Cellular Polarity Research Articles

Molecular pathological approach to cancer epigenomics and its clinical application.

Careful microscopic observation of histopathological specimens, accumulation of large numbers of high-quality tissue specimens, and analysis of molecular pathology in relation to morphological features are considered to yield realistic data on the nature of multistage carcinogenesis. Since the morphological hallmark of cancer is disruption of the normal histological structure maintained through cell-cell adhesiveness and cellular polarity, attempts have been made to investigate abnormalities of the cadherin-catenin cell adhesion system in human cancer cells. It has been shown that the CDH1 tumor suppressor gene encoding E-cadherin is silenced by DNA methylation, suggesting that a "double hit" involving DNA methylation and loss of heterozygosity leads to carcinogenesis. Therefore, in the 1990s, we focused on epigenomic mechanisms, which until then had not received much attention. In chronic hepatitis and liver cirrhosis associated with hepatitis virus infection, DNA methylation abnormalities were found to occur frequently, being one of the earliest indications that such abnormalities are present even in precancerous tissue. Aberrant expression and splicing of DNA methyltransferases, such as DNMT1 and DNMT3B, was found to underlie the mechanism of DNA methylation alterations in various organs. The CpG island methylator phenotype in renal cell carcinomawas identified for the first time, and its therapeutic targets were identified by multilayer omics analysis. Furthermore, the DNA methylation profile of nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinomawas clarified in groundbreaking studies. Since then, we have developed diagnostic markers for carcinogenesis risk in NASH patients and noninvasive diagnostic markers for upper urinary tract cancer, as well as developing a new high-performance liquid chromatography-based diagnostic system for DNA methylation diagnosis. Research on the cancer epigenome has revealed that DNA methylation alterations occur from the precancerous stage as a result of exposure to carcinogenic factors such as inflammation, smoking, and viral infections, and continuously contribute to multistage carcinogenesis through aberrant expression of cancer-related genes and genomic instability. DNA methylation alterations at the precancerous stages are inherited by or strengthened in cancers themselves and determine the clinicopathological aggressiveness of cancers as well as patient outcome. DNA methylation alterations have applications as biomarkers, and are expected to contribute to diagnosis, as well as preventive and preemptive medicine.

First person – Ann-Christin Groh and Annika Möller-Kerutt

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Annika Möller-Kerutt and Ann-Christin Groh are co-first authors on ‘ PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells’, published in JCS. Annika is a postdoc in the lab of Thomas Weide at University Hospital of Münster, Germany, and Ann-Christin undertook this work while a PhD student in the same laboratory lab and is now a Key Account Manager for Merck Healthcare, Germany. They are interested in how junction formation is dependent on cellular polarity.

CD73 mitigates ZEB1 expression in papillary thyroid carcinoma

BackgroundZEB1, a core transcription factor involved in epithelial-mesenchymal transition (EMT), is associated with aggressive cancer cell behavior, treatment resistance, and poor prognosis across various tumor types. Similarly, the expression and activity of CD73, an ectonucleotidase implicated in adenosine generation, is an important marker of tumor malignancy. Growing evidence suggests that EMT and the adenosinergic pathway are intricately linked and play a pivotal role in cancer development. Therefore, this study focuses on exploring the correlations between CD73 and ZEB1, considering their impact on tumor progression.MethodsWe employed CRISPR/Cas9 technology to silence CD73 expression in cell lines derived from papillary thyroid carcinoma. These same cells underwent lentiviral transduction of a reporter of ZEB1 non-coding RNA regulation. We conducted studies on cell migration using scratch assays and analyses of cellular speed and polarity. Additionally, we examined ZEB1 reporter expression through flow cytometry and immunocytochemistry, complemented by Western blot analysis for protein quantification. For further insights, we applied gene signatures representing different EMT states in an RNA-seq expression analysis of papillary thyroid carcinoma samples from The Cancer Genome Atlas.ResultsSilencing CD73 expression led to a reduction in ZEB1 non-coding RNA regulation reporter expression in a papillary thyroid carcinoma-derived cell line. Additionally, it also mitigated ZEB1 protein expression. Moreover, the expression of CD73 and ZEB1 was correlated with alterations in cell morphology characteristics crucial for cell migration, promoting an increase in cell polarity index and cell migration speed. RNA-seq analysis revealed higher expression of NT5E (CD73) in samples with BRAF mutations, accompanied by a prevalence of partial-EMT/hybrid state signature expression.ConclusionsCollectively, our findings suggest an association between CD73 expression and/or activity and the post-transcriptional regulation of ZEB1 by non-coding RNA, indicating a reduction in its absence. Further investigations are warranted to elucidate the relationship between CD73 and ZEB1, with the potential for targeting them as therapeutic alternatives for cancer treatment in the near future.

