Experimental Cell Research

Immune checkpoint blockade has revolutionized cancer therapy, but the therapeutic efficacy is limited. Clinical trials on blockade of newly identified immune checkpoints didn't show promising result, suggesting that it might be insufficient to understand the function of immune checkpoints in cancer merely in the context of immunity. Here, we found mutually exclusive expression patterns of the immune checkpoint VISTA (or VSIR) and the neural stemness factor SETDB1, an oncoprotein that promotes immunoevasion, in xenograft tumors, suggesting that cells with high VISTA expression represents a differentiated, and hence, less or non-malignant state in tumor. Non-neural differentiation factors HHEX, MYOD1 and PPARG promote, whereas oncoproteins KRAS (and the mutant KRAS(G12D)) and SOX2, both being embryonic neural factors, repress VISTA expression. This tendency can be inferred from the finding that neural stemness is the core property of cancer cell. Manipulated expression of VISTA in cancer cells generated no significant effect on cell tumorigenicity and differentiation state, but led to change in cell morphology and actin cytoskeleton. Mechanistically, VISTA regulates a key cytoskeleton regulator, WASF2, leading to the change in cell morphology, which might interfere with signal transduction of immune response. The results suggest that 1) high expression of a protein in tumor might represent a less or non-malignant state, targeting of which would leave malignant cells intact, and consequently, leading to weak or even no therapeutic efficacy, a key factor worth considering for target selection; 2) immune checkpoints might play other roles in cells that interfere with regulation of anti-tumor immunity.

Activated leukocyte cell adhesion molecule A (Alcama) plays a role in axonal guidance, cell differentiation, and retinal lamination in a developing retina and was identified as a marker for activated Muller glial cells in adult zebrafish. However, its spatiotemporal localization and its involvement in retina regeneration remains unclear. Here we induced focal photoreceptor damage in zebrafish using laser photocoagulation and examined the expression and localization of Alcama at different time points post lesion. Immunohistochemistry in wild type fish and Tg(kdrl-EGFP) fish showed Alcama localized to the blood retina barrier with increased expression in Muller glial end feet and radial processes in a regenerating retina. To confirm its role in retina regeneration, alcama expression was transiently knocked down using morpholinos in adult fish. Scanning laser ophthalmoscopy, Zpr1 immunostaining and EdU staining showed delayed retina regeneration in alcama knockdown fish, indicating a possible role for Alcama in zebrafish retina regeneration.

Human iPSC-derived brain organoids are revolutionizing tools to study layers biology, synergize disease modeling, and accelerate therapeutic discoveries that overcome obstacles in monolayer cell culture or animal models. The neurovascular unit including vasculature and microglia is critical for brain development, maintenance of synaptic plasticity and neural activity, and the high metabolic demands of long-term culture. We present a methodology to incorporate these important components during organoid generation and discuss potential approach, aiming consistent production of vascularized organoids for longitudinal study. We also demonstrate that this vascularized organoid is a versatile platform to model brain cancer and traumatic brain injury.

MLLT1 (also named ENL) is a chromatin reader protein whose encoding gene was originally identified as a chromosomal translocation partner with MLL(KMT2A) in acute leukemia. However, its role in normal hematopoiesis has not been investigated. This study uncovers a critical role of Mllt1 in normal B cell lymphopoiesis. We found Mllt1 to be essential for early B lymphocyte development using a conditional Mllt1 knockout mouse model that we developed. A significant decrease of bone marrow B-lineage progenitors, splenic transitional B cells and peripheral blood B cells were observed in Mllt1del mice compared to control Mllt1fl/fl mice. Similarly, Mllt1 deletion in in vitro cultured B-enriched progenitor cells from Mllt1fl/fl; Rosa26CreERT2/+ mice resulted in reduced B cells, demonstrating the cell-intrinsic role of Mllt1 in this process. Direct MLLT1 target genes including Il7r and critical B-lineage transcription factors, Ebf1 and Pax5, were decreased following Mllt1 deletion. Gene set enrichment, gene ontology, and functional analyses of Mllt1-deficient cells showed significant alterations related to B cell development, critical relevant signaling pathways, DNA replication, and mitochondrial function. In vitro complementation with MLLT1 rescued the B cell phenotype observed with endogenous Mllt1 deletion; however, specific MLLT1 YEATS domain mutants lacking chromatin reader and RNA-binding functions were unable to rescue the phenotype. Taken together, our research demonstrates a previously unappreciated role for MLLT1 as critical for maintenance of B cell lymphopoiesis.

