Effects of IKAP/hELP1 Deficiency on Gene Expression in Differentiating Neuroblastoma Cells: Implications for Familial Dysautonomia

Familial dysautonomia (FD) is a developmental neuropathy of the sensory and autonomous nervous systems. The IKBKAP gene, encoding the IKAP/hELP1 subunit of the RNA polymerase II Elongator complex is mutated in FD patients, leading to a tissue-specific mis-splicing of the gene and to the absence of the protein in neuronal tissues. To elucidate the function of IKAP/hELP1 in the development of neuronal cells, we have downregulated IKBKAP expression in SHSY5Y cells, a neuroblastoma cell line of a neural crest origin. We have previously shown that these cells exhibit abnormal cell adhesion when allowed to differentiate under defined culture conditions on laminin substratum. Here, we report results of a microarray expression analysis of IKAP/hELP1 downregulated cells that were grown on laminin under differentiation or non-differentiation growth conditions. It is shown that under non-differentiation growth conditions, IKAP/hELP1 downregulation affects genes important for early developmental stages of the nervous system, including cell signaling, cell adhesion and neural crest migration. IKAP/hELP1 downregulation during differentiation affects the expression of genes that play a role in late neuronal development, in axonal projection and synapse formation and function. We also show that IKAP/hELP1 deficiency affects the expression of genes involved in calcium metabolism before and after differentiation of the neuroblastoma cells. Hence, our data support IKAP/hELP1 importance in the development and function of neuronal cells and contribute to the understanding of the FD phenotype

Identification and Validation of Novel Cerebrospinal Fluid Biomarkers for Staging Early Alzheimer's Disease

Ideally, disease modifying therapies for Alzheimer disease (AD) will be applied during the 'preclinical' stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome.CSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aβ42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85-0.94 95% confidence interval [CI]) and 0.88 (0.81-0.94 CI), respectively.Four novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aβ42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions

Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host-adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development

Analysis of Nodular Cast Iron Microstructures for Micromechanical Model Development

One of the main factors in determining the different grades of ductile iron is the matrix structure. In the as-cast condition, the matrix will consist of varying proportions of pearlite and ferrite, and as the amount of pearlite increases, the strength and hardness of the iron also increase. Three different nodular cast irons are here considered and their microstructure characterised in detail using metallographic methods. Then micromechanics models based on the unit cell approach and the finite element method are introduced to describe the actual constitutive response of the materials and the predicted behaviours are compared with experiments

Usprawnienia rekultywacji podłoża w praktyce

The article deals with the innovation of substrate for land reclamation purposes made by the company OBSED a. s. It proposes a new and more economical composition of the reclamation substrate. It brings the results of the ecotoxicity tests applied on the pre-determined ratios of the individual components of the substrate. The ecotoxicity tests made used of white mustard (Sinapis alba) seeded in this reclamation substrate and its modifications. The article describes the manufacturing process of the original subsurface substrate for reclamation purposes and the carried out ecotoxicity tests on various quantitative proportions of the materials contained in the substrate. The aspect of innovation is grounded in altering the ratios of the input raw materials, including testing ecotoxicity of the new and more economical reclamation substrate, which still offers identical technical parameters as the previous reclamation substrate.W artykule opisano innowacyjne podłoże do celów melioracyjnych wykonanych przez firmę OBSED s.r. ekotoksyczności dla zaproponowanych różnych receptur podłoża. Testy ekotoksyczności wykonano używając nasion gorczycy białej (Sinapis alba) zasianych na podłożu do rekultywacji oraz jego modyfikacjach. W artykule przedstawiono proces produkcji oryginalnego podłoża w celach rekultywacji podpowierzchniowej i przeprowadzonych badań ekotoksyczności, w różnych proporcjach ilościowych materiałów zawartych w podłożu. Aspekt innowacji zawiera się w zmiennych proporcjach surowców wejściowych, oraz w badaniu ekotoksyczności nowego i bardziej ekonomicznego podłoża do rekultywacji, który charakteryzuje się takimi samymi parametrami technicznymi jak stosowane podłoża do rekultywacji

Influence of layer-wise fabrication and surface orientation on the notch fatigue behavior of as-built additively manufactured Ti6Al4V

Design and qualification of load-bearing metal parts produced by the additive manufacturing technology is a critical issue. Such metal parts are complex in geometry with notches that are critical locations under fatigue loading. Notch surfaces are typically in the as-built state because post-fabrication surface finishing is not a viable approach in most applications. Here the combined effect of an as-built surface and of a geometrical notch on the fatigue behavior of L-PBF Ti6Al4V was investigated using a new test methodology. Notches of different severity in specimens manufactured according to different directions with respect to the build direction were considered. The intrinsic as-built surface roughness of L-PBF process combined with the layer-wise fabrication of curved notch surfaces resulted in a surface quality strongly affecting notch fatigue behavior. An original interpretative framework of the notch fatigue behavior of L-PBF Ti6Al4V is proposed and discussed

New types of lead-free solders and their properties

The aim of this work is an experimental study of lead-free solders. Ternary and binary alloys with different ratios of individual elements Ag, Al, Bi, Cu, In, Mg, Sb, Sn and Zn were prepared experimentally. The study of low-fusing solder alloys was performed with the aspect of observing their selected physical, chemical, structural and technological properties. The following characteristics were studied: temperatures and enthalpies of phase transformations (DTA, TG, DSC) of individual solders at the rates of re-heating and cooling of specimens of about 4 degrees C/min, macro-and micro-structural analysis (optical metallography), micro-hardness, chemical analysis: ICP-AES, optical emission spectrometry (OES), X-ray micro-analysis of individual phases in the structure of solders (WDX, EDX), measurement of density and electrical resistivity of selected solders in dependence on the temperature, test of wettability with or without use of fluxes, measurement of corrosion properties

New types of lead-free solders and their properties

The aim of this work is an experimental study of lead-free solders. Ternary and binary alloys with different ratios of individual elements Ag, Al, Bi, Cu, In, Mg, Sb, Sn and Zn were prepared experimentally. The study of low-fusing solder alloys was performed with the aspect of observing their selected physical, chemical, structural and technological properties. The following characteristics were studied: temperatures and enthalpies of phase transformations (DTA, TG, DSC) of individual solders at the rates of re-heating and cooling of specimens of about 4 degrees C/min, macro-and micro-structural analysis (optical metallography), micro-hardness, chemical analysis: ICP-AES, optical emission spectrometry (OES), X-ray micro-analysis of individual phases in the structure of solders (WDX, EDX), measurement of density and electrical resistivity of selected solders in dependence on the temperature, test of wettability with or without use of fluxes, measurement of corrosion properties