Enhanced Vascular-like Network Formation of Encapsulated HUVECs and ADSCs Coculture in Growth Factors Conjugated GelMA Hydrogels.

Tissue engineering primarily aimed to alleviate the insufficiency of organ donations worldwide. Nonetheless, the survival of the engineered tissue is often compromised due to the complexity of the natural organ architectures, especially the vascular system inside the organ, which allows food-waste transfer. Thus, vascularization within the engineered tissue is of paramount importance. A critical aspect of this endeavor is the ability to replicate the intricacies of the extracellular matrix and promote the formation of functional vascular networks within engineered constructs. In this study, human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured in different types of gelatin methacrylate (GelMA). In brief, pro-angiogenic signaling growth factors (GFs), vascular endothelial growth factor (VEGF165) and basic fibroblast growth factor (bFGF), were conjugated onto GelMA via an EDC/NHS coupling reaction. The GelMA hydrogels conjugated with VEGF165 (GelMA@VEGF165) and bFGF (GelMA@bFGF) showed marginal changes in the chemical and physical characteristics of the GelMA hydrogels. Moreover, the conjugation of these growth factors demonstrated improved cell viability and cell proliferation within the hydrogel construct. Additionally, vascular-like network formation was observed predominantly on GelMA@GrowthFactor (GelMA@GF) hydrogels, particularly on GelMA@bFGF. This study suggests that growth factor-conjugated GelMA hydrogels would be a promising biomaterial for 3D vascular tissue engineering.

Coumarin-chalcone hybrid LM-021 and indole derivative NC009-1 targeting inflammation and oxidative stress to protect BE(2)-M17 cells against α-synuclein toxicity.

Parkinson's disease (PD) is featured mainly by the loss of dopaminergic neurons and the presence of α-synuclein-containing aggregates in the substantia nigra of brain. The α-synuclein fibrils and aggregates lead to increased oxidative stress and neural toxicity in PD. Chronic inflammation mediated by microglia is one of the hallmarks of PD pathophysiology. In this report, we showed that coumarin-chalcone hybrid LM-021 and indole derivative NC009-1 reduced the expression of major histocompatibility complex-II, NLR family pyrin domain containing (NLRP) 3, caspase-1, inducible nitric oxide synthase, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in α-synuclein-activated mouse BV-2 microglia. Release of pro-inflammatory mediators including nitric oxide, IL-1ß, IL-6 and TNF-α was also mitigated. In BE(2)-M17 cells expressing A53T α-synuclein aggregates, LM-021 and NC009-1 reduced α-synuclein aggregation, neuroinflammation, oxidative stress and apoptosis, and promoted neurite outgrowth. These protective effects were mediated by downregulating NLRP1, IL-1ß and IL-6, and their downstream pathways including nuclear factor (NF)-κB inhibitor alpha (IκBα)/NF-κB P65 subunit (P65), c-Jun N-terminal kinase (JNK)/proto-oncogene c-Jun (JUN), mitogen-activated protein kinase 14 (P38)/signal transducer and activator of transcription (STAT) 1, and Janus kinase 2 (JAK2)/STAT3. The study results indicate LM-021 and NC009-1 as potential new drug candidates for PD.

Using ΔK280 TauRD Folding Reporter Cells to Screen TRKB Agonists as Alzheimer's Disease Treatment Strategy.

