A new model for regulation of sphingosine kinase 1 translocation to the plasma membrane in breast cancer cells.

The translocation of sphingosine kinase 1 (SK1) to the plasma membrane (PM) is crucial in promoting oncogenesis. We have previously proposed that SK1 exists as both a monomer and dimer in equilibrium, although it is unclear whether these species translocate to the PM via the same or different mechanisms. We therefore investigated the structural determinants involved to better understand how translocation might potentially be targeted for therapeutic intervention. We report here that monomeric WT mouse SK1 (GFP-mSK1) translocates to the PM of MCF-7L cells stimulated with carbachol or phorbol 12-myristate 13-acetate, whereas the dimer translocates to the PM in response to sphingosine-1-phosphate; thus, the equilibrium between the monomer and dimer is sensitive to cellular stimulus. In addition, carbachol and phorbol 12-myristate 13-acetate induced translocation of monomeric GFP-mSK1 to lamellipodia, whereas sphingosine-1-phosphate induced translocation of dimeric GFP-mSK1 to filopodia, suggesting that SK1 regulates different cell biological processes dependent on dimerization. GFP-mSK1 mutants designed to modulate dimerization confirmed this difference in localization. Regulation by the C-terminal tail of SK1 was investigated using GFP-mSK1 truncations. Removal of the last five amino acids (PPEEP) prevented translocation of the enzyme to the PM, whereas removal of the last ten amino acids restored translocation. This suggests that the penultimate five amino acids (SRRGP) function as a translocation brake, which can be released by sequestration of the PPEEP sequence. We propose that these determinants alter the arrangement of N-terminal and C-terminal domains in SK1, leading to unique surfaces that promote differential translocation to the PM.

Proof of concept studies for siRNA delivery by nonionic surfactant vesicles: in vitro and in vivo evaluation of protein knockdown.

RNA interference is an effective and naturally occurring post-transcriptional gene regulatory mechanism. This mechanism involves the degradation of a target messenger RNA (mRNA) through the introduction of short interfering RNA (siRNA) that is complementary to the target mRNA. The application of siRNA-based therapeutics is limited by the development of an effective delivery system, as naked siRNA is unstable and cannot penetrate the cell membrane. In this study, we investigated the use of cationic niosomes (CN) prepared by microfluidic mixing for siRNA delivery. In an in vitro model, these vesicles were able to deliver anti-luciferase siRNA and effectively suppress luciferase expression in B16-F10 mouse melanoma cells. More importantly, in an in vivo mouse model, intratumoral administration of CN-carrying anti-luciferase siRNA led to significant suppression of luciferase expression compared with naked siRNA. Thus, we have established a novel and effective system for the delivery of siRNA both in vitro and in vivo, which shows high potential for future application of gene therapeutics.

Whole Genome Microarray Analysis of DUSP4-Deletion Reveals A Novel Role for MAP Kinase Phosphatase-2 (MKP-2) in Macrophage Gene Expression and Function.

BACKGROUND: Mitogen-activated protein kinase phosphatase-2 (MKP-2) is a type 1 nuclear dual specific phosphatase (DUSP-4). It plays an important role in macrophage inflammatory responses through the negative regulation of Mitogen activated protein kinase (MAPK) signalling. However, information on the effect of MKP-2 on other aspect of macrophage function is limited. METHODS: We investigated the impact of MKP-2 in the regulation of several genes that are involved in function while using comparative whole genome microarray analysis in macrophages from MKP-2 wild type (wt) and knock out (ko) mice. RESULTS: Our data showed that the lack of MKP-2 caused a significant down-regulation of colony-stimulating factor-2 (Csf2) and monocyte to macrophage-associated differentiation (Mmd) genes, suggesting a role of MKP-2 in macrophage development. When treated with macrophage colony stimulating factor (M-CSF), Mmd and Csf2 mRNA levels increased but significantly reduced in ko cells in comparison to wt counterparts. This effect of MKP-2 deletion on macrophage function was also observed by cell counting and DNA measurements. On the signalling level, M-CSF stimulation induced extracellular signal-regulated kinases (ERK) phosphorylation, which was significantly enhanced in the absence of MKP-2. Pharmacological inhibition of ERK reduced both Csf2 and Mmd genes in both wild type and ko cultures, which suggested that enhanced ERK activation in ko cultures may not explain effects on gene expression. Interestingly other functional markers were also shown to be reduced in ko macrophages in comparison to wt mice; the expression of CD115, which is a receptor for M-CSF, and CD34, a stem/progenitor cell marker, suggesting global regulation of gene expression by MKP-2. CONCLUSIONS: Transcriptome profiling reveals that MKP-2 regulates macrophage development showing candidate targets from monocyte-to-macrophage differentiation and macrophage proliferation. However, it is unclear whether effects upon ERK signalling are able to explain the effects of DUSP-4 deletion on macrophage function.

