The activity of methylene blue against asexual and sexual stages of Plasmodium vivax.

Methylene blue (MB) is an alternative for combating drug-resistant malaria parasites. Its transmission-blocking potential has been demonstrated in vivo in murine models, in vitro, and in clinical trials. MB shows high efficacy against Plasmodium vivax asexual stages; however, its efficacy in sexual stages is unknown. In this study, we evaluated the potential of MB against asexual and sexual forms of P. vivax isolated from the blood of patients residing in the Brazilian Amazon. An ex vivo schizont maturation assay, zygote to ookinete transformation assay, direct membrane feed assay (DMFA), and standard membrane feed assay (SMFA) using P. vivax gametocytes with MB exposure were performed. A cytotoxicity assay was also performed on freshly collected peripheral blood mononuclear cells (PBMCs) and the hepatocyte carcinoma cell line HepG2. MB inhibited the P. vivax schizont maturation and demonstrated an IC50 lower than that of chloroquine (control drug). In the sexual forms, the MB demonstrated a high level of inhibition in the transformation of the zygotes into ookinetes. In the DMFA, MB did not considerably affect the infection rate and showed low inhibition, but it demonstrated a slight decrease in the infection intensity in all tested concentrations. In contrast, in the SMFA, MB was able to completely block the transmission at the highest concentration (20 µM). MB demonstrated low cytotoxicity to fresh PBMCs but demonstrated higher cytotoxicity to the hepatocyte carcinoma cell line HepG2. These results show that MB may be a potential drug for vivax malaria treatment.

Bacteriostatic and Antibiofilm Efficacy of a Nisin Z Solution against Co-Cultures of Staphylococcus aureus and Pseudomonas aeruginosa from Diabetic Foot Infections.

Diabetes mellitus (DM) patients frequently develop diabetic foot ulcers (DFU) which are generally infected by a community of microorganisms, mainly Staphylococcus aureus and Pseudomonas aeruginosa. These bacteria exhibit a multi-drug resistance profile and biofilm-forming ability which represent a hurdle in the treatment of diabetic foot infections (DFI). We aimed to evaluate the potential of Nisin Z, an antimicrobial peptide (AMP), as an alternative treatment for severe DFI. Nisin Z shows antibacterial activity against Gram-positive and Gram-negative bacteria and an increased antibacterial effect against Gram-negatives when added to EDTA. As such, Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Minimum Biofilm Inhibitory Concentration (MBIC), and Minimum Biofilm Eradication Concentration (MBEC) were determined for Nisin Z, Nisin Z + EDTA (0.4%), and Nisin Z + EDTA incorporated into guar gum, in order to test its efficacy against S. aureus and P. aeruginosa isolated from the same DFU. Results showed that Nisin Z added to the chelation agent EDTA displayed higher antibacterial and bacteriostatic efficacy against mono and dual co-cultures of S. aureus and P. aeruginosa, and higher antibiofilm efficiency against monocultures. Nisin Z was moderately cytotoxic at 200 µg/mL. Prospect in vivo studies are needed to confirm the potential of Nisin Z supplemented with EDTA to be used as a complement to conventional antibiotic therapy for severe DFI.

Immunolocalization of Notch Signaling in Mouse Preimplantation Embryos.

The Notch signaling pathway is an important determinant of cell diversity and identity in most developing embryonic tissues. The pathway components are expressed dynamically, and their function is critical for embryonic survival.This protocol addresses the immunolocalization of Notch pathway components by confocal microscopy.

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening.