Emergent seesaw oscillations during cellular directional decision-making

Motile cells inside living tissues often encounter junctions, where their path branches into several alternative directions of migration. We present a theoretical model of cellular polarization for a cell migrating along a one-dimensional line, arriving at a symmetric Y junction and extending protrusions along the different paths that originate at the junction. The model predicts the spontaneous emergence of deterministic oscillations of growth and cellular polarization between competing protrusions during the directional decision-making process. The oscillations are modified by cellular noise but remain a dominant feature that affects the time it takes the cell to migrate across the junction. These predictions are confirmed experimentally for two different cell types (non-cancerous endothelial and cancerous glioma cells) migrating on a patterned network of thin adhesive lanes with junctions.

Molecular alterations associated with pathophysiology in liver-specific ZO-1 and ZO-2 knockout mice.

The liver is a complex organ with a highly organized structure in which tight junctions (TJs) play an important role in maintaining their function by regulating barrier properties and cellular polarity. Dysfunction of TJs is associated with liver diseases, including progressive familial intrahepatic cholestasis (PFIC). In this study, we investigated the molecular alterations in a liver-specific ZO-1 and ZO-2 double-knockout (DKO) mouse model, which exhibits features resembling those of PFIC4 patients with mutations in the ZO-2 gene. RNA-seq analysis revealed the upregulation of genes involved in the oxidative stress response, xenobiotic metabolism, and cholesterol metabolism in DKO livers. Conversely, the expression of genes regulated by HNF4α was lower in DKO livers than in the wild-type controls. Furthermore, age-associated analysis elucidated the timing and progression of these pathway changes as well as alterations in molecules related to TJs and apical polarity. Our research uncovered previously unknown implications of ZO-1 and ZO-2 in liver physiology and provides new insights into the molecular pathogenesis of PFIC4 and other tight junction-related liver diseases. These findings contribute to a better understanding of the complex mechanisms underlying liver function and dysfunction and may lead to the development of novel therapeutic strategies for liver diseases associated with tight junction impairment.Key words: tight junctions, ZO-1/ZO-2 knockout mouse, liver, transcriptome analysis, molecular pathological progression.

Studying Microtubule Dynamics in Human Neurons: Two-Dimensional Microtubule Tracing and Kymographs in iPSC- and SH-SY5Y-Derived Neurons for Tau Research.

The study of microtubule (MT) dynamics is essential for the understanding of cellular transport, cell polarity, axon formation, and other neurodevelopmental mechanisms. All these processes rely on the constant transition between assembly and disassembly of tubulin polymers to/from MTs, known as dynamic instability. This process is well-regulated, among others, by phosphorylation of microtubule-associated proteins (MAP), including the Tau protein. Protein kinases, in particular the microtubule affinity regulating kinase (MARK), regulate the MT-Tau interaction, inducingTau dissociation by phosphorylation. Phosphorylated Tau dissociates from microtubules forming insoluble aggregates known as neurofibrillary tangles. These accumulations of hyperphosphorylated Tau in the neurons disrupt the physiological MT-based transport machinery within the cell and can potentially lead to the development of neurodegenerative disorders, such as Alzheimer's disease (AD) and related tauopathies. Further investigations on the MT cytoskeleton dynamics are essential as they may elucidate pathomechanisms of neurodegenerative diseases - particularly tauopathies - as well as fundamental neurodevelopmental processes.The study of thedynamic assembly and disassembly of the MT network requires live-cell imaging rather than conventional immunocytochemistry based on fixed samples. To investigate MT dynamics, we perform live-cell imaging of neurons transfected with a fluorescently tagged version of the microtubule plus-end tracking protein (+TIP) EB3. This protein associates with the growing ends of MTs and thus visualizes MT growth in real time. Our imaging analysis protocol allows the determination of quantity, orientation, and velocity of MT growth in the soma and neurites of transfected neurons,using ImageJ-based tracking software and kymographs. Furthermore, functional effects of Tau and MARK kinases on the MT cytoskeleton can be assessed by overexpression or downregulation experiments of the respective protein prior to the live imaging assay. We use two different human neuronal cell models, naive and differentiated SH-SY5Y neuroblastoma cells, and neurons derived from induced pluripotent stem cells (iPSCs), both of which have shown success as models to study Tau-related pathologies.This protocol describes an optimized method for analysis of microtubule dynamics using fluorescent tagged EB3 protein as microtubule plus end marker. In this chapter, we outline the process of neuronal transfection, live-cell imaging, and necessary time-lapse image analysis based on ImageJ in two human-derived neuronal systems, which are suitable for the analysis of Tau trafficking and sorting studies.

