Safety and pharmacodynamic effects of a pharmacological chaperone on α-galactosidase A activity and globotriaosylceramide clearance in Fabry disease: report from two phase 2 clinical studies

Background

Fabry disease (FD) is a genetic disorder resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A), which leads to globotriaosylceramide (GL-3) accumulation in multiple tissues. We report on the safety and pharmacodynamics of migalastat hydrochloride, an investigational pharmacological chaperone given orally at 150 mg every-other-day.

Methods

Two open-label uncontrolled phase 2 studies of 12 and 24 weeks (NCT00283959 and NCT00283933) in 9 males with FD were combined. At multiple time points, α-Gal A activity and GL-3 levels were quantified in blood cells, kidney and skin. GL-3 levels were also evaluated through skin and renal histology.

Results

Compared to baseline, increased α-Gal A activity of at least 50% was demonstrated in blood, skin and kidney in 6 of 9 patients. Patients’ increased α-Gal A activities paralleled the α-Gal A increases observed in vitro in HEK-293 cells transfected with the corresponding mutant form of the enzyme. The same 6 patients who demonstrated increases of α-Gal A activity also had GL-3 reduction in skin, urine and/or kidney, and had α-Gal A mutations that responded in transfected cells incubated with the drug. The 3 patients who did not show a consistent response in vivo had α-Gal A mutations that did not respond to migalastat HCl in transfected cells. Migalastat HCl was well tolerated.

Conclusions

Migalastat HCl is a candidate pharmacological chaperone that provides a novel genotype-specific treatment for FD. It enhanced α-Gal A activity and resulted in GL-3 substrate decrease in patients with responsive GLA mutations. Phase 3 studies are ongoing.

Trial registration

Clinicaltrial.gov: NCT00283959 and NCT00283933

     

Genetic improvement of <Emphasis Type="Italic">Magnetospirillum gryphiswaldense</Emphasis> for enhanced biological removal of phosphate

Objectives

To improve its phosphate accumulating abilities for phosphate recycling from wastewater, a magnetotactic bacterium, Magnetospirillum gryphiswaldense, was genetically modified to over-express polyphosphate kinase.

Results

Polyphosphate kinase was over-expressed in the bacterium. The recombinant strain accumulated ninefold more polyphosphate from synthetic wastewater compared to original wild type. The magnetic property of the recombinant M. gryphiswaldense strain was retained.

Conclusions

The recombinant M. gryphiswaldense can be used for phosphate removal and recovery in bioremediation.

     

Imprinted and ancient gene: a potential mediator of cancer cell survival during tryptophan deprivation

Background

Depletion of tryptophan and the accumulation of tryptophan metabolites mediated by the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1), trigger immune cells to undergo apoptosis. However, cancer cells in the same microenvironment appear not to be affected. Mechanisms whereby cancer cells resist accelerated tryptophan degradation are not completely understood. We hypothesize that cancer cells co-opt IMPACT (the product of IMPrinted and AnCienT gene), to withstand periods of tryptophan deficiency.

Methods

A range of bioinformatic techniques including correlation and gene set variation analyses was applied to genomic datasets of cancer (The Cancer Genome Atlas) and normal (Genotype Tissue Expression Project) tissues to investigate IMPACT’s role in cancer. Survival of IMPACT-overexpressing GL261 glioma cells and their wild type counterparts cultured in low tryptophan media was assessed using fluorescence microscopy and MTT bio-reduction assay. Expression of the Integrated Stress Response proteins was measured using Western blotting.

Results

We found IMPACT to be upregulated and frequently amplified in a broad range of clinical cancers relative to their non-malignant tissue counterparts. In a subset of clinical cancers, high IMPACT expression associated with decreased activity of pathways and genes involved in stress response and with increased activity of translational regulation such as the mTOR pathway. Experimental studies using the GL261 glioma line showed that cells engineered to overexpress IMPACT, gained a survival advantage over wild-type lines when cultured under limiting tryptophan concentrations. No significant difference in the expression of proteins in the Integrated Stress Response pathway was detected in tryptophan-deprived GL261 IMPACT-overexpressors compared to that in wild-type cells. IMPACT-overexpressing GL261 cells but not their wild-type counterparts, showed marked enlargement of their nuclei and cytoplasmic area when stressed by tryptophan deprivation.

Conclusions

The bioinformatics data together with our laboratory studies, support the hypothesis that IMPACT mediates a protective mechanism allowing cancer cells to overcome microenvironmental stresses such as tryptophan deficiency.

