Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14.

Lipopolysaccharide (LPS) induces a strong pro-inflammatory reaction of macrophages upon activation of Toll-like receptor 4 (TLR4) with the assistance of CD14 protein. Considering a key role of plasma membrane rafts in CD14 and TLR4 activity and the significant impact exerted on that activity by endocytosis and intracellular trafficking of the both LPS acceptors, it seemed likely that the pro-inflammatory reaction could be modulated by flotillins. Flotillin-1 and -2 are scaffolding proteins associated with the plasma membrane and also with endo-membranes, affecting both the plasma membrane dynamics and intracellular protein trafficking. To verify the above hypothesis, a set of shRNA was used to down-regulate flotillin-2 in Raw264 cells, which were found to also become deficient in flotillin-1. The flotillin deficiency inhibited strongly the TRIF-dependent endosomal signaling of LPS-activated TLR4, and to a lower extent also the MyD88-dependent one, without affecting the cellular level of TLR4. The flotillin depletion also inhibited the pro-inflammatory activity of TLR2/TLR1 and TLR2/TLR6 but not TLR3. In agreement with those effects, the depletion of flotillins down-regulated the CD14 mRNA level and the cellular content of CD14 protein, and also inhibited constitutive CD14 endocytosis thereby facilitating its shedding. Ultimately, the cell-surface level of CD14 was markedly diminished. Concomitantly, CD14 recycling was enhanced via EEA1-positive early endosomes and golgin-97-positive trans-Golgi network, likely to compensate for the depletion of the cell-surface CD14. We propose that the paucity of surface CD14 is the reason for the down-regulated signaling of TLR4 and the other TLRs depending on CD14 for ligand binding.

Phytoalexin sakuranetin attenuates endocytosis and enhances resistance to rice blast.

Phytoalexin sakuranetin functions in resistance against rice blast. However, the mechanisms underlying the effects of sakuranetin remains elusive. Here, we report that rice lines expressing resistance (R) genes were found to contain high levels of sakuranetin, which correlates with attenuated endocytic trafficking of plasma membrane (PM) proteins. Exogenous and endogenous sakuranetin attenuates the endocytosis of various PM proteins and the fungal effector PWL2. Moreover, accumulation of the avirulence protein AvrCO39, resulting from uptake into rice cells by Magnaporthe oryzae, was reduced following treatment with sakuranetin. Pharmacological manipulation of clathrin-mediated endocytic (CME) suggests that this pathway is targeted by sakuranetin. Indeed, attenuation of CME by sakuranetin is sufficient to convey resistance against rice blast. Our data reveals a mechanism of rice against M. oryzae by increasing sakuranetin levels and repressing the CME of pathogen effectors, which is distinct from the action of many R genes that mainly function by modulating transcription.

The Chemical Inhibitors of Endocytosis: From Mechanisms to Potential Clinical Applications.

Endocytosis is one of the major ways cells communicate with their environment. This process is frequently hijacked by pathogens. Endocytosis also participates in the oncogenic transformation. Here, we review the approaches to inhibit endocytosis, discuss chemical inhibitors of this process, and discuss potential clinical applications of the endocytosis inhibitors.

Guanidine-modified albumin-MMAE conjugates with enhanced endocytosis ability.

Aiming to address the insufficient endocytosis ability of traditional albumin drug conjugates, this paper reports elegant guanidine modification to improve efficacy for the first time. A series of modified albumin drug conjugates were designed and synthesized with different structures, including guanidine (GA), biguanides (BGA) and phenyl (BA), and different quantities of modifications. Then, the endocytosis ability and in vitro/vivo potency of albumin drug conjugates were systematically studied. Finally, a preferred conjugate A4 was screened, which contained 15 BGA modifications. Conjugate A4 maintains spatial stability similar to that of the unmodified conjugate AVM and could significantly enhance endocytosis ability (p*** = 0.0009) compared with the unmodified conjugate AVM. Additionally, the in vitro potency of conjugate A4 (EC50 = 71.78 nmol in SKOV3 cells) was greatly enhanced (approximately 4 times) compared with that of the unmodified conjugate AVM (EC50 = 286.00 nmol in SKOV3 cells). The in vivo efficacy of conjugate A4 completely eliminated 50% of tumors at 33 mg/kg, which was significantly better than the efficacy of conjugate AVM at the same dose (P** = 0.0026). In addition, theranostic albumin drug conjugate A8 was designed to intuitively realize drug release and maintain antitumor activity similar to conjugate A4. In summary, the guanidine modification strategy could provide new ideas for the development of new generational albumin drug conjugates.