Mitochondrial plasticity, characterized by the dynamic balance between glycolysis and oxidative phos-phorylation in response to genetic and microenvironmental changes, is a hallmark of melanoma progression. Sphingolipids play a significant role in various aspects of cancer cell biology, including metabolic reprogramming. Previous observations had shown that the lysosomal sphingolipid-metabolizing enzyme {beta}-galactosylceramidase (GALC) rewires the lipid profile of mouse melanoma cells, exerting pro-oncogenic functions, gene silencing leading to a decreased oncogenic activity in murine and human melanoma cells. Here, we have focused on the mitochondrial sphingolipid composition and energetic metabolism in GALC knockout (KO) A2058 human melanoma cells. Targeted analysis of the mitochondrial sphingolipid profile, transcriptomic data, and mitochondrial structural and functional studies indicate that GALC loss drives a sphingolipid-mediated reprogramming of mitochondrial metabolism in absence of major structural alterations, characterized by bioenergetic insufficiency possibly due to ceramide- and sphingomyelin-driven impairment of respiratory chain function. Overall, these data indicate that GALC KO leads to a sphin-golipid-driven mitochondrial metabolic suppression and may provide novel information for the development of efficacious approaches in mitochondrial targeting melanoma therapies.

We report the generation and characterization of a human induced pluripotent stem cell (iPSC) line derived from dermal fibroblasts of a patient with Skogholt disease, a rare maternally inherited neurodegenerative syndrome associated with choroid plexus dysfunction and impaired cerebrospinal fluid (CSF) homeostasis. Patient fibroblasts were reprogrammed using the non-integrating Repro-OSKGM kit. The resulting iPSC line exhibited typical pluripotent morphology, expressed canonical pluripotency markers, maintained a normal karyotype, retained the disease-associated genetic variant, was mycoplasma-free, and demonstrated trilineage differentiation potential. We also made choroid plexus (ChP) like organoids from the generated iPSCs. This patient-specific iPSC line provides a valuable resource for generating choroid plexus organoids and neurons to investigate disease mechanisms and develop therapeutic strategies.

Regucalcin (RGN) plays diverse roles in cell biology, highlighting its importance in both physiological and pathological conditions. Prostate cancer patients with higher RGN expression exhibited significantly longer disease-free survival. Although RGN is a cell signaling suppressor, the molecular mechanisms underlying tumor suppression by RGN in the tumor microenvironment through cell-cell communication remain unclear. PC3 prostate cancer cell lines stably expressing RGN or a control vector were generated for this study. Extracellular vesicles (EVs) were isolated from these cell lines using differential ultracentrifugation. The murine macrophage cell line J7441 was treated with isolated EVs, and effects on M2 polarization were evaluated using qRT-PCR and western blot analysis. To assess the potential anti-tumor effects of EVs, PC3 parental cells were subcutaneously implanted at two sites per mouse, followed by intratumoral injection of the respective EVs. Tumor volume was monitored. Harvested fresh frozen tumor tissues underwent immunofluorescence staining for CD206, an M2 macrophage marker. RGN was detected in EVs from RGN-expressing cells, and treatment with these RGN-containing EVs was associated with reduced tumor growth and reduced M2 macrophage polarization in vitro and in vivo. Furthermore, recombinant RGN protein reduced the levels of p-AKT1 and p-ERK1/2. Moreover, the suppression of M2 macrophage polarization by RGN-containing EVs was accompanied by decreased p-AKT1 and p-ERK1/2 in vitro. This study describes an EV-associated mechanism that may contribute to the regulation of macrophage polarization and indicates that RGN-containing EVs merit further evaluation as a candidate approach for cancer treatment. Causal validation, such as macrophage depletion or CD206 knockdown, and evaluation in additional models remain to be addressed in future studies.

Transfection of cells with DNA plasmids typically involves the uptake of lipoparticles by endocytosis, followed by the inefficient escape of these particles from endosomes into the cytoplasm. We found that the expression of transfected plasmids was reduced in cells depleted of either SEPTIN 9 or proteins in the endosomal sorting complexes required for transport (ESCRT) pathway. The reduction in plasmid expression could not be fully explained by effects on endocytosis. SEPTIN 9 depletion appeared to reduce the acidification of plasmid-containing compartments, suggesting that it primarily affects the pH-sensitive escape of plasmids from endosomes. Depletion of the ESCRT proteins VPS36 or ALIX resulted in especially dramatic reductions in transfected plasmid expression, which were accompanied by reduced colocalization between the transfected DNA and CHMP4, an ESCRT protein important for endosomal membrane remodeling during intraluminal vesicle formation. Finally, transfected plasmid DNA was strongly colocalized with LC3B, suggesting that the default pathway for transfected material is autophagy.

