Relationship between white matter alteration and encoding related brain activation in connected brain regions

Background: Aging is associated with alterations of white matter and brain activation. Functional MRI studies in elderly subjects showed changes in encoding related brain activation such as hyperactivation in frontal areas and hypoactivation in occipital gyrus in comparison to a younger control group. A contributing factor could be alterations of white matter integrity, resulting from age-related small vessel disease (SVD), a pathology that effects the small vessels of the brain or Wallerian Degeneration (WD) that explains axonal degeneration distal of an injury. These processes lead to changes such as white matter hyperintensities (WMH) detected by MRI or microstructural change assessed by the diffusion tensor imaging (DTI), marker mean diffusivity (MD) or peak width (PW). It was hypothesized that the directionality of the structure-function relationship of the brain is dependent on the investigated brain region. We aim to verify this assumption. Other studies focused on frontal brain regions. Instead we implemented a whole-brain analysis of the structure-function relationship. Therefore, the goal of this study was to investigate changes in white matter as a predictor of changed encoding-related brain activation in anatomically connected brain regions in cognitively normal performing older subjects. Furthermore, there are different theories that try to explain the changed brain activation in association with white matter change, such as compensatory mechanisms, dedifferentiation theory or inefficient neuronal processes. To gain a better understanding we examined the association between decreased brain activation respectively increased white matter changes in relation to cognitive performance of the subjects. Methods: Cognitively healthy elderly subjects (N = 35) performed a face-name matching paradigm within the fMRI scanner with the encoding phase being relevant in the present study. The integrity of white matter was determined with measurement of WMH volume and DTI based markers such as MD and PW. We performed ANOVAs with DTI-markers as dependent and activation as the independent variable. Furthermore, we performed ANOVAs with white matter change or brain activation as dependent variable and cognitive performance as independent variable. Since we assumed that there are local differences of white matter change we created boxplots for the chosen MD, PW and WMH-ratio within the chosen fiber tracts and global MD, PW and WMH-ratio. Additionally, we computed a correlation matrix between tract-specific MD or PW for a comparison of these two markers. Results: We could demonstrate a significant positive association between PW in the inferior fronto-occipital fasciculus left (IFOF L) and the activation in the left frontal gyrus as well as PW in the inferior longitudinal fasciculus right (ILF R) and activation in the occipital gyrus. Furthermore, the data revealed no significant result for the relationship between white matter change and cognition or brain activation and cognition. The boxplot showed a significant difference between the white matter tracts when using MD and PW as marker. Because of its low burden we had to exclude WMH-ratio as a marker for white matter change. The correlation-matrix revealed that PW within the tracts correlated less with each other than MD. Conclusion: These results suggest that microstructural changes lead to increased brain activation due to decreased white matter connectivity and reduced fidelity of data transmission. Additionally, the subjects' cognitive performance appears not to benefit from the increased brain activation. Thus, the negative structure-function relationship seems not to be based on a compensatory mechanism or dedifferentiation theory but most likely on an inefficient neuronal response. White matter change can be considered as regionally variable as revealed by the boxplots and the correlation matrix. However, the structure-function relation seems not be dependent on brain region, because the whole brain analysis showed a consistent directionality of the structure-function relationship

Cell surface marker mediated purification of iPS cell intermediates from a reprogrammable mouse model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).Christian M. Nefzger, Sara Alaei, Anja S. Knaupp, Melissa L. Holmes, Jose M. Pol

Affinity capillary electrophoresis - mass spectrometry permits direct binding assessment of IgG and FcγRIIa in a glycoform-resolved manner

