Extensive Histopathological Characterization of Inflamed Bowel in the Dextran Sulfate Sodium Mouse Model with Emphasis on Clinically Relevant Biomarkers and Targets for Drug Development.

This study aims to develop a reliable and reproducible inflammatory bowel disease (IBD) murine model based on a careful spatial-temporal histological characterization. Secondary aims included extensive preclinical studies focused on the in situ expression of clinically relevant biomarkers and targets involved in IBD. C57BL/6 female mice were used to establish the IBD model. Colitis was induced by the oral administration of 2% Dextran Sulfate Sodium (DSS) for 5 days, followed by 2, 4 or 9 days of water. Histological analysis was performed by sectioning the whole colon into rings of 5 mm each. Immunohistochemical analyses were performed for molecular targets of interest for monitoring disease activity, treatment response and predicting outcome. Data reported here allowed us to develop an original scoring method useful as a tool for the histological assessment of preclinical models of DSS-induced IBD. Immunohistochemical data showed a significant increase in TNF-α, α4ß7, VEGFRII, GR-1, CD25, CD3 and IL-12p40 expression in DSS mice if compared to controls. No difference was observed for IL-17, IL-23R, IL-36R or F480. Knowledge of the spatial-temporal pattern distribution of the pathological lesions of a well-characterized disease model lays the foundation for the study of the tissue expression of meaningful predictive biomarkers, thereby improving translational success rates of preclinical studies for a personalized management of IBD patients.

Targeting the vascular endothelial growth factor receptor-1 by the monoclonal antibody D16F7 to increase the activity of immune checkpoint inhibitors against cutaneous melanoma.

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a membrane receptor for VEGF-A, placenta growth factor (PlGF) and VEGF-B that plays a crucial role in melanoma invasiveness, vasculogenic mimicry and tumor-associated angiogenesis. Furthermore, activation of VEGFR-1 is involved in the mobilization of myeloid progenitors from the bone marrow that infiltrate the tumor. Myeloid-derived suppressor cells and tumor-associated macrophages have been involved in tumor progression and resistance to cancer treatment with immune checkpoint inhibitors (ICIs). We have recently demonstrated that the anti-VEGFR-1 monoclonal antibody (mAb) D16F7 developed in our laboratories is able to inhibit melanoma growth in preclinical in vivo models and to reduce monocyte/macrophage progenitor mobilization and tumor infiltration by myeloid cells. Aim of the study was to investigate whether the anti-VEGFR-1 mAb D16F7 affects the activity of protumoral M2 macrophages in vitro in response to PlGF and inhibits the recruitment of these cells to the melanoma site in vivo. Finally, we tested whether, through its multi-targeted action, D16F7 mAb might increase the efficacy of ICIs against melanoma. The results indicated that VEGFR-1 expression is up-regulated in human activated M2 macrophages compared to activated M1 cells and exposure to the D16F7 mAb decreases in vitro chemotaxis of activated M2 macrophages. In vivo treatment with the anti-VEGFR-1 mAb D16F7 of B6D2F1 mice injected with syngeneic B16F10 melanoma cells resulted in tumor growth inhibition associated with the modification of tumor microenvironment that involves a decrease of melanoma infiltration by M2 macrophages and PD-1+ and FoxP3+ cells. These alterations result in increased M1/M2 and CD8+/FoxP3+ ratios, which favor an antitumor and immunostimulating milieu. Accordingly, D16F7 mAb increased the antitumor activity of the ICIs anti-CTLA-4 and anti-PD-1 mAbs. Overall, these data reinforce the role of VEGFR-1-mediated-signalling as a valid target for reducing tumor infiltration by protumoral macrophages and for improving the efficacy of immunotherapy with ICIs.

Glutathione transferase P silencing promotes neuronal differentiation of retinal R28 cells.

