Alkaline lignins from Morinda citrifolia leaves are potential immunomodulatory, antitumor, and antimicrobial agents.

Morinda citrifolia, commonly known as noni, is a plant belonging to the Rubiaceae family. This plant has a high biological potential, which has different biological properties, including antioxidant, antibacterial, antiviral, antifungal, antitumor and anti-inflammatory. In this work, the immunomodulatory, antitumor and antimicrobial activities of lignin isolated from Morinda citrifolia leaves were investigated. The results showed that this lignin was not cytotoxic and that it was able to promote activation and differentiation of immune cells in addition to inducing the production of anti-inflammatory cytokines. Furthermore, it was able to inhibit the growth of different tumor and microbial cells in vitro. This pioneering study on these different activities shows that the lignin isolated in this study can be used as a raw material to obtain biomedical and pharmaceutical products.

Stearoyl CoA Desaturase-1 Silencing in Glioblastoma Cells: Phospholipid Remodeling and Cytotoxicity Enhanced upon Autophagy Inhibition.

Modulation of lipid metabolism is a well-established cancer hallmark, and SCD1 has been recognized as a key enzyme in promoting cancer cell growth, including in glioblastoma (GBM), the deadliest brain tumor and a paradigm of cancer resistance. The central goal of this work was to identify, by MS, the phospholipidome alterations resulting from the silencing of SCD1 in human GBM cells, in order to implement an innovative therapy to fight GBM cell resistance. With this purpose, RNAi technology was employed, and low serum-containing medium was used to mimic nutrient deficiency conditions, at which SCD1 is overexpressed. Besides the expected increase in the saturated to unsaturated fatty acid ratio in SCD1 silenced-GBM cells, a striking increase in polyunsaturated chains, particularly in phosphatidylethanolamine and cardiolipin species, was noticed and tentatively correlated with an increase in autophagy (evidenced by the increase in LC3BII/I ratio). The contribution of autophagy to mitigate the impact of SCD1 silencing on GBM cell viability and growth, whose modest inhibition could be correlated with the maintenance of energetically associated mitochondria, was evidenced by using autophagy inhibitors. In conclusion, SCD1 silencing could constitute an important tool to halt GBM resistance to the available treatments, especially when coupled with a mitochondria disrupter chemotherapeutic.

MiR-200c-based metabolic modulation in glioblastoma cells as a strategy to overcome tumor chemoresistance.

Glioblastoma (GB) is the most aggressive and common form of primary brain tumor characterized by fast proliferation, high invasion and resistance to current standard treatment. The average survival rate post-diagnosis is 14.6 months, despite the aggressive standard post-surgery radiotherapy concomitant with chemotherapy with temozolomide (TMZ). Currently, efforts are being endowed to develop new and more efficient therapeutic approaches capable to overcome chemoresistance, inhibit tumor progression and improve overall patient survival rate. Abnormal microRNA (miRNA) expression has been correlated with chemoresistance, proliferation and resistance to apoptosis, which result from their master regulatory role of gene expression. Altered cell metabolism, favoring glycolysis, was identified as an emerging cancer hallmark and has been described in GB, thus offering a new target for innovative GB therapies. In this work, we hypothesized that a gene therapy-based strategy consisting of the overexpression of a miRNA downregulated in GB and predicted to target crucial metabolic enzymes might promote a shift of GB cell metabolism, decreasing the glycolytic dependence of tumor cells and contributing to their sensitization to chemotherapy with TMZ. The increase of miR-200c levels in DBTRG cells resulted in downregulation of messenger RNA of enzymes involved in bioenergetics pathways and impaired cell metabolism and mobility. In addition, miR-200c overexpression prior to DBTRG cell exposure to TMZ resulted in cell cycle arrest. Overall, our results show that miR-200c overexpression could offer a way to overcome chemoresistance developed by GB cells in response to current standard chemotherapy, providing an improvement to current GB standard treatment, with benefit for patient outcome.

