Junction Assembly Research Articles

Exploring susceptibility and therapeutic targets for kidney stones through proteome-wide Mendelian randomization.

Given the high recurrence rate of kidney stones, surgical lithotripsy and stone removal are not the ultimate treatments for kidney stones. There's an urgent need to explore the genetic mechanisms behind the susceptibility to kidney stones and to identify potential targets for prevention, to reduce the renal damage caused by recurrent stone formation. In this study, we screened 4548 circulating proteins using proteome-wide Mendelian Randomization (MR) to find proteins with a causal relationship to kidney stone risk. Additionally, proteome-wide association study (PWAS) and colocalization analysis were used to validate and prioritize candidate proteins. Moreover, downstream analyses including single-cell analysis, enrichment analysis, protein-protein interaction (PPI), and druggability analysis were conducted on the proteins causally related to kidney stones, to further explore the genetic mechanisms of susceptibility and the potential of proteins as drug targets. Ultimately, 22 target proteins associated with the risk of kidney stones were identified. Six plasma proteins (COLGALT1, CLMP, LECT1, ITIH1, CDHR3, CPLX2) were negatively correlated with kidney stone risk, while the genetic overexpression of 16 target proteins (GJA1, STOM, IRF9, F9, TMPRSS11D, ADH1B, SPINK13, CRYBB2, TNS2, DOCK9, OXSM, MST1, IL2, LMAN2, ITIH3, KLRF1) increased the risk of kidney stones. Based on the PWAS and colocalization analysis results, the 22 target proteins were classified into 3 tiers: IL2, CPLX2, and LMAN2 as tier 1 proteins with the most compelling evidence, MST1, ITIH1, and ITIH3 as tier 2 proteins, and the rest as tier 3 proteins. Enrichment analysis and PPI showed that target proteins mainly affect the occurrence of kidney stones through leukocyte activation and cell junction assembly. Druggability analysis suggested that IL2, MST1, and ITIH1 have potential as drug targets, and potential drugs were evaluated through molecular docking. In summary, this study employed multiple analytical methods to screen plasma proteins related to susceptibility to kidney stones, providing new insights into the genetic mechanisms of kidney stones and potential targets for treatment and prevention.

Loss of Cdc42 causes abnormal optic cup morphogenesis and microphthalmia in mouse.

Congenital ocular malformations originate from defective morphogenesis during early eye development and cause 25% of childhood blindness. Formation of the eye is a multi-step, dynamic process; it involves evagination of the optic vesicle, followed by distal and ventral invagination, leading to the formation of a two-layered optic cup with a transient optic fissure. These tissue folding events require extensive changes in cell shape and tissue growth mediated by cytoskeleton mechanics and intercellular adhesion. We hypothesized that the Rho GTPase Cdc42 may be an essential, convergent effector downstream of key regulatory factors required for ocular morphogenesis. CDC42 controls actin remodeling, apicobasal polarity, and junction assembly. Here we identify a novel essential function for Cdc42 during eye morphogenesis in mouse; in Cdc42 mutant eyes expansion of the ventral optic cup is arrested, resulting in microphthalmia and a wide coloboma. Our analyses show that Cdc42 is required for expression of the polarity effector proteins PRKCZ and PARD6, intercellular junction protein tight junction protein 1, β-catenin, actin cytoskeleton F-actin, and contractile protein phospho myosin light chain 2. Expression of RPE fate determinants OTX2 and MITF, and formation of the RPE layer are severely affected in the temporal domain of the proximal optic cup. EdU incorporation is significantly downregulated. In addition, mitotic retinal progenitor cells mis-localized deeper, basal regions, likely contributing to decreased proliferation. We propose that morphogenesis of the ventral optic cup requires Cdc42 function for coordinated optic cup expansion and establishment of subretinal space, tissue tension, and differentiation of the ventral RPE layer.

Targeting AMP-activated protein kinase in sepsis.

Sepsis is a global health challenge marked by limited clinical options and high mortality rates. AMP-activated protein kinase (AMPK) is a cellular energy sensor that mediates multiple crucial metabolic pathways that may be an attractive therapeutic target in sepsis. Pre-clinical experimental studies have demonstrated that pharmacological activation of AMPK can offer multiple potential benefits during sepsis, including anti-inflammatory effects, induction of autophagy, promotion of mitochondrial biogenesis, enhanced phagocytosis, antimicrobial properties, and regulation of tight junction assembly. This review aims to discuss the existing evidence supporting the therapeutic potential of AMPK activation in sepsis management.

Intestinal epithelial differentiation and barrier function is promoted in vitro by a Cynara cardunculus L. leaf extract through AMPK pathway activation

Gut epithelial barrier perturbation leads to leaky gut syndrome and permeation of substances activating immune response. Polyphenols can improve intestinal barrier function and represent candidates for preventing development of leaky gut. Herein, we evaluated in vitro the molecular mechanisms involved in the protective effects of a polyphenol-rich extract from leaves of Cynara cardunculus L. (CCLE) on intestinal barrier function and integrity on Caco-2 human epithelial cells. Treatment with CCLE from seeding until complete differentiation improved intestinal function by increasing trans-epithelial electrical resistance (TEER), reducing paracellular permeability to fluorescein, and promoting faster recovery of tight junctions (TJ) assembly in the Ca2+ switch assay. CCLE stimulated epithelial cell differentiation inducing alkaline phosphatase activity and TJ proteins. These CCLE-induced effects were attributed to activation of AMP-activated protein kinase (AMPK) pathway. Our data support the use of Cynara cardunculus L. leaves, an agricultural co-product rich in bioactive polyphenols, for the health of intestinal epithelium.

Coactosin-like protein 1 regulates integrity and repair of model intestinal epithelial barriers via actin binding dependent and independent mechanisms.

The actin cytoskeleton regulates the integrity and repair of epithelial barriers by mediating the assembly of tight junctions (TJs), and adherens junctions (AJs), and driving epithelial wound healing. Actin filaments undergo a constant turnover guided by numerous actin-binding proteins, however, the roles of actin filament dynamics in regulating intestinal epithelial barrier integrity and repair remain poorly understood. Coactosin-like protein 1 (COTL1) is a member of the ADF/cofilin homology domain protein superfamily that binds and stabilizes actin filaments. COTL1 is essential for neuronal and cancer cell migration, however, its functions in epithelia remain unknown. The goal of this study is to investigate the roles of COTL1 in regulating the structure, permeability, and repair of the epithelial barrier in human intestinal epithelial cells (IEC). COTL1 was found to be enriched at apical junctions in polarized IEC monolayers in vitro. The knockdown of COTL1 in IEC significantly increased paracellular permeability, impaired the steady state TJ and AJ integrity, and attenuated junctional reassembly in a calcium-switch model. Consistently, downregulation of COTL1 expression in Drosophila melanogaster increased gut permeability. Loss of COTL1 attenuated collective IEC migration and decreased cell-matrix attachment. The observed junctional abnormalities in COTL1-depleted IEC were accompanied by the impaired assembly of the cortical actomyosin cytoskeleton. Overexpression of either wild-type COTL1 or its actin-binding deficient mutant tightened the paracellular barrier and activated junction-associated myosin II. Furthermore, the actin-uncoupled COTL1 mutant inhibited epithelial migration and matrix attachment. These findings highlight COTL1 as a novel regulator of the intestinal epithelial barrier integrity and repair.

Identification of hub genes and key modules in laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC) is the prominent cancer in head and neck, which greatly affects life quality of patients. The pathogenesis of LSCC is not clear. Presently, the LSCC treatments include chemotherapy, surgery and radiotherapy; however, these methods have poor efficacy in patients with recurrent and persistent cancer. Therefore, the study identified the hub genes accompanied with LSCC, which may be a potential therapeutic target in the future. We extracted whole transcriptome high-throughput sequencing (HTS) LSCC data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and calculate differentially expressed genes (DEGs) between LSCC and normal samples using statistical software RStudio. Through weighted gene co-expression network analysis (WGCNA), enrichment examination of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) functions, and examination of protein-protein interaction (PPI) network, we obtained network hub genes and validated the hub genes prognostic value and expression levels of protein. Through analysis of differential gene expression, from the GEO and TCGA databases 2,139 and 2,774 DEGs were obtained, respectively, 13 and 15 modules were screened from TCGA-LSCC and GSE127165 datasets by WGCNA, respectively. The most significant positive and negative correlation modules in the WGCNA and DEG lists were overlapped, and overall 36 co-expressed overlapping genes were retrieved. Through enrichment analysis of GO and KEGG, it was found that the gene functions were highly concentrated in cell junction assembly, basement membrane, extracellular matrix (ECM) structural constituent etc., and the pathways were mainly concentrated in ECM receptor interaction, focal adhesion, small cell lung cancer, and toxoplasmosis. Through analysis of PPI network analysis, 10 network hub genes (SNAI2, ITGA6, LAMB3, LAMC2, CAV1, COL7A1, GJA1, EHF, OAT, and GPT) were obtained. Finally, survival analysis and protein expression validation of these genes confirmed that low OAT expression and high CAV1 expression remarkably influenced the survival of patient's prognosis with LSCC. We recognized the hub genes and key modules nearly associated to LSCC and these genes were validated by survival analysis and the database of Human Protein Atlas (HPA), which is of high importance for unveiling the pathogenesis of LSCC and probing for new precise biological marker and potential therapeutic targets.

Characterization of a new arrhythmogenic cardiomyopathy mouse model due to filamin c haploinsufficiency

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Spanish Ministry of Universities FPU fellowship (FPU19/04044), Spanish Ministry of Science and Innovation Digital Transition call (TED2021-129774B-C22), EU HORIZON EIC Pathfinder Challenges 2022 Cardiogenomics call (Project 101115416). Background Filamin C (FLNC) is widely expressed in heart and skeletal muscle. It participates in cell junction and sarcomere assembly and function. We have previously shown that heterozygous FLNC truncating variants (FLNCtv) cause arrhythmogenic/dilated cardiomyopathy (ACM/DCM) in patients. Diverse studies have suggested haploinsufficiency as the potential mechanism of this disease. However, animal models of FLNCtv have only been reported to develop ACM/DCM in homozygosity. Purpose To develop a new model of ACM/DCM caused by a FLNCtv and study the progression of the disease in heterozygosity. Methods Two gRNAs flanking exon 15 of the Flnc gene and SpCas9 protein were microinjected into mouse zygotes to generate a mouse line with a constitutive deletion of Flnc exon 15 (FlncWt/Ex15del). mRNA and protein stability were determined by qRT-PCR and western blot, respectively. Cardiac function was analysed by ECG and echocardiography every 2 months, starting at 8 weeks of age (Fig. 1B). The mean ECG values were calculated using 3 time intervals and standard ECG waves. Results FlncWt/Ex15del mice showed a reduction in Flnc mRNA and protein similar to that observed in a human ACM/DCM patient bearing a similar mutation, suggesting that this FLNCtv causes haploinsufficiency. Both male and female FlncWt/Ex15del mice developed cardiac electrophysiological defects, including reduced amplitude of the QRS complex and the P-wave, notched R and/or S waves, and increased QT interval times, and these defects worsened with time. At 40 weeks of age, FlncWt/Ex15del mice showed some initial signs of left ventricular dilatation. Conclusions Mice carrying FLNCtv in heterozygosity develop electrophysiological changes resembling pathological features of ACM/DCM patients due to haploinsufficiency. The development of this pathological phenotype in heterozygosity, as opposed to previous models, paves the way for the development of new gene therapies using this model.

Kidney Organoid Modeling of WT1 Mutations Reveals Key Regulatory Paths Underlying Podocyte Development.

Wilms tumor-1(WT1) is a crucial transcription factor that regulates podocyte development. However, the epigenomic mechanism underlying the function of WT1 during podocyte development has yet to be fully elucidated. Here, single-cell chromatin accessibilityand gene expressionmaps of foetal kidneys and kidney organoids are generated. Functional implications of WT1-targeted genes, which are crucial for the development of podocytes and the maintenance of their structure, including BMPER/PAX2/MAGI2 that regulates WNT signaling pathway, MYH9 that maintains actin filament organization and NPHS1 that modulates cell junction assembly are identified. To further illustrate the functional importance of WT1-mediated transcriptional regulation during podocyte development, cultured and implanted patient-derived kidney organoids derived from the Induced Pluripotent Stem Cell (iPSCs) of a patient with a heterozygous missense mutation in WT1 are generated. Results from single-cell RNA sequencing (scRNA-seq)and functional assays confirm that the WT1 mutation leads to delays in podocyte development and causes damage to cell structures, due to its failure to activate the targeting genes MAGI2, MYH9, and NPHS1. Notably, correcting the mutation in the patient iPSCs using CRISPR-Cas9 gene editing rescues the podocyte phenotype. Collectively, this work elucidates the WT1-related epigenomic landscape with respect to human podocyte development and identifies the disease-causing role of a WT1 mutation.

Prioritizing cervical cancer candidate genes using chaos game and fractal-based time series approach.

Cervical cancer is one of the most severe threats to women worldwide and holds fourth rank in lethality. It is estimated that 604, 127 cervical cancer cases have been reported in 2020 globally. With advancements in high throughput technologies and bioinformatics, several cervical candidate genes have been proposed for better therapeutic strategies. In this paper, we intend to prioritize the candidate genes that are involved in cervical cancer progression through a fractal time series-based cross-correlations approach. we apply the chaos game representation theory combining a two-dimensional multifractal detrended cross-correlations approach among the known and candidate genes involved in cervical cancer progression to prioritize the candidate genes. We obtained 16 candidate genes that showed cross-correlation with known cancer genes. Functional enrichment analysis of the candidate genes shows that they involve GO terms: biological processes, cell-cell junction assembly, cell-cell junction organization, regulation of cell shape, cortical actin cytoskeleton organization, and actomyosin structure organization. KEGG pathway analysis revealed genes' role in Rap1 signaling pathway, ErbB signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, mTOR signaling pathway, Acute myeloid leukemia, chronic myeloid leukemia, Breast cancer, Thyroid cancer, Bladder cancer, and Gastric cancer. Further, we performed survival analysis and prioritized six genes CDH2, PAIP1, BRAF, EPB41L3, OSMR, and RUNX1 as potential candidate genes for cervical cancer that has a crucial role in tumor progression. We found that our study through this integrative approach an efficient tool and paved a new way to prioritize the candidate genes and these genes could be evaluated experimentally for potential validation. We suggest this may be useful in analyzing the nucleotide sequences and protein sequences for clustering, classification, class affiliation, etc.

Identification of an FMNL2 Interactome by Quantitative Mass Spectrometry.

Formin Homology Proteins (Formins) are a highly conserved family of cytoskeletal regulatory proteins that participate in a diverse range of cellular processes. FMNL2 is a member of the Diaphanous-Related Formin sub-group, and previous reports suggest FMNL2's role in filopodia assembly, force generation at lamellipodia, subcellular trafficking, cell-cell junction assembly, and focal adhesion formation. How FMNL2 is recruited to these sites of action is not well understood. To shed light on how FMNL2 activity is partitioned between subcellular locations, we used biotin proximity labeling and proteomic analysis to identify an FMNL2 interactome. The interactome identified known and new FMNL2 interacting proteins with functions related to previously described FMNL2 activities. In addition, our interactome predicts a novel connection between FMNL2 and extracellular vesicle assembly. We show directly that FMNL2 protein is present in exosomes.

#2305 Mice with a PAX2 missense variant are susceptible to experimental focal segmental glomerulosclerosis (FSGS) and display impaired glomerular repair

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a histopathologic lesion observed in chronic kidney disease characterized by scarring of the glomerulus due to the injury and irreversible loss of highly specialized glomerular epithelial cells called podocytes. Recent studies suggest that podocytes may be regenerated by another population of glomerular epithelial cells called parietal epithelial cells (PECs), although this remains controversial. PAX2 is a transcription factor protein that regulates glomerular development and its expression persists in adult parietal epithelial cells (PECs), postulated to serve as a reservoir for podocytes in the event of injury and loss. Our team previously found that PAX2 pathogenic missense variants account for 4% of adult FSGS. In our present study, our aim is to determine whether PEC-mediated podocyte regeneration is impaired in mice with a pathogenic Pax2 missense variant (Pax2-MV). Method FSGS was induced in wildtype and Pax2-MV mice using Adriamycin. Urine samples were collected from mice every week for 28 days to measure proteinuria. At 28 days after Adriamycin injury, kidneys were collected for histopathology, transmission electron microscopy, and immunofluorescence staining for PAX2 and podocyte marker WT-1. Glomeruli from Adriamycin-injured mice were also isolated through intra-cardiac perfusion with Dynabeads and subjected to mass-spectrometry for proteomics analysis. Proteins with statistically-significant fold changes in expression levels were further analyzed by Gene Ontology to determine biological and molecular processes involved in pathogenesis. Kidney tissue from an FSGS patient with a pathogenic PAX2 missense variant was also examined by histopathology and PAX2 immunohistochemistry. Results Compared to wildtype, Pax2-MV mice were more susceptible to worsened FSGS after Adriamycin, as demonstrated by worsened glomerular scarring, increased podocyte injury and loss, and increased proteinuria, associated with increased morality in Pax2-MV mice (Fig. 1A-B). Wildtype mice showed fewer PAX2-expressing PECs after injury, accompanied by the occurrence of PAX2/WT-1 co-expressing glomerular tuft cells (Fig. 2A-B). These suggest that in Adriamycin-injured wildtype, PAX2-expressing PECs move to the glomerular tuft, where they trans-differentiate to a podocyte fate. In contrast, Pax2-MV mice showed no changes in numbers of PAX2-expressing PECs after injury, associated with fewer PAX2/WT-1 co-expressing glomerular tuft cells compared to injured wildtype (Fig. 2A-B). These suggest that in Adriamycin-injured Pax2-MV mice, PAX2-expressing PECs are unable to move to the glomerular tuft, resulting in decreased numbers of cells undergoing podocyte trans-differentiation. A subset of ectopic PAX2-expressing glomerular tuft cells after injury was increased in Pax2-MV mice (Fig. 2A), suggesting a pathologic process given the worse outcomes observed in these mice. Consistent with findings from injured Pax2-MV mice, we found that in an FSGS patient with a pathogenic PAX2 missense variant, PAX2-expressing glomerular tuft cells overlap with areas of glomerular scarring, while in a normal human kidney biopsy, PAX2 expression is only observed in PECs but not within glomerular tuft cells (Fig. 2A). Lastly, proteomics Gene Ontology analysis of isolated glomeruli demonstrated biological and molecular processes indicative of maladaptive glomerular repair in injured Pax2-MV mice, including decreased regulation of cell shape, decreased cell-cell junction assembly and decreased actin and cadherin binding compared to wildtype (Fig. 2C). Conclusion Altogether, our findings suggest that PAX2-expressing PECs migrate to the glomerular tuft to assist in podocyte regeneration in wildtype. However, this mechanism is impaired in Pax2-MV mice, leading to worsened podocyte loss and injury and further exacerbation of glomerular disease (Fig. 2C). Our findings provide a further understanding of normal and impaired regeneration mechanisms in FSGS, and our identified pathogenic mechanisms can be further harnessed for future therapeutic development.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice

BackgroundRetinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation.MethodsWe first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR.ResultsASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2.ConclusionThese data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

Actin Cytoskeleton Remodeling Accompanied by Redistribution of Adhesion Proteins Drives Migration of Cells in Different EMT States.

Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of β-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-β-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.

Dsg2 ectodomain organization increases throughout desmosome assembly

ABSTRACT Desmosomes are intercellular junctions that regulate mechanical integrity in epithelia and cardiac muscle. Dynamic desmosome remodeling is essential for wound healing and development, yet the mechanisms governing junction assembly remain elusive. While we and others have shown that cadherin ectodomains are highly organized, how this ordered architecture emerges during assembly is unknown. Using fluorescence polarization microscopy, we show that desmoglein 2 (Dsg2) ectodomain order gradually increases during 8 h of assembly, coinciding with increasing adhesive strength. In a scratch wound assay, we observed a similar increase in order in desmosomes assembling at the leading edge of migratory cells. Together, our findings indicate that cadherin organization is a hallmark of desmosome maturity and may play a role in conferring adhesive strength.

Application of direct electric current to the corneal and conjunctival epithelia regulates the tight junctional assembly for ocular iontophoretic drug delivery

Objectives: In this study, we determined how iontophoresis (IP) affects tight junctions (TJs) in isolated rabbit corneas and conjunctiva.
 Methods: Direct electric current in the range of 0.5–2.0 and 0.5–10 mA/cm2 were applied to the cornea and conjunctiva, respectively, for 30 min. The localization and expression levels of TJ-associated proteins were assessed before and after the application of the electric currents using immunostaining and western blotting.
 Results: In both corneal and conjunctival epithelia, the localization of proteins, such as claudin-1, claudin-4, occludin, and ZO-1, was temporarily altered by anodal and cathodal IP; however, the protein relocalization was slower at higher currents. Additionally, in both anodal and cathodal IP, the expression levels of claudin-1 and occludin in the cornea and conjunctiva remained unchanged after the application of the electric currents compared with those before.
 Conclusion: Our results indicated that the application of a direct electric current temporarily regulated TJ assemblies without altering the levels of TJ-associated proteins in both the cornea and conjunctiva. This temporary weakening of the paracellular barrier by the current may be responsible for the enhanced drug transport across the cornea and conjunctiva induced by ocular IP.
 Keywords: ocular drug delivery, iontophoresis, electric current, cornea, conjunctiva, tight junction

Palmitoylation of proteolipid protein M6 promotes tricellular junction assembly in epithelia of Drosophila.

Tricellular junctions (TCJs) seal epithelial cell vertices and are essential for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka, also known as Bark), Gliotactin (Gli) and M6 organize occluding TCJs. Aka and M6 localize in an interdependent manner to vertices and act jointly to localize Gli, but how these proteins interact to assemble TCJs was not previously known. Here, we show that the proteolipid protein M6 physically interacts with Aka and with itself, and that M6 is palmitoylated on conserved juxta-membrane cysteine residues. This modification promotes vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed localization of M6 and Aka but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, whereas subsequent TCJ growth relies on different mechanisms that are independent of M6 palmitoylation.

Transcriptomic analysis identifies B-lymphocyte kinase as a therapeutic target for desmoplastic small round cell tumor cancer stem cell-like cells

Desmoplastic small round cell tumor (DSRCT) is an aggressive pediatric cancer caused by the EWSR1-WT1 fusion oncoprotein. The tumor is refractory to treatment with a 5-year survival rate of only 15–25%, necessitating the development of novel therapeutics, especially those able to target chemoresistant subpopulations. Novel in vitro cancer stem cell-like (CSC-like) culture conditions increase the expression of stemness markers (SOX2, NANOG) and reduce DSRCT cell line susceptibility to chemotherapy while maintaining the ability of DSRCT cells to form xenografts. To gain insights into this chemoresistant model, RNA-seq was performed to elucidate transcriptional alterations between DSRCT cells grown in CSC-like spheres and normal 2-dimensional adherent state. Commonly upregulated and downregulated genes were identified and utilized in pathway analysis revealing upregulation of pathways related to chromatin assembly and disassembly and downregulation of pathways including cell junction assembly and extracellular matrix organization. Alterations in chromatin assembly suggest a role for epigenetics in the DSRCT CSC-like state, which was further investigated with ATAC-seq, identifying over 10,000 differentially accessible peaks, including 4444 sphere accessible peaks and 6,120 adherent accessible peaks. Accessible regions were associated with higher gene expression, including increased accessibility of the CSC marker SOX2 in CSC-like culture conditions. These analyses were further utilized to identify potential CSC therapeutic targets, leading to the identification of B-lymphocyte kinase (BLK) as a CSC-enriched, EWSR1-WT1-regulated, druggable target. BLK inhibition and knockdown reduced CSC-like properties, including abrogation of tumorsphere formation and stemness marker expression. Importantly, BLK knockdown reduced DSRCT CSC-like cell chemoresistance, making its inhibition a promising target for future combination therapy.

Conductance and assembly of quasi-1D coordination chain molecular junctions with triazole derivatives.

Incorporating transition metal atoms into metal-molecule-metal junctions presents opportunities for exploring the electronic properties of coordination complexes, organometallics and metal-organic materials on the single molecule level. Recent single molecule conductance studies have shown that in situ incorporation of electrode metal atoms into coordination chains formed in the junction can occur with deprotonated, negatively charged organic ligands, such as the imidazolate (Im-) anion. However, the mechanism and chemical principles, such as the role of the charge state of the ligand, for the construction of such coordination chains are still debated. Here, we probe the role of the ligand charge state and electronic structure in single-molecule conductance and formation of metal-molecule coordination chains. We perform break junction measurements with triazole isomers, which can bridge junctions both in their neutral and charged forms, and find that prior deprotonation of the ligands is not required for coordination complex assembly, but can affect the molecular conductance and junction formation probability. Our results indicate that coordination chains can form with neutral ligands, as long as the electron density in the frontier MOs is concentrated at the binding sites and along the direction of pulling, promoting ligand binding and incorporation of gold atoms into the junction during elongation. Our findings may provide insight into design principles for in situ assembled molecular wires with transition metal atoms and open the door to electronic and spintronic studies of such materials.

Influence of postruminal casein infusion and exogenous glucagon-like peptide 2 administration on the jejunal mucosal transcriptome in cattle.

