Inhibition of cholesterol transport impairs Cav-1 trafficking and small extracellular vesicles secretion, promoting amphisome formation in melanoma cells.

Caveolin-1 (Cav-1) is a fundamental constituent of caveolae, whose functionality and structure are strictly dependent on cholesterol. In this work the U18666A inhibitor was used to study the role of cholesterol transport in the endosomal degradative-secretory system in a metastatic human melanoma cell line (WM266-4). We found that U18666A induces a shift of Cav-1 from the plasma membrane to the endolysosomal compartment, which is involved, through Multi Vesicular Bodies (MVBs), in the formation and release of small extracellular vesicles (sEVs). Moreover, this inhibitor induces an increase in the production of sEVs with chemical-physical characteristics similar to control sEVs but with a different protein composition (lower expression of Cav-1 and increase of LC3II) and reduced transfer capacity on target cells. Furthermore, we determined that U18666A affects mitochondrial function and also cancer cell aggressive features, such as migration and invasion. Taken together, these results indicate that the blockage of cholesterol transport, determining the internalization of Cav-1, may modify sEVs secretory pathways through an increased fusion between autophagosomes and MVBs to form amphisome, which in turn fuses with the plasma membrane releasing a heterogeneous population of sEVs to maintain homeostasis and ensure correct cellular functionality.

The Antihypertensive Drug Telmisartan Protects Oligodendrocytes from Cholesterol Accumulation and Promotes Differentiation by a PPAR-γ-Mediated Mechanism.

Our previous studies have demonstrated that specific peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists play a fundamental role in oligodendrocyte progenitor (OP) differentiation, protecting them against oxidative and inflammatory damage. The antihypertensive drug Telmisartan (TLM) was shown to act as a PPAR-γ modulator. This study investigates the TLM effect on OP differentiation and validates its capability to restore damage in a pharmacological model of Niemann-Pick type C (NPC) disease through a PPAR-γ-mediated mechanism. For the first time in purified OPs, we demonstrate that TLM-induced PPAR-γ activation downregulates the type 1 angiotensin II receptor (AT1), the level of which naturally decreases during differentiation. Like other PPAR-γ agonists, we show that TLM promotes peroxisomal proliferation and promotes OP differentiation. Furthermore, TLM can offset the OP maturation arrest induced by a lysosomal cholesterol transport inhibitor (U18666A), which reproduces an NPC1-like phenotype. In the NPC1 model, TLM also reduces cholesterol accumulation within peroxisomal and lysosomal compartments and the contacts between lysosomes and peroxisomes, revealing that TLM can regulate intracellular cholesterol transport, crucial for myelin formation. Altogether, these data indicate a new potential use of TLM in hypomyelination pathologies such as NPC1, underlining the possible repositioning of the drug already used in other pathologies.

Syk inhibitors interfere with erythrocyte membrane modification during P falciparum growth and suppress parasite egress.

Band 3 (also known as the anion exchanger, SLCA1, AE1) constitutes the major attachment site of the spectrin-based cytoskeleton to the erythrocyte's lipid bilayer and thereby contributes critically to the stability of the red cell membrane. During the intraerythrocytic stage of Plasmodium falciparum's lifecycle, band 3 becomes tyrosine phosphorylated in response to oxidative stress, leading to a decrease in its affinity for the spectrin/actin cytoskeleton and causing global membrane destabilization. Because this membrane weakening is hypothesized to facilitate parasite egress and the consequent dissemination of released merozoites throughout the bloodstream, we decided to explore which tyrosine kinase inhibitors might block the kinase-induced membrane destabilization. We demonstrate here that multiple Syk kinase inhibitors both prevent parasite-induced band 3 tyrosine phosphorylation and inhibit parasite-promoted membrane destabilization. We also show that the same Syk kinase inhibitors suppress merozoite egress near the end of the parasite's intraerythrocytic lifecycle. Because the entrapped merozoites die when prevented from escaping their host erythrocytes and because some Syk inhibitors have displayed long-term safety in human clinical trials, we suggest Syk kinase inhibitors constitute a promising class of antimalarial drugs that can suppress parasitemia by inhibiting a host target that cannot be mutated by the parasite to evolve drug resistance.

