�����ƽ��

Oxygen tension is a key regulator of early human neurogenesis; however, quantifying intra-tissue O2 in 3D models for an extended period remains difficult. Existing approaches, such as needle-type fiber microsensors and intensity-based oxygen probes or time-domain lifetime imaging, either perturb the organoids or require high excitation doses that limit the measurement period. Here, we present a step-by-step protocol to measure intra-organoid oxygen in human cerebral organoids (hCOs) using embedded ruthenium-based CPOx microbeads and widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM). The workflow covers dorsal/ventral cerebral organoid patterning, organoid fusion at day 12 with co-embedded CPOx beads, standardized FD-FLIM acquisition (470-nm external modulation, 16 phases at 50 kHz, dual-tap camera), automated bead detection and lifetime extraction in MATLAB, and session-matched Stern–Volmer calibration with Ru(dpp)3(ClO4)2 to convert lifetimes to oxygen concentration. The protocol outputs per-bead oxygen maps and longitudinal patterns stratified by bead location (intra-organoid vs. gel) and sample state (healthy vs. abnormal), enabling direct linkage between developmental growth and oxygen dynamics. Key features • End-to-end workflow linking hCOs generation, on-gel bead embedding, and FD-FLIM oxygen readout.• Longitudinal single-organoid tracking of oxygen tension with bead-level metadata.• Reference-based lifetime calibration and reproducible camera/LED settings.• Ready-to-reuse materials, recipes, timing, and analysis logic.

Effective communication between multipotent mesenchymal stromal cells (MSCs) and endothelial cells (ECs) plays a critical role in the regulation of angiogenesis, especially under conditions of hypoxia. In addition to paracrine stimulation, direct intercellular contacts play an important role in the angiogenic interaction between MSCs and ECs, making them an important target for modulating vascular network restoration under ischemic conditions. The aim of this study was to determine the contribution of gap junctions (GJs) to the angiogenic response of MSCs and the EA.hy926 cell line (an Endothelial Cell Model) under acute hypoxic stress. In a cell co-culture model at 0.1% O2 using a specific GJ inhibitor (carbenoxolone), molecular, cellular, and functional tests were performed: assessment of viability, proliferation, migration, secretion of angiogenic mediators, and expression of crucial genes. GJ blockade was accompanied by decreases in the proliferation and migration activity and angiogenic potential of the conditioned medium in in vitro and in ovo tests. These data highlight the importance of the GJ in coordinating the angiogenic response in conditions of acute hypoxia and can be used to develop protocols for regenerative medicine.

Semaphorin3A (Sema3A) is a regulatory protein found to be expressed on regulatory T and B cells and also secreted into peripheral blood. It has been identified as a potent immune regulator; however, not all its regulatory mechanisms have been evaluated. In this respect, we aim to investigate how Sema3A affects key metabolic pathways in T cells during homeostasis and rheumatoid arthritis (RA), and on the AKT/mTORC1 signaling axis. In this study, peripheral blood samples were collected from 119 healthy donors and 32 rheumatoid arthritis patients. T cells were subjected to Seahorse analysis to evaluate OXPHOS and glycolysis, live cell TMRE staining to evaluate mitochondrial activity, mass spectrometry for metabolite profiling, ATP determination to study ATP production, and Western blot analysis to investigate the signaling pathway activity. This study presents evidence showing that Sema3A inhibits the AKT/mTORC1 pathway, leading to a decreased glucose uptake and glycolysis disruption. Furthermore, we show that Sema3A reduces mitochondrial capacity and OXPHOS in activated T cells of healthy and RA donors, leading to a decreased ATP production. In contrast, Sema3A upregulates fatty acid oxidation (FA), probably as a backup pathway to ensure cell survival. Results with p values of <0.05 were considered significant. Our data may point to Sema3A’s ability to convert activated T cells’ metabolic profile back to its non-activated state. This may suggest that Sema3A might be a beneficial treatment for immune-mediated diseases by metabolically reprogramming activated T cells.