Colorectal cancer cells increase glycolysis to help meet the metabolic demands required for cell growth. Many factors, both endogenous and exogenous, likely drive cellular metabolism and enhance glycolytic flux in colorectal cells. Interleukin-1 beta (IL-1{beta}) is a pro-inflammatory cytokine that is elevated in colorectal cancer. In this study, we investigated the effect of IL-1{beta} toward driving the cancer cell to increase glycolysis, while also suppressing the oxidation of the fiber-derived nutrient butyrate. The results presented here demonstrate that IL-1{beta} stimulated glycolysis and inhibited maximal mitochondrial respiration. IL-1{beta} also increased the phosphorylation of AKT and hypoxia-inducible factor 1 alpha (HIF1) levels. Utilizing colorectal cancer cells with AKT1/2 or HIF1 knocked out showed the requirement of these proteins in mediating the increase in glycolysis following IL-1{beta} treatment. Importantly, AKT1/2 was identified as upstream of HIF1, as IL-1{beta} still increased phosphorylation of AKT even in the absence of HIF1. However, loss of AKT1/2 completely abolished the ability of IL-1{beta} to increase HIF1 protein levels. Tumor necrosis factor alpha (TNF), another cytokine found to be elevated in colorectal cancer, also increased glycolysis in an AKT and HIF1-dependent manner. Our data point to a common pathway through AKT activation and HIF1 upregulation, by which pro-inflammatory cytokines increase glycolysis in colorectal cancer cells to help promote cancer progression.

Abstract Background: The stem cell factor receptor or c-Kit is a type III receptor tyrosine kinase, activated by its ligand Stem cell factor (SCF). Up on activation, c-kit induces signaling pathways that regulates blood cell proliferation, survival, differentiation, and migration. Several studies reported that c-Kit/SCF signaling, contributes to the development and progression of acute myeloid leukemia (AML) in patients. However, the downstream proteins regulated by c-kit activation and their clinical significance in AML remain poorly explored. Methods: Human Acute megakaryoblastic leukemia (Mo7e) cells, were-stimulated with SCF and global protein expression were profiled using two-dimensional gel electrophoresis coupled with MALDI-TOF and LC-MS/MS. Differentially expressed proteins were functionally characterized and validated using patient data from the TCGA-LAML and matched normal data from GTEx, GEO datasets, and quantitative RT-PCR. Their diagnostic and prognostic significance was assessed using ROC, Cox regression, LASSO, Kaplan Meier survival analyses, and a prognostic nomogram model. Results: Proteomic profiling identified 14 differentially expressed proteins in SCF-stimulated Mo7e cells, which are predicted to involved in cytoskeletal organization, protein folding, metabolism, vesicular trafficking, and translational regulation. Transcriptomic analysis of the TCGA-LAML cohort revealed significant dysregulation of CFL1, CCT8, HSP90B1, MDH2, EIF5A, GSN, and TPI1. Integrated ROC, Cox regression, and LASSO analyses identified CFL1, CCT8, and GSN as the most robust prognostic biomarkers associated with poor overall survival in LAML patients. Their expression patterns were validated in independent GEO datasets and by qRT-PCR in SCF stimulated Mo7e cells. Finally, a three-gene nomogram model was developed and validated to predict the overall survival probability of AML patients at 1-, 3-, and 5-year time points. Conclusions: This study identifies CFL1, CCT8, and GSN as key downstream effectors of c-Kit signaling as prognostic biomarkers for AML. These findings provide mechanistic insights into c-Kit-driven leukemogenesis and establish a clinically relevant three-gene signature for AML risk stratification and potential therapeutic targeting.

