Increases in cytoplasmic free Ca2+ ions ([Ca2+]) are a critical signal in pancreatic islet beta-cells and are usually required for insulin secretion in response to glucose or other secretagogues. Changes in Ca2+, monitored using high-speed imaging across individual or multiple planes of the islet, can be used to explore the functional networks of beta-cells required for the precise regulation of insulin secretion. These networks are composed of functionally distinct beta-cell subpopulations: first-responders, highly connected hubs, and leader beta-cells, which initiate, connect, and dictate the pattern of spatially organized Ca2+ oscillations, respectively. Alterations in Ca2+ coordination among beta-cells contribute to defective insulin secretion, which underlies all forms of diabetes mellitus. Here, we provide a detailed protocol to perform Ca2+ imaging in isolated rodent islets, focusing on mouse islets expressing the genetic Ca2+ sensor, GCaMP6. We provide a step-by-step guide to evaluate general parameters of islet Ca2+dynamics, coordination, connectivity, and identification of specific functional subpopulations. This approach can be applied to investigate the role of Ca2+ dynamics and coordination in tissues where coordination is critical for normal function.
KIR+NKG2A- natural killer (NK) cells can detect and eliminate malignant and infected cells that have downregulated single HLA class I molecules to escape T cell recognition. So far, these KIRonly NK cells cannot be efficiently expanded in vitro without concomitant co-expression of NKG2A, which modulates their specificity. In this context, we recently demonstrated that circulating innate lymphoid cells 1 (cILC1s) have NK cell progenitor potential and can be differentiated into KIRonly NK cells using murine feeder cells. Here, we established an animal-free culture system enabling the generation and expansion of NK cells from cord blood (CB)-derived cILC1s using human mesenchymal stem cells (MSCs) as feeder cells. Compared to the murine niche provided by the OP9-DL1 cell line, human MSCs generally enabled a much more efficient generation of NK cells, resulting in significantly higher yields of KIR+ NK cells. The frequency of KIRonly NK cells could be further increased by addition of the soluble NOTCH ligand DLL1. Furthermore, we utilized the cILC1/MSC platform to study education of KIRonly NK cells by HLA-C-encoded ligands in a human stem cell niche. This effect was strongest for homozygous (C1/C1) compared to heterozygous (C1/C2) donors, suggesting that cognate KIR/KIR ligand interaction mediates a gene-dosage-dependent education effect. Altogether, this optimized culture protocol overcomes previous limitations by enabling efficient generation of KIR-expressing NK cells in an animal-free, GMP-compatible system. The presented approach may facilitate the clinical translation of NK cell-based strategies for cellular immunotherapy and in addition provides a platform for mechanistic studies of NK cell education.
B cells are key contributors to the pathogenesis of many autoimmune diseases (AID), including multiple sclerosis (MS), and appear to evade the peripheral tolerance checkpoints that normally maintain immune homeostasis. The fate of B cells at these checkpoints is believed to be regulated by intracellular Ca2+ signaling cascades triggered through engagement of B cell receptors (BCR), and by the suppressive effects of regulatory T cells (Tregs). However, most of the current knowledge about Treg-B cell interaction comes from animal studies, while data from human studies, particularly in the context of AID, are sparse. In contrast, impaired Treg-mediated inhibition of conventional T cells (Tcons) has already been described for several AID, including MS. To assess the ability of Tregs to suppress activated B cells in healthy individuals and patients with MS. B and T cell populations were isolated from 40 MS patients and 98 age- and sex-matched healthy donors (HD). Single-cell live Ca²⁺ imaging was used to assess early activation signals in B cells. In vitro proliferation assays and coculture experiments were employed to evaluate downstream responses, including proliferation, transcription factor activation (NFATc1, NF-ĸB), interleukin 6 (IL-6) release, and surface expression levels of antigen-presenting capacity (APC) markers both in anti-IgM/anti-CD40-stimulated B cells alone, and in the presence of Tregs. We demonstrate that Tregs exert a robust suppressive effect on B cell proliferation, IL-6 secretion and NFATc1 which is [1] independent of Ca2+ signaling [2], dependent on direct cell contact, and [3] impaired in MS. In contrast, early Ca2+ responses and downstream effects of anti-IgM/anti-CD40 stimulation, including activation of NFATc1 and NF-κB, as well as proliferation, did not differ between MS- and HD-derived B cells. This study provides new data on Treg-mediated suppression of B cells in humans, including at single-cell level. Our findings show that the Treg dysfunction in MS previously described in the context of Tcon regulation extends to B cell regulation. Given the critical role of B cells in MS pathogenesis, this impaired Treg-B cell interaction may represent a previously underappreciated disease mechanism with potentially important therapeutic implications.
