Real-time PCR Research Articles

Expression evaluation of exogenous and endogenous alcohol dehydrogenase genes in transgenic Arabidopsis

A lot of endogenous genes, as well as genes from related species, are transformed back into crops for overexpression to improve their corresponding traits. However, almost all of these transgenic events remain at the testing stage. Most of the singular transgenic events of crops approved for commercial release are developed by the transformation and heterologous expression of exogenous genes from distant species. To detect the differences in expression, protein accumulation, and enzyme activity between transformed exogenous and endogenous genes, the coding sequences (CDSs) of the alcohol dehydrogenase (ADH) genes were cloned from dicotyledonous Arabidopsis, monocotyledonous maize, and prokaryotic Escherichia coli, constructed into expression vector pBI121-cMycNY, and used to transform wild-type Arabidopsis, respectively. Three homozygous T3 lines with a single integration site were screened for each of the three transformed genes by antibiotic screening, polymerase chain reaction (PCR) identification, and genomic DNA resequencing. Real-time quantitative PCR (RT-qPCR) analysis showed that the relative expression levels of the transformed exogenous ZmADH and EcADH genes were ten or tens of times higher than that of the transformed endogenous AtADH gene. After confirming the encoded proteins of these transformed genes by Western blotting, enzyme-linked immunosorbent assay (ELISA) showed that the accumulation levels of the proteins encoded by the transformed genes ZmADH and EcADH were significantly higher than that encoded by the transformed endogenous gene AtADH. Enzyme reaction assay showed that the ADH activities of the T3 lines transformed by the exogenous genes ZmADH and EcADH were also significantly higher than that transformed by the endogenous gene AtADH as well as the wild-type control. These results indicated that exogenous genes were more conducive to transgenic improvement of crops.

Sex dimorphic associations of Prader-Willi imprinted gene expressions in umbilical cord with prenatal and postnatal growth in healthy infants.

The impact of Prader-Willi syndrome (PWS) domain gene expression on the growth of healthy children is not well understood. This study investigated associations between PWS domain gene expression in umbilical cord tissue and prenatal and postnatal growth, considering potential sex differences. Relative gene expression of paternally expressed MAGEL2, NDN, and SNURF-SNRPN, and the small nucleolar RNAs SNORD116 and SNORD115 were determined by real-time quantitative polymerase chain reaction in umbilical cord tissue from 122 healthy newborns (59 girls and 63 boys). Gene expression levels were correlated with auxological measures at birth, infancy, and childhood (ages 2, 4, and 6years). MAGEL2, NDN, SNORD116, and SNORD115 expression in the umbilical cord was negatively associated with birth weight, length, and placental weight (P < 0.001). Postnatally, these genes were positively associated with weight and length at 3months (P < 0.001) and weight gain from birth to ages 1, 2, and 4years (P < 0.01). Negative associations at birth were stronger in girls (P < 0.001), while positive associations during infancy and childhood were stronger in boys (P < 0.001). MAGEL2, SNORD116, and SNORD115 expression predicted early-postnatal growth, explaining the higher growth rate in boys compared to girls and accounting for sex differences up to 1.5kg in weight and 3cm in height during infancy. Paternally expressed PWS domain gene expression in the umbilical cord was negatively associated with prenatal growth and positively with early-postnatal growth in healthy infants. This gene expression may predict early human postnatal growth and promote the well-known sex dimorphism in growth. These results can also help in understanding the etiology of PWS, which remains unclear.

Relationship between transmembrane emp24 domain containing 2 expression and tumor stem cell characteristics in oral cancer

Objective: Transmembrane Emp24 Domain Containing 2 (TMED2) is a mediator of membrane protein trafficking involved in intracellular protein transport. Recent research suggests that TMED2 plays an important role in the development and metastasis of tumors; however, its exact mechanisms in oral cancer (OC) remain unclear. This study aims to elucidate the role and possible mechanisms of TMED2 in OC. Material and Methods: We investigated the impact of TMED2 knockdown on the invasion, migration, and proliferation capabilities of OC cells. Furthermore, we analyzed the in vitro and in vivo interactions between TMED2 and polypeptide-N-acetylgalactosaminyltransferase 7 (GALNT7) as well as explored the regulatory function of TMED2 on GALNT7. The alterations in stem cell markers were assessed using clone formation assays, western blot, and quantitative real-time polymerase chain reaction. Results: The upregulation of TMED2 promoted the proliferation and invasion abilities of OC cells. Further analysis revealed that TMED2 enhanced the stem-like properties and tumorigenicity of OC cells by directly regulating the expression of GALNT7. In vivo and in vitro results suggested that silencing TMED2 expression reduced the incidence of OC. Conclusion: Our data imply that TMED2 stimulates GALNT7 transcription, which in turn amplifies the stem-like characteristics and carcinogenic potential of OC cells. Moreover, the block of TMED2 prevents cancers from growing and spreading in vivo. This finding provides a new therapeutic target for the treatment of OC and highlights the critical role of TMED2 in the condition.