We previously demonstrated that postruminal casein infusion and exogenous glucagon-like peptide 2 (GLP-2) administration independently stimulated growth and carbohydrase activity of the pancreas and jejunal mucosa in cattle. The objective of the current study was to profile the jejunal mucosal transcriptome of cattle using next-generation RNA sequencing in response to postruminal casein infusion and exogenous GLP-2. Twenty-four Holstein steers [250 ± 23.1 kg body weight (BW)] received a continuous abomasal infusion of 3.94 g raw corn starch/kg of BW combined with either 0 or 1.30 g casein/kg of BW for 7 d. Steers received subcutaneous injections at 0800 and 2000 h to provide either 0 or 100 μg GLP-2/kg of BW per day. At the end of the 7-d treatment period, steers were slaughtered for collection of the jejunal mucosa. Total RNA was extracted from jejunal mucosal tissue, strand-specific cDNA libraries were prepared, and RNA sequencing was conducted to generate 150-bp paired-end reads at a depth of 40 M reads per sample. Differentially expressed genes (DEG), KEGG pathway enrichment, and gene ontology enrichment were determined based on the FDR-corrected P-value (padj). Exogenous GLP-2 administration upregulated (padj < 0.05) 667 genes and downregulated 1,101 genes of the jejunal mucosa. Sphingolipid metabolism, bile secretion, adherens junction, and galactose metabolism were among the top KEGG pathways enriched with upregulated DEG (padj < 0.05) in response to exogenous GLP-2 administration. The top gene ontologies enriched with upregulated DEG (padj < 0.05) in response to exogenous GLP-2 administration included nutrient metabolic processes, brush border and bicellular tight junction assembly, and enzyme and transporter activities. Exogenous GLP-2 administration increased or tended to increase (padj < 0.10) brush border carbohydrase (MGAM, LCT, TREH), hexose transporter (SLC5A1, SLC2A2), and associated transcription factor (HNF1, GATA4, KAT2B) mRNA expression of the jejunal mucosa. Gene ontologies and KEGG pathways that were downregulated (padj < 0.05) in response to exogenous GLP-2 were related to genetic information processing. Postruminal casein infusion downregulated (padj < 0.05) 7 jejunal mucosal genes that collectively did not result in enriched KEGG pathways or gene ontologies. This study highlights some of the transcriptional mechanisms associated with increased growth, starch assimilation capacity, and barrier function of the jejunal mucosa in response to exogenous GLP-2 administration.

Relevance Analysis of TPM2 and Clinicopathological Characteristics in Breast Cancer.

The function of tropomyosin 2 (TPM2) in breast cancer is still far understudied. In this study, we aim to explore the roles of TPM2 in breast cancer progression. This research included 155 breast cancer tissues. The expression of TPM2 was analyzed by immunohistochemical staining and grading. The mRNA expression of TPM2 in pan-cancer was analyzed with The Cancer Genome Atlas (TCGA) data plate form. The differential expression of TPM2 protein and the differential promoter methylation level of TPM2 between breast cancer tissues and normal breast tissues were analyzed by the UALCAN online database. The relationship between TPM2 and signaling pathways was interpreted by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) pathway enrichment analyses. The survival curve of TPM2 was analyzed across the Kaplan-Meier plotter online database. Furthermore, the relationship between TPM2 expression and infiltrating macrophages was validated through in vitro co-culture experiments. TPM2 expression was significantly down-regulated in breast cancer samples. In addition, TPM2 expression was correlated with lymph node metastasis and high-grade histopathological morphology. The receiver operating characteristic (ROC) curve indicated that TPM2 expression could well distinguish between normal breast tissue and breast cancer tissue. TPM2 may have potential value in breast cancer diagnosis. Bioinformatics analysis illustrated that TPM2 was mainly involved in extracellular matrix organization, collagen fibril organization, cell junction assembly, focal adhesion, cAMP signaling pathway, estrogen signaling pathway, Wnt signaling pathway, and adaptive immune system. TPM2 expression was correlated with immune infiltrating cells and immune checkpoint molecules. Our in vitro co-culture experiments showed that the M2 macrophages could upregulate the expression of TPM2. TPM2 may play key roles in breast cancer occurrence and development, especially in cancer metastasis. TPM2 may be a potential biomarker for breast cancer diagnosis.