Changes In Protein Expression Research Articles

O08 Exploring the effect of skin ageing-related basement membrane collagen loss on nuclear mechanics

Abstract Introduction and aims Skin ageing is a universal process that can increase susceptibility to inflammation and disease. Basement membrane collagens (BMCs) are reduced during ageing and the consequences are poorly understood. BMC sensing of mechanical stress, vital for skin homeostasis, elicits signalling sensed via cytoskeletal actin filaments to the linker of nucleoskeleton and cytoskeleton (LINC) complex (composed of nesprin and sun proteins), nuclear lamina and lamina-associated chromatin. Our hypothesis is that BMC loss during ageing leads to a deformation of the nucleocytoskeleton and disrupts chromatin organization in keratinocytes, leading to impairment of epidermal differentiation and inability to withstand mechanical stress. Methods To explore the role of BMC in mechanical stress response, stable lentiviral short hairpin (sh)RNA knockdowns of Col7 (shCol7) and Col17 (shCol17) were generated in immortalized N/TERT keratinocytes. Western blot (WB) validated knockdowns were compared with shC nontargeting control. IncuCyte assays showed shCol17 had a hyperproliferative phenotype compared with shC. Sun2 was reduced in shCol17 (P < 0.001) compared with shC (n = 5 experimental replicates). The mechanical stretch effect on cells on a flexible membrane was studied using immunofluorescence and WB analysis. Images taken from the IN Cell Analyzer confocal microscope were analysed to detect changes in protein expression and localization. Nuclear and cytoplasmic fractions made before and after stretch were analysed by WB. Results We observed collagen-specific responses to stretch-induced stress with changes to actin, components of the nuclear membrane, and LINC complex proteins. Additionally, work on investigating chromatin changes with BMC knockdown using Cleavage Under Targets & Tagmentation and Cleavage Under Targeted Accessible Chromatin assays is ongoing. Conclusions Our data show an altered nucleocytoskeleton in the context of BMC loss and mechanotransduction in skin. These data could provide novel insights into epidermal regulation by BMC in ageing.

P11 A role for keratinocyte arginase in skin wound healing and barrier function

Abstract Introduction and aims Delayed cutaneous wound healing is a major problem that can result in social isolation and significant morbidity including amputation. There are many changes in cellular function in a chronic wound. Notably, the enzyme arginase (ARG)1 is markedly changed in wound healing. We have identified a role for ARG1 in keratinocyte re-epithelialization during wound closure, but the underlying mechanism driving re-epithelialization was not known. We aimed to determine the role of ARG1 in keratinocyte migration, differentiation and barrier formation. Methods Two-dimensional scratch and differentiation assays using immortalized keratinocytes (NTERT cells) treated with the ARG1 inhibitor, nor-NOHA, and short hairpin RNA-based knockdown cells were assessed. Gene and protein expression changes were highlighted. Expression of proteins was further validated using immunohistochemical profiling of human skin equivalents and human wound lesions by spatial transcriptomics. Results ARG1 was strongly expressed both 2–4h postscratching and at late stages of keratinocyte differentiation. Dysregulation of ARG1 expression was spatially highlighted in chronic lesions, further suggesting an involvement in keratinocyte migration and differentiation. ARG1 inhibition led to a significant delay in postscratch keratinocyte migration. Furthermore, upon ARG1 inhibition, a significant decrease was seen in the expression of late differentiation markers involucrin and filaggrin. ARG1 inhibition significantly decreased the expression of host defence genes β-defensin 3, lipocalin 2 and kallikrein 6. ARG1 triggers production of multiple downstream products and we confirmed a role for the pathway involving production of putrescine and urea, as the addition of these metabolites led to the rescue of these phenotypes. Conclusions ARG1 and its downstream metabolites play a major role in keratinocyte migration, differentiation, and barrier formation, which are vital functions for re-epithelialization and wound closure. Therefore, manipulation of the ARG1 pathway may be crucial in the management of chronic lesions.

TPX2 upregulates MMP13 to promote the progression of lipopolysaccharide-induced osteoarthritis.

