Cell Populations Research Articles

Spatial transcriptomic applications in orthopedics.

This review highlights the transformative impact of spatial transcriptomics on orthopedic research, focusing on its application in deciphering intricate gene expression patterns within musculoskeletal tissues. The paper reviews literature for spatial transcriptomic methods, specifically 10X Visium, 10X Xenium, seqFISH+, MERFISH, NanoString GeoMx DSP, used on musculoskeletal tissues (cartilage, joints, bone, tendon, ligament, and synovium). Searches identified 29 published manuscripts reporting spatial transcriptomic data in cartilage, bone, tendon, synovium, and intervertebral disc. Most publications of spatial transcriptomic data are from tendon and synovium. 10X Visium has been used in 22 of the 29 papers identified. Spatial transcriptomics has been used to identify novel cell populations and cell signaling pathways that regulate development and disease. Imaging-based spatial transcriptomic methods may be better for hypothesis testing as they generally have subcellular resolution but sequence fewer genes. Sequencing methods may be better for untargeted, shotgun approaches that can generate useful hypotheses from the spatial data from unimpaired tissue sections. Spatial transcriptomic methods could become useful for clinical diagnostics and precision medicine approaches.

Anti-Müllerian hormone signalling sustains circadian homeostasis in zebrafish

Circadian clocks temporally orchestrate the behavioural and physiological rhythms. The core molecules establishing the circadian clock are clear; however, the critical signalling pathways that cause or favour the homeostasis are poorly understood. Here, we report that anti-Müllerian hormone (Amh)-mediated signalling plays an important role in sustaining circadian homeostasis in zebrafish. Notably, amh knockout dampens molecular clock oscillations and disrupts both behavioural and hormonal circadian rhythms, which are recapitulated in bmpr2a null mutants. Somatotropes and gonadotropes are identified as Amh-targeted pituitary cell populations. Single-cell transcriptome analysis further reveals a lineage-specific regulation of pituitary clock by Amh. Moreover, Amh-induced effect on clock gene expression can be abolished by blocking Smad1/5/9 phosphorylation and bmpr2a knockout. Mechanistically, Amh binds to its receptors, Bmpr2a/Bmpr1bb, which in turn activate Smad1/5/9 by phosphorylation and promote circadian gene expression. Our findings reveal a key hormone signalling pathway for circadian homeostasis in zebrafish with implications for rhythmic organ functions and circadian health.

Innate and Adaptive Immunity is not Impacted by Inflammatory Bowel Disease Medications in Pregnant Women and Their Offspring

Abstract Background and Aims Inflammatory bowel disease (IBD) therapeutics and pregnancy itself are known to impact immune cell populations, but a detailed understanding of the effect of specific therapies is lacking, particularly for infants exposed in utero. With comprehensive flow cytometric assessment, we aimed to explore how IBD and its treatment impact both maternal and infant immunophenotypes and cytokine production. Methods Peripheral blood was taken for flow cytometry immunophenotyping and quantification of cytokine responses to stimulation from women with IBD and healthy controls throughout pregnancy, at delivery, and postpartum. Blood samples were also taken from the umbilical cord and peripherally from the resultant offspring at the age of 6 weeks. Results Eighteen participants (16 with IBD and 2 healthy controls) were recruited to this single-center prospective cohort study. Basal T regulatory cell population proportions were 46% lower (0.21% vs 0.39%, P = .027) in those participants exposed to ustekinumab (n = 4). No other significant differences were noted in immunophenotype according to drug therapy exposure. Basal (1.75% vs 0.47%, P = .036) and lipopolysaccharide-stimulated (72.2% vs 64.7%, P = .028) production of tumour necrosis factor by classical dendritic cells were increased 3.7- and 1.1-fold, respectively, in those with an elevated fecal calprotectin (n = 6). Basal CD4+α4β7+ (41.4% to 66.5%, P = .0043) and CD8+α4β7+ (81.2% to 93.6%, P = .007) population proportions increased 1.6- and 1.2-fold, respectively, in infants from birth to 6 weeks. Conclusions This study provides the foundation for ongoing investigation to more definitively extricate the impact of maternal IBD activity, drug exposures, and disease phenotype on infant immune system development and function. These novel data suggest that IBD pharmacotherapies may not significantly impact maternal or infant immune regulation.

