Expression Of Differentiation Markers Research Articles

What Are the Reliable Plasma Biomarkers for Mild Cognitive Impairment? A Clinical 4D Proteomics Study and Validation

Objective. At present, Alzheimer’s disease (AD) lacks effective treatment means, and early diagnosis and intervention are the keys to treatment. Therefore, for mild cognitive impairment (MCI) and AD patients, blood sample analysis using the 4D nonstandard (label-free) proteomic in-depth quantitative analysis, looking for specific protein marker expression differences, is important. These marker levels change as AD progresses, and the analysis of these biomarkers changes with this method, which has the potential to show the degree of disease progression and can be used for the diagnosis and preventive treatment of MCI and AD. Materials and Methods. Patients were recruited according to the inclusion and exclusion criteria and divided into three groups according to scale scores. Elderly patients diagnosed with AD were selected as the AD group (n = 9). Patients diagnosed with MCI were classified into the MCI group (n = 10). Cognitively healthy elderly patients were included in the normal cognition control group (n = 10). Patients’ blood samples were used for 4D label-free proteomic in-depth quantitative analysis to identify potential blood biomarkers. The sample size of each group was expanded (n = 30), and the selected biomarkers were verified by enzyme-linked immunosorbent assay (ELISA) to verify the accuracy of the proteomic prediction. Results. Six specific blood markers, namely, APOE, MMP9, UBR5, PLA2G7, STAT5B, and S100A8, were detected by 4D label-free proteomic quantitative analysis. These markers showed a statistically significant upregulation trend in the MCI and AD groups compared with the normal cognition control group (P<0.05). ELISA results showed that the levels of these six proteins in the MCI group were significantly higher than those in the normal cognition control group, and the levels of these six proteins in the AD group were significantly higher than those in the MCI group (P<0.05). Conclusion. The plasma levels of APOE, MMP9, UBR5, PLA2G7, STAT5B, and S100A8 in cognitively healthy elderly patients and patients with MCI and AD were significantly different and, more importantly, showed a trend of increasing expression. These results indicate that these six human plasma markers have important diagnostic and therapeutic potential in the identification of cognitive impairment and have value for in-depth research and clinical application.

Abstract A023: Stratification of bladder cancer patients with the use of flow cytometry is increasing the diagnostic potential of molecular classifiers

Abstract Recent research suggests the potential categorization of bladder cancer (BLCa) patients into molecular subtypes to aid in the selection of appropriate treatment strategies, be it conventional, targeted, or a combination thereof. However, the practical implementation of molecular classifiers has faced obstacles due to the intricate nature of gene regulation, tumor heterogeneity, and challenges related to tumor sampling caused by substantial stromal infiltration and tumor multifocality. Hence, there is a pressing need for more straightforward marker-based methodologies to assist in genomic and transcriptomic subtyping efforts. Our study addressed this gap by establishing and validating a panel of antibodies for application in flow cytometry (FCM) to enhance the stratification of BLCa patients. We curated a set of 17 FCM markers capable of delineating four cell clusters (epithelial, immune, endothelial, and stromal) and defining tumor classification (basal and luminal), immune infiltration, and hot-target expression on BLCa tissue samples. This FCM panel was deployed on ex vivo tissue samples treated with chemotherapy and targeted therapy agents. Initially tested on a cohort of 10 BLCa tissue samples, our validation of the FCM panel revealed correlations between marker expression and clinical-pathological features, such as the presence of metastasis and heightened immune and stromal infiltration. Notably, samples exhibiting lower claudin-4 levels displayed increased immune infiltration, while those with metastases at diagnosis showcased elevated basal marker expression. The translational potential of the FCM panel was demonstrated in ex vivo experiments where tissue slices from six patients were subjected to chemotherapy and targeted therapy agents, leading to observable shifts in marker expression post-treatment, particularly pronounced with chemotherapy. Noteworthy effects included reductions in epithelial cells expressing FGFR3 and EGFR following targeted therapy with erdafitinib or lapatinib, alongside alterations in the basal/luminal cell ratio indicated by increased CD44, CD90, and CD239 marker expressions. Our findings underscore the value of FCM in categorizing BLCa patients based on specific marker expression, supplementing conventional histological analyses to enhance cancer diagnosis, guide personalized therapeutic approaches, and monitor treatment efficacy. Citation Format: Martina Radic, Federico La Manna, Martina Minoli, Panagiotis Chouvardas, George N. Thalmann, Roland Seiler-Blarer, Marianna Kruithof-de Julio, Bernhard Kiss. Stratification of bladder cancer patients with the use of flow cytometry is increasing the diagnostic potential of molecular classifiers [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A023.

Wuliangye strong aroma baijiu promotes intestinal homeostasis by improving gut microbiota and regulating intestinal stem cell proliferation and differentiation.

