Intracellular galectin interactions in health and disease.

In the galectin family, a group of lectins is united by their evolutionarily conserved carbohydrate recognition domains. These polypeptides play a role in various cellular processes and are implicated in disease mechanisms such as cancer, fibrosis, infection, and inflammation. Following synthesis in the cytosol, manifold interactions of galectins have been described both extracellularly and intracellularly. Extracellular galectins frequently engage with glycoproteins or glycolipids in a carbohydrate-dependent manner. Intracellularly, galectins bind to non-glycosylated proteins situated in distinct cellular compartments, each with multiple cellular functions. This diversity complicates attempts to form a comprehensive understanding of the role of galectin molecules within the cell. This review enumerates intracellular galectin interaction partners and outlines their involvement in cellular processes. The intricate connections between galectin functions and pathomechanisms are illustrated through discussions of intracellular galectin assemblies in immune and cancer cells. This underscores the imperative need to fully comprehend the interplay of galectins with the cellular machinery and to devise therapeutic strategies aimed at counteracting the establishment of galectin-based disease mechanisms.

Inhibition and Mechanism of Citral on Bacillus cereus Vegetative Cells, Spores, and Biofilms.

Bacillus cereus causes food poisoning by producing toxins that cause diarrhea and vomiting and, in severe cases, endocarditis, meningitis, and other diseases. It also tends to form biofilms and spores that lead to contamination of the food production environment. Citral is a potent natural antibacterial agent, but its antibacterial activity against B. cereus has not been extensively studied. In this study, we first determined the minimum inhibitory concentrations and minimum bactericidal concentrations, growth curves, killing effect in different media, membrane potential, intracellular adenosine triphosphate (ATP), reactive oxygen species levels, and morphology of vegetative cells, followed by germination rate, morphology, germination state of spores, and finally biofilm clearance effect. The results showed that the minimum inhibitory concentrations and minimum bactericidal concentrations of citral against bacteria ranged from 100 to 800 µg/mL. The lag phase of bacteria was effectively prolonged by citral, and the growth rate of bacteria was slowed down. Bacteria in Luria-Bertani broth were reduced to below the detection limit by citral at 800 µg/mL within 0.5 h. Bacteria in rice were reduced to 3 log CFU/g by citral at 4000 µg/mL within 0.5 h. After treatment with citral, intracellular ATP concentration was reduced, membrane potential was altered, intracellular reactive oxygen species concentration was increased, and normal cell morphology was altered. After treatment with citral at 400 µg/mL, spore germination rate was reduced to 16.71%, spore morphology was affected, and spore germination state was altered. It also had a good effect on biofilm removal. The present study showed that citral had good bacteriostatic activity against B. cereus vegetative cells and its spores and also had a good clearance effect on its biofilm. Citral has the potential to be used as a bacteriostatic substance for the control of B. cereus in food industry production.

Multiscale topology classifies cells in subcellular spatial transcriptomics.

Spatial transcriptomics measures in situ gene expression at millions of locations within a tissue1, hitherto with some trade-off between transcriptome depth, spatial resolution and sample size2. Although integration of image-based segmentation has enabled impactful work in this context, it is limited by imaging quality and tissue heterogeneity. By contrast, recent array-based technologies offer the ability to measure the entire transcriptome at subcellular resolution across large samples3-6. Presently, there exist no approaches for cell type identification that directly leverage this information to annotate individual cells. Here we propose a multiscale approach to automatically classify cell types at this subcellular level, using both transcriptomic information and spatial context. We showcase this on both targeted and whole-transcriptome spatial platforms, improving cell classification and morphology for human kidney tissue and pinpointing individual sparsely distributed renal mouse immune cells without reliance on image data. By integrating these predictions into a topological pipeline based on multiparameter persistent homology7-9, we identify cell spatial relationships characteristic of a mouse model of lupus nephritis, which we validate experimentally by immunofluorescence. The proposed framework readily generalizes to new platforms, providing a comprehensive pipeline bridging different levels of biological organization from genes through to tissues.

Influence of intersignaling crosstalk on the intracellular localization of YAP/TAZ in lung cells.

A cell is a dynamic system in which various processes occur simultaneously. In particular, intra- and intercellular signaling pathway crosstalk has a significant impact on a cell's life cycle, differentiation, proliferation, growth, regeneration, and, consequently, on the normal functioning of an entire organ. Hippo signaling and YAP/TAZ nucleocytoplasmic shuttling play a pivotal role in normal development, homeostasis, and tissue regeneration, particularly in lung cells. Intersignaling communication has a significant impact on the core components of the Hippo pathway and on YAP/TAZ localization. This review describes the crosstalk between Hippo signaling and key lung signaling pathways (WNT, SHH, TGFß, Notch, Rho, and mTOR) using lung cells as an example and highlights the remaining unanswered questions.