The impact of antibody glycoforms on FcγRIIa activation and immune responses is poorly understood. Yet, glycoform binding assessment remains one of the major analytical challenges requiring long enrichment or glycoengineering steps. Here, we developed and applied an affinity capillary electrophoresis-mass spectrometry approach to selectively assess the binding of different antibody glycoforms to the FcγIIa receptor without the need of glycoengineering. The approach required only low microgram amounts of antibody and receptor and enables assessing the binding of high and low-abundance glycoforms. The approach indicated clear differences in binging between doubly-, hemi-glycosylated and non-glycosylated antibodies as well as for mutated (Leu234Ala, Leu235Ala - Pro329-Gly (LALA-PG)) IgG1 antibodies silenced for Fcγ binding. The LALA-PG mutated antibody showed no binding to the FcγIIa receptor (excluding potential non-specific binding effects) while the non-glycosylated IgG1 showed a strongly reduced, but still minor binding. The highest binding affinity was for the antibody carrying two complex-type glycans. Man5 glycans resulted in decreased binding compared to complex-type glycans, with the lowest binding for the IgG containing two Man5. For complex-type glycans, galactosylation showed a subtle increase in binding to the FcγIIa receptor, and sialylation showed an increase in binding for lower sialylated species. Fucosylation did not influence binding to the FcγIIa receptor. Finally, the assay was evaluated for the two variants of the FcγRIIa receptor (allotypes H131 and R131) showing highly comparable glycoform selectivity. Overall, the proposed approach allows the direct comparison of binding affinities of different antibody species in mixtures promising a fast establishment of their structure-function relationships

Evaluación de sistema de guiado para la toma de muestras de compactación edáfica a campo

La adopción del uso de la agricultura de precisión, al igual que el manejo por ambientes de los lotes, se ha visto incrementada en los últimos años y en particular en la última década, debido al desarrollo masivo de dispositivos electrónicos confiables y también a una baja relativa en los costos de los mismos. En el marco de los proyectos de investigación dependientes de la Secretaria de Investigación y Vinculación Tecnológica de la Universidad Nacional de Moreno (UNM) se llevó a cabo, en conjunto con el INTA, el desarrollo de un sistema de medición de compactación edáfica “Penetrómetro Digital” (Figura 1). Dicho dispositivo desarrollado enteramente entre la UNM y el Laboratorio de Agroelectrónica del Instituto de Ingeniería Rural del INTA permite registrar el índice de cono a intervalos de profundidad de 1cm. Para poder adaptar este dispositivo de diagnóstico básico de compactación a la agricultura moderna, se lo dotó de un GPS de forma nativa y se le agregaron varias funcionalidades que facilitan la toma de muestras en los lotes. El equipo permite la carga de una lista georeferenciada de los puntos en donde se deben realizar las mediciones y mediante esta información asiste al usuario, guiándolo dentro del lote para que pueda encontrar estos puntos y realizar las mediciones.Instituto de Ingeniería RuralFil: Moltoni, Andrés Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Ingeniería Rural; ArgentinaFil: Masiá, Gerardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Ingeniería Rural; ArgentinaFil: Fiorini, Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Ingeniería Rural; ArgentinaFil: Knaupp, Lucas. Universidad Nacional de Moreno; ArgentinaFil: Clemares, Nicolás. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Ingeniería Rural; ArgentinaFil: Duró, Sebastián. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Ingeniería Rural; ArgentinaFil: Moltoni, Luciana A. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Ingeniería Rural; Argentin

Impact of the 237th Residue on the Folding of Human Carbonic Anhydrase II

The deficiency of human carbonic anhydrase II (HCAII) has been recognized to be associated with a disease called CAII deficiency syndrome (CADS). Among the many mutations, the P237H mutation has been characterized to lead to a significant decrease in the activity of the enzyme and in the Gibbs free energy of folding. However, sequence alignment indicated that the 237th residue of CAII is not fully conserved across all species. The FoldX theoretical calculations suggested that this residue did not significantly contribute to the overall folding of HCAII, since all mutants had small ΔΔG values (around 1 kcal/mol). The experimental determination indicated that at least three mutations affect HCAII folding significantly and the P237H mutation was the most deleterious one, suggesting that Pro237 was important to HCAII folding. The discrepancy between theoretical and experimental results suggested that caution should be taken when using the prediction methods to evaluate the details of disease-related mutations

Affinity capillary electrophoresis-mass spectrometry as a tool to unravel proteoform-specific antibody-receptor interactions

