MT1-MMP and TIMP-2 are first expressed in the colon glands after a single dose of azoxymethane (AOM).

Tissue inhibitor of metalloproteinase-2 (TIMP-2) and membrane-type 1-matrix metalloproteinase (MT1-MMP) are always expressed during the cancer process. The aim was to identify which regions of the colon mucosa MT1-MMP and TIMP-2 begin to express themselves, as well as to establish their expression in relation to cell proliferation and mucin production. After intraperitoneal injection of 15 mg/kg of azoxymethane (AOM) at 4, 12, and 20 weeks, histological sections of the middle segment of the rat colon mucosa were evaluated by immunohistochemistry for cell proliferation and expression of MT1-MMP and TIMP-2 and histochemistry for mucin. As a result, a single dose of AOM initially increased the intensity of MT1-MMP and TIMP-2 expression in the conjunctive cells and glands, concurrently with alterations in the distribution of the mucin produced in the gland of the large intestine mucosa and cell proliferation. As a result, at 4 and 12 weeks, a single dose of AOM initially stimulated the expression of MT1-MMP and TIMP-2 in the conjunctive cells and glands with greater intensity. Changes in the cell proliferation and distribution of the mucin produced in the large intestine mucosa gland were observed. We conclude that MT1-MMP and TIMP-2 were first and strongly expressed in all cells of the colon glands, concurrently with an increase in cell proliferation and a diffuse dispersion of mucin, indicating the onset of the dysplasia process following a single dosage of AOM.

Long non-coding RNA CASC2 regulates osteoblasts matrix mineralization.

Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases in the bone tissue, including osteoporosis and tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (hMSCs). CASC2 knockdown, using small interfering RNA against CASC2 (siCASC2), increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining level nor the expression of early osteogenic transcripts, including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at both transcriptional and protein level. On the other hand, inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP. The results indicate that lncRNA CASC2 regulates late osteogenic differentiation and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests that targeting lncRNA CASC2 could be a potential approach for modulating bone mineralization.

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages.

Introduction: Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3'UTR (3'UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, particularly in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells affect 3'UTR-APA and IPA in primary human macrophages was unclear. Methods: In this study, we isolated primary human monocytes from healthy donors, differentiated and polarized them into a pro-inflammatory state and performed indirect co-cultures with CRC cells. ChrRNA-Seq and 3'RNA-Seq was performed to quantify gene expression and characterize new 3'UTR-APA and IPA mRNA isoforms. Results: Our results show that polarization of human macrophages from naïve to a pro-inflammatory state causes a marked increase of proximal polyA site selection in the 3'UTR and IPA events in genes relevant to macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3'UTR-APA and IPA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induces 3'UTR-APA alterations. Notably, some of these gene expression differences were also found in tumor-associated macrophages of CRC patients, indicating that they are physiologically relevant. Upon macrophage pro-inflammatory polarization, SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Discussion: Our results reveal new 3'UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools. Furthermore, our results highlight a function for SRSF12 in pro-inflammatory macrophages, key cells in the tumor response.

Long non-coding RNA H19 regulates matrisome signature and impacts cell behavior on MSC-engineered extracellular matrices.