Formulation of Nonionic Surfactant Vesicles (NISV) Prepared by Microfluidics for Therapeutic Delivery of siRNA into Cancer Cells.

Small interfering RNAs (siRNA) have a broad potential as therapeutic agents to reversibly silence any target gene of interest. The clinical application of siRNA requires the use of safe and effective delivery systems. In this study, we investigated the use of nonionic surfactant vesicles (NISV) for the delivery of siRNA. Different types of NISV formulations were synthesized by microfluidic mixing and then evaluated for their physiochemical properties and cytotoxicity. The ability of the NISV to carry and transfect siRNA targeting green fluorescent protein (GFP) into A549 that stably express GFP (copGFP-A549) was evaluated. Flow cytometry and Western blotting were used to study the GFP expression knockdown, and significant knockdown was observed as a result of siRNA delivery to the cells by NISV. This occurred in particular when using Tween 85, which was able to achieve more than 70% GFP knockdown. NISV were thus demonstrated to provide a promising and effective platform for therapeutic delivery of siRNA.

Metabolomic Tools to Assess the Chemistry and Bioactivity of Endophytic Aspergillus Strain.

Endophytic fungi associated with medicinal plants are a potential source of novel chemistry and biology that may find applications as pharmaceutical and agrochemical drugs. In this study, a combination of metabolomics and bioactivity-guided approaches were employed to isolate secondary metabolites with cytotoxicity against cancer cells from an endophytic Aspergillus aculeatus. The endophyte was isolated from the Egyptian medicinal plant Terminalia laxiflora and identified using molecular biological methods. Metabolomics and dereplication studies were accomplished by utilizing the MZmine software coupled with the universal Dictionary of Natural Products database. Metabolic profiling, with aid of multivariate data analysis, was performed at different stages of the growth curve to choose the optimized method suitable for up-scaling. The optimized culture method yielded a crude extract abundant with biologically-active secondary metabolites. Crude extracts were fractionated using different high-throughput chromatographic techniques. Purified compounds were identified by HR-ESI-MS, 1D- and 2D-NMR. This study introduced a new method of dereplication utilizing both high-resolution mass spectrometry and NMR spectroscopy. The metabolites were putatively identified by applying a chemotaxonomic filter. We also present a short review on the diverse chemistry of terrestrial endophytic strains of Aspergillus, which has become a part of our dereplication work and this will be of wide interest to those working in this field.

Tumor regression following intravenous administration of lactoferrin- and lactoferricin-bearing dendriplexes.