Improving pollutants environmental risk assessment using a multi model toxicity determination with in vitro, bacterial, animal and plant model systems: The case of the herbicide alachlor.

Several environmental pollutants, including pesticides, herbicides and persistent organic pollutants play an important role in the development of chronic diseases. However, most studies have examined environmental pollutants toxicity in target organisms or using a specific toxicological test, losing the real effect throughout the ecosystem. In this sense an integrative environmental risk of pollutants assessment, using different model organisms is necessary to predict the real impact in the ecosystem and implications for target and non-target organisms. The objective of this study was to use alachlor, a chloroacetanilide herbicide responsible for chronic toxicity, to understand its impact in target and non-target organisms and at different levels of biological organization by using several model organisms, including membranes of dipalmitoylphosphatidylcholine (DPPC), rat liver mitochondria, bacterial (Bacillus stearothermophilus), plant (Lemna gibba) and mammalian cell lines (HeLa and neuro2a). Our results demonstrated that alachlor strongly interacted with membranes of DPPC and interfered with mitochondrial bioenergetics by reducing the respiratory control ratio and the transmembrane potential. Moreover, alachlor also decreased the growth of B. stearothermophilus and its respiratory activity, as well as decreased the viability of both mammalian cell lines. The values of TC50 increased in the following order: Lemna gibba < neuro2a < HeLa cells < Bacillus stearothermophilus. Together, the results suggest that biological membranes constitute a putative target for the toxic action of this lipophilic herbicide and point out the risks of its dissemination on environment, compromising ecosystem equilibrium and human health.

Anti-inflammatory activity of novel thiosemicarbazone compounds indole-based as COX inhibitors.

BACKGROUND: In this article, a series of 20 new thiosemicarbazone derivatives containing indole were synthesized and evaluated for their anti-inflammatory potential. METHODS: The compounds were obtained through a synthetic route of only two steps, with yields that varied between 33.6 and 90.4%, and characterized by spectroscopic and spectrometric techniques. RESULTS: An initial screening through the lymphoproliferation assay revealed that compounds LT76, LT81, and LT87 were able to inhibit lymphocyte proliferation, with CC50 of 0.56 ± 0.036, 0.9 ± 0.01 and 0.5 ± 0.07 µM, respectively, better results than indomethacin (CC50 > 12 µM). In addition, these compounds were able to suppress the in-vitro production of TNF-α and NO, in addition to stimulating the production of IL-4. Reinforcing in-vitro assays, the compounds were able to inhibit COX-2 similar to Celecoxib showing greater selectivity for this isoform (LT81 SI: 23.06 versus Celecoxib SI: 11.88). Animal studies showed that compounds LT76 (64.8% inhibition after 6 h), LT81 (89% inhibition after 6 h) and LT87 (100% inhibition after 4 h) were able to suppress edema in mice after inoculation carrageenan with greater potency than indomethacin, and immunohistochemistry revealed that the groups treated with LT76, LT81 and LT87 reduced the expression of COX-2, similar or better results when compared to indomethacin. Complementarily, in-silico studies have shown that these compounds have a good pharmacokinetic profile, for respecting the parameters of Lipinski and Veber, showing their good bioavailability. CONCLUSIONS: These results demonstrate the potency of thiosemicarbazone derivatives containing indole and confirm their importance as scaffolds of molecules with notorious anti-inflammatory activity.

Differentiation of glioblastoma stem cells promoted by miR-128 or miR-302a overexpression enhances senescence-associated cytotoxicity of axitinib.