BACKGROUND: The vast and promising class of long non-coding RNAs (lncRNAs) has been under investigation for distinct therapeutic applications. Nevertheless, their role as molecular drivers of bone regeneration remains poorly studied. The lncRNA H19 mediates osteogenic differentiation of Mesenchymal Stem/Stromal Cells (MSCs) through the control of intracellular pathways. However, the effect of H19 on the extracellular matrix (ECM) components is still largely unknown. This research study was designed to decode the H19-mediated ECM regulatory network, and to reveal how the decellularized siH19-engineered matrices influence MSC proliferation and fate. This is particularly relevant for diseases in which the ECM regulation and remodeling processes are disrupted, such as osteoporosis. METHODS: Mass spectrometry-based quantitative proteomics analysis was used to identify ECM components, after oligonucleotides delivery to osteoporosis-derived hMSCs. Moreover, qRT-PCR, immunofluorescence and proliferation, differentiation and apoptosis assays were performed. Engineered matrices were decellularized, characterized by atomic force microscopy and repopulated with hMSC and pre-adipocytes. Clinical bone samples were characterized by histomorphometry analysis. RESULTS: Our study provides an in-depth proteome-wide and matrisome-specific analysis of the ECM proteins controlled by the lncRNA H19. Using bone marrow-isolated MSC from patients with osteoporosis, we identified fibrillin-1 (FBN1), vitronectin (VTN) and collagen triple helix repeat containing 1 (CTHRC1), among others, as having different pattern levels following H19 silencing. Decellularized siH19-engineered matrices are less dense and have a decreased collagen content compared with control matrices. Repopulation with naïve MSCs promotes a shift towards the adipogenic lineage in detriment of the osteogenic lineage and inhibits proliferation. In pre-adipocytes, these siH19-matrices enhance lipid droplets formation. Mechanistically, H19 is targeted by miR-29c, whose expression is decreased in osteoporotic bone clinical samples. Accordingly, miR-29c impacts MSC proliferation and collagen production, but does not influence ALP staining or mineralization, revealing that H19 silencing and miR-29c mimics have complementary but not overlapping functions. CONCLUSION: Our data suggest H19 as a therapeutic target to engineer the bone ECM and to control cell behavior.

Neuroprotective effects on microglia and insights into the structure-activity relationship of an antioxidant peptide isolated from Pelophylax perezi.

Tryptophyllins constitute a heterogeneous group of peptides that are one of the first classes of peptides identified from amphibian's skin secretions. Here, we report the structural characterization and antioxidant properties of a novel tryptophyllin-like peptide, named PpT-2, isolated from the Iberian green frog Pelophylax perezi. The skin secretion of P. perezi was obtained by electrical stimulation and fractionated using RP-HPLC. De novo peptide sequencing was conducted using MALDI MS/MS. The primary structure of PpT-2 (FPWLLS-NH2 ) was confirmed by Edman degradation and subsequently investigated using in silico tools. PpT-2 shared physicochemical properties with other well-known antioxidants. To test PpT-2 for antioxidant activity in vitro, the peptide was synthesized by solid phase and assessed in the chemical-based ABTS and DPPH scavenging assays. Then, a flow cytometry experiment was conducted to assess PpT-2 antioxidant activity in oxidatively challenged murine microglial cells. As predicted by the in silico analyses, PpT-2 scavenged free radicals in vitro and suppressed the generation of reactive species in PMA-stimulated BV-2 microglia cells. We further explored possible bioactivities of PpT-2 against prostate cancer cells and bacteria, against which the peptide exerted a moderate antiproliferative effect and negligible antimicrobial activity. The biocompatibility of PpT-2 was evaluated in cytotoxicity assays and in vivo toxicity with Galleria mellonella. No toxicity was detected in cells treated with up to 512 µg/ml and in G. mellonella treated with up to 40 mg/kg PpT-2. This novel peptide, PpT-2, stands as a promising peptide with potential therapeutic and biotechnological applications, mainly for the treatment/prevention of neurodegenerative disorders.

TNF-alpha-induced microglia activation requires miR-342: impact on NF-kB signaling and neurotoxicity.

Growing evidences suggest that sustained neuroinflammation, caused by microglia overactivation, is implicated in the development and aggravation of several neurological and psychiatric disorders. In some pathological conditions, microglia produce increased levels of cytotoxic and inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), which can reactivate microglia in a positive feedback mechanism. However, specific molecular mediators that can be effectively targeted to control TNF-α-mediated microglia overactivation, are yet to be uncovered. In this context, we aim to identify novel TNF-α-mediated micro(mi)RNAs and to dissect their roles in microglia activation, as well as to explore their impact on the cellular communication with neurons. A miRNA microarray, followed by RT-qPCR validation, was performed on TNF-α-stimulated primary rat microglia. Gain- and loss-of-function in vitro assays and proteomic analysis were used to dissect the role of miR-342 in microglia activation. Co-cultures of microglia with hippocampal neurons, using a microfluidic system, were performed to understand the impact on neurotoxicity. Stimulation of primary rat microglia with TNF-α led to an upregulation of Nos2, Tnf, and Il1b mRNAs. In addition, ph-NF-kB p65 levels were also increased. miRNA microarray analysis followed by RT-qPCR validation revealed that TNF-α stimulation induced the upregulation of miR-342. Interestingly, miR-342 overexpression in N9 microglia was sufficient to activate the NF-kB pathway by inhibiting BAG-1, leading to increased secretion of TNF-α and IL-1ß. Conversely, miR-342 inhibition led to a strong decrease in the levels of these cytokines after TNF-α activation. In fact, both TNF-α-stimulated and miR-342-overexpressing microglia drastically affected neuron viability. Remarkably, increased levels of nitrites were detected in the supernatants of these co-cultures. Globally, our findings show that miR-342 is a crucial mediator of TNF-α-mediated microglia activation and a potential target to tackle microglia-driven neuroinflammation.

The Antioxidant Peptide Salamandrin-I: First Bioactive Peptide Identified from Skin Secretion of Salamandra Genus (Salamandra salamandra).

Amphibian skin is a multifunctional organ that plays key roles in defense, breathing, and water balance. In this study, skin secretion samples of the fire salamander (Salamandra salamandra) were separated using RP-HPLC and de novo sequenced using MALDI-TOF MS/MS. Next, we used an in silico platform to screen antioxidant molecules in the framework of density functional theory. One of the identified peptides, salamandrin-I, [M + H]+ = 1406.6 Da, was selected for solid-phase synthesis; it showed free radical scavenging activity against DPPH and ABTS radicals. Salamandrin-I did not show antimicrobial activity against Gram-positive and -negative bacteria. In vitro assays using human microglia and red blood cells showed that salamandrin-I has no cytotoxicity up to the concentration of 100 µM. In addition, in vivo toxicity tests on Galleria mellonella larvae resulted in no mortality at 20 and 40 mg/kg. Antioxidant peptides derived from natural sources are increasingly attracting interest. Among several applications, these peptides, such as salamandrin-I, can be used as templates in the design of novel antioxidant molecules that may contribute to devising strategies for more effective control of neurological disease.

miR-99a in bone homeostasis: Regulating osteogenic lineage commitment and osteoclast differentiation.

BACKGROUND: The tight coupling between osteoblasts and osteoclasts is essential to maintain bone homeostasis. Deregulation of this process leads to loss and deterioration of the bone tissue causing diseases, such as osteoporosis. MicroRNAs are able to control bone-related mechanisms and have been explored as therapeutic tools. In this study, we investigated the potential of miR-99a-5p to modulate osteogenic differentiation, osteoclastogenesis, and the osteoblasts-osteoclasts crosstalk. METHODS: To achieve this goal, human primary Mesenchymal Stem/Stromal Cells (MSC) were differentiated into osteoblasts and adipocytes, and miR-99a-5p expression was evaluated by RT-qPCR. Knockdown and overexpression experiments were conducted to modulate miR-99a-5p expression in MC3T3 cells. Cell proliferation and cell death/apoptosis were evaluated by resazurin assay and flow cytometry, respectively. Proteomic analysis was used to identify the miR-99a-5p regulatory network, and ELISA to evaluate OPG levels in the cell culture supernatant. Conditioned media from MC3T3-transfected cells was used to culture RAW 264.7 cells and the effect on osteoclast differentiation was assessed. Human primary monocytes were isolated to induce osteoclastogenesis and evaluate miR-99a-5p expression. Finally, levels of miR-99a-5p were modulated in RAW 264.7 cells to understand the impact on osteoclastogenesis. RESULTS: The results show that miR-99a-5p is significantly downregulated during the early stages of human primary MSCs osteogenic differentiation and during MC3T3 osteogenic differentiation. On the other hand, miR-99a-5p levels are increased during the initial stages of adipogenic differentiation. Inhibition of miR-99a-5p in MC3T3 pre-osteoblastic cells promoted osteogenic differentiation, whereas its overexpression suppressed the levels of osteogenic specific genes (Runx2 and Alpl), as well as mineralization, with no effect on proliferation or apoptosis. Proteomic analysis of miR-99a-5p-transfected cells showed that numerous proteins known to be involved in cell differentiation were altered, including osteogenic differentiation markers and extracellular matrix proteins. While inhibition of miR-99a-5p increased the Tnfrsf11b (OPG encoding gene)/Tnfsf11 (RANKL encoding gene) mRNA expression ratio, in addition to increasing OPG secretion, miR-99a-5p overexpression resulted in the opposite effect. The cell culture supernatant of miR-99a-5p-inhibited MC3T3 cells impaired the osteoclastogenic potential of RAW 264.7 cells by decreasing the number of multinucleated cells and reducing the expression of osteoclastogenic markers. Interestingly, miR-99a-5p expression is increased during osteoclasts differentiation, both in human primary monocytes and RAW 264.7. These results show that miR-99a-5p per se is a positive regulator of osteoclastogenic differentiation. CONCLUSIONS: Globally, our findings show that miR-99a-5p inhibition promotes the commitment into osteogenic differentiation, impairs osteoclastogenic differentiation, and control bone cells communication. Ultimately, it supports miR-99a-5p as a target candidate for future miRNA-based therapies for bone diseases associated with bone remodeling deregulation.

Genetically Engineered-MSC Therapies for Non-unions, Delayed Unions and Critical-size Bone Defects.

The normal bone regeneration process is a complex and coordinated series of events involving different cell types and molecules. However, this process is impaired in critical-size/large bone defects, with non-unions or delayed unions remaining a major clinical problem. Novel strategies are needed to aid the current therapeutic approaches. Mesenchymal stem/stromal cells (MSCs) are able to promote bone regeneration. Their beneficial effects can be improved by modulating the expression levels of specific genes with the purpose of stimulating MSC proliferation, osteogenic differentiation or their immunomodulatory capacity. In this context, the genetic engineering of MSCs is expected to further enhance their pro-regenerative properties and accelerate bone healing. Herein, we review the most promising molecular candidates (protein-coding and non-coding transcripts) and discuss the different methodologies to engineer and deliver MSCs, mainly focusing on in vivo animal studies. Considering the potential of the MSC secretome for bone repair, this topic has also been addressed. Furthermore, the promising results of clinical studies using MSC for bone regeneration are discussed. Finally, we debate the advantages and limitations of using MSCs, or genetically-engineered MSCs, and their potential as promoters of bone fracture regeneration/repair.

Cell Cycle Kinase Polo Is Controlled by a Widespread 3' Untranslated Region Regulatory Sequence in Drosophila melanogaster.

Alternative polyadenylation generates transcriptomic diversity, although the physiological impact and regulatory mechanisms involved are still poorly understood. The cell cycle kinase Polo is controlled by alternative polyadenylation in the 3' untranslated region (3'UTR), with critical physiological consequences. Here, we characterized the molecular mechanisms required for polo alternative polyadenylation. We identified a conserved upstream sequence element (USE) close to the polo proximal poly(A) signal. Transgenic flies without this sequence show incorrect selection of polo poly(A) signals with consequent downregulation of Polo expression levels and insufficient/defective activation of Polo kinetochore targets Mps1 and Aurora B. Deletion of the USE results in abnormal mitoses in neuroblasts, revealing a role for this sequence in vivo We found that Hephaestus binds to the USE RNA and that hephaestus mutants display defects in polo alternative polyadenylation concomitant with a striking reduction in Polo protein levels, leading to mitotic errors and aneuploidy. Bioinformatic analyses show that the USE is preferentially localized upstream of noncanonical polyadenylation signals in Drosophila melanogaster genes. Taken together, our results revealed the molecular mechanisms involved in polo alternative polyadenylation, with remarkable physiological functions in Polo expression and activity at the kinetochores, and disclosed a new in vivo function for USEs in Drosophila melanogaster.

Xanthohumol inhibits cell proliferation and induces apoptosis in human thyroid cells.