BACKGROUND: Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES: Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS: The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION: Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Transmission-blocking compound candidates against Plasmodium vivax using P. berghei as an initial screening

BACKGROUND Different strategies for improvement of malaria control and elimination are based on the blockage of malaria parasite transmission to the mosquito vector. These strategies include the drugs that target the plasmodial sexual stages in humans and the early developmental stages inside mosquitoes. OBJECTIVES Here we tested Malaria Box compounds in order to evaluate their activity against male and female gametocytes in Plasmodium berghei, mosquito infection in P. vivax and ookinete formation in both species. METHODS/FINDINGS The membrane feeding assay and the development of ookinetes by a 24 h ex vivo culture and the ookinete yield per 1000 erythrocytes were used to test transmission-blocking potential of the Malaria Box compounds in P. vivax. For P. berghei we used flow cytometry to evaluate male and female gametocyte time course and fluorescence microscopy to check the ookinete development. The two species used in this study showed similar results concerning the compounds' activity against gametocytes and ookinetes, which were different from those in P. falciparum. In addition, from the eight Malaria Box compounds tested in both species, compounds MMV665830, MMV665878 and MMV665941 were selected as a hit compounds due the high inhibition observed. CONCLUSION Our results showed that P. berghei is suitable as an initial screening system to test compounds against P. vivax.

Influence of Storage on the Antimicrobial and Cytotoxic Activities of a Nisin-biogel with Potential to be Applied to Diabetic Foot Infections Treatment.

Staphylococcus aureus is the most prevalent pathogen in diabetic foot infections (DFIs). In addition to its ability to express several virulence factors, including the formation of recalcitrant biofilms, S. aureus is also becoming increasingly resistant to most antibiotics used in clinical practice. The search for alternative treatment strategies for DFI is urgently needed. Antimicrobial peptides (AMPs), namely, nisin, are emerging as potential new therapeutics for managing DFIs. Our team has developed a nisin-guar gum biogel to be applied to DFIs. In this study, to confirm its future in vivo applicability, we evaluated the influence of four storage temperatures (-20 °C, 4 °C, 22 °C, and 37 °C) during a 24 months storage period on its antimicrobial activity towards DFI S. aureus, and its cytotoxicity, to a human keratinocyte cell line. When stored at temperatures below 22 °C, the biogel antimicrobial activity was not significantly influenced by storage duration or temperature. Moreover, nisin incorporated within the guar gum biogel exhibited no significant levels of cytotoxicity on human keratinocyte cells, confirming its potential for DFIs therapeutics. In conclusion, results confirm that the nisin-biogel is a potential candidate to be used as an alternative or complement compound for conventional DFI therapeutics.

Notch Signaling Function in the Angiocrine Regulation of Tumor Development.

The concept of tumor growth being angiogenesis dependent had its origin in the observations of Judah Folkman in 1969 of a retinoblastoma in a child. Tumor angiogenesis is initiated when endothelial cells (ECs) respond to local stimuli and migrate towards the growing mass, which results in the formation of tubular structures surrounded by perivascular support cells that transport blood to the inner tumor. In turn, the neo-vasculature supports tumor development and eventual metastasis. This process is highly regulated by several signaling pathways. Central to this process is the Notch signaling pathway. Beyond the role of Notch signaling in tumor angiogenesis, a major hallmark of cancer development, it has also been implicated in the regulation of tumor cell proliferation and survival, in epithelial-to-mesenchymal transition, invasion and metastasis and in the regulation of cancer stem cells, in a variety of hematologic and solid malignancies. There is increasing evidence for the tumor vasculature being important in roles other than those linked to blood perfusion. Namely, endothelial cells act on and influence neighboring tumor cells by use of angiocrine factors to generate a unique cellular microenvironment, thereby regulating tumor stem-like cells' homeostasis, modulating tumor progression, invasiveness, trafficking and metastasis. This review will focus on Notch signaling components that play a part in angiocrine signaling in a tumor setting.

Fragment-oriented synthesis: ß-elaboration of cyclic amine fragments using enecarbamates as platform intermediates.

A strategy for the ß-sp3 functionalisation of cyclic amines is described. Regioselective conversion of protected amines to enecarbamates is achieved through electrochemical oxidation; these intermediates can be derivatised by functionalised alkyl halides under photoredox catalysis. The potential of the methods is highlighted by direct growth of a DCP2B-binding fragment.

Creating Diversity from Biomass: A Tandem Bio/Metal-Catalysis towards Chemoselective Synthesis of Densely Substituted Furans.

A new chemoselective (enzymatic desymmetrization/Ru-catalyzed C-H activation) sequence to obtain differently substituted furans from the largely available 2,5-furandicarboxylic acid (FDCA) was developed. Series of di- and trisubstituted furans were prepared in very good yields and excellent chemoselectivity. This study discloses a new approach towards valorization of the furanics platform through the use of FDCA as a stable intermediate, thus circumventing the chemical instability of the parent 5-hydroxymethylfurfural.

Metastasis is impaired by endothelial-specific Dll4 loss-of-function through inhibition of epithelial-to-mesenchymal transition and reduction of cancer stem cells and circulating tumor cells.

Systemic inhibition of Dll4 has been shown to thoroughly reduce cancer metastasis. The exact cause of this effect and whether it is endothelial mediated remains to be clarified. Therefore, we proposed to analyze the impact of endothelial Dll4 loss-of-function on metastasis induction on three early steps of the metastatic process, regulation of epithelial-to-mesenchymal transition (EMT), cancer stem cell (CSC) frequency and circulating tumor cell (CTC) number. For this, Lewis Lung Carcinoma (LLC) cells were used to model mouse tumor metastasis in vivo, by subcutaneous transplantation into endothelial-specific Dll4 loss-of-function mice. We observed that endothelial-specific Dll4 loss-of-function is responsible for the tumor vascular regression that leads to the reduction of tumor burden. It induces an increase in tumoral blood vessel density, but the neovessels are poorly perfused, with increased leakage and reduced perivascular maturation. Unexpectedly, although hypoxia was increased in the tumor, the number and burden of macro-metastasis was significantly reduced. This is likely to be a consequence of the observed reduction in both EMT and CSC numbers caused by the endothelial-specific Dll4 loss-of-function. This multifactorial context may explain the concomitantly observed reduction of the circulating tumor cell count. Furthermore, our results suggest that endothelial Dll4/Notch-function mediates tumor hypoxia-driven increase of EMT. Therefore, it appears that endothelial Dll4 may constitute a promising target to prevent metastasis.

Bioconjugation with Maleimides: A Useful Tool for Chemical Biology.

Maleimide chemistry stands out in the bioconjugation toolbox by virtue of its synthetic accessibility, excellent reactivity, and practicability. The second-generation of clinically approved antibody-drug conjugates (ADC) and much of the current ADC pipeline in clinical trials contain the maleimide linkage. However, thiosuccinimide linkages are now known to be less robust than once thought, and ergo, are correlated with suboptimal pharmacodynamics, pharmacokinetics, and safety profiles in some ADC constructs. Rational design of novel generations of maleimides and maleimide-type reagents have been reported to address the shortcomings of classical maleimides, allowing for the formation of robust bioconjugate linkages. This review highlights the main strategies for rational reagent design that have allowed irreversible bioconjugations in cysteines, reversible labelling strategies and disulfide re-bridging.

Methods to Study Angiogenesis in a Mouse Model of Prostate Cancer.

Angiogenesis is one important hallmark of cancer progression which explains the relevance of developing methods to efficiently analyze the neo-angiogenic process. In this report we make use of the transgenic adenocarcinoma of the murine prostate (TRAMP) model, considered a good model for studying clinical prostate cancer progression, to describe in detail the methods used to study angiogenesis in this type of solid tumor development. In this report we provide step-by-step procedures on the basis of previous work in our laboratory for: the mouse urogenital sinus (UGS) collection; microdissection of the prostate; preparation of the prostatic samples for immunofluorescence (to analyze vascular density, morphology, maturation, functionality, hypoxia, and others); preparation of prostatic samples to histopathological analysis and/or immunohistochemistry; and endothelial and vascular mural cell sorting and isolation by fluorescent associated cell sorting (FACS) to further analysis (mRNA, protein, or other) or to maintain in culture.

miR-21 ablation and obeticholic acid ameliorate nonalcoholic steatohepatitis in mice.

