The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma.

Despite recent advances in therapeutic treatments, multiple myeloma (MM) remains an incurable malignancy. Epigenetic factors contribute to the initiation, progression, relapse, and clonal heterogeneity in MM, but our knowledge on epigenetic mechanisms underlying MM development is far from complete. The SAGA complex serves as a coactivator in transcription and catalyzes acetylation and deubiquitylation. Analyses of datasets in the Cancer Dependency Map Project revealed many SAGA components are selective dependencies in MM. To define SAGA-specific functions, we focused on ADA2B, the only subunit in the lysine acetyltransferase (KAT) module that specifically functions in SAGA. Integration of RNA-seq, ATAC-seq, and CUT&RUN results identified pathways directly regulated by ADA2B include MTORC1 signaling, MYC, E2F, and MM-specific MAF oncogenic programs. We discovered that ADA2B is recruited to MAF and MYC gene targets, and that MAF shares a majority of its targets with MYC in MM cells. Furthermore, we found the SANT domain of ADA2B is required for interaction with both GCN5 and PCAF acetyltransferases, incorporation into SAGA, and ADA2B protein stability. Our findings uncover previously unknown SAGA KAT module-dependent mechanisms controlling MM cell growth, revealing a vulnerability that might be exploited for future development of MM therapy.

A reconstructed genome-scale metabolic model of Helicobacter pylori for predicting putative drug targets in clarithromycin and rifampicin resistance conditions.

BACKGROUND: Helicobacter pylori is considered a true human pathogen for which rising drug resistance constitutes a drastic concern globally. The present study aimed to reconstruct a genome-scale metabolic model (GSMM) to decipher the metabolic capability of H. pylori strains in response to clarithromycin and rifampicin along with identification of novel drug targets. MATERIALS AND METHODS: The iIT341 model of H. pylori was updated based on genome annotation data, and biochemical knowledge from literature and databases. Context-specific models were generated by integrating the transcriptomic data of clarithromycin and rifampicin resistance into the model. Flux balance analysis was employed for identifying essential genes in each strain, which were further prioritized upon being nonhomologs to humans, virulence factor analysis, druggability, and broad-spectrum analysis. Additionally, metabolic differences between sensitive and resistant strains were also investigated based on flux variability analysis and pathway enrichment analysis of transcriptomic data. RESULTS: The reconstructed GSMM was named as HpM485 model. Pathway enrichment and flux variability analyses demonstrated reduced activity in the ribosomal pathway in both clarithromycin- and rifampicin-resistant strains. Also, a significant decrease was detected in the activity of metabolic pathways of clarithromycin-resistant strain. Moreover, 23 and 16 essential genes were exclusively detected in clarithromycin- and rifampicin-resistant strains, respectively. Based on prioritization analysis, cyclopropane fatty acid synthase and phosphoenolpyruvate synthase were identified as putative drug targets in clarithromycin- and rifampicin-resistant strains, respectively. CONCLUSIONS: We present a robust and reliable metabolic model of H. pylori. This model can predict novel drug targets to combat drug resistance and explore the metabolic capability of H. pylori in various conditions.

The role of gender differences in the outcome of juvenile social isolation: Emphasis on changes in behavioral, biochemical and expression of nitric oxide synthase genes alteration.

Social isolation can cause serious problem in performance of individuals in community. As gender differences may cause variation results in the severity of depressive behavior and response of patients to therapy, the impact of gender and the interaction of the level of endocrine secretion in depression were investigated in this study. Wistar rats of both sexes were subjected to post-weaning social isolation (PWSI) conditions and, together with the control group, experienced several behavioral tests including open-field Test (OFT), elevated plus maze (EPM), force swimming test (FST), splash test and novel object recognition test (NOR). Hippocampal tissue was isolated to measure biochemical factors such as nitric oxide level, FRAP amount, MDA level. In addition, real-time-PCR test was used to quantify the genes expression level of inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS). On the other hand, sexual hormone levels in blood were measured. Both cognitive and behavioral f unctions were declined as the result of PWSI induction in male and diestrus female rats. The consequent surge of estradiol during estrous phase seems to suppress the accumulation of reactive oxygen species (ROS), and modulate iNOS and nNOS expression. In conclusion, while the pattern of PWSI in surge cellular antioxidants, raising cellular ROS level is gender-specific, this alleviation was in relation with the drop of estradiol and unrelated with testosterone level.