Monoclonal antibody (mAb) pharmaceuticals consist of a plethora of different proteoforms with different functional characteristics, including pharmacokinetics and pharmacodynamics, requiring their individual assessment. Current binding techniques do not distinguish between coexisting proteoforms requiring tedious production of enriched proteoforms. Here, we have developed an approach based on mobility shift-affinity capillary electrophoresis-mass spectrometry (ACE-MS), which permitted us to determine the binding of coexisting mAb proteoforms to Fc receptors (FcRs). For high-sensitivity MS analysis, we used a sheathless interface providing adequate mAb sensitivity allowing functional characterization of mAbs with a high sensitivity and dynamic range. As a model system, we focused on the interaction with the neonatal FcR (FcRn), which determines the half-life of mAbs. Depending on the oxidation status, proteoforms exhibited different electrophoretic mobility shifts in the presence of FcRn, which could be used to determine their affinity. We confirmed the decrease of the FcRn affinity with antibody oxidation and observed a minor glycosylation effect, with higher affinities for galactosylated glycoforms. Next to relative binding, the approach permits the determination of individual K-D values in solution resulting in values of 422 and 139 nM for double-oxidized and non-oxidized variants. Hyphenation with native MS provides unique capabilities for simultaneous heterogeneity assessment for mAbs, FcRn, and complexes formed. The latter provides information on binding stoichiometry revealing 1:1 and 1:2 for antibody/FcRn complexes. The use of differently engineered Fc-only constructs allowed distinguishing between symmetric and asymmetric binding. The approach opens up unique possibilities for proteoform-resolved antibody binding studies to FcRn and can be extended to other FcRs and protein interactions.Proteomic

A versatile strategy for isolating a highly enriched population of intestinal stem cells

The isolation of pure populations of mouse intestinal stem cells (ISCs) is essential to facilitate functional studies of tissue homeostasis, tissue regeneration, and intestinal diseases. However, the purification of ISCs has relied predominantly on the use of transgenic reporter alleles in mice. Here, we introduce a combinational cell surface marker-mediated strategy that allows the isolation of an ISC population transcriptionally and functionally equivalent to the gold standard Lgr5-GFP ISCs. Used on reporter-free mice, this strategy allows the isolation of functional, transcriptionally distinct ISCs uncompromised by Lgr5 haploinsufficiency.Christian M. Nefzger, Thierry Jardé, Fernando J.Rossello, Katja Horvay, Anja S.Knaupp, David R.Powell ... et al

The pathological Trento variant of alpha-1-antitrypsin (E75V) shows non-classical behaviour during polymerization

Severe alpha-1-antitrypsin deficiency (AATD) is most frequently associated with the alpha-1-antitrypsin (AAT) Z variant (E342K). ZZ homozygotes exhibit accumulation of AAT as polymers in the endoplasmic reticulum of hepatocytes. This protein deposition can lead to liver disease, with the resulting low circulating levels of AAT predisposing to early-onset emphysema due to dysregulation of elastinolytic activity in the lungs. An increasing number of rare AAT alleles have been identified in patients with severe AATD, typically in combination with the Z allele. Here we report a new mutation (E75V) in a patient with severe plasma deficiency, which we designate Trento. In contrast to the Z mutant, Trento AAT was secreted efficiently when expressed in cellular models but showed compromised conformational stability. PAGE and ELISA-based analyses of the secreted protein revealed the presence of oligomeric species with electrophoretic and immunorecognition profiles different from those of Z and S (E264V) AAT polymers, including reduced recognition by conformational monoclonal antibodies 2C1 and 4B12. This altered recognition was not due to direct effects on the epitope of the 2C1 monoclonal antibody which we localised between helices E and F. Structural analyses indicate the likely basis for polymer formation is the loss of a highly conserved stabilising interaction between helix C and the post-helix I loop. These results highlight this region as important for maintaining native state stability and, when compromised, results in the formation of pathological polymers that are different from those produced by Z and S AAT. This article is protected by copyright. All rights reserved