Here we describe the establishment of a new canine mammary tumour (CMT) cell line, FR37-CMT that does not show dependence on female hormonal signaling to induce tumour xenografts in NOD-SCID mice. FR37-CMT cell line has a stellate or fusiform shape, displays the ability to reorganize the collagen matrix, expresses vimentin, CD44 and shows the loss of E-cadherin which is considered a fundamental event in epithelial to mesenchymal transition (EMT). The up-regulation of ZEB1, the detection of phosphorylated ERK1/2 and the downregulation of DICER1 and miR-200c are also in accordance with the mesenchymal characteristics of FR37-CMT cell line. FR37-CMT shows a higher resistance to cisplatin (IC50>50 µM) and to doxorubicin (IC50>5.3 µM) compared with other CMT cell lines. These results support the use of FR37-CMT as a new CMT model that may assist the understanding of the molecular mechanisms underlying EMT, CMT drug resistance, fostering the development of novel therapies targeting CMT.

The syntheses of the heterometallic sodium and potassium-dioxidovanadium 2D polymers, [NaVO2(1kappaNOO';2kappaO"-L)(H2O)]n(1) and [KVO2(1kappaNOO';2kappaO';3kappaO"-L)(EtOH)]n(2) (where the kappa notation indicates the coordinating atoms of the polydentate ligand L) derived from (3,5-di-tert-butyl-2-hydroxybenzylidene)-2-hydroxybenzohydrazide (H2L) are reported. The polymers were characterized by IR, NMR, elemental analysis and single crystal X-ray diffraction analysis. The antiproliferative potential of 1 and 2 was examined towards four human cancer cell lines (ovarian carcinoma, A2780, colorectal carcinoma, HCT116, prostate carcinoma, PC3 and breast adenocarcinoma, MCF-7cell lines) and normal human fibroblasts. Complex 1 and 2 showed the highest cytotoxic activity against A2780 cell line (IC50 8.2 and 11.3muM, respectively) with 1>2 and an IC50 in the same range as cisplatin (IC50 3.4muM; obtained in the same experimental conditions) but, interestingly, with no cytotoxicity to healthy human fibroblasts for concentrations up to 75muM. This high cytotoxicity of 1 in ovarian cancer cells and its low cytotoxicity in healthy cells demonstrates its potential for further biological studies. Our results suggest that both complexes induce ovarian carcinoma cell death via apoptosis and autophagy, but autophagy is the main biological cause of the reduction of viability observed and that ROS (reactive oxygen species) may play an important role in triggering cell death.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), a Lancefield group C streptococci (GCS), is a frequent cause of bovine mastitis. This highly prevalent disease is the costliest in dairy industry. Adherence and biofilm production are important factors in streptoccocal pathogenesis. We have previously described the adhesion and internalization of SDSD isolates in human cells and now we describe the biofilm production capability of this bacterium. In this work we integrated microbiology, imaging and computational methods to evaluate the biofilm production capability of SDSD isolates; to assess the presence of biofilm regulatory protein BrpA homolog in the biofilm producers; and to predict a structural model of BrpA-like protein and its binding to putative inhibitors. Our results show that SDSD isolates form biofilms on abiotic surface such as glass (hydrophilic) and polystyrene (hydrophobic), with the strongest biofilm formation observed in glass. This ability was mainly associated with a proteinaceous extracellular matrix, confirmed by the dispersion of the biofilms after proteinase K and trypsin treatment. The biofilm formation in SDSD isolates was also confirmed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Under SEM observation, VSD16 isolate formed cell aggregates during biofilm growth while VSD9 and VSD10 formed smooth and filmy layers. We show that brpA-like gene is present and expressed in SDSD biofilm-producing isolates and its expression levels correlated with the biofilm production capability, being more expressed in the late exponential phase of planktonic growth compared to biofilm growth. Fisetin, a known biofilm inhibitor and a putative BrpA binding molecule, dramatically inhibited biofilm formation by the SDSD isolates but did not affect planktonic growth, at the tested concentrations. Homology modeling was used to predict the 3D structure of BrpA-like protein. Using high throughput virtual screening and molecular docking, we selected five ligand molecules with strong binding affinity to the hydrophobic cleft of the protein, making them potential inhibitor candidates of the SDSD BrpA-like protein. These results warrant further investigations for developing novel strategies for SDSD anti-biofilm therapy.