The cell growth inhibitory potential of xanthohumol (XN), a natural prenylflavonoid present in hops and beer, on human papillary thyroid cancer cells is reported. We demonstrate that XN decreases the proliferation of TPC-1 cancer cells in a dose and time dependent manners. At low concentration (10 µM) XN was shown to significantly inhibit carcinogenesis by a mechanism that stops or slows down cell division, preserving the viability of the cells. At higher concentration (100 µM) a decrease of cell viability was observed by induction of apoptosis. As evidenced, XN induced DNA fragmentation in TPC-1 cells and promoted cell cycle arrest, which decreased the percentage of cells in G1 phase and increased in S phase after 72 h of treatment. Furthermore, XN exposure triggered an increase in caspase-3 and caspase-7 activity, supporting its role in the activation of apoptosis. Cell-free studies demonstrated that high concentrations of XN are responsible for an increase of free radicals generated in a Fenton system which may mediate apoptosis through a pro-oxidant pathway. Altogether, our data show that XN induces the apoptosis of TPC-1 cancer cells in a concentration-dependent manner, suggesting XN to be a promising candidate for thyroid cancer therapy.

Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster.

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster, expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis-element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate.

Neutral PEGylated liposomal formulation for efficient folate-mediated delivery of MCL1 siRNA to activated macrophages.

Cationic liposomes are efficient vectors for systemic delivery of therapeutic small interfering RNA (siRNA), taking advantage of RNA interference (RNAi), a naturally occurring gene-silencing mechanism in mammalian cells. However, toxicity at high concentrations, short circulating half-lives and lack of specificity restrict their successful application in a wider scale. The purpose of this study was to evaluate the efficiency of neutral liposomes containing polyethylene glycol (PEG) to encapsulate siRNA in their aqueous core. This formulation will reduce drastically the toxicity associated to cationic liposomes by bringing surface charge to almost zero, increasing stealth degree and therefore circulation time. In this study, we evaluate the efficiency of folate-targeted liposomes for specific delivery of siRNA to activated macrophages, key effector cells in rheumatoid arthritis (RA) pathology which specifically express folate receptor ß (FRß). Myeloid cell leukaemia-1 (Mcl-1) is a protein essential for synovial macrophage survival, since Mcl-1 suppression results in the induction of apoptosis. The effect of MCL1 siRNA incorporated in liposomal formulation was assessed in primary human macrophages and successful inhibition of Mcl-1 expression was achieved. Here we show that the neutral liposomal derived from DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) formulation developed is efficient to encapsulate MCL1 siRNA and silencing gene expression in activated human macrophages.

Expression of Rac1 alternative 3' UTRs is a cell specific mechanism with a function in dendrite outgrowth in cortical neurons.

The differential expression of mRNAs containing tandem alternative 3' UTRs, achieved by mechanisms of alternative polyadenylation and post-transcriptional regulation, has been correlated with a variety of cellular states. In differentiated cells and brain tissues there is a general use of distal polyadenylation signals, originating mRNAs with longer 3' UTRs, in contrast with proliferating cells and other tissues such as testis, where most mRNAs contain shorter 3' UTRs. Although cell type and state are relevant in many biological processes, how these mechanisms occur in specific brain cell types is still poorly understood. Rac1 is a member of the Rho family of small GTPases with essential roles in multiple cellular processes, including cell differentiation and axonal growth. Here we used different brain cell types and tissues, including oligodendrocytes, microglia, astrocytes, cortical and hippocampal neurons, and optical nerve, to show that classical Rho GTPases express mRNAs with alternative 3' UTRs differently, by gene- and cell- specific mechanisms. In particular, we show that Rac1 originate mRNA isoforms with longer 3' UTRs specifically during neurite growth of cortical, but not hippocampal neurons. Furthermore, we demonstrate that the longest Rac1 3' UTR is necessary for driving the mRNA to the neurites, and also for neurite outgrowth in cortical neurons. Our results indicate that the expression of Rac1 longer 3' UTR is a gene and cell-type specific mechanism in the brain, with a new physiological function in cortical neuron differentiation.

Assessment of liposome disruption to quantify drug delivery in vitro.

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.

Enhancing Methotrexate Tolerance with Folate Tagged Liposomes in Arthritic Mice.