Glutathione transferases (GSTs) play an important role in retinal pathophysiology. Within this family, the GSTP isoform is known as an endogenous regulator of cell survival and proliferation pathways and of cellular responses to oxidative stress. In the present study we silenced GSTP in R28 cells, a retinal precursor cell line with markers of both glial and neuronal origin, and obtained stable clones which were viable and, unexpectedly, characterized by a more neuronal phenotype. The degree of neuronal differentiation was inversely correlated with GSTP residual expression levels. The clone with the lowest expression of GSTP showed metabolic reprogramming, a more favorable redox status and, despite its neuronal phenotype, a sensitivity to glutamate and 4-hydroxynonenal toxicity comparable to that of control cells. Altogether, our evidence shows that near full depletion of GSTP in retinal precursor cells, triggers neuronal differentiation and prosurvival metabolic changes.

Glutathione S-transferase P1-1 as a target for mesothelioma treatment.

Malignant pleural mesothelioma is a poorly responsive tumor known to overexpress the phase II detoxification enzyme glutathione-S-transferase, which catalyzes the conjugation between glutathione and platinum(II)-containing drugs. Therefore, we evaluated the effect of the strong glutathione S-transferase inhibitor NBDHEX on human mesothelioma cell lines (MSTO-211H, MPP89, MM-B1 and Mero 48a) featuring the most common mesothelioma phenotypes: epithelioid and biphasic. Even though a different response to NBDHEX was observed, the molecule was very effective on all cell lines tested, triggering a sustained activation of both JNK and p38, followed by caspase activation and apoptosis. NBDHEX also caused severe oxidative stress in the MPP89 cells and, to a lesser extent, in the MMB1 cells, while it did not cause a significant redox imbalance in the other cell lines. The efficacy of the drug was found to be comparable or even higher than that of cisplatin. Moreover, it showed synergistic or additive effects when used in combination with cisplatin. In conclusion, NBDHEX was effective on mesothelioma cell lines, with IC(50) values in the low micromolar range (IC(50) between 1 and 4 µM). These findings indicate that NBDHEX, alone or in combination with cisplatin, is a promising new strategy for treating this rare and aggressive malignancy.

Retinoic acid and proteotoxic stress induce AML cell death overcoming stromal cell protection.

BACKGROUND: Acute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A). METHODS: We treated FLT3-ITD+ AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD+ AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML. RESULTS: The combination RBA exerts strong cytotoxic activity on FLT3-ITD+ AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD+ AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs. CONCLUSIONS: Our findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD+ AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling.

The Anti-Vascular Endothelial Growth Factor Receptor 1 (VEGFR-1) D16F7 Monoclonal Antibody Inhibits Melanoma Adhesion to Soluble VEGFR-1 and Tissue Invasion in Response to Placenta Growth Factor.

Placenta growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family involved in tumor-associated angiogenesis and melanoma invasion of the extra-cellular matrix (ECM) through activation of membrane VEGF receptor 1 (VEGFR-1). A soluble VEGFR-1 (sVEGFR-1) form is released in the ECM, where it sequesters proangiogenic factors and stimulates endothelial or tumor cell adhesion and chemotaxis through interaction with α5ß1 integrin. The anti-VEGFR-1 monoclonal antibody (D16F7 mAb) inhibits VEGF-A or PlGF-mediated signal transduction without affecting ligand interaction, thus preserving sVEGFR-1 decoy function. The aim of this study was to investigate whether D16F7 mAb hampers melanoma spread by in vitro analysis of cell adhesion to sVEGFR-1, ECM invasion, transmigration through an endothelial cell monolayer and in vivo evaluation of tumor infiltrative potential in a syngeneic murine model. Results indicate that D16F7 mAb significantly inhibits melanoma adhesion to sVEGFR-1 and ECM invasion, as well as transmigration in response to PlGF. Moreover, treatment of melanoma-bearing mice with the anti-VEGFR-1 mAb not only inhibits tumor growth but also induces a significant reduction in bone infiltration associated with a decrease in PlGF-positive melanoma cells. Furthermore, D16F7 mAb reduces PlGF production by melanoma cells. Therefore, blockade of PLGF/VEGFR-1 signaling represents a suitable strategy to counteract the metastatic potential of melanoma.