Despite the intense global efforts towards an effective treatment of glioblastoma (GB), current therapeutic options are unsatisfactory with a median survival time of 12-15 months after diagnosis, which has not improved significantly over more than a decade. The high tumoral heterogeneity confers resistance to therapies, which has hindered a successful clinical outcome, GB remaining among the deadliest cancers. A hallmark of GB is its high recurrence rate, which has been attributed to the presence of a small subpopulation of tumor cells called GB stem-like cells (GSC). In the present work, the efficacy of a multimodal strategy combining microRNA (miRNA) modulation with new generation multitargeted tyrosine kinase inhibitors (imatinib and axitinib) was investigated aiming at tackling this subpopulation of GB cells. MiR-128 and miR-302a were selected as attractive therapeutic candidates on the basis of previous findings reporting that reestablishment of their decreased expression levels in GSC resulted in cell differentiation, which could represent a possible strategy to sensitize GSC to chemotherapy. Our results show that overexpression of miR-128 or miR-302a induced GSC differentiation, which enhanced senescence mediated by axitinib treatment, thus further impairing GSC proliferation. We also provided evidence for the capacity of GSC to efficiently internalize functionalized stable nucleic acid lipid particles, previously developed and successfully applied in our laboratory to target GB. Taken together, our findings will be important in the future design of a GB-targeted multimodal miRNA-based gene therapy, combining overexpression of miR-128 or miR-302a with axitinib treatment, endowed with the ability to overcome drug resistance.

Downregulation of long non-protein coding RNA MVIH impairs glioblastoma cell proliferation and invasion through an miR-302a-dependent mechanism.

Glioblastoma (GB) is the most frequent and malignant type of brain tumor, for which no effective therapy exists. The high proliferative and invasive nature of GB, as well as its acquired resistance to chemotherapy, makes this type of cancer extremely lethal shortly after diagnosis. Long non-protein coding RNAs (lncRNA) are a class of regulatory RNAs whose levels can be dysregulated in the context of diseases, unbalancing several physiological processes. The lncRNA associated with microvascular invasion in hepatocellular carcinoma (lncRNA-MVIH), overexpressed in several cancers, was described to co-precipitate with phosphoglycerate kinase 1 (PGK1), preventing secretion of this enzyme to the extracellular environment and promoting cell migration and invasion. We hypothesized that, by silencing the expression of lncRNA-MVIH, the secretion of PGK1 would increase, reducing GB cell migration and invasion capabilities. We observed that lncRNA-MVIH silencing in human GB cells significantly decreased glycolysis, cell growth, migration, and invasion and sensitized GB cells to cediranib. However, no increase in extracellular PGK1 was observed as a consequence of lncRNA-MVIH silencing, and therefore, we investigated the possibility of a mechanism of miRNA sponge of lncRNA-MVIH being in place. We found that the levels of miR-302a loaded onto RISC increased in GB cells after lncRNA-MVIH silencing, with the consequent downregulation of several miR-302a molecular targets. Our findings suggest a new mechanism of action of lncRNA-MVIH as a sponge of miR-302a. We suggest that lncRNA-MVIH knockdown may be a promising strategy to address GB invasiveness and chemoresistance, holding potential towards its future application in a clinical context.

Secondary Metabolites with Antioxidant Activities for the Putative Treatment of Amyotrophic Lateral Sclerosis (ALS): "Experimental Evidences".

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder that is characterized by progressive loss of the upper and lower motor neurons at the spinal or bulbar level. Oxidative stress (OS) associated with mitochondrial dysfunction and the deterioration of the electron transport chain are factors that contribute to neurodegeneration and perform a potential role in the pathogenesis of ALS. Natural antioxidant molecules have been proposed as an alternative form of treatment for the prevention of age-related neurological diseases, in which ALS is included. Researches support that regulations in cellular reduction/oxidation (redox) processes are being increasingly implicated in this disease, and antioxidant drugs are aimed at a promising pathway to treatment. Among the strategies used for obtaining new drugs, we can highlight the isolation of secondary metabolite compounds from natural sources that, along with semisynthetic derivatives, correspond to approximately 40% of the drugs found on the market. Among these compounds, we emphasize oxygenated and nitrogenous compounds, such as flavonoids, coumarins, and alkaloids, in addition to the fatty acids, that already stand out in the literature for their antioxidant properties, consisting in a part of the diets of millions of people worldwide. Therefore, this review is aimed at presenting and summarizing the main articles published within the last years, which represent the therapeutic potential of antioxidant compounds of natural origin for the treatment of ALS.