This study seeks to identify potential clinical biomarkers for osteoarthritis (OA) using bioinformatics and investigate OA mechanisms through cellular assays. Differentially Expressed Genes (DEGs) from GSE52042 (four OA samples, four control samples) were screened and analyzed with protein-protein interaction (PPI) analysis. Overlapping genes in GSE52042 and GSE206848 (seven OA samples, and seven control samples) were identified and evaluated using Gene Set Enrichment Analysis (GSEA) and clinical diagnostic value analysis to determine the hub gene. Finally, whether and how the hub gene impacts LPS-induced OA progression was explored by in vitro experiments, including Western blotting (WB), co-immunoprecipitation (Co-IP), flow cytometry, etc. Bioinformatics analysis of DEGs (142 up-regulated and 171 down-regulated) in GSE52042 identified two overlapping genes (U2AF2, TPX2) that exhibit significant clinical diagnostic value. These genes are up-regulated in OA samples from both GSE52042 and GSE206848 datasets. Notably, TPX2, which AUC = 0.873 was identified as the hub gene. In vitro experiments have demonstrated that silencing TPX2 can alleviate damage to chondrocytes induced by lipopolysaccharide (LPS). Furthermore, there is a protein interaction between TPX2 and MMP13 in OA. Excessive MMP13 can attenuate the effects of TPX2 knockdown on LPS-induced changes in OA protein expression, cell growth, and apoptosis. In conclusion, our findings shed light on the molecular mechanisms of OA and suggested TPX2 as a potential therapeutic target. TPX2 could promote the progression of LPS-induced OA by up-regulating the expression of MMP13, which provides some implications for clinical research.

Glycyrrhizin Ameliorates Cardiac Injury in Rats with Severe Acute Pancreatitis by Inhibiting Ferroptosis via the Keap1/Nrf2/HO-1 Pathway.

Severe acute pancreatitis (SAP) is a potential fatal gastrointestinal disease that is usually complicated by myocardial injury and dysfunction. Due to the lack of understanding of the mechanism of SAP-associated cardiac injury (SACI), there is still no complete treatment. To explore the alleviative effect and anti-ferroptosis mechanism against SACI of glycyrrhizin (GL), an inhibitor of oxidative stress. The SAP model was established by perfusing 5% sodium taurocholate into biliopancreatic duct in rats. H&E staining and serum assays were used to assess the injury changes of pancreas and heart. Echocardiography was used to evaluate the cardiac function. Transmission electron microscopy (TEM) and oxidative stress assays were used to investigate the ferroptosis-related morphological and biochemical changes. Western blot and immunofluorescence were performed to analyzed the expression of ferroptosis-related proteins. Significant myocardial impairment was found in SAP rats according to increased histopathological scores, serum creatine kinase-MB (CK-MB) and cardiac troponin-I (cTnI) levels, and a decreased fractional shortening and ejection fraction. The decreased mitochondrial cristae and significant expression changes of ferroptosis-related proteins confirmed the presence of ferroptosis in SACI. GL treatment attenuated above-mentioned cardiac tissues damage by inhibiting ferroptosis via restoring the expression of Nrf2 and HO-1 in vivo and in vitro. Treating with ML385 (a Nrf2 inhibitor) or transfecting with siRNA-Nrf2 reversed the protective effect of GL. Our findings demonstrate the involvement of ferroptosis in SACI and suggest a potential role for GL in the treatment of SACI by supressing ferroptosis via Keap1/Nrf2/HO-1 pathway.

Modeling of host PDZ-dependent interactions with SARS-CoV-2 Envelope protein and changes in PDZ proteins expression in macrophages and dendritic cells.