Wuliangye strong aroma baijiu (hereafter, Wuliangye baijiu) is a traditional Chinese grain liquor containing short-chain fatty acids, ethyl caproate, ethyl lactate, other trace components, and a large proportion of ethanol. The effects of Wuliangye baijiu on intestinal stem cells and intestinal epithelial development have not been elucidated. Here, the role of Wuliangye baijiu in intestinal epithelial regeneration and gut microbiota modulation was investigated by administering a Lieber-DeCarli chronic ethanol liquid diet in a mouse model to mimic long-term (8 weeks') light/moderate alcohol consumption (1.6 g kg-1 day-1) in healthy human adults. Wuliangye baijiu promoted colonic crypt proliferation in mice. According to immunofluorescence and reverse transcription-quantitative polymerase chain reaction analyses, compared with the ethanol-only treatment, Wuliangye baijiu increased the number of intestinal stem cells and goblet cells and the expression of enteroendocrine cell differentiation markers in the mouse colon. Furthermore, gut microbiota analysis showed an increase in the relative abundance of microbiota related to intestinal homeostasis following Wuliangye baijiu administration. Notably, increased abundance of Bacteroidota, Faecalibaculum, Lachnospiraceae, and Blautia may play an essential role in promoting stem-cell-mediated intestinal epithelial development and maintaining intestinal homeostasis. In summary, these findings suggest that Wuliangye baijiu can be used to regulate intestinal stem cell proliferation and differentiation in mice and to alter gut microbiota distributions, thereby promoting intestinal homeostasis. This research elucidates the mechanism by which Wuliangye baijiu promotes intestinal health. © 2024 Society of Chemical Industry.

Abstract A011: Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition

Abstract Introduction: Epithelial changes can extend beyond the boundaries of an incipient tumor, leading to cancer recurrence even after effective excision of the primary tumor. This ‘field cancerization’ and consequent high recurrence rate is especially pronounced in bladder cancer. The purpose of this study is to characterize the urothelium in the context of ‘field cancerization’ in a mouse model of bladder cancer as well as in patients with bladder cancer and to determine if reversal of the field effect will prevent tumor formation. Results: We investigated field cancerization by single cell transcriptomics (scRNA-Seq) in a murine (male FVB mice) bladder carcinogenesis model using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and found that prior to overt tumor formation urothelial cells throughout the entire bladder take on a strongly basal phenotype, characterized by reduced expression of differentiation markers (i.e. Upk1a, Upk2, and Upk3a) and increased expression of basal markers (i.e. Krt14, Krt6a, Krt5). Using monocle, we applied single cells from a BBN treated mouse bladder library to a pseudotime trajectory generated from a control mouse bladder library in order to unbiasedly characterize urothelial differentiation in the non-tumor urothelium of BBN treated mouse bladders. scRNA-Seq analysis of non-tumor urothelium from bladder cancer patients revealed a similar ‘basalization’ among non-tumor urothelial cells. In order to apply our single cell findings to bulk RNA-seq data, we developed a basal index from differentially expressed genes in basal clusters versus non-basal clusters from healthy control human urothelium single cell libraries, and we applied this basal index to non-tumor bladder samples from the The Cancer Genome Atlas (TCGA). We found that among a small subset of patients with paired tumor and non-tumor urothelium in the bladder TCGA dataset, the basal index, when applied to the non-tumor urothelium, was prognostic of survival. Finally, we found that basalization in BBN treated mice could be reversed pharmacologically by epidermal growth factor (EGF) pathway antagonism using erlotinib and trametinib and that over a 4-month time course this reversal dramatically decreased tumor incidence and improved survival, suggesting a new, more effective approach using mechanism-based cancer drugs for the prevention of bladder cancer recurrence in patients. Conclusions: The non-tumor urothelium in BBN treated mouse bladders, prior to overt tumor formation, demonstrates a basal phenotype which we characterize with scRNA-Seq and pseudotime analysis. Similarly, in patients with bladder cancer we observe that the non-tumor urothelium also takes on a basal phenotype. This ‘basalization’ of the non-tumor urothelium may potentially be prognostic, and pharmacologic reversal of the basal phenotype in the non-tumor urothelium using EGF pathway inhibitors prevents tumors in BBN treated mice. Citation Format: Aaron M. Kershner, Kris B. Prado, Bernhard M. Kiss, Karim Mrouj, Lolita Penland, Wan-Jin Lu, Jiyun Choi, Edward Diaz, Rovin Lachmansingh, Bertha Chen, Chia-Sui Kao, Joseph C. Liao, Philip A. Beachy. Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A011.

Sequential Fabrication of a Three-Layer Retina-like Structure.

Tissue engineering is considered a promising approach to treating advanced degenerative maculopathies such as nonexudative age-related macular degeneration (AMD), the leading cause of blindness worldwide. The retina consists of several hierarchical tissue layers, each of which is supported by a layer underneath. Each of these layers has a different morphology and requires distinct conditions for proper assembly. In fact, a prerequisite step for the assembly of each of these layers is the organization of the layer underneath. Advanced retinal degeneration includes degeneration of the other retina layers, including the choroid, the retinal pigmented epithelium (RPE), and the photoreceptors. Here, we report a step-by-step fabrication process of a three-layer retina-like structure. The process included the 3D printing of a choroid-like structure in an extracellular matrix (ECM) hydrogel, followed by deposition of the RPE monolayer. After the formation of the blood vessel-RPE interface, the photoreceptor cells were deposited to interact with the RPE layer. At the end of the fabrication process, each layer was characterized for its morphology and expression of specific markers, and the integration of the three-layer retina was evaluated. We envision that such a retina-like structure may be able to attenuate the deterioration of a degenerated retina and improve engraftment and regeneration. This retinal implant may potentially be suitable for a spectrum of macular degenerative diseases for which there are currently no cures and may save millions from complete blindness.