This study proposes a novel therapeutic strategy to suppress cancer growth by modulating the MDM2-p53 axis via Alternative Polyadenylation (APA). MDM2 normally promotes tumorigenesis by ubiquitinating and degrading the tumor suppressor p53. In cancer cells, preferential use of proximal polyadenylation signals (PAS) results in shortened 3'UTRs, allowing oncogenic transcripts like MDM2 to evade nuclear sequestration mediated by Inverted Alu (IRAlu) double-stranded RNA structures. We hypothesized that forcing distal PAS usage would elongate the MDM2 mRNA, promoting its nuclear retention and reducing protein translation, thereby restoring p53 activity. Using CRISPR-Cas9, we targeted and deleted the most frequent proximal PAS in the MDM2 3'UTR of A549 cells. Successful genome editing was confirmed via PCR. As expected, Western blot analysis showed a significant reduction in MDM2 expression in PAS-edited cells. However, experimental outcomes contradicted our initial hypothesis: edited cells exhibited higher viability under doxorubicin treatment compared to wild-type cells. Furthermore, despite decreased MDM2 levels, a concurrent reduction in phosphorylated p53 (p-p53) was observed. These unexpected results suggest that MDM2 3'UTR elongation may trigger a non-canonical regulatory mechanism that bypasses the traditional MDM2-p53 interaction. This study highlights the complexity of post-transcriptional regulation and suggests that APA-mediated gene modulation can induce unforeseen compensatory survival pathways in cancer cells, necessitating further investigation into the broader functional landscape of elongated 3'UTRs.

Mitofusin 2 (MFN2), a key outer mitochondrial membrane GTPase, regulates mitochondrial fusion, mitophagy, calcium homeostasis, and cellular bioenergetics. This study investigated the role of MFN2 variants in patients with Dilated Cardiomyopathy (DCM) using whole-exome sequencing (WES) of 5 familial and 10 sporadic DCM cases. A rare de-novo MFN2 variant, c.932A>G (p. N311S), was identified in a DCM patient, which is absent in 100 healthy controls as well as in the 1000 Genomes, IndiGenomes, databases while it shows very low MAF (0.0000081) in gnomAD. Structural modelling predicted the variant to be highly deleterious and revealed marked conformational distortion of the mutant protein (RMSD = 8.95 A). Molecular docking further showed a weakened interaction between MFN2-N311S and PRKN (Parkin), indicating impaired mitophagy and defective mitochondrial quality control. Moreover, functional analysis in stable H9c2 cardiomyoblast cell lines demonstrated significantly reduced MFN2 mutant protein expression, extensive mitochondrial clustering and fragmentation. The mutant protein also indicated significant reduction in mitochondrial membrane potential, ATP production, and oxygen consumption rate (OCR), together with elevated cytosolic Ca2+ and reactive oxygen species (ROS) levels. qRT-PCR analysis further revealed activation of the PI3K/AKT/mTOR signalling pathway and increased expression of hypertrophic markers Myh6, Nppa, Nfatc1, and Nfatc2. The above findings collectively highlight the significant impact of the MFN2 mutation on mitochondrial dynamics and cellular health, suggesting a significant correlation with the pathogenesis of DCM. This finding could further open a door to develop a potential therapeutic target for DCM.

Background/Objectives: Cancer survivors frequently experience long-term neurocognitive impairments following chemotherapy and cranial irradiation, yet experimental models that enable mechanistic investigation of therapy-induced neurotoxicity at the transcriptional level remain limited. This study aimed to develop a human three-dimensional (3D) neural tissue model derived from L-Myc immortalized neural stem cells (LMNSCs) and use transcriptomic profiling to identify molecular pathways underlying chemotherapy- and radiation-induced neural injury and extracellular vesicle (EV)-mediated recovery. Methods: LMNSCs were differentiated in a 3D, methylcellulose-based culture to generate neural tissue containing neurons, astrocytes, and oligodendrocytes. Cultures were exposed to methotrexate (MTX) or ionizing radiation to induce neural injury and subsequently treated with LMNSC-derived EVs. Neural injury and repair mechanisms were evaluated by immunocytochemistry and bulk transcriptomics. Results: MTX and irradiation induced dose-dependent injury, exhibited by loss of neuronal complexity and reduced glial populations. LMNSC-EV treatment promoted recovery of neuronal and glial populations following MTX- and irradiation-induced injury. Transcriptomic analysis of irradiated cultures revealed activation of inflammation, DNA damage, and stress-response pathways, which were attenuated after treatment with LMNSC-EVs. Conclusions: LMNSC-based 3D neural tissue provides a human-relevant platform for modeling cancer therapy-induced neurotoxicity. Furthermore, LMNSC-EVs represent a promising cell-free regenerative therapeutic that restores injury-associated inflammatory, stress, and metabol-ic transcriptional programs after radiation-induced neural injury.

