Cells

Background. Cancer cachexia is a multifactorial syndrome characterized by substantial metabolic disturbances including systemic inflammation, and muscle atrophy, which may affect the regulation of iron metabolism in patients. This study aimed to characterize this regulation in two murine models of colorectal and pancreatic cancer, both associated with a high prevalence of cachexia. Methods. Histological, biochemical and molecular analyses were performed in serum, skeletal muscle, liver, spleen and tumor from UN-KC-6141 pancreatic tumor bearing mice and from ApcMin/+ mice (colorectal cancer model), both models exhibiting variable cachexia severity. Results. Both models induce cachexia, but diverge markedly in their regulation of iron metabolism. UNKC mice display reduced plasma iron availability (-9.6% transferrin saturation, p<0.001) without concomitant anemia, while hepatic hepcidin synthesis is upregulated (+100.6%, p<0.001) by inflammation, promoting ferroportin (FPN) downregulation (-42.4%, p<0.001) and iron sequestration in the spleen (+134% total iron content, p<0.001). In the atrophied gastrocnemius, iron concentration is preserved through increased ferritin (+86.6%, p=0.010) and hemoproteins (i.e., myoglobin, CYSC, COXIV) expression. Conversely, ApcMin/+ mice exhibit anemia together with hepatic and splenic iron depletion (-48.4%, p<0.001 and -84.9%, p<0.001, respectively). Hepcidin synthesis is downregulated (-99.9%, p<0.001) despite persistent inflammation, indicating that hypoxia and/or iron-deficiency signals probably predominate over the inflammatory pathway. FPN is markedly decreased (-94.2%, p<0.001) in the iron-depleted spleen despite low hepcidin. In the atrophied quadriceps of ApcMin/+ mice, iron deficiency is associated with decreased FPN and ferritin (-87.9%, p<0.001, and -38.4%, p=0.016, respectively), and increased TfR1 expression (+305%, p=0.107). Finally, tumor iron import may play a significant role in cachexia progression, as suggested by the association between tumor weight, tumor TfR1 protein levels and body weight loss in UNKC mice. Conclusion. These findings reveal cancer type-specific signatures of iron regulation in cachexia, which appear to be more driven by cancer type specific features than by cachexia severity itself, with skeletal muscle iron metabolism impaired mainly when cachexia is associated with anemia. This suggests that the clinical management of iron metabolism in cachectic patients should be guided by the underlying cancer type.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

Abstract Background Migrasomes, the newly discovered vesicular organelles, have long been speculated to originate from membrane dynamics during cell migration. Here we present integrated imaging and multi-omics evidence supporting a ciliary origin of migrasomes. The shared vesicular trafficking pathways between ciliogenesis and migrasome biogenesis (trans-Golgi/endosomal systems) prompted us to investigate their spatiotemporal coupling mechanism in disease models. Methods Using high-resolution SEM and fluorescence confocal microscopy (FCM), we visualized cilium-like structures with enlarged tips adjacent to migrasome-like vesicles across multiple cell types. TMT-based quantitative proteomics was used to identify proteins shared among migrasomes from different cell types and examine their associations with ciliary proteins. DMBA-induced breast cancer and femoral fracture models were used to investigate the cilia-migrasome association during tumor metastasis and fracture healing. Results Bioinformatics analysis showed a highly positive incidence correlation between cilia and migrasome genes in 31 types of 7858 human normal tissues and organs and various cell types. In 32 cancer tissues, cilia and migrasome genes were still strongly but abnormally correlated. Signaling enrichment by ARL13B and TSPAN4 showed that cilia-migrasome association is related to epithelial-mesenchymal transition (EMT). By analyzing the ARL13B and PIGK expression, we found the abnormal cilia-migrasome association in metastatic breast cancer and positive cilia-migrasome association in fracture healing bone. Conclusion Our findings support a ciliary origin of migrasomes, pointing to a previously unrecognized pathway of organelle biogenesis. Notably, the ciliary-migrasomal association correlates with two distinct pathological/physiological contexts: (1) aberrant epithelial-mesenchymal transition (EMT) in metastatic breast cancer, and (2) accelerated regeneration during fracture healing and tissue repair. This dual correlation not only establishes a mechanistic link between ciliary function and migrasome biogenesis but also highlights their divergent roles in disease progression versus tissue homeostasis. Investigating the spatiotemporal dynamics of this association may provide novel therapeutic targets for metastatic malignancies and regenerative medicine.