Cytocompatibility of electrospun poly-L-lactic acid membranes for Bruch's membrane regeneration using human embryonic stem cell-derived retinal pigment epithelial cells.

Cell replacement therapy is under development for dry age-related macular degeneration (AMD). A thin membrane resembling the Bruch's membrane is required to form a cell-on-membrane construct with retinal pigment epithelial (RPE) cells. These cells have been differentiated from human embryonic stem cells (hESCs) in vitro. A carrier membrane is required for cell implantation, which is biocompatible for cell growth and has dimensions and physical properties resembling the Bruch's membrane. Here a nanofiber electrospun poly-L-lactic acid (PLLA) membrane is tested for capacity to support cell growth and maturation. The requirements for laminin coating of the membrane are identified here. A porous electrospun nanofibrous PLLA membrane of ∼50 nm fiber diameter was developed as a prototype support for functional RPE cells grown as a monolayer. The need for laminin coating applied to the membrane following treatment with poly-L-ornithine (PLO), was identified in terms of cell growth and survival. Test membranes were compared in terms of hydrophilicity after laminin coating, mechanical properties of surface roughness and Young's modulus, porosity and ability to promote the attachment and proliferation of hESC-RPE cells in culture for up to 8 weeks. Over this time, RPE cell proliferation, morphology, and marker and gene expression, were monitored. The functional capacity of cell monolayers was identified in terms of transepithelial electrical resistance (TEER), phagocytosis of cells, as well as expression of the cytokines, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PLLA polymer fibers are naturally hydrophobic, so their hydrophilicity was improved by pretreatment with PLO for subsequent coating with the bioactive protein laminin. They were then assessed for amount of laminin adsorbed, contact angle and uniformity of coating using scanning electron microscopy (SEM). Pretreatment with 100% PLO gave the best result over 10% PLO treatment or no treatment prior to laminin adsorption with significantly greater surface stiffness and modulus. By 6 weeks after cell plating, the coated membranes could support a mature RPE monolayer showing a dense apical microvillus structure and pigmented 3D polygonal cell morphology. After 8 weeks, PLO (100%)-Lam coated membranes exhibited the highest cell number, cell proliferation, and RPE barrier function measured as TEER. RPE cells showed the higher levels of specific surface marker and gene expression. Microphthalmia-associated transcription factor expression was highly upregulated indicating maturation of cells. Functionality of cells was indicated by expression of VEGF and PEDF genes as well as phagocytic capacity. In conclusion, electrospun PLLA membranes coated with PLO-Lam have the physical and biological properties to support the distribution and migration of hESC-RPE cells throughout the whole structure. They represent a good membrane candidate for preparation of hESC-RPE cells as a monolayer for implantation into the subretinal space of AMD patients.

Expression changes of RNA m6A regulators in mouse cerebellum affected by hypobaric hypoxia stimulation

Objective: To investigate the role of RNA m6A methylation in mediating cerebellar dysplasia through analyzing the phenotypes of the mouse cerebella and the expression of several key m6A regulators upon hypobaric hypoxia treatment. Methods: Five-day old C57/BL6 mice were exposed to hypobaric hypoxia for 9 days. The status of mouse cerebellar development was analyzed by comparing the body weights, brain weights and histological features. Immunostaining of cell-type-specific markers was performed to analyze the cerebellar morphology. Real-time PCR, Western blot and immunohistochemical staining were performed to detect the expression of key m6A regulators in the mouse cerebella. Results: Compared with the control, the body weights, brain weights and cerebellar volumes of hypobaric hypoxic mice were significantly reduced (P<0.01). The expression of specific markers in different cells, including NeuN (mature neuron), Calbindin-D28K (Purkinje cell) and GFAP (astrocyte), was decreased in hypobaric hypoxic mouse cerebella (P<0.01), accompanied with disorganized cellular structure. The expression of methyltransferase METTL3 was significantly down-regulated in the cerebella of hypobaric hypoxic mice (P<0.05). Conclusions: Hypobaric hypoxia stimulation causes mouse cerebellar dysplasia, with structural abnormalities in mature granular neurons, Purkinje cells and astrocytes. Expression of METTL3 is decreased in hypobaric hypoxic mice cerebellum compared with that of normobaric normoxic mice, suggesting that its mediated RNA m6A methylation may play an important role in hypobaric hypoxia-induced mouse cerebellar dysplasia.

Integrating Network Pharmacology, Quantitative Metabolic Network Analysis, In Vitro Experiments, and Molecular Dynamics to Explore the Mechanism of Angelica Sinensis for Regulating Bone Metabolism.