Comprehensive and quantitative analysis of intracellular structure polarization at the apical–basal axis in elongating Arabidopsis zygotes

A comprehensive and quantitative evaluation of multiple intracellular structures or proteins is a promising approach to provide a deeper understanding of and new insights into cellular polarity. In this study, we developed an image analysis pipeline to obtain intensity profiles of fluorescent probes along the apical–basal axis in elongating Arabidopsis thaliana zygotes based on two-photon live-cell imaging data. This technique showed the intracellular distribution of actin filaments, mitochondria, microtubules, and vacuolar membranes along the apical–basal axis in elongating zygotes from the onset of cell elongation to just before asymmetric cell division. Hierarchical cluster analysis of the quantitative data on intracellular distribution revealed that the zygote may be compartmentalized into two parts, with a boundary located 43.6% from the cell tip, immediately after fertilization. To explore the biological significance of this compartmentalization, we examined the positions of the asymmetric cell divisions from the dataset used in this distribution analysis. We found that the cell division plane was reproducibly inserted 20.5% from the cell tip. This position corresponded well with the midpoint of the compartmentalized apical region, suggesting a potential relationship between the zygote compartmentalization, which begins with cell elongation, and the position of the asymmetric cell division.

Dictionary of immune responses to cytokines at single-cell resolution

Cytokines mediate cell–cell communication in the immune system and represent important therapeutic targets1–3. A myriad of studies have highlighted their central role in immune function4–13, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap, we created the Immune Dictionary, a compendium of single-cell transcriptomic profiles of more than 17 immune cell types in response to each of 86 cytokines (>1,400 cytokine–cell type combinations) in mouse lymph nodes in vivo. A cytokine-centric view of the dictionary revealed that most cytokines induce highly cell-type-specific responses. For example, the inflammatory cytokine interleukin-1β induces distinct gene programmes in almost every cell type. A cell-type-centric view of the dictionary identified more than 66 cytokine-driven cellular polarization states across immune cell types, including previously uncharacterized states such as an interleukin-18-induced polyfunctional natural killer cell state. Based on this dictionary, we developed companion software, Immune Response Enrichment Analysis, for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumours following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell–cell communication networks in any immune response.

Centrosome clustering control in osteoclasts through CCR5-mediated signaling

Osteoclasts uniquely resorb calcified bone matrices. To exert their function, mature osteoclasts maintain the cellular polarity and directional vesicle trafficking to and from the resorbing bone surface. However, the regulatory mechanisms and pathophysiological relevance of these processes remain largely unexplored. Bone histomorphometric analyses in Ccr5-deficient mice showed abnormalities in the morphology and functional phenotype of their osteoclasts, compared to wild type mice. We observed disorganized clustering of nuclei, as well as centrosomes that organize the microtubule network, which was concomitant with impaired cathepsin K secretion in cultured Ccr5-deficient osteoclasts. Intriguingly, forced expression of constitutively active Rho or Rac restored these cytoskeletal phenotypes with recovery of cathepsin K secretion. Furthermore, a gene-disease enrichment analysis identified that PLEKHM1, a responsible gene for osteopetrosis, which regulates lysosomal trafficking in osteoclasts, was regulated by CCR5. These experimental results highlighted that CCR5-mediated signaling served as an intracellular organizer for centrosome clustering in osteoclasts, which was involved in the pathophysiology of bone metabolism.

Iron inhibits glioblastoma cell migration and polarization.

Glioblastoma is one of the deadliest malignancies facing modern oncology today. The ability of glioblastoma cells to diffusely spread into neighboring healthy brain makes complete surgical resection nearly impossible and contributes to the recurrent disease faced by most patients. Although research into the impact of iron on glioblastoma has addressed proliferation, there has been little investigation into how cellular iron impacts the ability of glioblastoma cells to migrate-a key question, especially in the context of the diffuse spread observed in these tumors. Herein, we show that increasing cellular iron content results in decreased migratory capacity of human glioblastoma cells. The decrease in migratory capacity was accompanied by a decrease in cellular polarization in the direction of movement. Expression of CDC42, a Rho GTPase that is essential for both cellular migration and establishment of polarity in the direction of cell movement, was reduced upon iron treatment. We then analyzed a single-cell RNA-seq dataset of human glioblastoma samples and found that cells at the tumor periphery had a gene signature that is consistent with having lower levels of cellular iron. Altogether, our results suggest that cellular iron content is impacting glioblastoma cell migratory capacity and that cells with higher iron levels exhibit reduced motility.

Quantifying spatial dynamics of Mycobacterium tuberculosis infection of human macrophages using microfabricated patterns

Macrophages provide a first line of defense against invading pathogens, including the leading cause of bacterial mortality, Mycobacterium tuberculosis (Mtb). A challenge for quantitative characterization of host-pathogen processes in differentially polarized primary human monocyte-derived macrophages (MDMs) is their heterogeneous morphology. Here, we describe the use of microfabricated patterns that constrain the size and shape of cells, mimicking the physiological spatial confinement cells experience in tissues, to quantitatively characterize interactions during and after phagocytosis at the single-cell level at high resolution. Comparing pro-inflammatory (M1) and anti-inflammatory (M2) MDMs, we find interferon-γ stimulation increases the phagocytic contraction, while contraction and bacterial uptake decrease following silencing of phagocytosis regulator NHLRC2 or bacterial surface lipid removal. We identify host organelle position alterations within infected MDMs and differences in Mtb subcellular localization in line with M1 and M2 cellular polarity. Our approach can be adapted to study other host-pathogen interactions and coupled with downstream automated analytical approaches.

Alteration of genome-wide DNA methylation in non-uranium miners induced by high level radon exposure.

In China, according to statistics about underground non-uranium mine radon levels, 15% exceed the national standard intervention level of 1000Bq/m3, and some mines may exceed 10,000Bq/m3. The relationship between radon exposure in underground miners and lung cancer has already been established, but the mechanisms and biological processes underlying it are poorly understood. In order to identify the genome-wide DNA methylation profile associated with long-term radon exposure, we performed the Infinium Human Methylation 850K BeadChip measurement in whole blood samples obtained from 15 underground non-uranium miners and 10 matched aboveground control workers. Radon concentrations in the air of workplaces and living environments were measured by CR-39 radon detectors, and annual effective doses were calculated using the detection data. Under the high radon concentration with an average value of 12,700Bq·m-3, a total of 165 significant differentially methylated positions (127 hypermethylated sites and 38 hypomethylated sites) annotated to 71 genes were identified in underground miners (|Δβ| ≥ 0.10, p<0.05), and the average DNA methylation level of 165 DMPs was significantly higher than that of the control workers. Most DMPs were found on chromosome 1, and approximately one-quarter of them were located in genomic promoter regions. Through bioinformatics analysis and pyrosequencing validation, five candidate genes differentially methylated by radon, including TIMP2, EMP2, CPT1B, AMD1 and SLC43A2 were identified. GO and KEGG analysis implicated that long term radon exposure could induce the lung cancer related biological processes such as cell adhesion and cellular polarity maintenance. Our study provides evidence for the alterations of genome-wide DNA methylation profiles induced by long-term high level radon exposure, and new insights into searching for carcinogenic biomarkers of high radon exposure in future studies.