[The off-target effects of dynamin inhibitor Dyngo-4a during the internalization of dextran by bone marrow mesenchymal stem cells in rat].

Objective To investigate the possible off-target effects of dynamin (DNM) inhibitor Dyngo-4a in dynamin-dependent endocytic pathways. Methods Bone marrow mesenchymal stem cells (BMSCs) obtained from SD rats were isolated and cultured, and identified by flow cytometry. The cells were divided into inhibitor control group, Dyngo-4a-treated group, negative control siRNA (si-NC) transfection group, DNM2 siRNA transfection (si-DNM2) group, si-DNM2 and Dyngo-4a co-treated group. Real time quantitative PCR and Western blot analysis were used to verify the silencing efficiencies of DNM2 gene and CCK-8 assay were used to detect the cell viability after Dyngo-4a treatment. Confocal microscopy was used to detect the number and mean fluorescence intensity (MFI) of transferrin-Dylight649-positive and dextran-TMR-positive vesicles. Results The mRNA and protein expression levels of DNM2 were down-regulated using small interfering RNA. The number of transferrin-Dylight649-positive vesicles significantly decreased in si-DNM2 group compared with si-NC group. For the number and MFI of dextran-TMR-positive vesicles, no significant change was observed between the si-DNM2 group and the si-NC group, but there was a significant reduction in the si-DNM2 and Dyngo-4a co-treated group compared with the si-DNM2 group. A significant decrease was also found in the Dyngo-4a-treated group compared with the inhibitor control group. Conclusion The off-target effects of dynamin inhibitor Dyngo-4a presents in the internalization of dextran by BMSCs.

The Mood Stabilizer Lithium Slows Down Synaptic Vesicle Cycling at Glutamatergic Synapses.

Lithium is a mood stabilizer broadly used to prevent and treat symptoms of mania and depression in people with bipolar disorder (BD). Little is known, however, about its mode of action. Here, we analyzed the impact of lithium on synaptic vesicle (SV) cycling at presynaptic terminals releasing glutamate, a neurotransmitter previously implicated in BD and other neuropsychiatric conditions. We used the pHluorin-based synaptic tracer vGpH and a fully automated image processing pipeline to quantify the effect of lithium on both SV exocytosis and endocytosis in hippocampal neurons. We found that lithium selectively reduces SV exocytic rates during electrical stimulation, and markedly slows down SV recycling post-stimulation. Analysis of single-bouton responses revealed the existence of functionally distinct excitatory synapses with varying sensitivity to lithium-some terminals show responses similar to untreated cells, while others are markedly impaired in their ability to recycle SVs. While the cause of this heterogeneity is unclear, these data indicate that lithium interacts with the SV machinery and influences glutamate release in a large fraction of excitatory synapses. Together, our findings show that lithium down modulates SV cycling, an effect consistent with clinical reports indicating hyperactivation of glutamate neurotransmission in BD.

Bile Acid Conjugation on Solid Nanoparticles Enhances ASBT-Mediated Endocytosis and Chylomicron Pathway but Weakens the Transcytosis by Inducing Transport Flow in a Cellular Negative Feedback Loop.

Bile acid-modified nanoparticles provide a convenient strategy to improve oral bioavailability of poorly permeable drugs by exploiting specific interactions with bile acid transporters. However, the underlying mechanisms are unknown, especially considering the different absorption sites of free bile acids (ileum) and digested fat molecules from bile acid-emulsified fat droplets (duodenum). Here, glycocholic acid (GCA)-conjugated polystyrene nanoparticles (GCPNs) are synthesized and their transport in Caco-2 cell models is studied. GCA conjugation enhances the uptake by interactions with apical sodium-dependent bile acid transporter (ASBT). A new pathway correlated with both ASBT and chylomicron pathways is identified. Meanwhile, the higher uptake of GCPNs does not lead to higher transcytosis to the same degree compared with unmodified nanoparticles (CPNs). The pharmacological and genomics study confirm that GCA conjugation changes the endocytosis mechanisms and downregulates the cellular response to the transport at gene levels, which works as a negative feedback loop and explains the higher cellular retention of GCPNs. These findings offer a solid foundation in the bile acid-based nanomedicine design, with utilizing advantages of the ASBT-mediated uptake, as well as inspiration to take comprehensive consideration of the cellular response with more developed technologies.