FMR1 deletion in rats induces hyperactivity with no changes in striatal dopamine transporter availability.

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder emerging in early life characterized by impairments in social interaction, poor verbal and non-verbal communication, and repetitive patterns of behaviors. Among the best-known genetic risk factors for ASD, there are mutations causing the loss of the Fragile X Messenger Ribonucleoprotein 1 (FMRP) leading to Fragile X syndrome (FXS), a common form of inherited intellectual disability and the leading monogenic cause of ASD. Being a pivotal regulator of motor activity, motivation, attention, and reward processing, dopaminergic neurotransmission has a key role in several neuropsychiatric disorders, including ASD. Fmr1 Δexon 8 rats have been validated as a genetic model of ASD based on FMR1 deletion, and they are also a rat model of FXS. Here, we performed behavioral, biochemical and in vivo SPECT neuroimaging experiments to investigate whether Fmr1 Δexon 8 rats display ASD-like repetitive behaviors associated with changes in striatal dopamine transporter (DAT) availability assessed through in vivo SPECT neuroimaging. At the behavioral level, Fmr1 Δexon 8 rats displayed hyperactivity in the open field test in the absence of repetitive behaviors in the hole board test. However, these behavioral alterations were not associated with changes in striatal DAT availability as assessed by non-invasive in vivo SPECT and Western blot analyses.

Human adipose-derived stromal cells transplantation prolongs reproductive lifespan on mouse models of mild and severe premature ovarian insufficiency.

BACKGROUND: Although recent studies have investigated the ability of Mesenchymal Stromal Cells (MSCs) to alleviate short-term ovarian damage in animal models of chemotherapy-induced Premature Ovarian Insufficiency (POI), no data are available on reproductive lifespan recovery, especially in a severe POI condition. For this reason, we investigated the potential of MSCs isolated from human adipose tissue (hASCs), since they are easy to harvest and abundant, in ameliorating the length and performance of reproductive life in both mild and severe chemotherapy-induced murine POI models. METHODS: Mild and severe POI models were established by intraperitoneally administering a light (12 mg/kg busulfan + 120 mg/kg cyclophosphamide) or heavy (30 mg/kg busulfan + 120 mg/kg cyclophosphamide) dose of chemotherapy, respectively, in CD1 mice. In both cases, a week later, 1 × 106 hASCs were transplanted systemically through the tail vein. After four additional weeks, some females were sacrificed to collect ovaries for morphological evaluation. H&E staining was performed to assess stroma alteration and to count follicle numbers; immunofluorescence staining for αSMA was used to analyse vascularization. Of the remaining females, some were mated after superovulation to collect 2-cell embryos in order to evaluate their pre-implantation developmental capacity in vitro, while others were naturally mated to monitor litters and reproductive lifespan length. F1 litters' weight, ovaries and reproductive lifespan were also analysed. RESULTS: hASC transplantation alleviated ovarian weight loss and size decrease and reduced alterations on ovarian stroma and vasculature, concurrently preventing the progressive follicle stockpile depletion caused by chemotherapy. These effects were associated with the preservation of the oocyte competence to develop into blastocyst in vitro and, more interestingly, with a significant decrease of chemotherapy-induced POI features, like shortness of reproductive lifespan, reduced number of litters and longer time to plug (the latter only presented in the severe POI model). CONCLUSION: Human ASC transplantation was able to significantly reduce all the alterations induced by the chemotherapeutic treatment, while improving oocyte quality and prolonging reproductive functions, thus counteracting infertility. These results, strengthened by the use of an outbred model, support the potential applications of hASCs in women with POI, nowadays mainly induced by anticancer therapies.

Recombinant BCG-Rv1767 amount determines, in vivo, antigen-specific T cells location, frequency, and protective outcome.

