Primary Cell Research Articles

Exosomes derived from Mouse Bone Marrow Mesenchymal Stem Cells Attenuate Nucleus Pulposus Cell Apoptosis via the miR-155- 5p/Trim32 Axis.

Lower back pain, shown to be strongly associated with IVDD, affects approximately 60%-80% of adults and has a considerable societal and economic impact. Evidence suggests that IVDD, caused by abnormal apoptosis of nucleus pulposus cells (NPCs), can be treated using MSC-derived exosomes. This study aimed to evaluate the role of miR155-5p/Trim32 in intervertebral disc disease (IVDD) and elucidate the underlying molecular mechanisms. Deregulating miR-155 has been shown to promote Fas-mediated apoptosis in human IVDD. Evidence also suggests that tripartite motif (TRIM)-containing protein 32 (Trim32) is regulated by miR-155. However, the role of miR155-5p/Trim32 in IVDD remains unclear. Cell viability was checked using CCK-8 kits, and flow cytometry was used to analyze cell cycle and apoptosis. Cell migration was measured with a Transwell assay, while a luciferase assay was adopted to study how miR-155-5p interacts with Trim32. The roles of Trim32 and miR-155-5p were studied by silencing or up-regulating them in NPCs, while qPCR and immunoblots were used to evaluate mRNA and protein changes, respectively. TNF-α treatment significantly inhibited cell viability but promoted Trim32 expression in primary mouse NPCs. Administration of bone marrow mesenchymal stem cells (BMSCs) attenuated primary NPC cell cycle arrest and apoptosis induced by TNFα. BMSCs-derived exosomes could be taken up by NPCs to inhibit TNF-α-induced cell cycle arrest and apoptosis through miR-155-5p. Examination of the underlying mechanism showed that miR-155-5p targeted Trim32. Moreover, Trim32 overexpression inhibited the effect of BMSCs-derived exosomes on primary mouse NPC cell apoptosis induced by TNF-α. Overall, these findings suggest that exosomes from BMSCs can suppress TNF-α-induced cell cycle arrest and apoptosis in primary mouse NPCs through the delivery of miR-155-5p by targeting Trim32. This study provides a promising therapeutic strategy for IVDD.

Investigation on Vision System: Digital FPGA Implementation in Case of Retina Rod Cells.

The development of prostheses and treatments for illnesses and recovery has recently been centered on hardware modeling for various delicate biological components, including the nervous system, brain, eyes, and heart. The retina, being the thinnest and deepest layer of the eye, is of particular interest. In this study, we employ the Nyquist-Based Approximation of Retina Rod Cell (NBAoRRC) approach, which has been adapted to utilize Look-Up Tables (LUTs) rather than original functions, to implement rod cells in the retina using cost-effective hardware. In modern mathematical models, numerous nonlinear functions are used to represent the activity of these cells. However, these nonlinear functions would require a substantial amount of hardware for direct implementation and may not meet the required speed constraints. The proposed method eliminates the need for multiplication functions and utilizes a fast, cost-effective rod cell device. Simulation results demonstrate the extent to which the proposed model aligns with the behavior of the primary rod cell model, particularly in terms of dynamic behavior. Based on the results of hardware implementation using the Field-Programmable Gate Arrays (FPGA) board Virtex-5, the proposed model is shown to be reliable, consume 30 percent less power than the primary model, and have reduced hardware resource requirements. Based on the results of hardware implementation using the reconfigurable FPGA board Virtex-5, the proposed model is reliable, uses 30% less power consumption than the primary model in the worth state of the set of approximation method, and has a reduced hardware resource requirement. In fact, using the proposed model, this reduction in the power consumption can be achieved. Finally, in this article, by using the LUT which is systematically sampled (Nyquist rate), we were able to remove all costly operators in terms of hardware (digital) realization and achieve very good results in the field of digital implementation in two scales of network and single neuron.

Abstract 5415: Fibroblast mediated head and neck tumor cell invasion in 3D culture is independent of partial epithelial to mesenchymal transition

