Interaction of the retinoic acid signaling pathway with spicule formation in the marine sponge Suberites domuncula through activation of bone morphogenetic protein-1

Background

The formation of the spicules in siliceous sponges involves the formation of cylinder-like structures in the extraspicular space, composed of the enzyme silicatein and the calcium-dependent lectin.

Scope of review

Molecular cloning of the cDNAs (carotene dioxygenase, retinal dehydrogenase, and BMB-1 [bone morphogenic protein-1]) from the demosponge Suberites domuncula was performed. These tools were used to understand the retinoid metabolism in the animal by qRT-PCR, immunoblotting and TEM.

Major conclusions

We demonstrate that silintaphin-2, a silicatein-interacting protein, is processed from a longer-sized 15-kDa precursor to a truncated, shorter-sized 13 kDa calcium-binding protein via proteolytic cleavage at the dipeptide Ala↓Asp, mediated by BMP-1. The expression of this protease as well as the expression of two key enzymes of the carotinoid metabolism, the β,β-carotene-15,15′-dioxygenase and the retinal dehydrogenase/reductase, were found to be strongly up-regulated by retinoic acid. Hence retinoic acid turned out to be a key factor in skeletogenesis in the most ancient still existing metazoans, the sponges.

General significance

It is shown that retinoic acid regulates the formation of the organic cylinder that surrounds the axis of the spicules and enables, as a scaffold, the radial apposition of new silica layers and hence the growth of the spicules.     

Isolation of the silicatein-α interactor silintaphin-2 by a novel solid-phase pull-down assay

The skeleton of siliceous sponges consists of amorphous biogenous silica (biosilica). Biosilica formation is driven enzymatically by means of silicatein(s). During this unique process of enzymatic polycondensation, skeletal elements (spicules) that enfold a central proteinaceous structure (axial filament), mainly comprising silicatein, are formed. However, only the concerted action of silicatein and other proteins can explain the genetically controlled diversity of spicular morphotypes, from simple rods with pointed ends to intricate structures with up to six rays. With the scaffold protein silintaphin-1, a first silicatein interactor that facilitates the formation of the axial filament and, consequently, of the growing spicule was discovered. In this study, a new interactor has been identified by both a conventional yeast two-hybrid library screening and a newly established pull-down assay. For the latter approach, silicatein-α has been bioengineered to carry a Glu tag, which confers binding affinity to hydroxyapatite. After immobilization on a solid-phase matrix (hydroxyapatite), the Glu-tagged silicatein was used as bait for the identification of interactors. Both approaches revealed a 15 kDa polypeptide, and its identity was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Colocalization of silintaphin-2 and silicatein-α within the axial filament and on the spicule surface was shown by immunohistological analyses. Subsequent autoradiography demonstrated the Ca(2+) binding affinity of this silicatein interactor. These findings indicate that both proteins operate in concert during spiculogenesis. Besides binding of calcium, silintaphin-2 shares several structural features with certain acidic, secreted extracellular matrix proteins that facilitate tissue mineralization in Metazoa. Hence, silintaphin-2 might mediate signal transduction during spiculogenesis or may play a more direct role during biosilica formation, in concert with silicatein.     

Apposition of silica lamellae during growth of spicules in the demosponge Suberites domuncula: biological/biochemical studies and chemical/biomimetical confirmation

Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; their surfaces are formed by silicatein. The extracellularly located spicules are surrounded by sclerocytes which are filled with both electron-dense and electron-poor vesicles; energy dispersive X-ray analysis/scanning electron microscopical studies revealed that the electron-dense vesicles are filled of silicon/silica and therefore termed silicasomes. The release of the content of the silicasomes into the hollow cylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicatein molecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silicatein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]-poly(acetoxime methacrylate) [reactive ester polymer]-N(epsilon)-benzyloxycarbonyl L-lysine tert-butyl ester-Ni(II); finally His-tagged silicatein is immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for a technical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis of nanostructured tubes.     