Agrobacterium-Mediated Transient Gene Expression Optimized for the Bioenergy Crop Camelina sativa.

Camelina sativa, a Brassicaceae family crop, is used for fodder, human food, and biofuels. Its relatively high resistance to abiotic and biotic stresses, as well as being a climate-resilient oilseed crop, has contributed to its popularity. Camelina's seed yield and oil contents have been improved using various technologies like RNAi and CRISPR/Cas9 genome editing. A stable transformation system for protein localization and other cell autonomous investigations, on the other hand, is tedious and time consuming. This study describes a transient gene expression protocol for Camelina sativa cultivar DH55 leaves using Agrobacterium strain C58C1. The method is suitable for subcellular protein localization and colocalization studies and can be used with both constitutive and chemically induced genes. We report the subcellular localization of the N-terminal ER membrane signal anchor region (1-32 aa) of the At3G28580 gene-encoded protein from Arabidopsis in intact leaves and the expression and localization of other known organelle markers. This method offers a fast and convenient way to study proteins in the commercially important Camelina crop system. Key features • This method is based on the approach of Zhang et al. [1] and has been optimized for bioenergy crop Camelina species. • A constitutive and inducible transient gene expression in the hexaploid species Camelina sativa cultivar DH55. • Requires only 16-18 days to complete with high efficacy. Graphical overview.

Integrating Multi-omics Data for Alzheimer's Disease to Explore Its Biomarkers Via the Hypergraph-Regularized Joint Deep Semi-Non-Negative Matrix Factorization Algorithm.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Its etiology may be associated with genetic, environmental, and lifestyle factors. With the advancement of technology, the integration of genomics, transcriptomics, and imaging data related to AD allows simultaneous exploration of molecular information at different levels and their interaction within the organism. This paper proposes a hypergraph-regularized joint deep semi-non-negative matrix factorization (HR-JDSNMF) algorithm to integrate positron emission tomography (PET), single-nucleotide polymorphism (SNP), and gene expression data for AD. The method employs matrix factorization techniques to nonlinearly decompose the original data at multiple layers, extracting deep features from different omics data, and utilizes hypergraph mining to uncover high-order correlations among the three types of data. Experimental results demonstrate that this approach outperforms several matrix factorization-based algorithms and effectively identifies multi-omics biomarkers for AD. Additionally, single-cell RNA sequencing (scRNA-seq) data for AD were collected, and genes within significant modules were used to categorize different types of cell clusters into high and low-risk cell groups. Finally, the study extensively explores the differences in differentiation and communication between these two cell types. The multi-omics biomarkers unearthed in this study can serve as valuable references for the clinical diagnosis and drug target discovery for AD. The realization of the algorithm in this paper code is available at https://github.com/ShubingKong/HR-JDSNMF .

Distinct members of the Caenorhabditis elegans CeMbio reference microbiota exert cryptic virulence that is masked by host defense.

Microbiotas are complex microbial communities that colonize specific niches in the host and provide essential organismal functions that are important in health and disease. Understanding the ability of each distinct community member to promote or impair host health, alone or in the context of the community, is imperative for understanding how differences in community structure affect host health and vice versa. Recently, a reference 12-member microbiota for the model organism Caenorhabditis elegans, known as CeMbio, was defined. Here, we show the differential ability of each CeMbio bacterial species to activate innate immunity through the conserved PMK-1/p38 MAPK, ACh-WNT, and HLH-30/TFEB pathways. Although distinct CeMbio members differed in their ability to activate the PMK-1/p38 pathway, the ability to do so did not correlate with bacterial-induced lifespan reduction in wild-type or immunodeficient animals. In contrast, most species activated HLH-30/TFEB and showed virulence toward hlh-30-deficient animals. These results suggest that the microbiota of C. elegans is rife with bacteria that can shorten the host's lifespan if host defense is compromised and that HLH-30/TFEB is a fundamental and key host protective factor.

Dataset used to develop soft computing models that predict the stiffness modulus of bituminous mixtures.