Amino-terminated carbon dots for imaging Golgi apparatus polarity in live cells

Cellular polarity plays a crucial role in numerous pathological and biological processes. For example, the polarity of the Golgi apparatus strongly influences the intracellular protein processing and transport. The development of fluorescent probes for tracking of subcellular levels of polarity changes remains a great challenge. Herein, amino-terminated carbon dots (CDs) which have superior Golgi apparatus targeting ability and polarity-sensitive property were designed and fabricated by the mixing of p-phenylenediamine and ethylenediamine through a simple solvothermal route. CDs allowed effectively imaging of Golgi apparatus morphological dynamics during cell division and cell death, while folic acid-conjugated CDs possessed high folate-receptor binding affinity and could be used to identify cancer cells. Importantly, the Golgi polarity in apoptotic cells was found to be higher than that of non-apoptotic cells. This work provided a reliable and robust way to real-time track the changes of Golgi apparatus and the local polarity variation, implying the great potential of CDs for the apoptosis evaluation and therapeutic drug screening.

Striped Expression of Leucine-Rich Repeat Proteins Coordinates Cell Intercalation and Compartment Boundary Formation in the Early Drosophila Embryo.

Planar polarity is a commonly observed phenomenon in which proteins display a consistent asymmetry in their subcellular localization or activity across the plane of a tissue. During animal development, planar polarity is a fundamental mechanism for coordinating the behaviors of groups of cells to achieve anisotropic tissue remodeling, growth, and organization. Therefore, a primary focus of developmental biology research has been to understand the molecular mechanisms underlying planar polarity in a variety of systems to identify conserved principles of tissue organization. In the early Drosophila embryo, the germband neuroectoderm epithelium rapidly doubles in length along the anterior-posterior axis through a process known as convergent extension (CE); it also becomes subdivided into tandem tissue compartments through the formation of compartment boundaries (CBs). Both processes are dependent on the planar polarity of proteins involved in cellular tension and adhesion. The enrichment of actomyosin-based tension and adherens junction-based adhesion at specific cell-cell contacts is required for coordinated cell intercalation, which drives CE, and the creation of highly stable cell-cell contacts at CBs. Recent studies have revealed a system for rapid cellular polarization triggered by the expression of leucine-rich-repeat (LRR) cell-surface proteins in striped patterns. In particular, the non-uniform expression of Toll-2, Toll-6, Toll-8, and Tartan generates local cellular asymmetries that allow cells to distinguish between cell-cell contacts oriented parallel or perpendicular to the anterior-posterior axis. In this review, we discuss (1) the biomechanical underpinnings of CE and CB formation, (2) how the initial symmetry-breaking events of anterior-posterior patterning culminate in planar polarity, and (3) recent advances in understanding the molecular mechanisms downstream of LRR receptors that lead to planar polarized tension and junctional adhesion.

Aquaporin water channels: roles beyond renal water handling.

Aquaporin (AQP) water channels are pivotal to renal water handling and therefore in the regulation of body water homeostasis. However, beyond the kidney, AQPs facilitate water reabsorption and secretion in other cells and tissues, including sweat and salivary glands and the gastrointestinal tract. A growing body of evidence has also revealed that AQPs not only facilitate the transport of water but also the transport of several small molecules and gases such as glycerol, H2O2, ions and CO2. Moreover, AQPs are increasingly understood to contribute to various cellular processes, including cellular migration, adhesion and polarity, and to act upstream of several intracellular and intercellular signalling pathways to regulate processes such as cell proliferation, apoptosis and cell invasiveness. Of note, several AQPs are highly expressed in multiple cancers, where their expression can correlate with the spread of cancerous cells to lymph nodes and alter the response of cancers to conventional chemotherapeutics. These data suggest that AQPs have diverse roles in various homeostatic and physiological systems and may be exploited for prognostics and therapeutic interventions.