Genome wide identification of MATE and ALMT gene family in lentil (Lens culinaris Medikus) and expression profiling under Al stress condition

BackgroundThe membrane transporters viz. multidrug and toxic compound extrusion (MATE) and aluminum-activated malate transporter (ALMT) are associated with aluminum (Al) tolerance by accelerating secretion of organic acids, which can influence nutrient availability and stress response. However, such transporter families have not yet been reported in lentil under Al stress condition.Method and resultsIn this study, 90 MATE and 14 ALMT genes were identified and clustered into four (MATE) and five (ALMT) subfamilies/clades with smaller subgroups. All the MATE and ALMT genes were unevenly dispersed across lentil chromosomes. Duplication analysis suggested that LcMATE gene family has expanded primarily through tandem duplication event. Collinearity of lentil with soybean suggested a close relationship between the MATE genes. The MATE promoter regions harboured many stress responsive as well as Al resistance transcription factor 1 related cis-regulatory elements. Predicted 3D (three-dimensional) structure and molecular docking revealed that 5 LcMATE proteins could bind citrate and contain amino acids related to its secretion via citrate exuding motif and other neighbouring sites. Expression analyses of LcMATE and LcALMT genes were performed using quantitative real-time polymerase chain reaction (qRT-PCR). Six genes namely, LcM1, LcM42, LcM46, LcM47, LcALMT8 and LcALMT14 responded to Al stress with varying levels of expression patterns at different time points (3, 6, 12 and 24 h).ConclusionOur findings offer thorough details on the MATE and ALMT transporters in lentils and will aid in valuable understanding for future functional studies of these transporters in generating Al tolerant cultivars.

Clinical significance of stratifying prostate cancer patients through specific circulating genes.

Patient stratification remains a challenge for optimal treatment of prostate cancer (PCa). This clinical heterogeneity implies intra-tumoural heterogeneity, with different prostate epithelial cell subtypes not all targeted by current treatments. We reported that such cell subtypes are traceable in liquid biopsies through representative transcripts. Expanding on this concept, we included 57 genes representing cell subtypes, drug targets and relevant to resistance as non-invasive biomarkers for stratification. This panel was tested by RT-qPCR (quantitative reverse transcription polymerase chain reaction) in blood of controls and different categories of PCa patients. Overall, circulating transcripts showed predictive value throughout the disease. Those with aggressive pathological features such as intra-ductal carcinoma at diagnosis showed more genes over-expressed. In metastatic patients, signatures of subtypes or resistance were associated with treatments, progression-free survival and overall survival. Altogether, testing markers of cell diversity, an intrinsic feature of tumours, and drug targets via liquid biopsies represents a valuable means to stratify patients and predict responses to current or new therapeutic modalities. Over-expressed drug target genes suggest potential benefit from targeted treatments, justifying new clinical trials to offer patient-tailored strategies to eventually impact on PCa mortality.

LINC02418 suppresses endometrial cancer progression via regulating miR-494-3p/RASGRF1 axis.

Long non-coding RNAs (lncRNAs) have emerged as pivotal regulatory molecules in cancer biology. Among these, long intergenic non-protein coding RNA 02418 (LINC02418), a recently identified lncRNA, has been linked to endometrial cancer (EC), although its function and operational mechanisms are largely unclear. The present investigation aims to elucidate the molecular mechanism through which LINC02418 influences EC pathogenesis. We employed Western blotting and quantitative real-time PCR to analyze Ras protein specific guanine nucleotide releasing factor 1 (RASGRF1) and LINC02418 expression profiles in EC tissues and cell lines. Functional analyses, including cell proliferation, migration, and invasion assays, were conducted to evaluate the impact of LINC02418 overexpression on EC cells. Xenograft mouse models were established for in vivo validation. The molecular interactions between LINC02418, miR-494-3p, and RASGRF1 were characterized using luciferase reporter and RNA pull-down assays. LINC02418 expression was significantly downregulated in EC tissues and cell lines compared to their normal counterparts. Forced expression of LINC02418 significantly suppressed EC cell proliferation, migration, and invasion in vitro. In xenograft models, LINC02418 overexpression resulted in reduced tumor burden and enhanced cell death. Mechanistically, LINC02418 enhanced RASGRF1 expression by sequestering miR-494-3p, a finding substantiated by RNA pull-down assays. The tumor-suppressive effects of LINC02418 were partially reversed by RASGRF1 silencing and miR-494-3p overexpression. Clinical analyses revealed that reduced RASGRF1 expression correlated with poor histological differentiation, advanced tumor stages, and decreased overall survival in EC patients. Our findings establish LINC02418 as a tumor suppressor that regulates EC progression through modulation of the miR-494-3p/RASGRF1 axis, highlighting its potential as a therapeutic target in EC treatment.