This data article presents information on the measurement of Indirect Tensile Stiffness Modulus of laboratory and field asphalt mixtures. The asphalt mixes are composed of three distinct binders that were categorised by their penetration grade (40/55-TLA, 60/75-TLA, and 60/70-MB) and aggregates (limestone, sharp sand, and filler). The asphalt mixtures are called dense-graded hot mix asphalt (HMA) and gap-graded stone matrix asphalt (SMA). The variables in the dataset were selected in accordance with the specifications of the dynamic modulus models that are currently in use as well as the needs for the quality control and assurance (QC & QA) assessment of asphalt concrete mixes. The data parameters included are temperature, asphalt content, and binder viscosity, air void content, cumulative percent retained on 19, 12.5, and 4.75 mm sieves, maximum theoretical specific gravity, aggregate passing #200 sieve, effective asphalt content, density, flow, marshal stability, coarse-to-fine particle ratio and the Indirect Tensile Stiffness Modulus (ITSM). Utilising soft computing techniques, models were developed utilising the data thus eliminating the requirement for complex and time-consuming laboratory testing.

Community cohesion looseness in gene networks reveals individualized drug targets and resistance.

Community cohesion plays a critical role in the determination of an individual's health in social science. Intriguingly, a community structure of gene networks indicates that the concept of community cohesion could be applied between the genes as well to overcome the limitations of single gene-based biomarkers for precision oncology. Here, we develop community cohesion scores which precisely quantify the community ability to retain the interactions between the genes and their cellular functions in each individualized gene network. Using breast cancer as a proof-of-concept study, we measure the community cohesion score profiles of 950 case samples and predict the individualized therapeutic targets in 2-fold. First, we prioritize them by finding druggable genes present in the community with the most and relatively decreased scores in each individual. Then, we pinpoint more individualized therapeutic targets by discovering the genes which greatly contribute to the community cohesion looseness in each individualized gene network. Compared with the previous approaches, the community cohesion scores show at least four times higher performance in predicting effective individualized chemotherapy targets based on drug sensitivity data. Furthermore, the community cohesion scores successfully discover the known breast cancer subtypes and we suggest new targeted therapy targets for triple negative breast cancer (e.g. KIT and GABRP). Lastly, we demonstrate that the community cohesion scores can predict tamoxifen responses in ER+ breast cancer and suggest potential combination therapies (e.g. NAMPT and RXRA inhibitors) to reduce endocrine therapy resistance based on individualized characteristics. Our method opens new perspectives for the biomarker development in precision oncology.

PLCζ, WBP2NL and TNF-α expression in spermatozoa is associated with stallion fertility and seminal quality?

This study aims to investigate the gene expression of sperm-borne phospholipase C zeta (PLCζ), WW domain-binding protein 2N-Terminal Like (WBP2NL), and Tumor necrosis factor (TNF-α), as a negative control, in spermatozoa and their relationship with fertility and seminal quality in stallions. Ejaculates from 40 Criollo stallions were used, whose fertility was assessed on the basis of their pregnancy rate per cycle in at least two breeding seasons. Pregnancy rates ranged from 20% to 90% and were used to divide the stallions into two groups: High rates (≥ 50%) (n = 25), and Low rates (< 50%) (n = 15). A computer-assisted sperm analysis system - (CASA) analyzed semen after collection. Also were evaluated the physical and functional integrity of the plasmatic membrane and sperm morphology alterations. All stallions expressed PLCζ, WBP2NL, and TNF-α. PLCζ positively correlates with conception rate, total motility (TM), progressive motility (PM), plasmatic membrane functionality, and integrity. A simple linear regression was detected between pregnancy rate and PLCζ expression (P = 0.003), TM (P < 0.001) and PM (P < 0.001). PLCζ gene expression was higher (P = 0,012) in the High rates group than in the Low group. WBP2NL and TNF-α did not correlate with seminal quality and stallion's fertility. It was concluded that PLCζ gene expression in the spermatozoa might be used as a biomarker of fertility and seminal quality in stallions. Parameters of sperm kinetics also showed, positive correlation between TM, PM and pregnancy rate.

Immunomodulatory-associated gene transcripts to multipotency of bovine amniotic fluid mesenchymal stem cells.

The adnexa fetal tissues are sources of mesenchymal stromal cells (MSCs) due to their noninvasive harvest, with all biological material discarded most of the time. MSCs are a promise regarding to their plasticity, self-renewal, differentiation potentials, immunomodulatory and anti-inflammatory properties, which have made clinical stem cell therapy a reality. The present study aimed to characterize and evaluate the immunomodulation ability of bovine mesenchymal cells collected from bovine amniotic fluid (bAFMSCs) isolated and subjected to sixth consecutive culture passages in vitro. The multilineage properties of the bAFMSCs collections confirmed the ability to undergo adipogenic, chondrogenic and osteogenic differentiation. The mesenchymal gene transcription CD106, CD73, CD29, CD90 and CD166 were detected in bAFMSCs, whereas CD34 and CD45 were not detected. Regarding cytokine mRNA expression, IL2, IL6, INFα, INFß, INFγ, TNFα and TNFß were downregulated, while IL10 was highly regulated in all studied passages. The present study demonstrated the immunological properties and multipotency of in vitro bAFMSCs collections, and thus, they can be tested in cattle pathological treatments or multiplication by nuclear transfer cloning.

