Disease-related changes in the cerebrospinal fluid metabolome in amyotrophic lateral sclerosis detected by GC/TOFMS

Background/Aim

The changes in the cerebrospinal fluid (CSF) metabolome associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are poorly understood and earlier smaller studies have shown conflicting results. The metabolomic methodology is suitable for screening large cohorts of samples. Global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as CSF.

Methodology

Using gas chromatography coupled to mass spectrometry (GC/TOFMS) and multivariate statistical modeling, we simultaneously studied the metabolome signature of ∼120 small metabolites in the CSF of patients with ALS, stratified according to hereditary disposition and clinical subtypes of ALS in relation to controls.

Principal Findings

The study is the first to report data validated over two sub-sets of ALS vs. control patients for a large set of metabolites analyzed by GC/TOFMS. We find that patients with sporadic amyotrophic lateral sclerosis (SALS) have a heterogeneous metabolite signature in the cerebrospinal fluid, in some patients being almost identical to controls. However, familial amyotrophic lateral sclerosis (FALS) without superoxide dismutase-1 gene (SOD1) mutation is less heterogeneous than SALS. The metabolome of the cerebrospinal fluid of 17 ALS patients with a SOD1 gene mutation was found to form a separate homogeneous group. Analysis of metabolites revealed that glutamate and glutamine were reduced, in particular in patients with a familial predisposition. There are significant differences in the metabolite profile and composition among patients with FALS, SALS and patients carrying a mutation in the SOD1 gene suggesting that the neurodegenerative process in different subtypes of ALS may be partially dissimilar.

Conclusions/Significance

Patients with a genetic predisposition to amyotrophic lateral sclerosis have a more distinct and homogeneous signature than patients with a sporadic disease.     

Different capacity of monocyte subsets to phagocytose iron-oxide nanoparticles

Objective

To explore the capacity of human CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes to phagocyte iron-oxide nanoparticles in vitro.

Methods

Human monocytes were labeled with four different magnetic nanoparticle preparations (Ferumoxides, SHU 555C, CLIO-680, MION-48) exhibiting distinct properties and cellular uptake was quantitatively assessed by flow cytometry, fluorescence microscopy, atomic absorption spectrometry and Magnetic Resonance Imaging (MRI). Additionally we determined whether cellular uptake of the nanoparticles resulted in phenotypic changes of cell surface markers.

Results

Cellular uptake differed between the four nanoparticle preparations. However for each nanoparticle tested, CD14⁺⁺CD16^- monocytes displayed a significantly higher uptake compared to CD14⁺CD16⁺⁺ monocytes, this resulted in significantly lower T1 and T2 relaxation times of these cells. The uptake of iron-oxide nanoparticles further resulted in a remarkable shift of expression of cell surface proteins indicating that the labeling procedure affects the phenotype of CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes differently.

Conclusion

Human monocyte subsets internalize different magnetic nanoparticle preparations differently, resulting in variable loading capacities, imaging phenotypes and likely biological properties.     

Workers dominate male production in the neotropical bumblebee Bombus wilmattae (Hymenoptera: Apidae)

Background

Cooperation and conflict in social insects are closely linked to the genetic structure of the colony. Kin selection theory predicts conflict over the production of males between the workers and the queen and between the workers themselves, depending on intra-colonial relatedness but also on other factors like colony efficiency, sex ratios, cost of worker reproduction and worker dominance behaviour. In most bumblebee (Bombus) species the queen wins this conflict and often dominates male production. However, most studies in bumblebees have been conducted with only a few selected, mostly single mated species from temperate climate regions. Here we study the genetic colony composition of the facultative polyandrous neotropical bumblebee Bombus wilmattae, to assess the outcome of the queen-worker conflict over male production and to detect potential worker policing.

Results

A total of 120 males from five colonies were genotyped with up to nine microsatellite markers to infer their parentage. Four of the five colonies were queen right at point of time of male sampling, while one had an uncertain queen status. The workers clearly dominated production of males with an average of 84.9% +/- 14.3% of males being worker sons. In the two doubly mated colonies 62.5% and 96.7% of the male offspring originated from workers and both patrilines participated in male production. Inferring the mother genotypes from the male offspring, between four to eight workers participated in the production of males.

Conclusions

In this study we show that the workers clearly win the queen-worker conflict over male production in B. wilmattae, which sets them apart from the temperate bumblebee species studied so far. Workers clearly dominated male production in the singly as well the doubly mated colonies, with up to eight workers producing male offspring in a single colony. Moreover no monopolization of reproduction by single workers occurred.     

Towards automated production and drug sensitivity testing using scaffold-free spherical tumor microtissues

Although the relevance of three-dimensional (3-D) culture has been recognized for years and exploited at an academic level, its translation to industrial applications has been slow. The development of reliable high-throughput technologies is clearly a prerequisite for the industrial implementation of 3-D models. In this study the robustness of spherical microtissue production and drug testing in a 96-well hanging-drop multiwell plate format was assessed on a standard 96-well channel robotic platform. Microtissue models derived from six different cell lines were produced and characterized according to their growth profile and morphology displaying high-density tissue-like reformation and growth over at least 15 days. The colon cancer cell line HCT116 was chosen as a model to assess microtissue-based assay reproducibility. Within three individual production batches the size variations of the produced microtissues were below 5%. Reliability of the microtissue-based assay was tested using two reference compounds, staurosporine and chlorambucil. In four independent drug testings the calculated IC(50) values were benchmarked against 2-D multiwell testings displaying similar consistency. The technology presented here for the automated production of a variety of microtissues for efficacy testing in a standard 96-well format will aid the implementation of more organotypic models at an early time point in the drug discovery process.     

High genetic diversity despite the potential for stepping-stone colonizations in an invasive species of gecko on Moorea, French Polynesia

Invasive species often have reduced genetic diversity, but the opposite can be true if there have been multiple introductions and genetic admixture. Reduced diversity is most likely soon after establishment, in remote locations, when there is lower propagule pressure and with stepping-stone colonizations. The common house gecko (Hemidactylus frenatus) was introduced to Moorea, French Polynesia in the remote eastern Pacific within the last two decades and accordingly is expected to exhibit low diversity. In contrast, we show that H. frenatus on Moorea has exceptionally high genetic diversity, similar to that near the native range in Asia and much higher than reported for other Pacific island reptiles. The high diversity in this recently founded population likely reflects extensive genetic admixture in source population(s) and a life history that promotes retention of diversity. These observations point to the importance of understanding range-wide dynamics of genetic admixture in highly invasive species.     

Fetal renal and blood pressure responses to steroid infusion after early prenatal treatment with dexamethasone

Maternal infusion of dexamethasone for 48 h early in gestation results in upregulation of mRNA for mineralocorticoid and glucocorticoid (MR and GR) receptors and angiotensin II receptors in ovine fetal kidneys late in gestation. This study sought to determine whether dexamethasone exposure results in changes in renal function and blood pressure responsiveness to infused cortisol or aldosterone in the late-gestation fetus. Merino ewes carrying single fetuses were infused with isotonic saline (Sal; n = 9) or dexamethasone (Dex, 0.48 mg/h; n = 10) for 48 h between days 26 and 28 of gestation (term = 150 days). At 115-122 days, renal function and blood pressure were measured in fetuses during a 4-h infusion of saline, cortisol (100 microg/h), or aldosterone (5 microg/h). Infusions were given in random order at least 2 days apart. Basal blood pressure and renal function were similar in Sal and Dex groups and did not change over the course of saline infusion. Cortisol infusion caused similar increases in blood pressure, urine flow, and glomerular filtration rate (GFR) in the groups. Aldosterone infusion caused a significantly different GFR response between the groups [P(treatment x time) < 0.05], but increase in K excretion and decrease in Na-to-K ratio were similar in the groups. The similar results obtained with cortisol and aldosterone infusion suggest no increased renal functional maturity to those hormones after early prenatal dexamethasone exposure. This suggests that changes in mRNA for MR and GR in kidneys of dexamethasone-exposed fetuses do not result in functional differences and highlights the renin-angiotensin system, as reported previously, as more important in this model.     

Tissue-specific localization of gibberellins and expression of gibberellin-biosynthetic and signaling genes in wood-forming tissues in aspen

Bioactive gibberellins (GAs) are known regulators of shoot growth and development in plants. In an attempt to identify where GAs are formed, we have analyzed the expression patterns of six GA biosynthesis genes and two genes with predicted roles in GA signaling and responses in relation to measured levels of GAs. The analysis was based on tangential sections, giving tissue-specific resolution across the cambial region of aspen trees (Populus tremula). Gibberellin quantification by GC/MS-SRM showed that the bioactive GA1 and GA4 were predominantly located in the zone of expansion of xylem cells. Based on co-localization of the expression of the late GA biosynthesis gene GA 20-oxidase 1 and bioactive GAs, we suggest that de novo GA biosynthesis occurs in the expanding xylem. However, expression levels of the first committed GA biosynthesis enzyme, ent-copalyl diphosphate synthase, were high in the phloem, suggesting that a GA precursor(s) may be transported to the xylem. The expression of the GA signaling and response genes DELLA-like1 and GIP-like1 coincided well with sites of high bioactive GA levels. We therefore suggest that the main role of GA during wood formation is to regulate early stages of xylem differentiation, including cell elongation.     

In-vitro analysis of the expression of TGFbeta -superfamily-members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during dedifferentiation in cell culture

Traditional surgical methods for the reconstruction of cartilage defects rely on the transplantation of autologous and allogenous tissues. The disadvantages of these techniques are the limited availability of suitable tissues and the donor site morbidity of transplants. In addition, in cultured chondrocytes, the dedifferentiation of cells seems unavoidable during multiplication. In this study, we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) and human mesenchymal stem cells (MSC) in cell culture using microarray technique, immunohistochemistry and RT-PCR. Transforming growth factor beta (TGFbeta) is a multifunctional peptide that plays play a crucial role in inducing and maintaining chondrogenic differentiation. In dedifferentiating chondrocytes, the gene for TGFbeta1 was constantly expressed, while the gene for TGFbeta2 was never expressed. The genes for TGFalpha, TGFbeta4 and TGFbetai were activated with ongoing dedifferentiation. TGFbeta-receptor 3 was constantly expressed, while the genes for the TGFbeta-receptors 1 and 2 were never expressed. Immunohistochemical staining for TGFbeta beta 3 revealed upregulation in the course of dedifferentiation. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation, whereas the gene for LTBP3 was constantly expressed, and negative results were obtained for the gene for LTBP4. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation. During chondrogenic differentiation, the MSCs constantly expressed TGFbeta1, beta2, beta3 and beta4. LTBP1, LTBP2 and TGFbeta-R3 were downregulated. In conclusion, TGFbeta3, TGFbeta4, TGFbetai, LTBP1 and LTBP2 may assist the process of dedifferentiation, while TGFbeta1 and beta2 might not be involved in this process. Of the TGFbeta-receptors, only type 3 might be involved in dedifferentiation.     

Plasmacytoid dendritic cells control TLR7 sensitivity of naive B cells via type I IFN

Detailed information of human B cell activation via TLR may lead to a better understanding of B cell involvement in autoimmunity and malignancy. In this study we identified a fundamental difference in the regulation of TLR7- and TLR9-mediated B cell stimulation: whereas the induction of polyclonal naive B cell proliferation by the TLR7 ligands resiquimod (R848) and loxoribine required the presence of plasmacytoid dendritic cells (PDCs), activation via the TLR9 ligand CpG was independent of PDCs. We found that PDC-derived type I IFN enhanced TLR7 sensitivity of B cells by selectively up-regulating TLR7 expression. In contrast the expression levels of TLR9 and of other TLRs studied remained unchanged. In the presence of type I IFN, TLR7 ligation triggered polyclonal B cell expansion and B cell differentiation toward Ig-producing plasma cells; notably, this occurred independently of T cell help and B cell Ag. Human B cells did not respond to ligands of other TLRs including TLR2, TLR4 and TLR6 with and without type I IFN. In conclusion, our results reveal a distinct regulation of TLR7 and TLR9 function in human B cells and highlight TLR7 and TLR9 as unique targets for therapeutic intervention in B cell-mediated immunity and disease.