STIM2β is a Ca2+ signaling modulator for the regulation of mitotic clonal expansion and PPARG2 transcription in adipogenesis.

Intracellular Ca2+ is crucial in the regulation of adipocyte lipid metabolism and adipogenesis. In this study, we aimed to investigate the regulation mechanism of intracellular Ca2+ levels ([Ca2+]i) during adipocyte differentiation. We found that the expression of stromal interaction molecule 2 beta (STIM2β), which is the inhibitor of store-operated Ca2+ entry (SOCE), is upregulated throughout the differentiation process. Evaluation of [Ca2+]i in 3 T3-L1 and primary stromal vascular fraction (SVF) cells revealed that the basal Ca2+ level is downregulated after differentiation. Knockout (KO) of STIM2β in 3T3-L1 and primary SVF cells showed increased [Ca2+]i, indicating the involvement of STIM2β in the regulation of [Ca2+]i during adipogenesis. We further evaluated the function of STIM2β-mediated [Ca2+]i in early and terminal differentiation of adipogenesis. Analysis of cell proliferation rate during mitotic clonal expansion (MCE) in wild-type and STIM2β KO 3T3-L1 cell lines revealed that a larger population of KO cells underwent G1 arrest, suggesting that reduced [Ca2+]i by STIM2β induces MCE. Additionally, ablation of STIM2β increased differentiation efficiency, with more lipid accumulation and rapid transcriptional activation of adipogenic genes, especially proliferator-activator receptor γ2 (PPARG2). We found that PPARG2 transcription is regulated by store-operated calcium entry (SOCE) downstream transcription factors, confirming that increased [Ca2+]i by STIM2β ablation promotes PPARG2 transcription during adipogenesis. Additionally, STIM2β KO mice showed hypertrophic adipose tissue development. Our data suggest that STIM2β-mediated [Ca2+]i plays a pivotal role in the regulation of mitotic clonal expansion and PPARG2 gene activation and provides evidence that MCE is not a prerequisite process for terminal differentiation during adipogenesis.

Parent-of-origin regulation by maternal auts2 shapes neurodevelopment and behavior in fish

BackgroundParental experience can influence progeny behavior through gamete-mediated non-genetic inheritance, that is, mechanisms that do not involve changes in inherited DNA sequence. However, underlying mechanisms remain poorly understood in vertebrates, especially for maternal effects. Here, we use the medaka, a model fish species, to investigate the role of auts2a, the ortholog of human AUTS2, a gene repressed in the fish oocyte following maternal stress and associated with neurodevelopmental disorders.ResultsWe show that auts2a expression in the oocyte influences long-term progeny behavior, including anxiety-like behavior and environment recognition capabilities. Using single-nuclei RNA-sequencing, we reveal that maternal auts2a influences gene expression in neural cell populations during neurodevelopment. We also show that maternal auts2a knock-out triggers differences in maternally inherited factors, including early embryonic transcriptional and post-transcriptional regulators.ConclusionsTogether, our results reveal the unsuspected role of an autism-related gene expressed in the mother’s oocyte in shaping progeny neurodevelopment and behavior. Finally, we report that auts2a/AUTS2 is part of a group of evolutionarily conserved genes associated with human neurodevelopmental disorders and expressed in oocytes across species, from fish to mammals. These findings raise important questions about their potential role in the non-genetic regulation of progeny neurodevelopment and behavior in vertebrates.

Changes in glycinergic neurotransmission alter mammalian retinal information processing

Glycine, along with GABA, constitutes the major inhibitory neurotransmitter in the central nervous system. In the retina, glycinergic neurotransmission is primarily used by amacrine cells that are involved in the lateral processing of visual stimuli in the inner retina. We have previously shown that the high-affinity glycine transporter 1 (GlyT1), that is commonly used as a reliable marker for glycinergic amacrine cells in the retina, is essential for glycinergic neurotransmission by these cells. Abolishment of retinal GlyT1 expression results in a breakdown of glycinergic neurotransmission by AII amacrine cells, but most likely also by other glycinergic amacrine cell populations. However, the impact of loss of glycinergic neurotransmission on retinal signal processing and visually guided behavior, has not yet been elucidated. In this study, the effects of loss of retinal GlyT1 expression in glycinergic amacrine cells on the optomotor reflex and on the photopic and scotopic electroretinogram (ERG) responses were analyzed. We show that retinal GlyT1-deficient mice have normal optomotor responses to rotating black and white stripes. When stimuli with sawtooth luminance profiles were used, thereby differentially activating ON and OFF pathways, the GlyT1 deficient mice showed facilitated responses to ON preferring stimuli, whereas responses to OFF preferring stimuli were unchanged. These findings were corroborated by ERG recordings that showed undistinguishable responses after flash stimulation but revealed differences in the differential processing of ON and OFF preferring stimuli. To determine if the function of retinal GlyT1 is conserved in humans, we analyzed ERG recordings from a patient diagnosed with GlyT1 encephalopathy. We show that GlyT1 deficiency results in marked ERG changes, characterized by an almost complete loss of the “photopic hill” phenomenon, a hill-like appearance of the relationship between the b-wave amplitude and log light stimulus strength under background illumination conditions, and reductions in the ERG oscillatory potentials in the dark- and light-adapted states. Both findings are consistent with an altered interaction between ON- and OFF pathways in the retina. Taken together our data show that glycinergic neurotransmission in the retina has important functions in retinal ON and OFF processing both in mice and humans.

Tumor cell-intrinsic BIN1 deficiency promotes the immunosuppression and impedes ferroptosis of non-small cell lung cancer via G3BP1-mediated degradation of STAT1

BackgroundTumors often evade immune surveillance by limiting T cell infiltration. In non-small cell lung cancer (NSCLC), increased infiltration of CD8+ T cells is associated with a favorable response to immunotherapy. While BIN1 is recognized as a tumor suppressor gene, its role in shaping the tumor microenvironment in NSCLC has yet to be fully clarified.MethodsTo investigate the relationship between BIN1 expression and CD8+T cell infiltration in NSCLC, we performed a comprehensive data analysis utilizing clinical information from NSCLC patients. BIN1 expression levels in NSCLC tissues were evaluated, and their correlation with CD8+T cells infiltration and patient survival outcomes was examined. Loss-of-function strategies targeting BIN1 were applied in syngeneic NSCLC mouse models to assess its functional significance. Tumor growth was monitored, and immune cell populations were analyzed in terms of frequency and functionality through mass cytometry and flow cytometry techniques. Cytokine secretion was profiled using multiplex assays. Additionally, RNA sequencing, immunoprecipitation-mass spectrometry, and molecular docking were employed to confirm direct interactions between BIN1 and cytokine-encoding genes. Finally, the regulatory role of BIN1 in ferroptosis in NSCLC cells were explored using metabolomics analysis, ROS measurement, and MDA detection.ResultsWe observed that BIN1 expression is downregulated in NSCLC tumor tissues, with its reduced expression strongly associated with advanced disease progression and poor prognosis. Bioinformatics analysis of immune infiltration in human NSCLC samples revealed a positive correlation between BIN1 expression in NSCLC tissues and CD8+ T cell infiltration. Furthermore, the prognostic impact of BIN1 on NSCLC patients is strongly linked to the level of CD8+ T cell infiltration. In syngeneic mouse models, the knockout of BIN1 in NSCLC cells significantly inhibited CD8+ T cell infiltration and impaired their cytotoxic function, facilitating tumor immune evasion. Mechanistically, we demonstrated that BIN1 directly interacts with G3BP1, and its knockout stabilizes G3BP1. This, in turn, promotes STAT1 degradation and reduces the secretion of T cell-recruiting chemokines such as CXCL10 and CCL5. Finally, our findings reveal that BIN1 influences ferroptosis in NSCLC cells through the G3BP1/STAT1/GSH pathway, thereby regulating NSCLC cell proliferation, migration, and invasion.ConclusionThis study highlights the crucial role of the BIN1/G3BP1/STAT1/CD8+ tumor-infiltrating lymphocyte signaling pathway in the progression of NSCLC and its mechanisms of immune evasion. This fundings lay a foundation for the development of BIN1-targeted therapies aimed at improving tumor immunogenicity and transforming immunologically “cold” NSCLC into a more responsive disease.