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with a five-year-survival of 61-64%. Recent studies have demonstrated tumor keratinocytes with features of a partial transition from epithelial to a mesenchymal phenotype, also known as partial-EMT (pEMT), at the edge of invading tumor islands associates with metastatic disease. As such, pEMT likely results from direct interactions between tumor cells and their surrounding microenvironment. To test if specific secreted factors or cell interactions drive the acquisition of pEMT, we first analyzed a series of primary and established HNSCC cell populations to determine TGF-β1 responsiveness since TGF-β1 is a known driver of pEMT. Of the eight populations tested, six displayed an increased level of pSMAD3 upon incubation with 5 ng/mL of TGF-β1 (p < 0.0001). To determine if this TGF-β1 response leads to an increase in pEMT phenotype, a pEMT-score (derived from mRNA expression of 9 pEMT markers) for each HNSCC population was determined following TGF-β1 treatment. As expected, the two non-responsive HNSCC populations showed no increase in pEMT-score, whilst five of the six TGF-β1-responsive HNSCC populations demonstrated a significant increase in pEMT-score (p = 0.006). To assess if TGF-β induced pEMT phenotype correlates with increased invasive potential, we produced 3D skin equivalent models using the eight HNSCC keratinocyte populations, in the presence or absence of primary fibroblasts and/or TGF-β1 treatment. TGF-β1 treatment alone caused a significant increase in invasion of all five TGF-β1-induced pEMT HNSCC populations (r2 = 0.449, p = 0.0045), whilst the three populations previously shown to be pEMT unresponsive to TGF-β1 treatment showed no increase. Interestingly, addition of primary fibroblasts without TGF-β1 treatment caused a universal dramatic increase in invasion in 3D organ culture (p = 0.0064, 34-fold increase), regardless of the previously determined TGF-β1-induced pEMT status. Unexpectedly, TGF-β1 treatment in the presence of fibroblasts caused no significant increase in invasion in 3D cultures (r2 = 0.00001, p = 0.9906). We next compared levels of pEMT markers in 3D cultures using immunofluorescence and observed a significant increase in PDPN in six of the eight HNSCC populations (p = 0.0136) with the addition of fibroblasts suggesting that factors independent of TGF-β1 contributes to pEMT marker expression and invasion in 3D culture. Overall, these data demonstrate fibroblasts induce HNSCC keratinocyte invasion regardless of the population’s ability to respond to TGF-β1-induced pEMT and that pEMT may be driven by factors independent of TGF-β1 in 3D culture. Citation Format: Pyung Hun Park, Michael M. Alexander, Joseph M. Curry, Adam J. Luginbuhl, Andrew P. South. Fibroblast mediated head and neck tumor cell invasion in 3D culture is independent of partial epithelial to mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5415.

Abstract 1338: Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells

Abstract Human leukocyte antigen-G (HLA-G) suppresses lymphoid and myeloid immune systems by binding ILT2 and ILT4 respectively and activating ITIM signaling. HLA-G is abundantly expressed in placental cytotrophoblasts to block fetal rejection. HLA-G is not expressed in most normal cells but is derepressed in in about 50% of malignancies. We engineered ILT2 and ILT4 to produce activating signals as Chimeric ILT Receptors or CIR. Preliminary results indicated robust killing of Acute Myeloid Leukemia (AML) cells by Natural Killer (NK) cells expressing either ILT2 or ILT4 CIRs. Here, we evaluated the effect of alteration of activating domains in the CIR constructs to produce signals that mimic the effect of IL-18 signaling in CIR-NK cells to enhance cytotoxicity, NK cell growth and persistence. Further, HLA-G expression and CIR-NK cell targeting of primary human cell types were rigorously screened. Activated NK cells derived from peripheral blood (2 to 5 donors) were transduced with γ-retroviruses directing expression of CIR proteins (CIR.X.Y or CIR.Y.X) where X stimulates cytotoxicity through CD3ζ, DAP10 or DAP12, and Y directs coactivation through 4-1BB, fusion of the MyD88 death domain or recruitment of endogenous MyD88 with receptor-derived TIR domains and soluble IL-15. CIR-NK cells were cocultured with GFPffluc-expressing AML and solid tumor target cell lines or normal primary human cells. NK cell proliferation and cytotoxicity of CIR-NK cells was monitored by Incucyte microscopy and supernatants were analyzed for cytokine release by ELISA. At low E:T (1:20 - 1:40) in 7-day cocultures, mock-transduced NK cells displayed innate killing activity against HLA-G+ Molm13 and Kasumi1 AML cells that was augmented by CIR constructs containing direct fusions of MyD88 and DAP10 or DAP12 (mock = 2.06E7 ± 4.53E6 vs. CIR-ILT4.MyD88.DAP10 = 1.03E7 ± 5.46E6) with 7-fold elevation of IFN-γ production. CIR constructs that recruited endogenous MyD88 signaling through the TLR2 TIR domain (4-1BB.DAP10.TLR2) also exhibited enhanced anti-HLA-G activity (9.13E6 ± 5.33E6). Against HLA-G+ HT-1376 bladder cancer cells (E:T = 1:10-1:20), CIR-NK cells also displayed enhanced tumor cell killing with enhanced NK cell growth. Conversely, CIR-enhanced killing was not observed against HLA-G− HCT-116 colorectal cancer cells. The persistence of CIR-NK cell potency after 4 weeks of expansion varied markedly with different activation domains employed. When screened against primary human cells (cardiomyocytes, hepatocytes, hepatic endothelial cells, corneal, colon and lung epithelial cells), no CIR-specific targeting was observed at high E:T ratios (1:1 and 1:5). In conclusion, CIR-NK cells exhibit potent and specific anti-tumor activity against HLA-G+ solid and leukemia cells. Alteration of CIR-NK cell intracellular signaling can optimize the potency and persistence of NK cell anti-tumor activity. Citation Format: MyLinh Duong, Jihyun Park, Raphel Ognar, Simon Wain-Hobson, Henri Bayle. Novel activation domains coupled to chimeric ILT receptors (CIR) enhance NK cell targeting of HLA-G+leukemic and solid tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1338.