Background: Epidemiological studies have highlighted an inverse association between cancer and Alzheimers disease (AD), but the mechanisms underlying this association are not currently understood. Amyloid precursor protein (APP) and its cleavage may represent one potential mechanism. Its importance has been heavily researched due to it giving rise to the production of amyloid-beta, a pathological hallmark of AD. APP, however, has not been extensively investigated in cancer. Methods: FFPE prostate tumour and adjacent benign tissue immunohistochemically stained for APP, androgen receptor and the insulin-like growth factor-I receptor (IGF1R). Normal prostate PNT2, androgen sensitive LNCaP and androgen insensitive PC3 in vitro were used to assess the effects of androgen, IGF-I and their inhibitors on APP and its cleavage; changes in protein abundance were measured by western blot; interactions between proteins using co-immunoprecipitation and effects on cell proliferation, apoptosis and cell signalling of APP silenced cells using a BrdU proliferation assay, Muse Cell Analyser and by TMT proteomic analysis respectively. Results: Data showed that APP was upregulated in prostate tumour tissue compared to adjacent benign. Furthermore, in cancer cell models, in contrast to normal prostate epithelial cells, it was observed that APP was critical for prostate cancer cell survival by interacting with androgen and IGF-I signalling pathways. Conclusions: Drugs that target APP have already been developed for AD and our data suggests they offer an exciting opportunity for repurposing in the treatment of prostate cancer.

Background: Low-density lipoprotein cholesterol (LDL-C) is an established risk factor for atherosclerotic cerebrovascular disease, but its effects on the cerebral microcirculation are poorly defined. We investigated whether LDL-C is associated with pericyte dysfunction in acute ischemic stroke (AIS) and explored underlying mechanisms. Methods: Serum soluble platelet-derived growth factor receptor-{beta} (sPDGFR{beta}) was measured in two independent AIS cohorts (n=97 and n=86). Cerebral pericyte transcriptional responses to LDL-C were analyzed by single-cell RNA sequencing in Apoe-knockout mice fed control or high-fat diet (n=4 [male/female: 2/2]/group), and cultured human brain pericytes were exposed to purified human LDL cholesterol with or without N-acetyl-L-cysteine. Results: Higher serum sPDGFR{beta} was associated with greater neurological deficit and circulating neurofilament light chain. LDL-C positively correlated with sPDGFR{beta} in both clinical cohorts. High-fat diet induced coordinated suppression of type-I interferon pathways, and a positive shift in reactive oxygen species-related transcriptional programs in mouse pericytes; exploratory sex-by-diet analyses suggested larger transcriptional responses in females. In cultured human pericytes, LDL-C induced oxidative-stress, lipid-handling, and ferroptosis-associated transcriptional programs while suppressing type-I interferon signaling. Cross-species enrichment analysis showed that LDL-C-induced and -repressed signatures in cultured pericytes aligned with the sex-by-diet interaction in Apoe-knockout mice. LDL-C also reduced cell-surface PDGFR{beta} and increased sPDGFR{beta} release in cultured pericytes, which were significantly attenuated by N-acetyl-L-cysteine, whereas LDH activity increased numerically but not significantly. Conclusions: Clinical and experimental findings support an LDL-pericyte axis linking atherogenic lipid stress to cerebral microvascular dysfunction, with possible sex-dependent modulation of oxidative stress and type-I interferon signaling as potential mechanisms.