Periodontitis Exacerbates Colorectal Cancer by Altering Gut Microbiota-Derived Metabolomics in Mice.

The correlation between periodontitis and colorectal cancer (CRC) has drawn widespread attention. However, how periodontitis affects CRC progression remains unclear. C57BL/6 mice were used to establish experimental periodontitis and CRC model. Histological alterations of periodontium and colon were observed by hematoxylin and eosin staining. Micro-computed tomography (micro-CT) was applied to evaluate alveolar bone loss (ABL). Tumor growth was detected by immunofluorescence. Gut bacteria were analyzed using 16S rRNA sequencing. Gas chromatography-mass spectrometry (GC-MS) was performed to observe the alterations of gut microbial metabolites. The detection of associated pathways was carried out using quantitative real-time PCR (qRT-PCR). Experimental periodontitis significantly induced increases in tumor number in mice with CRC. Double immunofluorescence for Ki67 and β-catenin, as well as Cyclin D1 and β-catenin, indicated that experimental periodontitis observably promoted tumor growth. 16S rRNA sequencing and untargeted metabolomics analysis displayed that experimental periodontitis altered gut microbial community and metabolite profiles in CRC mice. Notably, we found that experimental periodontitis dramatically increased the level of three oncometabolites (serotonin, adenosine, and spermine) in mice with CRC. Alterations of gut microbial community and metabolites might be relevant in experimental periodontitis deteriorating CRC.

Transcriptome Analysis Reveals the Key Genes and Pathways for Growth, Ion Transport, and Oxidative Stress in Post-Larval Black Tiger Shrimp (Penaeus monodon) Under Acute Low Salt Stress.

As an abiotic stress factor, salinity significantly affects the physiological activities of crustaceans. In this study, transcriptome sequencing was used to evaluate the mechanism of ion transport and the physiological response of black tiger shrimp (Penaeus monodon) under low salt stress. Four hundred post larval (PL) stage P. monodon were distributed in eight experimental tanks and exposed to 3 or 5 ppt salt concentrations for 96h. Low salinity significantly reduced the survival rate of shrimp but simultaneously activated the activity of ion transporter enzymes Na+/K+-ATPase (NKA) and Ca2+/Mg2+-ATPase), the expression of NKA, galectin 10, and cytochrompe c peroxidase genes, and the activity and expression of antioxidant-related genes (superoxide dismutase, catalase, heat shock protein 60). Low salt stress activated the urea cycle but significantly inhibited glutathione metabolization-related indicators (glutamate dehydrogenase, glutaminase, glutamic acid). RNA-seq analysis identified 221 differentially expressed genes (78 up-regulated and 143 down-regulated). Quantitative real-time PCR and RNA-seq results of 11 of them were consistent, illustrating the validity of the transcriptomic predictions. Gene set enrichment analysis results showed that calcium ion transmembrane transport, calmodulin binding, the stress-activated protein kinase signaling cascade, and regulation of the cytosolic calcium ion concentration process were significantly enriched. These results showed that low salt stress activated the calcium-dominated ion transport pathway and promoted molting growth of P. monodon. They also indicate that there is potential for larval rearing shrimp under low salt conditions.

