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Neuroinflammation is an established factor contributing to amyotrophic lateral sclerosis (ALS) pathology, implicating the possible detrimental effects of inflammatory cytokines on motor neurons. The RNA/DNA-binding protein TDP-43 has emerged as a pivotal actor in ALS, because TDP-43 mutations cause familial ALS and loss of nuclear TDP-43, associated with its redistribution into cytoplasmic aggregates (TDP-43 proteinopathy) in motor neurons occurs in 97% of ALS cases. However, mechanisms linking neuroinflammation to TDP-43 mislocalization have not been described. Programmed death-ligand 1 (PD-L1) is an immune-modulatory protein, highly expressed on cell surfaces following acute inflammatory stress. To determine which inflammatory cytokines might impact motor neuron function, seven cytokines known to be elevated in ALS patients’ cerebrospinal fluid were tested for their effects on PD-L1 expression in human iPSC-derived motor neurons. Among the tested cytokines, only interferon-γ (IFN-γ) was found to strongly promote PD-L1 expression. Thus, we hypothesized that excessive exposure to IFN-γ may contribute to motor neuron degeneration in ALS. We observed that neuronal populations exposed to IFN-γ exhibited severe TDP-43 cytoplasmic aggregation and excitotoxic behavior correlated with impaired neural firing activity, hallmarks of ALS pathology, in both normal and ALS mutant (TARDB1K+/-) neurons. Single-cell RNA sequencing revealed possible mechanisms for these effects. Motor neurons exposed to IFN-γ exhibited an extensive shift of their gene expression profile toward a neurodegenerative phenotype. Notably, IFN-γ treatment induced aberrant expression levels for 70 genes that are listed in the recent literature as being dysregulated in various ALS subtypes. Additionally, we found that genes related to neuronal electrophysiology, protein aggregation, and TDP-43 misregulation were abnormally expressed in IFN-γ treated cells. Moreover, IFN-γ induced a significant reduction in the expression of genes that encode indispensable proteins for neuromuscular synapse development and maintenance, implying that the continuous cytokine exposure could directly impair signal transmission between motor axons and muscle membranes. Our findings suggest that IFN-γ could be a potent upstream pathogenic driver of ALS and provide potential candidates for future therapeutic targets to treat sporadic forms of ALS, which account for roughly 90% of reported cases.

Cells are exquisitely receptive to the physical cues present within their native microenvironment. Manipulation of substrate topography is therefore a simple strategy to promote the development of cells in vitro toward a phenotype that is more representative of their in vivo counterparts. In the cases of cardiac and skeletal muscle, substrate topographies have been used to promote uniaxial alignment, myofibrillar development, and cytoskeletal organization in cultured cells for downstream applications in basic biological studies, disease modelling, and drug screening. In this chapter, we review the advantages conferred on muscle cultures by topographic patterns, discuss methods for producing patterns of different dimensions, and provide a perspective on the role these technologies could play in enhancing the predictive power of next generation preclinical assays.

Charcot-Marie-Tooth disease type 2D (CMT2D), is a hereditary peripheral neuropathy caused by mutations in the gene encoding glycyl-tRNA synthetase (GARS1). Here, human induced pluripotent stem cell (hiPSC)-based models of CMT2D bearing mutations in GARS1 and their use for the identification of predictive biomarkers amenable to therapeutic efficacy screening is described. Cultures containing spinal cord motor neurons generated from this line exhibited network activity marked by significant deficiencies in spontaneous action potential firing and burst fire behavior. This result matched clinical data collected from a patient bearing a GARS1(P724H) mutation and was coupled with significant decreases in acetylated α-tubulin levels and mitochondrial movement within axons. Treatment with HDAC6 inhibitors, tubastatin A and CKD504, improved mitochondrial movement and α-tubulin acetylation in these cells. Furthermore, CKD504 treatment enhanced population-level electrophysiological activity, highlighting its potential as an effective treatment for CMT2D.

Neurons derived from human induced pluripotent stem cells (hiPSCs) are powerful tools for modeling neural pathophysiology and preclinical efficacy/toxicity screening of novel therapeutic compounds. However, human neurons cultured in vitro typically do not fully recapitulate the physiology of the human nervous system, especially in terms of exhibiting morphological maturation, longevity, and electrochemical signaling ability comparable to that of adult human neurons. In this study, we investigated the potential for astrocyte-derived extracellular vesicles (EVs) to modulate survival and electrophysiological function of human neurons in vitro. Specifically, we demonstrate that EVs obtained from human astrocytes promote enhanced single cell electrophysiological function and anti-apoptotic behavior in a homogeneous population of human iPSC-derived cortical neurons. Furthermore, EV-proteomic analysis was performed to identify cargo proteins with the potential to promote the physiological enhancement observed. EV cargos were found to include neuroprotective proteins such as heat shock proteins, alpha-synuclein, and lipoprotein receptor-related protein 1 (LRP1), as well as apolipoprotein E (APOE), which negatively regulates neuronal apoptosis, and a peroxidasin homolog that supports neuronal oxidative stress management. Proteins that positively regulate neuronal excitability and synaptic development were also detected, such as potassium channel tetramerization domain containing 12 (KCTD12), glucose-6- phosphate dehydrogenase (G6PD), kinesin family member 5B (KIF5B), spectrin-alpha non-erythrocytic1 (SPTAN1). The remarkable improvements in electrophysiological function and evident inhibition of apoptotic signaling in cultured neurons exposed to these cargos may hold significance for improving preclinical in vitro screening modalities. In addition, our collected data highlight the potential for EV-based therapeutics as a potential class of future clinical treatment for tackling inveterate central and peripheral neuropathies.