Lauroylated Histidine-Enriched S413-PV Peptide as an Efficient Gene Silencing Mediator in Cancer Cells.

PURPOSE: This study aimed to endow the cell-penetrating peptide (CPP) S413-PV with adequate features towards a safe and effective application in cancer gene therapy. METHODS: Peptide/siRNA complexes were prepared with two new derivatives of the CPP S413-PV, which combine a lauroyl group attached to the N- or C-terminus with a histidine-enrichment in the N-terminus of the S413-PV peptide, being named C12-H5-S413-PV and H5-S413-PV-C12, respectively. Physicochemical characterization of siRNA complexes was performed and their cytotoxicity and efficiency to mediate siRNA delivery and gene silencing in cancer cells were assessed in the absence and presence of serum. RESULTS: Peptide/siRNA complexes prepared with the C12-H5-S413-PV derivative showed a nanoscale (ca. 100 nm) particle size, as revealed by TEM, and efficiently mediated gene silencing (37%) in human U87 glioblastoma cells in the presence of 30% serum. In addition, the new C12-H5-S413-PV-based siRNA delivery system efficiently downregulated stearoyl-CoA desaturase-1, a key-enzyme of lipid metabolism overexpressed in cancer, which resulted in a significant decrease in the viability of U87 cells. Importantly, these complexes were able to spare healthy human astrocytes. CONCLUSIONS: These encouraging results pave the way for a potential application of the C12-H5-S413-PV peptide as a promising tool in cancer gene therapy.

Lysosomal Storage Disease-Associated Neuropathy: Targeting Stable Nucleic Acid Lipid Particle (SNALP)-Formulated siRNAs to the Brain as a Therapeutic Approach.

More than two thirds of Lysosomal Storage Diseases (LSDs) present central nervous system involvement. Nevertheless, only one of the currently approved therapies has an impact on neuropathology. Therefore, alternative approaches are under development, either addressing the underlying enzymatic defect or its downstream consequences. Also under study is the possibility to block substrate accumulation upstream, by promoting a decrease of its synthesis. This concept is known as substrate reduction therapy and may be triggered by several molecules, such as small interfering RNAs (siRNAs). siRNAs promote RNA interference, a naturally occurring sequence-specific post-transcriptional gene-silencing mechanism, and may target virtually any gene of interest, inhibiting its expression. Still, naked siRNAs have limited cellular uptake, low biological stability, and unfavorable pharmacokinetics. Thus, their translation into clinics requires proper delivery methods. One promising platform is a special class of liposomes called stable nucleic acid lipid particles (SNALPs), which are characterized by high cargo encapsulation efficiency and may be engineered to promote targeted delivery to specific receptors. Here, we review the concept of SNALPs, presenting a series of examples on their efficacy as siRNA nanodelivery systems. By doing so, we hope to unveil the therapeutic potential of these nanosystems for targeted brain delivery of siRNAs in LSDs.

Effect of Vitamin K3 Inhibiting the Function of NorA Efflux Pump and Its Gene Expression on Staphylococcus aureus.

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus, is synthesized by the expression of the norA gene. Menadione, also known as vitamin K3, is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

In vitro activity of aryl-thiazole derivatives against Schistosoma mansoni schistosomula and adult worms.