One possibility to improve the efficacy of BCG vaccine against TB is to create a recombinant BCG (r-BCG), increasing the expression of mycobacterial antigens, to ameliorate the response to BCG. Here we describe a new r-BCG expressing the gene Rv1767, induced by Mycobacterium tuberculosis during its survival in human macrophages. The r-BCG elicited a specific T cells response in Balb/c mice higher than wt BCG. The r-BCG amount used to immunise mice determined diverse Th1/Th2 equilibriums, which was not the same in spleen and Lymph Nodes. Differences in cytokines production were found for IL-10, IL-4, TNF-alpha, and Arginase-1, which, in some conditions, resulted higher in r-BCG as compared to wt BCG-immunised mice. The immunisation with r-BCG-Rv1767 induced a lesser protective activity than wt BCG in a mouse model of TB. This reduction might likely be explained by the specific T cells phenotype and setting existing before MTB challenge, induced by either the single or the triple dose of r-BCG. The use of this model may help to highlight the capacity of different M. tuberculosis antigens to induce a protective immune response, actually not necessarily embodied by an increased frequency of Antigen-specific effector memory T cells.

HIV replication leads to skewed maturation of CD8-positive T-cell responses in infected children.

HIV-1 infection causes a severe T-cell impairment with alteration of immune response. However, in children the natural decline of lymphocytes and CD4 cells in early life makes it more difficult to monitor immunocompetence and progression of HIV-infection. Aim of this study was to characterize the CD8 response in non-vertically HIV-infected children exposed persistently to viremia and in HIV-infected children controlling efficiently viremia by ART, by analysing the effect of persistent viremia on CD4 and CD8 T-cells count, HIV-specific immune-response and naive/memory pattern of CD8 T-cell. Whereas, no differences of CD4 count between viremic patients and viral controllers were observed (1046.9 +/- 472.1 cells/microl vs 1101.3 +/- 415.4 cells/microl; p > 0.05), CD8 count was higher in the viremic patients (1080.6 +/- 652.1 cells/microl vs 747.5 +/- 389.9 cells/microl, p < 0.05). In viremic patients, HIV-specific CD8 T-cells correlated with viral load. However, in this group a loss of HIV-specific CD8 response was associated with a 7 fold decrease of naïve and increase of pre-effector CD8 T-cells (62.8% +/- 10.21% vs 10.37% +/- 7.91%, p < 0.03). Persistent exposure to viremia alters HIV-specific CD8 response possibly through a persistent immune activation process leading to exhaustion of naive CD8 T-cells and skewed maturation of memory subset. Therefore, memory CD8 T-cells might lose the ability to respond correctly and efficiently to HIV-antigen exposure.

Theranostic Designed Near-Infrared Fluorescent Poly (Lactic-co-Glycolic Acid) Nanoparticles and Preliminary Studies with Functionalized VEGF-Nanoparticles.

Poly-lactic-co-glycolic acid nanoparticles (PLGA-NPs) were approved by the Food and Drug Administration (FDA) for drug delivery in cancer. The enhanced permeability and retention (EPR) effect drives their accumulation minimizing the side effects of chemotherapeutics. Our aim was to develop a new theranostic tool for cancer diagnosis and therapy based on PLGA-NPs and to evaluate the added value of vascular endothelial growth factor (VEGF) for enhanced tumor targeting. In vitro and in vivo properties of PLGA-NPs were tested and compared with VEGF-PLGA-NPs. Dynamic light scattering (DLS) was performed to evaluate the particle size, polydispersity index (PDI), and zeta potential of both preparations. Spectroscopy was used to confirm the absorption spectra in the near-infrared (NIR). In vivo, in BALB/c mice bearing a syngeneic tumor in the right thigh, intravenously injected PLGA-NPs showed a high target-to-muscle ratio (4.2 T/M at 24 h post-injection) that increased over time, with a maximum uptake at 72 h and a retention of the NPs up to 240 h. VEGF-PLGA-NPs accumulated in tumors 1.75 times more than PLGA-NPs with a tumor-to-muscle ratio of 7.90 ± 1.61 (versus 4.49 ± 0.54 of PLGA-NPs). Our study highlights the tumor-targeting potential of PLGA-NPs for diagnostic and therapeutic applications. Such NPs can be conjugated with proteins such as VEGF to increase accumulation in tumor lesions.