Background: Cryptococcus-associated immune reconstitution inflammatory syndrome (C-IRIS) is a life-threatening complication of immune recovery, often triggered by antiretroviral therapy and characterized by Th1-skewed CD4+ T cell hyperactivation, neuroinflammation, and pulmonary dysfunction. Methods: Using a validated murine model of unmasking C-IRIS, we assessed the therapeutic potential of star-shaped glatiramer acetate (sGA), a structurally enhanced derivative of the FDA-approved immunomodulator glatiramer acetate (GA). sGA was administered intraperitoneally on days 1 and 3 post-CD4+ T cell reconstitution. Results: sGA significantly ameliorated C-IRIS-associated respiratory dysfunction, including increasing breaths per minute by ~35% and improved minute volume, total respiratory cycle time, expiration time, and inspiration time. Survival rate grew to 75% on day 14 for sGA-treated C-IRIS mice. In both the lung and the brain, sGA reduced total CD4+ T cells and selectively diminished Th1 cells by 50–60% and Th17 cells by 40–50%. Activated microglia decreased by 45% within the brain, indicating attenuated innate immune activation. Golgi-Cox analysis revealed region-specific neuroprotection: neuronal loss in the prefrontal cortex, lateral hypothalamus, and periaqueductal gray was rescued by 25–40%, whereas hippocampal neurons were relatively preserved, and basolateral amygdala neurons showed no significant recovery. Conclusion: Collectively, our findings suggest that sGA exerts neuroprotection in C-IRIS by limiting peripheral CD4+ T cell effector activity and suppressing CNS-resident immune activation. This study supports the use of sGA as a promising preclinical therapeutic candidate for C-IRIS and other Th1-mediated neuroinflammatory conditions.

Acute myeloid leukemia is a genetically and cellularly heterogeneous disease. We characterize 120 AMLs using genomic and transcriptomic analyses, including single-cell RNA sequencing. Our results reveal an extensive cellular heterogeneity that distorts the bulk transcriptomic profiles. Selective examination of the transcriptional signatures of >90,000 immature AML cells identifies four main clusters, thereby extending current genomic classification of AML. Notably, NPM1-mutated AML can be stratified into two clinically relevant classes, with NPM1class I associated with downregulation of MHC class II and excellent survival following hematopoietic stem cell transplantation. NPM1class II is instead associated with resistance to allogeneic T cells in an ex vivo co-culture assay, and importantly, dismal survival following hematopoietic stem cell transplantation. These findings provide insights into the cellular state space of AML, define diagnostic entities, and highlight potential therapeutic intervention points. The clinical outcomes and treatment responses of acute myeloid leukemia (AML) patients are heterogeneous. Here, the authors use bulk and single-cell sequencing approaches and identify two transcriptomic subtypes within NPM1-mutated AML with distinct immune evasion properties and responses to hematopoietic stem cell transplantation.

Reactive oxygen species (ROS), particularly superoxide anion (O2•-) and hydrogen peroxide (H2O2), originating from mitochondria, are increasingly recognized as critical mediators of physiological signaling and cellular function. While in the adult brain, mitochondrial ROS, specifically mitochondrial H2O2, modulate metabolism and sustains cognitive processes, their role in the developing cerebral cortex remains undefined. Here, we leverage a knock-in mouse model constitutively expressing mitochondrially targeted catalase (mCAT) to attenuate mitochondrial H2O2 levels and investigate their impact during cortical development. In neurosphere cultures derived from embryonic day 14.5 (E14.5) mCAT mice, reduced mitochondrial H2O2 altered glutathione redox homeostasis and glucose metabolism leading to suppressed progenitor cell proliferation, without compromising viability. In vivo, neural progenitor cell (NPC) proliferation, neuronal differentiation and cortical layering were disrupted starting at gestational day E15. Together, these data uncover a physiological role for mitochondrial hydrogen peroxide in orchestrating neural precursor proliferation and differentiation, ultimately influencing mammalian cerebral cortex formation.

Persistence of the HIV-1 reservoir is the major barrier to a cure. Little is known about the dynamics of the proviral integration site landscape and inducibility of the viral reservoir in early-treated individuals. Here, we perform a longitudinal analysis of the viral reservoir in individuals who started treatment during acute infection and compare these findings to chronically-treated individuals. Even in early-treated individuals, clonal expansion contributes to reservoir persistence. Integration site analysis reveals similar distributions after one year of antiretroviral therapy (ART), irrespective of treatment initiation timing. Notably, proviruses integrated in heterochromatin regions are already detected in early-treated individuals after one year on ART and are progressively enriched after five years on ART, suggesting post-integration selection mechanisms. Using a lipid nanoparticle containing Tat mRNA (Tat-LNP) in combination with phorbol myristate acetate (PMA), we detect for the first time the inducible reservoir in individuals treated during acute infection with small reservoir sizes. Furthermore, we show that, in both the acute and chronic cohorts, the inducible reservoir shifts towards a more differentiated T cell compartment over time. Collectively, these findings indicate that clonal expansion and integration site selection contribute to reservoir persistence early after ART initiation in individuals treated shortly after seroconversion. Author summaryDespite ART, HIV persists in a small pool of infected cells. This reservoir is the main barrier to curing HIV. We studied how this reservoir changes over time in people who started treatment very early after infection and compared them to people who started later. We found that, even with early treatment, the infected cells persist and expand over time. After 5 years on ART, in people who started treatment early, HIV becomes enriched in regions of the genome that are less transcriptionally active, suggesting that selective pressures shape which infected cells persist. We also applied a new combination of latency reversal agents, allowing us for the first time to detect the inducible reservoir even in people with very small reservoir sizes. Finally, we observed that the inducible reservoir shifts towards more differentiated types of immune cells over time on ART. Together, our findings provide new insights into how HIV persists despite early treatment.