BackgroundPremature ovarian insufficiency (POI) is a condition affecting female fertility e.g., in the context of autoimmune disease. A recent proof-of-concept study showed that B-cell depletion with rituximab (RTX) temporarily restored ovarian function in patients with autoimmune POI. However, the underlying immunological mechanisms behind this response is unknown. ObjectivesTo characterize B- and T-cell phenotypes before and after RTX therapy in the above-mentioned patients and associations with autoantibody levels and treatment response. MethodsCryopreserved cell samples were available from six autoimmune patients with POI, four of whom were positive for autoantibodies against steroidogenic enzymes (21OH, 17OH and/or SCC). Patients received RTX and blood samples were collected at baseline, second infusion (2 weeks), 3, 8 and 12 months. Peripheral B- and T-cell phenotypes were analysed using spectral flow cytometry and correlated with autoantibody levels. ResultsAt baseline, patients displayed signs of B-cell activation by alteration in subset composition compared to matched controls. Upon closer analysis, patients positive for steroidogenic enzyme autoantibodies had significantly increased frequency of CXCR3+switched memory (SWM) B-cells than autoantibody-negative patients, which correlated positively with anti-17OH and-SCC autoantibody levels. In parallel, increased Th1 cell frequencies were also observed in the same patient group. Longitudinal analyses showed expected B-cell depletion followed by repopulation after RTX. Changes in T-cell subsets were observed at 8 months with reduced frequencies of Th1 and Tfh cells. ConclusionAutoimmune POI displayed altered B- and T-cell subsets implicating cell crosstalk, immune cell infiltration to sites of inflammation such as ovaries, and presence of Th1-driven immunopathogenesis. Key messagesO_LIIncreased CXCR3+SWM B cells and Th1 cells were found in patients with autoimmune POI who were positive for autoantibodies against steroidogenic enzymes. C_LIO_LICXCR3 expression on SWM B cells positively associated with anti-17OH and anti-SCC autoantibody levels. C_LIO_LIRTX induced rapid B-cell depletion followed by repopulation and a delayed T-cell effect at 8 months with reduced Th1 and Tfh cell frequencies. C_LI Capsule summaryRTX has recently been shown to restore fertility in autoimmune POI. Lymphocyte profiling identified Th1 bias accompanied by CXCR3+memory B cells that correlate with autoantibody levels. B-cell depletion appears to temporarily alleviate ovarian autoimmunity and ameliorate physiological function.

Purpose: Age is the leading factor for age-related macular degeneration (AMD), in which retinal pigmented epithelium (RPE) atrophy is a hallmark. The nuclear factor-erythroid 2-related factor 2 (Nrf2), a major anti-stress transcription factor, decreases in AMD, suggesting bioenergetic stress interacts with age during AMD pathogenesis. We profiled visual function and gene expression of the retina and RPE-choroid with Nrf2 deletion and aging. Methods: We tested 3mo and 14mo Nrf2 null (Nrf2 KO) and Nrf2 WT mice for optokinetic tracking (OKT) and dark-adapted electroretinography (ERG) responses. Eyes were harvested for retina outer nuclear layer (ONL) thickness analysis and retina and RPE-choroid cup RNA isolation for RNAseq profiling. Results: Visual acuity was reduced with aging with lower values in the Nrf2 KO vs Nrf2 WT comparison at 14mo. We observed aged-related reduction of a-, b-, and c-wave ERG amplitudes in both WT and KO mice. Age-related c-wave decline was exacerbated by Nrf2 loss. By RNAseq, two-way ANOVA of RPE-choroid samples revealed differentially expressed genes (DEGs) with age and genotype main effects, as well as DEGs with age and genotype interaction effects. Age DEGs were over-represented in pathways related to inflammation and phagocytosis. Genotype DEGs were associated with alterations in antioxidant and inflammatory processes. Retina RNAseq showed age and genotype DEGs. Conclusions: Ablation of Nrf2 exacerbates age-related declines suggesting that decrease in Nrf2 with aging has functional consequences for inflammation/resiliency. We provide retina and RPE-choroid bulk-RNAseq datasets from the same cohort of mice that can be interrogated to better understand degeneration of retina/RPE-choroid.