Inhibition of Mek 1/2 kinase activity and stimulation of melanogenesis by 5,7-dimethoxycoumarin treatment of melanoma cells.

In this study, the processes of differentiation and melanogenesis induced by 5,7-dimethoxycoumarin in murine (B16) and human (A375) melanoma cells were investigated. Taking into account the previously demonstrated antiproliferative and differentiation activities of this compound, we examined Ras/Raf/Mek/Erk mitogen-activated protein kinase activity following treatment; inhibition of Mek 1/2 kinase activity and subsequent reduction in Erk 1/2 activation were detected in both cell types. We observed melanogenesis induction associated to an increase in cAMP-response element-binding protein (CREB) and microphthalmia-associated transcription factor (Mitf) expression, both involved in its regulation. Mitf is fundamental for development, survival and differentiation of melanocyte and melanoma, since it regulates transcription of genes encoding for proteins involved in cell cycle progression or in melanogenesis, such as the enzyme tyrosinase. A significant increase of tyrosinase activity was revealed following treatment in B16 but not in A375 cells, although a strong synthesis of melanin was induced by 5,7-dimethoxycoumarin in both cell lines. The treatment induced protoporphyrine IX accumulation involved in melanogenesis since it promotes stability of cAMP. Finally, the Mek 1/2 inhibitor U0126 significantly potentiated growth inhibition of B16 cells triggered by 5,7-dimethoxycoumarin, suggesting that down-regulation of Raf/Mek/Erk pathway sensitizes melanoma cells to 5,7-dimethoxycoumarin treatment.

Paternal activation of CB2 cannabinoid receptor impairs placental and embryonic growth via an epigenetic mechanism.

The cannabinoid receptor type 2 (CB2) is the peripheral receptor for cannabinoids, involved in the homeostatic control of several physiological functions. Male mitotic germ cells express a high level of CB2, whose activation promotes their differentiation in both in vitro and in vivo experiments, controlling the correct progression of spermatogenesis. However, it remains elusive if CB2 activation in spermatogonia could affect reproductive success in terms of fertility and healthy pregnancy outcomes. In this study, we explored the effects of male CB2 activation on sperm number and quality and its influence on next generation health. We show that exposure of male mice to JWH-133, a selective CB2 agonist, decreased sperm count, impaired placental development and reduced offspring growth. These defects were associated with altered DNA methylation/hydroxymethylation levels at imprinted genes in sperm and conserved in placenta. Our findings reveal that paternal selective activation of CB2 alters the sperm epigenome and compromises offspring growth. This study demonstrates, for the first time, a new role of CB2 signaling in male gametes in causing epigenetic alterations that can be transmitted to the next generation by sperm, highlighting potential risks induced by recreational cannabinoid exposure.

Targeting LOX-1 Inhibits Colorectal Cancer Metastasis in an Animal Model.

Recurrence and metastasis are the primary causes of mortality in patients with colorectal cancer (CRC), and therefore effective tools to reduce morbidity and mortality of CRC patients are necessary. LOX-1, the ox-LDL receptor, is strongly involved in inflammation, obesity, and atherosclerosis, and several studies have assessed its role in the carcinogenesis process linking ROS, metabolic disorders and cancer. We have already demonstrated in vitro that LOX-1 expression correlates to the aggressiveness of human colon cancer and its downregulation weakens the tumoral phenotype, indicating its potential function as a biomarker and a target in CRC therapy. Here we further investigate in vivo the role of LOX-1 in colon tumorigenesis by xenografting procedures, injecting nude mice both subcutaneously and intravenously with human high grade metastatic colorectal cancer cells, DLD-1, in which LOX-1 expression has been downregulated by shRNA (LOX-1RNAi cells). Histopathological and immunohistochemical evaluations have been performed on xenograft tumors. The experiments have been complemented by the analysis of the volatile compounds (VOCs) collected from the cages of injected mice and analyzed by gas-chromatography and gas sensors. After intravenous injection of LOX-1RNAi cells, we found that LOX-1 silencing influences both the engraftment of the tumor and the metastasis development, acting by angiogenesis. For the first time, we have observed that LOX-1 inhibition significantly prevents metastasis formation in injected mice and, at the same time, induces a downregulation of VEGF-A165, HIF-1α, and ß-catenin whose expression is involved in cell migration and metastasis, and a variation of histone H4 acetylation pattern suggesting also a role of LOX-1 in regulating gene transcription. The analysis of the volatile compounds (VOCs) collected from the cages of injected mice has evidenced a specific profile in those xenograft mice in which metastasis originates. These findings underline the role of LOX-1 as a potential target for inhibition of tumor progression and metastasis, enhancing current therapeutic strategies against colorectal cancer.