Metallosphaera sedula bifurcates into two sizes when it is cultured mixotrophically on soluble iron

Metallosphaera sedula is a thermoacidophilic archaeon that obtains all of its energy for growth from aerobic respiration and oxidative phosphorylation at the expense of selected organic and inorganic sources of electrons. Initial velocities for the oxidation of soluble ferrous ions by intact cells at 60 °C and pH 1.5 were determined using an integrating cavity absorption meter that permitted accurate absorbance measurements to quantify the increase in soluble ferric iron in the presence of turbid suspensions of the live organisms. M. sedula that was cultured on yeast extract either in the absence or the presence of 20 mM soluble ferrous iron exhibited turnover numbers for soluble iron oxidation of 304 ± 26 and 333 ± 31 attamoles/cell/min, respectively. These functional data were consistent with the transcriptomic evidence presented by others, that the proteins presumably responsible for aerobic respiration on soluble iron are expressed constitutively in M. sedula. Intact cells of M. sedula were characterized by electrical impedance, laser light diffraction, and transmission electron microscopic measurements. All three types of measurements were consistent with the surprising observation that cells cultured on yeast extract in the presence of soluble iron bifurcated into approximately equal numbers of coccoidal cells of two sizes, smaller cells with an average diameter of 0.6 μm and larger cells with an average diameter of 1.35 μm. Cells cultured on the same concentration of yeast extract but in the absence of soluble iron comprised a single cell size with an intermediate average diameter of 1.06 μm. This unexpected bifurcation of a clonal cell population into two demonstrably different sizes when the extracellular nutrient environment changes has not previously been reported for M. sedula, or any other single-celled archaeon or eubacterium.

Most human DNA replication initiation is dispersed throughout the genome with only a minority within previously identified initiation zones

BackgroundThe identification of sites of DNA replication initiation in mammalian cells has been challenging. Here, we present unbiased detection of replication initiation events in human cells using BrdU incorporation and single-molecule nanopore sequencing.ResultsIncreases in BrdU incorporation allow us to measure DNA replication dynamics, including identification of replication initiation, fork direction, and termination on individual nanopore sequencing reads. Importantly, initiation and termination events are identified on single molecules with high resolution, throughout S-phase, genome-wide, and at high coverage at specific loci using targeted enrichment. We find a significant enrichment of initiation sites within the broad initiation zones identified by population-level studies. However, these focused initiation sites only account for ~ 20% of all identified replication initiation events. Most initiation events are dispersed throughout the genome and are missed by cell population approaches. This indicates that most initiation occurs at sites that, individually, are rarely used. These dispersed initiation sites contrast with the focused sites identified by population studies, in that they do not show a strong relationship to transcription or a particular epigenetic signature.ConclusionsWe show here that single-molecule sequencing enables unbiased detection and characterization of DNA replication initiation events, including the numerous dispersed initiation events that replicate most of the human genome.

Single-cell RNA sequencing reveals adrb1 as a sympathetic nerve-regulated immune checkpoint driving T cell exhaustion and impacting immunotherapy in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) presents significant health challenges due to its aggressive nature and poor prognosis from late-stage diagnosis. Despite these challenges, emerging therapies like immune checkpoint inhibitors offer hope. β1-adrenergic signaling has been implicated in T cell exhaustion, which weakens the immune response in ESCC. Blocking this pathway could restore T cell function. Recent advances in single-cell RNA sequencing (scRNA-seq) have enabled deeper insights into tumor heterogeneity and the immune landscape, opening the door for personalized treatment strategies that may improve survival and reduce resistance to therapy.MethodsWe combined scRNA-seq with bulk RNA analysis to explore adrenergic receptor signaling in ESCC, focusing on changes before and after neoadjuvant therapy. We identified ADRB1+ T cells through data analysis and experimental validation. Copy number variation (CNV) analysis detected malignant cells within scRNA-seq data, while intercellular interaction analysis examined communication between cell populations. Deconvolution of TCGA data revealed key immune populations, which were integrated into a prognostic model based on the adrenergic receptor signaling pathway and differentially expressed genes.ResultsThe adrenergic receptor signaling pathway was found in various immune cells, including T cells. scRNA-seq analysis revealed increased ADRB1 expression in T cells after neoadjuvant therapy. Immunofluorescence confirmed colocalization of ADRB1 with T cells, and fluorescence-activated cell sorting (FACS) showed that ADRB1 expression was elevated alongside exhaustion markers, while immune function markers were reduced. CNV analysis highlighted malignant cells in the tumor microenvironment, and intercellular interaction analysis explored ADRB1+ T cells’ role in immune support. Deconvolution of TCGA data identified ADRB1+ T cells, SPP1+ macrophages, and CD44+ malignant cells, all of which were prognostically significant. A prognostic model constructed from the intersection of the adrenergic receptor signaling pathway and differentially expressed genes following neoadjuvant therapy showed a significant prognostic effect.ConclusionsADRB1 expression increases after neoadjuvant therapy in ESCC and correlates with poor prognosis. Our findings suggest ADRB1 as a potential prognostic biomarker and therapeutic target for post-neoadjuvant immunotherapy.