Ependymal (E1) cells, with their tufts of [~]50 motile cilia, line the walls of the brain ventricles and help propel the cerebrospinal fluid (CSF). The CSF is rich in signaling molecules, but the cellular targets that detect these signals and their function remain unknown. Here, we describe a distinct population of ependymal cells (E2) in the forebrain of mice and humans, the majority having only 1 or 2 cilia. These cilia were motile, but unlike E1 cells cilia, their pattern of motility and high expression of Arl13b and Inpp5e suggest a sensory function. E2 cells were characterized by an enormous, donut-like basal body that contained an increased number and size of subdistal appendages. In mice, E2 cells were mostly born in the embryo, but completed their differentiation in juveniles and young adults; they were found at higher densities in regions of high CSF flow and neurogenesis. E2 cilia contained the G protein-coupled receptor Smoothened, which accumulated in their cilia upon exposure to Sonic Hedgehog (Shh). Together, these findings identify E2 cells as a novel CSF-sensing ependymal cell type and provide a cellular target for the CSF signaling.

Chondrosarcoma is a hyper-vascularised, chemoresistant cartilage malignancy driven by VEGF-centred angiogenesis, and local adipose depots are increasingly recognised as paracrine drivers of tumour angiogenesis via adipokines and extracellular vesicles (EVs). The infrapatellar fat pad (IFP), an inflammatory adipose depot within the articular joint in direct cartilage contact, is a key local source of adipose-derived EVs, and thus a candidate driver of angiogenesis in chondrosarcoma. The aim of this study was to determine whether the IFP is a productive source of EVs, and whether IFP-derived EVs induce angiogenesis in articular chondrocytes. The IFP released significantly more EVs than subcutaneous fat (n = 8 per depot; p = 0.027). Treating primary human articular chondrocytes with IFP EVs for 24 h upregulated VEGFA (+1.6-fold, p = 0.036) and downregulated BMP4 (-2.4-fold, p = 0.011), engaging the VEGF/eNOS/ERK axis that drives chondrosarcoma angiogenesis. Re-analysis of a previously published phospho-kinase dataset from the same donor EVs, corroborated by a pooled donor-group analysis (n = 3), supported activation of eNOS, ERK1/2, PLC-{gamma}1 and HSP27. These findings identify the IFP as a dominant source of EVs within the articular joint, which can induce a pro-angiogenic, VEGF-axis switch in articular cartilage cells, supporting a signalling model relevant to chondrosarcoma angiogenesis.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

At the onset of intervertebral disc degeneration, within the disc core, the pH and osmolarity decrease, and the residing notochordal cells (NCs) gradually transition towards nucleus pulposus cells (NPCs). How these microenvironmental cues shape the NCs extracellular vesicles (EV)-enriched secretome, and thus EV-mediated communication with NPCs during this transition, remains poorly understood. To study this, we collected the secretome from pig NC-rich tissue cultured for 4 days in either healthy or degenerate disc media to mimic these changes. In both conditions, NC-rich tissues were largely comparable at the histological and biochemical levels. Despite, tissues released glycosaminoglycans (GAGs), depleting the extracellular matrix. Surprisingly, degenerative media did not differentially release inflammatory regulators, though it reduced PGE2 release. We asked whether this extended to EV-enriched secretome media (SM_EV+), and found that the degenerative media reduced the number of EVs without altering their morphology or size. We then determined NC-EV association of inflammatory and matrix regulators. NC-EV isolation enriched MMP1, IL6 and IL10 and depleted soluble GAGs. Conversely, EV-depletion (SM_EV-) removed most GAGs without affecting MMP1, IL6, and IL10, suggesting that they contribute to the NC-EV soft corona. Functionally, healthy SM_EV+ improved GAG production by NPCs, but attenuated TBXT expression. Degenerate SM_EV+ did not elicit detectable EV-specific effects. These findings suggest that, in health, secretome-mediated communication from NCs to NPCs is only partially EV-mediated. At the onset of IVD degeneration, low pH and osmolarity impair the release of NC-EVs and negate the EV-specific beneficial matrix-anabolic effects on NPCs, contributing to the NC-to-NPC transition.