Genome-Wide Identification of the Kinesin Gene Family in Soybean and Its Response to Salt Stress

The kinesin (KIN) gene family is a subgroup of motor proteins. It plays a critical role in plant development and responses to environmental stresses. However, their function in soybean salt tolerance has yet to be clearly defined. This study employed bioinformatics approaches and identified 139 kinesin family members in the soybean genome. These 139 genes were classified into 10 subgroups, unevenly distributed across the chromosomes. The promoter regions of GmKIN genes harbored several stress-responsive elements, and segmental duplication was the primary driver of the expansion of the GmKIN gene family. Based on publicly available RNA-seq data, we studied the response patterns of 139 GmKIN genes to salt stress and found that 20 KIN genes in soybeans were upregulated after salt stress, with GmKIN114, GmKIN102, GmKIN109, and GmKIN99 showing more than a threefold increase in their expression under salt stress. Using quantitative fluorescence PCR, transgenic yeast, and a transgenic hairy root system, we preliminarily validated the salt tolerance functions of the four KIN genes in soybeans. This study probed into the GmKIN gene family in soybean, offering valuable insights into the functional roles of these genes in stress adaptation.

Glutamine-glutamate centered metabolism as the potential therapeutic target against Japanese encephalitis virus-induced encephalitis

BackgroundJapanese encephalitis (JE) induced by Japanese encephalitis virus (JEV) infection is the most prevalent diagnosed epidemic viral encephalitis globally. The underlying pathological mechanisms remain largely unknown. Given that viruses are obligate intracellular parasites, cellular metabolic reprogramming triggered by viral infection is intricately related to the establishment of infection and progression of disease. Therefore, uncovering and manipulating the metabolic reprogramming that underlies viral infection will help elucidate the pathogenic mechanisms and develop novel therapeutic strategies.MethodsMetabolomics analysis was performed to comprehensively delineate the metabolic profiles in JEV-infected mice brains and neurons. Metabolic flux analysis, quantitative real-time PCR, western blotting and fluorescence immunohistochemistry were utilized to describe detailed glutamine-glutamate metabolic profiles during JEV infection. Exogenous addition of metabolites and associated compounds and RNA interference were employed to manipulate glutamine-glutamate metabolism to clarify its effects on viral replication. The survival rate, severity of neuroinflammation, and levels of viral replication were assessed to determine the efficacy of glutamine supplementation in JEV-challenged mice.ResultsHere, we have delineated a novel perspective on the pathogenesis of JE by identifying an aberrant low flux in glutamine-glutamate metabolism both in vivo and in vitro, which was critical in the establishment of JEV infection and progression of JE. The perturbed glutamine-glutamate metabolism induced neurotransmitter imbalance and created an immune-inhibitory state with increased gamma-aminobutyric acid/glutamate ratio, thus facilitating efficient viral replication both in JEV-infected neurons and the brain of JEV-infected mice. In addition, viral infection restrained the utilization of glutamine via the glutamate-α-ketoglutaric acid axis in neurons, thus avoiding the adverse effects of glutamine oxidation on viral propagation. As the conversion of glutamine to glutamate was inhibited after JEV infection, the metabolism of glutathione (GSH) was simultaneously impaired, exacerbating oxidative stress in JEV-infected neurons and mice brains and promoting the progression of JE. Importantly, the supplementation of glutamine in vivo alleviated the intracranial inflammation and enhanced the survival of JEV-challenged mice.ConclusionAltogether, our study highlights an aberrant glutamine-glutamate metabolism during JEV infection and unveils how this facilitates viral replication and promotes JE progression. Manipulation of these metabolic alterations may potentially be exploited to develop therapeutic approaches for JEV infection.Graphical

Resveratrol Reduces Cisplatin-induced Cochlear Hair Cell Pyroptosis by Inhibiting the mtROS/TXNIP/NLRP3 Pathway.