Silintaphin-1--interaction with silicatein during structure-guiding bio-silica formation

Silicateins are unique enzymes of sponges (phylum Porifera) that template and catalyze the polymerization of nanoscale silicate to siliceous skeletal elements. These multifunctional spicules are often elaborately shaped, with complex symmetries. They carry an axial proteinaceous filament, consisting of silicatein and the scaffold protein silintaphin-1, which guides silica deposition and subsequent spicular morphogenesis. In vivo, the synthesis of the axial filament very likely proceeds in three steps: (a) assembly of silicatein monomers to form one pentamer; (b) assembly of pentamers to form fractal-like structures; and finally (c) assembly of fractal-like structures to form filaments. The present study was aimed at exploring the effect of self-assembled complexes of silicatein and silintaphin-1 on biosilica synthesis in vitro. Hence, in a comparative approach, recombinant silicatein and recombinant silintaphin-1 were used at different stoichiometric ratios to form axial filaments and to synthesize biosilica. Whereas recombinant silicatein-α reaggregates to randomly organized structures, coincubation of silicatein-α and silintaphin-1 (molecular ratio 4 : 1) resulted in synthetic filaments via fractal-like patterned self-assemblies, as observed by electron microscopy. Concurrently, owing to the concerted action of both proteins, the enzymatic activity of silicatein-α strongly increased by 5.3-fold (with the substrate tetraethyl orthosilicate), leading to significantly enhanced synthesis of biosilica. These results indicate that silicatein-α-mediated biosilicification depends on the concomitant presence of silicatein-α and silintaphin-1. Accordingly, silintaphin-1 might not only enhance the enzymatic activity of silicatein-α, but also accelerate the nonenzymatic polycondensation of the silica product before releasing the fully synthesized biosiliceous polymer.     

Acquisition of structure-guiding and structure-forming properties during maturation from the pro-silicatein to the silicatein form

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties.     

Histochemical and electron microscopic analysis of spiculogenesis in the demosponge Suberites domuncula.

The skeleton of demosponges is built of spicules consisting of biosilica. Using the primmorph system from Suberites domuncula, we demonstrate that silicatein, the biosilica-synthesizing enzyme, and silicase, the catabolic enzyme, are colocalized at the surface of growing spicules as well as in the axial filament located in the axial canal. It is assumed that these two enzymes are responsible for the deposition of biosilica. In search of additional potential structural molecules that might guide the mineralization process during spiculogenesis to species-specific spicules, electron microscopic studies with antibodies against galectin and silicatein were performed. These studies showed that silicatein forms a complex with galectin; the strings/bundles of this complex are intimately associated with the surface of the spicules and arranged concentrically around them. Collagen fibers are near the silactein/galectin complexes. The strings/bundles formed from silicatein/galectin display a lower degree of orientation than the collagen fibers arranged in a highly ordered pattern around the spicules. These data indicate that species-specific formation of spicules involves a network of (diffusible) regulatory factor(s) controlling enzymatic silica deposition; this mineralization process proceeds on a galectin/collagen organic matrix.     

Sponge spicules as blueprints for the biofabrication of inorganic–organic composites and biomaterials

While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms complement their body plan with silica. However, of all recent animals, only sponges (phylum Porifera) are able to polymerize silica enzymatically mediated in order to generate massive siliceous skeletal elements (spicules) during a unique reaction, at ambient temperature and pressure. During this biomineralization process (i.e., biosilicification) hydrated, amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometers to meters. Spicules lend structural stability to the sponge body, deter predators, and transmit light similar to optic fibers. This peculiar phenomenon has been comprehensively studied in recent years and in several approaches, the molecular background was explored to create tools that might be employed for novel bioinspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it obtains final size and shape. Again, this process is mediated by silicatein and silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nanostructured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics. In this context, first bioinspired approaches implement recombinant silicatein and silintaphin-1 for applications in the field of biomedicine (biosilica-mediated regeneration of tooth and bone defects) or micro-optics (in vitro synthesis of light waveguides) with promising results.     