     

Hyperthermic treatment of DMBA-induced rat mammary cancer using magnetic nanoparticles

Background

We have developed magnetite cationic liposomes (MCLs) and applied them as a mediator of local hyperthermia. MCLs can generate heat under an alternating magnetic field (AMF). In this study, the in vivo effect of hyperthermia mediated by MCLs was examined using 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary cancer as a spontaneous cancer model.

Method

MCLs were injected into the mammary cancer and then subjected to an AMF.

Results

Four rats in 20 developed mammary tumors at more than 1 site in the body. The first-developed tumor in each of these 4 rats was selected and heated to over 43°C following administration of MCLs by an infusion pump. After a series of 3 hyperthermia treatments, treated tumors in 3 of the 4 rats were well controlled over a 30-day observation period. One of the 4 rats exhibited regrowth after 2 weeks. In this rat, there were 3 sites of tumor regrowth. Two of these regrowths were reduced in volume and regressed completely after 31 days, although the remaining one grew rapidly. These results indicated hyperthermia-induced immunological antitumor activity mediated by the MCLs.

Conclusion

Our results suggest that hyperthermic treatment using MCLs is effective in a spontaneous cancer model.

     

Size dependent heat generation of magnetite nanoparticles under AC magnetic field for cancer therapy

Background

We have developed magnetic cationic liposomes (MCLs) that contained magnetic nanoparticles as heating mediator for applying them to local hyperthermia. The heating performance of the MCLs is significantly affected by the property of the incorporated magnetite nanoparticles. We estimated heating capacity of magnetite nanoparticles by measuring its specific absorption rate (SAR) against irradiation of the alternating magnetic field (AMF).

Method

Magnetite nanoparticles which have various specific-surface-area (SSA) are dispersed in the sample tubes, subjected to various AMF and studied SAR.

Result

Heat generation of magnetite particles under variable AMF conditions was summarized by the SSA. There were two maximum SAR values locally between 12 m²/g to 190 m²/g of the SSA in all ranges of applied AMF frequency and those values increased followed by the intensity of AMF power. One of the maximum values was observed at approximately 90 m²/g of the SSA particles and the other was observed at approximately 120 m²/g of the SSA particles. A boundary value of the SAR for heat generation was observed around 110 m²/g of SSA particles and the effects of the AMF power were different on both hand. Smaller SSA particles showed strong correlation of the SAR value to the intensity of the AMF power though larger SSA particles showed weaker correlation.

Conclusion

Those results suggest that two maximum SAR value stand for the heating mechanism of magnetite nanoparticles represented by hysteresis loss and relaxation loss.

     

Improving the power generation of microbial fuel cells by modifying the anode with single-wall carbon nanohorns

Objectives

To increase the power generation of microbial fuel cells (MFCs), anode modification with carbon materials (activated carbon, carbon nanotubes, and carbon nanohorns) was investigated.

Results

Maximum power densities of a stainless-steel anode MFC with a non-modified electrode (SS-MFC), an activated carbon-modified electrode (AC-MFC), a carbon nanotube-modified electrode (CNT-MFC) and a carbon nanohorn-modified electrode (CNH-MFC) were 72, 244, 261 and 327 mW m^?2, respectively. The total polarization resistance measured by electrochemical impedance spectroscopy were 3610 Ω for SS-MFC, 283 Ω for AC-MFC, 231 Ω for CNTs-MFC, and 136 Ω for CNHs-MFC, consistent with the anode resistances obtained by fitting the anode polarization curves.

Conclusions

Single-wall carbon nanohorns are better than activated carbon and carbon nanotubes as a new anode modification material for improving anode performance.

     

Crosstalk between glial and glioblastoma cells triggers the “go-or-grow” phenotype of tumor cells

Background

Glioblastoma (GBM), the most malignant primary brain tumor, leads to poor and unpredictable clinical outcomes. Recent studies showed the tumor microenvironment has a critical role in regulating tumor growth by establishing a complex network of interactions with tumor cells. In this context, we investigated how GBM cells modulate resident glial cells, particularly their paracrine activity, and how this modulation can influence back on the malignant phenotype of GBM cells.

Methods

Conditioned media (CM) of primary mouse glial cultures unexposed (unprimed) or exposed (primed) to the secretome of GL261 GBM cells were analyzed by proteomic analysis. Additionally, these CM were used in GBM cells to evaluate their impact in glioma cell viability, migration capacity and activation of tumor-related intracellular pathways.