Down-regulation of wheat Rubisco activase isoforms expression by virus-induced gene silencing.

Rubisco activase (Rca) is an essential photosynthetic enzyme that removes inhibitors from the catalytic sites of the carboxylating enzyme Rubisco. In wheat, Rca is composed of one longer 46 kDa α-isoform and two shorter 42 kDa ß-isoforms encoded by the genes TaRca1 and TaRca2. TaRca1 produces a single transcript from which a short 1ß-isoform is expressed, whereas two alternative transcripts are generated from TaRca2 directing expression of either a long 2α-isoform or a short 2ß-isoform. The 2ß isoform is similar but not identical to 1ß. Here, virus-induced gene silencing (VIGS) was used to silence the different TaRca transcripts. Abundance of the transcripts and the respective protein isoforms was then evaluated in the VIGS-treated and control plants. Remarkably, treatment with the construct specifically targeting TaRca1 efficiently decreased expression not only of TaRca1 but also of the two alternative TaRca2 transcripts. Similarly, specific targeting of the TaRca2 transcript encoding a long isoform TaRca2α resulted in silencing of both TaRca2 alternative transcripts. The corresponding protein isoforms decreased in abundance. These findings indicate concomitant down-regulation of TaRca1 and TaRca2 at both transcript and protein levels and may impact the feasibility of altering the relative abundance of Rca isoforms in wheat.

A novel model based on ubiquitination-related gene to predict prognosis and immunotherapy response in hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is a common cancer that is increasingly becoming a global health problem and a major public health concern. In order to improve patient outcomes, additional biomarkers and targets must be explored. Ubiquitination-related genes (URGs), as tumor regulators, exhibit multiple functions in tumor development. Our objective was to examine the influence of URGs on the prognosis of patients with HCC. Methods: By utilizing unsupervised cluster analysis, we were able to identify URGs in the database and create a risk score profile for predicting the prognosis of patients with HCC. The model's clinical application was explored using subject operating characteristic curves, survival analysis, and correlation analysis. We additionally examined the variances in clinical traits, immune infiltration, somatic genetic alterations, and responsiveness to treatment among high- and low-risk populations identified by the prognostic model. Scores for immune cell infiltration and immune-related pathway activity were determined by performing ssGSEA enrichment analysis. Additionally, to investigate potential mechanisms, we utilized GO, KEGG and GSVA analyses. Results: We developed a risk scoring model that relies on genes associated with ubiquitination. As the risk score increased, the malignancy and prognosis of the tumor worsened. The high-risk and low-risk groups exhibited notable disparities in relation to the immune microenvironment, genes associated with immune checkpoints, sensitivity to drugs, and response to immunotherapy. Conclusion: The utilization of a risk model that relies on genes associated with ubiquitination can serve as a biomarker to assess the prognosis of patients with HCC, and aid in the selection of suitable therapeutic agents.

Expression profiles and characterization of microRNAs responding to chitin in Arthrobotrys oligospora.

Extracellular proteases, such as chitinases secreted by Arthrobotrys oligospora (A. oligospora), play a crucial role in the process of nematode infection. However, post-transcriptional regulation of gene expression involving microRNAs (miRNAs) in A. oligospora remains scarcely described. Hereto, transcriptome sequencing was carried out to analyze the expression profiles of chitin-responsive miRNAs in A. oligospora. Based on the RNA-seq data, the differential expression of miRNAs (DEmiRNAs) in response to chitin was screened, identified and characterized in A. oligospora. Meanwhile, the potential target genes were predicted by the online tools miRanda and Targetscan, respectively. Furthermore, the interaction of DEmiRNA with it's target gene was validated by a dual-luciferase reporter assay system. Among 85 novel miRNAs identified, 25 miRNAs displayed significant differences in expression in A. oligospora in response to chitin. Gene Ontology (GO) analysis showed that the potential genes targeted by DEmiRNAs were enriched in the biological processes such as bio-degradation, extracellular components and cell cycle. KEGG analysis revealed that the target genes were mainly involved in Hippo, carbon and riboflavin metabolic pathway. Outstandingly, chitinase AOL_s00004g379, which is involved in the hydrolysis metabolic pathway of chitin, was confirmed to be a target gene of differential miR_70. These findings suggest that chitin-responsive miRNAs are involved in the regulation of cell proliferation, predator hyphae growth and chitinase expression through the mechanisms of post-transcriptional regulation, which provides a new perspective to the molecular mechanisms underlying miRNAs-mediated control of gene expression in A. oligospora.