Combined ascorbic acid and T3 produce better healing compared to bone marrow mesenchymal stem cells in an Achilles tendon injury rat model: a proof of concept study.

BACKGROUND: This pilot study aimed to ascertain whether the local application of ascorbic acid (AA), of T3, and of rat (r) bone marrow mesenchymal stem cells (BMSCs), alone or in all possible combinations, promoted healing after an Achilles tendon injury in a rat model. METHODS: An Achilles tendon defect was produced in 24 6-8-week-old male inbred Lewis rats. The animals were then randomly divided into eight groups of three rats each. The tendon defect was filled with 50 µL of phosphate-buffered saline (PBS) containing (1) 50 µg/mL AA (AA group), (2) 10-7 M T3 (T3 group), (3) 4 × 106 rBMSCs (rBMSC group), (4) 50 µg/mL AA + 10-7 M T3 (AA + T3 group), (5) 4 × 106 rBMSCs + 50 µg/mL AA (rBMSC + AA group), (6) 4 × 106 rBMSCs + 10-7 M T3 (rBMSC + T3 group), (7) 4 × 106 rBMSCS + 50 µg/mL AA + 10-7 M T3 (rBMSC + AA + T3 group), and (8) PBS only (control group: CTRL). All treatments were administered by local injection immediately after the tendons had been damaged; additionally, AA was injected also on the second and fourth day from the first injection (for groups 1, 4, 5, and 7), and T3 was injected again every day for 4 days (for groups 2, 4, 6, and 7). At 30 days from initial treatment, tendon samples were harvested, and the quality of tendon repair was evaluated using histological and histomorphological analysis. The structure and morphology of the injured Achilles tendons were evaluated using the modified Svensson, Soslowsky, and Cook score, and the collagen type I and III ratio was calculated. RESULTS: The group treated with AA combined with T3 displayed the lowest Svensson, Soslowsky, and Cook total score value of all tissue sections at histopathological examination, with fiber structure close to regular orientation, normal-like tendon vasculature, and no cartilage formation. AA + T3 also showed the highest collagen I and the lowest collagen III values compared to all other treatments including the CTRL. CONCLUSION: There are potential benefits using a combination of AA and T3 to accelerate tendon healing.

The RNA binding protein Sam68 controls T helper 1 differentiation and anti-mycobacterial response through modulation of miR-29.

Polarization of naive T cells into interferon (IFN)-γ-producing T helper 1 (Th1) cells is an essential event in the inflammatory response to pathogens. Herein, we identify the RNA binding protein Sam68 as a specific modulator of Th1 differentiation. Sam68-knockout (ko) naive T cells are strongly defective in IL-12-mediated Th1 polarization and express low levels of T-bet and Eomes. Consequently, Sam68-ko Th1 cells are significantly impaired in IFN-γ production. Moreover, we found that Sam68 is required for the induction of an inflammatory Th1 response during Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection, thus limiting bacterial dissemination in the lungs. Mechanistically, Sam68 directly binds to the microRNA miR-29, a negative regulator of Th1 response, and inhibits its expression during BCG infection. These findings uncover a novel post-transcriptional mechanism required for the Th1-mediated defense against intracellular pathogens and identify a new function for Sam68 in the regulation of the immune response.