Comparative Proteomics and Functional Analysis Reveal a Role of Plasmodium falciparum Osmiophilic Bodies in Malaria Parasite Transmission.

An essential step in the transmission of the malaria parasite to the Anopheles vector is the transformation of the mature gametocytes into gametes in the mosquito gut, where they egress from the erythrocytes and mate to produce a zygote, which matures into a motile ookinete. Osmiophilic bodies are electron dense secretory organelles of the female gametocytes which discharge their contents during gamete formation, suggestive of a role in gamete egress. Only one protein with no functional annotation, Pfg377, is described to specifically reside in osmiophilic bodies in Plasmodium falciparum Importantly, Pfg377 defective gametocytes lack osmiophilic bodies and fail to infect mosquitoes, as confirmed here with newly produced pfg377 disrupted parasites. The unique feature of Pfg377 defective gametocytes of lacking osmiophilic bodies was here exploited to perform comparative, label free, global and affinity proteomics analyses of mutant and wild type gametocytes to identify components of these organelles. Subcellular localization studies with fluorescent reporter gene fusions and specific antibodies revealed an osmiophilic body localization for four out of five candidate gene products analyzed: the proteases PfSUB2 (subtilisin 2) and PfDPAP2 (Dipeptidyl aminopeptidase 2), the ortholog of the osmiophilic body component of the rodent malaria gametocytes PbGEST and a previously nonannotated 13 kDa protein. These results establish that osmiophilic bodies and their components are dispensable or marginally contribute (PfDPAP2) to gamete egress. Instead, this work reveals a previously unsuspected role of these organelles in P. falciparum development in the mosquito vector.

CRISPR-Cas9-modified pfmdr1 protects Plasmodium falciparum asexual blood stages and gametocytes against a class of piperazine-containing compounds but potentiates artemisinin-based combination therapy partner drugs.

Emerging resistance to first-line antimalarial combination therapies threatens malaria treatment and the global elimination campaign. Improved therapeutic strategies are required to protect existing drugs and enhance treatment efficacy. We report that the piperazine-containing compound ACT-451840 exhibits single-digit nanomolar inhibition of the Plasmodium falciparum asexual blood stages and transmissible gametocyte forms. Genome sequence analyses of in vitro-derived ACT-451840-resistant parasites revealed single nucleotide polymorphisms in pfmdr1, which encodes a digestive vacuole membrane-bound ATP-binding cassette transporter known to alter P. falciparum susceptibility to multiple first-line antimalarials. CRISPR-Cas9 based gene editing confirmed that PfMDR1 point mutations mediated ACT-451840 resistance. Resistant parasites demonstrated increased susceptibility to the clinical drugs lumefantrine, mefloquine, quinine and amodiaquine. Stage V gametocytes harboring Cas9-introduced pfmdr1 mutations also acquired ACT-451840 resistance. These findings reveal that PfMDR1 mutations can impart resistance to compounds active against asexual blood stages and mature gametocytes. Exploiting PfMDR1 resistance mechanisms provides new opportunities for developing disease-relieving and transmission-blocking antimalarials.

Distinct properties of the egress-related osmiophilic bodies in male and female gametocytes of the rodent malaria parasite Plasmodium berghei.

Gametogenesis is the earliest event after uptake of malaria parasites by the mosquito vector, with a decisive impact on colonization of the mosquito midgut. This process is triggered by a drop in temperature and contact with mosquito molecules. In a few minutes, male and female gametocytes escape from the host erythrocyte by rupturing the parasitophorous vacuole and the erythrocyte membranes. Electron-dense, oval-shaped organelles, the osmiophilic bodies (OB), have been implicated in the egress of female gametocytes. By comparative electron microscopy and electron tomography analyses combined with immunolocalization experiments, we here define the morphological features distinctive of male secretory organelles, hereafter named MOB (male osmiophilic bodies). These organelles appear as club-shaped, electron-dense vesicles, smaller than female OB. We found that a drop in temperature triggers MOB clustering, independently of exposure to other stimuli. MDV1/PEG3, a protein associated with OB in Plasmodium berghei females, localizes to both non-clustered and clustered MOB, suggesting that clustering precedes vesicle discharge. A P. berghei mutant lacking the OB-resident female-specific protein Pbg377 displays a dramatic reduction in size of the OB, accompanied by a delay in female gamete egress efficiency, while female gamete fertility is not affected. Immunolocalization experiments indicated that MDV1/PEG3 is still recruited to OB-remnant structures.