Differential RNA Expression Between Metastatic and Primary Neuroblastoma Cells.

INTRODUCTION: Neuroblastoma (NB) is the most common extra-cranial malignancy in children. Poor survival in high-risk NB is attributed to recurrent metastatic disease. To better study metastatic disease, we used a novel mouse model to investigate differential gene expression between primary tumor cells and metastatic cells. We hypothesized that metastatic NB cells have a different gene expression profile from primary tumor cells and cultured cells. METHODS: Using three human NB cell lines (NGP, CHLA255, and SH-SY5Y), orthotopic xenografts were established in immunodeficient nod/scid gamma mice via subcapsular renal injection. Mice were sacrificed and NB cells were isolated from the primary tumor and from sites of metastasis (bone marrow, liver). RNA sequencing, gene set analysis, and pathway analysis were performed to identify differentially expressed genes and molecular pathways in the metastatic cells compared to primary tumor cells. RESULTS: There were 266 differentially expressed genes in metastatic tumor cells (bone marrow and liver combined) compared to primary tumor cells. The top upregulated gene was KCNK1 and the top downregulated genes were PDE7B and NEBL. Top upregulated pathways in the metastatic cells were involved in ion transport, cell signaling, and cell proliferation. Top downregulated pathways were involved in DNA synthesis, transcription, and cellular metabolism. CONCLUSIONS: In metastatic NB cells, our study identified the upregulation of biologic processes involved in cell cycle regulation, cell proliferation, migration, and invasion. Ongoing studies aim to validate downstream translation of these genomic alterations, as well as target these pathways to more effectively suppress and inhibit recurrent metastatic disease in NB.

The effects of a glyphosate-based herbicide on the bovine gametes during an in vitro embryo production model.

Roundup® (R), while it is the most used herbicide globally, and its residues are ubiquitous in urban and suburban areas, its impact on vertebrates' safety remains highly debated. Here, in three in vitro experiments, we investigated the effects of a very low dose (1 ppm) of R on the fertilization capacity and embryo development in cattle. In the first experiment, frozen-thawed bull semen exposed to R for 1 h exhibited reduced motility parameters but unaffected fertilization ability. However, after in vitro fertilization, the rates of embryo formation were significantly lower compared to the untreated controls. In the second experiment, oocytes exposed to R during in vitro maturation showed reduced cleavage rates, and the embryo yield on days 7, 8, and 9 of embryo culture was significantly lower than that of the controls. In the third experiment, oocytes were matured in the presence of R and in a medium containing both R and Zinc, chosen to offer antioxidant protection to the oocytes. Day-7 blastocysts were analyzed for the expression of genes associated with oxidative stress, apoptosis, and epigenetic reprogramming. Exposure to R markedly suppressed embryo formation rates compared to the controls. The combination of R with Zinc restored the blastocyst yield, which on days 8 and 9 was comparable to that of the controls and higher than the groups exposed only to R on all days. The gene expression analysis revealed that R promotes oxidative stress development, triggers apoptosis, and induces epigenetic changes in developing embryos, while zinc presence alleviates these adverse effects of R. These findings imply that even at very low doses, R could be highly toxic, leading to functional abnormalities in both gametes, potentially affecting fertility in both genders.

Comparative investigation on chemical and green synthesized titanium dioxide nanoparticles against chromium (VI) stress eliciting differential physiological, biochemical, and cellular attributes in Helianthus annuus L.

