specifying transcription factor SOX17 became strongly upregulated in vivo. Notably, whereas SOX17 loss did not affect AKP organoid propagation in vitro, its loss markedly reduced the ability of AKP tumours to persist in vivo. The small fraction of SOX17-null tumours that grew displayed notable interferon-γ (IFNγ)-producing effector-like CD8+ T cell infiltrates in contrast to the immune-suppressive microenvironment in wild-type counterparts. Mechanistically, in both endogenous Apc-null pre-malignant adenomas and transplanted organoid-derived AKP CRCs, SOX17 suppresses the ability of tumour cells to sense and respond to IFNγ, preventing anti-tumour T cell responses. Finally, SOX17 engages a fetal intestinal programme that drives differentiation away from LGR5+ tumour cells to produce immune-evasive LGR5- tumour cells with lower expression of major histocompatibility complex class I (MHC-I). We propose that SOX17 is a transcription factor that is engaged during the early steps of colon cancer to orchestrate an immune-evasive programme that permits CRC initiation and progression.

Fine-tuning GPT-3 for machine learning electronic and functional properties of organic molecules.

We evaluate the effectiveness of fine-tuning GPT-3 for the prediction of electronic and functional properties of organic molecules. Our findings show that fine-tuned GPT-3 can successfully identify and distinguish between chemically meaningful patterns, and discern subtle differences among them, exhibiting robust predictive performance for the prediction of molecular properties. We focus on assessing the fine-tuned models' resilience to information loss, resulting from the absence of atoms or chemical groups, and to noise that we introduce via random alterations in atomic identities. We discuss the challenges and limitations inherent to the use of GPT-3 in molecular machine-learning tasks and suggest potential directions for future research and improvements to address these issues.

Chronic metabolic stress drives developmental programs and loss of tissue functions in non-transformed liver that mirror tumor states and stratify survival.

Under chronic stress, cells must balance competing demands between cellular survival and tissue function. In metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD/NASH), hepatocytes cooperate with structural and immune cells to perform crucial metabolic, synthetic, and detoxification functions despite nutrient imbalances. While prior work has emphasized stress-induced drivers of cell death, the dynamic adaptations of surviving cells and their functional repercussions remain unclear. Namely, we do not know which pathways and programs define cellular responses, what regulatory factors mediate (mal)adaptations, and how this aberrant activity connects to tissue-scale dysfunction and long-term disease outcomes. Here, by applying longitudinal single-cell multi -omics to a mouse model of chronic metabolic stress and extending to human cohorts, we show that stress drives survival-linked tradeoffs and metabolic rewiring, manifesting as shifts towards development-associated states in non-transformed hepatocytes with accompanying decreases in their professional functionality. Diet-induced adaptations occur significantly prior to tumorigenesis but parallel tumorigenesis-induced phenotypes and predict worsened human cancer survival. Through the development of a multi -omic computational gene regulatory inference framework and human in vitro and mouse in vivo genetic perturbations, we validate transcriptional (RELB, SOX4) and metabolic (HMGCS2) mediators that co-regulate and couple the balance between developmental state and hepatocyte functional identity programming. Our work defines cellular features of liver adaptation to chronic stress as well as their links to long-term disease outcomes and cancer hallmarks, unifying diverse axes of cellular dysfunction around core causal mechanisms.

Beta-Hydroxybutyrate Augments Oxaliplatin-Induced Cytotoxicity by Altering Energy Metabolism in Colorectal Cancer Organoids.

Deregulation of cellular metabolism has recently emerged as a notable cancer characteristic. This reprogramming of key metabolic pathways supports tumor growth. Targeting cancer metabolism demonstrates the potential for managing colorectal cancer. Beta-hydroxybutyrate (BOHB) acts as an acetyl-CoA source for the tricarboxylic acid (TCA) cycle, possibly redirecting energy metabolic pathways towards the TCA cycle that could enhance sensitivity to oxaliplatin, through the generation of reactive oxygen species (ROS). This study explores the potential of BOHB to enhance oxaliplatin's cytotoxic effect by altering the energy metabolism in colorectal cancer. The study employed advanced in vitro organoid technology, which successfully emulates in vivo physiology. The combination treatment efficacy of BOHB and oxaliplatin was evaluated via cell viability assay. The levels of key proteins involved in energy metabolism, apoptotic pathways, DNA damage markers, and histone acetylation were analyzed via Western Blot. ROS levels were evaluated via flow cytometer. Non-toxic doses of BOHB with oxaliplatin significantly amplified cytotoxicity in colorectal cancer organoids. Treatment with BOHB and/or melatonin resulted in significantly decreased lactate dehydrogenase A and increased mitochondrial carrier protein 2 levels, indicating inhibited aerobic glycolysis and an increased oxidative phosphorylation rate. This metabolic shift induced apoptotic cell death mediated by oxaliplatin, owing to high levels of ROS. Melatonin counteracted this effect by protecting cancer cells from high oxidative stress conditions. BOHB may enhance the efficacy of chemotherapeutics with a similar mechanism of action to oxaliplatin in colorectal cancer treatment. These innovative combinations could improve treatment outcomes for colorectal cancer patients.

Zaxinone mimics (MiZax) efficiently promote growth and production of potato and strawberry plants under desert climate conditions.

Climate changes and the rapid expanding human population have become critical concerns for global food security. One of the promising solutions is the employment of plant growth regulators (PGRs) for increasing crop yield and overcoming adverse growth conditions, such as desert climate. Recently, the apocarotenoid zaxinone and its two mimics (MiZax3 and MiZax5) have shown a promising growth-promoting activity in cereals and vegetable crops under greenhouse and field conditions. Herein, we further investigated the effect of MiZax3 and MiZax5, at different concentrations (5 and 10 µM in 2021; 2.5 and 5 µM in 2022), on the growth and yield of the two valuable vegetable crops, potato and strawberry, in the Kingdom of Saudi of Arabia. Application of both MiZax significantly increased plant agronomic traits, yield components and total yield, in five independent field trials from 2021 to 2022. Remarkably, the amount of applied MiZax was far less than humic acid, a widely applied commercial compound used here for comparison. Hence, our results indicate that MiZax are very promising PGRs that can be applied to promote the growth and yield of vegetable crops even under desert conditions and at relatively low concentrations.

Ultrasensitive Detection of Circulating LINE-1 ORF1p as a Specific Multicancer Biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring. SIGNIFICANCE: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 µL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489.

Dietary fat and lipid metabolism in the tumor microenvironment.

Metabolic reprogramming has been considered a core hallmark of cancer, in which excessive accumulation of lipids promote cancer initiation, progression and metastasis. Lipid metabolism often includes the digestion and absorption of dietary fat, and the ways in which cancer cells utilize lipids are often influenced by the complex interactions within the tumor microenvironment. Among multiple cancer risk factors, obesity has a positive association with multiple cancer types, while diets like calorie restriction and fasting improve health and delay cancer. Impact of these diets on tumorigenesis or cancer prevention are generally studied on cancer cells, despite heterogeneity of the tumor microenvironment. Cancer cells regularly interact with these heterogeneous microenvironmental components, including immune and stromal cells, to promote cancer progression and metastasis, and there is an intricate metabolic crosstalk between these compartments. Here, we focus on discussing fat metabolism and response to dietary fat in the tumor microenvironment, focusing on both immune and stromal components and shedding light on therapeutic strategies surrounding lipid metabolic and signaling pathways.

Identification of Unknown Inverted Singlet-Triplet Cores by High-Throughput Virtual Screening.

Molecules where the energy of the lowest excited singlet state is found below the energy of the lowest triplet state (inverted singlet-triplet molecules) are extremely rare. It is particularly challenging to discover new ones through virtual screening because the required wavefunction-based methods are expensive and unsuitable for high-throughput calculations. Here, we devised a virtual screening approach where the molecules to be considered with advanced methods are pre-selected with increasingly more sophisticated filters that include the evaluation of the HOMO-LUMO exchange integral and approximate CASSCF calculations. A final set of 7 candidates (0.05% of the initial 15 000) were verified to possess inversion between singlet and triplet states with state-of-the-art multireference methods (MS-CASPT2). One of them is deemed of particular interest because it is unrelated to other proposals made in the literature.

Ablative radiotherapy improves survival but does not cure autochthonous mouse models of prostate and colorectal cancer.

BACKGROUND: Genetically engineered mouse models (GEMMs) of cancer are powerful tools to study mechanisms of disease progression and therapy response, yet little is known about how these models respond to multimodality therapy used in patients. Radiation therapy (RT) is frequently used to treat localized cancers with curative intent, delay progression of oligometastases, and palliate symptoms of metastatic disease. METHODS: Here we report the development, testing, and validation of a platform to immobilize and target tumors in mice with stereotactic ablative RT (SART). Xenograft and autochthonous tumor models were treated with hypofractionated ablative doses of radiotherapy. RESULTS: We demonstrate that hypofractionated regimens used in clinical practice can be effectively delivered in mouse models. SART alters tumor stroma and the immune environment, improves survival in GEMMs of primary prostate and colorectal cancer, and synergizes with androgen deprivation in prostate cancer. Complete pathologic responses were achieved in xenograft models, but not in GEMMs. CONCLUSIONS: While SART is capable of fully ablating xenografts, it is unable to completely eradicate disease in GEMMs, arguing that resistance to potentially curative therapy can be modeled in GEMMs.

Mice can be used to model the types of cancer seen in people to investigate the effects of cancer therapies, such as radiation. Here, we apply radiation therapy treatments that are able to cure cancer in humans to mice that have cancer of the prostate or colorectum. We show that the mice do not experience many side effects and that the tumours reduce in size, but in some cases show progression after treatment. Our study demonstrates that mice can be used to better understand how human cancers respond to radiation treatment, which can lead to the development of improved treatments and treatment schedules.

Post-Transplantation Cyclophosphamide-Based Graft-versus-Host Disease Prophylaxis.

BACKGROUND: In patients undergoing allogeneic hematopoietic stem-cell transplantation (HSCT), a calcineurin inhibitor plus methotrexate has been a standard prophylaxis against graft-versus-host disease (GVHD). A phase 2 study indicated the potential superiority of a post-transplantation regimen of cyclophosphamide, tacrolimus, and mycophenolate mofetil. METHODS: In a phase 3 trial, we randomly assigned adults with hematologic cancers in a 1:1 ratio to receive cyclophosphamide-tacrolimus-mycophenolate mofetil (experimental prophylaxis) or tacrolimus-methotrexate (standard prophylaxis). The patients underwent HSCT from an HLA-matched related donor or a matched or 7/8 mismatched (i.e., mismatched at only one of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci) unrelated donor, after reduced-intensity conditioning. The primary end point was GVHD-free, relapse-free survival at 1 year, assessed in a time-to-event analysis, with events defined as grade III or IV acute GVHD, chronic GVHD warranting systemic immunosuppression, disease relapse or progression, and death from any cause. RESULTS: In a multivariate Cox regression analysis, GVHD-free, relapse-free survival was significantly more common among the 214 patients in the experimental-prophylaxis group than among the 217 patients in the standard-prophylaxis group (hazard ratio for grade III or IV acute GVHD, chronic GVHD, disease relapse or progression, or death, 0.64; 95% confidence interval [CI], 0.49 to 0.83; P = 0.001). At 1 year, the adjusted GVHD-free, relapse-free survival was 52.7% (95% CI, 45.8 to 59.2) with experimental prophylaxis and 34.9% (95% CI, 28.6 to 41.3) with standard prophylaxis. Patients in the experimental-prophylaxis group appeared to have less severe acute or chronic GVHD and a higher incidence of immunosuppression-free survival at 1 year. Overall and disease-free survival, relapse, transplantation-related death, and engraftment did not differ substantially between the groups. CONCLUSIONS: Among patients undergoing allogeneic HLA-matched HSCT with reduced-intensity conditioning, GVHD-free, relapse-free survival at 1 year was significantly more common among those who received cyclophosphamide-tacrolimus-mycophenolate mofetil than among those who received tacrolimus-methotrexate. (Funded by the National Heart, Lung, and Blood Institute and others; BMT CTN 1703 ClinicalTrials.gov number, NCT03959241.).

Nanocellulose-Based Passivated-Carbon Quantum Dots (P-CQDs) for Antimicrobial Applications: A Practical Review.

Passivated-carbon quantum dots (P-CQDs) have been attracting great interest as an antimicrobial therapy tool due to their bright fluorescence, lack of toxicity, eco-friendly nature, simple synthetic schemes, and possession of photocatalytic functions comparable to those present in traditional nanometric semiconductors. Besides synthetic precursors, CQDs can be synthesized from a plethora of natural resources including microcrystalline cellulose (MCC) and nanocrystalline cellulose (NCC). Converting MCC into NCC is performed chemically via the top-down route, while synthesizing CODs from NCC can be performed via the bottom-up route. Due to the good surface charge status with the NCC precursor, we focused in this review on synthesizing CQDs from nanocelluloses (MCC and NCC) since they could become a potential source for fabricating carbon quantum dots that are affected by pyrolysis temperature. There are several P-CQDs synthesized with a wide spectrum of featured properties, namely functionalized carbon quantum dots (F-CQDs) and passivated carbon quantum dots (P-CQDs). There are two different important P-CQDs, namely 2,2'-ethylenedioxy-bis-ethylamine (EDA-CQDs) and 3-ethoxypropylamine (EPA-CQDs), that have achieved desirable results in the antiviral therapy field. Since NoV is the most common dangerous cause of nonbacterial, acute gastroenteritis outbreaks worldwide, this review deals with NoV in detail. The surficial charge status (SCS) of the P-CQDs plays an important role in their interactions with NoVs. The EDA-CQDs were found to be more effective than EPA-CQDs in inhibiting the NoV binding. This difference may be attributed to their SCS as well as the virus surface. EDA-CQDs with surficial terminal amino (-NH2) groups are positively charged at physiological pH (-NH3+), whereas EPA-CQDs with surficial terminal methyl groups (-CH3) are not charged. Since the NoV particles are negatively charged, they are attracted to the positively charged EDA-CQDs, resulting in enhancing the P-CQDs concentration around the virus particles. The carbon nanotubes (CNTs) were found to be comparable to the P-CQDs in the non-specific binding with NoV capsid proteins, through complementary charges, π-π stacking, and/or hydrophobic interactions.

Epigenetic and Transcriptional Dynamics of Notch Program in Intestinal Differentiation.

The equilibrium between stem cell self-renewal and differentiation followed by proper lineage specification of progenitor cells is considered imperative for maintaining intestinal homeostasis. In the hierarchical model, intestinal differentiation is defined by the stepwise acquisition of lineage-specific mature cell features, where Notch signaling and lateral inhibition instructively regulate the cell-fate decisions. Recent studies reveal a broadly permissive intestinal chromatin underlies the lineage plasticity and adaptation to diet mediated by Notch transcriptional program. Here, we review the conventional understanding of Notch programming in intestinal differentiation and describe how new data from epigenetic and transcriptional analyses may refine or revise the current view. We provide instructions on sample preparation and data analysis and explain how to use ChIP-seq and scRNA-seq in combination of lineage tracing assay to determine the dynamics of Notch program and intestinal differentiation in the context of dietary and metabolic regulation of cell-fate decisions.

A Novel Microwave Hot Pressing Machine for Production of Fixed Oils from Different Biopolymeric Structured Tissues.

A microwave hot pressing machine (MHPM) was used to heat the colander to produce fixed oils from each of castor, sunflower, rapeseed, and moringa seed and compared them to those obtained using an ordinary electric hot pressing machine (EHPM). The physical properties, namely the moisture content of seed (MCs), the seed content of fixed oil (Scfo), the yield of the main fixed oil (Ymfo), the yield of recovered fixed oil (Yrfo), extraction loss (EL), six Efficiency of fixed oil extraction (Efoe), specific gravity (SGfo), refractive index (RI) as well as chemical properties, namely iodine number (IN), saponification value (SV), acid value (AV), and the yield of fatty acid (Yfa) of the four oils extracted by the MHPM and EHPM were determined. Chemical constituents of the resultant oil were identified using GC/MS after saponification and methylation processes. The Ymfo and SV obtained using the MHPM were higher than those for the EHPM for all four fixed oils studied. On the other hand, each of the SGfo, RI, IN, AV, and pH of the fixed oils did not alter statistically due to changing the heating tool from electric band heaters into a microwave beam. The qualities of the four fixed oils extracted by the MHPM were very encouraging as a pivot of the industrial fixed oil projects compared to the EHPM. The prominent fatty acid of the castor fixed oil was found to be ricinoleic acid, making up 76.41% and 71.99% contents of oils extracted using the MHPM and EHPM, respectively. In addition, the oleic acid was the prominent fatty acid in each of the fixed oils of sunflower, rapeseed, and moringa species, and its yield by using the MHPM was higher than that for the EHPM. The role of microwave irradiation in facilitating fixed oil extrusion from the biopolymeric structured organelles (lipid bodies) was protruded. Since it was confirmed by the present study that using microwave irradiation is simple, facile, more eco-friendly, cost-effective, retains parent quality of oils, and allows for the warming of bigger machines and spaces, we think it will make an industrial revolution in oil extraction field.