Erythrocyte remodeling in Plasmodium berghei infection: the contribution of SEP family members.

The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine-rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep-coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein-protein interactions.

Early gametocytes of the malaria parasite Plasmodium falciparum specifically remodel the adhesive properties of infected erythrocyte surface.

In Plasmodium falciparum infections the parasite transmission stages, the gametocytes, mature in 10 days sequestered in internal organs. Recent studies suggest that cell mechanical properties rather than adhesive interactions play a role in sequestration during gametocyte maturation. It remains instead obscure how sequestration is established, and how the earliest sexual stages, morphologically similar to asexual trophozoites, modify the infected erythrocytes and their cytoadhesive properties at the onset of gametocytogenesis. Here, purified P. falciparum early gametocytes were used to ultrastructurally and biochemically analyse parasite-induced modifications on the red blood cell surface and to measure their functional consequences on adhesion to human endothelial cells. This work revealed that stage I gametocytes are able to deform the infected erythrocytes like asexual parasites, but do not modify its surface with adhesive 'knob' structures and associated proteins. Reduced levels of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins are exposed on the red blood cell surface by these parasites, and the expression of the var gene family, which encodes 50-60 variants of PfEMP1, is dramatically downregulated in the transition from asexual development to gametocytogenesis. Cytoadhesion assays show that such gene expression changes and host cell surface modifications functionally result in the inability of stage I gametocytes to bind the host ligands used by the asexual parasite to bind endothelial cells. In conclusion, these results identify specific differences in molecular and cellular mechanisms of host cell remodelling and in adhesive properties, leading to clearly distinct host parasite interplays in the establishment of sequestration of stage I gametocytes and of asexual trophozoites.

A perforin-like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes.

Successful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin-like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore-forming toxin, we conclude that rupture of the erythrocyte membrane is blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforin-like protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.

Unveiling Cryptosporidium parvum sporozoite-derived extracellular vesicles: profiling, origin, and protein composition.

Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after its ingestion, the oocyst excysts and releases four sporozoites into the host intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) that was not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. Fluorescence flow cytometry and transmission electron microscopy (TEM) experiments identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs). Nanoparticle tracking analysis (NTA) revealed mode diameter of 181 nm for LEVs and 105 nm for SEVs, respectively. Immunodetection experiments proved the presence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy trials demonstrated the localization of CpRom1 on the LEVs surface. TEM and scanning electron microscopy (SEM) showed that LEVs were generated by means of the budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Distinct protein compositions were observed between LEVs and SEVs as evidenced by their corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 and 16 proteins unique for LEVs and SEVs, respectively. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. and this last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.

Anaerobic treatment of groundwater co-contaminated by toluene and copper in a single chamber bioelectrochemical system.

Addressing the simultaneous removal of multiple coexisting groundwater contaminants poses a significant challenge, primarily because of their different physicochemical properties. Indeed, different chemical compounds may necessitate establishing distinct, and sometimes conflicting, (bio)degradation and/or removal pathways. In this work, we investigated the concomitant anaerobic treatment of toluene and copper in a single-chamber bioelectrochemical cell with a potential difference of 1 V applied between the anode and the cathode. As a result, the electric current generated by the bioelectrocatalytic oxidation of toluene at the anode caused the abiotic reduction and precipitation of copper at the cathode, until the complete removal of both contaminants was achieved. Open circuit potential (OCP) experiments confirmed that the removal of copper and toluene was primarily associated with polarization. Analogously, abiotic experiments, at an applied potential of 1 V, confirmed that neither toluene was oxidized nor copper was reduced in the absence of microbial activity. At the end of each experiment, both electrodes were characterized by means of a comprehensive suite of chemical and microbiological analyses, evidencing a highly selected microbial community competent in the biodegradation of toluene in the anodic biofilm, and a uniform electrodeposition of spherical Cu2O nanoparticles over the cathode surface.