Oral administration of D-aspartate,but not L-aspartate,depresses rectal temperature and alters plasma metabolites in chicks

Aims

L-Aspartate (L-Asp) and D-aspartate (D-Asp) are physiologically important amino acids in mammals and birds. However, the functions of these amino acids have not yet been fully understood. In this study, we therefore examined the effects of L-Asp and D-Asp in terms of regulating body temperature, plasma metabolites and catecholamines in chicks.

Main methods

Chicks were first orally administered with different doses (0, 3.75, 7.5 and 15 mmol/kg body weight) of L- or D-Asp to monitor the effects of these amino acids on rectal temperature during 120 min of the experimental period.

Key findings

Oral administration of D-Asp, but not of L-Asp, linearly decreased the rectal temperature in chicks. Importantly, orally administered D-Asp led to a significant reduction in body temperature in chicks even under high ambient temperature (HT) conditions. However, centrally administered D-Asp did not significantly influence the body temperature in chicks. As for plasma metabolites and catecholamines, orally administered D-Asp led to decreased triacylglycerol and uric acid concentrations and increased glucose and chlorine concentrations but did not alter plasma catecholamines.

Significance

These results suggest that oral administration of D-Asp may play a potent role in reducing body temperature under both normal and HT conditions. The alteration of plasma metabolites further indicates that D-Asp may contribute to the regulation of metabolic activity in chicks.     

Identification of silicateins in freshwater sponge <Emphasis Type="Italic">Lubomirskia baicalensis</Emphasis>

Siliceous spicules of Baikal freshwater sponge Lubomirskia baicalensis contain several proteins, including silicateins. Analysis of a L. baicalensis cDNA library revealed four different mRNAs coding for proteins related to marine sponge silicatein α (α1, α2, α3, and α4). The intron-exon structure was determined forthe genomic α1 silicatein gene. The gene is 1988 bp from the initiation to the termination codon and consists of six intron (total size 1007 bp) and seven exons (total size 981 bp). Mass spectrometry of a tryptic digest of spicule proteins revealed peptides of two silicateins α.     

Amino acid facilitates absorption of copper in the Caco-2 cell culture model

Aim

Copper deficiency could cause fatal hematological and neurological disorders or other diseases. Amino acids are involved in the absorption of copper ions. The purpose of this study is to evaluate the absorption of copper in amino acid complex forms and determine its mechanism in the Caco-2 cell culture model.

Main methods

The human colonic adenocarcinoma cell line Caco-2 culture model was used to determine the permeability of copper ions in inorganic form (CuSO₄) and the amino acid complex forms. Lysine and methionine, as well as carboplatin were used to determine the possible involvement of amino acid transporters or copper transporter 1 (CTR1).

Key findings

The results showed that all of the amino acid complex forms facilitated copper absorption. The apparent permeabilities of copper ions in these complex forms were at least 7.6 fold higher than those in the CuSO₄ form. The permeability rank order of copper in various amino acid complex forms was Cu-glutamate < Cu-lysine = Cu-aspartic acid = Cu methionine < Cu-arginine < Cu-(lysine/glutamate). Mechanistic studies revealed that the enhanced absorption of copper in copper amino acid complexes could be the result of enhanced uptake (as in Cu-methionine complex) or enhanced basolateral efflux (as in Cu-lysine complex). Copper transporter 1 (or CTR1) inhibitor carboplatin did not affect the absorption of copper in Cu-methionine complex, suggesting that the dominant pathway for copper amino acid complexes is not CTR1.

Significance

Enhanced absorption of copper ions in amino acid complex appears to be mediated by amino acid transporters.     