Identification of the distinct immune microenvironment features associated with progression following high dose melphalan and autologous stem cell transplant in multiple myeloma.

A key treatment for patients with multiple myeloma is high-dose melphalan followed by autologous stem cell transplant (ASCT). It can provide a deep response with long-term remission. However, some patients progress quickly, and it is not clear why that is. Here, we performed single-cell RNA and T-cell receptor (TCR) sequencing of the immune microenvironment of 40 patients before and after ASCT to determine if differences in the immune composition could define those who would progress. Clear differences in cell populations were identified in progressors, including increased T-cell infiltration, decreased TCR diversity, and decreased frequency of monocytes and CD56bright NK cells. We identified cell interactions that predicted progression including increased frequency of CD8+ exhausted T cells and stromal cells and decreased frequency of CD56bright NK cells and plasmacytoid dendritic cells. We propose and validate a model of progression that can also be determined by flow cytometry. Together these data highlight the importance of the immune microenvironment in understanding responses to ASCT.

Rhinovirus as a driver of airway T cell dynamics in children with treatment-refractory recurrent wheeze.

Severe asthma in children is notoriously difficult to treat, and its immunopathogenesis is complex. In particular, the contribution of T cells and relationships to antiviral immunity remain enigmatic. Here, we coupled deep phenotyping with machine learning methods to elucidate the dynamics of T cells in the lower airways of children with treatment-refractory recurrent wheeze, and examine rhinovirus (RV) as a driver. Our strategy revealed a T cell landscape dominated by type 1 and type 17 CD8+ signatures. Interrogation of phenotypic relationships coupled with trajectory mapping identified T cell migratory and differentiation pathways spanning the blood and airways that culminated in tissue residency, and involved transitions between type 1 and type 17 tissue-resident types. These dynamics were reflected in cytokine polyfunctionality. Use of machine learning tools to cross-compare T cell populations that were enriched in the airways of RV-positive children with those induced in the blood following experimental RV challenge precisely pinpointed RV-responsive signatures that contributed to T cell migratory and differentiation pathways. Despite their rarity, these signatures were also detected in the airways of RV-negative children. Together, our results underscore the aberrant nature of type 1 immunity in the airways of children with recurrent wheeze, and implicate an important viral trigger as a driver.

A comprehensive analysis of induced pluripotent stem cell (iPSC) production and applications

The ability to reprogram mature, differentiated cells into induced pluripotent stem cells (iPSCs) using exogenous pluripotency factors opened up unprecedented opportunities for their application in biomedicine. iPSCs are already successfully used in cell and regenerative therapy, as various drug discovery platforms and for in vitro disease modeling. However, even though already 20 years have passed since their discovery, the production of iPSC-based therapies is still associated with a number of hurdles due to low reprogramming efficiency, the complexity of accurate characterization of the resulting colonies, and the concerns associated with the safety of this approach. However, significant progress in many areas of molecular biology facilitated the production, characterization, and thorough assessment of the safety profile of iPSCs. The number of iPSC-based studies has been steadily increasing in recent years, leading to the accumulation of significant knowledge in this area. In this review, we aimed to provide a comprehensive analysis of methods used for reprogramming and subsequent characterization of iPSCs, discussed barriers towards achieving these goals, and various approaches to improve the efficiency of reprogramming of different cell populations. In addition, we focused on the analysis of iPSC application in preclinical and clinical studies. The accumulated breadth of data helps to draw conclusions about the future of this technology in biomedicine.

Cross-talk between microbiota-gut-brain axis and blood pressure regulation.