Despite advances in surgery, chemotherapy and radiotherapy, the outcomes of patients with GBM have not significantly improved. Tumor recurrence in the resection margins occurs in more than 80 % of cases indicating aggressive treatment modalities, such as gene therapy are warranted. We have examined photochemical internalization (PCI) as a method for the non-viral transfection of the cytosine deaminase (CD) suicide gene into glioma cells. The CD gene encodes an enzyme that can convert the nontoxic antifungal agent, 5-fluorocytosine, into the chemotherapeutic drug, 5-fluorouracil. Multicell tumor spheroids derived from established rat and human glioma cell lines were used as in vitro tumor models. Plasmids containing either the CD gene alone or together with the uracil phosphoribosyl transferase (UPRT) gene combined with the gene carrier protamine sulfate were employed in all experiments. PCI was performed with the photosensitizer AlPcS2a and 670 nm laser irradiance. Protamine sulfate/CD DNA polyplexes proved nontoxic but inefficient transfection agents due to endosomal entrapment. In contrast, PCI mediated CD gene transfection resulted in a significant inhibition of spheroid growth in the presence of, but not in the absence of, 5-FC. Repetitive PCI induced transfection was more efficient at low CD plasmid concentration than single treatment. The results clearly indicate that AlPcS2a-mediated PCI can be used to enhance transfection of a tumor suicide gene such as CD, in malignant glioma cells and cells transfected with both the CD and UPRT genes had a pronounced bystander effect.

The Wando grand bridge has been designed in 2003 and main structure has constructed in 2010. The bridge is composed of 75 m steel tower, 500 m steel girder and 32 cables. This paper presents the design, fabrication, erection, and cable installation of the bridge.The main bridge is 500m long and composed of 70 m, 90 m, 200 m, and 140 m spans. Super structure of Wando grand bridge is orthotropic steel deck with U-rib. The width of the deck is 24.8 m and is composed of four lanes for vehicle + one lane for pedestrian. Diaphragm is located at every 6 m, and interval of U-rib is 0.64 m. The material for main plates is SM520B(tensile strength is 520 MPa) which is used widely for bridge superstructure in Korea. Multi strand cables are installed at 32 points in a 2-face fan type. Total length of cable is 3,587 m and the total length of strand is 188,266 m. The ultimate tensile strength of strand, Fpu is 1,770 MPa. Allowable strength of cable is 0.45 Fpu at completed stage, 0.65 Fpu at erection stage, respectively.The verticality of tower was given as 1/2000 at construction site. Considering construction error, the target for verticality was set to 1/5000. To get the accurate straightness, every structural element at the internal joint of pylon was milled using facing machine. Girder was diveded to nine segments in which 4 segments have been erected by crawler crane and others have been erected by floating crane. As the bridge was constructed at shallow sea zone, special floating crane having 2 booms and 4 hooks was utilized.Geometric control was utilized to make accurate erection. At the first step, weight of some blocks were measured in factory using load cell and compared with shop drawing net weight. The target value of geometric control of the bridge is ± 60mm for girder deflection, ± 35mm for tower horizontal displacement, and 10% for cable tension. Construction has been performed successfully, and there is no adjustment for cable tension. Cable has been installed at average three days erection cycle. As the cable is mono strand type of Freyssinet, ISO tensioning method has been used.The pavement for the bridge was finished last year and bridge has been opened.

A dynamic light scattering (DLS) method was adopted for measuring the corrosion of iron nanoparticles. The average diameter of the nanoparticles in a sodium chloride suspension increased linearly with time as iron oxide layers formed around the nanoparticles. The nanoparticle corrosion rate determined by DLS was found to be almost identical to the value obtained by conventional immersion tests (ASTM G31). The DLS method offers the advantage that measurements may be completed within several hours under natural corrosion conditions whereas the conventional immersion method requires several months. Application of the DLS method to alloy nanoparticles with a variety of chromium compositions showed that the nanoparticle sizes changed nonlinearly over time, and the curves were best fit by a first order exponential function. The first order time constants were found to be linearly related to the corrosion rates determined by ASTM G31.

A facile and sensitive immunoassay protocol for the detection of alpha-fetoprotein (AFP) was developed using gold-coated iron oxide magnetic nanoclusters and dynamic light scattering (DLS) methods. The increase in the average particle size due to AFP-mediated aggregation was measured using DLS, and the detection limit was better than 0.01 ng mL(-1).