The possibility of using gene therapy for the treatment of cancer is limited by the lack of safe, intravenously administered delivery systems able to selectively deliver therapeutic genes to tumors. In this study, we investigated if the conjugation of the polypropylenimine dendrimer to lactoferrin and lactoferricin, whose receptors are overexpressed on cancer cells, could result in a selective gene delivery to tumors and a subsequently enhanced therapeutic efficacy. The conjugation of lactoferrin and lactoferricin to the dendrimer significantly increased the gene expression in the tumor while decreasing the non-specific gene expression in the liver. Consequently, the intravenous administration of the targeted dendriplexes encoding TNFα led to the complete suppression of 60% of A431 tumors and up to 50% of B16-F10 tumors over one month. The treatment was well tolerated by the animals. These results suggest that these novel lactoferrin- and lactoferricin-bearing dendrimers are promising gene delivery systems for cancer therapy. FROM THE CLINICAL EDITOR: Specific targeting of cancer cells should enhance the delivery of chemotherapeutic agents. This is especially true for gene delivery. In this article, the authors utilized a dendrimer-based system and conjugated this with lactoferrin and lactoferricin to deliver anti-tumor genes. The positive findings in animal studies should provide the basis for further clinical studies.

CoCr wear particles generated from CoCr alloy metal-on-metal hip replacements, and cobalt ions stimulate apoptosis and expression of general toxicology-related genes in monocyte-like U937 cells.

Cobalt-chromium (CoCr) particles in the nanometre size range and their concomitant release of Co and Cr ions into the patients' circulation are produced by wear at the articulating surfaces of metal-on-metal (MoM) implants. This process is associated with inflammation, bone loss and implant loosening and led to the withdrawal from the market of the DePuy ASR™ MoM hip replacements in 2010. Ions released from CoCr particles derived from a resurfacing implant in vitro and their subsequent cellular up-take were measured by ICP-MS. Moreover, the ability of such metal debris and Co ions to induce both apoptosis was evaluated with both FACS and immunoblotting. qRT-PCR was used to assess the effects on the expression of lymphotoxin alpha (LTA), BCL2-associated athanogene (BAG1), nitric oxide synthase 2 inducible (NOS2), FBJ murine osteosarcoma viral oncogene homolog (FOS), growth arrest and DNA-damage-inducible alpha (GADD45A). ICP-MS showed that the wear debris released significant (p<0.05) amounts of Co and Cr ions into the culture medium, and significant (p<0.05) cellular uptake of both ions. There was also an increase (p<0.05) in apoptosis after a 48h exposure to wear debris. Analysis of qRT-PCR results found significant up-regulation (p<0.05) particularly of NOS2 and BAG1 in Co pre-treated cells which were subsequently exposed to Co ions+debris. Metal debris was more effective as an inducer of apoptosis and gene expression when cells had been pre-treated with Co ions. This suggests that if a patient receives sequential bilateral CoCr implants, the second implant may be more likely to produce adverse effects than the first one.

Kisspeptin a potential therapeutic target in treatment of both metabolic and reproductive dysfunction.

Kisspeptins (KPs) are proteins that were first recognized to have antimetastatic action. Later, the critical role of this peptide in the regulation of reproduction was proved. In recent years, evidence has been accumulated supporting a role for KPs in regulating metabolic processes in a sexual dimorphic manner. It has been proposed that KPs regulate metabolism both indirectly via gonadal hormones and/or directly via the kisspeptin receptor in the brain, brown adipose tissue, and pancreas. The aim of the review is to provide both experimental and clinical evidence indicating that KPs are peptides linking metabolism and reproduction. We propose that KPs could be used as a potential target to treat both metabolic and reproductive abnormalities. Thus, we focus on the consequences of disruptions in KPs and their receptors in metabolic conditions such as diabetes, undernutrition, obesity, and reproductive disorders (hypogonadotropic hypogonadism and polycystic ovary syndrome). Data from both animal models and human subjects indicate that alterations in KPs in the case of metabolic imbalance lead also to disruptions in reproductive functions. Changes both in the hypothalamic and peripheral KP systems in animal models of the aforementioned disorders are discussed. Finally, an overview of current clinical studies involving KP in fertility and metabolism show fewer studies on metabolism (15%) and only one to date on both. Presented data indicate a dynamic and emerging field of KP studies as possible therapeutic targets in treatments of both reproductive and metabolic dysfunctions.