A pair of paralogs, Chchd3 and Chchd6, two components of mitochondrial contact site and cristae organizing system (MICOS), have been identified to be candidate pathogenetic genes in congenital heart disease (CHD). Previous research found that knockdown (KD) of the single Chchd3/6 (Chchd3) gene and other MICOS components in Drosophila impaired heart function, likely due to a deficit in mitochondrial organization, ATP production, actomyosin levels, and thus severely diminished contractility. However, the underlying mechanisms of how MICOS deficiency leads to these defects are not clear. Here, we performed genetic manipulations in the Drosophila heart to probe for possible interactions between MICOS-compromised mitochondria and other organelles and processes. We found that moderate reduction in Pink1/parkin-mediated mitophagy synergistically aggravated cardiac Chchd3 KD phenotypes, indicating a major interaction. Further, Chchd3 KD increased the level of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Interestingly, KD of catalase (CAT) also elevated cardiac ROS levels, but surprisingly did not compromise contractility either by itself or in combination with Chchd3 KD to aggravate the cardiac phenotype. However, CAT overexpression (OE) in Chchd3 KD hearts restored contractility, but only partially, even though elevated ROS due to Chchd3 KD was fully normalized. Similarly, counteracting ER stress by overexpressing Xbp1 (or spliced mouse Xbp1) also partially rescued the heart function defects induced by Chchd3 KD. Overall, these data indicate a critical role of mitophagy and ER/oxidative stress in cardiac homeostasis involving Chchd3, which suggests that deficiency of MICOS function contributes to heart dysfunction via multiple stress responsive pathways.

Adipose-derived mesenchymal stem cells (gAD-MSCs) are promising candidates for veterinary regenerative medicine, yet the characterization of gAD-MSCs from locally adapted Indonesian goat breeds remains limited. This study aimed to isolate and characterize gAD-MSCs from Peranakan Ettawa (PE) goats using tissue explant culture. Subcutaneous adipose tissue was collected from the base of the tail of healthy PE goats (n=3). Primary cell outgrowth from explants was observed by Day 5, displaying characteristic fibroblast-like, spindle-shaped morphology and strong plastic adherence. Serial passaging to Passage 3 (P3) yielded a morphologically stable, homogeneous cell population. Assessment of cellular metabolic activity via the resazurin assay demonstrated sustained cell viability and a statistically significant increase in metabolic activity between Day 3 and Day 5 (p < 0.05). Furthermore, functional clonogenic capacity, evaluated using the colony-forming unit (CFU) assay, showed continuous temporal expansion of colonies over 14 days, yielding an average of 52.0 + - 4.1 colonies per dish. These findings confirm that expanded gAD-MSCs P3from PE goats maintain characteristic mesenchymal morphology, sustained metabolic activity, and clonogenic capacity. This work provides a baseline cellular profile of PE goat gAD-MSCs, supporting their potential use in veterinary regenerative medicine and tissue engineering.

Introduction. Hypertrophic cardiomyopathy (HCM) is characterized by clinical and genetic heterogeneity. Age of manifestation, clinical and anatomical phenotypes of HCM vary significantly. This study discusses genetic causes and reconstructive surgery results for patients with particular intracardiac phenotype - diffused generalized HCM (DG-HCM). Methods: personal and familial medical history, general examination, 12-lead resting ECG, 24-hour ECG Holter monitoring, transthoracic and transesophageal EchoCG, cardiac MRI with gadolinium enhancement. A ten-gene panel was sequenced by IonTorrent PGM. Mutational screening in patients with suspected multisystemic diseases was performed by Sanger sequencing. Results: 170 patients with obstructive HCM (oHCM) requesting genetic counseling and surgical correction of HCM were evaluated. We distinguished particular DG-HCM subtype of oHCM (diffuse hypertrophy of IVS, LV free walls, papillary muscles displaced towards the LV apex) in 34 patients; 31 out of 34 underwent open heart reconstructive surgery. Patients with DG-HCM were younger at the time of surgery, had higher risk of SCD, and connective tissue dysplasia of the mitral valve. Hemodynamics normalization was observed in 1, 3, and 5 years after surgery. Eighteen ICDs were implanted; five patients experienced appropriate shocks. The genetic spectrum was enriched up to 30% by multisystem disorders. Mutations in "sarcomeric" genes were detected in 15%. Conclusion: Intracardiac phenotype of HCM may correlate with genetic cause and long-term prognosis. DG-HCM phenotype accounts for 20% oHCM patients and indications for open-heart surgery. Extended myectomy with parietal resection of papillary muscles and correction of mitral valve insufficiency provides long-term benefits for DG-HCM patients. Multisystem disorders in patients with DG-HCM should be of special attention. Study was supported by research project FURG-2024-0004.