Bone metabolic diseases are serious health issues worldwide. Angelica sinensis (AS) is traditionally used in Chinese medicine for treating bone metabolism diseases clinically. However, the mechanism of AS in regulating bone metabolism remains uncertain. The current investigation was structured to elucidate the potential mechanisms of AS for modulating bone metabolism. Firstly, targets of AS regulating bone metabolism were collected by network pharmacology. Then, the transcriptional regulation of RUNX2 was enriched as one of the key pathways for AS to regulate bone metabolism, constructing its metabolic network. Secondly, combining molecular docking, network efficiency, and network flux analyses, we conducted a quantitative evaluation of the metabolic network to reveal the potential mechanisms and components of AS regulating bone metabolism. Finally, we explored the effect of AS on the differentiation of osteoclasts from M-CSF and RANKL-induced RAW264.7 cells, as well as its impact on the osteogenic induction of MC3T3-E1 cells. We verified the mechanism and key targets of AS on bone metabolism using qRT-PCR. Furthermore, the key component was preliminarily validated through molecular dynamics simulation. Quantitative metabolic network of the transcriptional regulation of RUNX2 was constructed to illustrate the potential mechanism of AS for regulating bone metabolism, indicating that ferulic acid may be a pharmacological component of AS that interferes with bone metabolism. AS suppressed osteoclast differentiation in M-CSF and RANKL-induced RAW264.7 cells and reversed the expressions of osteoclastic differentiation markers, including RUNX2 and SRC. Additionally, AS induced osteogenic generation in MC3T3-E1 cells and reversed the expressions of markers associated with osteoblastic generation, such as RUNX2 and HDAC4. Molecular dynamics simulation displayed a strong binding affinity among ferulic acid, HDAC4 and SRC. This study reveals a systematic perspective on the intervention bone mechanism of AS by transcriptive regulation by RUNX2, guiding the clinical use of AS in treating diseases of the skeletal system.

Monocytes prevent apoptosis of iPSCs and promote differentiation of kidney organoids

BackgroundInduced pluripotent stem cells (iPSCs)-derived kidney organoids are a promising model for studying disease mechanisms and renal development. Despite several protocols having been developed, further improvements are needed to overcome existing limitations and enable a wider application of this model. One of the approaches to improve the differentiation of renal organoids in vitro is to include in the system cell types important for kidney organogenesis in vivo, such as macrophages. Another approach could be to improve cell survival. Mesodermal lineage differentiation is the common initial step of the reported protocols. The glycogen synthase kinase-3 (GSK-3) activity inhibitor, CHIR99021 (CHIR), is applied to induce mesodermal differentiation. It has been reported that CHIR simultaneously induces iPSCs apoptosis that can compromise cell differentiation. We thought to interfere with CHIR-induced apoptosis of iPSCs using rapamycin.MethodsDifferentiation of kidney organoids from human iPSCs was performed. Cell survival and autophagy were analyzed using Cell counting kit 8 (CCK8) kit and Autophagy detection kit. Cells were treated with rapamycin or co-cultured with human monocytes isolated from peripheral blood or iPSCs-macrophages using a transwell co-culture system. Monocyte-derived extracellular vesicles (EVs) were isolated using polyethylene glycol precipitation. Expression of apoptotic markers cleaved Caspase 3, Poly [ADP-ribose] polymerase 1 (PARP-1) and markers of differentiation T-Box Transcription Factor 6 (TBX6), odd-skipped related 1 (OSR1), Nephrin, E-Cadherin, Paired box gene 2 (Pax2) and GATA Binding Protein 3 (Gata3) was assessed by RT-PCR and western blotting. Organoids were imaged by 3D-confocal microscopy.ResultsWe observed that CHIR induced apoptosis of iPSCs during the initial stage of renal organoid differentiation. Underlying mechanisms implied the accumulation of reactive oxygen species and decreased autophagy. Activation of autophagy by rapamacin and by an indirect co-culture of differentiating iPSCs with iPSCs-macrophages and human peripheral blood monocytes prevented apoptosis induced by CHIR. Furthermore, monocytes (but not rapamycin) strongly promoted expression of renal differentiation markers and organoids development via released extracellular vesicles.ConclusionOur data suggest that co-culturing of iPSCs with human monocytes strongly improves differentiation of kidney organoids. An underlying mechanism of monocytic action implies, but not limited to, an increased autophagy in CHIR-treated iPSCs. Our findings enhance the utility of kidney organoid models.