Hydrophobicity-enhanced ferritin nanoparticles for efficient encapsulation and targeted delivery of hydrophobic drugs to tumor cells.

Ferritin, a naturally occurring iron storage protein, has gained significant attention as a drug delivery platform due to its inherent biocompatibility and capacity to encapsulate therapeutic agents. In this study, we successfully genetically engineered human H ferritin by incorporating 4 or 6 tryptophan residues per subunit, strategically oriented towards the inner cavity of the nanoparticle. This modification aimed to enhance the encapsulation of hydrophobic drugs into the ferritin cage. Comprehensive characterization of the mutants revealed that only the variant carrying four tryptophan substitutions per subunit retained the ability to disassemble and reassemble properly. As a proof of concept, we evaluated the loading capacity of this mutant with ellipticine, a natural hydrophobic indole alkaloid with multimodal anticancer activity. Our data demonstrated that this specific mutant exhibited significantly higher efficiency in loading ellipticine compared to human H ferritin. Furthermore, to evaluate the versatility of this hydrophobicity-enhanced ferritin nanoparticle as a drug carrier, we conducted a comparative study by also encapsulating doxorubicin, a commonly used anticancer drug. Subsequently, we tested both ellipticine and doxorubicin-loaded nanoparticles on a promyelocytic leukemia cell line, demonstrating efficient uptake by these cells and resulting in the expected cytotoxic effect.

Simulated Microgravity Exposure Induces Antioxidant Barrier Deregulation and Mitochondria Enlargement in TCam-2 Cell Spheroids.

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of reactive oxygen species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes in gravitational force, even though published data on germ cell models are scarce. We previously studied the effects of simulated microgravity (s-microgravity) on a 2D cultured TCam-2 seminoma-derived cell line, considered the only human cell line available to study in vitro mitotically active human male germ cells. In this study, we used a corresponding TCam-2 3D cell culture model that mimics cell-cell contacts in organ tissue to test the possible effects induced by s-microgravity exposure. TCam-2 cell spheroids were cultured for 24 h under unitary gravity (Ctr) or s-microgravity conditions, the latter obtained using a random positioning machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels was observed after RPM exposure. In line with these results, a trend of protein and lipid oxidation increase and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis via transmission electron microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, with GPX1, NCF1, and CYBB being downregulated, whereas NOX4 and HMOX1 are upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated with the observed mitochondria alteration. Altogether, these data demonstrate TCam-2 spheroid sensitivity to acute s-microgravity exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow them to overcome the exposure to altered gravitational force.

The recurrent pathogenic Pro890Leu substitution in CLTC causes a generalized defect in synaptic transmission in Caenorhabditis elegans.

De novo CLTC mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. CLTC encodes the widely expressed heavy polypeptide of clathrin, a major component of the coated vesicles mediating endocytosis, intracellular trafficking, and synaptic vesicle recycling. The underlying pathogenic mechanism is largely unknown. Here, we assessed the functional impact of the recurrent c.2669C > T (p.P890L) substitution, which is associated with a relatively mild ID/MD phenotype. Primary fibroblasts endogenously expressing the mutated protein show reduced transferrin uptake compared to fibroblast lines obtained from three unrelated healthy donors, suggesting defective clathrin-mediated endocytosis. In vitro studies also reveal a block in cell cycle transition from G0/G1 to the S phase in patient's cells compared to control cells. To demonstrate the causative role of the p.P890L substitution, the pathogenic missense change was introduced at the orthologous position of the Caenorhabditis elegans gene, chc-1 (p.P892L), via CRISPR/Cas9. The resulting homozygous gene-edited strain displays resistance to aldicarb and hypersensitivity to PTZ, indicating defective release of acetylcholine and GABA by ventral cord motor neurons. Consistently, mutant animals show synaptic vesicle depletion at the sublateral nerve cords, and slightly defective dopamine signaling, highlighting a generalized deficit in synaptic transmission. This defective release of neurotransmitters is associated with their secondary accumulation at the presynaptic membrane. Automated analysis of C. elegans locomotion indicates that chc-1 mutants move slower than their isogenic controls and display defective synaptic plasticity. Phenotypic profiling of chc-1 (+/P892L) heterozygous animals and transgenic overexpression experiments document a mild dominant-negative behavior for the mutant allele. Finally, a more severe phenotype resembling that of chc-1 null mutants is observed in animals harboring the c.3146 T > C substitution (p.L1049P), homologs of the pathogenic c.3140 T > C (p.L1047P) change associated with a severe epileptic phenotype. Overall, our findings provide novel insights into disease mechanisms and genotype-phenotype correlations of CLTC-related disorders.

Metabolic labelling of a subpopulation of small extracellular vesicles using a fluorescent palmitic acid analogue.

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

Critical role for a stage-specific actin in male exflagellation of the malaria parasite.

Male gametogenesis occurs directly after uptake of malaria parasites by the mosquito vector and leads to the release of eight nucleated flagellar gametes. Here, we report that one of the two parasite actin isoforms, named actin II, is essential for this process. Disruption of actin II in Plasmodium berghei resulted in viable asexual blood stages, but male gametogenesis was specifically inhibited. Upon activation, male gametocyte DNA was replicated normally and axonemes assembled, but egress from the host cell was inhibited, and axoneme motility abolished. The major actin isoform, actin I, displayed dual localization to the cytoplasm and the nucleus in male gametocytes. After activation actin I was found to be restricted to the cytoplasm. In actII(-) mutant parasites, this re-localization was abolished and actin I remained in both cellular compartments. These findings reveal vital and pleiotropic functions for the actin II isoform in male gametogenesis of the malaria parasite.

Specific tagging of the egress-related osmiophilic bodies in the gametocytes of Plasmodium falciparum.

BACKGROUND: Gametocytes, the blood stages responsible for Plasmodium falciparum transmission, contain electron dense organelles, traditionally named osmiophilic bodies, that are believed to be involved in gamete egress from the host cell. In order to provide novel tools in the cellular and molecular studies of osmiophilic body biology, a P. falciparum transgenic line in which these organelles are specifically marked by a reporter protein was produced and characterized. METHODOLOGY: A P. falciparum transgenic line expressing an 80-residue N-terminal fragment of the osmiophilic body protein Pfg377 fused to the reporter protein DsRed, under the control of pfg377 upstream and downstream regulatory regions, was produced. RESULTS: The transgenic fusion protein is expressed at the appropriate time and stage of sexual differentiation and is trafficked to osmiophilic bodies as the endogenous Pfg377 protein. These results indicate that a relatively small N-terminal portion of Pfg377 is sufficient to target the DsRed reporter to the gametocyte osmiophilic bodies. CONCLUSIONS: This is the first identification of a P. falciparum aminoacid sequence able to mediate trafficking to such organelles. To fluorescently tag such poorly characterized organelles opens novel avenues in cellular and imaging studies on their biogenesis and on their role in gamete egress.

Towards the Standardization of Intestinal In Vitro Advanced Barrier Model for Nanoparticles Uptake and Crossing: The SiO2 Case Study.

Increasing interest is being addressed to the development of a reliable, reproducible and relevant in vitro model of intestinal barrier, mainly for engineered nanomaterials hazard and risk assessment, in order to meet regulatory and scientific demands. Starting from the consolidated Caco-2 cell model, widely used for determining translocation of drugs and chemicals, the establishment of an advanced intestinal barrier model with different level of complexity is important for overcoming Caco-2 monoculture limitations. For this purpose, a tri-culture model, consisting of two human intestinal epithelial cells (Caco-2 and HT29-MTX) and a human lymphocyte B cell (Raji B), was developed by several research groups to mimic the in vivo intestinal epithelium, furnishing appropriate tools for nanotoxicological studies. However, tri-culture model shows high levels of variability in ENM uptake/translocation studies. With the aim of implementing the standardization and optimization of this tri-culture for ENM translocation studies, the present paper intends to identify and discuss such relevant parameters involved in model establishment as: tri-culture condition set-up, barrier integrity evaluation, mucus characterization, M-cell induction. SiO2 fluorescent nanoparticles were used to compare the different models. Although a low level of SiO2 translocation is reported for all the different culture conditions. a relevant role of mucus and M-cells in NPs uptake/translocation has been highlighted.

Functional Characterization of the Thrombospondin-Related Paralogous Proteins Rhoptry Discharge Factors 1 and 2 Unveils Phenotypic Plasticity in Toxoplasma gondii Rhoptry Exocytosis.

To gain access to the intracellular cytoplasmic niche essential for their growth and replication, apicomplexan parasites such as Toxoplasma gondii rely on the timely secretion of two types of apical organelles named micronemes and rhoptries. Rhoptry proteins are key to host cell invasion and remodeling, however, the molecular mechanisms underlying the tight control of rhoptry discharge are poorly understood. Here, we report the identification and functional characterization of two novel T. gondii thrombospondin-related proteins implicated in rhoptry exocytosis. The two proteins, already annotated as MIC15 and MIC14, were renamed rhoptry discharge factor 1 (RDF1) and rhoptry discharge factor 2 (RDF2) and found to be exclusive of the Coccidia class of apicomplexan parasites. Furthermore, they were shown to have a paralogous relationship and share a C-terminal transmembrane domain followed by a short cytoplasmic tail. Immunofluorescence analysis of T. gondii tachyzoites revealed that RDF1 presents a diffuse punctate localization not reminiscent of any know subcellular compartment, whereas RDF2 was not detected. Using a conditional knockdown approach, we demonstrated that RDF1 loss caused a marked growth defect. The lack of the protein did not affect parasite gliding motility, host cell attachment, replication and egress, whereas invasion was dramatically reduced. Notably, while RDF1 depletion did not result in altered microneme exocytosis, rhoptry discharge was found to be heavily impaired. Interestingly, rhoptry secretion was reversed by spontaneous upregulation of the RDF2 gene in knockdown parasites grown under constant RDF1 repression. Collectively, our results identify RDF1 and RDF2 as additional key players in the pathway controlling rhoptry discharge. Furthermore, this study unveils a new example of compensatory mechanism contributing to phenotypic plasticity in T. gondii.

Myelin like electrogenic filamentation and Liquid Microbial Fuel Cells Dataset.

Biofilm at water-oil interface of hypoxic water columns of microcosms, prepared from a lacustrine sample, that used diesel as a carbon source was found to show electrogenic properties. These microcosms named, Liquid Microbial Fuel Cells (L-MFCs) were electrically characterized using a custom electronic analyzer; accurate determination of voltage (V), power density (W/m 2), and current density (A/m2) for both charge and discharge phases was carried out. The instrument made it possible to carry out cell characterizations using resistive loads between 0 Ω (Ohm) and 10 kΩ. During the hypoxic and electrogenic phase, the synthesis of a system of "bacterial piping induction", produced filaments of hundreds of micrometers in which the microbial cells are hosted. Ultrastructural microscopy collected by scanning (SEM), transmission (TEM), immunofluorescence, Thunder Imager 3D, confocal laser scanning (CLSM) microscopy revealed a "myelin like" structure during filamentation processes; this "myelin like" structure exhibited cross-reactivity towards different epitopes of the myelin basic protein (MBP) and Claudin 11 (O4) of human oligodendrocytes. The disclosure of these filamentation processes could be helpful to describe further unconventional microbial structures in aquatic ecosystems and of the animal world. The data that support the findings of this study are openly available in at https://data.mendeley.com/datasets/7d35tj3j96/1.