Stimulation of the Po-shen and Shen-hun scalp-acupuncture bands modifies levels of inhibitory and excitatory amino acids in the immature rat brain

Objective

In the present study, the effect of stimulation of the Po-shen and Shen-hun scalp-acupuncture bands on tissue amino acid concentrations in several brain regions in awake and pentobarbital-sedated immature rats was evaluated.

Materials and methods

Sprague–Dawley rats (aged 15 days) were organized in four groups of at least eight animals: control groups received saline solution 0.9% or sodium pentobarbital at 30 mg/kg dosage via intraperitoneal. Experimental groups received saline solution or sodium pentobarbital plus stimulation in Po-shen and Shen-hun scalp-acupuncture bands for one continuous hour during 10 sessions by using scalp-acupuncture.

Results

As compared to rats receiving saline solution, scalp-acupuncture produced significant changes in amino acid concentrations, depending on the analyzed region, as follows: in inhibitory amino acids, a GABA increase was observed in amygdala and hippocampus (491 and 184%, respectively), but a decrease in the substantia nigra (80%); glycine showed decrease in all the analyzed regions, except for an increase in brainstem(78%); glutamine presented an increase in hippocampus and cortex (42 and 149%, respectively). In the case of excitatory amino acids, glutamate decreased in all the analyzed regions; whereas aspartate decreased in substantia nigra and brainstem (77.08 and 35%, correspondingly) but increased in hippocampus and cortex (32 and 54%, respectively). The combined treatment of scalp-acupuncture and a GABAergic depressant drug like pentobarbital resulted in almost all changes induced in amino acids for scalp-acupuncture alone being significantly reverted.

Conclusion

Stimulation of the Po-shen and Shen-hun scalp-acupuncture bands by using scalp-acupuncture alone might produce depressant activity by changes in amino acids, but the combination with a GABAergic tranquilizer like sodium pentobarbital can interfere with this response.     

Conserved sequence in the aggrecan interglobular domain modulates cleavage by ADAMTS-4 and ADAMTS-5

Background

Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.

Methods

To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.

Results

Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.

Conclusion

We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme–substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.

General significance

Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis.     

Inhibition of calcium-calmodulin complex formation by vasorelaxant basic dipeptides demonstrated by in vitro and in silico analyses

Background

Tryptophan-histidine (Trp-His) was found to suppress the activity of the Ca^2 +/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca^2 +-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca^2 +-CaM complex formation, a CaMKII activator.

Methods

The ability of Trp-His and other peptides to inhibit Ca^2 +-CaM complex formation was investigated by a Ca^2 +-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation.

Results

We showed that Trp-His inhibited Ca^2 +-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca^2 +-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp + His, 3-methyl His-Trp, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silico studies suggested the possibility that Trp-His and His-Trp interacted with the Ca^2 +-binding site of CaM by forming hydrogen bonds with key Ca^2 +-binding residues of CaM, with a binding free energy of − 49.1 and − 68.0 kJ/mol, respectively.

Conclusions

This is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca^2 +-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study.

General significance

The results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca^2 +-CaM complex formation in the future.     

Klotho gene polymorphism of rs3752472 is associated with the risk of urinary calculi in the population of Han nationality in Eastern China

Background/aims

The incidence of urolithiasis has considerably increased throughout the world in the last two decades. Clinical researches have showed an association between oxidative stress and stone formation. Emerging evidence indicated a novel function for klotho protein in anti-oxidative stress. In this study, we aimed at investigating a possible relationship between klotho gene polymorphisms and the risk of calcium oxalate urolithiasis in the population of Han nationality in Eastern China.

Methods

Klotho gene polymorphisms rs3752472 in exon3, rs650439 in intron 4 and rs577912 in intron 1 were investigated in 426 patients with calcium oxalate stones compared with 282 age-matched healthy volunteers with no history of stone formation, using TaqMan SNP Genotyping Assays.

