Measuring mineralised tissue formation and resorption in a human 3D osteoblast-osteoclast co-culture model.

In vitro tissue engineered bone constructs have been developed, but models which mimic both formation and resorption in parallel are still lacking. To be used as a model for the bone remodeling process, the formation and resorption of mineralised tissue volume over time needs to be visualised, localised and quantified. The goal of this study was to develop a human 3D osteoblast-osteoclast co-culture in which 1) osteoblasts deposit mineralised matrix, 2) monocytes differentiate into resorbing osteoclasts, and 3) the formation and resorption of mineralised matrix could be quantified over time using micro-computed tomography (µCT). Mesenchymal stromal cells were seeded on silk fibroin scaffolds and differentiated towards osteoblasts to create mineralised constructs. Thereafter, monocytes were added and differentiated towards osteoclasts. The presence of osteoblasts and osteoclasts was confirmed using immunohistochemistry. Osteoclastic activity was confirmed by measuring the increased release of osteoclast marker tartrate resistant acid phosphatase (TRAP), suggesting that osteoclasts were actively resorbing mineralised tissue. Resorption pits were visualised using scanning electron microscopy. Mineralised matrix formation and resorption were quantified using µCT and subsequent scans were registered to visualise remodelling. Both formation and resorption occurred in parallel in the co-culture. The resorbed tissue volume exceeded the formed tissue volume after day 12. In conclusion, the current model was able to visualise, localise and quantify mineralised matrix formation and resorption. Such a model could be used to facilitate fundamental research on bone remodeling, facilitate drug testing and may have clinical implications in personalised medicine by allowing the use of patient cells.

Localisation of mineralised tissue in a complex spinner flask environment correlates with predicted wall shear stress level localisation.

Spinner flask bioreactors have often been employed for bone tissue engineering. However, the reasons for their success in facilitating bone growth remain inconclusive. It was hypothesised that engineered bone tissue formation can be attributed to mechanical stimuli, which can be predicted in the tissue engineered construct. To test the hypothesis and draw conclusions as to how mechanical stimulation affects cell behaviour, a multi- disciplinary approach using cell culture experiments and computational fluid dynamics (CFD) to simulate the complex flow within the spinner flask and scaffold was employed. Micro-computed tomography and histology showed that statically cultured human bone marrow derived stromal cells on silk fibroin scaffolds did not form extracellular matrix (ECM) or deposit minerals. However, constructs cultured at 60 rpm resulted in ECM formation and mineralisation, mainly at the bottom of the scaffold (bottom: 78 ± 7 %, middle: 17 ± 5 %, top: 5 ± 2 % of total mineralised volume). Culturing at 300 rpm led to a more homogeneously distributed ECM (bottom: 40 ± 14 %, middle: 33 ± 1 %, top: 27 ± 14 % of total mineralised volume). These observations were in agreement (Pearson correlation coefficient: 97 %) with the computational simulations that predicted maximal scaffold mineralisation, based on wall shear stress stimulation, in the bottom at 60 rpm and in the main body at 300 rpm. Such combinations of CFD modelling and experimentation could advance our knowledge of the mechanical stimuli that cells experience in vitro and link them to biological responses.

Antisocial behavior and polymorphisms in the oxytocin receptor gene: findings in two independent samples.

The quantitative genetic contribution to antisocial behavior is well established, but few, if any, genetic variants are established as risk factors. Emerging evidence suggests that the neuropeptide oxytocin (OXT) may modulate interpersonal aggression. We here investigated whether single-nucleotide polymorphisms (SNPs) in the OXT receptor gene (OXTR) are associated with the expression of antisocial behavior. A discovery sample, including both sexes, was drawn from the Child and Adolescent Twin Study in Sweden (CATSS; n=2372), and a sample from the Twin Study of Child and Adolescent Development (TCHAD; n=1232) was used for replication. Eight SNPs in OXTR, selected on previous associations with social and antisocial behavior, were genotyped in the participants of CATSS. Significant polymorphisms were subsequently genotyped in TCHAD for replication. Participants completed self-assessment questionnaires-Life History of Aggression (LHA; available only in CATSS), and Self-Reported Delinquency (SRD; available in both samples)-designed to capture antisocial behavior as continuous traits. In the discovery sample, the rs7632287 AA genotype was associated with higher frequency of antisocial behavior in boys, and this was then replicated in the second sample. In particular, overt aggression (directly targeting another individual) was strongly associated with this genotype in boys (P=6.2 × 10(-7) in the discovery sample). Meta-analysis of the results for antisocial behavior from both samples yielded P=2.5 × 10(-5). Furthermore, an association between rs4564970 and LHA (P=0.00013) survived correction in the discovery sample, but there was no association with the SRD in the replication sample. We conclude that the rs7632287 and rs4564970 polymorphisms in OXTR may independently influence antisocial behavior in adolescent boys. Further replication of our results will be crucial to understanding how aberrant social behavior arises, and would support the OXT receptor as one potential target in the treatment of aggressive antisocial behavior.