Schistosomiasis is caused by a trematode of the genus Schistosoma and affects over 200 million people worldwide. The only drug recommended by the World Health Organization for treatment and control of schistosomiasis is praziquantel. Development of new drugs is therefore of great importance. Thiazoles are regarded as privileged structures with a broad spectrum of activities and are potential sources of new drug prototypes, since they can act through interactions with DNA and inhibition of DNA synthesis. In this context, we report the synthesis of a series of thiazole derivatives and their in vitro schistosomicidal activity by testing eight molecules (NJ03-08; NJ11-12) containing thiazole structures. Parameters such as motility and mortality, egg laying, pairing and parasite viability by ATP quantification, which were influenced by these compounds, were evaluated during the assays. Scanning electron microscopy (SEM) was utilized for evaluation of morphological changes in the tegument. Schistosomula and adult worms were treated in vitro with different concentrations (6.25 to 50 µM) of the thiazoles for up to 5 and 3 days, respectively. After in vitro treatment for five days with 6.25 µM NJ05 or NJ07 separately, we observed a decrease of 30% in schistosomula viability, whilst treatment with NJ05+NJ07 lead to a reduction of 75% in viability measured by ATP quantitation and propidium iodide labeling. Adult worms' treatment with 50 µM NJ05, NJ07 or NJ05 + NJ07 showed decreased motility to 30-50% compared with controls. Compound NJ05 was more effective than NJ07, and adult worm viability after three days was reduced to 25% in parasites treated with 50 µM NJ05, compared with a viability reduction to 40% with 50 µM NJ07. SEM analysis showed severe alterations in adult worms with formation of bulges and blisters throughout the dorsal region of parasites treated with NJ05 or NJ07. Oviposition was extremely affected by treatment with the NJ series compounds; at concentrations of 25 µM and 50 µM, oviposition reached almost zero with NJ05, NJ07 or NJ05 + NJ07 already at day one. Tested genes involved in egg biosynthesis were all confirmed by qPCR as downregulated in females treated with 25 µM NJ05 for 2 days, with a significant reduction in expression of p14, Tyrosinase 2, p48 and fs800. NJ05, NJ07 or NJ05+NJ07 treatment of HEK293 (human embryonic cell line) and HES (human epithelial cell line) showed EC50 in the range of 18.42 to 145.20 µM. Overall, our results demonstrate that those molecules are suitable targets for further development into new drugs for schistosomiasis treatment, although progress is needed to lessen the cytotoxic effects on human cells. According to the present study, thiazole derivatives have schistosomicidal activities and may be part of a possible new arsenal of compounds against schistosomiasis.

Glucosylceramide synthase silencing combined with the receptor tyrosine kinase inhibitor axitinib as a new multimodal strategy for glioblastoma.

A great deal of evidence revealing that lipid metabolism is drastically altered during tumorigenesis has been accumulated. In this work, glucosylceramide synthase (GCS) was targeted, using RNA interference technology (siRNAs), in U87 and DBTRG human glioblastoma (GBM) cells, as in both cell types GCS showed to be overexpressed with respect to normal human astrocytes. The efficacy of a combined therapy to tackle GBM, allying GCS silencing to the new generation chemotherapeutics sunitinib and axitinib, or to the alkylating drugs etoposide and temozolomide, is evaluated here for the first time. With this purpose, studies addressing GBM cell viability and proliferation, cell cycle and apoptosis were performed, which revealed that combination of GCS silencing with axitinib treatment represents a promising therapeutic approach. The reduction of cell viability induced by this combined therapy is proposed to be mediated by excessive production of reactive oxygen species. This work, identifying GCS as a key molecular target to increase GBM susceptibility to a new generation chemotherapeutic, opens windows to the development of innovative strategies to halt GBM recurrence after surgical resection.

Suicide Gene Therapy for Oral Squamous Cell Carcinoma.

Suicide gene therapy has been tested for the treatment of a variety of cancers, including oral cancer. Among the various suicide gene therapy approaches that have been reported, the Herpes Simplex Virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system is one of the most extensively studied systems, holding great promise in cancer therapy. In this chapter, we describe methods to use the HSV-tk/GCV system to achieve antitumor activity, both in cultured oral cancer cells and in orthotopic and subcutaneous murine models of oral squamous cell carcinoma, using ligand-associated lipoplexes for enhancing therapeutic delivery.

In vitro activity of aryl-thiazole derivatives against Schistosoma mansoni schistosomula and adult worms

Schistosomiasis is caused by a trematode of the genus Schistosoma and affects over 200 million people worldwide. The only drug recommended by the World Health Organization for treatment and control of schistosomiasis is praziquantel. Development of new drugs is therefore of great importance. Thiazoles are regarded as privileged structures with a broad spectrum of activities and are potential sources of new drug prototypes, since they can act through interactions with DNA and inhibition of DNA synthesis. In this context, we report the synthesis of a series of thiazole derivatives and their in vitro schistosomicidal activity by testing eight molecules (NJ03-08; NJ11-12) containing thiazole structures. Parameters such as motility and mortality, egg laying, pairing and parasite viability by ATP quantification, which were influenced by these compounds, were evaluated during the assays. Scanning electron microscopy (SEM) was utilized for evaluation of morphological changes in the tegument. Schistosomula and adult worms were treated in vitro with different concentrations (6.25 to 50 µM) of the thiazoles for up to 5 and 3 days, respectively. After in vitro treatment for five days with 6.25 µM NJ05 or NJ07 separately, we observed a decrease of 30% in schistosomula viability, whilst treatment with NJ05+NJ07 lead to a reduction of 75% in viability measured by ATP quantitation and propidium iodide labeling. Adult worms’ treatment with 50 µM NJ05, NJ07 or NJ05 + NJ07 showed decreased motility to 30–50% compared with controls. Compound NJ05 was more effective than NJ07, and adult worm viability after three days was reduced to 25% in parasites treated with 50 µM NJ05, compared with a viability reduction to 40% with 50 µM NJ07. SEM analysis showed severe alterations in adult worms with formation of bulges and blisters throughout the dorsal region of parasites treated with NJ05 or NJ07. Oviposition was extremely affected by treatment with the NJ series compounds; at concentrations of 25 µM and 50 µM, oviposition reached almost zero with NJ05, NJ07 or NJ05 + NJ07 already at day one. Tested genes involved in egg biosynthesis were all confirmed by qPCR as downregulated in females treated with 25 µM NJ05 for 2 days, with a significant reduction in expression of p14, Tyrosinase 2, p48 and fs800. NJ05, NJ07 or NJ05+NJ07 treatment of HEK293 (human embryonic cell line) and HES (human epithelial cell line) showed EC50 in the range of 18.42 to 145.20 µM. Overall, our results demonstrate that those molecules are suitable targets for further development into new drugs for schistosomiasis treatment, although progress is needed to lessen the cytotoxic effects on human cells. According to the present study, thiazole derivatives have schistosomicidal activities and may be part of a possible new arsenal of compounds against schistosomiasis.

In vitro activity of aryl-thiazole derivatives against Schistosoma mansoni schistosomula and adult worms

Schistosomiasis is caused by a trematode of the genus Schistosoma and affects over 200 million people worldwide. The only drug recommended by the World Health Organization for treatment and control of schistosomiasis is praziquantel. Development of new drugs is therefore of great importance. Thiazoles are regarded as privileged structures with a broad spectrum of activities and are potential sources of new drug prototypes, since they can act through interactions with DNA and inhibition of DNA synthesis. In this context, we report the synthesis of a series of thiazole derivatives and their in vitro schistosomicidal activity by testing eight molecules (NJ03-08; NJ11-12) containing thiazole structures. Parameters such as motility and mortality, egg laying, pairing and parasite viability by ATP quantification, which were influenced by these compounds, were evaluated during the assays. Scanning electron microscopy (SEM) was utilized for evaluation of morphological changes in the tegument. Schistosomula and adult worms were treated in vitro with different concentrations (6.25 to 50 µM) of the thiazoles for up to 5 and 3 days, respectively. After in vitro treatment for five days with 6.25 µM NJ05 or NJ07 separately, we observed a decrease of 30% in schistosomula viability, whilst treatment with NJ05+NJ07 lead to a reduction of 75% in viability measured by ATP quantitation and propidium iodide labeling. Adult worms’ treatment with 50 µM NJ05, NJ07 or NJ05 + NJ07 showed decreased motility to 30–50% compared with controls. Compound NJ05 was more effective than NJ07, and adult worm viability after three days was reduced to 25% in parasites treated with 50 µM NJ05, compared with a viability reduction to 40% with 50 µM NJ07. SEM analysis showed severe alterations in adult worms with formation of bulges and blisters throughout the dorsal region of parasites treated with NJ05 or NJ07. Oviposition was extremely affected by treatment with the NJ series compounds; at concentrations of 25 µM and 50 µM, oviposition reached almost zero with NJ05, NJ07 or NJ05 + NJ07 already at day one. Tested genes involved in egg biosynthesis were all confirmed by qPCR as downregulated in females treated with 25 µM NJ05 for 2 days, with a significant reduction in expression of p14, Tyrosinase 2, p48 and fs800. NJ05, NJ07 or NJ05+NJ07 treatment of HEK293 (human embryonic cell line) and HES (human epithelial cell line) showed EC50 in the range of 18.42 to 145.20 µM. Overall, our results demonstrate that those molecules are suitable targets for further development into new drugs for schistosomiasis treatment, although progress is needed to lessen the cytotoxic effects on human cells. According to the present study, thiazole derivatives have schistosomicidal activities and may be part of a possible new arsenal of compounds against schistosomiasis.