Effects of mindful breathing training combined with diary-based rehabilitation guidance in lung cancer patients undergoing surgery: A randomized controlled trial

Background and purposeLung cancer surgery patients experience severe physical and mental symptoms, which seriously affect their quality of life and prognosis. Mindful breathing training is a promising strategy to improve their symptoms, but its effectiveness is affected by training compliance, and diary-based rehabilitation instruction has been shown to help improve training compliance. Therefore, the aim of this study was to evaluate the effects of mindful breathing training combined with diary-based rehabilitation guidance on improving perioperative outcomes in lung cancer surgery patients. Materials and methodsThis single-center, assessor-blinded, prospective, three-arm randomized controlled trial was conducted from November 1, 2021 to November 1, 2022. Patients diagnosed with primary non-small cell lung cancer and scheduled for thoracoscopic surgery were randomly allocated to the combined intervention group, the mindful breathing group or the control group, with 34 patients in each group. The control group received routine care, while the mindful breathing group received mindful breathing training and routine care. The combined intervention group received both mindful breathing training and diary-based rehabilitation guidance, along with routine care. ResultsThe per-protocol analysis revealed that patients in the mindful breathing group experienced statistically significant improvements in dyspnea, fatigue and anxiety. Patients in the combined intervention group had statistically significant improvements in dyspnea, fatigue, anxiety, depression, exercise self-efficacy and training compliance. ConclusionThis study provides evidence that mindful breathing training combined with diary-based rehabilitation guidance can be effective in improving perioperative outcomes in lung cancer patients. It can be applied in clinical practice in the future.

P53 Overexpression May Represent an Early Marker of Clinicopathologic Progression in Vasculogenic Mesenchymal Lesions of Germ Cell Tumor Origin.

Vasculogenic mesenchymal lesions (VMLs) of germ cell tumor origin are thought to originate in postpubertal-type yolk sac tumor componentsand include a spectrum of lesions from teratoma with vasculogenic stroma (TVS), to low and high-grade vasculogenic mesenchymal tumors (VMTs). VMLs exhibit rudimentary to well-developed neoplastic vessels within primitive mesenchyme, being considered a neoplastic reiteration of embryonic vasculogenesis in the splanchnic mesoderm of the yolk sac. They occur in patients with primary mediastinal germ cell tumors after chemotherapy, and a subset progresses to "somatic-type" sarcomas [including angiosarcoma (AS)], with high-grade VMTs likely portending a higher risk. Recently, we encountered a low-grade VMT that progressed to metastatic AS during follow-up. In this case, both the low-grade VMT and the subsequent AS demonstrated p53 overexpression, suggesting that p53 alterations may precede histopathologic transformation. To test this hypothesis, we evaluated neoplasms representing the entire spectrum of VMLs using p53 immunohistochemistry (IHC; clone DO-7, Dako). Overexpression was defined as nuclear positivity in > 80% of neoplastic cells. Because the distinction between high-grade VMT and AS can be subjective in some cases, they were grouped together in a single category. Thirty-nine VMLs were assessed: 16 high-grade VMT/AS, 19 low-grade VMT, and 4 TVS. Patient age ranged from 19 to 46 years (mean, 30 years; male = 97%). Four high-grade VMT/AS and one low-grade VMT showed p53 overexpression (5/39 VMLs, 13%; 4/16 high-grade VMT/AS, 25%). These tumors included 1 unequivocal AS and 1 high-grade VMT/AS with progression to rhabdomyosarcoma. The only low-grade VMT with p53 overexpression demonstrated progression to AS. Another high-grade VMT that progressed to sarcoma demonstrated p53 overexpression in the sarcoma component, but it was excluded because the VMT was not represented in the material availableat the time of the study. Lesions with intratumoral grade heterogeneity (classified based the highest grade), demonstrated more pronounced p53 overexpression in the high-grade components. P53 overexpression is associated with disease progression in a subset of VMTs and may precede morphologic transformation to sarcoma. Routine evaluation of VMTs with p53 IHC seems justified, with overexpressors likely requiring an close clinical surveillance.