Results

The proteomic analysis revealed that the pre-exposure of glial cells to CM from GBM cells led to the upregulation of several proteins related to inflammatory response, cell adhesion and extracellular structure organization within the secretome of primed glial cells. At the functional levels, CM derived from unprimed glial cells favored an increase in GBM cell migration capacity, while CM from primed glial cells promoted cells viability. These effects on GBM cells were accompanied by activation of particular intracellular cancer-related pathways, mainly the MAPK/ERK pathway, which is a known regulator of cell proliferation.

Conclusions

Together, our results suggest that glial cells can impact on the pathophysiology of GBM tumors, and that the secretome of GBM cells is able to modulate the secretome of neighboring glial cells, in a way that regulates the “go-or-grow” phenotypic switch of GBM cells.

     

Untargeted and stable isotope-assisted metabolomic analysis of MDA-MB-231 cells under hypoxia

Introduction

Hypoxia commonly occurs in cancers and is highly related with the occurrence, development and metastasis of cancer. Treatment of triple negative breast cancer remains challenge. Knowledge about the metabolic status of triple negative breast cancer cell lines in hypoxia is valuable for the understanding of molecular mechanisms of this tumor subtype to develop effective therapeutics.

Objectives

Comprehensively characterize the metabolic profiles of triple negative breast cancer cell line MDA-MB-231 in normoxia and hypoxia and the pathways involved in metabolic changes in hypoxia.

Methods

Differences in metabolic profiles affected pathways of MDA-MB-231 cells in normoxia and hypoxia were characterized using GC–MS based untargeted and stable isotope assisted metabolomic techniques.

Results

Thirty-three metabolites were significantly changed in hypoxia and nine pathways were involved. Hypoxia increased glycolysis, inhibited TCA cycle, pentose phosphate pathway and pyruvate carboxylation, while increased glutaminolysis in MDA-MB-231 cells.

Conclusion

The current results provide metabolic differences of MDA-MB-231 cells in normoxia and hypoxia conditions as well as the involved metabolic pathways, demonstrating the power of combined use of untargeted and stable isotope-assisted metabolomic methods in comprehensive metabolomic analysis.

     

The function and magnetic resonance imaging of immature dendritic cells under ultrasmall superparamagnetic iron oxide (USPIO)-labeling

Objective

To investigate the effects of ultrasmall superparamagnetic iron oxide (USPIO) labeling on the maturity or immune tolerance of immature dendritic cells (imDCs) as the success of immunotherapy with immature dendritic cells is highly dependent on immune tolerance.

Results

The feasibility of tracking implanted USPIO-labeled imDCs in vivo by magnetic resonance imaging (MRI) was explored. The effects of USPIO labeling on the immune tolerance of imDCs was examined. USPIO when higher than 200 μg/ml caused considerable damage to imDCs, induced imDC maturation, and impacted the immune tolerance of imDCs. USPIO labeling caused a dose-dependent increase in autophagosome formation in imDCs, and autophagy inhibitors prevented the maturation of imDCs while stimulating their immune tolerance.

Conclusions

We speculate that high concentrations of USPIO can be used to induce imDC maturation, and that this process is likely mediated through an autophagy-related pathway.

     

Collagen and fibronectin surface modification of nanoporous anodic alumina and macroporous silicon for endothelial cell cultures

Background

The ability to direct the cellular response by means of biomaterial surface topography is important for biomedical applications. Substrate surface topography has been shown to be an effective cue for the regulation of cellular response. Here, the response of human aortic endothelial cells to nanoporous anodic alumina and macroporous silicon with collagen and fibronectin functionalization has been studied.

Methods

Confocal microscopy and scanning electron microscopy were employed to analyse the effects of the material and the porosity on the adhesion, morphology, and proliferation of the cells. Cell spreading and filopodia formation on macro- and nanoporous material was characterized by atomic force microscopy. We have also studied the influence of the protein on the adhesion.

Results

It was obtained the best results when the material is functionalized with fibronectin, regarding cells adhesion, morphology, and proliferation.

Conclusion

These results permit to obtain chemical modified 3D structures for several biotechnology applications such as tissue engineering, organ-on-chip or regenerative medicine.