Hypertension, or high blood pressure (BP), is a widespread condition affecting one in three adults globally. Despite the availability of treatment options, 50% of hypertensive patients in countries such as Australia fail to achieve adequate BP control, often due to a lack of response to current therapies. Diet plays a crucial role in BP regulation. A high-fibre diet reduces BP through the gut microbiome and the production of microbial metabolites known as short-chain fatty acids (SCFAs). However, the mechanisms of BP regulation by SCFAs remained still unclear. A novel hypothesis we explore in this review is that these microbial metabolites may regulate BP via the activation of central mechanisms, a phenomenon called the gut-brain axis. While substantial evidence in animal models and humans supports the protective role of SCFAs in hypertension, the precise mechanisms remain unclear. SCFA stimulates the release of neurotransmitters and hormones such as serotonin, cholecystokinin, glucagon-like peptide 1 and peptide YY by enteroendocrine cells, a rare population of cells lining the gastrointestinal tract. These hormones bind to their receptors on the peripheral nervous system nerves, such as the vagus and spinal nerves, conveying information to the brain. The mechanisms by which information is relayed from the gut microbiome to the brain likely involve the immune system and gut-derived neurotransmitters and hormones. A deeper understanding of these pathways and mechanisms will facilitate the development of novel therapeutics for hypertension and other cardiovascular diseases.

Exploring the role of cell cycle regulation in human mature adipocyte dedifferentiation

BackgroundDedifferentiated fat (DFAT) cells have been used in regenerative medicine due to their multipotent potential. According to the literature, the process of adipocyte dedifferentiation is characterized by liposecretion which results in a fibroblastlike, proliferating cell population, with increased expression of genes related to cell cycle. A number of pathways have been implicated in the process, but the role of the cell cycle in adipocyte dedifferentiation has yet to be investigated. Here we characterize the process of liposecretion, the cellular features of DFAT cells and the role of the cell cycle.MethodsPrimary adipocytes and adipocyte-derived pluripotent cells (APC) were isolated from human adipose tissue and mature adipocytes were dedifferentiated in ceiling culture. The intracellular organization of DFAT and APC were compared using transmission electron microscopy (TEM), and the changes of intracellular lipid content over time were tracked with Oil Red O. Finally, we tested whether liposecretion is a cell cycle-dependent phenomenon by cultivating mature adipocytes in ceiling culture with or without four different inhibitors of the cell cycle (AraC, Irinotecan, Vincristine and RO-3306).ResultsDFAT cells were enriched in intracellular lipids, which are stored in small lipid droplets. In addition, liposecretion, which characterizes mature adipocyte dedifferentiation, is characterized by the rapid secretion of a large lipid droplet that is coated by a membrane. This phenomenon seems to be hindered by the presence of cyclin dependent kinase 1 (CDK1) inhibitor RO-3306.ConclusionBoth human adipose tissue depots undergo dedifferentiation in vitro, but visceral adipose tissue DFAT cells retain more lipids than subcutaneous-derived DFAT cells. Liposecretion is characterized by the rapid ejection of a membrane-wrapped lipid droplet. This phenomenon is dependent on CDK1 and likely relies on the presence of integrin-mediated cellular adherence.

Hepatic stellate cell heterogeneity: Functional aspects and therapeutic implications.

Hepatic stellate cell heterogeneity: Functional aspects and therapeutic implications.

Dual-responsive nanoparticles targeting ACE-II senescence for therapeutic mitigation of acute lung injury

Acute lung injury (ALI) is a life-threatening condition characterized by severe pulmonary dysfunction, with alveolar type II epithelial cell (ACE-II) senescence playing a pivotal role in its progression. In this study, we developed pH/reactive oxygen species (ROS) dual-responsive nanoparticles (GNPsanti-SP-C) for the targeted delivery of Growth Differentiation Factor 15 (GDF15) to counteract ACE-II senescence. These nanoparticles (NPs) effectively activate the AMP-activated protein kinase (AMPK)/Sirtuin 1 (SIRT1) signaling pathway, inducing the mitochondrial unfolded protein response (UPRmt) and reversing senescence-associated cellular dysfunction. GNPsanti-SP-C were systematically engineered and demonstrated robust pH/ROS sensitivity, efficient GDF15 release, and precise ACE-II targeting. In lipopolysaccharide (LPS)-induced ALI mouse model, GNPsanti-SP-C treatment significantly mitigated lung injury, reduced inflammatory responses, and enhanced pulmonary function, as evidenced by decreased inflammatory markers, lung edema, and improved histopathology. Single-cell transcriptomic and proteomic analyses revealed increased ACE-II cell populations, reduced expression of senescence markers, and upregulation of AMPK/SIRT1 signaling. In vitro studies further demonstrated that UPRmt activation is associated with the NPs’ therapeutic effects, suggesting a potential role in their mechanism of action. These findings demonstrate the potential of GDF15-loaded dual-responsive NPs as an innovative strategy to address cellular senescence and alleviate ALI-associated pulmonary damage.Graphical abstract