Cisplatin is an effective anti-cancer drug with limited clinical applications due to ototoxicity. Resveratrol, known for its antioxidant and anti-inflammatory properties, has been reported to mitigate these adverse effects, although the underlying mechanism remains under-researched. This study aimed to investigate the effects and underlying mechanisms of resveratrol on cisplatin-induced ototoxicity. Ototoxicity was modeled in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells by cisplatin exposure, followed by interventions using thioredoxin-interacting protein (TXNIP) siRNA transfection, MitoQ, or resveratrol. Apoptosis and proliferation were quantitatively assessed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and Ki67 immunostaining. Quantitative real-time PCR (qRT-PCR) and western blotting were used to measure the changes in mRNA and protein levels. Flow cytometry and enzyme- linked immunosorbent assay (ELISA) were used to analyze pyroptotic cells and inflammatory responses. Reactive oxygen species (ROS) production was tracked using 2', 7'- dichlorofluorescein diacetate (DCFH-DA) staining and flow cytometry. Mitochondrial Membrane Potential (MMP) and mitochondrial permeability transition pore (MPTP) opening levels were analyzed through tetramethylrhodamine ethyl ester (TMRE) staining and specific reagent kits, respectively. Lastly, immunofluorescence staining and co-immunoprecipitation were employed to investigate the co-localization and interactions between TXNIP and thioredoxin (TRX)/NOD-like receptor family pyrin domain-containing 3 (NLRP3) proteins. Cisplatin exacerbated apoptosis, suppressed cell proliferation, and upregulated NLRP3, pro-Caspase-1, cleaved Caspase-1, Gasdermin D (GSDMD), GSDMD-N, and TXNIP expression. Concurrently, cisplatin resulted in increased pyroptotic cells and increased interleukin-6 (IL-6), IL-18, IL-1β, and tumor necrosis factor-α (TNF-α) levels. These effects were mitigated by TXNIP knockdown. Furthermore, cisplatin led to elevated cellular ROS and mitochondrial ROS (mtROS), decreased MMP, and inhibited MPTP opening. Cisplatin reduced the colocalization and interaction between TRX and TXNIP while enhancing those between TXNIP and NLRP3. These changes were attenuated by MitoQ. Resveratrol displayed effects similar to those of TXNIP knockdown and MitoQ treatment. Resveratrol alleviated the toxic effects of cisplatin on cochlear hair cells by inhibiting cell pyroptosis process mediated by the mtROS/TXNIP/NLRP3 pathway.

Apoptosis antagonizing transcription factor expression and its validation as a potential diagnostic and prognostic biomarker in oral squamous cell carcinoma

BackgroundOral squamous cell carcinoma (OSCC) is characterized by a high degree of malignancy and poor prognosis. This study aimed to investigate the expression of apoptosis antagonizing transcription factor (AATF) in OSCC, examine its correlation with clinicopathological features, assess its prognostic implications, and explore its potential role in OSCC progression.MethodsExpression profiles and clinical data of OSCC patients were obtained from The Cancer Genome Atlas (TCGA). Immunohistochemical analysis on tissue microarrays was performed to assess AATF expression in OSCC. Functional enrichment analyses were conducted to identify potential signaling pathways and biological functions associated with AATF. Logistic regression analyses were employed to evaluate the relationship between AATF expression and clinicopathological features. Immune cell infiltration was assessed using single-sample gene set enrichment analysis (ssGSEA). The prognostic value of AATF was determined using Kaplan-Meier and Cox regression analyses. A nomogram was developed to predict overall survival (OS) rates at one, three-, and five years post-cancer diagnosis. Validation of AATF expression was performed using quantitative real-time PCR (qRT-PCR)ResultsAATF was significantly overexpressed in OSCC, and high AATF expression correlated with adverse clinicopathological features, including histologic grade and lymph node metastasis. Functional enrichment analysis revealed several enriched pathways, including epidermis development, immunoglobulin complex, antigen binding and IL-17 signaling pathway. Notably, AATF overexpression was negatively correlated with the infiltration levels of mast cells, interdigitating dendritic cells and Th 17 cells. High AATF expression significantly predicted poorer overall survival (OS) and disease-specific survival (DSS). Multivariate Cox analysis confirmed AATF as an independent negative prognostic marker of OS. Validation via qRT-PCR confirmed the overexpression of AATF in OSCC tissues.ConclusionElevated expression of AATF in OSCC correlates with adverse clinicopathological features and negatively impacts immune cell infiltration. High AATF levels serve as an independent marker of poor OS and DSS. These findings support AATF as a valuable prognostic biomarker and a potential therapeutic target in OSCC, warranting further investigation.

Labeling tumor-associated extracellular vesicles with antibody-DNA conjugates for quantitative analysis

IntroductionExtracellular vesicles (EVs) shed from tumor cells into peripheral circulation or other body fluids are promising biomarkers for cancer diagnosis with enormously long circulation. Consequently, precise methods for differentiating normal and tumor-associated EVs (TAEs) are required.MethodsThis study used quantifiable antibody-DNA conjugate-assisted quantitative methods combined with proximity ligation technology to detect TAEs. The antibody-DNA conjugate contained one antibody associated with three oligonucleotides for signal amplification. The antibody in the conjugate can recognize the surface tumor antigens of TAEs. Simultaneously, DNA in the conjugate is attached to the surfaces of TAEs and holds the signal amplification post, converting protein identities to DNA amplification for protein detection, even at the molecular level.ResultsThese findings revealed that TAEs can be quantitatively detected using DNA-mediated quantitative polymerase chain reaction (qPCR). Antibody-DNA conjugates were used to recognize the epithelial cell adhesion molecule (EpCAM) antigen on the TAE surface and quantify the antigen using qPCR for cancer analysis.DiscussionThis method proposed a new quantitative detection approach for TAEs, which aim to identify specific EV-associated markers for diagnostic or therapeutic, this method could inspire a new idea for tumor diagnosis and detection of other diseases.