Core–shell structure of carbon nanohorns and pH-sensitive liposome for doxorubicin and tumor-associated macrophage polarization factor interleukin-21 codelivery

The combination of immunotherapy and chemotherapy is becoming a very promising cancer treatment method. However, the effective delivery of chemical drug or immune molecule to targeting tissues is a scientific issue to be solved urgently. A novel core–shell structure of single-walled carbon nanohorns (SWCNHs) and pH-sensitive liposome (PSL) was constructed for doxorubicin (DOX) and tumor-associated macrophage (TAM) polarization factor interleukin-21 (IL-21) codelivery in this study. The physicochemical properties, cytotoxicity, cellular uptake, macrophage polarization, and antitumor efficacy of functionalized nanocarriers were fully evaluated. DOX was incorporated into oxidized SWCNHs via π–π interaction (DOX-O-SWCNH) and then coated with IL-21-loaded PSL to obtain IL-21-loaded PSL (IL-21-PSL)-DOX-O-SWCNHs. The release rates of IL-21 and DOX from functionalized nanocarriers at the pH 7.4 of were lower than those at the pH of 5.5, thereby indicating a pH-sensitive drug release. Blank nanocarriers showed relatively low cytotoxicity to A549 cells and 293T cells. The half maximal inhibitory concentration (IC50) value of PSL-DOX-O-SWCNHs for A549 cells was 164.85 μg/mL. The PSL-DOX-O-SWCNHs can be effectively uptaken by A549 cells and the IL-21-PSL-DOX-O-SWCNHs can significantly downregulate the expression of M2 macrophage-related factors (IL-10 levels) and upregulate that of M1 macrophage-related factors (iNOs levels). The IL-21-PSL-DOX-O-SWCNHs also showed significantly high tumor targeting and distribution and significantly low tumor volume of A549 lung cancer cell-bearing nude mice, showing the cooperating effects of DOX and IL-21. The functionalized nanocarriers were mainly distributed in the tumor tissues and liver and less in the spleen, lung, and heart of mice. In conclusion, the functionalized IL-21-PSL-DOX-O-SWCNHs showed high drug loading, excellent cellular uptake, pH-sensitive release, TAM polarization, and remarkable antitumor efficacy in vivo.

Hitting the mark: Localization of mRNA and biomolecular condensates in health and disease.

Subcellular mRNA localization is critical to a multitude of biological processes such as development of cellular polarity, embryogenesis, tissue differentiation, protein complex formation, cell migration, and rapid responses to environmental stimuli and synaptic depolarization. Our understanding of the mechanisms of mRNA localization must now be revised to include formation and trafficking of biomolecular condensates, as several biomolecular condensates that transport and localize mRNA have recently been discovered. Disruptions in mRNA localization can have catastrophic effects on developmental processes and biomolecular condensate biology and have been shown to contribute to diverse diseases. A fundamental understanding of mRNA localization is essential to understanding how aberrations in this biology contribute the etiology of numerous cancers though support of cancer cell migration and biomolecular condensate dysregulation, as well as many neurodegenerative diseases, through misregulation of mRNA localization and biomolecular condensate biology. This article is categorized under: RNA Export and Localization > RNA Localization RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

Dictionary of immune responses to 86 cytokines in vivoat single-cell resolution

Abstract Cytokines mediate cell-cell communication in the immune system and represent important therapeutic targets. A myriad of studies have underscored their central role in immune function, yet we lack a global view of the cellular responses of each immune cell type to each cytokine. To address this gap we created the Immune Dictionary – a compendium of single-cell transcriptomic profiles of over 20 cell types in response to each of 86 cytokines in murine lymph nodes in vivo, representing over 1,500 cytokine-cell type combinations. A cytokine-centric view of the dictionary revealed that most cytokines induced highly cell type-specific responses. For example, the inflammatory cytokine IL-1β induced distinct gene programs in almost every cell type. A cell type-centric view of the dictionary identified multiple cytokine-driven cellular polarization states in each immune cell type, including previously uncharacterized states such as an IL-18-induced polyfunctional NK cell state. Based on the dictionary, we developed companion software, Immune Response Enrichment Analysis (IREA), for assessing cytokine activities and immune cell polarization from gene expression data, and applied it to reveal cytokine networks in tumors following immune checkpoint blockade therapy. Our dictionary generates new hypotheses for cytokine functions, illuminates pleiotropic effects of cytokines, expands our knowledge of activation states of each immune cell type, and provides a framework to deduce the roles of specific cytokines and cell-cell communication networks in any immune response. This work was supported by the NIH Grant RM1HG006193 and an Adelson Medical Research Foundation Grant to N.H. This work was also supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Doctoral Fellowship, Whitaker Health Sciences Fund Fellowship, and Wellington and Irene Loh Fund Fellowship to A.C., and NCI Research Specialist Award (R50CA251956) to S.L.