This corrects the article DOI: 10.1038/cddis.2017.172.

miR-21 ablation and obeticholic acid ameliorate nonalcoholic steatohepatitis in mice.

microRNAs were recently suggested to contribute to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a disease lacking specific pharmacological treatments. In that regard, nuclear receptors are arising as key molecular targets for the treatment of nonalcoholic steatohepatitis (NASH). Here we show that, in a typical model of NASH-associated liver damage, microRNA-21 (miR-21) ablation results in a progressive decrease in steatosis, inflammation and lipoapoptosis, with impairment of fibrosis. In a complementary fast food (FF) diet NASH model, mimicking features of the metabolic syndrome, miR-21 levels increase in both liver and muscle, concomitantly with decreased expression of peroxisome proliferator-activated receptor α (PPARα), a key miR-21 target. Strikingly, miR-21 knockout mice fed the FF diet supplemented with farnesoid X receptor (FXR) agonist obeticholic acid (OCA) display minimal steatosis, inflammation, oxidative stress and cholesterol accumulation. In addition, lipoprotein metabolism was restored, including decreased fatty acid uptake and polyunsaturation, and liver and muscle insulin sensitivity fully reinstated. Finally, the miR-21/PPARα axis was found amplified in liver and muscle biopsies, and in serum, of NAFLD patients, co-substantiating its role in the development of the metabolic syndrome. By unveiling that miR-21 abrogation, together with FXR activation by OCA, significantly improves whole body metabolic parameters in NASH, our results highlight the therapeutic potential of nuclear receptor multi-targeting therapies for NAFLD.

Endothelial Dll4 overexpression reduces vascular response and inhibits tumor growth and metastasization in vivo.

BACKGROUND: The inhibition of Delta-like 4 (Dll4)/Notch signaling has been shown to result in excessive, nonfunctional vessel proliferation and significant tumor growth suppression. However, safety concerns emerged with the identification of side effects resulting from chronic Dll4/Notch blockade. Alternatively, we explored the endothelial Dll4 overexpression using different mouse tumor models. METHODS: We used a transgenic mouse model of endothelial-specific Dll4 overexpression, previously produced. Growth kinetics and vascular histopathology of several types of solid tumors was evaluated, namely Lewis Lung Carcinoma xenografts, chemically-induced skin papillomas and RIP1-Tag2 insulinomas. RESULTS: We found that increased Dll4/Notch signaling reduces tumor growth by reducing vascular endothelial growth factor (VEGF)-induced endothelial proliferation, tumor vessel density and overall tumor blood supply. In addition, Dll4 overexpression consistently improved tumor vascular maturation and functionality, as indicated by increased vessel calibers, enhanced mural cell recruitment and increased network perfusion. Importantly, the tumor vessel normalization is not more effective than restricted vessel proliferation, but was found to prevent metastasis formation and allow for increased delivery to the tumor of concomitant chemotherapy, improving its efficacy. CONCLUSIONS: By reducing endothelial sensitivity to VEGF, these results imply that Dll4/Notch stimulation in tumor microenvironment could be beneficial to solid cancer patient treatment by reducing primary tumor size, improving tumor drug delivery and reducing metastization. Endothelial specific Dll4 overexpression thus appears as a promising anti-angiogenic modality that might improve cancer control.

Delta-like 4/Notch signaling promotes Apc Min/+ tumor initiation through angiogenic and non-angiogenic related mechanisms.