Evaluating HPV Viral Load and Multiple Infections for Enhanced Cervical Cancer Risk-Based Assessment

Human papillomavirus (HPV) is a major cause of cervical cancer, a significant health concern worldwide. Despite advances in screening methods, including the Pap test and the HPV DNA test, limitations remain in accurately predicting which HPV infections will progress to cervical intraepithelial neoplasia (CIN) and, eventually, invasive cancer. This study evaluates the usefulness in real life of assessing HPV viral load and the presence of multiple HPV genotypes in enhancing the diagnostic accuracy of triage in cervical cancer screening. A retrospective analysis was performed on 55 formalin-fixed paraffin-embedded cervical samples collected from women who underwent colposcopy with a biopsy or conization at San Martino Hospital, Genova, Italy, between January and June 2021. Histological diagnoses were compared with molecular analyses (HPV genotyping, viral load quantification and co-infection) using a multiplex real-time PCR platform. Of the samples analyzed, 56.4% were HPV DNA positive, while 40% tested negative. The molecular analysis identified more HPV-negative cases than the histological analysis (p &lt; 0.05). Higher viral loads and HPV co-infections were more frequent in high-grade CIN lesions. These markers may help identify patients at an elevated risk for persistent infections and cancer progression. These findings support the potential of integrating HPV viral load and genotype co-infection assessments into routine cervical cancer screening protocols to improve early detection and reduce overtreatment and unnecessary interventions.

P0198 Comparative Transcriptomic Analysis of Tonsil-derived and Adipose-derived Mesenchymal Stem Cells: Proliferation, Migration, and Anti-inflammatory Potential in Murine Colitis Model

Abstract Background Tonsil-derived mesenchymal stem cells (TMSCs) have emerged as a promising alternative to traditional MSCs due to their rapid proliferation and accessibility. This study explores the unique transcriptomic and cellular properties of TMSCs compared with adipose-derived MSCs (AMSCs). Methods TMSCs and AMSCs were cultured under identical conditions in low-glucose DMEM with 10% FBS. Proliferation was evaluated via CCK-8 assays, and migration potential was assessed using scratch assays. Transcriptomic differences were analyzed using RNA sequencing, with key findings validated by qPCR. Results TMSCs displayed distinct morphological features compared to AMSCs, with TMSCs showing a spindle shape, while AMSCs appeared more irregular and rounded shape. In terms of proliferation, TMSCs demonstrated a significantly higher proliferation rate by day 7 (245.3% of control vs. 134.4% of control, p &amp;lt; 0.001). However, there was no significant difference in migration capacity between TMSCs and AMSCs over a 24-hour period (50.8 ± 8.2 vs. 53.8 ± 9.8, p = 0.537). RNA sequencing analysis revealed notable differences in gene expression, with TMSCs showing enrichment in pathways related to extracellular matrix, cell adhesion, and immune response, particularly with upregulation of the MAPK signaling pathway compared with AMSCs. For anti-inflammatory cytokines, IL-33, FOXF1, and HGF were upregulated in TMSCs relative to AMSCs. In relation to proliferation and migration, CCL2, CXCL12, and STC1 levels were also higher in TMSCs. Additionally, TMSCs exhibited increased expression of RAB27B, suggesting a greater potential for exosome release. In a DSS-induced acute colitis model, both TMSCs and AMSCs alleviated colitis symptoms with significant improvements in colon length (p &amp;lt; 0.05). Real-time PCR analysis of mouse colonic tissue also demonstrated a reduction in pro-inflammatory cytokines, such as IL-1β, with TMSCs showing a more pronounced reduction. Conclusion This study demonstrates that TMSCs possess unique characteristics, including a higher proliferation rate and distinct gene expression profiles compared to AMSCs. While both MSC types exhibited similar anti-inflammatory effects in murine colitis model, the enhanced anti-inflammatory potential and increased exosome release capacity of TMSCs, combined with their easy availability, highlight their promise for clinical application.