Preparation of graphene oxide-stabilized Pickering emulsion adjuvant for Pgp3 recombinant vaccine and enhanced immunoprotection against Chlamydia Trachomatis infection.

Traditional emulsion adjuvants are limited in clinical application because of their surfactant dependence. Graphene oxide (GO) has unique amphiphilic properties and therefore has potential to be used as a surfactant substitute to stabilize Pickering emulsions. In this study, GO-stabilized Pickering emulsion (GPE) was prepared and used as an adjuvant to facilitate an enhanced immune response to the Chlamydia trachomatis (Ct) Pgp3 recombinant vaccine. Firstly, GPE was prepared by optimizing the sonication conditions, pH, salinity, GO concentration, and water/oil ratio. GPE with small-size droplets was characterized and chosen as the candidate. Subsequently, controlled-release antigen delivery by GPE was explored. Cellular uptake behaviors, M1 polarization, and cytokine stimulation by GPE + Pgp3 was considered in terms of the production of macrophages. Finally, GPE's adjuvant effect was evaluated by vaccination with Pgp3 recombinant in BALB/c mouse models. GPE with the smallest droplet sizes was prepared by sonication under 163 W for 2 min at 1 mg/mL GO in natural salinity with a pH of 2 when the water/oil ratio was 10:1 (w/w). The optimized average GPE droplet size was 1.8 μm and the zeta potential was -25.0 ± 1.3 mv. GPE delivered antigens by adsorption onto the droplet surface, demonstrating the controlled release of antigens both in vitro and in vivo. In addition, GPE promoted antigen uptake, which stimulated proinflammatory tumor necrosis factor alpha (TNF-α), enhancing the M1 polarization of macrophages in vitro. Macrophage recruitment was also significantly promoted by GPE at the injection site. In the GPE + Pgp3 treatment group, higher levels of immunoglobin (IgG), immunoglobin G1 (IgG1), immunoglobin G2a (IgG2a) sera, and immunoglobin A (IgA) were detected in vaginal fluid, and higher levels of IFN-γ and IL-2 secretion were stimulated, than in the Pgp3 group, showing a significant type 1 T helper (Th1)-type cellular immune response. Chlamydia muridarum challenging showed that GPE enhanced Pgp3's immunoprotection through its advanced clearance of bacterial burden and alleviation of chronic pathological damage in the genital tract. This study enabled the rational design of small-size GPE, shedding light on antigen adsorption and control release, macrophage uptake, polarization and recruitment, which enhanced augmented humoral and cellular immunity and ameliorated chlamydial-induced tissue damage in the genital tract.

Effects of environmental stress factors on the actin cytoskeleton of fungi and plants: Ionizing radiation and ROS.

Actin is an abundant and multifaceted protein in eukaryotic cells that has been detected in the cytoplasm as well as in the nucleus. In cooperation with numerous interacting accessory-proteins, monomeric actin (G-actin) polymerizes into microfilaments (F-actin) which constitute ubiquitous subcellular higher order structures. Considering the extensive spatial dimensions and multifunctionality of actin superarrays, the present study analyses the issue if and to what extent environmental stress factors, specifically ionizing radiation (IR) and reactive oxygen species (ROS), affect the cellular actin-entity. In that context, this review particularly surveys IR-response of fungi and plants. It examines in detail which actin-related cellular constituents and molecular pathways are influenced by IR and related ROS. This comprehensive survey concludes that the general integrity of the total cellular actin cytoskeleton is a requirement for IR-tolerance. Actin's functions in genome organization and nuclear events like chromatin remodeling, DNA-repair, and transcription play a key role. Beyond that, it is highly significant that the macromolecular cytoplasmic and cortical actin-frameworks are affected by IR as well. In response to IR, actin-filament bundling proteins (fimbrins) are required to stabilize cables or patches. In addition, the actin-associated factors mediating cellular polarity are essential for IR-survivability. Moreover, it is concluded that a cellular homeostasis system comprising ROS, ROS-scavengers, NADPH-oxidases, and the actin cytoskeleton plays an essential role here. Consequently, besides the actin-fraction which controls crucial genome-integrity, also the portion which facilitates orderly cellular transport and polarized growth has to be maintained in order to survive IR.