Cardiac function may be compromised in patients with elevated blood cobalt levels secondary to metal-on-metal hip implants.

Aims: Elevated blood cobalt levels secondary to metal-on-metal (MoM) hip arthroplasties are a suggested risk factor for developing cardiovascular complications including cardiomyopathy. Clinical studies assessing patients with MoM hips using left ventricular ejection fraction (LVEF) have found conflicting evidence of cobalt-induced cardiomyopathy. Global longitudinal strain (GLS) is an echocardiography measurement known to be more sensitive than LVEF when diagnosing early cardiomyopathies. The extent of cardiovascular injury, as measured by GLS, in patients with elevated blood cobalt levels has not previously been examined. Methods: A total of 16 patients with documented blood cobalt ion levels above 13 µg/l (13 ppb, 221 nmol/l) were identified from a regional arthroplasty database. They were matched with eight patients awaiting hip arthroplasty. All patients underwent echocardiography, including GLS, investigating potential signs of cardiomyopathy. Results: Patients with MoM hip arthroplasties had a mean blood cobalt level of 29 µg/l (495 nmol/l) compared to 0.01 µg/l (0.2 nmol/l) in the control group. GLS readings were available for seven of the MoM cohort, and were significantly lower when compared with controls (-15.5% vs -18% (MoM vs control); p = 0.025)). Pearson correlation demonstrated that GLS significantly correlated with blood cobalt level (r = 0.8521; p < 0.001). However, there were no differences or correlations for other echocardiography measurements, including LVEF (64.3% vs 63.7% (MoM vs control); p = 0.845). Conclusion: This study supports the hypothesis that patients with elevated blood cobalt levels above 13 µg/l in the presence of a MoM hip implant may have impaired cardiac function compared to a control group of patients awaiting hip arthroplasty. It is the first study to use the more sensitive parameter of GLS to assess for any cardiac contractile dysfunction in patients with a MoM hip implant and a normal LVEF. Larger studies should be performed to determine the potential of GLS as a predictor of cardiac complications in patients with MoM arthroplasties.

Transferrin-Bearing, Zein-Based Hybrid Lipid Nanoparticles for Drug and Gene Delivery to Prostate Cancer Cells.

Gene therapy holds great promise for treating prostate cancer unresponsive to conventional therapies. However, the lack of delivery systems that can transport therapeutic DNA and drugs while targeting tumors without harming healthy tissues presents a significant challenge. This study aimed to explore the potential of novel hybrid lipid nanoparticles, composed of biocompatible zein and conjugated to the cancer-targeting ligand transferrin. These nanoparticles were designed to entrap the anti-cancer drug docetaxel and carry plasmid DNA, with the objective of improving the delivery of therapeutic payloads to prostate cancer cells, thereby enhancing their anti-proliferative efficacy and gene expression levels. These transferrin-bearing, zein-based hybrid lipid nanoparticles efficiently entrapped docetaxel, leading to increased uptake by PC-3 and LNCaP cancer cells and significantly enhancing anti-proliferative efficacy at docetaxel concentrations exceeding 1 µg/mL. Furthermore, they demonstrated proficient DNA condensation, exceeding 80% at polymer-DNA weight ratios of 1500:1 and 2000:1. This resulted in increased gene expression across all tested cell lines, with the highest transfection levels up to 11-fold higher than those observed with controls, in LNCaP cells. These novel transferrin-bearing, zein-based hybrid lipid nanoparticles therefore exhibit promising potential as drug and gene delivery systems for prostate cancer therapy.

Cobalt Neurotoxicity: Transcriptional Effect of Elevated Cobalt Blood Levels in the Rodent Brain.