Purpose. To determine which proposed keratoconus (KC) mechanisms carry its genetic susceptibility and how it relates to corneal structure, and which describe its corneal tissue. Methods. Six mechanism modules and two attribution sets (transforming growth factor-{beta} [TGF-{beta}] and canonical type-2 atopy) were tested for enrichment in three independent European KC genome-wide association studies (GWAS) and in GWAS of central corneal thickness (CCT) and corneal resistance factor (CRF), with colocalization at matrix loci. Tissue expression was pooled across nine arms from eight studies with control-source meta-regression; activator protein-1 (AP-1)/immediate-early regulatory activity was scored in 107 donors, spatial expression in 21, and serum responses in human and rabbit stromal cells. Results. Extracellular matrix (ECM) was the only module enriched in all three KC sources (P [&le;] 5.46 x 10-) and for CCT, CRF, and thickness-independent CRF (P [&le;] 2.07 x 10-). COL5A1 shared causal variants with CCT in all three sources (posterior probability 0.985-0.991), and FNDC3B and COL1A1 with CRF or its thickness-independent component (0.961-0.986). Pooled, KC corneas had lower general and corneal-local inflammatory and stress/immediate-early expression (log2 fold-change -0.97, -0.98, -0.82; P [&le;] 0.049). In whole-cornea and stromal tissue, ECM expression reversed between postmortem-donor and pathological controls (log2 fold-change +0.36, -2.34), whereas inflammatory and TGF-{beta} programs were lower against both. AP-1/immediate-early regulatory activity was lower in four donor cohorts (Hedges' g -1.50) and stress/immediate-early expression in two spatial cohorts; serum raised immediate-early readouts in human and rabbit cells. Conclusions. KC combines matrix-mechanical genetic susceptibility with a low-inflammatory, low-immediate-early corneal state whose matrix expression direction depends on the control source. Because this susceptibility is genetically shared with corneal thickness and resistance, interventions on the inherited disease are judged by corneal structure and mechanics.

The transport of about 70 enzymes to lysosomes depends on mannose 6-phosphate signals formed by GNPTAB. Editing of Gnptab in an intestinal mouse cell line revealed the loss of multiple lysosomal enzymes associated with the accumulation of sphingomyelins, ceramides, and cholesterol, and reduced levels of bis(monoacylglycero)phosphate (BMP) in lysosomal proteomes and lipidomes. By cross-correlation we identified two subsets of lysosomal lipid-modifying enzymes linked with mixed unsaturated or di-monosaturated BMP. Autophagy-related proteins functioning in early stages of autophagosome formation associated with neutral ceramides, whereas proteins involved in autophagosome-lysosome fusion correlated with negatively charged BMPs. Therefore, the high cholesterol and low BMP level of lysosomes might be causal for impaired autophagic flux in GNPTAB deficient cells. We propose a functional axis of three lysosomal proteins as potential target to improve the autophagic flux in cells with dysfunctional lysosomes and in a newly established intestinal organoid model suitable as novel experimental tool.

Fibroblasts are readily obtained and expanded and may provide an alternative cell source for cultivated seafood if their latent myogenic potential can be accessed. We examined whether transient exposure to 5-azacytidine (AZA), a cytidine analogue known to convert mouse embryonic fibroblasts into myoblasts, elicits a persistent muscle gene program in embryo-derived zebrafish (Danio rerio) Z3 fibroblasts. Cells were exposed to 0, 2.5, 5 or 10 M AZA for 48 h and maintained in drug-free medium for 3 weeks. RNA-seq identified 744 genes that increased and 670 that decreased consistently across the concentration series. Increased genes encompassed myogenic regulators, fusion factors, sarcomeric components and excitation-contraction machinery, whereas decreased genes were enriched for extracellular-matrix and adhesion functions. In a separate six-dose RT-qPCR experiment (0-20 M AZA) with three biological replicates per concentration, all 19 selected muscle genes were significantly increased at 1.25 M, the lowest concentration tested, whereas 9 of 12 extracellular-matrix/fibroblast genes were significantly decreased. Several muscle genes plateaued or declined at higher concentrations, and multinucleated myotubes were not observed. A separate 5-aza-2'-deoxycytidine (decitabine) series showed a comparable muscle-associated response at 0.25 M. These findings indicate that zebrafish fibroblasts retain access to a myogenic-like transcriptional program, and they provide a model for identifying food-compatible approaches to control fish-cell lineage plasticity for cultivated seafood.