The presence of bone marrow (BM) disseminated tumor cells (DTCbm) identifies early-stage breast cancer patients at increased risk of recurrence and poorer overall survival. However, limitations in detecting DTCbm by standard immunohistochemical approaches have hampered clinical application. To address this gap, we developed a flow cytometry-based method, DTC-Flow, that enables the sensitive and efficient detection and molecular characterization of breast cancer DTCbm. Our analysis identified HER2 as a sensitive marker for detecting breast cancer cells, including those lacking HER2 amplification are claudin-low. DTC-Flow using a HER2/EpCAM/CD45 marker panel enabled >90% cancer cell recovery and sensitivity of one cancer cell per million nucleated BM cells across a range of breast cancer subtypes. Molecular analyses of DTC-Flow-sorted DTCbm from metastatic patients suggested a quiescent state and demonstrated their close genomic relationship to primary/metastatic tumors, as well as continued genetic evolution. In early-stage breast cancer patients, DTC-Flow detected DTCbm with greater sensitivity than cytokeratin-based immunohistochemical approaches. Our data support the development of DTC-Flow as a sensitive and specific platform to identify breast cancer patients harboring DTCbm and better understand the biology of minimal residual disease. Ultimately, this platform could enable the selection of personalized therapeutic approaches based on molecular features of DTCbm, monitoring of DTCbm to assess the efficacy of such therapies, and the development of novel therapeutic approaches targeting unique biological vulnerabilities of DTCs in order to eradicate these cells before they can give rise to lethal recurrent cancers.

ABSTRACTNeural stem cell (NSC)‐based therapies offer a promising strategy to promote brain repair by delivering neurotrophic factors, supporting cell replacement, and stimulating endogenous neurogenesis following injury. While numerous studies have highlighted the protective and regenerative potential of NSCs and their extracellular vesicles (EVs), progress toward clinical translation remains hindered by limited molecular characterization of NSC lines and their EV cargo. To address this gap, we characterized two therapeutically relevant human fetal NSC lines, LMNSC01 and LMNSC02, both engineered to express the L‐MYC gene, along with their corresponding EVs. LMNSC01 cells primarily differentiated into neurones with limited glial populations, whereas LMNSC02 cells gave rise to all three major neural lineages: neural, glial and oligodendrocyte progenitor cells (OPCs). scRNA‐seq revealed distinct transcriptional profiles with minimal overlap between the two LMNSC lines. Using single extracellular vesicle nanoscopy, we observed that both lines released predominantly circular EVs, with LMNSC02‐EVs exhibiting higher levels of tetraspanins (CD9, CD63, and CD81) and a larger average diameter than LMNSC01‐EVs. Proteomic profiling revealed that LMNSC01‐EVs are enriched in proteins involved in cell adhesion, migration, junction formation, and neuronal projection development, while LMNSC02‐EVs are enriched in factors related to cytoplasmic translation initiation and biosynthesis. These LMNSC‐EVs (collected from undifferentiated LMNSCs) demonstrated neuroprotective effects in a brain organoid model of methotrexate‐induced toxicity when added to corresponding LMNSC01‐ or LMNSC02‐derived brain organoids. LMNSC01‐ and LMNSC02‐derived EVs restored neuronal and astrocytic populations but failed to rescue OPCs. These findings demonstrate the therapeutic potential of LMNSC‐derived EVs to counter chemotherapy‐induced neurotoxicity by preserving neurones and astrocytes, while highlighting the need for repeated or complementary interventions to restore oligodendrocyte populations.

BACKGROUND: Glioblastoma (GBM), the most aggressive primary brain tumor, has a dismal prognosis largely due to therapy-resistant stem-like cells that drive recurrence. While N6-methyladenosine modifications in GBM are well-studied, the role of 5-methylcytosine (m5C) modifications specifically in circular RNAs (circRNAs) remains poorly understood. METHODS: Bioinformatic analysis, qRT-PCR and Western blot assays were used to investigate the expression of NSUN7, circNTRK2, YBX3, STK31, IKZF1 in GBM tissues and cell lines. m5C dot blot assays, m5C-bisulfite sequence assays, Sanger’s sequencing, RNA pull down and RIP assays, in vitro kinase assays, chromatin immunoprecipitation and luciferase reporter gene assays are used to clarify the interaction between factors above. Colony formation assays, sphere formation assays, and stemness marker (OCT4, DCLK1) analysis were utilized to assess the impact of the factors above on GBM stemness. Subcutaneous heterotopic and orthotopic xenograft are utilized to demonstrate the function of YBX3/circNTRK2/STK31/IKZF1 axis in GBM in vivo. RESULTS: NSUN7 and YBX3 are both upregulated in GBM tissues and cell lines. NSUN7 catalyzes m5C modification of circNTRK2. YBX3 decreasing circNTRK2 stability and function via binding m5C of circNTRK2. CircNTRK2 binds to and activates STK31, leading to phosphorylation of IKZF1 at S63, which decreases the O-GlcNAc levels and transcriptional activity of IKZF1, thereby promoting the stem cell characteristics of GBM cells. CONCLUSIONS: This study unveils that NSUN7-mediated m5C modification of circNTRK2 fuels GBM stemness via circNTRK2-dependent STK31 activation, thus identifying potential biomarkers for targeted molecular therapy of GBM and providing novel therapeutic targets for GBM treatment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-025-07484-1.