Metal-on-metal (MoM) hip implants made of cobalt chromium (CoCr) alloy have shown early failure compared with other bearing materials. A consequence of the abnormal wear produced by these prostheses is elevated levels of cobalt in the blood of patients, which can lead to systemic conditions involving cardiac and neurological symptoms. In order to better understand the implications for patients with these implants, we carried out metal content and RNA-Seq analysis of excised tissue from rats treated intraperitonially for 28 days with low concentrations of cobalt. Cobalt blood levels in dosed rats were found to be similar to those seen in some patients with MoM implants (range: 4-38 µg/L Co in blood). Significant accumulation of cobalt was measured in a range of tissues including kidney, liver, and heart, but also in brain tissue. RNA-Seq analysis of neural tissue revealed that exposure to cobalt induces a transcriptional response in the prefrontal cortex (pref. cortex), cerebellum, and hippocampus. Many of the most up- and downregulated genes appear to correspond to choroid plexus transcripts. These results indicate that the choroid plexus could be the brain tissue most affected by cobalt. More specifically, the differentially expressed genes show a disruption of steroidogenesis and lipid metabolism. Several other transcripts also demonstrate that cobalt induces an immune response. In summary, cobalt exposure induces alterations in the brain transcriptome, more specifically, the choroid plexus, which is in direct contact with neurotoxicants at the blood-cerebrospinal fluid barrier.

Lactoferrin- and Dendrimer-Bearing Gold Nanocages for Stimulus-Free DNA Delivery to Prostate Cancer Cells.

Background: The use of gene therapy to treat prostate cancer is hampered by the lack of effective nanocarriers that can selectively deliver therapeutic genes to cancer cells. To overcome this, we hypothesize that conjugating lactoferrin, a tumor-targeting ligand, and the diaminobutyric polypropylenimine dendrimer into gold nanocages, followed by complexation with a plasmid DNA, would enhance gene expression and anti-proliferation activity in prostate cancer cells without the use of external stimuli. Methods: Novel gold nanocages bearing lactoferrin and conjugated to diaminobutyric polypropylenimine dendrimer (AuNCs-DAB-Lf) were synthesized and characterized. Following complexation with a plasmid DNA, their gene expression, cellular uptake and anti-proliferative efficacies were evaluated on PC-3 prostate cancer cells. Results: AuNCs-DAB-Lf was able to complex DNA at conjugate: DNA weight ratios 5:1 onwards. Gene expression was at its highest after treatment with AuNCs-DAB-Lf at a weight ratio of 10:1, as a result of a significant increase in DNA uptake mediated by the conjugate at that ratio in PC-3 cells. Consequently, the anti-proliferative activity of AuNCs-DAB-Lf-DNA encoding TNFα was significantly improved by up to 9-fold compared with DAB dendriplex encoding TNFα. Conclusion: Lactoferrin-bearing dendrimer-conjugated gold nanocages are highly promising gene delivery systems for the treatment of prostate cancer.

Targeting 17ß-estradiol biosynthesis in neural stem cells improves stroke outcome.

Dax-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital region on X-chromosome gene 1) blocks 17ß-estradiol biosynthesis and its knockdown would be expected to increase 17ß-estradiol production. We hypothesized that knockdown of Dax-1 in a conditionally immortalized neural stem cell (NSC) line, MHP36, is a useful approach to increase 17ß-estradiol production. Short hairpin (sh) RNA targeted to Dax-1 in NSCs, namely MHP36-Dax1KD cells, resulted in the degradation of Dax-1 RNA and attenuation of Dax-1 protein expression. In vitro, MHP36-Dax1KD cells exhibited overexpression of aromatase and increased 17ß-estradiol secretion compared to MHP36 cells. As 17ß-estradiol has been shown to promote the efficacy of cell therapy, we interrogated the application of 17ß-estradiol-enriched NSCs in a relevant in vivo disease model. We hypothesized that MHP36-Dax1KD cells will enhance functional recovery after transplantation in a stroke model. C57BL/6 male adult mice underwent ischemia/reperfusion by left middle cerebral artery occlusion for 45 min using an intraluminal thread. Two days later male mice randomly received vehicle, MHP36 cells, MHP36-Dax1KD cells, and MHP36 cells suspended in 17ß-estradiol (100 nm) or 17ß-estradiol alone (100 nm) with serial behavioral testing over 28 days followed by post-mortem histology and blinded analysis. Recovery of sensorimotor function was accelerated and enhanced, and lesion volume was reduced by MHP36-Dax1KD transplants. Regarding mechanisms, immunofluorescence indicated increased synaptic plasticity and neuronal differentiation after MHP36-Dax1KD transplants. In conclusion, knockdown of Dax-1 is a useful target to increase 17ß-estradiol biosynthesis in NSCs and improves functional recovery after stroke in vivo, possibly mediated through neuroprotection and improved synaptic plasticity. Therefore, targeting 17ß-estradiol biosynthesis in stem cells may be a promising therapeutic strategy for enhancing the efficacy of stem cell-based therapies for stroke.