NtGCN2 confers cadmium tolerance in Nicotiana tabacum L. by regulating cadmium uptake, efflux, and subcellular distribution.

General control non-derepressible-2 (GCN2) is widely expressed in eukaryotes and responds to biotic and abiotic stressors. However, the precise function and mechanism of action of GCN2 in response to cadmium (Cd) stress in Nicotiana tabacum L. (tobacco) remains unclear. We investigated the role of NtGCN2 in Cd tolerance and explored the mechanism by which NtGCN2 responds to Cd stress in tobacco by exposing NtGCN2 transgenic tobacco lines to different concentrations of CdCl2. NtGCN2 was activated under 50 µmol·L-1 CdCl2 stress and enhanced the Cd tolerance and photosynthetic capacities of tobacco by increasing chlorophyll content and antioxidant capacity by upregulating NtSOD, NtPOD, and NtCAT expression and corresponding enzyme activities and decreasing malondialdehyde and O2·- contents. NtGCN2 enhanced the osmoregulatory capacity of tobacco by elevating proline (Pro) and soluble sugar contents and maintaining low levels of relative conductivity. Finally, NtGCN2 enhanced Cd tolerance in tobacco by reducing Cd uptake and translocation, promoting Cd efflux, and regulating Cd subcellular distribution. In conclusion, NtGCN2 improves the tolerance of tobacco to Cd through a series of mechanisms, namely, increasing antioxidant, photosynthetic, and osmoregulation capacities and regulating Cd uptake, translocation, efflux, and subcellular distribution. This study provides a scientific basis for further exploration of the role of NtGCN2 in plant responses to Cd stress and enhancement of the Cd stress signaling network in tobacco.

Carboxymethylated Desmodium styracifolium polysaccharide reduces the risk of calcium oxalate kidney stone formation by inhibiting crystal adhesion and promoting crystal endocytosis.

The inhibition of cell surface crystal adhesion and an appropriate increase in crystal endocytosis contribute to the inhibition of kidney stone formation. In this study, we investigated the effects of different degrees of carboxymethylation on these processes. An injury model was established by treating human renal proximal tubular epithelial (HK-2) cells with 98.3 ± 8.1 nm calcium oxalate dihydrate (nanoCOD) crystals. The HK-2 cells were protected with carboxy (-COOH) Desmodium styracifolium polysaccharides at 1.17% (DSP0), 7.45% (CDSP1), 12.2% (CDSP2), and 17.7% (CDSP3). Changes in biochemical indexes and effects on nanoCOD adhesion and endocytosis were detected. The protection of HK-2 cells from nanoCOD-induced oxidative damage by carboxymethylated Desmodium styracifolium polysaccharides (CDSPs) is closely related to the protection of subcellular organelles, such as mitochondria. CDSPs can reduce crystal adhesion on the cell surface and maintain appropriate crystal endocytosis, thereby reducing the risk of kidney stone formation. CDSP2 with moderate -COOH content showed the strongest protective activity among the CDSPs.

Modulating Intracellular Dynamics for Optimized Intracellular Release and Transcytosis Equilibrium.

Active transcytosis-mediated nanomedicine transport presents considerable potential in overcoming diverse delivery barriers, thereby facilitating tumor accumulation and penetration. Nevertheless, the persistent challenge lies in achieving a nuanced equilibrium between intracellular interception for drug release and transcytosis for tumor penetration. In this study, a comprehensive exploration is conducted involving a series of polyglutamine-paclitaxel conjugates featuring distinct hydrophilic/hydrophobic ratios (HHR) and tertiary amine-oxide proportions (TP) (OPGA-PTX). The screening process, meticulously focused on delineating their subcellular distribution, transcytosis capability, and tumor penetration, unveils a particularly promising candidate denoted as OPPX, characterized by an HHR of 10:1 and a TP of 100%. OPPX, distinguished by its rapid cellular internalization through multiple endocytic pathways, selectively engages in trafficking to the Golgi apparatus for transcytosis to facilitate accumulation within and penetration throughout tumor tissues and simultaneously sorted to lysosomes for cathepsin B-activated drug release. This study not only identifies OPPX as an exemplary nanomedicine but also underscores the feasibility of modulating subcellular distribution to optimize the active transport capabilities and intracellular release mechanisms of nanomedicines, providing an alternative approach to designing efficient anticancer nanomedicines.

Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

Activating plant immunity: the hidden dance of intracellular Ca2+ stores.

Calcium ion (Ca2+) serves as a versatile and conserved second messenger in orchestrating immune responses. In plants, plasma membrane-localized Ca2+-permeable channels can be activated to induce Ca2+ influx from extracellular space to cytosol upon pathogen infection. Notably, different immune elicitors can induce dynamic Ca2+ signatures in the cytosol. During pattern-triggered immunity, there is a rapid and transient increase in cytosolic Ca2+, whereas in effector-triggered immunity, the elevation of cytosolic Ca2+ is strong and sustained. Numerous Ca2+ sensors are localized in the cytosol or different intracellular organelles, which are responsible for detecting and converting Ca2+ signals. In fact, Ca2+ signaling coordinated by cytosol and subcellular compartments plays a crucial role in activating plant immune responses. However, the complete Ca2+ signaling network in plant cells is still largely ambiguous. This review offers a comprehensive insight into the collaborative role of intracellular Ca2+ stores in shaping the Ca2+ signaling network during plant immunity, and several intriguing questions for future research are highlighted.

Nanoscale Methods for Longitudinal Extraction of Intracellular Contents.

Longitudinal analysis of intracellular contents including gene and protein expression is crucial for deciphering the fundamentally dynamic nature of cells. This offers invaluable insights into complex tissue composition and behavior, and drives progress in disease diagnosis, biomarker discovery, and drug development. Traditional longitudinal analysis workflows, involving the destruction of cells at various timepoints, limit insights to singular moments and fail to account for cellular heterogeneity. Current non-destructive approaches, like temporal modeling with single-cell ribonucleic acid sequencing (RNA-seq) and live-cell fluorescence imaging, either rely on biological assumptions or possess the risk of cellular perturbation. Recent advances in nanoscale technologies for non-destructive intracellular content extraction offer a promising solution to these challenges. These novel methods work at the nanoscale to non-destructively access cellular membranes and can be broadly classified into three mechanisms: tip-facilitated aspiration, membrane-based, and probe-based methods. This perspective focuses on these emerging nanotechnologies for repeated intracellular content extraction. Their potential in longitudinal analysis is discussed, the critical requirements for effective repeated sampling are addressed, and the suitability of each technique for various applications is explored. Furthermore, unresolved challenges in repeated sampling are highlighted to encourage further research in this growing field.

Heme metabolism in nonerythroid cells.

Heme is an iron-containing prosthetic group necessary for the function of several proteins termed "hemoproteins." Erythrocytes contain most of the body's heme in the form of hemoglobin and contain high concentrations of free heme. In nonerythroid cells, where cytosolic heme concentrations are 2 to 3 orders of magnitude lower, heme plays an essential and often overlooked role in a variety of cellular processes. Indeed, hemoproteins are found in almost every subcellular compartment and are integral in cellular operations such as oxidative phosphorylation, amino acid metabolism, xenobiotic metabolism, and transcriptional regulation. Growing evidence reveals the participation of heme in dynamic processes such as circadian rhythms, NO signaling, and the modulation of enzyme activity. This dynamic view of heme biology uncovers exciting possibilities as to how hemoproteins may participate in a range of physiologic systems. Here, we discuss how heme is regulated at the level of its synthesis, availability, redox state, transport, and degradation and highlight the implications for cellular function and whole organism physiology.

Inhibition of intracellular ATP synthesis impairs the recruitment of homologous recombination factors after ionizing radiation.

Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB repair requires adenosine-5'-triphosphate (ATP) for protein kinase activities in the multiple steps of DSB repair, such as DNA ligation, chromatin remodeling, and DNA damage signaling via protein kinase and ATPase activities. To investigate whether low ATP culture conditions affect the recruitment of repair proteins at DSB sites, IR-induced foci were examined in the presence of ATP synthesis inhibitors. We found that p53 binding protein 1 foci formation was modestly reduced under low ATP conditions after IR, although phosphorylated histone H2AX and mediator of DNA damage checkpoint 1 foci formation were not impaired. Next, we examined the foci formation of breast cancer susceptibility gene I (BRCA1), replication protein A (RPA) and radiation 51 (RAD51), which are HR factors, in G2 phase cells following IR. Interestingly, BRCA1 and RPA foci in the G2 phase were significantly reduced under low ATP conditions compared to that under normal culture conditions. Notably, RAD51 foci were drastically impaired under low ATP conditions. These results suggest that HR does not effectively progress under low ATP conditions; in particular, ATP shortages impair downstream steps in HR, such as RAD51 loading. Taken together, these results suggest that the maintenance of cellular ATP levels is critical for DNA damage response and HR progression after IR.

Nanosensors Monitor Intracellular GSH Depletion: GSH Triggers Cu(II) for Tumor Imaging and Inhibition.

Glutathione (GSH) is the primary antioxidant in cells, and GSH consumption will break the redox balance in cells. Based on this, a method that uses high concentrations of GSH in the tumor microenvironment to trigger the redox reaction of Cu(II) to generate copper nanoprobes with fluorescence and tumor growth inhibition properties is proposed. The nanoprobe mainly exists in the form of Cu(I) and catalyzes the decomposition of hydrogen peroxide into hydroxyl radicals. At the same time, a simple and controllable carbon micro-nano electrode is used to construct a single-cell sensing platform, which enable the detection of glutathione content in single living cells after Cu(II) treatment, providing an excellent example for detecting single-cell biomolecules.

Emergent Spatiotemporal Organization in Stochastic Intracellular Transport Dynamics.

The interior of a living cell is an active, fluctuating, and crowded environment, yet it maintains a high level of coherent organization. This dichotomy is readily apparent in the intracellular transport system of the cell. Membrane-bound compartments called endosomes play a key role in carrying cargo, in conjunction with myriad components including cargo adaptor proteins, membrane sculptors, motor proteins, and the cytoskeleton. These components coordinate to effectively navigate the crowded cell interior and transport cargo to specific intracellular locations, even though the underlying protein interactions and enzymatic reactions exhibit stochastic behavior. A major challenge is to measure, analyze, and understand how, despite the inherent stochasticity of the constituent processes, the collective outcomes show an emergent spatiotemporal order that is precise and robust. This review focuses on this intriguing dichotomy, providing insights into the known mechanisms of noise suppression and noise utilization in intracellular transport processes, and also identifies opportunities for future inquiry.

Functional diversification of cell signaling by GPCR localization.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and a critical class of regulators of mammalian physiology. Also known as seven transmembrane receptors (7TMs), GPCRs are ubiquitously expressed and versatile, detecting a diverse set of endogenous stimuli, including odorants, neurotransmitters, hormones, peptides, and lipids. Accordingly, GPCRs have emerged as the largest class of drug targets, accounting for upward of 30% of all prescription drugs. The view that ligand-induced GPCR responses originate exclusively from the cell surface has evolved to reflect accumulating evidence that receptors can elicit additional waves of signaling from intracellular compartments. These events in turn shape unique cellular and physiological outcomes. Here, we discuss our current understanding of the roles and regulation of compartmentalized GPCR signaling.

An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation.

Slow deactivation is a critical property of voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26-135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured "tip" (residues 1-12) and an amphipathic α-helical region (residues 13-25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this "hydrophobic nexus" is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.

standR: spatial transcriptomic analysis for GeoMx DSP data.

To gain a better understanding of the complexity of gene expression in normal and diseased tissues it is important to account for the spatial context and identity of cells in situ. State-of-the-art spatial profiling technologies, such as the Nanostring GeoMx Digital Spatial Profiler (DSP), now allow quantitative spatially resolved measurement of the transcriptome in tissues. However, the bioinformatics pipelines currently used to analyse GeoMx data often fail to successfully account for the technical variability within the data and the complexity of experimental designs, thus limiting the accuracy and reliability of the subsequent analysis. Carefully designed quality control workflows, that include in-depth experiment-specific investigations into technical variation and appropriate adjustment for such variation can address this issue. Here, we present standR, an R/Bioconductor package that enables an end-to-end analysis of GeoMx DSP data. With four case studies from previously published experiments, we demonstrate how the standR workflow can enhance the statistical power of GeoMx DSP data analysis and how the application of standR enables scientists to develop in-depth insights into the biology of interest.

Calcimycin mediates apoptosis in breast and cervical cancer cell lines by inducing intracellular calcium levels in a P2RX4-dependent manner.

BACKGROUND: Calcimycin (A23187) is a polyether antibiotic and divalent cation ionophore, extracted from Streptomyces chartrecensis. With wide variety of antimicrobial activities, it also exhibits cytotoxicity of tumor cells. Calcimycin exhibit therapeutic potential against tumor cell growth; however, the molecular mechanism remains to be fully elucidated. Present study explores the mechanism of calcimycin-induced apoptosis cancer cell lines. METHODS: Apoptotic induction in a dose-dependent manner were recorded with MTT assays, Phase contrast imaging, wound healing assay, fluorescence imaging by DAPI and AO/EB staining and FACS using cell line model. Mitochondrial potential was analyzed by TMRM assay as Ca2+ signaling is well known to be influenced and synchronized by mitochondria also. RESULTS: Calcimycin induces apoptosis in dose dependent manner, also accompanied by increased intracellular calcium-level and expression of purinergic receptor-P2RX4, a ligand-gated ion channel. CONCLUSION: Calcimycin tends to increase the intracellular calcium level, mRNA expression of ATP receptor P2RX4, and phosphorylation of p38. Blocking of either intracellular calcium by BAPTA-AM, P2RX4 expression by antagonist 5-BDBD, and phospho-p38 by SB203580, abrogated the apoptotic activity of calcimycin. GENERAL SIGNIFICANCE: Taken together, these results show that calcimycin induces apoptosis in P2RX4 and ATP mediated intracellular Ca2+ and p38 MAPK mediated pathway in both the cancer cell lines. This study explored a new mode of action for calcimycin in cancer that could be potentially employed in future studies for cancer therapeutic research. This study disentangles that the calcimycin-induced apoptotic cell death is P2RX4 and ATP involved, intracellular Ca2+ and p38 MAPK mediated pathway.

[Ru(dpp)3]Cl2-Embedded Oxygen Nano Polymeric Sensors: A Promising Tool for Monitoring Intracellular and Intratumoral Oxygen Gradients with High Quantum Yield and Long Lifetime.

Unraveling the intricacies between oxygen dynamics and cellular processes in the tumor microenvironment (TME) hinges upon precise monitoring of intracellular and intratumoral oxygen levels, which holds paramount significance. The majority of these reported oxygen nanoprobes suffer compromised lifetime and quantum yield when exposed to the robust ROS activities prevalent in TME, limiting their prolonged in vitro usability. Herein, the ruthenium-embedded oxygen nano polymeric sensor (Ru-ONPS) is proposed for precise oxygen gradient monitoring within the cellular environment and TME. Ru-ONPS (≈64±7 nm) incorporates [Ru(dpp)3]Cl2 dye into F-127 and crosslinks it with urea and paraformaldehyde, ensuring a prolonged lifetime (5.4 µs), high quantum yield (66.65 ± 2.43% in N2 and 49.80 ± 3.14% in O2), superior photostability (>30 min), and excellent stability in diverse environmental conditions. Based on the Stern-Volmer plot, the Ru-ONPS shows complete linearity for a wide dynamic range (0-23 mg L-1), with a detection limit of 10 µg mL-1. Confocal imaging reveals Ru-ONPS cellular uptake and intratumoral distribution. After 72 h, HCT-8 cells show 5.20±1.03% oxygen levels, while NIH3T3 cells have 7.07±1.90%. Co-culture spheroids display declining oxygen levels of 17.90±0.88%, 10.90±0.88%, and 5.10±1.18%, at 48, 120, and 216 h, respectively. Ru-ONPS advances cellular oxygen measurement and facilitates hypoxia-dependent metastatic research and therapeutic target identification.

Adenylate cyclase toxin of Bordetella parapertussis disrupts the epithelial barrier granting the bacterial access to the intracellular space of epithelial cells.

B. parapertussis is one of the etiological agents of whooping cough. Once inhaled, the bacteria bind to the respiratory epithelium and start the infection. Little is known about this first step of host colonization and the role of the human airway epithelial barrier on B. parapertussis infection. We here investigated the outcome of the interaction of B. parapertussis with a polarized monolayer of respiratory epithelial cells. Our results show that B. parapertussis preferentially attaches to the intercellular boundaries, and causes the disruption of the tight junction integrity through the action of adenylate cyclase toxin (CyaA). We further found evidence indicating that this disruption enables the bacterial access to components of the basolateral membrane of epithelial cells to which B. parapertussis efficiently attaches and gains access to the intracellular location, where it can survive and eventually spread back into the extracellular environment. Altogether, these results suggest that the adenylate cyclase toxin enables B. parapertussis to overcome the epithelial barrier and eventually establish a niche of persistence within the respiratory epithelial cells.