Background: Parkinson's disease (PD) is characterized by the progressive loss of midbrain dopaminergic (DA) neurons and aberrant alpha-synuclein (alpha-syn) aggregation, yet disease-modifying therapies that simultaneously retard neurodegeneration and foster regeneration remain lacking. Methods: In this study, we isolated extracellular vesicles (TH and FOXA2 double-positive cell-derived EVs) from TH and FOXA2 double-positive differentiated neural cells, a distinct midbrain floor-plate-derived neural cell population that co-expresses the floor-plate transcription factor FOXA2 together with TH and should therefore not be equated with conventional mature midbrain dopaminergic neurons, and systematically evaluated their therapeutic potential across three complementary models: A53T transgenic mice, 6-OHDA-lesioned rats, and 6-OHDA-treated human midbrain organoids. Integrative transcriptomic and single-cell sequencing analyses, together with toxicological evaluations in rodents and non-human primates, were performed to investigate the underlying mechanisms and safety profile. These TH and FOXA2 double-positive cells were generated using a fully suspension-based, matrix-free, and chemically defined differentiation system devoid of N2/B27 supplements and fetal bovine serum (FBS). This approach offers a simple, controllable, low-cost, and scalable platform with high batch-to-batch consistency for EV manufacturing. Throughout this study, the term "TH and FOXA2 double-positive cells" refers to this distinct TH and FOXA2 double-positive cell population and should not be interpreted as conventional terminally differentiated dopaminergic neurons. Results: Intranasal administration of TH and FOXA2 double-positive cell-derived EVs in A53T mice and 6-OHDA-lesioned rats significantly ameliorated motor deficits, increased nigral tyrosine hydroxylase (TH)-positive neuron counts, reduced alpha-syn aggregation, and suppressed gliosis. In human midbrain organoids, TH and FOXA2 double-positive cell-derived EVs preserved DA neuronal morphology and reduced GFAP and alpha-syn expression. Notably, when EVs were administered simultaneously with 6-OHDA modeling rather than after modeling was completed, the protective effect was stronger. Multi-omics analysis revealed that TH and FOXA2 double-positive cell-derived EVs reversed pathological signatures of interferon-responsive microglia and oxidative stress-adapted astrocytes, while restoring WNT- and non-canonical WNT-mediated intercellular communications. Safety evaluations showed no discernible organ toxicity. Conclusions: TH and FOXA2 double-positive cell-derived EVs exert neuroprotective effects across multiple PD models by modulating specific glial subpopulations and reinstating development-associated signaling pathways. Together with a fully suspension-based, matrix-free, and N2/B27-free differentiation process that enables low-cost, scalable, and highly reproducible manufacturing, TH and FOXA2 double-positive cell-derived EVs represent a promising therapeutic candidate for Parkinson's disease.

Mitochondrial transplantation has recently emerged as an alternative treatment for cardiovascular disease (CVD), aimed at increasing mitochondrial number and improving mitochondrial function. Though mitochondrial dysfunction is a key factor in the development of right ventricular hypertrophy/failure, the effects of mitochondrial transplantation on disease mitigation have been largely unexplored. Therefore, this in vitro study aimed to determine whether the transplantation of exogenous L6 myotube mitochondria mitigates negative outcomes in H9C2 cardiomyocytes that were stimulated to hypertrophy with phenylephrine. Control (CTL), control with mitochondrial transplantation (MitoTx), Phenylephrine only (Phe-only), and phenylephrine with mitochondrial transplantation (Phe+MitoTx) treatments were evaluated. Pilot experiments indicated mitochondrial transplantation into healthy cardiomyocytes acutely increased Complex I-linked oxidative phosphorylation (OXPHOS) capacity (p=0.005) and maximal respiratory capacity (p=0.022) within 24 hours, and these data alongside microscopic evidence of fluorescently labeled L6 mitochondria in H9C2 cardiomyocytes suggested successful transplantation. Regarding treatment comparisons, Phe-only showed a significant increase in cell area (p<0.05), while Phe+MitoTx blunted the hypertrophic cardiomyocyte response. Consistent with these results, proteomic analysis of 4,806 proteins showed that transplantation enriched the mitochondrial proteome, impacting pathways including OXPHOS, respiration, fatty acid and amino acid metabolism, while suppressing extracellular matrix remodeling and de-differentiation signatures. This response was observed in healthy and phenylephrine-stressed cardiomyocytes. In conclusion, our in vitro data indicates that transplantation of L6 skeletal muscle mitochondria mitigates negative effects induced by phenylephrine in H9C2 cardiomyocytes. However, more rigorous in vivo studies are needed to determine if this is a suitable approach for disease mitigation.