Understanding the mechanisms underlying cell-cell interactions necessitates a versatile method to assemble cells in a precisely-controlled, cell-specific environment, enabling analysis with high spatiotemporal resolution. Here, we introduce a microfluidic technique using stacked flows to create "virtual" channels under constant or pulsatile flow conditions, supporting various assembly geometries, e.g., doublets and triplets of cells with similar or different sizes, while maintaining independent access to each cell. We demonstrate the method using cell lines and primary human cells within the physiological context of immune cell interactions. Selective access to individual cell types is demonstrated with different molecules: fluorescent dyes for staining the nucleus and plasma membrane and to follow cell activity, antibodies for specific molecular targeting, and calcium ionophores to stimulate cellular activity. As shown by real-time observation of early immune-cell activation following immunological synapse formation between T lymphocytes and leukemic cells, this platform provides a powerful tool for analyzing cell-cell interactions and holds strong potential for fundamental research and clinical applications in precision medicine, drug testing, and disease monitoring.
Hepatocellular carcinoma (HCC) is a major contributor to cancer-related mortality worldwide. Store-operated calcium (Ca2+) entry (SOCE), the principal Ca2+ influx pathway in non-excitable cells, has been implicated in regulating tumor cell proliferation, migration, and survival. Although annexin A5 (ANXA5) has been implicated in several malignancies, its mechanistic contribution to Ca2+ signaling in HCC remains unclear. SOCE-related differentially expressed genes were identified through integrated bioinformatics analyses of The Cancer Genome Atlas and the Gene Expression Omnibus datasets. Huh-7 and HepG2 cells with stable ANXA5 knockdown were established using lentiviral transduction. Molecular interactions and functional alterations were examined by co-immunoprecipitation, enzymatic activity assays, inositol 1,4,5-trisphosphate (IP3) quantification, Ca2+ imaging, western blotting, quantitative polymerase chain reaction (PCR), and flow cytometry. Cellular phenotypes were assessed using proliferation and migration assays, whereas tumor growth was evaluated in subcutaneous xenograft models using nude mice. Notably, all in vitro experiments in this study were validated using both Huh-7 and HepG2 cells, whereas only Huh-7 cells were employed for in vivo experiments. ANXA5 was identified as an SOCE-associated gene whose elevated expression correlated with poor prognosis in HCC. Functional assays demonstrated that ANXA5 depletion significantly suppressed HCC cell proliferation and migration. Co-immunoprecipitation assays showed reduced levels of GAPDH co-precipitating with ANXA5 in ANXA5-deficient cells, suggesting impaired association between ANXA5 and GAPDH. Although ANXA5 knockdown did not alter GAPDH expression, it markedly reduced GAPDH enzymatic activity, leading to decreased IP3 production, impaired endoplasmic reticulum Ca2+ release, and attenuated SOCE-mediated Ca2+ influx. Importantly, pharmacological modulation of phospholipase C (PLC) activity with U73122 and its inactive analog U73443 further supported the involvement of PLC-IP3 signaling in SOCE impairment and malignant phenotypes following ANXA5 depletion. In vivo, ANXA5 silencing significantly inhibited tumor growth and was accompanied by reduced expression of Ki-67, vimentin, and the M2 macrophage marker cluster of differentiation 206 (CD206). These findings support a working model in which ANXA5 interacts with GAPDH and is associated with altered IP3 production and SOCE-dependent Ca2+ signaling, potentially contributing to HCC progression and immune modulation. Collectively, this ANXA5/GAPDH/IP3/SOCE axis may provide a mechanistic framework for understanding HCC development and suggests ANXA5 as a potential therapeutic target.
Intracellular calcium (Ca2+) dynamics drives contractile function in cardiac myocytes. In particular, L-type Calcium Channels (LCCs) and Ryanodine Receptors (RyRs) are organized in microdomains, where LCCs trigger substantial Ca2+ release from the Sarcoplasmic Reticulum (SR) via RyRs. Different microdomains can be coupled at different length scales by calcium diffusion or common external activation. We present a Scalable Aggregate Calcium Release Unit (SA-CaRU) model for human ventricular myocytes that integrates a recently developed Markov Chain (MC)-based description of LCCs, replacing classical Hodgkin-Huxley gates. Our approach is based on previously published MC-based frameworks for the human heart, enabling stochastic gating and reproducing evoked local Ca2+ release statistics across different effective levels of microdomain aggregation. Our single-SA-CaRU system captures, within a unified framework, key features of microscale and macroscale Ca2+ cycling and allows, for the first time, systematic exploration of variability in SR Ca2+ release as a function of effective microdomain size and coupling. Simulations with increasing numbers of channels reveal that the transition from stochastic to deterministic-like Ca2+ behavior is typically sharp at a specific cluster size. Under normal (healthy) conditions, this occurs at O ( 1 0 2 ) LCCs (with mild sensitivity to the RyR:LCC scaling). However, under high phosphorylation or LCC upregulation, stochasticity persists and convergence to deterministic-like behavior is absent or markedly delayed even for total LCC numbers as large as 20,000. In these conditions, whole-cell deterministic models become doubtful, since their behavior can be qualitatively different from that arising from any plausibly mediated coordination of subcellular calcium release units.
This systematic review aimed to answer the following PICO question: Does dentin conditioning with different intracanal medications alter the adhesion, migration, and differentiation of undifferentiated cells during regenerative endodontic therapy?. A comprehensive search was performed in PubMed, Embase, Scopus, LILACS, and Web of Science up to May 2025. Gray literature was screened through Google Scholar and ProQuest. In vitro studies evaluating the effects of dentin conditioning with intracanal medications on cell behavior (proliferation, adhesion, migration, and differentiation) were included. Two reviewers independently conducted study selection, data extraction, and risk of bias assessment using the RoBDEMAT tool. Data were qualitatively synthesized. From 2,620 retrieved records, 12 studies fulfilled the eligibility criteria. Most studies used human root dentin as a substrate and tested calcium hydroxide-based formulations, triple antibiotic paste (TAP), or double antibiotic paste (DAP); others evaluated silver nanoparticles, calcium hypochlorite, Bio-C Temp, or antibiotic-containing fibers. Dental pulp stem cells were most frequently used, followed by apical papilla, deciduous tooth, and periodontal ligament stem cells. Conditioning times ranged from 7 to 28 days. Calcium hydroxide showed the most favorable outcomes for cell adhesion, morphology, and proliferation. Lower antibiotic concentrations (1 mg/mL) demonstrated acceptable biocompatibility. Only one study assessed cell differentiation, showing increased DMP-1 and DSPP expression after calcium hydroxide conditioning; none evaluated cell migration. Calcium hydroxide exhibited the most consistent and favorable biological responses. TAP and DAP showed concentration-dependent cytotoxicity; however, more robust evidence is needed to clarify how intracanal medications influence cell behavior on conditioned dentin.
Calmodulin (CAM) is a highly conserved calcium ion sensor in eukaryotic cells. Its molecule contains four EF-hand domains, which can respond to changes in intracellular calcium ion concentration and conduct signals. As the core mediator of calcium signals, CAM further activates the downstream Calcium/calmodulin-dependent protein kinases (CAMK) family. CAMK is a type of serine/threonine protein kinase with catalytic and regulatory domains. Its classical activation method is the formation of a complex between intracellular calcium ions and CAM and binding to the regulatory domain of CAMK, inducing conformational changes, thereby phosphorylating downstream effect proteins. Besides calcium ions, its activity can also be regulated by small molecules such as citric acid. In the field of oncology, CAMK2 is overexpressed in various malignant tumors such as non-small cell lung cancer and colorectal cancer, and is closely related to tumor proliferation, invasion, and poor prognosis. The mechanism involves regulating tumor stem cells and AMPK and other signaling pathways. In addition, the CAMK family also participates in inflammatory responses by regulating NF-κB and intervenes in metabolic processes such as glycolysis and fatty acid β-oxidation, thereby playing a role in immune diseases and metabolic disorders. In summary, the CAMK family serves as a key hub of calcium signals and is widely involved in the processes of cardiovascular diseases, tumors, immune abnormalities, and metabolic disorders. In-depth exploration of its structure and regulatory mechanisms will provide new targets and strategies for the treatment of related diseases.