An RNA Damage Response Network Mediates the Lethality of 5-FU in Clinically Relevant Tumor Types.

5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug's efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug's DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

Global calibration for non-targeted fraud detection in quinoa flour using portable hyperspectral imaging and chemometrics.

Quinoa is one of the highest nutritious grains, and global consumption of quinoa flour has increased as people pay more attention to health. Due to its high value, quinoa flour is susceptible to adulteration. Cross-contamination between quinoa flour and other flour can be easily neglected due to their highly similar appearance. Therefore, detecting adulteration in quinoa flour is important to consumers, industries, and regulatory agencies. In this study, portable hyperspectral imaging in the visible near-infrared (VNIR) spectral range (400-1000 nm) was applied as a rapid tool to detect adulteration in quinoa flour. Quinoa flour was adulterated with wheat, rice, soybean, and corn in the range of 0-98% with 2% increments. Partial least squares regression (PLSR) models were developed, and the best model for detecting the % authentic flour (quinoa) was obtained by the raw spectral data with R2p of 0.99, RMSEP of 3.08%, RPD of 8.77, and RER of 25.32. The model was improved, by selecting only 13 wavelengths using bootstrapping soft shrinkage (BOSS), to R2p of 0.99, RMSEP of 2.93%, RPD of 9.18, and RER of 26.60. A visualization map was also generated to predict the level of quinoa in the adulterated samples. The results of this study demonstrate the ability of VNIR hyperspectral imaging for adulteration detection in quinoa flour as an alternative to the complicated traditional method.

Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells.

BACKGROUND & AIMS: Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). METHODS: We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs in vivo. RESULTS: We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed in vitro. CONCLUSIONS: Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.

Quantitative p53 immunostaining aids in the detection of prevalent dysplasia.

AIMS: The lack of accepted scoring criteria has precluded the use of p53 in routine practice. We evaluate the utility of automated quantitative p53 analysis in risk stratifying Barrett's oesophagus (BE) patients using non-dysplastic BE (NDBE) biopsies in a multicentric cohort of BE progressor (P) and non-progressor (NP) patients. METHODS: NDBE biopsies prior to the diagnosis of advanced neoplasia from 75 BE-P, and index and last surveillance biopsies from 148 BE-NP were stained for p53, and scored digitally as 1+, 2+ and 3+. A secondary cohort of 30 BE-P was evaluated. RESULTS: Compared with BE-NP, BE-P was predominantly men (p=0.001), ≥55 years of age (p=0.008), with longer BE segments (71% vs 33%; p<0.001). The mean number of 3+p53 positive cells and 3+ positive glands were significantly more in BE-P versus BE-NP NDBE biopsies (175 vs 9.7, p<0.001; 9.8 vs 0.1; p<0.001, respectively). At a cut-off of ≥10 p53 (3+) positive cells, the sensitivity and specificity of the assay to identify BE-P were 39% and 93%. On multivariate analysis, scoring p53 in NDBE biopsies, age, gender and length of BE were significantly associated with neoplastic progression. 54% of patients classified as prevalent dysplasia showed an abnormal p53 immunohistochemical stain. These findings were validated in the secondary cohort. CONCLUSIONS: Automated p53 analysis in NDBE biopsies serves as a promising tool for assessing BE neoplastic progression and risk stratification. Our study highlights the practical applicability of p53 assay to routine surveillance practice and its ability to detect prevalent dysplasia.

Post-fast refeeding enhances intestinal stem cell-mediated regeneration and tumourigenesis through mTORC1-dependent polyamine synthesis.

For more than a century, fasting regimens have improved health, lifespan, and tissue regeneration in diverse organisms, including humans. However, how fasting and post-fast refeeding impact adult stem cells and tumour formation has yet to be explored in depth. Here, we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation: Post-fast refeeding augments the regenerative capacity of Lgr5+ intestinal stem cells (ISCs), and loss of the tumour suppressor Apc in ISCs under post-fast refeeding leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum (AL) fed states. This demonstrates that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust induction of mTORC1 in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production, or protein synthesis abrogates the regenerative or tumourigenic effects of post-fast refeeding. Thus, fast-refeeding cycles must be carefully considered when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst not only in stem cell-driven regeneration but also in tumourigenicity.