Imaging plant cell walls using fluorescent stains: The beauty is in the details.

Plants continuously face various environmental stressors throughout their lifetime. To be able to grow and adapt in different environments, they developed specialized tissues that allowed them to maintain a protected yet interconnected body. These tissues undergo specific primary and secondary cell wall modifications that are essential to ensure normal plant growth, adaptation and successful land colonization. The composition of cell walls can vary among different plant species, organs and tissues. The ability to remodel their cell walls is fundamental for plants to be able to cope with multiple biotic and abiotic stressors. A better understanding of the changes taking place in plant cell walls may help identify and develop new strategies as well as tools to enhance plants' survival under environmental stresses or prevent pathogen attack. Since the invention of microscopy, numerous imaging techniques have been developed to determine the composition and dynamics of plant cell walls during normal growth and in response to environmental stimuli. In this review, we discuss the main advances in imaging plant cell walls, with a particular focus on fluorescent stains for different cell wall components and their compatibility with tissue clearing techniques. Lay Description: Plants are continuously subjected to various environmental stresses during their lifespan. They evolved specialized tissues that thrive in different environments, enabling them to maintain a protected yet interconnected body. Such tissues undergo distinct primary and secondary cell wall alterations essential to normal plant growth, their adaptability and successful land colonization. Cell wall composition may differ among various plant species, organs and even tissues. To deal with various biotic and abiotic stresses, plants must have the capacity to remodel their cell walls. Gaining insight into changes that take place in plant cell walls will help identify and create novel tools and strategies to improve plants' ability to withstand environmental challenges. Multiple imaging techniques have been developed since the introduction of microscopy to analyse the composition and dynamics of plant cell walls during growth and in response to environmental changes. Advancements in plant tissue cleaning procedures and their compatibility with cell wall stains have significantly enhanced our ability to perform high-resolution cell wall imaging. At the same time, several factors influence the effectiveness of cleaning and staining plant specimens, as well as the time necessary for the process, including the specimen's size, thickness, tissue complexity and the presence of autofluorescence. In this review, we will discuss the major advances in imaging plant cell walls, with a particular emphasis on fluorescent stains for diverse cell wall components and their compatibility with tissue clearing techniques. We hope that this review will assist readers in selecting the most appropriate stain or combination of stains to highlight specific cell wall components of interest.

Early Diagnosis of Rare Diaphragmatic Synovial Sarcoma in a Pediatric Patient With Epigastric Pain Using Point-of-Care Ultrasound.

Synovial cell sarcoma is a rare mesenchymal tumor that typically originates from the soft tissues of the extremities of young adults. Only 3 cases of primary diaphragmatic synovial cell sarcoma have been described in the literature: 2 in adult males and 1 in a 12-year-old pediatric patient.1-3 When this tumor is found in the mediastinum or pericardial region, prognosis is historically poor because of the advanced disease stage at time of diagnosis. The surgical course and pathology have been described in this 12-year-old boy.3 This is the first case, to our knowledge, of the use of cardiac point-of-care ultrasound in the early identification and diagnosis of a primary diaphragmatic synovial sarcoma in a pediatric patient.

Dynamic evolution of bone marrow adipocyte in B cell acute lymphoblastic leukemia: insights from diagnosis to post-chemotherapy

ABSTRACT Adipocyte is a unique and versatile component of bone marrow microenvironment (BMM). However, the dynamic evolution of Bone Marrow (BM) adipocytes from the diagnosis of B cell Acute Lymphoblastic Leukemia (B-ALL) to the post-treatment state, and how they affect the progression of leukemia, remains inadequately explicated. Primary patient-derived xenograft models (PDXs) and stromal cell co-culture system are employed in this study. We show that the dynamic evolution of BM adipocytes from initial diagnosis of B-ALL to the post-chemotherapy phase, transitioning from cellular depletion in the initial leukemia niche to a fully restored state upon remission. Increased BM adipocytes retards engraftment of B-ALL cells in PDX models and inhibits cells growth of B-ALL in vitro. Mechanistically, the proliferation arrest of B-ALL cells in the context of adipocytes-enrichment niche, might attribute to the presence of adiponectin secreted by adipocytes themselves and the absence of cytokines secreted by mesenchymal stem cell (MSCs). In summary, our findings offer a novel perspective for further in-depth understanding of the dynamic balance between BMM and B-ALL.