Abstract PO3-13-08: D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease

Abstract Background: Obesity is a recognized risk factor for the development of metabolic disease and breast cancer possibly due to chronic inflammation mediated by cytokines secreted by adipose tissue. Given this, our group examined differential expression of several markers of chronic inflammation in adipose tissue of both benign and malignant breast tissue from patients. The aryl hydrocarbon receptor (AhR) is an important cytosolic, ligand-dependent transcription factor found within hepatocytes and adipocytes that is upregulated in many types of cancer. AhR promotes the initiation, progression, and metastasis of cancer cells allowing certain cancers to evade immune recognition (Xue et al. Front. Immunology 2018). Indoleamine 2,3-dioxygenase 1 (IDO1) is an enzyme that generates kynurenine (KYN) from tryptophan (TRP), which acts as an AhR agonist. We sought to determine expression of IDO1 via immunohistochemistry (IHC) in mammary adipose tissue associated with benign or malignant breast. We hypothesized that adipose tissue adjacent to malignant breast tissue may have heightened expression of IDO1 in addition to Cyp1b1, and SPP1; both of which are also markers of increased aryl-hydrocarbon transcriptional activity, compared to adipose tissue noncontiguous to malignant lesions. KYN affects gene expression for insulin resistance and levels are elevated in plasma of older obese women compared to lean women; however, the relationship to breast malignancy is not known. Methods: IDO1 (Cell Signaling, 1:200), Cyp1b1 (Thermo Fisher, 1:300), and SPP1 (Thermo Fisher, 1:200) IHC was performed according to the manufacturers' protocols. Adipose tissue immediately adjacent to, and &amp;gt;2 mm away from, tumors (three DCIS, 11 invasive carcinomas) was evaluated in 14 patients. Adipose tissue in separate tissue blocks from benign breast tissue (three ipsilateral, 11 contralateral) in the same patients was also evaluated. Human plasma KYN levels were assayed by HPLC in the Dartmouth Clinical Pharmacology Shared Resource as described previously (Rojas et al. Obesity 2021). Results: In this preliminary pilot study, there was no difference in staining pattern for IDO1 in adipose tissue present in malignant and benign samples. Two patients showed increased Cyp1b1 and SPP1 staining immediately adjacent to the invasive carcinoma (Figure 1). Patients with benign breast disease did not show increased Cyp1b1 or SPP1 staining. Results will be confirmed using qPCR for mRNA. Plasma samples from patients with benign disease showed a lower level of KYN expression on average (0.370 ug/mL, N = 8) compared to plasma from patients with malignant disease (0.424, N = 10). Additionally, the ratio of KYN:TRP was lower in the benign group (0.065) compared to the cohort with malignancy (0.080). Univariate linear regression will be used to further assess relationships between continuous variables in the study population (e.g., patient age, BMI, KYN plasma levels, benign vs malignant breast disease). Regression lines and the associated slope estimates (b-value) and p-values for regression models will be provided on scatter plots. Conclusions: While there was no difference in IDO1 staining patterns in adipose tissue of malignant and benign samples in this group, tissue samples from patients with invasive carcinoma demonstrated increased Cyp1b1 and SPP1 staining adjacent to the malignant tumor; while benign tissue samples did not show a positive stain. This suggests increased aryl-hydrocarbon transcriptional activity in invasive breast carcinoma—further linking AhR signaling with adipose tissue immune dysregulation and invasive malignancy. Additionally, KYN plasma levels were higher in patients with malignant disease indicating a possible relationship between insulin resistance and breast malignancy. Additional research may offer new insights relating to this important pathway and ultimately treatments aimed at blunting its nefarious effects. Citation Format: Adam Ephraim, Jonathan Pirruccello, Scott Palisoul, Jonathan Marotti, Mary Chamberlin. D17104: Aryl Hydrocarbon Receptor Signaling and Other Biomarkers of Fat Metabolism in Human Mammary Adipose Tissue from Women with Malignant Breast Disease [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-08.

Targeting miR-31 represses tumourigenesis and dedifferentiation of BRAFV600E-associated thyroid carcinoma.

BRAFV600E is the most common genetic mutation in differentiated thyroid cancer (DTC) occurring in 60% of patients and drives malignant tumour cell phenotypes including proliferation, metastasis and immune-escape. BRAFV600E-mutated papillary thyroid cancer (PTC) also displays greatly reduced expression of thyroid differentiation markers, thus tendency to radioactive iodine (RAI) refractory and poor prognosis. Therefore, understanding the molecular mechanisms and main oncogenic events underlying BRAFV600E will guide future therapy development. Bioinformatics and clinical specimen analyses, genetic manipulation of BRAFV600E-induced PTC model, functional and mechanism exploration guided with transcriptomic screening, as well as systematic rescue experiments were applied to investigate miR-31 function within BRAFV600E-induced thyroid cancer development. Besides, nanoparticles carrying miR-31 antagomirs were testified to alleviate 131I iodide therapy on PTC models. We identify miR-31 as a significantly increased onco-miR in BRAFV600E-associated PTC that promotes tumour progression, metastasis and RAI refractoriness via sustained Wnt/β-catenin signalling. Mechanistically, highly activated BRAF/MAPK pathway induces miR-31 expression via c-Jun-mediated transcriptional regulation across in vitro and transgenic mouse models. MiR-31 in turn facilitates β-catenin stabilisation via directly repressing tumour suppressors CEBPA and DACH1, which direct the expression of multiple essential Wnt/β-catenin pathway inhibitors. Genetic functional assays showed that thyroid-specific knockout of miR-31 inhibited BRAFV600E-induced PTC progression, and strikingly, enhanced expression of sodium-iodide symporter and other thyroid differentiation markers, thus promoted 131I uptake. Nanoparticle-mediated application of anti-miR-31 antagomirs markedly elevated radio-sensitivity of BRAFV600E-induced PTC tumours to 131I therapy, and efficiently suppressed tumour progression in the pre-clinical mouse model. Our findings elucidate a novel BRAF/MAPK-miR-31-Wnt/β-catenin regulatory mechanism underlying clinically BRAFV600E-associated DTC tumourigenesis and dedifferentiation, also highlight a potential adjuvant therapeutic strategy for advanced DTC.

Histone demethylase KDM3B mediates matrix stiffness-induced osteogenic differentiation of adipose-derived stem cells

Biomechanical signals in the extracellular niche are considered promising for programming the lineage specification of stem cells. Recent studies have reported that biomechanics, such as the microstructure of nanomaterials, can induce adipose-derived stem cells (ASCs) to differentiate into osteoblasts, mediating gene regulation at the epigenetic level. Therefore, in this study, transcriptome expression levels of histone demethylases in ASCs were screened after treatment with different matrix stiffnesses, and histone lysine demethylase 3B (KDM3B) was found to promote osteogenic differentiation of ASCs in response to matrix stiffness, indicating a positive modulatory effect on this biological process. ASCs exhibited widespread and polygonal shapes with a distinct bundle-like expression of vinculin parallel to the axial cytoskeleton along the cell margins on the stiff matrix rather than round shapes with a smeared and shorter expression on the soft matrix. Comparatively rigid polydimethylsiloxane material directed ASCs into an osteogenic phenotype in inductive culture media via the upregulation of osteocalcin, alkaline phosphatase, and runt-related transcription factor 2. Treatment with KDM3B-siRNA decreased the expression of osteogenic differentiation markers and impaired mitochondrial dynamics and mitochondrial membrane potential. These results illustrate the critical role of KDM3B in the biomechanics-induced osteogenic commitment of ASCs and provide new avenues for the further application of stem cells as potential therapeutics for bone regeneration.

Novel regulation of kidney function by gastrointestinal hormones

The control of whole-body energy metabolism involves intricate crosstalk between multiple organ systems including the gut and the brain that play well-recognized roles in this process. However, the important contributions and mechanisms by other organs are poorly understood. Errors in metabolic regulation can lead to human metabolic diseases including diabetes and obesity that affect 75% of the population and can lead to significant comorbidities. Improving our mechanistic understanding of multi-organ metabolic crosstalk in health and disease is essential for developing highly effcient and new therapeutic approaches. Macula densa (MD) cells in the kidney are chief regulators of renal blood flow and tissue remodeling. We hypothesized that the kidney is a major player in body metabolism via a new mechanism of gut-kidney crosstalk that involves the gut hormones gastrin and cholecystokinine (CCK) activating intrarenal, MD-specific mechanisms to increase blood flow and filtration, progenitor cells and tissue growth factors. High-resolution MD bulk and single-cell transcriptome analysis was performed from freshly isolated, enriched MD cells using MD-GFP mouse kidneys. Single cell MD Ca2+ signaling was analyzed in vivo using intravital multiphoton microscopy (MPM) of Sox2-GT mice that express the ratiometric Ca2+ reporter GCaMP6f/tdTomato in all renal cells. The altered expression of MD specific markers in response to gastrin/CCK8 stimuli was studied using western blot and the novel MDgeo immortalized cell line cultured in vitro. RNA sequencing and transcriptome analysis identified the gastrin/CCK receptor Cckbr as one of the highest expressed genes in MD versus control kidney cells. Intravital multiphoton imaging of Sox2-GCaMP6 reporter mouse kidneys revealed robust and entirely MD cell-specific calcium signaling in response to systemic gastrin/CCK injection. Using the novel MDgeo immortalized cell line cultured in vitro, CCK8 treatment led to 3-fold increases in MD cell synthesis of Prostaglandin E2 (PGE2)-generating COX2 and CCN1 that are established mediators of MD-induced glomerular hyperfiltration and tissue remodeling, respectively. Chronic treatment of Ren1d-Confetti mice with Darunavir, an HIV protease inhibitor significantly increased plasma CCK levels, Ren+ mesenchymal progenitor cell number and clonality, renal cell proliferation and tissue hypertrophy. This study uncovered new regulatory mechanisms of systemic metabolism especially the role of MD cells in the kidney. Repurposing the commonly used HIV drug Darunavir for kidney and metabolic diseases may provide therapeutic benefit for millions of patients worldwide. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Characterization of Ano1 expressing cells in the mouse corpus cavernosum

Background/objectives: Highly coordinated contraction and relaxation of smooth muscle cells (SMCs) is critical to penile function. The Ca2+-activated Cl− channel, Anoctamin-1 (Ano1) plays a role in this as antagonists of this channel inhibit contractile, electrical and intracellular Ca2+ activity. mRNA encoding Ano1 is highly expressed in whole rabbit corpus cavernosum (CC). Despite its significant contribution to erectile function, the specific cell type that expresses Ano1 has not been determined, with previous studies relying on morphological identification of cells alone. Atypical SMCs (ASMCs) expressing platelet derived growth factor receptor-a (PDGFRa; Pdgfra), myosin heavy chain ( Myh11) and Ano1 have been observed in the renal pelvis where they are thought to drive the activity in adjacent SMCs (Grainger et al. 2020; PMID: 32415739). Therefore, the objective of this study is to identify the cell type that expresses Ano1 in the CC. Methods: Cells were isolated from the CC of mice selectively expressing eGFP in PDGFRa+ cells (PDGFRa+-eGFP mice) and SMCs ( Myh11-eGFP mice). Cell suspensions were sorted using fluorescence activated cell sorting (FACS) and evaluated for expression of cell specific markers ( Pdgfra and Myh11) by qPCR. Enriched PDGFRa+ cells and SMCs were examined with ddPCR to determine the expression of Ano1. Protein expression was evaluated using immunofluorescent labeling of frozen CC sections with an antibody targeting Ano1. Results: FACS sorted Myh11-eGFP+ cells expressed Myh11 and Pdgfra. Sorting of PDGFRa-eGFP+ cells revealed two subpopulations, bright and dim. Examination of these two PDGFRa-eGFP+ subpopulations revealed that while both expressed similar levels of Pdgfra, the dim population also expressed Myh11 suggesting that these cells may be similar to ASMCs of the renal pelvis. ddPCR revealed that the PDGFRa-eGFP+ dim population had higher gene copy numbers of Ano1 compared to Myh11-eGFP+ cells. Immunofluorescent labeling of CC sections revealed that Ano1 protein was present in a subpopulation of PDGFRa+ cells. Conclusion: The data presented in this study demonstrates that Ano1 is expressed in mouse CC PDGFRa-eGFP+ dim populations that also express Pdgfra and Myh11; this is similar to ASMCs in the renal pelvis. Future directions include examining the relationship of PDGFRa-eGFP+ dim cells to SMCs and nerve terminals in the CC and to determine if these cells are also present in human CC. This cell population could represent a novel target for the treatment of erectile dysfunction in patients who do not respond to phosphodiesterase inhibitors. Funding: AUA Research Scholar Award to KIH, NIH DK129528 to CAC, NIH P20GM130459 for imaging and molecular cores. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cytotoxicity effects and differentiation potential of ormocer-based and nanohybrid composite resins on human dental pulp stem cells

Objectiveto compare conventional nanohybrid (Ceram.x Spectra) and ormocer-based (Admira fusion) dental composite resins effects on human dental pulp stem cells (hDPSCs) in terms of cytotoxicity, self-renewal, migration and osteogenic differentiation. MethodshDPSCs were cultured in presence of different dilutions (undiluted, form 1:2 to 1:100) of CeramX (CX) and Admira fusion (AD) eluates and viability assay in standard or osteogenic conditions were performed. Samples and eluates were prepared according to ISO 10993–12. In addition, apoptosis, self-renewal and migration activity evaluations were carried out. Osteogenic differentiation potential was tested by Alkaline Phosphatase Activity, alizarin red staining and gene expression of specific markers (ALP, RUNX2, OCN, OPN and COL1α1). Statistical analysis was performed by means of a One-way analysis of variance (One-way ANOVA) followed by a Tukey’s test for multiple comparison; results were presented as mean ± standard error of mean (SEM). ResultsAdmira Fusion demonstrated to be highly biocompatible and showed positive effects on hDPSCs proliferation and differentiation; on the contrary, conventional nanohybrid composite showed to be more cytotoxic and without any notable effect on stem cells differentiation. Moreover, the obtained results were further corroborated by a significant upregulation of osteogenic differentiation markers obtained in presence of ormocer-based composite resin eluate. Specifically, in AD 1:50 group expression levels of ALP, Runx2, Col1α1 were double than control (ALP, p = 0.045; Runx2, p = 0.003; Col1α1, p = 0.001) and CX 1:50 (ALP, p = 0.006; RUNX2, p = 0.029; Col1α1, p = 0.005). Moreover, in the same group, OPN and OCN resulted about 5 times more expressed as compared to control (OPN, p = 0.009; OCN, p = 0.0005) and CX 1:50 (OPN, p = 0.012; OCN, p = 0.0006). SignificanceThe less cytotoxicity obtained by AD than conventional nanohybrid composite may be attributed to a reduced monomers release in the oral environment, supporting the hypothesis of limited adverse effect and enhanced healing potential, mainly when the material is positioned in close contact with pulp tissue.

Establishment of an induced pluripotent stem cell (iPSC) line (INNDSUi004-A) from a patient with Congenital Nemaline Myopathy

We used a non-integrated reprogramming approach to establish a human induced pluripotent stem cell (hiPSC) line (INNDSUi004-A) from the skin fibroblasts of a 13-year-old female individual with Congenital Nemaline Myopath. The cells obtained have typical characteristics of embryonic stem cells, show expression of specific pluripotency markers, and can differentiate into three germ layers in vitro. This iPSC cell line has the genetic information of the patient and is a good model for studying disease mechanisms and developing novel therapies.

Zebrafish nampt-a mutants are viable despite perturbed primitive hematopoiesis.