Identification of microRNAs and relative target genes in Moringa oleifera leaf and callus.

MicroRNAs, a class of small, non-coding RNAs, play important roles in plant growth, development and stress response by negatively regulating gene expression. Moringa oleifera Lam. plant has many medical and nutritional uses; however, little attention has been dedicated to its potential for the bio production of active compounds. In this study, 431 conserved and 392 novel microRNA families were identified and 9 novel small RNA libraries constructed from leaf, and cold stress treated callus, using high-throughput sequencing technology. Based on the M. oleifera genome, the microRNA repertoire of the seed was re-evaluated. qRT-PCR analysis confirmed the expression pattern of 11 conserved microRNAs in all groups. MicroRNA159 was found to be the most abundant conserved microRNA in leaf and callus, while microRNA393 was most abundantly expressed in the seed. The majority of predicted microRNA target genes were transcriptional factors involved in plant reproduction, growth/development and abiotic/biotic stress response. In conclusion, this is the first comprehensive analysis of microRNAs in M. oleifera leaf and callus which represents an important addition to the existing M. oleifera seed microRNA database and allows for possible exploitation of plant microRNAs induced with abiotic stress, as a tool for bio-enrichment with pharmacologically important phytochemicals.

Publisher Correction: Identification of microRNAs and relative target genes in Moringa oleifera leaf and callus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cell cycle arrest and differentiation induction by 5,7-dimethoxycoumarin in melanoma cell lines.

In the present study we investigated the antiproliferative activity of 5,7-dimethoxycoumarin on the murine B16 and human A375 melanoma cell lines. The inhibitory concentration 50 (IC50) was estimated for each cell line by preliminary assay of tetrazolium salt reduction (MTT). With Trypan blue exclusion test we detected a cytostatic but not cytotoxic effect of the treatment in melanoma cells: 5,7-dimethoxycoumarin significantly reduced cell proliferation in a time- and dose-dependent manner, blocking the cell cycle in the G0/G1 phase both in B16 and A375 cells. Melanoma growth reduction was coupled to a differentiation process detected by monitoring some specific markers: i) morphological changes with development of dendrite-like projections from the cell surface; ii) melanin synthesis; and iii) PpIX accumulation. Induction of the differentiation process was more significant in murine melanoma cells, where the treatment irreversibly reduced cell growth. Consistent with G0/G1 arrest and melanogenesis in B16 cells, 5,7-dimethoxycoumarin strongly decreased activation of the mitogen-activated protein kinase extracellular signal-related kinase 1/2, which is upregulated in many types of cancer. These findings suggest that 5,7-dimethoxycoumarin should be further investigated through studies both in vitro, to identify the binding partners for this compound, and in preclinical animal models.

In vivo biological fate of poly(vinylalcohol) microbubbles in mice.

Microbubbles (MBs) are used in clinical practice as vascular ultrasound contrast agents, and are gaining popularity as a platform supporting multimodal imaging and targeted therapy, facilitating drug delivery under ultrasound exposure. Here, we report on the in vivo biological impact of newly discovered MBs with promising features as a multimodal theranostic device. The shell of the air-filled MBs is made of the poly(vinyl alcohol) (PVA), a well-established, FDA-approved polymer. Nevertheless, as size, shape and dispersity can significantly impact the biological response of particulate systems, studying their fate after administration is crucial. The safety and the biodistribution of PVA MBs were analysed in vivo and ex vivo by coupling a near infrared (NIR) fluorophore on their shell: MBs accumulated mainly in liver and spleen at 24 hours post-injection with their clearance from the spleen 7 days post-dosing. A possible way of elimination was identified in macrophages ability to engulf MBs both in vitro and in vivo. One month post-dosing, transmission electron microscopy (TEM) highlighted the lack of relevant defects and the elimination of PVA MBs by Kupffer cells. This study is the first successful attempt to fill the lack of knowledge necessary to bring PVA MBs one step closer to their possible clinical use.