PDZ (PSD-95/Dlg/ZO-1) domain-containing proteins constitute a large family of scaffolds involved in a wide range of cellular tasks, and mainly studied in polarity functions. Diverse host PDZ proteins can be targeted by viral pathogens which express proteins containing PDZ-binding motifs (PDZbm). Previously, we have identified host PDZ-based interactions with the SARS-CoV-2 E protein (2E) in human monocytes. Here, we deepen the study of these interactions by docking and molecular dynamics analyses to identify the most favorable PDZ-PDZbm interaction of seven host PDZ proteins with the PDZbm of 2E. In addition, we analyzed changes in the expression of three of the PDZ proteins identified as 2E interactors in monocytes (syntenin, ZO-2, and IL-16), in human monocyte-derived macrophages (MΦ) and in dendritic cells (DCs) upon stimulation. Our results suggest that these PDZ proteins may have important functions in professional antigen-presenting cells (APCs), and their targeting by the PDZbm of 2E, a central virulence determinant of SARS-CoV-2, support the hypothesis that such PDZ-dependent interaction in immune cells may constitute a viral evasion mechanism. Inhibitor design based on the PDZbm of 2E in the development of drugs against a variety of diseases is discussed.

Construction and Validation of a Rat Model of Acute Necrotizing Pancreatitis-Associated Intestinal Injury.

Acute pancreatitis (AP) is an acute inflammatory reaction of the pancreatic tissue, which involves auto-digestion, oedema, haemorrhage, and necrosis. AP can be categorized into mild, moderately severe and severe AP, with severe pancreatitis also referred to as acute necrotizing pancreatitis (ANP). ANP is characterized by the accumulation of necrotic material in the peritoneal cavity. This can result in intestinal injury. However, the mechanism of ANP-associated intestinal injury remains unclear. We established an ANP-associated intestinal injury rat model (ANP-IR model) by injecting pancreatitis-associated ascites fluid (PAAF) and necrotic pancreatic tissue at various proportions into the triangular area formed by the left renal artery and ureter. The feasibility of the ANP-IR model was verified by comparing the similar changes in indicators of intestinal inflammation and barrier function between the two rat models. In addition, we detected changes in apoptosis levels and YAP protein expression in the ileal tissues of rats in each group and validated them in vitro in rat epithelial crypt cells (IEC-6) to further explore the potential injury mechanisms of ANP-associated intestinal injury. We also collected clinical data from patients with ANP to validate the effects of PAAF and pancreatic necrosis on intestinal injury. Our findings offer a theoretical basis for restricting the buildup of peritoneal necrosis in individuals with ANP, thus promoting the restoration of intestinal function and enhancing treatment efficacy. The use of the ANP-IR model in further studies can help us better understand the mechanism and treatment of ANP-associated intestinal injury.

Ontogeny of Human Liver Aldehyde Oxidase: Developmental Changes and Implications for Drug Metabolism.

Despite the increasing importance of aldehyde oxidase (AO) in the drug metabolism of clinical candidates, ontogeny data for AO are limited. The objective of our study was to characterize the age-dependent AO content and activity in the human liver cytosolic fraction (HLC) and human hepatocytes (HH). HLC (n = 121 donors) and HH (n = 50 donors) were analyzed for (1) AO protein content by quantitative proteomics and (2) enzyme activity using carbazeran as a probe substrate. AO activity showed high technical variability and poor correlation with the content in HLC samples, whereas hepatocyte samples showed a strong correlation between the content and activity. Similarly, AO content and activity showed no significant age-dependent differences in HLC samples, whereas the average AO content and activity in hepatocytes increased significantly (∼20-40-fold) from the neonatal levels (0-28 days). Based on the hepatocyte data, the age at which 50% of the adult AO content is reached (age50) was 3.15 years (0.32-13.97 years, 95% CI). Metabolite profiling of carbazeran revealed age-dependent metabolic switching and the role of non-AO mechanisms (glucuronidation and desmethylation) in carbazeran elimination. The content-activity correlation in hepatocytes improved significantly (R2 = 0.95; p < 0.0001) in samples showing <10% contribution of glucuronidation toward the overall metabolism, confirming that AO-mediated oxidation and glucuronidation are the key routes of carbazeran metabolism. Considering the confounding effect of glucuronidation on AO activity, AO content-based ontogeny data are a more direct reflection of developmental changes in protein expression. The comprehensive ontogeny data of AO in HH samples are more reliable than HLC data, which are important for developing robust physiologically based pharmacokinetic models for predicting AO-mediated metabolism in children.