Background. Progression of prostate cancer under androgen receptor (AR) pathway-targeted therapy spans a phenotypic spectrum, from tumors that sustain AR pathway output to tumors that become independent of AR signaling. Experimental models that occupy the intermediate portion of this spectrum, in which AR is still expressed but pathway output is reduced, are comparatively scarce. Methods. LNCaP cells expressing a triple-modality reporter (LNCaP-TR) were injected into nude mice by the intracardiac route, and a bone lesion that arose in the middle spine of one animal was recovered and propagated as LNCaP-TR-BoM. The subline and its parental line were compared by two-dimensional and three-dimensional culture assays, RNA-seq, qRT-PCR, and immunoblotting. AR pathway output was assessed using KLK3 expression under AR antagonist treatment. Public prostate cancer datasets provided clinical context. A CRISPR-Cas9 EPHA3-edited derivative was evaluated in aggregation and spheroid assays. Results. Relative to parental LNCaP-TR, LNCaP-TR-BoM formed more numerous and larger cell aggregates in monolayer culture and larger, less circular spheroids in both suspension and Matrigel three-dimensional culture. AR mRNA was modestly reduced and AR protein remained readily detectable, whereas KLK3 mRNA and protein were reduced disproportionately more than those of AR. Residual KLK3 expression remained suppressible by AR inhibitors enzalutamide and darolutamide. Transcriptome-based pseudotime placed LNCaP-TR-BoM between the parental line and the androgen-refractory LNCaP-RF variant. Subsequent differential gene expression (DGE) and pathway enrichment analysis revealed that LNCaP-TR-BoM exhibits transcriptomic rewiring concordant with that of clinical mCRPC, thus evidencing LNCaP-TR-BoM as a potentially clinically relevant model cell line. Finally, EPHA3 showed the largest fold change among the most significant differentially expressed genes in LNCaP-TR-BoM compared with the parental line. Therefore, EPHA3 was explored to demonstrate the utility of the LNCaP-TR-BoM cell line as a discovery system for genes that potentially drive the malignancy of bone-metastatic prostate cancer at the intermediate AR-attenuated progression stage. An EPHA3-silenced derivative of LNCaP-TR-BoM lacking detectable EPHA3 protein formed smaller aggregates and smaller, more circular spheroids, suggesting EPHA3 may play a role in driving the malignancy of AR-intermediate bone-metastatic prostate cancer. This finding provides support for the potential utility of the LNCaP-TR-BoM cell line as a discovery system for genes relevant to driving AR-intermediate bone-metastatic prostate cancer. Conclusions. LNCaP-TR-BoM is a bone lesion-derived prostate cancer cell line in which AR expression is retained but AR pathway output is attenuated, accompanied by an mCRPC-concordant transcriptomic shift and altered multicellular organization. It provides a paired discovery system for candidate genes such as EPHA3.

Background and aims: Cyclin E1 is a canonical regulator of cell cycle progression known to promote liver fibrosis and hepatocarcinogenesis. However, its precise contribution to hepatic immune responses remains poorly understood. We investigated whether Cyclin E1 regulates macrophage-associated inflammation and acute immune-mediated liver injury. Methods: Hepatic immune populations were analyzed in untreated and concanavalin A-treated wildtype and constitutive Ccne1-/- mice by flow cytometry. Liver injury was assessed by survival, histopathology, and caspase activation. Inflammatory mediators were quantified by qPCR and ELISA. Bone marrow-derived macrophages were examined during differentiation and pro-inflammatory polarization using flow cytometry, cytokine measurements, and RNA-sequencing. Lipid nanoparticle-mediated delivery of Ccne1-specific siRNA was evaluated in J774A.1 macrophage-like cells. Results: Under homeostatic conditions, Ccne1-/- mice exhibited reduced hepatic immune cell numbers, including Kupffer cells and CD4+ and CD8+ lymphoid cells. Following concanavalin A administration, Ccne1-/- mice showed improved survival, reduced hepatic necrosis, and diminished caspase-3 and caspase-8 activation. Protection was associated with reduced Kupffer and CD8+ lymphoid cell abundance and attenuated inflammatory mediator expression. Cyclin E1 deficiency did not substantially alter bone marrow-derived macrophage differentiation or cell cycle distribution. However, pro-inflammatory Ccne1-/- macrophages showed reduced interleukin-6 expression, attenuated inflammatory gene programs, and lower inferred IRF1 and NF-{kappa}B1 activity. Ccne1 knockdown similarly reduced interleukin-6 expression in pro-inflammatory J774A.1 cells. Conclusions: Cyclin E1 contributes to hepatic immune homeostasis and promotes macrophage-associated inflammatory signaling during immune-mediated hepatitis. Its inflammatory function is uncoupled from overt changes in macrophage cell cycle progression and may involve regulation of IRF1- and NF-{kappa}B-associated transcriptional programs. Targeting Cyclin E1 may therefore provide a strategy to attenuate selected inflammatory responses in liver disease.