Acylation of the S413-PV cell-penetrating peptide as a means of enhancing its capacity to mediate nucleic acid delivery: Relevance of peptide/lipid interactions.

BACKGROUND: Cell-penetrating peptides (CPPs) have been extensively exploited in gene therapy approaches as vectors for intracellular delivery of bioactive molecules. The ability of CPPs to be internalized into cells and their capacity to complex nucleic acids depend on their molecular structure, both primary and secondary, namely regarding hydrophobicity/hydrophilicity. CPP acylation has been used as a strategy to improve this structural feature. METHODS: Acyl groups (from 6 to 18 carbon atoms) were attached to the S413-PV peptide and their effects on the peptide competence to complex siRNAs and to mediate gene silencing in glioblastoma (GBM) cells were studied. A systematic characterization of membrane interactions with S413-PV acyl-derivatives was also conducted, using different biophysical techniques (surface pressure-area isotherms in Langmuir monolayers, DSC and 31P NMR) to unravel a relationship between CPP biological activity and CPP effects on membrane stability and lipid organization. RESULTS: A remarkable concordance was noticed between acylated-S413-PV peptide competence to promote gene silencing in GBM cells and disturbance induced in membrane models, the lauroyl- and myristoyl-S413-PV peptides being the most effective. A cut-off effect was described for the first time regarding the influence of acyl-chain length on CPP bioactivity. CONCLUSIONS: C12-S413-PV showed high capacity to destabilize lipid bilayers, to escape from lysosomal degradation and to mediate gene silencing without promoting cytotoxicity. GENERAL SIGNIFICANCE: Besides unraveling a new CPP with high potential to be employed as a gene delivery vector, this work emphasizes the benefit from allying biophysical and biological studies towards a proper CPP structural refinement for successful pre-clinical/clinical application.

Multimodal highly fluorescent-magnetic nanoplatform to target transferrin receptors in cancer cells.

BACKGROUND: Site-specific multimodal nanoplatforms with fluorescent-magnetic properties have great potential for biological sciences. For this reason, we developed a multimodal nanoprobe (BNPs-Tf), by covalently conjugating an optical-magnetically active bimodal nanosystem, based on quantum dots and iron oxide nanoparticles, with the human holo-transferrin (Tf). METHODS: The Tf bioconjugation efficiency was evaluated by the fluorescence microplate assay (FMA) and the amount of Tf immobilized on BNPs was quantified by fluorescence spectroscopy. Moreover, relaxometric and fluorescent properties of the BNPs-Tf were evaluated, as well as its ability to label specifically HeLa cells. Cytotoxicity was also performed by Alamar Blue assay. RESULTS: The FMA confirmed an efficient bioconjugation and the fluorescence spectroscopy analysis indicated that 98% of Tf was immobilized on BNPs. BNPs-Tf also presented a bright fluorescence and a transversal/longitudinal relaxivities ratio (r2/r1) of 65. Importantly, the developed BNPs-Tf were able to label, efficiently and specifically, the Tf receptors in HeLa cells, as shown by fluorescence and magnetic resonance imaging assays. Moreover, this multimodal system did not cause noteworthy cytotoxicity. CONCLUSIONS: The prepared BNPs-Tf hold great promise as an effective and specific multimodal, highly fluorescent-magnetic, nanoplatform for fluorescence analyses and T2-weighted images. GENERAL SIGNIFICANCE: This study developed an attractive and versatile multimodal nanoplatform that has potential to be applied in a variety of in vitro and in vivo studies, addressing biological processes, diagnostic, and therapeutics. Moreover, this work opens new possibilities for designing other efficient multimodal nanosystems, considering other biomolecules in their composition able to provide them important functional properties.