Methotrexate is the first line of treatment of rheumatoid arthritis. Since many patients become unresponsive to methotrexate treatment, only very expensive biological therapies are effective and increased methotrexate tolerance strategies need to be identified. Here we propose the encapsulation of methotrexate in a new liposomal formulation using a hydrophobic fragment of surfactant protein conjugated to a linker and folate to enhance their tolerance and efficacy. In this study we aim to evaluate the efficiency of this system to treat rheumatoid arthritis, by targeting folate receptor ß present at the surface of activated macrophages, key effector cells in this pathology. The specificity of our liposomal formulation to target folate receptor ß was investigated both in vitro as in vivo using a mouse model of arthritis (collagen-induced arthritis in DBA/1J mice strain). In both systems, the liposomal constructs were shown to be highly specific and efficient in targeting folate receptor ß. These liposomal formulations also significantly increase the clinical benefit of the encapsulated methotrexate in vivo in arthritic mice, together with reduced expression of CD39 and CD73 ectonucleotidases by joint-infiltrating macrophages. Thus, our formulation might be a promising cost effective way to treat rheumatoid arthritis and delay or reduce methotrexate intolerance.

Peptide Anchor for Folate-Targeted Liposomal Delivery.

Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform.

To adapt the use of GH3.TRE-Luc reporter gene cell line for a quantitative high-throughput screening (qHTS) platform, we miniaturized the reporter gene assay to a 1536-well plate format. 1280 chemicals from the Library of Pharmacologically Active Compounds (LOPAC) and the National Toxicology Program (NTP) 1408 compound collection were analyzed to identify potential thyroid hormone receptor (TR) agonists and antagonists. Of the 2688 compounds tested, eight scored as potential TR agonists when the positive hit cut-off was defined at ≥10% efficacy, relative to maximal triiodothyronine (T3) induction, and with only one of those compounds reaching ≥20% efficacy. One common class of compounds positive in the agonist assays were retinoids such as all-trans retinoic acid, which are likely acting via the retinoid-X receptor, the heterodimer partner with the TR. Five potential TR antagonists were identified, including the antiallergy drug tranilast and the anxiolytic drug SB 205384 but also some cytotoxic compounds like 5-fluorouracil. None of the inactive compounds were structurally related to T3, nor had been reported elsewhere to be thyroid hormone disruptors, so false negatives were not detected. None of the low potency (>100µM) TR agonists resembled T3 or T4, thus these may not bind directly in the ligand-binding pocket of the receptor. For TR agonists, in the qHTS, a hit cut-off of ≥20% efficacy at 100 µM may avoid identification of positives with low or no physiological relevance. The miniaturized GH3.TRE-Luc assay offers a promising addition to the in vitro test battery for endocrine disruption, and given the low percentage of compounds testing positive, its high-throughput nature is an important advantage for future toxicological screening.

Liposome and protein based stealth nanoparticles.

Liposomes and protein based nanoparticles were tuned with different polymers and glycolipids to improve stealth and thus decrease their clearance by macrophages. Liposomes were coated with polyethylene glycol (PEG) and brain-tissue-derived monosialoganglioside (GM1). Bovine serum albumin (BSA) nanoparticles were produced incorporating a PEGylated surfactant (PEG-surfactant). All obtained nanoparticles were monodisperse, with sizes ranging from 80 to 120 nm, with a zeta-potential close to zero. The presented stealth strategies lead to a decrease of internalization levels by macrophages. These surface modified nanoparticles could be used for production of new drug delivery nanosystems for systemic administration (e.g. intravenous application).

NanoSIMS50 - a powerful tool to elucidate cellular localization of halogenated organic compounds.

Persistent organic pollutants are widely distributed in the environment and lots of toxicological data are available. However, little is known on the intracellular fate of such compounds. Here a method applying secondary ion mass spectrometry is described that can be used to visualize cellular localization of halogenated compounds and to semi-quantitatively calculate concentrations of such compounds. Of the model compounds tested, TBBPA was homogenously distributed in the cell membrane of the H295R cells while PFOS accumulated in very distinct locations in the cell membrane. Relative intracellular concentrations of 4-OH-BDE69 and 4-OH-BDE121 in GH3.TRE were 61 % and 18 %, respectively, compared to the parent compounds. These differences may partly explain that observed effect concentrations for 4-OH-BDEs in in vitro experiments are usually lower than what would be expected based on receptor binding studies. NanoSIMS50 proved to be a powerful tool to describe the cellular distribution of halogenated compounds. The semi-quantitative data that can be obtained may help to further explain results from in vitro or in vivo experiments.