     

Bacterial magnetosomes as an efficient gene delivery platform for cancer theranostics

Background

Gene therapy has gained an increasing interest in its anti-tumor efficiency. However, numerous efforts are required to promote them to clinics. In this study, a novel and efficient delivery platform based on bacterial magnetosomes (BMs) were developed, and the efficiency of BMs in delivering small interfering ribonucleic acid (siRNA) as well as antiproliferative effects in vitro were investigated.

Results

Initially, we optimized the nitrogen/phosphate ratio and the BMs/siRNA mass ratio as 20 and 1:2, respectively, to prepare the BMs–PEI–siRNA composites. Furthermore, the prepared nanoconjugates were systematically characterized. The dynamic light scattering measurements indicated that the particle size and the zeta potential of BMs–PEI–siRNA are 196.5 nm and 49.5 ± 3.77 mV, respectively, which are optimum for cell internalization. Moreover, the confocal laser scanning microscope observations showed that these composites were at a proximity to the nucleus and led to an effective silencing effect. BMs–PEI–siRNA composites efficiently inhibited the growth of HeLa cells in a dose-as well as time-dependent manner. Eventually, a dual stain assay using acridine orange/ethidium bromide, revealed that these nanocomposites induced late apoptosis in cancer cells.

Conclusions

A novel and efficient gene delivery system based on BMs was successfully produced for cancer therapy, and these innovative carriers will potentially find widespread applications in the pharmaceutical field.

     

MicroRNA-190b confers radio-sensitivity through negative regulation of Bcl-2 in gastric cancer cells

Objectives

To determine the role of miR-190b in radio-sensitivity of gastric cancer (GC).

Results

In radio-resistant GC cells, down-regulation of miR-190b and up-regulation of Bcl-2 were observed. The protein expression of Bcl-2 was negatively regulated by miR-190b. Overexpression of miR-190b significantly decreased cell viability and enhanced radio-sensitivity of GC cells. Of note, these effects of miR-190b on GC cells radio-sensitivity were abolished by Bcl-2.

Conclusion

miR-190b confers radio-sensitivity of GC cells, possibly via negative regulation of Bcl-2.

     

PKM2 knockdown influences SREBP activation and lipid synthesis in bovine mammary-gland epithelial MAC-T cells

Objective

The purpose of the article is to evaluate the changes in lipid metabolism in bovine mammary-gland epithelial MAC-T cells after PKM2 knockdown.

Results

MAC-T cells stably expressing low levels of PKM2 were established with lentivirus-mediated small hairpin RNA. Although the knockdown of PKM2 had no effect on MAC-T cell growth, the reduced expression of PKM2 attenuated the mRNA and protein expression of key enzymes involved in sterol synthesis through the SREBP pathway.

Conclusions

The downregulation of PKM2 significantly influenced lipid synthesis in bovine mammary-gland epithelial MAC-T cells. These findings extend our understanding of the crosstalk between glycolysis and lipid metabolism in bovine mammary-gland epithelial cells.

     

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway

Objectives

To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing.

Results

Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased.

Conclusions

Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

     

The role of bone marrow mononuclear cell-conditioned medium in the proliferation and migration of human dermal fibroblasts

Background

Several recent studies have demonstrated the great potential of bone marrow cells in regenerative medicine, not only for their ability to differentiate to match a damaged cell type, but also because they synthesize and release various growth factors and cytokines.We examined the effect of bone marrow cell-conditioned medium in the healing process, especially in terms of fibroblast proliferation and migration.

Methods

These in vitro studies consisted of co-culture (without direct contact) of dermal fibroblasts with mononuclear bone marrow cells and the use of conditioned medium obtained from these cultures in a scratch wound model.

Results

Mononuclear cells were found to increase the proliferation of fibroblasts, and the conditioned medium showed a stimulatory effect on the migration of fibroblasts.

Conclusion

When considered together with the observed increase in growth factor levels in conditioned medium, it appears that these cells act through a paracrine mechanism.

     

Metabolic profiling identifies trehalose as an abundant and diurnally fluctuating metabolite in the microalga <Emphasis Type="Italic">Ostreococcus tauri</Emphasis>

Introduction

The picoeukaryotic alga Ostreococcus tauri (Chlorophyta) belongs to the widespread group of marine prasinophytes. Despite its ecological importance, little is known about the metabolism of this alga.

Objectives

In this work, changes in the metabolome were quantified when O. tauri was grown under alternating cycles of 12 h light and 12 h darkness.