In this work, biocompatible and biodegradable poly(d-l-lactide-co-glycolide) (PLGA) composite microparticles with potential use as carrier for vaccines and other drugs to the lungs were developed using supercritical CO2-assisted spray-drying (SASD). Bovine serum albumin (BSA) was chosen as model vaccine, and l-leucine as a dispersibility enhancer, and their effects on the particle characteristics were evaluated. The dry powder formulations (DPFs) were characterized in terms of their morphology and aerodynamic performance using an in vitro aerosolization study – Andersen cascade impactor (ACI) − to obtain data such as the fine particle fraction (FPF) with percentages up to 43.4%, and the mass median aerodynamic diameter (MMAD) values between the 1.7 and 3.5μm. Additionally, pharmacokinetic and cytotoxicity studies were performed confirming that the produced particles have all the necessary requirements for potential pulmonary delivery.

Silver nanoparticles (AgNPs) were prepared by GREEN chemistry relying on the reduction of AgNO3 by phytochemicals present in black tea extract. AgNPs were fully characterized by transmission electron microscopy (TEM), ultraviolet-visible spectroscopy ((UV-vis)), X-ray diffraction (XRD) and energy dispersive absorption spectroscopy (EDS). The synthesized AgNPs induced a decrease of the cell viability in a dose-dependent manner with a low IC50 (0.5+/-0.1muM) for an ovarian carcinoma cell line (A2780) compared to primary human fibroblasts (IC50 5.0+/-0.1muM). The DNA binding capability of CT (calf thymus) DNA was investigated using electronic absorption and fluorescence spectroscopies, circular dichroism and viscosity titration methods. Additionally, the AgNPs strongly quench the intrinsic fluorescence of BSA, as determined by synchronous fluorescence spectra.

Microfluidic (MF) advancements have been leveraged toward the development of state-of-the-art platforms for molecular diagnostics, where isothermal amplification schemes allow for further simplification of DNA detection and quantification protocols. The MF integration with loop-mediated isothermal amplification (LAMP) is today the focus of a new generation of chip-based devices for molecular detection, aiming at fast and automated nucleic acid analysis. Here, we combined MF with droplet digital LAMP (ddLAMP) on an all-in-one device that allows for droplet generation, target amplification, and absolute quantification. This multilayer 3D chip was developed in less than 30 minutes by using a low-cost and extremely adaptable production process that exploits direct laser writing technology in "Shrinky-dinks" polystyrene sheets. ddLAMP and target quantification were performed directly on-chip, showing a high correlation between target concentration and positive droplet score. We validated this integrated chip via the amplification of targets ranging from five to 500,000 copies/reaction. Furthermore, on-chip amplification was performed in a 10 microL volume, attaining a limit of detection of five copies/microL under 60 min. This technology was applied to quantify a cancer biomarker, c-MYC, but it can be further extended to any other disease biomarker.

Background: An ideal strategy for cancer treatment is the specific induction of tumor cell death, sparing normal cells. Marine sponges are rich biological reservoirs of biomolecules, especially lectins, which have attracted considerable attention due to potential biological effect on human cells. Lectins are proteins that bind specific carbohydrate signatures and some gained further interest for their capacity to bind tumor associated carbohydrates antigens and induce tumor cell apoptosis. Objective: This study aimed to evaluate the antitumor potential of H3, a lectin, recently reported from marine sponge Haliclona caerulea on the human breast cancer cell line MCF7. Results: H3 reduced MCF7 cell viability with an IC50 of 100 μg/ml, without a significant effect on normal cells. At 24h, H3 induced a significant arrest in the G1 cell cycle phase. Consistently, almost 50% of the cells were in early apoptosis and showed remarkable increased expression of caspase-9 (CASP 9). H3 impaired dramatically the adhesiveness of MCF7 cells in culture. Assays conducted with Lysotracker Red probe showed increased organelle acidity, suggesting autophagic cell death, which was further supported by increased expression of microtubule-associated protein light chain 3 (LC3) and observable conversion of LC3-I in LC3-II by western blot. Conclusion: The apoptotic effect of H3 may be related to a balance between apoptotic and autophagic cell death, mediated by increased expression of CASP 9 and LC3-II. To the best of our knowledge this is the first report about a sponge lectin triggering both apoptosis and autophagy in MCF7 cell.