BACKGROUND: Delta like 4 (Dll4)/Notch signaling is a key regulator of tumor angiogenesis. Additionally, the role of Dll4 has been studied on tumor stem cells. However, as these cells are implicated in tumor angiogenesis, it is conceivable that the effect of Dll4 on these cells may be a consequence of its angiogenic function. Our aim was to evaluate the expression and dissect the functions of Dll4 in the Apc Min/+ model of colorectal cancer. METHODS: We evaluated the protein expression pattern of Dll4 and other Notch members in the Apc Min/+ tumors relatively to the normal gut and compared endothelial-specific with ubiquitous Dll4 knockout mice on an Apc Min/+ background. RESULTS: All Notch pathway members were present in the normal small and large intestine and in the adenomas of the same regions. Dll4, all Notch receptors and Hes1 expression seemed upregulated in the tumors, with some regional differences. The same members and Hes5, instead of Hes1, presented ectopic expression in the tumor parenchyma. Dll4 expression was most pronounced in the tumor cells but it was also present in the tumor blood vessels and in other stromal cells. Ubiquitous and endothelial-specific Dll4 deletion led to an equivalent reduction of tumor growth because of a similarly marked tumoral angiogenic phenotype promoting non-productive vasculature and consequently hypoxia and apoptosis. The ubiquitous Dll4 inhibition led to a stronger decrease of tumor multiplicity than the endothelial-specific deletion by further reducing tumor proliferation and tumor stem cell density through upregulation of the cyclin-dependent kinase inhibitors 1C and 1B and downregulation of Myc, Cyclin D1 and D2 independently of ß-catenin activation. This phenotype was associated to the observed increased epithelial differentiation deviated towards the secretory lineages by Atoh1 and Klf4 upregulation only in the ubiquitous Dll4 mutants. CONCLUSIONS: Dll4 seems to promote Apc Min/+ tumorigenesis through both angiogenic and non-angiogenic related mechanisms.

Irreversible Conjugation of Aldehydes in Water To Form Stable 1,2,4-Oxadiazinan-5-ones.

A new, irreversible aldehyde conjugation reaction in aqueous media was developed. α-Aminooxy acetohydrazides undergo irreversible condensation reactions with aliphatic, aromatic, or unsaturated aldehydes and isatins in a mixture of acetonitrile and acetate buffer at pH 4 to yield 1,2,4-oxadiazinan-5-one heterocycles in excellent isolated yields (40-99%). This class of heterocycles proved to be hydrolytically stable throughout a wide range of temperatures and pH (4.5-7).

Notch signaling dynamics in the adult healthy prostate and in prostatic tumor development.

BACKGROUND: The Notch signaling pathway has been implicated in prostate development, maintenance and tumorigenesis by its key role in cell-fate determination, differentiation and proliferation. Therefore, we proposed to analyze Notch family members transcription and expression, including ligands (Dll1, 3, 4 and Jagged1 and 2), receptors (Notch1-4) and effectors (Hes1, 2, 5 and Hey1, 2, L), in both normal and tumor bearing mouse prostates to better understand the dynamics of Notch signaling in prostate tumorigenesis. METHODS: Wild type mice and transgenic adenocarcinoma of the mouse prostate model (TRAMP) mice were sacrificed at 18, 24 or 30 weeks of age and the prostates collected and processed for either whole prostate or prostate cell specific populations mRNA analysis and for protein expression analysis by immunohistochemistry and immunofluorescence. RESULTS: We observed that Dll1 and Dll4 are expressed in the luminal compartment of the mouse healthy prostate, whereas Jagged2 expression is restricted to the basal and stromal compartment. Additionally, Notch2 and Notch4 are normally expressed in the prostate luminal compartment while Notch2 and Notch3 are also expressed in the stromal layer of the healthy prostate. As prostate tumor development takes place, there is up-regulation of Notch components. Particularly, the prostate tumor lesions have increased expression of Jagged1 and 2, of Notch3 and of Hey1. We have also detected the presence of activated Notch3 in prostatic tumors that co-express Jagged1 and ultimately the Hey1 effector. CONCLUSIONS: Taken together our results point out the Notch axis Jagged1-2/Notch3/Hey1 to be important for prostate tumor development and worthy of additional functional studies and validation in human clinical disease.

Synthesis of Symmetric Bis(N-alkylaniline)triarylmethanes via Friedel-Crafts-Catalyzed Reaction between Secondary Anilines and Aldehydes.

The first general protocol for the preparation of symmetric triarylmethanes bearing secondary anilines by ytterbium-catalyzed Friedel-Crafts reaction of hetero(aryl) aldehydes and secondary anilines is reported. Mechanistic studies indicated that the iminium ion intermediate is the electrophilic partner. The reaction is greatly accelerated by high pressure (9 kbar) and showed a broad substrate scope on the hetero(aryl) aldehyde. The new triarylmethanes exhibited activity against HT-29 cancer cell lines, with the best result scoring an IC50 of 1.74 µM.