BackgroundBrain endothelial cells (ECs) lining blood vessels are essential for the normal function of the brain. They form the first layer of the blood-brain barrier (BBB) and regulate nutrient exchange, immune responses, and angiogenesis. Numerous studies have reported the disruption of the BBB in neurodegenerative diseases, including Alzheimer’s disease (AD). However, the impact of cell-intrinsic amyloid pathology on EC function remains to be clarified.MethodsTo optimize the method for producing functional ECs from human induced pluripotent stem cells (hiPSCs), we compared two different protocols. The first, a widely used method, relies on spontaneous differentiation after mesoderm specification. The second method involves transient overexpression of ETV2 to guide EC differentiation. To study the impact of beta-amyloid overproduction on EC function, we generated ECs from hiPSC lines carrying the APP Swedish mutation (APPswe), which causes AD. We assessed the functionality of both control and APPswe ECs using in vitro permeability assays, 2D and 3D vessel formation assays, and adhesion assays.ResultsECs generated using transient ETV2 overexpression exhibited higher levels of canonical EC markers, tight junction proteins, transporters, leukocyte adhesion molecules, and angiogenesis-associated receptors than ECs derived by spontaneous differentiation. Additionally, ETV2-ECs responded robustly to inflammatory and angiogenic stimuli, displaying functional and transcriptional changes, whereas spontaneously differentiated ECs did not. Consequently, we chose the ETV2 overexpression protocol to study the impact of APPswe mutation on endothelial function. We found that ETV2-ECs carrying the APPswe mutation displayed a reduced angiogenic potential following exposure to the sprouting mix and elevated expression of leukocyte adhesion molecules following inflammatory stimulation, leading to increased adhesion of monocyte-like cells.ConclusionsOverall, our study suggests that APPswe mutation in ECs impairs their response to inflammatory and angiogenic stimuli, potentially contributing to AD progression. Additionally, we confirmed that ETV2 overexpression during a critical window effectively guides hiPSCs toward the EC lineage, resulting in a stable and pure population of ECs suitable for disease modeling and drug screening.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-025-00728-8.

Rapid global warming is affecting the metabolic and immune responses of dairy cows, making heat stress a major challenge in the industry. This study aimed to develop nutritional strategies to mitigate these effects. Sucrose has been identified as a carbohydrate that not only provides immediate energy to the rumen. Specifically, we investigated whether sucrose supplementation could alleviate heat stress-induced changes in rumen metabolism and blood immune parameters. A total of 12 Holstein cows were used in this study. All cows were initially maintained under the optimal temperature period (OTP, n = 12). They were then assigned to either the high-temperature period control group (HTP-CON, n = 6) or the high-temperature period treatment group (HTP-TRT, n = 6). The feeding trial lasted for four weeks. Heat stress reduced milk yield by 22.36%, and sucrose supplementation tended to attenuate this reduction to 10.75%; heat stress decreased milk yield, whereas sucrose supplementation has minor mitigating effect on milk yield reduction. Furthermore, heat stress altered rumen metabolites, leading to metabolic imbalances, particularly in amino acid and fatty acid metabolism. Sucrose supplementation improved amino acid metabolic pathways, including phenylalanine and glutamate metabolism, which are essential for immune and metabolic homeostasis. Key metabolites such as aspartic acid, tyrosine, and isobutyrate were restored. Additionally, heat stress induced an inflammatory response, increasing the proportion of Th1 cells and upregulating IL-1β and IL-2 expression in PBMCs. Sucrose supplementation alleviated inflammation by reducing pro-inflammatory cytokine expression and normalizing the Th1 cell proportion. In conclusion, sucrose supplementation improved the resilience of dairy cows by alleviating metabolic imbalances and inflammatory responses caused by heat stress. These findings enhance our understanding of the relationship between immunity and metabolism under environmental stress and support the development of improved dairy management strategies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-21840-7.