VCP protects neurons from proteopathic seeding

Uptake and spread of proteopathic seeds, such as αS, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of αS seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase αS seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of αS seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to αS since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.

Airbrushed Nanofibrous Membranes to Control Stem Cell Infiltration In 3D Printed Scaffolds

Extrusion-based 3D printing of polymeric biomaterials has emerged as a promising approach for the fabrication of complex tissue engineering constructs. However, the large pore and feature size lead to low cell seeding efficiency and limited control of spatial distribution of cells within the scaffolds. We developed hybrid scaffolds that are composed of 3D printed layers and airbrushed fibrous membranes. Airbrushing time was adjusted to fabricate low (L), medium (M), and high (H) density membranes to effectively control stem cell infiltration. When two distinct populations of stem cells were seeded from top or bottom of the scaffolds, scaffolds composed of LLL membranes showed gradual mixing of the cells with depth whereas LHL membranes led to two distinct regions of cells separated by the H membrane. Our results demonstrate that fibrous membranes incorporated within 3D printed layers enable user-defined and spatially controlled cell compositions within hybrid scaffolds.

Seeding Propensity and Characteristics of Pathogenic αSyn Assemblies in Formalin-Fixed Human Tissue from the Enteric Nervous System, Olfactory Bulb, and Brainstem in Cases Staged for Parkinson’s Disease

We investigated α-synuclein’s (αSyn) seeding activity in tissue from the brain and enteric nervous system. Specifically, we assessed the seeding propensity of pathogenic αSyn in formalin-fixed tissue from the gastric cardia and five brain regions of 29 individuals (12 Parkinson’s disease, 8 incidental Lewy body disease, 9 controls) using a protein misfolding cyclic amplification assay. The structural characteristics of the resultant αSyn assemblies were determined by limited proteolysis and transmission electron microscopy. We show that fixed tissue from Parkinson’s disease (PD) and incidental Lewy body disease (ILBD) seeds the aggregation of monomeric αSyn into fibrillar assemblies. Significant variations in the characteristics of fibrillar assemblies derived from different regions even within the same individual were observed. This finding suggests that fixation stabilizes seeds with an otherwise limited seeding propensity, that yield assemblies with different intrinsic structures (i.e., strains). The lag phase preceding fibril assembly for patients ≥80 was significantly shorter than in other age groups, suggesting the existence of increased numbers of seeds or a higher seeding potential of pathogenic αSyn with time. Seeding activity did not diminish in late-stage disease. No statistically significant difference in the seeding efficiency of specific regions was found, nor was there a relationship between seeding efficiency and the load of pathogenic αSyn in a particular region at a given neuropathological stage.

Comparison of size distribution and (Pro249-Ser258) epitope exposure in in vitro and in vivo derived Tau fibrils

Background Although several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity. Methods Tau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors. Results We observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients. Conclusions This study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.

Effect of crop establishment methods on yield and economics of cultivation of wheat

Zero till seed-cum-ferti drill, rice-wheat seeder and broadcasting method, as treatments T1 , T2 & T3 respectively, were used for wheat sowing at 100.0 kg/ha seed rate in pulverized sandy loam soil. Rice-wheat seeder delivered optimum seed rate upon maintenance of minimum of 3.75 kg seed per box. At every 52.0 meter run of machine 0.5 kg seed was to be filled in seed boxes to maintain uniform seeding at 100.0 kg/ha seed rate. The machines in treatment T1 and T2 had field capacities 0.49 ha/h and 0.169 ha/h and field efficiencies 71% & 82% respectively. Rice wheat seeder had registered 96.19% seeding efficiency in comparison to 97.67% seeding efficiency of seed drill. Maximum yield (4.27 t) was obtained in treatment T2, followed by treatment T1 (4.186 t) and T3 (4.08 t). Benefit-cost ratio of treatment T3 was found highest 4.77:1 followed by treatment T2 (4.66:1) in spite of minimum net return per hectare (Rs.71607.0) among all treatments. Highest net return/ ha of Rs.74454.0 was in treatment T2 , followed by treatment T1 (Rs.72089.0). Obviously, yield and economics of use of rice wheat seeder equipment justifies its utility for wheat cultivation by small and marginal farmers.

Electrodeposition of Hydroxyapatite on a Metallic 3D-Woven Bioscaffold

In this study, we demonstrate that a uniform coating of hydroxyapatite (HAp, Ca10(PO4)6(OH)2) can be electrochemically deposited onto metallic 3D-woven bone scaffolds to enhance their bioactivity. The HAp coatings were deposited onto metallic scaffolds using an electrolyte containing Ca(NO3)2·4H2O, NH4H2PO4, and NaNO3. The deposition potential was varied to maximize the uniformity and adhesion of the coating. Using X-ray diffraction (XRD), Raman spectroscopy, and energy-dispersive spectroscopy (EDS), we found crystallized HAp on the 3D-woven lattice under all deposition potentials, while the −1.5 V mercury sulfate reference electrode potential provided the best local uniformity with a satisfactory deposition rate. The coatings generated under this optimized condition were approximately 5 µm thick and uniform throughout the internal and external sections of the woven lattice. We seeded and cultured both coated and uncoated scaffolds with human adipose-derived stromal/stem cells (ASCs) for 12 h and 4 days. We observed that the HAp coating increased the initial cell seeding efficiency by approximately 20%. Furthermore, after 4 days of culture, ASCs cultured on HAp-coated stainless-steel scaffolds increased by 32% compared to only 17% on the uncoated scaffold. Together, these results suggest that the HAp coating improves cellular adhesion.

Tissue Engineered Esophageal Patch by Mesenchymal Stromal Cells: Optimization of Electrospun Patch Engineering

Aim of work was to locate a simple, reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited, namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size, pore orientation, porosity, and pore distribution were produced using two different techniques, such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity, seeding efficiency, and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method, as these conditions successfully improved the cellularization of polymeric patches. Furthermore, the investigation provided interesting information on patches’ stability in physiological simulating experimental conditions. Considering the in vitro results, it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover, the protocol turned out to be simple, repeatable, and reproducible.