Magnetic hyperthermia is a cancer treatment based on the exposure of magnetic nanoparticles to an alternating magnetic field in order to generate local heat. In this work, 3D cell culture models were prepared to observe the effect that a different number of internalized particles had on the mechanisms of cell death triggered upon the magnetic hyperthermia treatment. Macrophages were selected by their high capacity to uptake nanoparticles. Intracellular nanoparticle concentrations up to 7.5 pg Fe/cell were measured both by elemental analysis and magnetic characterization techniques. Cell viability after the magnetic hyperthermia treatment was decreased to <25% for intracellular iron contents above 1 pg per cell. Theoretical calculations of the intracellular thermal effects that occurred during the alternating magnetic field application indicated a very low increase in the global cell temperature. Different apoptotic routes were triggered depending on the number of internalized particles. At low intracellular magnetic nanoparticle amounts (below 1 pg Fe/cell), the intrinsic route was the main mechanism to induce apoptosis, as observed by the high Bax/Bcl-2 mRNA ratio and low caspase-8 activity. In contrast, at higher concentrations of internalized magnetic nanoparticles (1-7.5 pg Fe/cell), the extrinsic route was observed through the increased activity of caspase-8. Nevertheless, both mechanisms may coexist at intermediate iron concentrations. Knowledge on the different mechanisms of cell death triggered after the magnetic hyperthermia treatment is fundamental to understand the biological events activated by this procedure and their role in its effectiveness.

Abstract Cancer is one of the leading causes of death in the world. To challenge this epidemic, there are growing demands for the development of new advanced and targeted therapeutics capable of effectively tackling cancer cells with improved selectivity. Nanomedicine has put forward several innovative therapeutics toward improving therapeutic efficacy while decreasing the deleterious side effects of current chemotherapy. Multifunctional gold nanoparticles (AuNPs) have been at the core of a plethora of advanced therapeutic strategies that provide selective targeting with their unique optical properties, capable to interact with the light of specific wavelength to deliver therapy with tremendous spatiotemporal precision. AuNPs have been exploited as photodynamic and photothermal therapeutic agents alone or in combination with other cancer treatment modalities with other cancer applications. Due to their exceptional physicochemical properties, they have been proven efficacious allies for photodynamic therapy and for photothermal therapy regimens. Herein, the rapidly progressing literature related to the use of these promising strategies against cancer is discussed, highlighting their possible future clinical translation.

Hematologic malignancies are the most common type of cancer affecting children and young adults, and encompass diseases, such as leukemia, lymphoma, and myeloma, all of which impact blood associated tissues such as the bone marrow, lymphatic system, and blood cells. Clinical diagnostics of these malignancies relies heavily on the use of bone marrow samples, which is painful, debilitating, and not free from risks for leukemia patients. Liquid biopsies are based on minimally invasive assessment of markers in the blood (and other fluids) and have the potential to improve the efficacy of diagnostic/therapeutic strategies in leukemia patients, providing a useful tool for the real time molecular profiling of patients. The most promising noninvasive biomarkers are circulating tumor cells, circulating tumor DNA, microRNAs, and exosomes. Herein, we discuss the role of assessing these circulating biomarkers for the understanding of tumor progression and metastasis, tumor progression dynamics through treatment and for follow-up.

Many diseases have their treatment options narrowed and end up being fatal if detected during later stages. As a consequence, point-of-care devices have an increasing importance for routine screening applications in the health sector due to their portability, fast analyses and decreased cost. For that purpose, a multifunctional chip was developed and tested using gold nanoprobes to perform RNA optical detection inside a microfluidic chip without the need of molecular amplification steps. As a proof-of-concept, this device was used for the rapid detection of chronic myeloid leukemia, a hemato-oncological disease that would benefit from early stage diagnostics and screening tests. The chip passively mixed target RNA from samples, gold nanoprobes and saline solution to infer a result from their final colorimetric properties. An optical fiber network was used to evaluate its transmitted spectra inside the chip. Trials provided accurate output results within 3 min, yielding signal-to-noise ratios up to 9 dB. When compared to actual state-of-art screening techniques of chronic myeloid leukemia, these results were, at microscale, at least 10 times faster than the reported detection methods for chronic myeloid leukemia. Concerning point-of-care applications, this work paves the way for other new and more complex versions of optical based genosensors.

Abstract POXylated polyurea dendrimer nanoparticles (PUREG4OOx48) are loaded with sildenafil (SDF) by a supercritical carbon dioxide–assisted (scCO2) impregnation. Further supercritical CO2-assisted spray drying (SASD) leads to hybrid nano-in-micro dry powder formulations that are investigated aiming at efficient pulmonary delivery of SDF in pulmonary arterial hypertension treatment. This is the first report of the production of poly(D,L-lactide-co-glycolide)-cholesterol (PLGA-Chol) microparticles processed by SASD. The optimized formulation of nano-in-microparticles is composed of PLGA, Chol, and PUREG4OOx48, loaded with SDF solutions in a 77:23 ratio (PLGA-Chol:dendrimer, w/w). The dry powders are fully characterized and found to be highly biodegradable and biocompatible, and the SDF release profile evaluates under different pH values. The median mass average diameter (MMAD) of the nano-in-micro systems varies between 2.57 and 5 µm and the fine particle fraction (FPF) between 36% and 29% for PUREG4OMeOx48[PLGA-Chol] and PUREG4OEtOx48[PLGA-Chol], respectively. The data validate the potential use of these new formulations in inhalation therapy. In vitro studies are also carried out in order to evaluate the effect of the free drug in cell viability and formulations cytotoxicity.