To investigate the compensatory role of α-cell-derived paracrine signaling through glucagon and GLP-1 receptors in maintaining β-cell function when insulin secretion is compromised. A β-cell-specific Nox4 knockout mouse (Nox4βKO) displays defective glucose-stimulated insulin secretion and develops a prediabetic phenotype. To uncover the adaptive changes, we ran a detailed analysis of Nox4βKO pancreatic islets. We analyzed their composition, hormone secretion dynamics, receptor expression profiles, and downstream signaling pathways by immunocytochemistry, flow cytometry, RNA sequencing, cAMP assays, and insulin or glucagon secretion assays using both isolated islets and pancreatic slices across different glucose levels and receptor-modulating conditions. Prediabetic Nox4βKO islets showed increased α-cell numbers, expansion of bihormonal cells, and elevated production of glucagon and GLP-1. Receptor profiling revealed a shift in receptor engagement: whereas GLP-1R dominated in wild-type islets, GCGR signaling gained prominence in Nox4βKO islets. This functional rebalancing is consistent with an adaptive response to emerging β-cell dysfunction. Functional assays demonstrated that insulin secretion in prediabetic islets became increasingly reliant on glucagon-driven potentiation of GLP-1R and cAMP-dependent pathways. Transcriptomic and signaling data confirmed enhanced expression of cAMP-related intermediates and calcium-handling components, indicating partial preservation of insulin secretory capacity despite underlying defects. α-cell remodeling and flexible engagement of glucagon and GLP-1 receptors act as key compensatory mechanisms that may help to sustain insulin secretion during early β-cell stress. The context-dependent plasticity of intra-islet receptor activation highlights a coordinated multicellular adaptation in prediabetes and suggests that targeting intra-islet endocrine crosstalk may help preserve β-cell function in prediabetes.
Spiral ganglion neurons (SGNs) relay auditory sensory information from the cochlea to the brain. Their loss results in permanent hearing impairment in humans due to their limited regenerative capacity. Progress in hearing restoration has been constrained by the inaccessibility of human inner ear tissue and challenges in generating functionally mature human SGN-like neurons from stem cells in vitro. To generate human SGN-like neurons from human induced pluripotent stem cells (hiPSCs), we recapitulated key signaling pathways involved in human inner ear development. On day (D) 11 of differentiation, nerve growth factor receptor-positive cells (precursors of pre-placodal ectoderm and neural crest) were isolated using magnetic sorting. From D18 to D25, cultures were treated with sonic hedgehogs to induce otic neural progenitors. Neuronal maturation was subsequently promoted by a cocktail of brain-derived neurotrophic factor, neurotrophin-3, and insulin-like growth factor-1, which supports SGN development. Cellular identity and functionality were assessed using single-cell RNA sequencing, immunocytochemistry, whole-cell patch-clamp electrophysiology, co-culture assays, and calcium ion (Ca²⁺) imaging. hiPSC-derived SGN-like neurons exhibited morphological, molecular, electrophysiological, and functional characteristics of SGNs in vivo. Neurons acquired bipolar morphology and were wrapped by glial cells. Transcriptomic analysis revealed that SGN-like neurons were distinct from other neuronal lineages and showed similarity to type I and type II SGNs based on expression of synaptic and intrinsic excitability-related genes. Electrophysiological recordings revealed progressive hyperpolarization of resting membrane potential and emergence of overshooting action potentials, consistent with neuronal maturation. In co-culture systems, human SGN-like neurons formed functional synaptic connections with mouse cochlear hair cells and cochlear nucleus neurons, evidenced by Ca2+ transients and induction of the immediate early gene c-Fos. This study reports a robust and reproducible protocol for generating human SGN-like neurons from hiPSCs, providing a versatile platform for studying human auditory development, disease modeling, drug screening, and cell-based therapies for hearing restoration.
Can single-cell, mass spectrometry-based proteomics identify proteins associated with reduced developmental competence of Patl2-/- Metaphase II (MII) mouse oocytes and reveal therapeutic targets for Patl2-related infertility? Abnormal protein content is detected in Patl2-/- MII oocytes, which can be rescued by spindle transfer (ST). PATL2 is an RNA-binding protein that represses maternal mRNA translation during oocyte maturation. PATL2 mutations in humans often cause germinal vesicle (GV) arrest, although some affected patients produce MII oocytes with reduced fertilization and embryo developmental potential. Consequently, oocyte donation is required. The Patl2-/- knockout mouse model offers a unique opportunity to study Patl2-related infertility and evaluate potential treatments. Patl2 -/- mice (C57BL/6NTac-Patl2tm1a), with deletion of exon 7, were bred from April 2021 to October 2023, yielding 36 homozygous females from 271 pups. To investigate the role of Patl2 at the MII stage, in vivo MII oocytes from Patl2-/- and Patl2+/+ females were collected for analysis of key quality markers and single-cell proteomics. Based on these results, maternal ST was tested to rescue abnormal embryo development. At least three replicates were conducted per experiment. Four- to 12-week-old mice underwent superovulation and oocyte collection to assess in vitro and in vivo maturation. In vivo-matured MII oocytes were used to evaluate activation (AR) and blastocyst rates (BR) after PIEZO-ICSI, spindle configuration, and calcium oscillatory patterns following SrCl2 exposure. Vitrified-warmed oocytes were used for single-cell proteomics using a timsTOF ultra mass spectrometer operated in diaPASEF mode. ST involved transferring the Patl2-/- spindle to Patl2+/+ enucleated cytoplasm, followed by parthenogenetic activation (PA) via SrCl2 exposure. Patl2 -/- females exhibit lower in vivo MII rates (79.63%) than Patl2+/+ females (89.39%, P = 0.0123) but similar in vitro maturation rates (GV-MII = 48.74%) compared to Patl2+/+ females (52.85%, P = 0.5230). After PIEZO-ICSI with wild-type sperm, reduced AR (Patl2-/- = 31.71%, Patl2+/+ = 76.74%, P < 0.0001) and BR (Patl2-/- = 7.69%, Patl2+/+ = 42.42%, P = 0.0237) were observed in knockout oocytes. However, Patl2-/- oocytes exhibited normal spindle rates (78.57%) as seen in Patl2+/+ oocytes (86.00%, P = 0.3491), as well as a similar capacity to sustain long-lasting calcium oscillations (A×F = 6.15 ± 4.80) compared to Patl2+/+ oocytes (A×F = 4.59 ± 2.96, P = 0.1453). Single-cell proteomics identified 4882 proteins and confirmed the absence of Patl2 in knockout oocytes, from analyzing 25 Patl2+/+ and 27 Patl2-/- MII oocytes. After filtering, 3747 proteins were used for statistical analysis, revealing 1508 differentially expressed proteins (q-value < 0.05; 992 downregulated, 516 upregulated in Patl2-/- oocytes). The levels of multiple RNA-binding proteins, some of which are proposed Patl2 interactors (Cpeb1, Eif4e1b), were found to be significantly reduced in Patl2-/- oocytes. Additionally, the protein products of several maternal effect genes (MEGs) implicated in mRNA regulation (Zar1, Igf2bp2) and cell cycle division (Tcl1a, Cdk1, Mos) were downregulated, while MEGs participating in epigenetic modifications (Zfp57, Trim28) were upregulated in the knockout group. Consistent with these observations, ST-PA treatment significantly increased AR (100%) and BR (75%) in the Patl2-/- oocytes in comparison to PA alone (AR = 75.95%, P = 0.0078; BR = 45.00%, P = 0.0128), effectively rescuing development to wild-type levels. Lastly, ST-PA treatment did not alter embryonic development in Patl2+/+ oocytes and produced outcomes comparable to PA alone, supporting the technical safety and applicability of the technique. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE massIVE partner repository with the dataset identifier MSV000100606. Patl2 -/- mice exhibit a less severe phenotype compared to patients carrying PATL2 variants. Patl2-/- female mice display a high MII rate without significant spindle abnormalities, which contrasts with a previously published report. Additionally, ST treatment was conducted using parthenogenetically activated oocytes, rather than biparental embryos. ST represents a promising treatment for PATL2-related female infertility in patients with MII oocytes, as it appears to restore cytoplasmic defects linked to abnormal RNA-binding proteins and MEGs identified by single-cell proteomics. In contrast, other proposed treatments for poor embryo development, such as assisted oocyte activation, is unlikely to be effective since Patl2-/- oocytes show a normal calcium response. This study was supported by the Special Research Fund (BOF) (starting grant BOF.STG.2021.0042.01 awarded to B.H.) and the Research Foundation-Flanders (FWO) (fellowship 1177425 N awarded to E.A.). B.H. has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). A.C.B., E.A., A.C., M.F-I-A., J.G., A.R., M.B., A.B., C.A., K.C.P., D.S., K.G., and F.V.M. have nothing to disclose. B.H. reports being board member of the Belgian Ethical Committee on embryo research.
Loading of calcium ions into the numerous carboxy-proximal binding sites in the Repeats in ToXin (RTX) domains drives the cooperative and vectorial folding of RTX β-roll structures involved in cell binding and membrane penetration of RTX cytolysins. Two additional binding sites for calcium ions, coordinated by the side chains of residues D880, D918, and N936, were identified in the structure of the acylated cap of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA). We show that this calcium-binding structure plays a key role in membrane insertion of the toxin. An N936L residue substitution did not impact toxin acylation or CR3 receptor binding but disrupted the calcium-driven folding of the acylated segment and ablated the membrane penetration capacity of the toxin. Similarly, substitution of the corresponding D639 residue of Escherichia coli α-hemolysin (HlyA) abolished its cytolytic capacity. Moreover, disruption of the β-turn structures in the calcium-binding sites of the acylated segment of CyaA (G934L) and HlyA (G637L) strongly impaired the cytotoxic capacities of both toxins. On the contrary, a D880L substitution yielded a toxin with an enhanced CR3-independent cell penetration and pore-forming capacity. Hydrogen/deuterium exchange probing revealed that the D880L substitution altered the fold of the acylated segment and the interaction of its two acylated β-hairpins. Hence, loading the calcium-binding sites in the acylated segment controls its structure and rules the interaction and functional cooperation of its two acylated β-hairpins that facilitate penetration of the CyaA polypeptide into the cell membrane.
Decellularized extracellular matrix (dECM) is a promising bioink because it replicates the biochemical and structural features of native tissues. However, tissue-derived dECM is limited by restricted availability and potential immunogenicity. To overcome these challenges, we developed a macromolecular crowding (MMC)-enhanced cell-derived extracellular matrix (CD-ECM) bioink with improved yield and biofunctionality. MC3T3-E1 pre-osteoblasts were cultured under MMC conditions, followed by decellularization and enzymatic processing to generate a printable CD-ECM bioink. The MMC strategy markedly increased extracellular matrix (ECM) yield, collagen and glycosaminoglycan (GAG) content, and mechanical stability compared to conventional cultures. The optimized CD-ECM bioink exhibited reliable printability in both extrusion-based and digital light processing (DLP) 3D bioprinting, enabling fabrication of constructs with high shape fidelity and cell viability. Incorporation of α-tricalcium phosphate (α-TCP) further enhanced osteogenic performance, resulting in elevated alkaline phosphatase (ALP) activity, increased calcium deposition, and upregulation of osteogenic markers, including runt-related transcription factor 2 (RUNX2), collagen type I alpha 1(COL1A1), ALP, and osteocalcin (OCN). These findings highlight the synergistic interaction between ECM-derived biochemical cues and α-TCP-mediated ionic signaling. Overall, the MMC-enhanced CD-ECM/α-TCP bioink offers a versatile, biologically active, and osteoinductive platform for advanced bone tissue engineering and regenerative applications.
Regenerating cementum remains a major unmet challenge in periodontal and peri-implant therapy, underscoring the need to understand how cementoblasts respond to engineered surface cues. This study examined the manner in which titanium nanosurfaces integrating anisotropic nanopatterns with three-dimensional (3D) nanospike architectures regulate mechanotransduction and matrix mineralization in human cementoblast-like cells (hCEM). Titanium surfaces with isotropic, anisotropic, and 3D anisotropic nanospike architectures were fabricated and characterized through quantitative analyses of nanoscale geometry and topographical organization. Surface chemistry and crystallinity were characterized using Fourier transform infrared spectroscopy, grazing-incidence X-ray diffraction, and X-ray photoelectron spectroscopy. hCEM cultures on each surface were evaluated for extracellular calcium (Ca) and phosphate (P) levels, Ca/P ratios, extracellular matrix crystallinity, cytomorphology, and phosphate metabolism-associated gene expression. Mechanotransduction activity was assessed through focal adhesion-Hippo pathway signaling. Relationships between nanoscale architecture, cell stimulation, morphology, and mineralization were examined using correlation and path analyses. Despite comparable wettability and oxide chemistry to that of other nanosurfaces, 3D anisotropic nanospike surfaces produced the highest mineralization and exhibited the highest Ca/P ratios, clear hydroxyapatite signatures, pronounced extracellular nodules, and coordinated activation of phosphate metabolism gene profiles. These surfaces induced prominent nanoscale vertex-cell interactions and distinct cytomorphological responses. Mineralization did not show association with vertical roughness, hydroxyl content, or crystallographic features but positively correlated (r = 0.94) with composite nanoscale architectural metrics capturing spatial heterogeneity and vertex density. The finding that anisotropic 3D nanospike architectures are associated with enhanced matrix mineralization in human cementoblast-like cells under osteogenic conditions provides mechanistic insight into how nanoscale architecture modulates mineralization responses and may inform the design of cementum-targeted bioactive titanium surfaces.
The low yield of high-activity calcium-binding peptides constitutes a technical bottleneck restricting their application. Herein, a static affinity chromatography approach using needle-shaped hydroxyapatite was developed for the isolation of a novel cod bone calcium-binding peptide, GRGNEGPQ (CBP-8). The calcium-binding mechanism was investigated using experimental and computational methods, while its digestive behavior and calcium transport effects were evaluated using an in vitro digestion model and Caco-2 cell monolayer model. Results indicated that the carboxyl group of Gln served as a potential calcium-binding site and the CBP-8-Ca complex formed through multiple chelation modes. Electrostatic interactions were critical for coordination bond formation, with a formal charge transfer of -2.13 e from Ca2+ in the CBP-8-Ca complex. CBP-8-Ca exhibited good gastric stability but degraded intestinally. Additionally, CBP-8 promoted Ca2+ transport via non-specific calcium channels and TRPV6 channels. This study offers a promising and cost-effective strategy for the efficient production of highly active calcium-binding peptides.
Macrophages, as a major immune cell population within the tumor microenvironment (TME), play a pivotal role in disease progression and therapeutic outcomes. This study aimed to identify key macrophage subsets associated with ESCC immunotherapy response. Macrophage-specific marker gene associated with immunotherapy response was identified through single-cell RNA sequencing (scRNA-seq) analysis. The clinical relevance of CXCL2+ macrophages in ESCC patients was assessed by immunofluorescence staining. RNA sequencing was utilized to explore the role of CXCL2 in modulating macrophage functional phenotypes. The impact of CXCL2 on ESCC immunotherapy was validated by ESCC mouse model. ScRNA-seq analysis showed that CXCL2 was predominantly expressed in macrophages within TME and significantly upregulated in immunotherapy-responsive ESCC patients. Low infiltration of intratumoral CXCL2+ macrophages was correlated with poor survival. Mechanistically, CXCL2 promoted the phenotypic transition of macrophages to an immune-activated state by facilitating cytosolic Ca2+ influx. Additionally, CXCL2 suppressed tumor growth and enhanced the efficacy of anti-PD-1 antibody therapy in ESCC mouse models. Macrophage-specific CXCL2 represents a novel biomarker for predicting immunotherapy efficacy and may potentiate the efficacy of anti-PD-1 therapy in ESCC patients.
Although some forms of epilepsy directly result from mutations in nicotinic acetylcholine receptors, none of the currently available antiepileptic drugs (AEDs) is specifically designed to target the cholinergic system. However, there is growing evidence that some established AEDs, which were primarily designed to modulate excitatory glutamatergic and/or inhibitory GABAergic currents, may also influence cholinergic signalling. This study therefore investigated whether topiramate (TPM), a second-generation AED, directly affects calcium signals and whether the deacetylase sirtuin-1 (Sirt-1) contributes to this effect. Calcium imaging in the human neuroblastoma cell line SH-SY5Y was used to quantify acetylcholine- and nicotine-induced calcium signals following TPM treatment. To evaluate the role of protein deacetylases, TPM effects were further analysed in the presence of the deacetylase inhibitors trichostatin A (TSA) and inauhzin. TPM treatment significantly enhanced acetylcholine- and nicotine-induced calcium signals. This effect of TPM was completely abolished in the presence of TSA. However, the presence of inauhzin resulted in an inhibitory effect of TPM on acetylcholine-induced calcium signals. These findings reveal a previously uninvestigated modulatory effect of TPM on cholinergic calcium signalling that is directly dependent on the activity of deacetylases, like Sirt-1. The results may contribute to a better understanding of TPM's anticonvulsive mechanisms of action.
