We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic.

Aggregation of α-synuclein, the hallmark of α-synucleinopathies such as Parkinson’s disease, occurs in various glycosphingolipidoses. Although α-synuclein aggregation correlates with deficiencies in the lysosomal degradation of glycosphingolipids (GSL), the mechanism(s) involved in this aggregation remains unclear. We previously described the aggregation of α-synuclein in Krabbe’s disease (KD), a neurodegenerative glycosphingolipidosis caused by lysosomal deficiency of galactosyl-ceramidase (GALC) and the accumulation of the GSL psychosine. Here, we used a multi-pronged approach including genetic, biophysical and biochemical techniques to determine the pathogenic contribution, reversibility, and molecular mechanism of aggregation of α-synuclein in KD. While genetic knock-out of α-synuclein reduces, but does not completely prevent, neurological signs in a mouse model of KD, genetic correction of GALC deficiency completely prevents α-synuclein aggregation. We show that psychosine forms hydrophilic clusters and binds the C-terminus of α-synuclein through its amino group and sugar moiety, suggesting that psychosine promotes an open/aggregation-prone conformation of α-synuclein. Dopamine and carbidopa reverse the structural changes of psychosine by mediating a closed/aggregation-resistant conformation of α-synuclein. Our results underscore the therapeutic potential of lysosomal correction and small molecules to reduce neuronal burden in α-synucleinopathies, and provide a mechanistic understanding of α-synuclein aggregation in glycosphingolipidoses.

Krabbe's disease (KD) is primarily a demyelinating disorder, but recent studies have identified the presence of neuronal protein aggregates in the brain, at least partially composed by alpha-synuclein (α-syn). The role of this protein aggregation in the pathogenesis of KD is largely unknown, but it has added KD to a growing list of lysosomal storage diseases that can be also be considered as proteinopathies. While the presence of these protein aggregates within the KD brain is now appreciated, the remainder of the central nervous system (CNS) remains uncharacterized. This study is the first to report the presence of thioflavin-S reactive inclusions throughout the spinal cord of both murine and human spinal tissue. Stereological analysis revealed the temporal and spatial accumulation of these inclusions within the neurons of the ventral spinal cord vs. those located in the dorsal cord. This study also confirmed that these thio-S positive accumulations are present within neuronal populations and are made up at least in part by α-syn in both the twitcher mouse and cord autopsied material from affected human patients. Significantly, neonatal gene therapy for galactosylceramidase, a treatment that strongly improves the survival and health of KD mice, but not bone marrow transplantation prevents the formation of these inclusions in spinal neurons. These results expand the understanding of α-syn protein aggregation within the CNS of individuals afflicted with KD and underlines the tractability of this problem via early gene therapy, with potential impact to other synucleinopathies such as PD.

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.

A mega-project is a major project or a group of projects of significant cost that attract a high level of public attention or political interest because of substantial direct and indirect impacts on the community, environment, and state budget. Capturing and sharing the knowledge from the performance of the current mega projects is essential in order to avoid losing vital corporate knowledge assets in the construction industry. The learned lessons are gained from experience, success, and failure for improving future performance. This research aims to review and read out the lessons learned from 77 research papers that have dealt with the barriers that hinder the successful performance of mega building construction projects in developing countries, identify and classify the main obstacles, and propose improvements for successful implementation and management of mega building construction projects. The results of this paper will help project owners, construction companies, and other stakeholders in developing countries to overcome the limitations in the execution of mega building construction projects.

Lumbar disc replacement has become an area of interest for many practicing spine surgeons. As newer technology emerges focusing on motion sparing devices, novel techniques are being employed to better serve patients and increase post-operative outcomes. Traditionally, the anterior approach is utilized in total disc arthroplasty of the lumbar spine, although many are beginning to favor the use of a lateral approach. The lateral approach not only allows for the maintenance of the Anterior Longitudinal Ligament (ALL), but also avoids the great vessels during surgery which are encountered in the traditional anterior approach. Research has shown the lateral approach in total disc replacements (TDR) of the lumbar spine to be a not inferior alternative to the anterior approach, with long-term pain relief and a low complication rate.

Concrete cracking and defect patterns can often indicate its causes. In this research paper, detailed description of cracks types, causes, and proper solutions is identified. The main objective of the paper is to measure the response of specialists about the probability of certain cracks causes and solutions, and to evaluate the effect of using epoxy materials in repairing concrete cracks. The present study shows that about 50% of responders indicated that cracks due to steel corrosion represent between 10-20% of building cracks. And about 80% of the responders indicated that crack due to load increasing is rarely occurred in the site. The interviewed specialists ensured that using epoxy materials are effective in solving most cracks’ problems. The experimental work results show that the flexural strength of epoxy repaired samples exceeded that of original samples. And the increase in flexural strength is inversely proportioned with sample depth.