Opportunities and Perspectives for Three Dimensional Culture of Mesenchymal Stem Cell-Derived Exosomes.

Exosomes are nanosized extracellular vesicles (EVs) that participate in intercellular communication through surface proteins and the delivery of internal cargo. The exosomes have gained attention for their potential as disease biomarkers and therapeutic agents. The therapeutic ability of exosomes has been verified by copious previous studies. Effective methods for extensive clinical applications are being researched for exosome-based regenerative therapies, including the application of 3D cultures to enhance exosome production and secretion, which can resolve limited exosome secretion from the parent cells. Cell culture has emerged as a crucial approach for biomedical research because of its many benefits. Both well-established continuous cell lines and primary cell cultures continue to be invaluable for basic research and clinical application. Previous studies have shown that three-dimensional cultured exosomes (3D-Exo) improve therapeutic properties and yields compared with traditional culture systems. Since the majority of studies have focused on exosomes derived from mesenchymal stem cells (MSC-Exo), this review will also concentrate on MSC-Exo. In this review, we will summarize the advantages of 3D-Exo and introduce the 3D culture system and methods of exosome isolation, providing scientific strategies for the diagnosis, treatment, and prognosis of a wide variety of diseases.

Quality and applicability of cadaveric donor eyes for molecular biology research: An Indian experience.

Human ocular tissue banking plays an important part in the advancement of translational research for identifying the molecular processes involved in disease etiology and pathogenesis. Timely obtaining a good-quality ocular tissue from a cadaveric donor is exceedingly difficult, especially in remote areas, with a variable transportation time (within 12-24 h), raising concerns about RNA quality and its subsequent applications. Therefore, we assessed the utility of retinal tissues from cadaver donor and enucleated eyes based on the RNA quality and gene expression by real-time polymerase chain reaction (PCR). Prospective study. Retina tissues were separated from the donor/enucleated eyes received in the eye bank within 24 h of death (n = 15) and within an hour from OR (n = 3), respectively, and stored immediately at -80 degree. RNA was isolated using trizol, and the quantity and quality were assessed using Qubit and agarose gel electrophoresis, respectively. QPCR was performed for measuring the expression of different retinal-specific genes. The cellular viability of the retina was assessed by establishing explant primary cell cultures. The data were calculated as an average of normalised Ct values ± standard error of the mean. RNA obtained from cadaveric tissues despite being partially degraded showed a uniform strong gene expression of several retinal-specific genes such as PAX6, RHO, TUBB3, CRX, and ALDH1L1. The primary cultures established from cadaveric tissues showed viable cells. The cadaver donor tissues collected within 24 hours of death can be effectively utilized for gene expression profiling.

The Interactive Impacts of Corn Particle Size and Conditioning Temperature on Performance, Carcass Traits, and Intestinal Morphology of Broiler Chickens.

This study aimed to investigate the interactions between corn particle size (PS) and conditioning temperature (CT) on the performance, carcass traits, intestinal morphology, and immune responses in broilers fed a corn-soybean meal-based diet. A total of 360 one-day-old male broiler chicks (Ross 308) were randomly allocated into six dietary treatments in a 2 × 3 factorial arrangement, consisting of two corn PS (finely ground with geometric mean diameter (GMD) of 357 µm (PSF) vs. coarsely ground corn with GMD of 737 µm (PSC), and three CT [unconditioned (CTU), conditioned at 75 °C (CT75) and 90 °C (CT90)]. Birds were accommodated in 30 pens with five replicates and 12 chicks per each pen. There was no interaction between corn PS and CT on the growth performance and immune response of broilers at any growth phases. However, during the starter (0-10 days) period, the average daily weight gain (ADWG) and feed conversion ratio (FCR) of PSF-fed birds were significantly improved compared to those fed PSC (p < 0.05). During the starter (0-10 days) and grower (11-24 days) periods, increasing the conditioning temperature of corn increased the ADWG, while in the starter phase only the CT75 caused a lower FCR (p < 0.05). Broilers fed PSF corn showed the lowest FCR during the finisher (25-42 days) period compared to those fed PSC (p < 0.05). Conditioning corn at 75 °C reduced FCR during the finisher (25-42 days) period compared to the birds fed CTU and CT90 corn (p < 0.05). In whole experimental periods (1-42 days), PSF and CT75 treatment increased the ADWG compared to the PSC and CTU (p < 0.05). The CT75 treatment improved primary total anti-sheep red blood cell (SRBCs) titer (IgT) and IgM and secondary IgT and IgG responses compared to the other experimental groups (CTU and CT90) (p < 0.05). No significant PS × CT interaction was found on the Newcastle disease (ND) antibody titer of broiler chickens (p > 0.05). Feeding CT75 corn reduced duodenum and jejunum relative lengths compared to the birds fed diets containing CTU corn. Significant PS × CT interactions (p < 0.05) were observed for villus height, villus height to crypt depth, crypt depth, muscle thickness, and absorption surface area of the jejunum. The highest carcass yield was observed in the PSF-CT75 group (p < 0.05). In conclusion, the use of finely ground corn (PSF) conditioned at 75 °C (CT75) was beneficial to growth performance, development of the digestive tract, jejunum histomorphometry and the immune responses of broilers.

Small extracellular vesicle microRNAs in pediatric myasthenia gravis plasma and skeletal muscle.

The diagnosis of myasthenia gravis (MG) in children remains difficult. Circulating small extracellular vesicle (sEV)-derived miRNAs (sEV-miRNAs) have been recognized as biomarkers of various diseases and can be excreted by different cell types. These biomarker candidates also play a vital role in autoimmune diseases via intercellular communication. In the present study, we used sEV isolation and purification methods to extract the plasma-derived sEV-miRNAs from children with MG and healthy controls. A small RNA sequencing analysis confirmed the miRNA expression features in plasma-derived sEVs from MG patients. The miRNA expression analysis in vitro was determined using microarray analysis. The enrichment and network analyses of altered sEV-miRNAs were performed using miRNA databases and Database for Annotation, Visualization, and Integrated Discovery website. Quantitative real-time polymerase chain reaction was performed for validation of sEV-miRNA. The diagnostic power of altered sEV-miRNAs was evaluated using receiver operating characteristic curve analyses. Twenty-four sEV-miRNAs with altered expression level were identified between groups by DESeq2 method. The miRNAs were extracted from the sEVs, which were isolated from human primary skeletal muscle cell culture treated with mAb198. The target genes and enriched pathways of sEV-miRNAs partially overlapped between cell supernatant and plasma samples. The significantly downregulated miR-143-3p was validated in quantitative real-time polymerase chain reaction analysis. For the first time, we report that plasma-derived sEV-miRNAs may act as novel circulating biomarkers and therapeutic targets in pediatric MG.

Abstract P04: Role of glucocorticoid receptor in normal and malignant germinal center B cells

Abstract We have reported that super-enhancers (SEs) are specifically hypermutated by Activation-Induced Deaminase (AID)-induced Aberrant Somatic Hypermutation in &amp;gt;90% of diffuse large B cell lymphoma (DLBCL) cases (Bal et al. Nature 2022). Analysis of 122 primary DLBCL biopsies and cell lines identified more than 80 SEs that are recurrently hypermutated, with an average of 12 hypermutated SE/case. Hypermutated SEs are predominantly linked to genes encoding regulators of B cell development and well-known lymphoma oncogenes, including BCL6, BCL2 and CXCR4. We showed that specific hotspot mutations prevent binding and transcriptional downregulation by transcriptional repressors leading to target gene dysregulation. Among these, hypermutation of the SEs linked to BCL2 and CXCR4 abrogates the binding of the glucocorticoid receptor (GR)/transcription factor encoded by NR3C1. As a result, BCL2 and CXCR4 escape GR-mediated transcriptional repression leading to their de-regulated expression in germinal center (GC) B cells. Together with the observation that the NR3C1 gene is genetically inactivated in a small fraction of DLBCL, and the relevance of glucocorticoid-based therapy in DLBCL, these results prompted a comprehensive analysis of the role of GR in normal and malignant B cells. We report that GR is detectable in the nucleus of the great majority of GC B cells, suggesting that it is active as a transcription factor. Similarly, DLBCL primary cases (including GCB-, ABC- and unclassified DLBCL) display GR nuclear expression. Analysis of a conditional GC-specific Nr3c1 knock-out (KO) mouse model showed that GC B cells form normally in the absence of Nr3c1. However, the transcriptional profile of Nr3c1-KO GC B cells was significantly altered compared to wild-type cells. By integrating the GR binding profile obtained in normal human GC B cells with the transcripts differentially expressed in Nr3c1-KO GC B cells, we identified a set of about 2,000 genes that are directly modulated by GR during the GC reaction. Pathway enrichment analysis showed that GR controls several signaling pathways, suggesting a modulatory role on the BCR and CXCR4 pathways. In addition, GR targets display a significant overlap with the BCL6 transcriptional network, suggesting a cooperative role in GC B cell development. Notably, GR was shown to regulate pathways involved in plasma cell differentiation, while repressing targets associated with memory B cell development. In DLBCL cases, several NR3C1 binding sites are recurrently targeted by mutations, suggesting that genetic impairment of GR activity is heterogeneous and extends beyond CXCR4 and BCL2. Together, these data support a role for the glucocorticoid pathway in GC physiology. Recurrent inactivation of the NR3C1 gene or part of its transcriptional regulatory program may contribute to DLBCL pathogenesis, with implications for presently unknown specific roles of glucocorticoids in the therapeutic regimens for DLBCL. Citation Format: Clarissa Corinaldesi, Antony B Holmes, Elodie Bal, Laura Pasqualucci, Katia Basso, Riccardo Dalla-Favera. Role of glucocorticoid receptor in normal and malignant germinal center B cells [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P04.

Holistic exploration of CHGA and hsa-miR-137 in colorectal cancer via multi-omic data Integration

Colorectal cancer (CRC) ranks among the most widespread malignancies globally, with early detection significantly influencing prognosis. Employing a systems biology approach, we aimed to unravel the intricate mRNA-miRNA network linked to CRC pathogenesis, potentially yielding diagnostic biomarkers. Through an integrative analysis of microarray, Bulk RNA-seq, and single-cell RNA-seq data, we explored CRC-related transcriptomes comprehensively. Differential gene expression analysis uncovered crucial genes, while Weighted Gene Co-expression Network Analysis (WGCNA) identified key modules closely linked to CRC. Remarkably, CRC manifested its strongest correlation with the turquoise module, signifying its pivotal role. From the cohort of genes showing high Gene Significance (GS) and Module Membership (MM), and Differential Expression Genes (DEGs), we highlighted the downregulated Chromogranin A (CHGA) as a notable hub gene in CRC. This finding was corroborated by the Human Protein Atlas database, which illustrated decreased CHGA expression in CRC tissues. Additionally, CHGA displayed elevated expression in primary versus metastatic cell lines, as evidenced by the CCLE database. Subsequent RT-qPCR validation substantiated the marked downregulation of CHGA in CRC tissues, reinforcing the significance of our differential expression analysis. Analyzing the Space-Time Gut Cell Atlas dataset underscored specific CHGA expression in epithelial cell subclusters, a trend persisting across developmental stages. Furthermore, our scrutiny of colon and small intestine Enteroendocrine cells uncovered distinct CHGA expression patterns, accentuating its role in CRC pathogenesis. Utilizing the WGCNA algorithm and TargetScan database, we validated the downregulation of hsa-miR-137 in CRC, and integrated assessment highlighted its interplay with CHGA. Our findings advocate hsa-miR-137 and CHGA as promising CRC biomarkers, offering valuable insights into diagnosis and prognosis. Despite proteomic analysis yielding no direct correlation, our multifaceted approach contributes comprehensive understanding of CRC's intricate regulatory mechanisms. In conclusion, this study advances hsa-miR-137 and CHGA as promising CRC biomarkers through an integrated analysis of diverse datasets and network interactions.

MiR-210 promotes immune- and suppresses oocyte meiosis-related genes in the zebrafish ovarian cells

microRNA-210 (miRNA), a well-documented miRNA, has been implicated in a myriad of biological processes, including responses to hypoxia, angiogenesis, cell proliferation, and male infertility in humans. However, a comprehensive understanding of its functions in fish requires further investigation. This study pursued to elucidate the downstream effect of dre-miR-210-5p on primary ovarian cell culture in zebrafish (Danio rerio), an animal model. A protocol was settled down by incubations with either an miR-210 mimic or a scrambled miRNA in the isolated ovaries. RNA-sequencing analysis identified ∼6000 differentially expressed target genes revealing that downregulated genes were associated with reproduction-related pathways while immune-related pathways displayed an upregulated pattern. To identify molecular markers, predicted target genes were classified into reproduction and immune cell types. These findings underscore the existence of a profound interplay between the reproductive and immune systems, with miR-210 emerging as a pivotal player in orchestrating transcriptomic alterations within fish ovaries.

Treatment with Conditioned Medium from Tenocytes Primary Cell Cultures Accelerates Histological and Functional Recovery of Achilles Tendon in Tenotomized Mice

Treatment with Conditioned Medium from Tenocytes Primary Cell Cultures Accelerates Histological and Functional Recovery of Achilles Tendon in Tenotomized Mice

Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

Abstract A010: Mitochondrial depletion and metabolic reprogramming is a novel phenotype of Lynch syndrome-related endometrial carcinogenesis