Endocytic trafficking of GAS6-AXL complexes is associated with sustained AKT activation.

AXL, a TAM receptor tyrosine kinase (RTK), and its ligand growth arrest-specific 6 (GAS6) are implicated in cancer metastasis and drug resistance, and cellular entry of viruses. Given this, AXL is an attractive therapeutic target, and its inhibitors are being tested in cancer and COVID-19 clinical trials. Still, astonishingly little is known about intracellular mechanisms that control its function. Here, we characterized endocytosis of AXL, a process known to regulate intracellular functions of RTKs. Consistent with the notion that AXL is a primary receptor for GAS6, its depletion was sufficient to block GAS6 internalization. We discovered that upon receptor ligation, GAS6-AXL complexes were rapidly internalized via several endocytic pathways including both clathrin-mediated and clathrin-independent routes, among the latter the CLIC/GEEC pathway and macropinocytosis. The internalization of AXL was strictly dependent on its kinase activity. In comparison to other RTKs, AXL was endocytosed faster and the majority of the internalized receptor was not degraded but rather recycled via SNX1-positive endosomes. This trafficking pattern coincided with sustained AKT activation upon GAS6 stimulation. Specifically, reduced internalization of GAS6-AXL upon the CLIC/GEEC downregulation intensified, whereas impaired recycling due to depletion of SNX1 and SNX2 attenuated AKT signaling. Altogether, our data uncover the coupling between AXL endocytic trafficking and AKT signaling upon GAS6 stimulation. Moreover, our study provides a rationale for pharmacological inhibition of AXL in antiviral therapy as viruses utilize GAS6-AXL-triggered endocytosis to enter cells.

Smart Lipid-Polysaccharide Nanoparticles for Targeted Delivery of Doxorubicin to Breast Cancer Cells.

In this study, actively-targeted (CD44-receptors) and dual stimuli (pH/redox)-responsive lipid-polymer nanoparticles were proposed as a delivery vehicle of doxorubicin hydrochloride in triple negative breast cancer cell lines. A phosphatidylcholine lipid film was hydrated with a solution of oxidized hyaluronic acid and doxorubicin, chosen as model drug, followed by a crosslinking reaction with cystamine hydrochloride. The obtained spherical nanoparticles (mean diameter of 30 nm) were found to be efficiently internalized in cancer cells by a receptor-mediated endocytosis process, and to modulate the drug release depending on the pH and redox potential of the surrounding medium. In vitro cytotoxicity assays demonstrated the safety and efficacy of the nanoparticles in enhancing the cytotoxic effect of the free anticancer drug, with the IC50 values being reduced by two and three times in MDA-MB-468 and MDA-MB-231, respectively. The combination of self-assembled phospholipid molecules with a polysaccharide counterpart acting as receptor ligand, and stimuli-responsive chemical moieties, was carried out on smart multifunctional nanoparticles able to actively target breast cancer cells and improve the in vitro anticancer activity of doxorubicin.

Co-delivery of doxorubicin and siRNA by all-trans retinoic acid conjugated chitosan-based nanocarriers for multiple synergistic antitumor efficacy.

To achieve the co-delivery of doxorubicin (DOX) and small interfering RNA (siRNA) targeting B-cell lymphoma 2 (siBcl-2), lactose acid (LA) and all-trans retinoic acid (ATRA) double grafted N,N,N-trimethyl chitosan (TMC) nanoparticles (GTA NPs) were developed. The relative viability of QGY-7703 cells was decreased to 81.3% when the concentration of GTA NPs was 0.1 mg/mL, but no toxicity to normal cells was observed, indicating that the GTA NPs selectively inhibited the proliferation of tumor cells. With DOX loaded into the hydrophobic core and siRNA condensed onto the hydrophilic shell, GTA/DOX/siRNA NPs were prepared. The GTA/DOX/siRNA NPs possessed high cellular uptake via receptor-mediated endocytosis. Owing to multiple cooperative antitumor effects of DOX, siBcl-2, and GTA NPs, GTA/DOX/siRNA NPs had superior in vitro and in vivo antitumor efficiency to other formulations. These findings provide a guideline for the combined applications of multiple synergistic antitumor manners.

Flurbiprofen inhibits cell proliferation in thyroid cancer through interrupting HIP1R-induced endocytosis of PTEN.

BACKGROUND: The incidence of thyroid cancer, a most common tumor in the endocrine system, has increased in recent years. A growing number of studies have focused on the molecular mechanisms of thyroid cancer subtypes, aiming to identify effective therapeutic targets. Endocytosis is of vital significance in the malignant development of tumors, although its involvement in thyroid cancer has been rarely reported. METHODS: HIP1R expressions in thyroid cancer from the TCGA database were analyzed by UALCAN software. Thyroid epithelial and cancer cell lines were cultured in vitro. Western blotting and quantitative PCR were used to analyze protein and mRNA levels, respectively. Cell viability was measured by CCK-8 assay. Immunofluorescence staining indicated protein distribution in cell. Co-immunoprecipitation was used to study protein-protein interaction. Immunohistochemical staining was used to analyze protein expression in clinical tissues. Differences between groups were compared using the two-tailed Student's t test, and those among three or more groups were compared by one-way or two-way ANOVA. RESULTS: In the present study, HIP1R (Huntingtin Interacting Protein 1 Related) was found upregulated in thyroid cancer tissues and cell lines compared with that in the controls, while knockdown of HIP1R significantly inhibited the proliferation of thyroid cancer cells. Since HIP1R is essential for the clathrin-dependent endocytic process, we thereafter explored the effect of HIP1R on the endocytosis of thyroid cancer cells. Interestingly, knockdown of HIP1R significantly reduced the number of clathrin-coated pits (CCPs) in thyroid cancer cells. In addition, the interaction between HIP1R and PTEN (phosphatase and tensin homolog) was identified in thyroid cancer cells. Knockdown of HIP1R downregulated intracellular PTEN in thyroid cancer cells, but upregulated membrane-binding PTEN. Notably, flurbiprofen, a commonly used analgesic, significantly inhibited the proliferation of thyroid cancer cells and interfered with the interaction between HIP1R and PTEN, thereby enhancing the binding of PTEN to cell membrane. However, the proliferation inhibitory effect of flurbiprofen was attenuated when knocking down HIP1R or PTEN. CONCLUSIONS: Upregulated HIP1R in thyroid cancer cells promotes cell proliferation and mediates the endocytosis of PTEN. Flurbiprofen may exert an anti-tumor effect on thyroid cancer by blocking the interaction between HIP1R and PTEN.

[(WR)8WKßA]-Doxorubicin Conjugate: A Delivery System to Overcome Multi-Drug Resistance against Doxorubicin.

Doxorubicin (Dox) is an anthracycline chemotherapeutic agent used to treat breast, leukemia, and lymphoma malignancies. However, cardiotoxicity and inherent acquired resistance are major drawbacks, limiting its clinical application. We have previously shown that cyclic peptide [WR]9 containing alternate tryptophan (W) and arginine (R) residues acts as an efficient molecular transporter. An amphiphilic cyclic peptide containing a lysine (K) residue and alternative W and R was conjugated through a free side chain amino group with Dox via a glutarate linker to afford [(WR)8WKßA]-Dox conjugate. Antiproliferative assays were performed in different cancer cell lines using the conjugate and the corresponding physical mixture of the peptide and Dox to evaluate the effectiveness of synthesized conjugate compared to the parent drug alone. [(WR)8WKßA]-Dox conjugate showed higher antiproliferative activity at 10 µM and 5 µM than Dox alone at 5 µM. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and the triple-negative breast cancer cells MDA-MB-231 and MCF-7 by 71% and 77%, respectively, at a concentration of 5 µM after 72 h of incubation. In contrast, Dox inhibited the proliferation of SK-OV-3, MDA-MB-231, and MCF-7 by 35%, 63%, and 57%, respectively. Furthermore, [(WR)8WKßA]-Dox conjugate (5 µM) inhibited the cell viability of Dox-resistant cells (MES-SA/MX2) by 92%, while the viability of cells incubated with free Dox was only 15% at 5 µM. Confocal microscopy images confirmed the ability of both Dox conjugate and the physical mixture of the peptide with the drug to deliver Dox through an endocytosis-independent pathway, as the uptake was not inhibited in the presence of endocytosis inhibitors. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. Half-life (t1/2) for [(WR)8WKßA]-Dox conjugate was (∼6 h), and more than 80% of the conjugate was degraded at 12 h. The release of free Dox was assessed intracellularly using the CCRF-CEM cell line. The experiment demonstrated that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h. These data confirm the ability of the cyclic cell-penetrating peptide containing tryptophan and arginine residues as an efficient tool for delivery of Dox and for overcoming resistance to it.