Misfolded aggregation of the hyperphosphorylated microtubule binding protein Tau in the brain is a pathological hallmark of Alzheimer's disease (AD). Tau aggregation downregulates brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB) signaling and leads to neurotoxicity. Therefore, enhancement of BDNF/TRKB signaling could be a strategy to alleviate Tau neurotoxicity. In this study, eight compounds were evaluated for the potential of inhibiting Tau misfolding in human neuroblastoma SH-SY5Y cells expressing the pro-aggregator Tau folding reporter (ΔK280 TauRD-DsRed). Among them, coumarin derivative ZN-015 and quinoline derivatives VB-030 and VB-037 displayed chemical chaperone activity to reduce ΔK280 TauRD aggregation and promote neurite outgrowth. Studies of TRKB signaling revealed that ZN-015, VB-030 and VB-037 treatments significantly increased phosphorylation of TRKB and downstream Ca2+/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase 1/2 (ERK) and AKT serine/threonine kinase (AKT), to activate ribosomal S6 kinase (RSK) and cAMP response element-binding protein (CREB). Subsequently, p-CREB enhanced the transcription of pro-survival BDNF and BCL2 apoptosis regulator (BCL2), accompanied with reduced expression of anti-survival BCL2-associated X protein (BAX) in ΔK280 TauRD-DsRed-expressing cells. The neurite outgrowth promotion effect of ZN-015, VB-030 and VB-037 was counteracted by a RNA interference-mediated knockdown of TRKB, suggesting the role of these compounds acting as TRKB agonists. Tryptophan fluorescence quenching analysis showed that ZN-015, VB-030 and VB-037 interacted directly with a Pichia pastoris-expressed TRKB extracellular domain, indirectly supporting the role through TRKB signaling. The results of up-regulation in TRKB signaling open up the therapeutic potentials of ZN-015, VB-030 and VB-037 for AD.

Investigating Therapeutic Effects of Indole Derivatives Targeting Inflammation and Oxidative Stress in Neurotoxin-Induced Cell and Mouse Models of Parkinson's Disease.

Neuroinflammation and oxidative stress have been emerging as important pathways contributing to Parkinson's disease (PD) pathogenesis. In PD brains, the activated microglia release inflammatory factors such as interleukin (IL)-ß, IL-6, tumor necrosis factor (TNF)-α, and nitric oxide (NO), which increase oxidative stress and mediate neurodegeneration. Using 1-methyl-4-phenylpyridinium (MPP+)-activated human microglial HMC3 cells and the sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, we found the potential of indole derivative NC009-1 against neuroinflammation, oxidative stress, and neurodegeneration for PD. In vitro, NC009-1 alleviated MPP+-induced cytotoxicity, reduced NO, IL-1ß, IL-6, and TNF-α production, and suppressed NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in MPP+-activated HMC3 cells. In vivo, NC009-1 ameliorated motor deficits and non-motor depression, increased dopamine and dopamine transporter levels in the striatum, and reduced oxidative stress as well as microglia and astrocyte reactivity in the ventral midbrain of MPTP-treated mice. These protective effects were achieved by down-regulating NLRP3, CASP1, iNOS, IL-1ß, IL-6, and TNF-α, and up-regulating SOD2, NRF2, and NQO1. These results strengthen the involvement of neuroinflammation and oxidative stress in PD pathogenic mechanism, and indicate NC009-1 as a potential drug candidate for PD treatment.

Virtual Screening and Testing of GSK-3 Inhibitors Using Human SH-SY5Y Cells Expressing Tau Folding Reporter and Mouse Hippocampal Primary Culture under Tau Cytotoxicity.

Glycogen synthase kinase-3ß (GSK-3ß) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3ß from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3ß activities ranged from 36.1% to 90.0% were detected at the IC50 of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 TauRD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3ß Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogen-activated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 TauRD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3ß kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.

Novel Synthetic Coumarin-Chalcone Derivative (E)-3-(3-(4-(Dimethylamino)Phenyl)Acryloyl)-4-Hydroxy-2H-Chromen-2-One Activates CREB-Mediated Neuroprotection in Aß and Tau Cell Models of Alzheimer's Disease.

Abnormal accumulations of misfolded Aß and tau proteins are major components of the hallmark plaques and neurofibrillary tangles in the brains of Alzheimer's disease (AD) patients. These abnormal protein deposits cause neurodegeneration through a number of proposed mechanisms, including downregulation of the cAMP-response-element (CRE) binding protein 1 (CREB) signaling pathway. Using CRE-GFP reporter cells, we investigated the effects of three coumarin-chalcone derivatives synthesized in our lab on CREB-mediated gene expression. Aß-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells were used to evaluate these agents for possible antiaggregative, antioxidative, and neuroprotective effects. Blood-brain barrier (BBB) penetration was assessed by pharmacokinetic studies in mice. Of the three tested compounds, (E)-3-(3-(4-(dimethylamino)phenyl)acryloyl)-4-hydroxy-2H-chromen-2-one (LM-021) was observed to increase CREB-mediated gene expression through protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and extracellular signal-regulated kinase (ERK) in CRE-GFP reporter cells. LM-021 exhibited antiaggregative, antioxidative, and neuroprotective effects mediated by the upregulation of CREB phosphorylation and its downstream brain-derived neurotrophic factor and BCL2 apoptosis regulator genes in Aß-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells. Blockage of the PKA, CaMKII, or ERK pathway counteracted the beneficial effects of LM-021. LM-021 also exhibited good BBB penetration ability, with brain to plasma ratio of 5.3%, in in vivo pharmacokinetic assessment. Our results indicate that LM-021 works as a CREB enhancer to reduce Aß and tau aggregation and provide neuroprotection. These findings suggest the therapeutic potential of LM-021 in treating AD.

From Nanoparticles to Cancer Nanomedicine: Old Problems with New Solutions.

Anticancer nanomedicines have been studied over 30 years, but fewer than 10 formulations have been approved for clinical therapy today. Despite abundant options of anticancer drugs, it remains challenging to have agents specifically target cancer cells while reducing collateral toxicity to healthy tissue. Nanocompartments that can be selective toward points deeply within malignant tissues are a promising concept, but the heterogeneity of tumor tissue, inefficiency of cargo loading and releasing, and low uniformity of manufacture required from preclinical to commercialization are major obstacles. Technological advances have been made in this field, creating engineered nanomaterials with improved uniformity, flexibility of cargo loading, diversity of surface modification, and less inducible immune responses. This review highlights the developmental process of approved nanomedicines and the opportunities for novel materials that combine insights of tumors and nanotechnology to develop a more effective nanomedicine for cancer patients.

Formulated Chinese medicine Shaoyao Gancao Tang reduces NLRP1 and NLRP3 in Alzheimer's disease cell and mouse models for neuroprotection and cognitive improvement.

Amyloid ß (Aß) plays a major role in the neurodegeneration of Alzheimer's disease (AD). The accumulation of misfolded Aß causes oxidative stress and inflammatory damage leading to apoptotic cell death. Traditional Chinese herbal medicine (CHM) has been widely used in treating neurodegenerative diseases by reducing oxidative stress and neuroinflammation. We examined the neuroprotective effect of formulated CHM Shaoyao Gancao Tang (SG-Tang, made of Paeonia lactiflora and Glycyrrhiza uralensis at 1:1 ratio) in AD cell and mouse models. In Aß-GFP SH-SY5Y cells, SG-Tang reduced Aß aggregation and reactive oxygen species (ROS) production, as well as improved neurite outgrowth. When the Aß-GFP-expressing cells were stimulated with conditioned medium from interferon (IFN)-γ-activated HMC3 microglia, SG-Tang suppressed expressions of inducible nitric oxide synthase (iNOS), NLR family pyrin domain containing 1 (NLRP1) and 3 (NLRP3), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6, attenuated caspase-1 activity and ROS production, and promoted neurite outgrowth. In streptozocin-induced hyperglycemic APP/PS1/Tau triple transgenic (3×Tg-AD) mice, SG-Tang also reduced expressions of NLRP1, NLRP3, Aß and Tau in hippocampus and cortex, as well as improved working and spatial memories in Y maze and Morris water maze. Collectively, our results demonstrate the potential of SG-Tang in treating AD by moderating neuroinflammation.

Pathomechanism Characterization and Potential Therapeutics Identification for Parkinson's Disease Targeting Neuroinflammation.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DAergic) neurons and the presence of α-synuclein-containing Lewy bodies. The unstructured α-synuclein forms insoluble fibrils and aggregates that result in increased reactive oxygen species (ROS) and cellular toxicity in PD. Neuroinflammation engaged by microglia actively contributes to the pathogenesis of PD. In this study, we showed that VB-037 (a quinoline compound), glycyrrhetic acid (a pentacyclic triterpenoid), Glycyrrhiza inflata (G. inflata, a Chinese herbal medicine), and Shaoyao Gancao Tang (SG-Tang, a formulated Chinese medicine) suppressed the nitric oxide (NO) production and interleukin (IL)-1ß maturation in α-synuclein-stimulated BV-2 cells. Mouse inflammation antibody array further revealed increased IL-1α, IL-1ß, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) expression in α-synuclein-inflamed BV-2 cells and compound pretreatment effectively reduced the expression and release of these pro-inflammatory mediators. The test compounds and herbal medicines further reduced α-synuclein aggregation and associated oxidative stress, and protected cells against α-synuclein-induced neurotoxicity by downregulating NLR family pyrin domain containing 1 (NLRP1) and 3 (NLRP3), caspase 1, IL-1ß, IL-6, and associated nuclear factor (NF)-κB inhibitor alpha (IκBα)/NF-κB P65 subunit (P65), c-Jun N-terminal kinase (JNK)/proto-oncogene c-Jun (JUN), mitogen-activated protein kinase 14 (P38)/signal transducer and activator of transcription 1 (STAT1) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathways in dopaminergic neurons derived from α-synuclein-expressing SH-SY5Y cells. Our findings indicate the potential of VB-037, glycyrrhetic acid, G. inflata, and SG-Tang through mitigating α-synuclein-stimulated neuroinflammation in PD, as new drug candidates for PD treatment.

Association of SOD2 p.V16A polymorphism with Parkinson's disease: A meta-analysis in Han Chinese.

BACKGROUND: Oxidative stress could participate in the pathogenesis of Parkinson's disease (PD). However, the role of genetic variation of superoxide dismutase 2 (SOD2), an important regulator against oxidative stress, in PD remains to be elucidated. METHODS: We screened SOD2 gene variation by sequencing cDNA from 72 patients with early onset PD. A cohort of PD (n = 609) and ethnically matched controls (n = 681) were further examined for the identified sequence variant by PCR and NaeI restriction analysis. RESULTS: Only a reported c.47T>C polymorphism (rs4880, SOD2 p.V16A) was found by cDNA sequencing. Case-control study of c.47T>C revealed that genotype and allele frequencies were in Hardy-Weinberg equilibrium in both patients and healthy controls. In a recessive model, those with CC genotype had a 2.61-fold increased risk of PD (95% CI: 1.08-6.30, P = 0.03) compared to subjects with TT and TC genotypes. Significant association between CC genotype and PD in non-smokers was also observed after stratification according to the history of smoking (3.54-fold increased risk of PD, 95% CI: 1.17-10.72, P = 0.02). Meta-analysis by combining studies of Chinese in China, Singapore, and Taiwan (total 2302 cases and 2029 controls) consistently showed CC genotype with increased risk of PD (OR = 1.77, 95% CI: 1.15-2.71, P = 0.01). CONCLUSION: Our findings demonstrate that SOD2 p.V16A may play a role in the susceptibility of PD in Han Chinese.

Pathomechanism characterization and potential therapeutics identification for SCA3 targeting neuroinflammation.

Polyglutamine (polyQ)-mediated spinocerebellar ataxias (SCA) are caused by mutant genes with expanded CAG repeats encoding polyQ tracts. The misfolding and aggregation of polyQ proteins result in increased reactive oxygen species (ROS) and cellular toxicity. Inflammation is a common manifestation of oxidative stress and inflammatory process further reduces cellular antioxidant capacity. Increase of activated microglia in the pons of SCA type 3 (SCA3) patients suggests the involvement of neuroinflammation in the disease pathogenesis. In this study, we evaluated the anti-inflammatory potentials of indole compound NC009-1, 4-aminophenol-arachidonic acid derivative AM404, quinoline compound VB-037 and chalcone-coumarin derivative LM-031 using human HMC3 microglia and SCA3 ATXN3/Q75-GFP SH-SY5Y cells. The four tested compounds displayed anti-inflammatory activity by suppressing NO, IL-1ß, TNF-α and IL-6 production and CD68 expression of IFN-γ-activated HMC3 microglia. In retinoic acid-differentiated ATXN3/Q75-GFP SH-SY5Y cells inflamed with IFN-γ-primed HMC3 conditioned medium, treatment with the tested compounds mitigated the increased caspase 1 activity and lactate dehydrogenase release, reduced polyQ aggregation and ROS and/or promoted neurite outgrowth. Examination of IL-1ß- and TNF-α-mediated signaling pathways revealed that the tested compounds decreased IκBα/P65, JNK/JUN and/or P38/STAT1 signaling. The study results suggest the potential of NC009-1, AM404, VB-037 and LM-031 in treating SCA3 and probable other polyQ diseases.

Machine Learning Uncovers Food- and Excipient-Drug Interactions.

Inactive ingredients and generally recognized as safe compounds are regarded by the US Food and Drug Administration (FDA) as benign for human consumption within specified dose ranges, but a growing body of research has revealed that many inactive ingredients might have unknown biological effects at these concentrations and might alter treatment outcomes. To speed up such discoveries, we apply state-of-the-art machine learning to delineate currently unknown biological effects of inactive ingredients-focusing on P-glycoprotein (P-gp) and uridine diphosphate-glucuronosyltransferase-2B7 (UGT2B7), two proteins that impact the pharmacokinetics of approximately 20% of FDA-approved drugs. Our platform identifies vitamin A palmitate and abietic acid as inhibitors of P-gp and UGT2B7, respectively; in silico, in vitro, ex vivo, and in vivo validations support these interactions. Our predictive framework can elucidate biological effects of commonly consumed chemical matter with implications on food- and excipient-drug interactions and functional drug formulation development.

Shaoyao Gancao Tang (SG-Tang), a formulated Chinese medicine, reduces aggregation and exerts neuroprotection in spinocerebellar ataxia type 17 (SCA17) cell and mouse models.

Spinocerebellar ataxia (SCA) type 17 is an autosomal dominant ataxia caused by expanded polyglutamine (polyQ) tract in the TATA-box binding protein (TBP). Substantial studies have shown involvement of compromised mitochondria biogenesis regulator peroxisome proliferator-activated receptor gamma-coactivator 1 alpha (PGC-1α), nuclear factor erythroid 2-related factor 2 (NRF2), nuclear factor-Y subunit A (NFYA), and their downstream target genes in the pathogenesis of polyQ-expansion diseases. The extracts of Paeonia lactiflora (P. lactiflora) and Glycyrrhiza uralensis (G. uralensis) have long been used as a Chinese herbal medicine (CHM). Shaoyao Gancao Tang (SG-Tang) is a formulated CHM made of P. lactiflora and G. uralensis at a 1:1 ratio. In the present study, we demonstrated the aggregate-inhibitory and anti-oxidative effect of SG-Tang in 293 TBP/Q79 cells. We then showed that SG-Tang reduced the aggregates and ameliorated the neurite outgrowth deficits in TBP/Q79 SH-SY5Y cells. SG-Tang upregulated expression levels of NFYA, PGC-1α, NRF2, and their downstream target genes in TBP/Q79 SH-SY5Y cells. Knock down of NFYA, PGC-1α, and NRF2 attenuated the neurite outgrowth promoting effect of SG-Tang on TBP/Q79 SH-SY5Y cells. Furthermore, SG-Tang inhibited aggregation and rescued motor-deficits in SCA17 mouse model. The study results suggest the potential of SG-Tang in treating SCA17 and probable other polyQ diseases.

Indole Compound NC009-1 Augments APOE and TRKA in Alzheimer's Disease Cell and Mouse Models for Neuroprotection and Cognitive Improvement.

Alzheimer's disease (AD), associated with abnormal accumulation of amyloid-ß (Aß), is the most common cause of dementia among older people. A few studies have identified substantial AD biomarkers in blood but their results were inconsistent. Here we screened gene expression alterations on Aß-GFP SH-SY5Y neuronal model for AD, and evaluated the findings on peripheral leukocytes from 78 patients with AD and 56 healthy controls. The therapeutic responses of identified biomarker candidates were further examined in Aß-GFP SH-SY5Y neuronal and APP/PS1/Tau triple transgenic (3×Tg-AD) mouse models. Downregulation of apolipoprotein E (APOE) and tropomyosin receptor kinase A (TRKA) were detected in Aß-GFP SH-SY5Y cells and validated by peripheral leukocytes from AD patients. Treatment with an in-house indole compound NC009-1 upregulated the expression of APOE and TRKA accompanied with improvement of neurite outgrowth in Aß-GFP SH-SY5Y cells. NC009-1 further rescued the downregulated APOE and TRKA and reduced Aß and tau levels in hippocampus and cortex, and ameliorated cognitive deficits in streptozocin-induced hyperglycemic 3×Tg-AD mice. These results suggest the role of APOE and TRKA as potential peripheral biomarkers in AD, and offer a new drug development target of AD treatment. Further studies of a large series of AD patients will be warranted to verify the findings and confirm the correlation between these markers and therapeutic efficacy.

Targeting Ubiquitin Proteasome Pathway with Traditional Chinese Medicine for Treatment of Spinocerebellar Ataxia Type 3.

Nine autosomal dominant spinocerebellar ataxias (SCAs) are caused by an abnormal expansion of CAG trinucleotide repeats that encodes a polyglutamine (polyQ) tract within different genes. Accumulation of aggregated mutant proteins is a common feature of polyQ diseases, leading to progressive neuronal dysfunction and degeneration. SCA type 3 (SCA3), the most common form of SCA worldwide, is characterized by a CAG triplet expansion in chromosome 14q32.1 ATXN3 gene. As accumulation of the mutated polyQ protein is a possible initial event in the pathogenic cascade, clearance of aggregated protein by ubiquitin proteasome system (UPS) has been proposed to inhibit downstream detrimental events and suppress neuronal cell death. In this study, Chinese herbal medicine (CHM) extracts were studied for their proteasome-activating, polyQ aggregation-inhibitory and neuroprotective effects in GFPu and ATXN3/Q 75 -GFP 293/SH-SY5Y cells. Among the 14 tested extracts, 8 displayed increased proteasome activity, which was confirmed by 20S proteasome activity assay and analysis of ubiquitinated and fused GFP proteins in GFPu cells. All the eight extracts displayed good aggregation-inhibitory potential when tested in ATXN3/Q 75 -GFP 293 cells. Among them, neuroprotective effects of five selected extracts were shown by analyses of polyQ aggregation, neurite outgrowth, caspase 3 and proteasome activities, and ATXN3-GFP, ubiquitin, BCL2 and BAX protein levels in neuronal differentiated ATXN3/Q 75 -GFP SH-SY5Y cells. Finally, enhanced proteasome function, anti-oxidative activity and neuroprotection of catalpol, puerarin and daidzein (active constituents of Rehmannia glutinosa and Pueraria lobata) were demonstrated in GFPu and/or ATXN3/Q 75 -GFP 293/SH-SY5Y cells. This study may have therapeutic implication in polyQ-mediated disorders.

Stiffness modification of photopolymerizable gelatin-methacrylate hydrogels influences endothelial differentiation of human mesenchymal stem cells.

For stem cell differentiation, the microenvironment can play an important role, and hydrogels can provide a three-dimensional microenvironment to allow native cell growth in vitro. A challenge is that the stem cell's differentiation can be influenced by the matrix stiffness. We demonstrate a low-toxicity method to create different stiffness matrices, by using a photopolymerizable gelatin methacrylate (GelMA) hydrogel cross-linked by blue light (440 nm). The stiffness and porosity of GelMA hydrogel is easily modified by altering its concentration. We used human bone marrow mesenchymal stem cells (MSCs) as a cell source and cultured the GelMA-encapsulated cells with EGM-2 medium to induce endothelial differentiation. In our GelMA blue light hydrogel system, we found that MSCs can be differentiated into both endothelial-like and osteogenic-like cells. The mRNA expressions of endothelial cell markers CD31, von Willebrand factor, vascular endothelial growth factor receptor-2, and CD34 were significantly increased in softer GelMA hydrogels (7.5% and 10%) compared with stiffer matrices (15% GelMA). On the other hand, the enhancements of osteogenic markers mRNA expressions (Alkaline phosphatase (ALP), Runx2, osteocalcin, and osteopontin) were highest in 10% GelMA. We also found that 10% GelMA hydrogel offered optimal conditions for MSCs to form capillary-like structures. These results suggest that the mechanical properties of the GelMA hydrogel can influence both endothelial and osteogenic differentiation of MSCs and sequent capillary-like formation.

The indole compound NC009-1 inhibits aggregation and promotes neurite outgrowth through enhancement of HSPB1 in SCA17 cells and ameliorates the behavioral deficits in SCA17 mice.

Spinocerebellar ataxia type 17 (SCA17) is caused by the expansion of translated CAG repeat in the TATA box binding protein (TBP) gene encoding a long polyglutamine (polyQ) tract in the TBP protein, which leads to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On cells with inducible SCA17 TBP/Q79-GFP expression to test five in-house NC009 indole compounds for neuroprotection. We found that both aggregation and polyQ-induced reactive oxygen species can be significantly prohibited by the tested NC009 compounds in Tet-On TBP/Q79 293 cells. Among the five indole compounds, NC009-1 up-regulated expression of heat shock protein family B (small) member 1 (HSPB1) chaperone to reduce polyQ aggregation and promote neurite outgrowth in neuronal differentiated TBP/Q79 SH-SY5Y cells. The increased HSPB1 thus ameliorated the increased BH3 interacting domain death agonist (BID), cytochrome c (CYCS) release, and caspase 3 (CASP3) activation which result in apoptosis. Knock down of HSPB1 attenuated the effects of NC009-1 on TBP/Q79 SH-SY5Y cells, suggesting that HSPB1 might be one of the major pathways involved for NC009-1 effects. NC009-1 further reduced polyQ aggregation in Purkinje cells and ameliorated behavioral deficits in SCA17 TBP/Q109 transgenic mice. Our results suggest that NC009-1 has a neuroprotective effect on SCA17 cell and mouse models to support its therapeutic potential in SCA17 treatment.

Synthesis and Characterization of Polycaprolactone-Based Polyurethanes for the Fabrication of Elastic Guided Bone Regeneration Membrane.

The aim of this research is to synthesize polycaprolactone-based polyurethanes (PCL-based PUs) that can be further used for the fabrication of guided bone regeneration (GBR) membranes with higher tensile strength and elongation at break than collagen and PTFE membranes. The PCL-based PUs were prepared by the polymerization of polycaprolactone (PCL) diol with 1,6-hexamethylene diisocyanate (HDI) at different ratios using either polyethylene glycol (PEG) or ethylenediamine (EDA) as chain extenders. The chemical, mechanical, and thermal properties of the synthesized polymers were determined using NMR, FTIR, GPC, DSC, and tensile tester. The PCL and polyurethanes were fabricated as nanofiber membranes by electrospinning, and their mechanical properties and SEM morphology were also investigated. In vitro tests, including WST-1 assay, SEM of cells, and phalloidin cytoskeleton staining, were also performed. It was shown that electrospun membranes made of PCL and PCL-HDI-PEG (2 : 3 : 1) possessed tensile strength of 19.84 MPa and 11.72 MPa and elongation at break of 627% and 362%, respectively. These numbers are equivalent or higher than most of the commercially available collagen and PTFE membrane. As a result, these membranes may have potential for future GBR applications.

Functional properties of LRRK2 mutations in Taiwanese Parkinson disease.

BACKGROUND/PURPOSE: Leucine-rich repeat kinase 2 (LRRK2) is a large protein encoding multiple functional domains. Mutations within different LRRK2 domains have been considered to be involved in the development of Parkinson disease by different mechanisms. Our previous study found three LRRK2 mutations-p.R767H, p.S885N, and p.R1441H-in Taiwanese patients with Parkinson disease. METHODS: We evaluated the functional properties of LRRK2 p.R767H, p.S885N, and p.R1441H mutations by overexpressing them in human embryonic kidney 293 and neuroblastoma SK-N-SH cells. The common p.G2019S mutation in the kinase domain was included for comparison. RESULTS: In 293 cells, overexpressed p.R1441H-but not p.R767H, p.S885N, or p.G2019-increased GTP binding affinity to prolong the active state. Overexpressed p.R1441H and p.G2019S generated inclusions in 293 cells. In SK-N-SH cells, the α-synuclein was coexpressed with wild type as well as mutated p.R767H, p.S885N, p.R1441H, and p.G2019 LRRK2 proteins. Part of the perinuclear inclusions formed by p.R1441H and p.G2019S were colocalized with α-synuclein. Additionally, p.S885N and p.R1441H mutations caused reduced interaction between LRRK2 and ARHGEF7, a putative guanine nucleotide exchange factor for LRRK2, whereas this interaction was well preserved in p.R767H and p.G2019S mutations. CONCLUSION: Our study suggests that p.R1441H protein facilitates the formation of intracellular inclusions, compromises GTP hydrolysis by increasing its affinity for GTP, and reduces its interaction with ARHGEF7.

Antioxidant N-Acetylcysteine and Glutathione Increase the Viability and Proliferation of MG63 Cells Encapsulated in the Gelatin Methacrylate/VA-086/Blue Light Hydrogel System.

Photoencapsulation of cells inside a hydrogel system can provide a suitable path to establish a gel in situ for soft tissue regeneration applications. However, the presence of photoinitiators and blue or UV light irradiation can result in cell damage and an increase of reactive oxygen species. We here evaluate the benefits of an antioxidant pretreatment on the photoencapsulated cells. We study this by evaluating proliferation and viability of MG63 cells, which we combined with a gelatin methacrylate (GelMA) hydrogel system, using the photoinitiator, VA-086, cured with 440 nm blue light. We found that blue light irradiation as well as the presence of 1% VA-086 reduced MG63 cell proliferation rates. Adding a short pretreatment step to the MG63 cells, consisting of the antioxidant molecules N-acetylcysteine (NAC) and reduced glutathione (GSH), and optimizing the GelMA encapsulation steps, we found that both NAC and GSH pretreatments of MG63 cells significantly increased both proliferation and viability of the cells, when using a 15% GelMA hydrogel, 1% VA-086, and 1-min blue light exposure. These findings suggest that the use of antioxidant pretreatment can counteract the negative presence of the photoinitiators and blue light exposure and result in a suitable environment for photoencapsulating cells in situ for tissue engineering and soft tissue applications.