Methods

Algal metabolism was analyzed by gas chromatography-mass spectrometry. Using fluorescence-activated cell sorting, the bacteria associated with O. tauri were depleted to below 0.1% of total cells at the time of metabolic profiling.

Results

Of 111 metabolites quantified over light–dark cycles, 20 (18%) showed clear diurnal variations. The strongest fluctuations were found for trehalose. With an intracellular concentration of 1.6 mM in the dark, this disaccharide was six times more abundant at night than during the day. This fluctuation pattern of trehalose may be a consequence of starch degradation or of the synchronized cell cycle. On the other hand, maltose (and also sucrose) was below the detection limit (~10 μM). Accumulation of glycine in the light is in agreement with the presence of a classical glycolate pathway of photorespiration. We also provide evidence for the presence of fatty acid methyl and ethyl esters in O. tauri.

Conclusions

This study shows how the metabolism of O. tauri adapts to day and night and gives new insights into the configuration of the carbon metabolism. In addition, several less common metabolites were identified.

     

Novel microbial photobioelectrochemical cell using an invasive ultramicroelectrode array and a microfluidic chamber

Objective

To fabricate a novel microbial photobioelectrochemical cell using silicon microfabrication techniques.

Results

High-density photosynthetic cells were immobilized in a microfluidic chamber, and ultra-microelectrodes in a microtip array were inserted into the cytosolic space of the cells to directly harvest photosynthetic electrons. In this way, the microbial photobioelectrochemical cell operated without the aid of electron mediators. Both short circuit current and open circuit voltage of the microbial photobioelectrochemical cell responded to light stimuli, and recorded as high as 250 pA and 45 mV, respectively.

Conclusion

A microbial photobioelectrochemical cell was fabricated with potential use in next-generation photosynthesis-based solar cells and sensors.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Synthesis of <Emphasis Type="Italic">scl</Emphasis>-poly (3-hydroxyalkanoates) by <Emphasis Type="Italic">Bacillus cereus</Emphasis> found in freshwater,from monosaccharides and disaccharides

Background

Polyhydroxyalkanoates are a good substitute for synthetic plastic because they are highly biocompatible, ecofriendly, and biodegradable. Bacteria in freshwater bodies such as rivers, tube wells, and canals are exposed to alternating high and low concentrations of substrates that induce PHA production.

Methods

Fresh water samples were collected for isolation of bacterial strains. Screening of PHA in bacterial cells was performed with Sudan and Nile Red staining. Extracted PHA was characterized by FTIR.

Results

In this study, nine bacterial isolates were selected for PHA production on the basis of phenotypic screening. Their ability to accumulate PHAs was determined using different monosaccharides and disaccharides. Two bacterial isolates Bacillus cereus T1 (KY746353) and Bacillus cereus R3 (KY746354) produced PHAs. Optimal growth of the bacterial strain (T1) was observed in the presence of glucose, followed by maximum production of PHAs (63% PHAs) during the logarithmic phase of growth. B. cereus R3 (KY746354) accumulated 60% PHAs by dry cell weight.

Conclusion

PHA accumulation was relatively less with fructose, but both strains showed increased production (up to 50%) with sucrose. The polymer produced was characterized by Fourier-transform infrared spectroscopy (FTIR), which showed that the compound contains short-chain PHAs.

     

NMR-based metabolic characterization of chicken tissues and biofluids: a model for avian research

Introduction

Poultry is one of the most consumed meat in the world and its related industry is always looking for ways to improve animal welfare and productivity. It is therefore essential to understand the metabolic response of the chicken to new feed formulas, various supplements, infections and treatments.

Objectives

As a basis for future research investigating the impact of diet and infections on chicken’s metabolism, we established a high-resolution proton nuclear magnetic resonance (NMR)-based metabolic atlas of the healthy chicken (Gallus gallus).

Methods

Metabolic extractions were performed prior to ¹H-NMR and 2D NMR spectra acquisition on twelve biological matrices: liver, kidney, spleen, plasma, egg yolk and white, colon, caecum, faecal water, ileum, pectoral muscle and brain of 6 chickens. Metabolic profiles were then exhaustively characterized.

Results

Nearly 80 metabolites were identified. A cross-comparison of these matrices was performed to determine metabolic variations between and within each section and highlighted that only eight core metabolites were systematically found in every matrice.

Conclusion

This work constitutes a database for future NMR-based metabolomic investigations in relation to avian production and health.