The sphingosine kinase 1 inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole induces proteasomal degradation of sphingosine kinase 1 in mammalian cells.

Sphingosine kinase 1 (SK1) is an enzyme that catalyzes the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that the SK1 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) induces the proteasomal degradation of SK1 in human pulmonary artery smooth muscle cells, androgen-sensitive LNCaP prostate cancer cells, MCF-7 and MCF-7 HER2 breast cancer cells and that this is likely mediated by ceramide as a consequence of catalytic inhibition of SK1 by SKi. Moreover, SK1 is polyubiquitinated under basal conditions, and SKi appears to increase the degradation of SK1 by activating the proteasome. In addition, the proteasomal degradation of SK1a and SK1b in androgen-sensitive LNCaP cells is associated with the induction of apoptosis. However, SK1b in LNCaP-AI cells (androgen-independent) is less sensitive to SKi-induced proteasomal degradation and these cells are resistant to SKi-induced apoptosis, thereby implicating the ubiquitin-proteasomal degradation of SK1 as an important mechanism controlling cell survival.

Enhanced gene expression in tumors after intravenous administration of arginine-, lysine- and leucine-bearing polyethylenimine polyplex.

The potential of gene therapy to treat cancer is currently limited by the low expression of therapeutic genes in the tumors. Because amino acids are known to have excellent properties in cell penetration and gene expression regulation, we investigated if the conjugation of arginine (Arg), lysine (Lys) and leucine (Leu) onto the surface of the gene delivery system polyethylenimine (PEI) could lead to an improved gene expression in tumors. The intravenous administration of Arg-, Lys- and Leu-bearing PEI polyplexes led to a significant increase of gene expression in the tumor, with a ß-galactosidase expression amount at least threefold higher than that obtained after treatment with unmodified PEI polyplex. The three amino acid-bearing PEI polyplexes led to similar levels of gene expression in the tumor. The treatments were well tolerated by the mice. Arg-, Lys- and Leu-bearing PEI polyplexes are therefore highly promising gene delivery systems for cancer therapy.

Lactoferrin-Bearing Gold Nanocages for Gene Delivery in Prostate Cancer Cells in vitro.

BACKGROUND: Gold nanocages have been widely used as multifunctional platforms for drug and gene delivery, as well as photothermal agents for cancer therapy. However, their potential as gene delivery systems for cancer treatment has been reported in combination with chemotherapeutics and photothermal therapy, but not in isolation so far. The purpose of this work was to investigate whether the conjugation of gold nanocages with the cancer targeting ligand lactoferrin, polyethylene glycol and polyethylenimine could lead to enhanced transfection efficiency on prostate cancer cells in vitro, without assistance of external stimulation. METHODS: Novel lactoferrin-bearing gold nanocages conjugated to polyethylenimine and polyethylene glycol have been synthesized and characterized. Their transfection efficacy and cytotoxicity were assessed on PC-3 prostate cancer cell line following complexation with a plasmid DNA. RESULTS: Lactoferrin-bearing gold nanocages, alone or conjugated with polyethylenimine and polyethylene glycol, were able to condense DNA at conjugate:DNA weight ratios 5:1 and higher. Among all gold conjugates, the highest gene expression was obtained following treatment with gold complex conjugated with polyethylenimine and lactoferrin, at weight ratio 40:1, which was 1.71-fold higher than with polyethylenimine. This might be due to the increased DNA cellular uptake observed with this conjugate, by up to 8.65-fold in comparison with naked DNA. CONCLUSION: Lactoferrin-bearing gold nanocages conjugates are highly promising gene delivery systems to prostate cancer cells.