P0173 The impact of dietary composition on metallothionein gene expression in the intestinal epithelium by exclusive enteral nutrition

Abstract Background The mechanism by which exclusive enteral nutrition (EEN) is effective in the treatment of Crohn’s disease remains elusive. Epidemiologic studies have shown that a substantial proportion of patients with Crohn’s disease are deficient in micronutrients such as zinc, which has been shown to play an important role in the integrity of intestinal epithelial barrier function. Zinc plays a critical role in the transcriptional regulation of metallothioneins (MT), cysteine rich binding proteins that regulate both epithelial and immune function where they are generally believed to play a beneficial role in reducing disease. Methods Transcriptomic analyses were performed by both bulk RNAseq as well as GeoMx spatial transcriptomics. Enzymatic food digests using both bile acids and pancreatic enzymes, analyzed by Proton NMR metabolomics, were used to generate cell culture media for studies in intestinal epithelial cells. Results RNAseq of rectal biopsies obtained from healthy human subjects consuming EEN (Modulen-IBD, Nestlé Health Science) vs. an omnivore diet revealed a very specific induction of genes involved in mineral transport, namely MTs such as MT1G and MT2A. Spatial transcriptomics revealed that the induction of MTs was specific to the colonic epithelium. To develop a cellular model to characterize the mechanism underlying this response, we investigated the impact of animal protein, vegetable, and EEN based foods on the transcriptome of intestinal epithelial cells in culture by both RNAseq and targeted quantitative RT-PCR. This model involved the development of a physiologically relevant modality to digest whole food to generate serum-free cell culture media that was characterized using NMR-based metabolomics. Animal protein and EEN diets induced the expression of specific MTs whereas vegetable diets reduced their expression. We also show that zinc induced MT gene expression in intestinal epithelial cells whereas phytic acid, a phytochemical with known anti-nutrient properties that chelates zinc, reduces the ability of protein-based foods and EEN to induce MT gene expression. Conclusion The consumption of EEN leads to a very specific transcriptomic signature of MT gene expression in the rectal epithelium of humans that can be reproduced in cell culture by exposure to both animal protein-based foods and EEN. By contrast, vegetable-based foods inhibit MT gene expression possibly through the chelation of zinc. These results are consistent with the notion that the exclusion of plant-based foods through the consumption of EEN may have beneficial effects in the treatment of Crohn’s disease by reducing exposure to anti-nutrient phytochemicals that reduce the absorption of beneficial micronutrients such as zinc.

Evidence of Rapid Infection of Four Sweetpotato Potyviruses in a Commercial Field in California.

Sweetpotato (Ipomoea batatas Lam.) is grown worldwide and is a staple food in many countries. One of the main constraints for sweetpotato production is cultivar decline, caused by the accumulation of viruses and subsequent losses of storage root yield and quality over years of vegetative propagation. Virus indexing using diagnostic molecular techniques is essential for the detection and identification of the major viruses to avoid cultivar decline, but little information is available on the fate of clean (virus-tested) plant materials after field propagation. In this study, cuttings of three sweetpotato cultivars (Beauregard, Bellevue, and Vermillion) were acquired from a clean propagative material program and planted in commercial field plots in Merced County, California, over multiple growing seasons. In 2021 and 2022, the phytosanitary status of storage roots from different generations of the materials were assessed by reverse transcription quantitative PCR (RT-qPCR) at harvest at the Foundation Plant Services, University of California, Davis. The results showed their rapid infection with four major potyviruses-sweet potato feathery mottle virus, sweet potato virus 2, sweet potato virus C, and sweet potato virus G-over the two seasons. The infection by two to four potyviruses was confirmed by RT-PCR, RT-qPCR, high-throughput sequencing, and biological indexing onto Ipomoea setosa plants for symptoms evaluation.

Factors secreted from the stem cells of human exfoliated deciduous teeth inhibit osteoclastogenesis through the activation of the endogenous antioxidant system.