Nicotinamide phosphoribosyltransferase (Nampt) is required for recycling NAD+ in numerous cellular contexts. Morpholino-based knockdown of zebrafish nampt-a has been shown to cause abnormal development and defective hematopoiesis concomitant with decreased NAD+ levels. However, surprisingly, nampt-a mutant zebrafish were recently found to be viable, suggesting a discrepancy between the phenotypes in knockdown and knockout conditions. Here, we address this discrepancy by directly comparing loss-of-function approaches that result in identical defective transcripts in morphants and mutants. Using CRISPR/Cas9-mediated mutagenesis, we generated nampt-a mutant lines that carry the same mis-spliced mRNA as nampt-a morphants. Despite reduced NAD+ levels and perturbed expression of specific blood markers, nampt-a mutants did not display obvious developmental defects and were found to be viable. In contrast, injection of nampt-a morpholinos into wild-type or mutant nampt-a embryos caused aberrant phenotypes. Moreover, nampt-a morpholinos caused additional reduction of blood-related markers in nampt-a mutants, suggesting that the defects observed in nampt-a morphants can be partially attributed to off-target effects of the morpholinos. Our findings show that zebrafish nampt-a mutants are viable despite reduced NAD+ levels and a perturbed hematopoietic gene expression program, indicating strong robustness of primitive hematopoiesis during early embryogenesis.

Suppression of bone resorption by Mori Radicis Cortex through NFATc1 and c-Fos signaling-mediated inhibition of osteoclast differentiation.

Mori Radicis Cortex (MRC) is the root bark of the mulberry family as Morus alba L. In Korea, it is known as "Sangbaegpi". While MRC has demonstrated anti-inflammatory and antioxidant effects, its specific mechanisms of action and impact on osteoporosis remain poorly understood. To investigate the anti-osteoporosis effect of MRC, we examined the level of osteoclast differentiation inhibition in receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced-RAW 264.7 cells and animal models of ovariectomy (OVX) with MRC. Serum analysis in OVX animals was investigated by enzyme-linked immunosorbent assay (ELISA), and bone density analysis was confirmed by micro-computed tomography (micro-CT). The expression analysis of NFATc1 was confirmed by immunohistochemistry (IHC) in femur tissue. In addition, osteoclast differentiation inhibition was measured using tartrate-resistant acid phosphatase (TRAP). mRNA analysis was performed using reverse transcription polymerase chain reaction (RT-PCR), and the protein expression analysis was investigated by western blot. Micro-CT analysis showed that MRC effectively inhibited bone loss in the OVX-induced rat model. MRC also inhibited the expression of alkaline phosphatase (ALP) and TRAP in serum. Histological analysis showed that MRC treatment increased bone density and IHC analysis showed that MRC significantly inhibited the expression of NFATc1. In RANKL-induced-RAW 264.7 cells, MRC significantly reduced TRAP activity and actin ring formation. In addition, MRC significantly inhibited the expression of nuclear factor of activated T cells 1 (NFATc1)/ and c-Fos, and suppressed the mRNA expression. Based on micro-CT, serum and histological analysis, MRC effectively inhibited bone loss in an OVX-induced rat model. In addition, MRC treatment suppressed the expression of osteoclast differentiation, fusion, and bone resorption markers through inhibition of NFATc1/c-Fos expression in RANKL-induced RAW 264.7 cells, ultimately resulting in a decrease in osteoclast activity. These results demonstrate that MRC is effective in preventing bone loss through inhibiting osteoclast differentiation and activity.

Hydrogel/microcarrier cell scaffolds for rapid expansion of satellite cells from large yellow croakers: Differential analysis between 2D and 3D cell culture

Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.

Novel Peptide Derived from Gadus morhua Stimulates Osteoblastic Differentiation and Mineralization through Wnt/β-Catenin and BMP Signaling Pathways.

Marine biodiversity offers a wide array of active ingredient resources. Gadus morhua peptides (GMPs) showed excellent osteoprotective effects in ovariectomized mice. However, the potential osteogenesis mechanisms of key osteogenic peptides in GMP were seldom reported. In this study, a novel osteogenic peptide (GETNPADSKPGSIR, P-GM-2) was screened from GMP. P-GM-2 has a high stability coefficient and a strong interaction with epidermal growth factor receptor. Cell culture experiments showed that P-GM-2 stimulated the expression of osteogenic differentiation markers to promote osteoblast proliferation, differentiation, and mineralization. Additionally, P-GM-2 phosphorylates GSK-3β, leading to the stabilization of β-catenin and its translocation to the nucleus, thus initiating the activation of the Wnt/β-catenin signaling pathway. Meanwhile, P-GM-2 could also regulate the osteogenic differentiation of preosteoblasts by triggering the BMP/Smad and mitogen-activated protein kinase signaling pathways. Further validation with specific inhibitors (ICG001 and Noggin) demonstrated that the osteogenic activity of P-GM-2 was revealed by the activation of the BMP and Wnt/β-catenin pathways. In summary, these results provide theoretical and practical insights into P-GM-2 as an effective antiosteoporosis active ingredient.