Octadecyl chain-bearing PEGylated poly(propyleneimine)-based dendrimersomes: physicochemical studies, redox-responsiveness, DNA condensation, cytotoxicity and gene delivery to cancer cells.

Stimuli-responsive nanocarriers have become increasingly important for nucleic acid and drug delivery in cancer therapy. Here, we report the synthesis, characterization and evaluation of disulphide-linked, octadecyl (C18 alkyl) chain-bearing PEGylated generation 3-diaminobutyric polypropylenimine dendrimer-based vesicles (or dendrimersomes) for gene delivery. The lipid-bearing PEGylated dendrimer was successfully synthesized through in situ two-step reaction. It was able to spontaneously self-assemble into stable, cationic, nanosized vesicles, with low critical aggregation concentration value, and also showed redox-responsiveness in presence of a glutathione concentration similar to that of the cytosolic reducing environment. In addition, it was able to condense more than 70% of DNA at dendrimer: DNA weight ratios of 5 : 1 and higher. This dendriplex resulted in an enhanced cellular uptake of DNA at dendrimer: DNA weight ratios of 10 : 1 and 20 : 1, by up to 16-fold and by up to 28-fold compared with naked DNA in PC-3 and DU145 prostate cancer cell lines respectively. At a dendrimer: DNA weight ratio of 20 : 1, it led to an increase in gene expression in PC-3 and DU145 cells, compared with DAB dendriplex. These octadecyl chain-bearing, PEGylated dendrimer-based vesicles are therefore promising redox-sensitive drug and gene delivery systems for potential applications in combination cancer therapy.

Metabolomics-Guided Isolation of Anti-Trypanosomal Compounds from Endophytic Fungi of the Mangrove plant Avicennia Lanata.

Endophytic fungi have been explored not just for their ecological functions but also for their secondary metabolites as a new source of these pharmacologically active natural products. Accordingly, many structurally unique and biologically active compounds have been obtained from the cultures of endophytic fungi. Fusarium sp. and Lasiodiplodia theobromae were isolated from the root and stem of the mangrove plant Avicennia lanata, respectively, collected from Terengganu, Malaysia. High-resolution mass spectrometry and NMR spectroscopy were used as metabolomics profiling tools to identify and optimize the production of bioactive secondary metabolites in both strains at different growth stages and culture media. The spectral data was processed by utilizing Mzmine 2, a quantitative expression analysis software and an in house MS-Excel macro coupled with the Dictionary of Natural Products databases for dereplication studies. The investigation for the potential bioactive metabolites from a 15-day rice culture of Fusarium sp. yielded four 1,4- naphthoquinone with naphthazarin structures (1-4). On the other hand, the endophytic fungus L. theobromae grown on the 15-day solid rice culture produced dihydroisocoumarins (5-8). All the isolated compounds (1-8) showed significant activity against Trypanosoma brucei brucei with MIC values of 0.32-12.5 µM. Preliminary cytotoxicity screening against normal prostate cells (PNT2A) was also performed. All compounds exhibited low cytotoxicity, with compounds 3 and 4 showing the lowest cytotoxicity of only 22.3% and 38.6% of the control values at 100 µg/mL, respectively. Structure elucidation of the isolated secondary metabolites was achieved using 2D-NMR and HRESI-MS as well as comparison with literature data.