Systemic administration of conditioned medium (CM) from stem cells derived from human exfoliated deciduous teeth (SHED-CM) in mouse models of rheumatoid arthritis, osteoporosis, and osteoarthritis suppresses excessive osteoclast activity and restores bone integrity. However, the mechanism through which SHED-CM regulates osteoclastogenesis remains largely unknown. In the present study, we examined the anti-osteoclastogenic mechanism of SHED-CM in vitro. Bone marrow macrophages and RAW264.7 cells were treated with receptor activator of nuclear factor kappa-Β ligand (RANKL) in the presence of SHED-CM or CM from bone marrow mesenchymal stem cells (BMSC-CM). Osteoclast differentiation was assessed using tartrate-resistant acid phosphatase staining, actin ring formation, and expression of osteoclast-specific markers. RANKL-induced reactive oxygen species (ROS) production was analyzed as a critical mediator of osteoclastogenesis. The activation of endogenous antioxidant gene expression was examined using reverse transcription quantitative PCR. Liquid chromatography with tandem mass spectrometry (LC-MS) was used to identify proteins enriched in SHED-CM, and neutralizing antibodies were used to evaluate their functional roles. Compared to BMSC-CM, SHED-CM effectively inhibited RANKL-induced early osteoclast differentiation and late maturation. Notably, SHED-CM but not BMSC-CM suppressed RANKL-induced ROS production. SHED-CM increased the expression of genes encoding antioxidant enzymes. The LC-MS analysis identified seven proteins uniquely enriched in SHED-CM that activated the endogenous antioxidant system. Neutralizing antibodies against some of these proteins restore RANKL-induced ROS production and osteoclast differentiation. SHED-CM inhibited osteoclastogenesis, partially through the activation of multiple antioxidant enzymes in osteoclast precursors, highlighting its potential for treating bone-destructive diseases.

P0191 The role of myeloid mineralocorticoid receptor and its target neutrophil gelatinase-associated lipocalin in mice with colitis

Abstract Background Intestinal fibrosis is a common complication in IBD patients with no specific treatment. In extra-intestinal conditions, mineralocorticoid receptor (MR) antagonism inhibits inflammation and fibrosis in different pathophysiological conditions. We previously showed that pharmacological antagonism of MR also inhibits experimental intestinal fibrosis through its target neutrophil gelatinase-associated lipocalin (NGAL). We now hypothesize that myeloid MR may promote the transition from inflammation to intestinal fibrosis by modulation of macrophage plasticity. We aimed to investigate whether myeloid MR or myeloid NGAL deletion prevents colitis development in mice. Methods Acute and chronic colitis were respectively induced by 1% dextran sodium sulfate (DSS) in myeloid MR knock-out (MyMR KO n=10, WT n= 5) and myeloid NGAL knock-out (MyNGAL KO n=6, WT n=8). Colon tissues were collected for quantitative PCR analysis of inflammatory (Il1, Il6, Arg1, Nos2, Retnla, Adgre1) and fibrotic markers (Col1a1, Tgfb1, Fn1, Mmp3, Mmp9, Igf1, Tnf, Timp1). Fecal calprotectin and lipocalin levels were analyzed by ELISA. Results Myeloid MR deletion significantly reduced colon TGFβ mRNA expression (P&amp;lt;0.05) but did not impact weight loss or intestinal permeability in mice. Myeloid NGAL deletion significantly reduced colitis-induced weight loss (P&amp;lt;0.05) and inhibited fibrosis markers such as collagen 1 and metalloproteinase mRNA expression in mice with chronic colitis (Figure 1). Myeloid MR or NGAL invalidation did not significantly affect fecal calprotectin and lipocalin levels in our experimental conditions. Conclusion In our pilot study, myeloid MR and myeloid NGAL deletion inhibits fibrosis development but further studies are required to confirm these findings.

Effect of one prophylactic dose of Azithromycin on Bifidobacterium infantis colonization in infants from the Mumta Trial.

The effects of antibiotics on the microbiome remain incompletely understood. Azithromycin (AZ) has been shown to improve child survival and infant growth outcomes. This study aimed to assess the impact of AZ on B. infantis colonization and bacterial enteropathogen count in the infant gut. We analyzed clinical, biomarker, B. infantis and enteropathogen data from 150 mother-infant dyads from the MUMTA Lactating Women study. Colonization of B. infantis was assessed using quantitative PCR of fecal samples. We utilized a customized PCR-based TaqMan Array Card (TAC) for enteropathogen detection. AZ administration was associated with a 1.99-fold (95% CI 1.33-2.97) increase in colonization by B. infantis. B. infantis colonization was highest when inflammatory biomarker levels were within normal range. Mode of delivery (RR 2.43; 95% CI: 1.58, 3.76) and colostrum (RR2.05; 95% CI: 1.41, 2.98) given to the infant within 24 hours of birth were associated with B. infantis colonization. A single dose of AZ on day 42 reduced bacterial enteropathogen count in the AZ group on day 56, as compared to the pre-AZ count. Bacterial enteropathogen count for infants with wasting (Weight for length Z-score WLZ<-2) was 1.43-fold higher (95% CI: 1.00-2.03) than for infants with WLZ≥ -2. Over 60% of infants harboured with the macrolide resistance mph(A) gene CONCLUSIONS: AZ administration increases B. infantis colonization and reduces bacterial enteropathogen count in infants.