While screening for polyhydroxyalkanoate (PHA) producing strains, using glycerol rich by-product as carbon source, it was observed that extracellular polymers were also secreted into the culture broth. The scope of this study was to characterize both intracellular and extracellular polymers, produced by Pseudomonas putida NRRL B-14875 and Pseudomonas chlororaphis DSM 50083, mostly focusing on those novel extracellular polymers. It was found that they fall into the class of bioemulsifiers (BE), as they showed excellent emulsion stability against different hydrocarbons/oils at various pH conditions, temperature and salinity concentrations. Cytotoxicity tests revealed that BE produced by P. chlororaphis inhibited the growth of highly pigmented human melanoma cells (MNT-1) by 50% at concentrations between 150 and 200mug/mL, while no effect was observed on normal skin primary keratinocytes and melanocytes. This is the first study reporting mcl-PHA production by P. putida NRRL B-14785 and bioemulsifier production from both P. putida and P. chlororaphis strains.

Abstract POxylated polyurea dendrimer (PUREG4OOx48)-based nanoparticles were loaded with paclitaxel (PTX) and doxorubicin (DOX) and micronized with chitosan (CHT) by using supercritical CO2-assisted spray drying (SASD). Respirable, biocompatible, and biodegradable dry powder formulations (DPFs) were produced to effectively transport and deliver the chemotherapeutics with a controlled rate to the deep lung. In vitro studies performed with the use of the lung adenocarcinoma cell line showed that DOX@PUREG4OOx48 nanoparticles were much more cytotoxic than the free drug. Additionally, the DPFs did not show higher cytotoxicity than the respective nanoparticles, and DOX-DPFs showed a higher chemotherapeutic effect than PTX formulations in adenocarcinoma cells.

The design and preparation of highly efficient drug delivery platforms using green methodologies is at the forefront of nanotherapeutics research. POxylated polyurea dendrimers are efficiently synthesized using a supercritical-assisted polymerization in carbon dioxide. These fluorescent, pH-responsive and water-soluble core-shell smart nanocarriers show low toxicity in terms of cell viability and absence of glutathione depletion, two of the major side effect limitations of current vectors. The materials are also found to act as good transfection agents, through a mechanism involving an endosomal pathway, being able to reduce 100-fold the IC50 of paclitaxel.

Biofilms are generally defined as communities of cells involved in a self-produced extracellular matrix adhered to a surface. In biofilms, the bacteria are less sensitive to host defense mechanisms and antimicrobial agents, due to multiple strategies, that involve modulation of gene expression, controlled metabolic rate, intercellular communication, composition, and 3D architecture of the extracellular matrix. These factors play a key role in streptococci pathogenesis, contributing to therapy failure and promoting persistent infections. The species of the pyogenic group together with Streptococcus pneumoniae are the major pathogens belonging the genus Streptococcus, and its biofilm growth has been investigated, but insights in the genetic origin of biofilm formation are limited. This review summarizes pyogenic streptococci biofilms with details on constitution, formation, and virulence factors associated with formation.

Once released to the extracellular space, exosomes enable the transfer of proteins, lipids and RNA between different cells, being able to modulate the recipient cells' phenotypes. Members of the Rab small GTP-binding protein family, such as RAB27A, are responsible for the coordination of several steps in vesicle trafficking, including budding, mobility, docking and fusion. The use of gold nanoparticles (AuNPs) for gene silencing is considered a cutting-edge technology. Here, AuNPs were functionalized with thiolated oligonucleotides anti-RAB27A (AuNP@PEG@anti-RAB27A) for selective silencing of the gene with a consequent decrease of exosomes´ release by MCF-7 and MDA-MB-453 cells. Furthermore, communication between tumor and normal cells was observed both in terms of alterations in c-Myc gene expression and transportation of the AuNPs, mediating gene silencing in secondary cells.

Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability.