ScMINER: a mutual information-based framework for clustering and hidden driver inference from single-cell transcriptomics data

Single-cell transcriptomics data present challenges due to their inherent stochasticity and sparsity, complicating both cell clustering and cell type-specific network inference. To address these challenges, we introduce scMINER (single-cell Mutual Information-based Network Engineering Ranger), an integrative framework for unsupervised cell clustering, transcription factor and signaling protein network inference, and identification of hidden drivers from single-cell transcriptomic data. scMINER demonstrates superior accuracy in cell clustering, outperforming five state-of-the-art algorithms and excelling in distinguishing closely related cell populations. For network inference, scMINER outperforms three established methods, as validated by ATAC-seq and CROP-seq. In particular, it surpasses SCENIC in revealing key transcription factor drivers involved in T cell exhaustion and Treg tissue specification. Moreover, scMINER enables the inference of signaling protein networks and drivers with high accuracy, which presents an advantage in multimodal single cell data analysis. In addition, we establish scMINER Portal, an interactive visualization tool to facilitate exploration of scMINER results.

Circadian clock–gated cell renewal controls time-dependent changes in taste sensitivity

Circadian regulation of the cell cycle progression generates a diurnal supply of newborn cells to replace those lost in organs and tissues. In this study, we analyzed circadian time-dependent changes in cell types within the mouse tongue epithelium. Using single-cell RNA sequencing, we observed circadian time-dependent changes in the populations of stem/progenitor cells and the differentiated cells in mice tongues. Notably, we observed time-dependent changes in the type II taste cell population, which were abolished by ablation of taste bud stem cells, thereby inhibiting cell proliferation within the taste cell population. Through experiments with taste bud organoids (TBOs), we found a 24-h cell cycle period, which was disrupted by the knockdown of the core-clock gene Bmal1. In TBOs, both cell divisions and apoptotic cells exhibited circadian time-dependent phenotypes. Interestingly, the time-dependent changes in cell death disappeared in the stem cell-ablated TBOs, indicating that the diurnal supply of newly born cells is essential for the rhythmic cell death phenotype. Additionally, taste tests conducted at different times of the day revealed time-dependent sensitivity changes originating from type II taste cells in mice. These findings suggest that the time-dependent changes in taste cell population are driven by circadian clock-regulated cell cycle progression and control time-dependent physiological regulation in the mouse tongue.

Optimal fractionation scheme for lymphocyte infiltration in glioblastoma multiforme radiotherapy

PurposeRadioresistant and immunosuppressive tumors, such as glioblastoma multiforme (GBM), remain a challenge, as current clinical approaches—surgical resection and chemoradiation—do not yet provide effective treatment. Immunotherapy (IT) has emerged as a powerful tool in cancer; however, phase III clinical trials in GBM have yielded unsuccessful results, likely due to its critical dependence on preexisting antitumor immunity. Given its immunomodulatory potential, radiotherapy (RT) could serve as a tool to induce tumor inflammation and enhance responsiveness to IT. However, the optimal radiation configuration required to achieve the critical level of tumor inflammation for IT success remains elusive. This study assessed the most effective dose fractionation scheme for maximizing immune cell infiltration into tumors.Materials and methodsTwo orthotopic rat glioma models with differing vascularization and immunogenicity were irradiated with three dose fractionation schemes. Tumor immune cell populations were analyzed by flow cytometry.ResultsA single high dose (25 Gy) or extreme hypofractionation is required to elicit a significant immune infiltration in tumors.ConclusionsUsing RT as an immune primer in GBM would require very high and toxic doses with conventional RT methods. While 25 Gy is used in conventional stereotactic radiosurgery, such a high dose is typically limited to small brain volumes. Novel approaches, such as FLASH-RT or minibeam RT, offer alternatives to mitigate toxicity while achieving the required doses.