Abstract Background: Lynch syndrome (LS), defined by mutations in DNA mismatch repair genes including MSH2, carries a 60% lifetime risk of developing endometrial cancer (EC). Our recent studies in Msh2-deficient mice revealed mitochondrial dysfunction in EC pathogenesis. We aimed to define mechanisms of mitochondrial dysfunction and impact on metabolism to identify targets for EC prevention. Methods: We assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2flox/flox, abbreviated Msh2KO), primary cell lines from this model, and human EC cells with isogenic MSH2-knockdown. mtDNA damage was measured using RT-PCR. Mitochondrial content was quantified in vitro by immunofluorescence (IF) for TOM20 and in vivo by anti-TOM20 immunohistochemistry (IHC). Mitochondrial stress tests (MSTs) assessed mitochondrial function in vitro. Metabolite profiling via high resolution ion chromatography mass spectrometry was conducted on Msh2KO EC and MSH2-intact EC from the Pten +/− mouse model. Glycolysis was suppressed in vitro by replacing glucose with galactose in culture media. Results: Baseline mtDNA damage was elevated in MSH2-deficient MFE280 and RL95-2 EC cell lines by 4.8- to 7.9-fold compared to MSH2-intact KLE and Hec50 cells (p&amp;lt;0.001). MSH2 knockdown increased mtDNA damage in KLE and Hec50 cells by 2.5- to 5.7-fold (p&amp;lt;0.05). MSH2-deficient mouse and human cell lines had lower mitochondrial content compared to MSH2-intact counterparts as indicated by TOM20 staining, with over 60% reductions in mitochondrial volume in MSH2-deficient mouse cell lines and over 95% reductions in MSH2-deficient human EC cells compared to respective controls (p&amp;lt;0.0001). IHC showed reduced mitochondrial content during EC development in Msh2KO mice and in human LS EC tissues. MSTs revealed decreased baseline and induced mitochondrial function in Msh2KO cells relative to controls and in MSH2-deficient human cells (MFE280, RL95-2, and Hec59) compared to MSH2-intact human cells (KLE, Hec1a, Hec50), with p&amp;lt;0.0001. Within the top 10 differentially modulated metabolites in Msh2KO EC compared to Pten +/− EC were glucose 6-phosphate (log2 fold-change (FC) -1.79), mannose 6-phosphate (log2FC -1.77), fructose 6-phosphate (log2FC -1.54), and lactate (log2FC 0.68), padj&amp;lt;0.05. Galactose reduced viability in MSH2-deficient mouse and human cells more than in their MSH2-intact counterparts (p&amp;lt;0.01). Conclusions: We identified mitochondrial depletion as a novel phenotype of MSH2-deficient EC. mtDNA damage in MSH2-deficient cells suggests a role for MSH2 in mtDNA integrity. MSH2-deficient EC is characterized by mitochondrial DNA damage, mitochondrial content reduction, and decreased mitochondrial function. Decreased mitochondrial respiration for energy production facilitates metabolic reprogramming toward glycolysis, signified by increased usage of glycolytic intermediates and dependence on glycolysis for survival. Mitochondrial and metabolic aberrations could be evaluated as novel biomarkers for EC development and targets for prevention in women with LS. Citation Format: Mikayla B. Bowen, Brenda Melendez, Anna Zal, Collene Jeter, Wai Kin Chan, Lin Tan, Philip Lorenzi, Diana Moreno, Leah Peralta, Qian Zhang, Nisha Gokul, Karen Lu, Hyun-Eui Kim, Melinda S. Yates. Mitochondrial depletion and metabolic reprogramming is a novel phenotype of Lynch syndrome-related endometrial carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A010.

The Relevance of Astrocytic Cell Culture Models for Neuroinflammation in Neurodegeneration Research

Astrocytes are the predominant glial cells that provide essential support to neurons and promote microenvironment changes in neuropathological states. Astrocyte and astrocytic-like cell culture have substantially contributed to elucidating the molecular pathways involved in key glial roles, including those relevant to neurodevelopment, brain physiology and metabolism, which are not readily accessible with traditional approaches. The in vitro methodology has also been applied to neuroinflammatory and neurodegeneration contexts, revealing cellular changes involved in brain dysfunction. Astrocytes studies in culture started with primary cell approaches using embryonic and postmortem tissue. Further developments included newborn rodent primary cells, cell lines and immortalized astrocytes, which resulted in homogeneous cell-type preparations grown on flat surfaces. To overcome some in vitro shortcomings, tridimensional bioprinted models and organoid culture enabled the mimicking of tissue cellular arrangements and, above these achievements, complex astrocyte cell culture can be generated from induced pluripotent stem cells (iPSCs) to model diseases. These unprecedented breakthroughs allowed the development of platforms to test new therapies in brain cells derived from human material noninvasively obtained from live patients. In this work, we reviewed the most studied astrocytic cell models for discussing limitations, advantages and reliable experimental readouts for neuroinflammation in neurodegeneration research.