Exploring the Impact of Preoperative Sleep Fragmentation on Cognitive Function in Mice: The Role of Microglial Activation and Iron Metabolism

Background. Perioperative neurocognitive disorders (PND) are a significant concern, particularly for aged individuals. Sleep fragmentation (SF), a common condition in older adults, is considered a risk factor for PND. The present study explored the impact of SF on cognitive function and its association with microglial activation and iron metabolism. Methods. Adult and aged C57BL/6J mice were subjected to tibial fracture surgery (TFS) and varying durations of SF. Cognitive function was assessed using the Morris water maze and fear conditioning experiments. Microglial activation was evaluated by measuring CD68 protein expression and inflammatory cytokine levels. Iron metabolism and ferroptosis-related proteins were also examined. Results. SF significantly impacted spatial memory and conditioned fear responses in mice, with aged mice showing greater susceptibility. Microglial activation, indicated by changes in CD68 protein expression and inflammatory cytokine levels, was observed in mice exposed to SF. Alterations in iron metabolism, as evidenced by changes in hippocampal iron content and expression of ferroptosis-related proteins, were also observed in these mice. Conclusion. SF can lead to significant cognitive impairment, particularly in aged mice, likely mediated through microglial activation and dysregulated iron metabolism. These findings provide novel insights into the pathogenesis of PND and suggest potential targets for intervention. Significance. This study illuminates the complex interactions between SF, microglial activation, and cognitive function. It highlights the importance of sleep quality for cognitive health in older adults and points to potential therapeutic strategies for preventing PND, including targeting microglial activation and iron metabolism.

Decrypting the molecular basis of cellular drug phenotypes by dose-resolved expression proteomics.

Proteomics is making important contributions to drug discovery, from target deconvolution to mechanism of action (MoA) elucidation and the identification of biomarkers of drug response. Here we introduce decryptE, a proteome-wide approach that measures the full dose-response characteristics of drug-induced protein expression changes that informs cellular drug MoA. Assaying 144 clinical drugs and research compounds against 8,000 proteins resulted in more than 1 million dose-response curves that can be interactively explored online in ProteomicsDB and a custom-built Shiny App. Analysis of the collective data provided molecular explanations for known phenotypic drug effects and uncovered new aspects of the MoA of human medicines. We found that histone deacetylase inhibitors potently and strongly down-regulated the T cell receptor complex resulting in impaired human T cell activation in vitro and ex vivo. This offers a rational explanation for the efficacy of histone deacetylase inhibitors in certain lymphomas and autoimmune diseases and explains their poor performance in treating solid tumors.

Glycolic acid-induced disruption of epidermal homeostasis in a skin equivalent model: Insights into temporal dynamics and mechanisms

Glycolic acid (GA) is extensively used in cosmetic formulations and skin peeling treatments but its adverse effects, notably severe disruption of epidermal structure, limit its clinical utility. However, the detailed impact of GA on epidermal homeostasis, including changes in structure and protein expression over time, is not fully understood. This study employed a reconstructed human epidermis (RHE) model to assess the effects of varying GA concentrations on epidermal proliferation, differentiation, and desquamation at different time points. Through histology, immunofluorescence, and immunohistochemistry, we observed that 35% GA concentration adversely caused abnormal epidermal homeostasis by affecting epidermal proliferation, differentiation and desquamation. Our findings reveal time-specific responses of key proteins to GA: Filaggrin, Involucrin, Loricrin, and Ki67 showed very early responses; KLK10 an early response; and AQP3 and K10 late responses. This research provides a detailed characterization of GA's effects in an RHE model, mimicking clinical superficial peeling and identifying optimal times for detecting GA-induced changes. Our results offer insights for designing interventions to mitigate GA's adverse effects on skin, enhancing the safety and efficacy of GA peeling treatments.

Evaluating the toxicity of estetrol, 17α-ethinylestradiol, and their combination with drospirenone on zebrafish larvae: A behavioural and proteomic study

ObjectiveTo characterise and compare the toxicity of estetrol (E4) and 17α-ethinylestradiol (EE2), and their respective mixture with the progestin drospirenone (DRSP) in zebrafish (Danio rerio) embryos. MethodsZebrafish embryos were exposed to E4, EE2, DRSP, E4+DRSP, and EE2+DRSP in a fish embryo acute toxicity (FET) test. A second test examined behavioural responses and, using label-free proteomics, identified changes in protein expression in response to hormonal treatments, across a range of concentrations, including those that are considered to be environmentally relevant. ResultsIn the FET test, no effects were found from E4 at concentrations ≤100 mg/L, while EE2 induced mortality and morphological abnormalities at concentrations of 1-2 mg/L. In the behavioural test, exposure to 30 ng/L EE2 (∼200 × predicted environmental concentration – PEC) resulted in hypoactivity in fish larvae and exposure to 0.3 ng/L EE2 (∼2 × PEC) led to quantitative changes in protein abundance, revealing potential impacts on RNA processing and protein synthesis machinery. Exposure to E4 did not alter behaviour, but several groups of proteins were modulated, mainly at 710 ng/L (∼200 × PEC), including proteins involved in oxidative phosphorylation. When combined with DRSP, EE2 induced reduced effects on behaviour and proteomic responses, suggesting an antagonistic effect of DRSP. E4+DRSP induced no significant effects on behaviour or proteomic profiles at tested concentrations. ConclusionsThese findings suggest that E4-based combined oral contraceptives present a more favourable environmental profile than EE2-based contraceptives, particularly during the early developmental stages of fish.

Proteomic characterization of the different stages of seed germination in Cupressus gigantea

Abstract Seed germination is a pivotal period of plant growth and development. This process can be divided into four major stages, swelling absorption, seed coat dehiscence, radicle emergence and radicle elongation. Cupressus gigantea, a tree native to Tibet, China, is characterized by its resistance to stresses such as cold, and drought, and has a high economic and ecological value. Nevertheless, given its unique geographic location, its seeds are difficult to germinate. Therefore, it is crucial to explore the mechanisms involved in seed germination in this species to improve the germination efficiency of its seeds, thereby protecting this high-quality resource. Here, our findings indicate that seed germination was enhanced when exposed to a 6-h/8-h light/dark photoperiod, coupled with a temperature of 20°C. Furthermore, the application of exogenous GA3 (1 mg/ml, about 2.9 mM) stimulated the germination of C. gigantea seeds. Subsequently, proteomics was used to detect changes in protein expression during the four stages of seed germination. We identified 34 differentially expressed proteins (DEPs), including 13 at the radicle pre-emergence stage, and 17 at the radicle elongation stage. These DEPs were classified into eight functional groups, cytoskeletal proteins, energy metabolism, membrane transport, stress response, molecular chaperones, amino acid metabolism, antioxidant system and ABA signalling pathway. Most of them were found to be closely associated with amino acid metabolism. Combined, these findings indicate that, along with temperature and light, exogenous GA3 can increase the germination efficiency of C. gigantea seeds. Our study also offers insights into the changes in protein expression patterns in C. gigantea seeds during germination.

Human macrophages infected with Egyptian Rousette bat-isolated Marburg virus display inter-individual susceptibility and antiviral responsiveness

Marburg virus (MARV) is a highly pathogenic filovirus and a causative agent of sporadic zoonotic viral hemorrhagic fever outbreaks with high case fatality rates. In humans, filoviruses like MARV and Zaire Ebola virus (EBOV) target, among others, innate immune cells like dendritic cells and macrophages (MΦs). Filovirus-infected dendritic cells display impaired maturation and antigen presentation, while MΦs become hyper-activated and secrete proinflammatory cytokines and chemokines. Our current understanding of human macrophage responses to MARV remains limited. Here, we used human monocyte-derived macrophages (moMΦs) to address how their phenotype, transcriptional profile, and protein expression change upon an in vitro infection with a bat isolate of MARV. Confirming its tropism for macrophages, we show that MARV induces notable shifts in their transcription distinct from responses induced by lipopolysaccharide (LPS), marked by upregulated gene expression of several chemokines, type I interferons, and IFN-stimulated genes. MARV infection also elicited pronounced inter-individually different transcriptional programs in moMΦs, the induction of Wnt signaling-associated genes, and the downregulation of multiple biological processes and molecular pathways.