Bisphenol S Impairs Invasion and Proliferation of Extravillous Trophoblasts Cells by Interfering with Epidermal Growth Factor Receptor Signaling.

The placenta supports fetal growth and is vulnerable to exogenous chemical exposures. We have previously demonstrated that exposure to the emerging chemical bisphenol S (BPS) can alter placental endocrine function. Mechanistically, we have demonstrated that BPS interferes with epidermal growth factor receptor (EGFR) signaling, reducing placenta cell fusion. Extravillous trophoblasts (EVTs), a placenta cell type that aids with vascular remodeling, require EGF to invade into the maternal endometrium. We hypothesized that BPS would impair EGF-mediated invasion and proliferation in EVTs. Using human EVTs (HTR-8/SVneo cells), we tested whether BPS could inhibit the EGF response by blocking EGFR activation. We also evaluated functional endpoints of EGFR signaling, including EGF endocytosis, cell invasion and proliferation, and endovascular differentiation. We demonstrated that BPS blocked EGF-induced phosphorylation of EGFR by acting as a competitive antagonist to EGFR. Transwell assay and a three-dimensional microfluidic chip invasion assay revealed that BPS exposure can block EGF-mediated cell invasion. BPS also blocked EGF-mediated proliferation and endovascular differentiation. In conclusion, BPS can prevent EGF-mediated EVT proliferation and invasion through EGFR antagonism. Given the role of EGFR in trophoblast proliferation and differentiation during placental development, our findings suggest that maternal exposure to BPS may contribute to placental dysfunction via EGFR-mediated mechanisms.

SARS-CoV-2 Omicron variant shows less efficient replication and fusion activity when compared with Delta variant in TMPRSS2-expressed cells.

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell-cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.

The inhibition of interaction with serum albumin enhances the physiological activity of curcumin by increasing its cellular uptake.

Based on the free drug hypothesis, we hypothesized that food compounds that bind stronger to BSA than CUR inhibit the binding between BSA and CUR, and that this results in an increase of the cellular uptake and physiological activities of CUR. To verify this hypothesis, food compounds that bind stronger to BSA than CUR were identified. When THP-1 monocytes were co-treated with the identified compounds (e.g., piperine) and CUR, cell viability significantly decreased, suggesting that the physiological activity of CUR was enhanced. Also, when THP-1 macrophages were co-treated with CUR and the identified compounds following LPS + IFNγ treatment, the decrement of TNF-α was higher compared to treatment with CUR only. Furthermore, the cellular uptake of CUR was increased during this co-treatment. Such results verify our hypothesis, and provide insights into the development of ways to enhance the physiological activities of various food compounds via focusing on their interaction with albumin.

Enhancing the Endocytosis of Phosphatidylserine-Containing Liposomes through Tim4 by Modulation of Membrane Fluidity.

Phosphatidylserine (PS) is a unique lipid that is recognized by the endogenetic receptor, T-cell immunoglobulin mucin protein 4 (Tim4), and PS-containing liposomes have potential use in therapeutic applications. We prepared PS-containing liposomes of various lipid compositions and examined how lipid membrane fluidity affects PS recognition by Tim4 and the resulting endocytosis efficiency into Hela cells. Surface plasmon resonance and laurdan studies showed that increasing lipid membrane fluidity increased the stability of the PS-Tim4 interaction but hampered the entry of liposomes into cells. These results show that endocytosis efficiency is determined by balancing opposing forces induced by membrane fluidity. We found that inclusion of the zwitterionic helper lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, into liposomes ensured efficient cellular internalization because the presence of this lipid provides an ideal balance of lipid fluidity and Tim4 affinity. The results showed that PS recognition by Tim4 and the resulting endocytosis efficiency can be maximized by modulating the membrane fluidity of liposomes by selecting a zwitterionic helper lipid. This study improves our understanding of how to rationally optimize nanotechnology for targeted drug delivery.

Steroid-sensitive nephrotic syndrome candidate gene CLVS1 regulates podocyte oxidative stress and endocytosis.

We performed next-generation sequencing in patients with familial steroid-sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 (CLVS1) in a consanguineous family with 3 affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that could be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR/Cas9-mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid responsiveness observed in patients with SSNS. The p.H310Y variant also disrupted binding of clavesin-1 to α-tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1-deficient podocytes. Treatment of CLVS1-knockout or homozygous H310Y-knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, these data identify CLVS1 as a candidate gene for SSNS, provide insight into therapeutic effects of corticosteroids on podocyte cellular dynamics, and add to the growing evidence of the importance of endocytosis and oxidative stress regulation to podocyte function.