Dehydroascorbic acid (DHA), the oxidized form of vitamin C, exhibits specific toxicity against KRAS-mutant cancer cells; however, the early metabolic reprogramming mechanisms leading to cell death remain incompletely understood. In this study, we reveal the non-linear, dose-dependent response of the intracellular metabolome during the ultra-early phase of 1-hour DHA exposure. Under 1 mM DHA exposure, an early significant accumulation of the primary catabolite erythrulose (p = 0.030) and moderate consumption of glutamate (p = 0.0086) were observed, while core TCA cycle homeostasis and cell viability were preserved. In contrast, under 10 mM DHA exposure, an acute and profound alteration of the metabolic profile was observed. Along with the significant accumulation of glycerate, indicative of a glycolytic blockade, and the terminal peroxidation product oxalate, core TCA cycle intermediates such as -ketoglutarate and malate showed rapid depletion (Log2FC < -2.0, p < 10-7). Furthermore, coupled with this acute energy depletion, a statistically significant accumulation of free methionine (Log2FC = 1.14, p = 0.011), which was below the limit of detection in the control, was identified alongside profound depletions of glutamate (p < 0.001) and cysteine (p = 0.0027). These results strongly suggest that the initial response to high-dose DHA is not merely a linear accumulation of oxidative stress, but rather an "abrupt metabolic switch" characterized by rapid TCA cycle dysfunction followed by the early signature of a methionine trap.

Whether the nodal vulnerability in dementia is solely represented by changes in cellular abundance or attrition of cellular transcriptomic landscape attribute to that transition in aging brain is yet to uncover with minutiae. To understand this complication, we have analyzed two snRNA-seq data specific to (I) dorsolateral prefrontal cortex (DLPFC): A comprehensive study of cellular profiling among cognitively normal and pathological ageing and dementia (AD) brain, (II) occipital cortex (OC) in the context of Nasu-Hakola disease along with a tissue level bulk RNA-seq data corresponds to temporal cortex. We pinpoint cellular subpopulations their proportional changes within the nodal points and their transcriptomic profiling. In DLPFC we observed alteration of mitochondrial and synaptic maintenance program in the pathological manifestation of disease. In OC we noticed depleted upper layer CUX2 neuron, PVALB and SST neurons on the contrary an enrichment of RORB and LAMP5 neurons with compromised metabolic states in the disease context. Glial populations are marked by representation of fibrous astrocyte, neuronal-supporting subpopulations associated with extracellular matrix remodeling programs. Proportional increment of intermediate myelinating oligodendrocyte and a decrease in mature myelinating subpopulation highlighted a compromised myelin repair attempt. In temporal cortex at tissue level we observed transcriptomic landscape adopted a neuroglial phenotypes in dementia brain. Cell-Chat analysis demonstrated a shift in cellular coordination towards glial population from neuron in NHD brain. A single cell-based drug reprofiling approach identified efatutazone and balaglitazone as broad-spectrum candidates for major cell types in occipital cortex, whereas major actionable biological targets belong to CDK subtypes and proteasome subunits. Overall, our findings imply cellular persistence do not necessarily substantiate molecular preservation, also the coexistence of cellular vulnerability alongside selective remodeling features, present a cellular framework on how to cope up with degenerative changes.