The water-soluble 1D helical coordination polymer [Ag(Tpms)]n (1) [Tpms=tris(pyrazolyl)methane sulfonate, (-)O3SC(pz)3; pz=pyrazolyl] was synthesized and fully characterized, its single-crystal X-ray diffraction analysis revealing the ligand acting as a bridging chelate N3-donor ligand. The antiproliferative potential of 1 was performed on two human tumour cell lines, A2780 and HCT116, and in normal fibroblasts, with a much higher effect in the former cell line (IC50 of 0.04muM) as compared to the latter cell line and to normal fibroblasts. Compound 1 does not alter cell cycle progression but interferes with the adherence of A2780 cells triggering cell apoptosis. Apoptosis appears to occur via the extrinsic pathway (no changes in mitochondria membrane potential, reactive oxygen species (ROS) and pro-apoptotic (B-cell lymphoma 2 (BCL-2) associated protein (BAX))/anti-apoptotic (BCL-2) ratio) being this hypothesis also supported by the presence of silver mainly in the supernatants of A2780 cells. Results also indicated that cell death via autophagy was triggered. Proteomic analysis allowed us to confirm that compound 1 is able to induce a stress response in A2780 cells that is related with its antiproliferative activity and the trigger of apoptosis.

A new and never before reported hetero-arylidene-9(10H)-anthrone structure (4) was unexpectedly isolated on reaction of 1,2-dimethyl-3-ethylimidazolium iodide (2) and 9-anthracenecarboxaldehyde (3) under basic conditions. Its structure was unequivocally confirmed by X-ray crystallography. No cytotoxicity in human healthy fibroblasts and in two different cancer cell lines was observed, indicating its applicability in biological systems. Compound 4 interacts with CT-DNA by intercalation between the adjacent base pairs of DNA with a high binding affinity [Kb = 2.0 (±0.20) × 105 m–1], which is 10 × higher than that described for doxorubicin [Kb = 3.2 (±0.23) × 104 m–1]. Furthermore, compound 4 quenches the fluorescence emission of a GelRed–CT-DNA system with a quenching constant (KSV) of 3.3 (±0.3) × 103 m–1 calculated by the Stern–Volmer equation.

The pyogenic streptococci group includes pathogenic species for humans and other animals and has been associated with enduring morbidity and high mortality. The main reason for the treatment failure of streptococcal infections is the increased resistance to antibiotics. In recent years, infectious diseases caused by pyogenic streptococci resistant to multiple antibiotics have been raising with a significant impact to public health and veterinary industry. The rise of antibiotic-resistant streptococci has been associated to diverse mechanisms, such as efflux pumps and modifications of the antimicrobial target. Among streptococci, antibiotic resistance emerges from previously sensitive populations as result of horizontal gene transfer or chromosomal point mutations due to excessive use of antimicrobials. Streptococci strains are also recognized as biofilm producers. The increased resistance of biofilms to antibiotics among streptococci promote persistent infection, which comprise circa 80% of microbial infections in humans. Therefore, to overcome drug resistance, new strategies, including new antibacterial and antibiofilm agents, have been studied. Interestingly, the use of systems based on nanoparticles have been applied to tackle infection and reduce the emergence of drug resistance. Herein, we present a synopsis of mechanisms associated to drug resistance in (pyogenic) streptococci and discuss some innovative strategies as alternative to conventional antibiotics, such as bacteriocins, bacteriophage, and phage lysins, and metal nanoparticles. We shall provide focused discussion on the advantages and limitations of agents considering application, efficacy and safety in the context of impact to the host and evolution of bacterial resistance.

The rise in antibiotic resistance coupled with the gap in the discovery of active molecules has driven the need for more effective antimicrobials while focusing the attention into the repurpose of already existing drugs. Here, we evaluated the potential antibacterial activity of one cobalt and two zinc metallic compounds previously reported as having anticancer properties. Compounds were tested against a range of Gram-positive and -negative bacteria. The determination of the minimum inhibitory and bactericidal concentrations (MIC/MBC) of the drugs were used to assess their potential antibacterial activity and their effect on bacterial growth. Motility assays were conducted by exposing the bacteria to sub-MIC of each of the compounds. The effect of sub-MIC of the compounds on the membrane permeability was measured by ethidium bromide (EtBr) accumulation assay. Cell viability assays were performed in human cells. Compound TS262 was the most active against the range of bacteria tested. No effect was observed on the motility or accumulation of EtBr for any of the bacteria tested. Cell viability assays demonstrated that the compounds showed a decrease in cell viability at the MIC. These results are promising, and further studies on these compounds can lead to the development of new effective antimicrobials.