Following ORR intermediates adsorbed on a Pt cathode catalyst during break-in of a PEM fuel cell by in operando X-ray absorption spectroscopy.

In operando X-ray absorption spectroscopy data using the Δµ X-ray Absorption Near Edge Spectroscopy (XANES) analysis procedure is used to follow the ORR intermediate adsorbate coverage on a working catalyst in a PEMFC during initial activation and break-in. The adsorbate coverage and log i (Tafel) curves reveal a strong correlation, i.e., an increase in adsorbate intermediate coverage poisons Pt sites thereby decreasing the current. A decrease in Pt-O bond strength commensurate with decrease in potential causes a sequence of different dominant adsorbate volcano curves to exist, namely first O, then OH, and then OOH exactly as predicted by the different ORR kinetics mechanisms. During break-in, the incipient O coverage coming from exposure to air during storage and MEA preparation is rather quickly removed, compared to the slower and more subtle nanoparticle morphological changes, such as the rounding of the Pt nanoparticle edges/corners and smoothing of the planar surfaces, driven by the nanoparticle's tendency to lower its surface energy. These morphological changes increase the Pt-Pt average coordination number, decrease the average Pt-O bond strength, and thereby decrease the coverage of ORR intermediates, allowing increase in the current.

Non-ammoniagenic proliferation and differentiation media for cultivated adipose tissue.

Ammonia (Amm), and its aqueous solved state, ammonium, which is produced from glutamine (Gln) metabolism, is a known inhibitor of stem cell proliferation in vitro. In the context of cultivated beef, primary bovine fibro-adipogenic progenitor cells (FAPs) need to be grown and differentiated for several weeks in vitro for the production of cultivated fat. In this study, the ammonium sensitivity of these cells was investigated by introducing ammonium chloride, which was found to inhibit their proliferation when above 5 mM and their adipogenic differentiation when above 2 mM. Novel serum-free proliferation and differentiation media were hence developed with the aim to suppress Amm production during expansion and adipogenesis. Glutamine substitutes, such as a-ketoglutarate (aKG), glutamate (Glt) and pyruvate (Pyr) were investigated. It was found that aKG based proliferation medium (PM) was the most effective in promoting and maintaining FAPs growth over several passages while the specific Amm production rate was reduced more than 5-fold. In terms of differentiation capacity, the substitution of glucose (Gluc) and Gln with galactose (Gal) and Pyr was shown to be the most effective in promoting FAPs differentiation into mature adipocytes, resulting in over 2-fold increase of fat volume per cell, while suppressing Amm production. Our findings suggest that FAPs do not require Gln as an essential nutrient but, on the contrary, possess all the necessary metabolic pathways to proliferate and subsequently differentiate in a Gln-free medium, resulting in decreased Amm production rates and seemingly synthesising glutamine de novo. These findings are important for prolonging the lifespan of culture medium, allowing for reduced costs and process interventions.

Cytochrome P450 2E1 gene polymorphisms/haplotypes and Parkinson's disease in a Swedish population.

Cytochrome P450 2E1 (CYP2E1), which inter alia is located in dopamine containing neurons in the substantia nigra, has been hypothesized to be of importance for the pathophysiology of Parkinson's disease (PD), either by its production of reactive oxygen species (ROS) or by its capability to detoxify putative neurotoxins. Numerous polymorphisms in the coding and non-coding regions of the gene for this enzyme have been reported. Different variants may account for inter-individual differences in the activity of the enzyme or production of ROS. In this study, the CYP2E1 gene was examined in a control population (n = 272) and a population with PD (n = 347), using a tag-single nucleotide polymorphism (tSNP) approach founded on HapMap Data. Six tSNPs were used in the analysis and haplotype block data were obtained. In case of significance, the SNP was further examined regarding early/late age of disease onset and presence of relatives with PD. We found an association between allele and genotype frequencies of the C/G polymorphism at intron 7 (rs2070676) of this gene and PD (P value of 0.026 and 0.027, respectively). Furthermore, analysis of the rs2070676 polymorphism in subgroups of patients with age of disease onset higher than 50 years and those not having a relative with PD also demonstrated a significant difference with controls. This was seen in both genotype (corresponding to P value = 0.039 and 0.032) and allele (P = 0.027 and 0.017 respectively) frequency. As a representative of many polymorphisms or in possible linkage disequilibrium with other functional variants, it is possible that rs2070676 could influence the regulation of the enzyme. In conclusion, our results display an association between the rs2070676 polymorphism and PD. Additional investigations are needed to elucidate the importance of this polymorphism for the activity of CYP2E1 and PD susceptibility.

The serotonin reuptake inhibitor fluoxetine reduces sex steroid-related aggression in female rats: an animal model of premenstrual irritability?

The aggressive behavior displayed by some (but not all) female Wistar rats when an unfamiliar rat is being introduced into their home cage (the resident intruder paradigm) was found to be higher in non-receptive phases (metestrus, diestrus) than in the receptive phases (proestrus, estrus) of the estrus cycle, and effectively reduced by ovariectomy. When removal of the ovaries was followed by administration of estradiol and progesterone, in a regimen mimicking the normal cyclical release of these hormones, aggressive behavior was elicited, two days after estrus, in animals that had displayed aggressive behavior before ovariectomy, but not in those that had not. Short-term administration of a serotonin reuptake inhibitor (fluoxetine hydrochloride; 10 mg/kg, i.p.; 4-5 days) reduced both the aggressive behavior displayed during the diestrus phase by normally cycling rats, and the aggressive behavior elicited by administration of estradiol plus progesterone after ovariectomy. It is suggested that the aggressive behavior displayed by the female Wistar rat in the resident intruder paradigm may serve as an animal model of premenstrual dysphoria.

Serotonin transporter gene polymorphisms are associated with anxiety-related personality traits in women.

Several studies have reported an association between anxiety-related personality traits and a promoter polymorphism in the human serotonin transporter (5-HTT) gene (5-HTT gene-linked polymorphic region, 5-HTTLPR). In the present study, a population of 251 subjects was assessed with the Karolinska Scales of Personality (KSP) and genotyped both for the 5-HTTLPR and for a variable number of tandem repeats polymorphism in the second intron of the same gene. The interpretation of previous studies has to some extent been confounded by the studied subjects differing with respect to ethnicity, sex, and age. To circumvent this problem, all included subjects were Caucasians, women, and born in the same year (1956). Associations were found between the 5-HTTLPR and four of the five anxiety-related KSP scales (psychic anxiety, muscular tension, psychasthenia, and lack of assertiveness), subjects being homozygous for the short allele displaying higher anxiety scores than those of the long/long or long/short genotype. In addition, an association was found between the intron 2 polymorphism and one anxiety-related personality trait (somatic anxiety).

Serotonin transporter gene polymorphisms and platelet [3H] paroxetine binding in premenstrual dysphoria.

The purpose of this study was to investigate if women with premenstrual dysphoria differ from controls with respect to the number of platelet serotonin transporters, and with respect to three polymorphisms in the gene coding for the serotonin transporter: a 44 base pair insertion/deletion in the promoter region, a variable number of tandem repeats in the second intron, and a single nucleotide polymorphism in the 3' untranslated region. Also, the possible relationship between the three polymorphisms and platelet serotonin transporter density was analyzed. The density of platelet [(3)H]paroxetine binding sites was significantly lower in women with premenstrual dysphoria than in controls, but patients and controls did not differ with respect to allele or genotype frequency for any of the three polymorphisms examined. A significant association between the number of platelet serotonin transporters and the promoter polymorphism was observed, subjects being homozygous for the short (deletion) variant having higher platelet serotonin transporter density than subjects carrying the long (insertion) allele. The results support the assumption that serotonin-related psychiatric disorders-such as premenstrual dysphoria-may be associated with a reduction in platelet [(3)H]paroxetine binding, but argue against the notion that this reduction is due to certain variants of the serotonin transporter gene being more common in patients than in controls.

Association between the estrogen receptor beta gene and age of onset of Parkinson's disease.

The purpose of this study was to investigate the potential contribution of genetic variants in the estrogen receptor beta gene to the aetiology of Parkinson's disease (PD). Several lines of evidence from human and animal studies suggest a protective role for estrogen in PD. Recently the estrogen receptor beta subtype was reported to be an important mediator of estrogen actions in the nigrostriatal dopamine system. Two single nucleotide polymorphisms at position 1730 and 1082 in the ER beta gene were genotyped, using pyrosequencing, in 260 patients with PD and 308 controls recruited from the Swedish population. Neither of the two estrogen receptor beta polymorphisms was associated with an increased risk for PD. However, the G allele of the A1730G polymorphism was more frequent in patients with an early age of onset than in patients with a late age of onset of PD (P = 0.006). Patients carrying the GG genotype had an odds ratio of 2.2 for having an early onset of PD compared to non-carriers. In conclusion, our results indicate that genetic variation in the estrogen receptor beta gene may influence the age of onset of PD.

Maternal transmission disequilibrium of the glutamate receptor GRIK2 in schizophrenia.

Schizophrenia is characterized by thought disorders, hallucinations and delusions. Genetic studies have shown a high linkage at chromosome 6q16-21. Among the genes located in this region is the glutamate receptor ionotropic kainate 2 gene (GRIK2 or GLUR6), a functional candidate for susceptibility to schizophrenia. In this study, transmission of GRIK2 was evaluated in 356 schizophrenic patients from three different clinical centers. Whereas paternal transmission shows equilibrium, we observed maternal transmission disequilibrium of GRIK2 in the largest population (p=0.03), which was still significant when all populations were added (p=0.05). These results are similar to the maternal GRIK2 transmission disequilibrium previously reported for autism, and support the presence of a susceptibility gene for schizophrenia at 6q16.

Catalase activity in arctic microfungi grown at different temperatures.

The total of 98 strains of moulds were isolated from soils collected in arctic tundra (Spitzbergen). Among these strains Penicillium cyclopium 1, the most effective for production of catalase, was selected by the method of test-tube microculture. The time course of growth and catalase production by this strain showed the intracellular activity of this enzyme to be about 3-fold higher than its extracellular level Some properties of crude catalase preparation, isolated from postculture liquids by lyophilization, were also examined. Catalase activity showed its maximum at 15 degrees C, indicating adaptation of the enzyme to lower temperatures of the arctic environment.

Abnormal melatonin synthesis in autism spectrum disorders.

Melatonin is produced in the dark by the pineal gland and is a key regulator of circadian and seasonal rhythms. A low melatonin level has been reported in individuals with autism spectrum disorders (ASD), but the underlying cause of this deficit was unknown. The ASMT gene, encoding the last enzyme of melatonin synthesis, is located on the pseudo-autosomal region 1 of the sex chromosomes, deleted in several individuals with ASD. In this study, we sequenced all ASMT exons and promoters in individuals with ASD (n=250) and compared the allelic frequencies with controls (n=255). Non-conservative variations of ASMT were identified, including a splicing mutation present in two families with ASD, but not in controls. Two polymorphisms located in the promoter (rs4446909 and rs5989681) were more frequent in ASD compared to controls (P=0.0006) and were associated with a dramatic decrease in ASMT transcripts in blood cell lines (P=2 x 10(-10)). Biochemical analyses performed on blood platelets and/or cultured cells revealed a highly significant decrease in ASMT activity (P=2 x 10(-12)) and melatonin level (P=3 x 10(-11)) in individuals with ASD. These results indicate that a low melatonin level, caused by a primary deficit in ASMT activity, is a risk factor for ASD. They also support ASMT as a susceptibility gene for ASD and highlight the crucial role of melatonin in human cognition and behavior.

Spectrophotometric determination of aluminium in pharmaceutical preparations by azo dyes of 1,2,4-triazole series.

Azodyes, derivatives of 1,2,4-triazole and pyrocatechine: 3-(3',4'-dihydroxyphenylazo-1')-1,2,4-triazole (TRIAP) and 3-(3',4'-dihydroxyphenylazo-1')-5-mercapto-1,2,4-triazole (METRIAP), were used for spectrophotometric determination of Al. in composite pharmaceutical preparations. In aqueous-methanolic solution at pH 6.20-6.50 Al ions form stabile, orange chelates. Molar ratio L:Al. is 2:1 (TRIAP) or 3:1 (METRIAP) and stability constants, expressed by log K, are 12,604 (METRIAP) and 8,440 (TRIAP). Other components of these preparations, particularly Mg, do not disturb Al determination. The determination results were statistically calculated and compared with those obtained by the method of atomic absorption spectrophotometry (AAS). The advantage of the developed method is easy synthesis of reagents, simple analytical procedure, stability of formed complexes, good reproducibility and accuracy of results. Using TRIAP or METRIAP the elaborated spectrophotometric method is more accurate than AAS method, for defined purpose.

Association between a dinucleotide repeat polymorphism of the estrogen receptor alpha gene and personality traits in women.

Estrogens are known to play a key role in the regulation of various aspects of behavior. In order to study the potential contribution of genetic variation in the estrogen receptor (ER) alpha to specific personality traits, we investigated a repeat polymorphism in the ER alpha gene in 172 42-year-old women who had been assessed using the Karolinska Scales of Personality (KSP). Based on the hypothesis that there is a relationship between the length of a repeat polymorphism and gene function,(1) the alleles were divided into two groups: short and long. In order to elucidate the possible influence of the ER alpha gene on the different aspects of personality measured by means of the KSP, the possible association between this gene and four different factors ('neuroticism', 'psychoticism', 'non-conformity', and 'extraversion') was analysed. 'Neuroticism', 'psychoticism', and 'non-conformity' all appeared to be associated with the ER alpha gene. After correction for multiple comparisons by means of permutation analysis, the associations with the factor 'non-conformity'--including the subscales 'indirect aggression' and 'irritability'--and the factor 'psychoticism'--including the subscale 'suspicion'--remained significant. The results suggest that the studied dinucleotide repeat polymorphism of the ER alpha gene may contribute to specific components of personality.