A human pluripotent stem cell-derived in vitro model of the blood–brain barrier in cerebral malaria

BackgroundBlood–brain barrier (BBB) disruption is a central feature of cerebral malaria (CM), a severe complication of Plasmodium falciparum (Pf) infections. In CM, sequestration of Pf-infected red blood cells (Pf-iRBCs) to brain endothelial cells combined with inflammation, hemolysis, microvasculature obstruction and endothelial dysfunction mediates BBB disruption, resulting in severe neurologic symptoms including coma and seizures, potentially leading to death or long-term sequelae. In vitro models have advanced our knowledge of CM-mediated BBB disruption, but their physiological relevance remains uncertain. Using human induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs), we aimed to develop a novel in vitro model of the BBB in CM, exhibiting enhanced barrier properties.MethodshiPSC-BMECs were co-cultured with HB3var03 strain Pf-iRBCs up to 9 h. Barrier integrity was measured using transendothelial electrical resistance (TEER) and sodium fluorescein permeability assays. Localization and expression of tight junction (TJ) proteins (occludin, zonula occludens-1, claudin-5), cellular adhesion molecules (ICAM-1, VCAM-1), and endothelial surface markers (EPCR) were determined using immunofluorescence imaging (IF) and western blotting (WB). Expression of angiogenic and cell stress markers were measured using multiplex proteome profiler arrays.ResultsAfter 6-h of co-culture with Pf-iRBCs, hiPSC-BMECs showed reduced TEER and increased sodium fluorescein permeability compared to co-culture with uninfected RBCs, indicative of a leaky barrier. We observed disruptions in localization of occludin, zonula occludens-1, and claudin-5 by IF, but no change in protein expression by WB in Pf-iRBC co-cultures. Expression of ICAM-1 and VCAM-1 but not EPCR was elevated in hiPSC-BMECs with Pf-iRBC co-culture compared to uninfected RBC co-culture. In addition, there was an increase in expression of angiogenin, platelet factor-4, and phospho-heat shock protein-27 in the Pf-iRBCs co-culture compared to uninfected RBC co-culture.ConclusionThese findings demonstrate the validity of our hiPSC-BMECs based model of the BBB, that displays enhanced barrier integrity and appropriate TJ protein localization. In the hiPSC-BMEC co-culture with Pf-iRBCs, reduced TEER, increased paracellular permeability, changes in TJ protein localization, increase in expression of adhesion molecules, and markers of angiogenesis and cellular stress all point towards a novel model with enhanced barrier properties, suitable for investigating pathogenic mechanisms underlying BBB disruption in CM.

WNT5B drives osteosarcoma stemness, chemoresistance and metastasis.

Treatment for osteosarcoma, a paediatric bone cancer with no therapeutic advances in over three decades, is limited by a lack of targeted therapies. Osteosarcoma frequently metastasises to the lungs, and only 20% of patients survive 5 years after the diagnosis of metastatic disease. We found that WNT5B is the most abundant WNT expressed in osteosarcoma tumours and its expression correlates with metastasis, histologic subtype and reduced survival. Using tumor-spheroids to model cancer stem-like cells, we performed qPCR, immunoblotting, and immunofluorescence to monitor changes in gene and protein expression. Additionally, we measured sphere size, migration and forming efficiency to monitor phenotypic changes. Therefore, we characterised WNT5B's relevance to cancer stem-like cells, metastasis, and chemoresistance and evaluated its potential as a therapeutic target. In osteosarcoma cell lines and patient-derived spheres, WNT5B is enriched in stem cells and induces the expression of the stemness gene SOX2. WNT5B promotes sphere size, sphere-forming efficiency, and cell proliferation, migration, and chemoresistance to methotrexate (but not cisplatin or doxorubicin) in spheres formed from conventional cell lines and patient-derived xenografts. In vivo, WNT5B increased osteosarcoma lung and liver metastasis and inhibited the glycosaminoglycan hyaluronic acid via upregulation of hyaluronidase 1 (HYAL1), leading to changes in the tumour microenvironment. Further, we identified that WNT5B mRNA and protein correlate with the receptor ROR1 in primary tumours. Targeting WNT5B through inhibition of WNT/ROR1 signalling with an antibody to ROR1 reduced stemness properties, including chemoresistance, sphere size and SOX2 expression. Together, these data define WNT5B's role in driving osteosarcoma cancer stem cell expansion and methotrexate resistance and provide evidence that the WNT5B pathway is a promising candidate for treating osteosarcoma patients. WNT5B expression is high in osteosarcoma stem cells leading to increased stem cell proliferation and migration through SOX2. WNT5B expression in stem cells increases rates of osteosarcoma metastasis to the lungs and liver in vivo. The hyaluronic acid degradation enzyme HYAL1 is regulated by WNT5B in osteosarcoma contributing to metastasis. Inhibition of WNT5B with a ROR1 antibody decreases osteosarcoma stemness.

Ketogenic Diet Protects from Experimental Colitis in a Mouse Model Regardless of Dietary Fat Source.

While ketogenic diets (KDs) may have potential as adjunct treatments for gastrointestinal diseases, there is little knowledge on how the fat source of these diets impacts intestinal health. The objective of this study was to investigate how the source of dietary fat of KD influences experimental colitis. We fed nine-week-old male C57BL/6J mice (n = 36) with a low-fat control diet or KD high either in saturated fatty acids (SFA-KD) or polyunsaturated linoleic acid (LA-KD) for four weeks and then induced colitis with dextran sodium sulfate (DSS). To compare the diets, we analyzed macroscopic and histological changes in the colon, intestinal permeability to fluorescein isothiocyanate-dextran (FITC-dextran), and the colonic expression of tight junction proteins and inflammatory markers. While the effects were more pronounced with LA-KD, both KDs markedly alleviated DSS-induced histological lesions. LA-KD prevented inflammation-related weight loss and the shortening of the colon, as well as preserved Il1b and Tnf expression at a healthy level. Despite no significant between-group differences in permeability to FITC-dextran, LA-KD mitigated changes in tight junction protein expression. Thus, KDs may have preventive potential against intestinal inflammation, with the level of the effect being dependent on the dietary fat source.

Iron Supplementation Increases Tumor Burden and Alters Protein Expression in a Mouse Model of Human Intestinal Cancer.

Iron supplements are widely consumed. However, excess iron may accelerate intestinal tumorigenesis. To determine the effect of excess iron on intestinal tumor burden and protein expression changes between tumor and normal tissues, ApcMin/+ mice were fed control (adequate) and excess iron (45 and 450 mg iron/kg diet, respectively; n = 9/group) for 10 wk. Tumor burden was measured, and two-dimensional fluorescence difference gel electrophoresis was used to identify differentially expressed proteins in tumor and normal intestinal tissues. There was a significant increase (78.3%; p ≤ 0.05) in intestinal tumor burden (mm2/cm) with excess iron at wk 10. Of 980 analyzed protein spots, 69 differentially expressed (p ≤ 0.05) protein isoforms were identified, representing 55 genes. Of the isoforms, 56 differed (p ≤ 0.05) between tumor vs. normal tissues from the adequate iron group and 23 differed (p ≤ 0.05) between tumors from the adequate vs. excess iron. Differentially expressed proteins include those involved in cell integrity and adaptive response to reactive oxygen species (including, by gene ID: ANPEP, DPP7, ITGB1, PSMA1 HSPA5). Biochemical pathway analysis found that iron supplementation modulated four highly significant (p ≤ 0.05) functional networks. These findings enhance our understanding of interplay between dietary iron and intestinal tumorigenesis and may help develop more specific dietary guidelines regarding trace element intake.

The contributions of Pichia kudriavzevii EP1 to sourdough: Acid utilization, oligosaccharides and peptides production and aroma enhancement

Previous studies have demonstrated that co-fermenting Pichia kudriavzevii EP1 (EP) and Fructilactobacillus sanfranciscensis can enhance the aroma of sourdough bread. This research aimed to examine the fermentation mechanism of EP compared to Saccharomyces cerevisiae (N) in sourdough and investigate the pathways involved in aroma production. Gas chromatography was used to analyze volatile organic compounds (VOCs) and their correlative essential metabolites. Metabolomics and proteomics were utilized to track changes in metabolic pathways and protein expression during fermentation. The findings revealed that EP fermentation resulted in a significant decrease in VOCs like 2-phenylethanol and octanoic acid, while showing a notable increase (384%) in hexanol production. Essential metabolites revealed reduced utilization of sugars and amino acids, and decreased generation of organic acids like lactic and acetic acid. Specifically, EP fermentation led to decreased levels of glucose-6-phosphate dehydrogenase, pyruvate kinase, NAD+, and NADH, while ATP showed a significant increase. Metabolomics and proteomics analyses highlighted reduced utilization of sugars and amino acids for VOCs production. Consequently, sourdough fermented with EP was rich in oligosaccharides and small peptide molecules. EP utilized organic acids not only to reduce sourdough acidity but also to generate acetyl-CoA. Acetyl-CoA served as energy sources for metabolic activities, such as catalyzing transformations between asparagine, aspartate, glutamate, and glutamine, and played a role in producing higher alcohols like hexanol to enhance sourdough aroma. These findings suggest that EP prefers acid utilization over sugars and amino acids, offering a potential strategy for enhancing sourdough aroma through individual or combined fermentations with other microbiotas.

Proteins Involved in Focal Cell Adhesion and Podosome Formation Are Differentially Expressed during Colorectal Tumorigenesis in AOM-Treated Rats.

Colorectal tumorigenesis involves the development of aberrant crypt foci (ACF) or preneoplastic lesions, representing the earliest morphological lesion visible in colon cancer. The purpose of this study was to determine changes in protein expression in carcinogen-induced ACF as they mature and transform into adenomas. Protein expression profiles of azoxymethane (AOM)-induced F344 rat colon ACF and adenomas were compared at four time points, 4 (control), 8, 16, and 24 weeks post AOM administration (n = 9/group), with time points correlating with induction and transformation events. At each time point, micro-dissected ACF and/or adenoma tissues were analyzed across multiple quantitative two-dimensional (2D-DIGE) gels using a Cy-dye labeling technique and a pooled internal standard to quantify expression changes with statistical confidence. Western blot and subsequent network pathway mapping were used to confirm and elucidate differentially expressed (p ≤ 0.05) proteins, including changes in vinculin (Vcl; p = 0.007), scinderin (Scin; p = 0.02), and profilin (Pfn1; p = 0.01), By determining protein expression changes in ACF as they mature and transform into adenomas, a "baseline" of altered regulatory proteins associated with adenocarcinoma development in this model has been elucidated. These data will enable future studies aimed at biomarker identification and understanding the molecular biology of intestinal tumorigenesis and adenocarcinoma maturation under varying intestinal conditions.

Influence of Human Bone Marrow Mesenchymal Stem Cells Secretome from Acute Myeloid Leukemia Patients on the Proliferation and Death of K562 and K562-Lucena Leukemia Cell Lineages.

Leukemias are among the most prevalent types of cancer worldwide. Bone marrow mesenchymal stem cells (MSCs) participate in the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases such as leukemias, to a yet unknown extent. Here we described the effect of secretome of bone marrow MSCs obtained from healthy donors and from patients with acute myeloid leukemia (AML) on leukemic cell lineages, sensitive (K562) or resistant (K562-Lucena) to chemotherapy drugs. Cell proliferation, viability and death were evaluated, together with cell cycle, cytokine production and gene expression of ABC transporters and cyclins. The secretome of healthy MSCs decreased proliferation and viability of both K562 and K562-Lucena cells; moreover, an increase in apoptosis and necrosis rates was observed, together with the activation of caspase 3/7, cell cycle arrest in G0/G1 phase and changes in expression of several ABC proteins and cyclins D1 and D2. These effects were not observed using the secretome of MSCs derived from AML patients. In conclusion, the secretome of healthy MSCs have the capacity to inhibit the development of leukemia cells, at least in the studied conditions. However, MSCs from AML patients seem to have lost this capacity, and could therefore contribute to the development of leukemia.