3D Texture Analysis Reveals Imperceptible MRI Textural Alterations in the Thalamus and Putamen in Progressive Myoclonic Epilepsy Type 1, EPM1

Progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessively inherited neurodegenerative disorder characterized by young onset age, myoclonus and tonic-clonic epileptic seizures. At the time of diagnosis, the visual assessment of the brain MRI is usually normal, with no major changes found later. Therefore, we utilized texture analysis (TA) to characterize and classify the underlying properties of the affected brain tissue by means of 3D texture features. Sixteen genetically verified patients with EPM1 and 16 healthy controls were included in the study. TA was performed upon 3D volumes of interest that were placed bilaterally in the thalamus, amygdala, hippocampus, caudate nucleus and putamen. Compared to the healthy controls, EPM1 patients had significant textural differences especially in the thalamus and right putamen. The most significantly differing texture features included parameters that measure the complexity and heterogeneity of the tissue, such as the co-occurrence matrix-based entropy and angular second moment, and also the run-length matrix-based parameters of gray-level non-uniformity, short run emphasis and long run emphasis. This study demonstrates the usability of 3D TA for extracting additional information from MR images. Textural alterations which suggest complex, coarse and heterogeneous appearance were found bilaterally in the thalamus, supporting the previous literature on thalamic pathology in EPM1. The observed putamenal involvement is a novel finding. Our results encourage further studies on the clinical applications, feasibility, reproducibility and reliability of 3D TA.     

Utility of plasma apelin and other indices of cardiac dysfunction in the clinical assessment of patients with dilated cardiomyopathy

Apelin is a recently discovered peptide ligand reported to be involved in the regulation of cardiovascular homeostasis. The exact role of apelin in the pathophysiology of congestive heart failure has remained obscure, and the reported circulating levels of apelin in patients with heart failure have been contradictory. To establish the role of apelin in the assessment of cardiac dysfunction we measured plasma apelin levels in 65 patients with congestive heart failure caused by idiopathic dilated cardiomyopathy (IDC) and 14 healthy volunteers by specific radioimmunoassay. IDC patients were carefully examined including echocardiography, both-sided cardiac catheterization and cardiopulmonary exercise test. In addition, plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), N-terminal pro-atrial natriuretic peptide (NT-proANP), interleukin (IL)-6, tumor necrosis factor alpha (TNF-), epinephrine and norepinephrine were determined. Plasma apelin levels were similar in IDC patients (median 26.5 pg/ml, range < 3.40–97.6 pg/ml) and in control subjects (median 24.1 pg/ml, range 19.0–28.7 pg/ml; p = NS). Unlike the levels of NT-proBNP, IL-6, TNF-, and norepinephrine, plasma apelin levels did not reflect the severity of heart failure. Our study demonstrates that although disturbed apelin–APJ signalling in heart may play a role in the pathophysiology of heart failure, circulating apelin levels cannot be applied in the clinical assessment of patients with chronic left ventricular dysfunction.     

Verification and quantification of saxitoxin from algal samples using fast and validated hydrophilic interaction liquid chromatography-tandem mass spectrometry method

Hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was validated with algal samples for verification and quantification of saxitoxin (STX), a potent neurotoxin which is listed in the Chemical Weapons Convention (CWC) in Schedule 1A. Isocratic elution, conventional bore HILIC column and high flow rate together with accurate post-column splitter provided detection of STX in 6.5 min with total analysis time of 9 min per sample. STX analogue, gonyautoxin 1 (GTX 1) was used as an internal standard. Sample preparation of freeze-dried algae included liquid extraction and centrifugal filtering with mean recovery of 99.9% at concentration level of 10 ng/ml (n=3). Retention times for STX and GTX 1 were 6.47±0.03 min and 4.44±0.01 min (n=45), respectively. Four diagnostic product ions were used for reliable verification of saxitoxin. Method was found to be precise and linear (R(2)=0.9714 and R(2)=0.9768) in concentration ranges of 5-50 ng/ml and 25-200 ng/ml, respectively. For saxitoxin, calculated LOD was 3 ng/ml and LLOQ 11 ng/ml. Validation was conducted using spiked algal matrix since this method is not only needed for verification analysis for the CWC but also for safety analysis of other environmental samples for presence of STX. Identification criteria for verification of STX with HILIC-MS/MS method are discussed.     

Interactions of hydrophobin proteins in solution studied by small-angle X-ray scattering

Hydrophobins are a group of very surface-active, fungal proteins known to self-assemble on various hydrophobic/hydrophilic interfaces. The self-assembled films coat fungal structures and mediate their attachment to surfaces. Hydrophobins are also soluble in water. Here, the association of hydrophobins HFBI and HFBII from Trichoderma reesei in aqueous solution was studied using small-angle x-ray scattering. Both HFBI and HFBII exist mainly as tetramers in solution in the concentration range 0.5-10 mg/ml. The assemblies of HFBII dissociate more easily than those of HFBI, which can tolerate changes of pH from 3 to 9 and temperatures in the range 5°C-60°C. The self-association of HFBI and HFBII is mainly driven by the hydrophobic effect, and addition of salts along the Hofmeister series promotes the formation of larger assemblies, whereas ethanol breaks the tetramers into monomers. The possibility that the oligomers in solution form the building blocks of the self-assembled film at the air/water interface is discussed.     

Studies of crystallinity of Scots pine and Norway spruce cellulose

The variation in the mass fraction of crystalline cellulose (crystallinity of wood), the intrinsic crystallinity of cellulose, and the thickness of cellulose crystallites in early wood of Norway spruce [Picea abies (L.) Karst.], and Scots pine (Pinus sylvestris L.) grown in Finland were studied using wide angle X-ray scattering and nuclear magnetic resonance spectroscopy. The mass fraction of crystalline cellulose in wood increased slightly with the distance from the pith and was about 30±4% in mature wood of both species. The crystallinity of cellulose and the thickness of cellulose crystallites were almost constant for both species. The crystallinity of cellulose was 52±3% for both species and the average thickness of the cellulose crystallites was 32±1 Å and 31±1 Å for Norway spruce and Scots pine, respectively. The mass fraction of cellulose in wood, calculated from the crystallinity values, increased with the distance from the pith for both species.     

X-ray microdiffraction reveals the orientation of cellulose microfibrils and the size of cellulose crystallites in single Norway spruce tracheids

The microfibril angle (MFA) distribution and the size of cellulose crystallites in isolated double cell walls of Norway spruce (Picea abies [L.] Karst.) tracheids were determined by synchrotron X-ray microdiffraction using the reflections 200 and 004. Samples were 25 μm thick longitudinal sections of earlywood from annual rings 6–18 of several stems. The asymmetric MFA distributions extended from ?20° to 90°. The mean MFA of tangential cell walls decreased from an average of 24° into 19° from the pith to the bark. The mode of the MFA distribution was about 10° smaller than the mean MFA. The standard deviation of the MFA distribution varied between 18° and 25°. The mean MFA and the mode of the MFA distribution were larger in radial than in tangential cell walls. MFA distributions of mature wood samples exhibited a separate small peak at around 90°. The average width and length of cellulose crystallites varied between 28.9–30.9 Å and 192–284 Å, respectively. Both increased slightly as a function of annual ring number from the pith up to the 15th annual ring. An irrigation–fertilisation treatment of some of the stems resulted in longer cellulose crystallites compared to the untreated stems.     

Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.     

A Cryptic Frizzled Module in Cell Surface Collagen 18 Inhibits Wnt/β?Catenin Signaling