Results

Significant differences were found between rs3752472 and the risk of nephrolithiasis as CC genotype of rs3752472 klotho polymorphism had almost 2-fold increased stone risk compared with the heterozygote genotype CT and homozygous genotype TT(95% CI = 1.013–2.255, OR = 1.512,p = 0.043).

Conclusion

Our results showed that the rs3752472 polymorphism of klotho gene is associated with the risk of calcium oxalate urolithiasis and may act as a risk factor during stone formation in our study population.     

Mutational analysis of NHAoc/NHA2 in Saccharomyces cerevisiae

Background

NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes.

Methods

The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth.

Results

Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 cannot grow in 0.4 M NaCl, suggesting that these residues are also essential for antiporter activity.

Conclusions

Evolutionarily conserved amino acids are required for full antiporter function.

General Significance

Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo.     

Manipulating the bioactivity of hydroxyapatite nano-rods structured networks: Effects on mineral coating morphology and growth kinetic

Background

Nano-hydroxyapatite particles have better bioactivity than the coarse crystals. So, they can be utilized for engineered tissue implants with improved efficiency over other materials. The development of materials with specific bioactive characteristics is still under investigation.

Methods

The surface properties of four hydroxyapatite materials templated by different micelle-polymer structured network are studied. The synergistic interaction of each block copolymer in contact with CTAB rod-like micelles results in crystalline HAp nano-rods of 25–50 nm length organized in hierarchical structures with different micro-rough characteristics.

Results

It was observed that the material in vitro bioactivity strongly depends on the surface structure while in a minor extent on their Ca/P ratio. So, MIII and MIV materials with Skewness parameter R_sk > 2.62 favored the formation on their surfaces of net-like phase with a high growth kinetic constant; while MI and MII (R_sk ≤ 2.62) induced the appearance of spherulitic-like structures and a growth rate 1.75 times inferior. Material biocompatibility was confirmed by interaction with rat calvarial osteoblasts.

Conclusions

The different structures growth is attributed to a dissimilar matching of crystal planes in the material and the apatite layer formed. In specific synthesis conditions, a biocompatible material with a Ca/P ratio close to that for the trabecular bone and a morphology that are considered essential for bone-bonding was obtained.

General significance

The creation of implantable devices with a specific bioactive characteristic may be useful to manipulate the attachment of cells on mineral coating directly affecting the stability and life of the implant.     

The polymorphism − 1131T > C in apolipoprotein A5 gene is associated with dyslipidemia in Brazilian subjects

Background

Polymorphisms in apolipoprotein A5 gene (APOA5) have been associated with higher triglyceride levels in many populations. The aim of the study was to determine the allelic and genotypic distribution of the APOA5 − 1131T > C polymorphism and to identify the association of the genetic variant and the risk for dyslipidemia.

Methods

We genotyped 109 dyslipidemic subjects and 107 controls. The total cholesterol, triglycerides and HDL-c were determined enzymatically. Comparison of means among groups was calculated by ANOVA. Significant differences among groups were evaluated by Student–Newman–Keuls test.

Results

The minor allele C was more frequent in dyslipidemic subjects than controls (p = 0.019) and confers an increased individual risk for dyslipidemia (OR = 1.726, CI 95% = 1.095–2.721). The genotype analysis by gender showed that this allele was more frequent in dyslipidemic males (p = 0.037; OR = 2.050, CI 95% = 1.042–4.023). When participants were analyzed according to genotypes TT and TC/CC, C-carriers presented higher cholesterol and triglycerides levels than TT homozygous (p = 0.046 and 0.049, respectively).

Conclusions

The allele C confers higher total cholesterol and triglycerides levels in dyslipidemic adults. The APOA5 − 1131T > C polymorphism is associated with dyslipidemia in male subjects.     

Microencapsulation of dopamine neurons derived from human induced pluripotent stem cells

Background

Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress.

Methods

hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons.

Results

Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved.

Conclusion

hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads.

General significance

Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons.