PEP-1-GLRX1 Reduces Dopaminergic Neuronal Cell Loss by Modulating MAPK and Apoptosis Signaling in Parkinson's Disease.

Parkinson's disease (PD) is characterized mainly by the loss of dopaminergic neurons in the substantia nigra (SN) mediated via oxidative stress. Although glutaredoxin-1 (GLRX1) is known as one of the antioxidants involved in cell survival, the effects of GLRX1 on PD are still unclear. In this study, we investigated whether cell-permeable PEP-1-GLRX1 inhibits dopaminergic neuronal cell death induced by 1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We showed that PEP-1-GLRX1 protects cell death and DNA damage in MPP+-exposed SH-SY5Y cells via the inhibition of MAPK, Akt, and NF-κB activation and the regulation of apoptosis-related protein expression. Furthermore, we found that PEP-1-GLRX1 was delivered to the SN via the blood-brain barrier (BBB) and reduced the loss of dopaminergic neurons in the MPTP-induced PD model. These results indicate that PEP-1-GLRX1 markedly inhibited the loss of dopaminergic neurons in MPP+- and MPTP-induced cytotoxicity, suggesting that this fusion protein may represent a novel therapeutic agent against PD.

All-in-One Fusogenic Nanoreactor for the Rapid Detection of Exosomal MicroRNAs for Breast Cancer Diagnosis.

Molecular-profiling-based cancer diagnosis has significant implications for predicting disease prognosis and selecting targeted therapeutic interventions. The analysis of cancer-derived extracellular vesicles (EVs) provides a noninvasive and sequential method to assess the molecular landscape of cancer. Here, we developed an all-in-one fusogenic nanoreactor (FNR) encapsulating DNA-fueled molecular machines (DMMs) for the rapid and direct detection of EV-associated microRNAs (EV miRNAs) in a single step. This platform was strategically designed to interact selectively with EVs and induce membrane fusion under a specific trigger. After fusion, the DMMs recognized the target miRNA and initiated nonenzymatic signal amplification within a well-defined reaction volume, thus producing an amplified fluorescent signal within 30 min. We used the FNRs to analyze the unique expression levels of three EV miRNAs in various biofluids, including cell culture, urine, and plasma, and obtained an accuracy of 86.7% in the classification of three major breast cancer (BC) cell lines and a diagnostic accuracy of 86.4% in the distinction between patients with cancer and healthy donors. Notably, a linear discriminant analysis revealed that increasing the number of miRNAs from one to three improved the accuracy of BC patient discrimination from 78.8 to 95.4%. Therefore, this all-in-one diagnostic platform performs nondestructive EV processing and signal amplification in one step, providing a straightforward, accurate, and effective individual EV miRNA analysis strategy for personalized BC treatment.

Bonding of Flexible Membranes for Perfusable Vascularized Networks Patch.

BACKGROUND: In vitro generation of three-dimensional vessel network is crucial to investigate and possibly improve vascularization after implantation in vivo. This work has the purpose of engineering complex tissue regeneration of a vascular network including multiple cell-type, an extracellular matrix, and perfusability for clinical application. METHODS: The two electrospun membranes bonded with the vascular network shape are cultured with endothelial cells and medium flow through the engineered vascular network. The flexible membranes are bonded by amine-epoxy reaction and examined the perfusability with fluorescent beads. Also, the perfusion culture for 7 days of the endothelial cells is compared with static culture on the engineered vascular network membrane. RESULTS: The engineered membranes are showed perfusability through the vascular network, and the perfused network resulted in more cell proliferation and variation of the shear stress-related genes expression compared to the static culture. Also, for the generation of the complex vascularized network, pericytes are co-cultured with the engineered vascular network, which results in the Collagen I is expressed on the outer surface of the engineered structure. CONCLUSION: This study is showing the perfusable in vitro engineered vascular network with electrospun membrane. In further, the 3D vascularized network module can be expected as a platform for drug screening and regenerative medicine.

Febrile Seizures Cause Depression and Anxiogenic Behaviors in Rats.

Febrile seizure (FS) is a common type of seizure occurring in human during infancy and childhood. Although an epileptic seizure is associated with psychiatric disorders and comorbid diseases such as depression, anxiety, autism spectrum disorders, sleep disorders, attention deficits, cognitive impairment, and migraine, the causal relationship between FS and psychiatric disorders is poorly understood. The objective of the current study was to investigate the relationship of FS occurrence in childhood with the pathogenesis of anxiety disorder and depression using an FS rat model. We induced febrile seizures in infantile rats (11 days postnatal) using a mercury vapor lamp. At 3 weeks and 12 weeks after FS induction, we examined behaviors and recorded local field potentials (LFPs) to assess anxiety and depression disorder. Interestingly, after FS induction in infantile rats, anxiogenic behaviors and depression-like phenotypes were found in both adult and juvenile FS rats. The analysis of LFPs revealed that 4-7 Hz hippocampal theta rhythm, a neural oscillatory marker for anxiety disorder, was significantly increased in FS rats compared with their wild-type littermates. Taken together, our findings suggest that FS occurrence in infants is causally related to increased levels of anxiety-related behaviors and depression-like symptoms in juvenile and adult rodents.

Phosphodiesterase-5 Inhibitor Attenuates Anxious Phenotypes and Movement Disorder Induced by Mild Ischemic Stroke in Rats.

OBJECTIVE: Patients with mild ischemic stroke experience various sequela and residual symptoms, such as anxious behavior and deficits in movement. Few approaches have been proved to be effective and safe therapeutic approaches for patients with mild ischemic stroke by acute stroke. Sildenafil (SIL), a phosphodiesterase-5 inhibitor (PDE5i), is a known remedy for neurodegenerative disorders and vascular dementia through its angiogenesis and neurogenesis effects. In this study, we investigated the efficacy of PDE5i in the emotional and behavioral abnormalities in rats with mild ischemic stroke. METHODS: We divided the rats into four groups as follows (n=20, respectively) : group 1, naïve; group 2, middle cerebral artery occlusion (MCAo30); group 3, MCAo30+SIL-pre; and group 4, MCAo30+SIL-post. In the case of drug administration groups, single dose of PDE5i (sildenafil citrate, 20 mg/kg) was given at 30-minute before and after reperfusion of MCAo in rats. After surgery, we investigated and confirmed the therapeutic effect of sildenafil on histology, immunofluorescence, behavioral assays and neural oscillations. RESULTS: Sildenafil alleviated a neuronal loss and reduced the infarction volume. And results of behavior task and immunofluorescence shown possibility that anti-inflammation process and improve motor deficits sildenafil treatment after mild ischemic stroke. Furthermore, sildenafil treatment attenuated the alteration of theta-frequency rhythm in the CA1 region of the hippocampus, a known neural oscillatory marker for anxiety disorder in rodents, induced by mild ischemic stroke. CONCLUSION: PDE5i as effective therapeutic agents for anxiety and movement disorders and provide robust preclinical evidence to support the development and use of PDE5i for the treatment of mild ischemic stroke residual disorders.

Isolation and compositional analysis of galactoglycerolipids from perilla [Perilla frutescens (L.) Britton] leaves and comparison to the galactoglycerolipids from spinach and parsley.

The aim of this study was to isolate monogalactosyldiacylglycerols (MGDGs) and digalactosyldiacylglycerols (DGDGs) from perilla [Perilla frutescens (L.) Britton] and to investigate their fatty acid profiles. Perilla displayed the greatest total MGDG and DGDG content among the three types of leaf vegetables tested, that is, spinach, parsley, and perilla, containing 0.16 g/100 g MGDG and 0.04 g/100 g DGDG (on wet weight basis). High purity MGDG (approximately 97 g/100 g) and DGDG (approximately 86 g/100 g) were isolated from perilla chloroform/methanol (2:1, v/v) extracts by two-step silica gel column chromatography. MGDGs were primarily composed of 18:3n-3 and 16:3n-3, predominantly located at the sn-1 and sn-2 positions, respectively. In DGDG, 18:3n-3 and 16:0 were the most abundant fatty acids and were primarily found at the sn-1 and sn-2 positions, respectively. PRACTICAL APPLICATION: MGDGs and DGDGs are the most prevalent forms of galactoglycerolipids found in leaf vegetables including perilla and have been shown to exert health-beneficial effects, such as antitumor, anti-inflammatory, anticancer, and appetite-suppressing activities. Both MGDGs and DGDGs possess emulsifying properties. The present study may help better understand the health-beneficial effects of MGDG and DGDG from perilla, by providing total composition and positional distribution of the fatty acids. The present study also successfully established a protocol to isolate high purity MGDG and DGDG from perilla, thereby increasing their possible use as an ingredient in foods and nutraceuticals.

Enzymatic preparation of food-grade l-α-glycerylphosphorylcholine from soy phosphatidylcholine or fractionated soy lecithin.

l-α-Glycerylphosphorylcholine (l-α-GPC) is a biosynthetic precursor for the neurotransmitter acetylcholine in humans, making it a useful as a cognitive enhancer for treating patients with stroke and dementia, including Alzheimer's disease. The aim of this study was to prepare l-α-GPC via Novozym 435 (an immobilized Candida antarctica lipase B)-catalyzed hydrolysis of soy phosphatidylcholine or a fractionated soy lecithin, from which triacylglycerols were completely removed, followed by food-grade solvent extraction of l-α-GPC from the reaction products. The reaction was performed in n-hexane-water biphasic media in a stirred-batch reactor. Phosphatidylcholine was completely hydrolyzed to l-α-GPC under optimal conditions: temperature, 55°C; water content, 100 wt% of the substrate weight; enzyme loading, 10 wt% of the substrate weight; and reaction time of 6 hr (for soy phosphatidylcholine) or 8 hr (for fractionated soy lecithin). Water-soluble fractions of the reaction products containing 98.6 area% l-α-GPC (from soy phosphatidylcholine) or 52.4 area% glycerophosphodiesters, including l-α-GPC (from fractionated soy lecithin), were obtained after phase separation of the media. The resulting products would be suitable for use as food-grade cognitive enhancers because of the use of enzymatic reaction and food-grade solvent extraction.

Immobilized Phospholipase A1-Catalyzed Preparation of l-α-Glycerylphosphorylcholine from Phosphatidylcholine.

This study sought to prepare a cognitive enhancer l-α-glycerylphosphorylcholine (l-α-GPC) using an immobilized Lecitase Ultra (LU, phospholipase A1) to catalyze the hydrolysis of soy phosphatidylcholine (PC). Immobilization of LU on Lewatit VP OC 1600 provided the highest fixation level (83.1 g/100 g) and greatest catalytic activity achieving 100 g/100 g l-α-GPC within 20 h and was therefore selected as the optimal system for biocatalysis. Immobilization of LU increased its positional specificity compared to free LU, as shown by a decrease in the production of the phosphocholine byproduct. Under the optimal conditions determined by response surface methodology, PC was completely hydrolyzed to l-α-GPC and required a simple purification via phase separation of the biphasic media to obtain a yield of ∼26.4 g l-α-GPC from 100 g PC, with a purity of 98.5 g/100 g. Our findings suggest a possibility of using the immobilized LU as a new biocatalyst for the l-α-GPC production.

Blood Oxygenation Using Fluoropolymer-Based Artificial Lung Membranes.

Artificial lung (AL) membranes are used for blood oxygenation for patients undergoing open-heart surgery or acute lung failures. Current AL technology employs polypropylene and polymethylpentene membranes. Although effective, these membranes suffer from low biocompatibility, leading to undesired blood coagulation and hemolysis over a long term. In this work, we propose a new generation of AL membranes based on amphiphobic fluoropolymers. We employed poly(vinylidene-co-hexafluoropropylene), or PVDF-co-HFP, to fabricate macrovoid-free membranes with an optimal pore size range of 30-50 nm. The phase inversion behavior of PVDF-co-HFP was investigated in detail for structural optimization. To improve the wetting stability of the membranes, the fabricated membranes were coated using Hyflon AD60X, a type of fluoropolymer with an extremely low surface energy. Hyflon-coated materials displayed very low protein adsorption and a high contact angle for both water and blood. In the hydrophobic spectrum, the data showed an inverse relationship between the surface free energy and protein adsorption, suggesting an appropriate direction with respect to biocompatibility for AL research. The blood oxygenation performance was assessed using animal sheep blood, and the fabricated fluoropolymer membranes showed competitive performance to that of commercial polyolefin membranes without any detectable hemolysis. The data also confirmed that the bottleneck in the blood oxygenation performance was not the membrane permeance but rather the rate of mass transfer in the blood phase, highlighting the importance of efficient module design.

Biometric Image Analysis for Quantitation of Dividing Platelets.

(1) Background: Quantification of platelet division is challenging because automated Coulter cell counters produce equivocal platelet counts. (2) Methods: We applied the flow cytometric cell tracking dye dilution assay as a popular immunological method to evaluate lymphocyte proliferation to prove and quantitate platelet division. We also devised a method relying on platelet culture in a semisolid medium which enabled dividing platelets to be identified by limiting the diffusive movement of platelets. Mixing platelets of different labeling colors in semisolid medium and counting the platelet doublets of each color combination enabled us to prove and quantitate platelet division. (3) Results: The tracking dye dilution assay revealed that 75.5 to 85.6% of platelets were dividing after 20 hours in culture. Platelets labeled with two different tracking dyes were mixed and cultured in semisolid medium for differential doublet counting. We counted platelet singlets and doublets of each color and color combination using confocal microscopy after six hours of culture and compared the relative number of two-colored doublets with binomial prediction to prove platelet division (P < 0.01). Division was suppressed by taxol, nocodazole, or cytochalasin D treatment. We derived a formula for determining the fraction of dividing platelets using the numbers of singlets and doublets of each color and color combination. The platelet division fraction ranged from 8.8 to 17.5%. (4) Conclusion: We successfully measured platelet division using a simple biometric image analysis method with possible future application to microfluidic devices.