Coal is a major global energy resource, but its extraction raises significant environmental and health concerns. Mining activities release large amounts of particulate matter, including nanoparticles (NPs), into the environment. These NPs are hazardous due to their content of polycyclic aromatic hydrocarbons (PAHs), metals, oxides, and other compounds capable of disrupting cellular and molecular processes. This study evaluated the cytotoxic and genotoxic effects of coal-derived NPs on V79 and HaCaT cell lines, focusing on their impact on DNA stability and the mechanisms responsible for cellular damage. NPs were isolated using an acid-based separation method and applied to cells at concentrations of 50, 150, and 300 μg/mL. Atomic force microscopy (AFM) provided topographical characterization, while dynamic light scattering (DLS) confirmed their tendency to agglomerate. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) confirmed NP morphology and elemental composition, including carbon, oxygen, iron, calcium, silicon, aluminum, and copper. Cytotoxicity was assessed using resazurin and sulforhodamine B assays, and genotoxicity was evaluated using the comet assay, micronucleus test, and γH2AX immunostaining. Results showed a clear dose-dependent effect, with coal NPs inducing genomic instability and increased cell mortality, mainly through apoptosis. These findings highlight the importance of characterizing coal-derived NPs to better assess their environmental and health risks, particularly regarding respiratory diseases.
Chimeric antigen receptor (CAR) T-cell therapy is increasingly used for hematologic malignancies, yet the spectrum and clinical significance of its endocrine adverse events (AEs) remain poorly characterized. To identify and clinically contextualize signals of endocrine AEs associated with CAR T-cell therapy, with a focus on their overlap with cytokine release syndrome (CRS). We performed a retrospective disproportionality analysis using the FDA Adverse Event Reporting System (FAERS) database (2017 to Q2 2025). Reporting odds ratio (ROR) and information component (IC) were calculated for endocrine AEs associated with six CAR T-cell products. To validate and elaborate these pharmacovigilance signals, a structured literature review was conducted to identify published clinical evidence. The FAERS analysis identified 269 endocrine AE reports, yielding 14 significant disproportionality signals. Hyperglycemia was the most frequently reported event, while estrogen deficiency showed the strongest signal strength (ROR₀₂₅ = 14.93). Signals for adrenal insufficiency and hypothalamo-pituitary disorders were primarily associated with axicabtagene. Critically, mortality was frequently reported among cases with positive endocrine signals, particularly when the endocrine AE co-occurred with CRS. The literature review provided direct clinical validation: a retrospective study confirmed a 39% incidence of CRS-associated hyperglycemia, and independent case reports documented the first instances of CAR T-cell therapy-induced Hashimoto's thyroiditis and central diabetes insipidus. These clinical cases corroborated the FAERS signals and suggested immune-inflammatory mechanisms, often independent of corticosteroid use. This integrated analysis reveals a distinct spectrum of CAR T-cell-related endocrine toxicities, encompassing glucose and calcium dysregulation, pituitary axis disorders, and autoimmune phenomena. The frequent and potentially fatal overlap with CRS underscores the need for enhanced clinical vigilance. Proactive monitoring of endocrine function, especially in patients experiencing CRS, is warranted to mitigate these underrecognized complications.
Plant-derived extracellular vesicles (PDEVs) are emerging as bioactive dietary particles with the capacity to modulate mammalian physiology. Here, we characterize the structure and functional properties of apple-derived extracellular vesicles (ADEVs) and evaluate their relevance to neuroimmune and gut-brain communication. ADEVs exhibit canonical PDEV features and elicit rapid IP₃-dependent Calcium (Ca²⁺) signaling in fibroblasts while preserving blood-brain barrier integrity. Neural assays reveal marked cell-type specificity: ADEVs are efficiently internalized by glial cells and activate glial Ca²⁺ signaling yet display minimal neuronal uptake and no detectable Ca²⁺ response in differentiated SH-SY5Y neurons. Consistent with this selectivity, ADEVs attenuate TNF-α-induced cytokine secretion in activated glia but remain inert in resting neural cells. Although capable of encapsulating L-DOPA efficiently, ADEVs fail to deliver functional neuroprotection against rotenone toxicity, indicating limited neuronal compatibility for dopaminergic cargo. In parallel, using advanced in vitro colon simulation platforms, ADEVs modulate the colonic microbiome in a dose-dependent manner, promoting carbohydrate fermentation and short-chain fatty acid production while reducing proteolytic metabolism under physiologically relevant conditions, with sustained, region-specific effects during prolonged exposure. In vivo, ADEV administration in dogs with chronic intestinal inflammation is associated with altered circulating serotonin levels, suggesting engagement of gut-brain neurochemical pathways with potential implications for mood regulation. Collectively, these findings identify ADEVs as biocompatible, glia-responsive plant vesicles with potential neuromodulatory activity, while delineating intrinsic constraints in their use as neuronal drug-delivery systems.