Collagens contain cryptic polypeptide modules that regulate major cell functions, such as cell proliferation or death. Collagen XVIII (C18) exists as three amino terminal end variants with specific amino terminal polypeptide modules. We investigated the function of the variant 3 of C18 (V3C18) containing a frizzled module (FZC18), which carries structural identity with the extracellular cysteine-rich domain of the frizzled receptors. We show that V3C18 is a cell surface heparan sulfate proteoglycan, its topology being mediated by the FZC18 module. V3C18 mRNA was expressed at low levels in 21 normal adult human tissues. Its expression was up-regulated in fibrogenesis and in small well-differentiated liver tumors, but decreased in advanced human liver cancers. Low FZC18 immunostaining in liver cancer nodules correlated with markers of high Wnt/β−catenin activity. V3C18 (M_r = 170 kD) was proteolytically processed into a cell surface FZC18-containing 50 kD glycoprotein precursor that bound Wnt3a in vitro through FZC18 and suppressed Wnt3a-induced stabilization of β−catenin. Ectopic expression of either FZC18 (35 kD) or its 50 kD precursor inhibited Wnt/β−catenin signaling in colorectal and liver cancer cell lines, thus downregulating major cell cycle checkpoint gatekeepers cyclin D1 and c-myc and reducing tumor cell growth. By contrast, full-length V3C18 was unable to inhibit Wnt signaling. In summary, we identified a cell-surface signaling pathway whereby FZC18 inhibits Wnt/β−catenin signaling. The signal, encrypted within cell-surface C18, is released by enzymatic processing as an active frizzled cysteine-rich domain (CRD) that reduces cancer cell growth. Thus, extracellular matrix controls Wnt signaling through a collagen-embedded CRD behaving as a cell-surface sensor of proteolysis, conveying feedback cues to control cancer cell fate.     

Bacterial Exposures and Associations with Atopy and Asthma in Children

Background

The increase in prevalence of asthma and atopic diseases in Western countries has been linked to aspects of microbial exposure patterns of people. It remains unclear which microbial aspects contribute to the protective farm effect.

Objective

The objective of this study was to identify bacterial groups associated with prevalence of asthma and atopy, and to quantify indoor exposure to some of these bacterial groups.

Methods

A DNA fingerprinting technique, denaturing gradient gel electrophoresis (DGGE), was applied to mattress dust samples of farm children and control children in the context of the GABRIEL Advanced study. Associations between signals in DGGE and atopy, asthma and other allergic health outcomes were analyzed. Quantitative DNA based assays (qPCR) for four bacterial groups were applied on the dust samples to seek quantitative confirmation of associations indicated in DNA fingerprinting.

Results

Several statistically significant associations between individual bacterial signals and also bacterial diversity in DGGE and health outcomes in children were observed. The majority of these associations showed inverse relationships with atopy, less so with asthma. Also, in a subsequent confirmation study using a quantitative method (qPCR), higher mattress levels of specifically targeted bacterial groups - Mycobacterium spp., Bifidobacteriaceae spp. and two different clusters of Clostridium spp. - were associated with a lower prevalence of atopy.

Conclusion

DNA fingerprinting proved useful in identifying bacterial signals that were associated with atopy in particular. These findings were quantitatively confirmed for selected bacterial groups with a second method. High correlations between the different bacterial exposures impede a clear attribution of protective effects to one specific bacterial group. More diverse bacterial flora in mattress dust may link to microbial exposure patterns that protect against development of atopic diseases.     

Polarized transport of Alzheimer amyloid precursor protein is mediated by adaptor protein complex AP1-1B

Alzheimer amyloid precursor protein (APP) is the precursor for the Abeta peptide involved in pathogenesis of Alzheimer's disease. The soluble ectodomain fragment of APP (sAPP) functions as a growth factor for epithelial cells, suggesting an important function for APP outside neuronal tissue. Previous studies have shown that in polarized epithelial cells, APP is targeted to the basolateral domain. Tyr653 within the cytoplasmic tail of APP mediates the basolateral targeting of APP, but the sorting machinery that binds to this residue has largely remained unknown. In this study, we analyzed the role of adaptor complexes in the polarized sorting of APP. We show that the medium subunit mu1B of the epithelia-specific adaptor protein (AP)-1B binds onto the cytoplasmic tail of APP in a Tyr653-dependent way. Moreover, ectopic expression of mu1B in cells lacking AP-1B resulted in correction of apical missorting of wild-type but not Tyr653Ala APP. Basolateral secretion of sAPP was found to be independent of Tyr653. We propose a model for polarized targeting of APP according to which sorting of APP to basolateral domain is dependent on binding of AP-1B on Tyr653 in basolateral endosomes. This model is in accordance with the current understanding of sorting mechanisms mediating polarized targeting of membrane proteins.