Nanotechnology is a new scientific area that promotes unique concepts to comprehend the optimal mechanics of nanoparticles (NPs) in plants under heavy metal stress. The present investigation focuses on effects of synthetic and green synthesized titanium dioxide nanoparticles (TiO2 NPs and gTiO2 NPs) against Cr(VI). Green TiO2 NPs have been produced from plant leaf extract (Ricinus communis L.). Synthesis was confirmed employing an array of optical spectroscopic and electron microscopic techniques. Chromium strongly accelerated H2O2 and MDA productions by 227 % and 266 % at highest chromium concentration (60 mg/kg of soil), respectively, and also caused DNA damage, and decline in photosynthesis. Additionally, anomalies were observed in stomatal cells with gradual increment in chromium concentrations. Conversely, foliar applications of TiO2 NPs and gTiO2 NPs considerably mitigated chromium stress. Sunflower plants treated with modest amounts of green TiO2 NPs had significantly better growth index compared to chemically synthesized ones. Principal component analysis highlighted the variations among photosynthetic attributes, oxidative stress markers, and antioxidant defense systems. Notably, gTiO2 supplementation to the Cr(VI) strained plants minimized PC3 production which is a rare report so far. Conclusively, gTiO2 NPs have been identified to be promising nano-based nutrition resource for farming applications.

Exploring Bacillus mycoides PM35 efficacy in enhancing rice (Oryza sativa L.) response to different types of microplastics through gene regulation and cellular fractionation.

Soil contamination with microplastics (MPs) is a persistent threat to crop production worldwide. With a wide range of MP types, including polystyrene (PS), polyvinyl chloride (PVC) and polyethylene (PE), contaminating our environment, it is important to understand their impact on agricultural productivity. The present study was conducted to investigate the effects of different types of MPs (PS, PVC and PE) on various aspects of plant growth. Specifically, we examined growth and biomass, photosynthetic pigments, gas exchange attributes, oxidative stress responses, antioxidant compound activity (both enzymatic and non-enzymatic), gene expression, proline metabolism, the AsA-GSH cycle and cellular fractionation and nutritional status, in different parts of rice (Oryza sativa L.) seedlings, which were also exposed to plant growth promoting rhizobacteria (PGPR), i.e. Bacillus mycoides PM35, i.e. 20 µL. The research outcomes indicated that the different types of MPs in the soil notably reduced plant growth and biomass, photosynthetic pigments and gas exchange attributes. However, MP stress also induced oxidative stress in the roots and shoots of the plants by increasing malondialdehyde (MDA), hydrogen peroxide (H2O2) and electrolyte leakage (EL) which also induced increased compounds of various enzymatic and non-enzymatic antioxidants and also the gene expression. Furthermore, a significant increase in proline metabolism, the AsA-GSH cycle, and the fractionations of cellular components was observed. Although the application of B. mycoides PM35 showed a significant increase in plant growth and biomass, gas exchange characteristics, enzymatic and non-enzymatic compounds and their gene expression and also decreased oxidative stress. In addition, the application of B. mycoides PM35 enhanced cellular fractionation and decreased the proline metabolism and AsA-GSH cycle in O. sativa plants. These results open new insights for sustainable agriculture practices and hold immense promise in addressing the pressing challenges of MP contamination in agricultural soils.

In vitro aging alters the gene expression and secretome composition of canine adipose-derived mesenchymal stem cells.

Introduction: Canine adipose-derived mesenchymal stem cells (cAD-MSCs) hold therapeutic promise due to their regenerative potential, particularly within their secretome. However, concerns arise regarding the impact of in vitro cultivation necessitated for storing therapeutic doses, prompting this study to comprehensively explore the impact of in vitro aging on gene expression and secretome composition. Methods: The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling using RT-qPCR array, and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during in vitro culture. Results and Discussion: The results demonstrate that the gene expression and secretome composition of cAD-MSCs were impacted by in vitro aging. Among many alterations in gene expression between two passages, two significant downregulations were noted in the MSC-associated PTPRC and IL10 genes. While the majority of proteins and their functional characteristics were shared between passages, the influence of cell aging on secretome composition is highlighted by 10% of proteins being distinctively expressed in each passage, along with 21 significant up- and downregulations. The functional attributes of proteins detected in passage 3 demonstrated a greater inclination towards supporting the regenerative capacity of cAD-MSCs. Moreover, proteins in passage 6 exhibited a noteworthy correlation with the blood coagulation pathway, suggesting an elevated likelihood of coagulation events. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age in vitro. Furthermore, it contributes to broadening the currently restricted knowledge base concerning the secretome of cAD-MSCs. In conclusion, our findings show that the regenerative potential of cAD-MSCs, as well as their secretome, may be compromised by in vitro aging. Therefore, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.

Evaluation of EGFR and COX pathway inhibition in human colon organoids of serrated polyposis and other hereditary cancer syndromes.

Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs.