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.

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.

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.

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.

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.

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.

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.

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.

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.

Use of CRISPR/CAS9 Technologies to Study the Role of TLR in Dendritic Cell Subsets.

Dendritic cells (DCs) have a significant role in coordinating both innate and adaptive immunity by serving as sentinels that detect invaders and initiate immune responses to eliminate them, as well as presenting antigens to activate adaptive immune responses that are specific to the antigen and the context in which it was detected. The regulation of DC functions is complex and involves intracellular drivers such as transcription factors and signaling pathways, as well as intercellular interactions with adhesion molecules, chemokines, and their receptors in the microenvironment. Toll-like receptors (TLRs) are crucial for DCs to detect pathogen-associated molecular patterns (PAMPs) and initiate downstream signaling pathways that lead to DC maturation and education in bridging with adaptive immunity, including the upregulation of MHC class II expression, induction of CD80, CD86, and CD40, and production of innate cytokines. Understanding the TLR pathways that DCs use to respond to innate immune stimuli and convert them into adaptive responses is important for new therapeutic targets identification.We present a novel platform that offers a fast and affordable CRISPR-Cas9 screening of genes that are involved in dendritic cells' TLR-dependent activation. Using CRISPR/Cas9 screening to target individual TLR genes in different dendritic cell subsets allows the identification of TLR-dependent pathways that regulate dendritic cell activation and cytokine production. This approach offers the efficient targeting of TLR driver genes to modulate the immune response and identify novel immune response regulators, establishing a causal link between these regulators and functional phenotypes based on genotypes.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae.

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass spectrometry-based profiling, metabolomics, expression assays, and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds, termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor superfamily of transcriptional regulators, where RSS and hydrogen peroxide sensors segregate into distinct clusters of sequences. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity or DNA dissociation following treatment with glutathione disulfide or hydrogen peroxide. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Origin and functional diversification of PAS domain, a ubiquitous intracellular sensor.

Signal perception is a key function in regulating biological activities and adapting to changing environments. Per-Arnt-Sim (PAS) domains are ubiquitous sensors found in diverse receptors in bacteria, archaea, and eukaryotes, but their origins, distribution across the tree of life, and extent of their functional diversity are not fully characterized. Here, we show that using sequence conservation and structural information, it is possible to propose specific and potential functions for a large portion of nearly 3 million PAS domains. Our analysis suggests that PAS domains originated in bacteria and were horizontally transferred to archaea and eukaryotes. We reveal that gas sensing via a heme cofactor evolved independently in several lineages, whereas redox and light sensing via flavin adenine dinucleotide and flavin mononucleotide cofactors have the same origin. The close relatedness of human PAS domains to those in bacteria provides an opportunity for drug design by exploring potential natural ligands and cofactors for bacterial homologs.

The Current State of Charcot-Marie-Tooth Disease Treatment.

Charcot-Marie-Tooth disease (CMT) and associated neuropathies are the most predominant genetically transmitted neuromuscular conditions; however, effective pharmacological treatments have not established. The extensive genetic heterogeneity of CMT, which impacts the peripheral nerves and causes lifelong disability, presents a significant barrier to the development of comprehensive treatments. An estimated 100 loci within the human genome are linked to various forms of CMT and its related inherited neuropathies. This review delves into prospective therapeutic strategies used for the most frequently encountered CMT variants, namely CMT1A, CMT1B, CMTX1, and CMT2A. Compounds such as PXT3003, which are being clinically and preclinically investigated, and a broad array of therapeutic agents and their corresponding mechanisms are discussed. Furthermore, the progress in established gene therapy techniques, including gene replacement via viral vectors, exon skipping using antisense oligonucleotides, splicing modification, and gene knockdown, are appraised. Each of these gene therapies has the potential for substantial advancements in future research.

A genetic system for Akkermansia muciniphila reveals a role for mucin foraging in gut colonization and host sterol biosynthesis gene expression.

Akkermansia muciniphila, a mucophilic member of the gut microbiota, protects its host against metabolic disorders. Because it is genetically intractable, the mechanisms underlying mucin metabolism, gut colonization and its impact on host physiology are not well understood. Here we developed and applied transposon mutagenesis to identify genes important for intestinal colonization and for the use of mucin. An analysis of transposon mutants indicated that de novo biosynthesis of amino acids was required for A. muciniphila growth on mucin medium and that many glycoside hydrolases are redundant. We observed that mucin degradation products accumulate in internal compartments within bacteria in a process that requires genes encoding pili and a periplasmic protein complex, which we term mucin utilization locus (MUL) genes. We determined that MUL genes were required for intestinal colonization in mice but only when competing with other microbes. In germ-free mice, MUL genes were required for A. muciniphila to repress genes important for cholesterol biosynthesis in the colon. Our genetic system for A. muciniphila provides an important tool with which to uncover molecular links between the metabolism of mucins, regulation of lipid homeostasis and potential probiotic activities.