Posaconazole inhibits multiple steps of the alphavirus replication cycle.

Repurposing drugs is a promising strategy to identify therapeutic interventions against novel and re-emerging viruses. Posaconazole is an antifungal drug used to treat invasive aspergillosis and candidiasis. Recently, posaconazole and its structural analog, itraconazole were shown to inhibit replication of multiple viruses by modifying intracellular cholesterol homeostasis. Here, we show that posaconazole inhibits replication of the alphaviruses Semliki Forest virus (SFV), Sindbis virus and chikungunya virus with EC50 values ranging from 1.4 µM to 9.5 µM. Posaconazole treatment led to a significant reduction of virus entry in an assay using a temperature-sensitive SFV mutant, but time-of-addition and RNA transfection assays indicated that posaconazole also inhibits post-entry stages of the viral replication cycle. Virus replication in the presence of posaconazole was partially rescued by the addition of exogenous cholesterol. A transferrin uptake assay revealed that posaconazole considerably slowed down cellular endocytosis. A single point mutation in the SFV E2 glycoprotein, H255R, provided partial resistance to posaconazole as well as to methyl-ß-cyclodextrin, corroborating the effect of posaconazole on cholesterol and viral entry. Our results indicate that posaconazole inhibits multiple steps of the alphavirus replication cycle and broaden the spectrum of viruses that can be targeted in vitro by posaconazole, which could be further explored as a therapeutic agent against emerging viruses.

IRF6 Regulates the Delivery of E-Cadherin to the Plasma Membrane.

IRF6 is a transcription factor that is required for craniofacial development and epidermal morphogenesis. Specifically, Irf6-deficient mice lack the terminally differentiated epidermal layers, leading to an absence of barrier function. This phenotype also includes intraoral adhesions due to the absence of the oral periderm, leading to the mislocalization of E-cadherin and other cell‒cell adhesion proteins of the oral epithelium. However, the mechanisms by which IRF6 controls the localization of cell adhesion proteins are not understood. In this study, we show that in human and murine keratinocytes, loss of IRF6 leads to a breakdown of epidermal sheets after mechanical stress. This defect is due to a reduction of adhesion proteins at the plasma membrane. Dynamin inhibitors rescued the IRF6-dependent resistance of epidermal sheets to mechanical stress, but only inhibition of clathrin-mediated endocytosis rescued the localization of junctional proteins at the membrane. Our data show that E-cadherin recycling but not its endocytosis is affected by loss of IRF6. Overall, we demonstrate a role for IRF6 in the delivery of adhesion proteins to the cell membrane.

Exposure of Caenorhabditis elegans to Dietary Nε-Carboxymethyllysine Emphasizes Endocytosis as a New Route for Intestinal Absorption of Advanced Glycation End Products.

The impact of dietary advanced glycation end products (dAGEs) on human health has been discussed in many studies but, to date, no consensual pathophysiological process has been demonstrated. The intestinal absorption pathways which have so far been described for dAGEs, the passive diffusion of free AGE adducts and transport of glycated di-tripeptides by the peptide transporter 1 (PEPT-1), are not compatible with certain pathophysiological processes described. To get new insight into the intestinal absorption pathways and the pathophysiological mechanisms of dAGEs, we initiated an in vivo study with a so-called simple animal model with a complete digestive tract, Caenorhabditis elegans. Dietary bacteria were chemically modified with glyoxylic acid to mainly produce Nε-carboxymethyllysine (CML) and used to feed the worms. We performed different immunotechniques using an anti-CML antibody for the relative quantification of ingested CML and localization of this AGE in the worms' intestine. The relative expression of genes encoding different biological processes such as response to stresses and intestinal digestion were determined. The physiological development of the worms was verified. All the results were compared with those obtained with the control bacteria. The results revealed a new route for the intestinal absorption of dietary CML (dCML), endocytosis, which could be mediated by scavenger receptors. The exposure of worms to dCML induced a reproductive defect and a transcriptional response reflecting oxidative, carbonyl and protein folding stresses. These data, in particular the demonstration of endocytosis of dCML by enterocytes, open up new perspectives to better characterize the pathophysiological mechanisms of dAGEs.