High-throughput screening uncovers miRNAs enhancing glioblastoma cell susceptibility to tyrosine kinase inhibitors.

Glioblastoma (GBM) is a deadly and therapy resistant malignant brain tumour, characterized by an aggressive and diffuse growth pattern, which prevents complete surgical resection. Despite advances in the identification of genomic and molecular alterations that fuel the tumour, average patient survival post-diagnosis remains very low (∼14.6-months). In addition to being highly heterogeneous, GBM tumour cells exhibit high adaptive capacity to targeted molecular therapies owing to an established network of signalling cascades with functional redundancy, which provides them with robust compensatory survival mechanisms. Here, we investigated whether a multimodal strategy combining multitargeted tyrosine kinase inhibitors (MTKIs) and microRNA (miRNA) modulation could overcome the signalling pathway redundancy in GBM and, hence, promote tumour cell death. By performing a high-throughput screening, we identified a myriad of miRNAs, including those belonging to the miR-302-3p/372-3p/373-3p/520-3p family, which coordinately act with the MTKI sunitinib to decrease GBM cell viability. Two members of this family, hsa-miRNA-302a-3p and hsa-miRNA-520 b, were found to modulate the expression of receptor tyrosine kinase mediators (including AKT1, PIK3CA and SOS1) in U87 and DBTRG human GBM cells. Importantly, administration of mimics of these miRNAs with sunitinib or axitinib resulted in decreased tumour cell proliferation and enhanced cell death, whereas no significant effect was observed when coupling miRNA modulation with temozolomide, the first-line drug for GBM therapy. Overall, our results provide evidence that combining the 'horizontal' inhibition of signalling pathways promoted by MTKIs with the 'vertical' inhibition of the downstream signalling cascade promoted by hsa-miR-302a-3p and hsa-miR-520 b constitutes a promising approach towards GBM treatment.

Resveratrol stimulates the metabolic reprogramming of human CD4+ T cells to enhance effector function.

The polyphenol resveratrol activates the deacetylase Sirt1, resulting in various antioxidant, chemoprotectant, neuroprotective, cardioprotective, and anti-inflammatory properties. We found that at high concentrations of resveratrol, human CD4+ T cells showed defective antigen receptor signaling and arrest at the G1 stage of the cell cycle, whereas at low concentrations, cells were readily activated and exhibited enhanced Sirt1 deacetylase activity. Nevertheless, low-dose resveratrol rapidly stimulated genotoxic stress in the T cells, which resulted in engagement of a DNA damage response pathway that depended on the kinase ATR [ataxia telangiectasia-mutated (ATM) and Rad3-related], but not ATM, and subsequently in premitotic cell cycle arrest. The concomitant activation of p53 was coupled to the expression of gene products that regulate cell metabolism, leading to a metabolic reprogramming that was characterized by decreased glycolysis, increased glutamine consumption, and a shift to oxidative phosphorylation. These alterations in the bioenergetic homeostasis of CD4+ T cells resulted in enhanced effector function, with both naïve and memory CD4+ T cells secreting increased amounts of the inflammatory cytokine interferon-γ. Thus, our data highlight the wide range of metabolic adaptations that CD4+ T lymphocytes undergo in response to genomic stress.