Background: Human induced pluripotent stem cell-derived cardiomyocytes are valuable for cardiac disease modelling and drug testing but remain limited by immature structural and electrophysiological properties in conventional two-dimensional cultures. Three-dimensional cardiac models, including cardioids, provide a more physiological environment through multicellular organization and enable tissue-level functional analysis. Their electrophysiological properties and maturation, however, remain incompletely characterized. Methods: We performed an integrated electrophysiological characterization of human cardioids over the first three weeks of maturation using sharp microelectrode recordings, calcium transient measurements, whole-cell patch clamp recordings, immunofluorescence, transcriptomic analysis, and pharmacological interventions. Results: During maturation, cardioids exhibited progressive electrophysiological maturation, characterized by more physiological resting membrane potentials (-54.0{+/-}3.1mV to -68.1{+/-}1.2mV), increased action potential amplitude (73.5{+/-}4.8mV to 101.7{+/-}2.1mV) and upstroke velocity (44.3{+/-}5mV/ms to 86.6{+/-}8.4mV/ms), and shorter action potential duration (178.9{+/-}8.7ms to 144.0{+/-}4.8ms at 1Hz stimulation). These changes were associated with progressively increased INa density and enhanced SCN5A mRNA and Nav1.5 protein expression, consistent with progressive maturation of cardiac excitability. IKr current and KCNH2/hERG expression were already present at early maturation stages and remained stable, as evidenced by preserved sensitivity to dofetilide-blockade. Pharmacological inhibition of barium- and HMR-sensitive currents altered resting membrane and/or action potential characteristics in intact cardioids, whereas in isolated cardiomyocytes classical IK1 remained undetectable and IKs was small, highlighting a certain degree of persisting immaturity of the model. Conclusions: These findings demonstrate that human cardioids undergo progressive electrophysiological maturation and provide complementary tissue- and cellular-level insights into cardiac function, establishing their suitability for investigating INa and IKr-related cardiac pathologies.

As the main microtubule network organisers of animal cells, centrosomes orchestrate diverse cellular processes. Centrosomes have been linked to cancer when numerically or structurally aberrant, yet their precise roles at different cancer stages remains to be defined. Here, we study centrosome function in the epithelial-to-mesenchymal transition (EMT), a key driver of cancer initiation, progression and metastasis. We first depleted centrosomes in a breast cancer mesenchymal-like model, resulting in insignificant cellular or nuclear rearrangements. We then investigated non-carcinogenic breast cells undergoing the EMT via TGF{beta} signalling, and removed the centrosomes before triggering this transition. Stimulated epithelial cells acquired a mesenchymal phenotype and rearranged their cytoskeleton. By contrast, stimulated cells lacking centrosomes retained epithelial morphology, showed little cytoskeletal remodelling, yet still altered nuclear shape and upregulated key EMT markers. Transcriptome analysis revealed reduced expression of important genes involved in extracellular matrix organisation and a downregulation of cell junction components when lacking centrosomes; further analysis revealed paxillin showing reduced colocalisation with actin focal adhesions. Centrosomes thus prove essential for cytoskeletal remodelling in the EMT of breast cancer cells.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

Maternal hyperglycemia can affect offspring development and metabolism, but how its effects on skeletal muscle change during postnatal life remains unclear. We investigated skeletal muscle phenotypes in male offspring of streptozotocin-induced hyperglycemic mice at 4 and 8 weeks of age, representing early post-weaning and later postnatal stages, respectively. Maternal hyperglycemia resulted in lower offspring body and skeletal muscle weights at both time points. Myofiber cross-sectional area was reduced without a reduction in total myofiber number, indicating impaired myofiber growth rather than myofiber loss. At 4 weeks, skeletal muscle showed type I/IIA hybrid myofibers and increased expression of fetal, myogenic, and proliferation-associated genes. This immature profile was not maintained at 8 weeks; instead, the transcriptional profile shifted toward reduced mitochondrial and oxidative metabolism. DNA methylation changes at 4 weeks showed limited correspondence with transcriptional alterations. Taken together, maternal hyperglycemia impaired postnatal myofiber growth at both time points, with distinct molecular profiles at 4 and 8 weeks.