Registration study for the prevention and treatment of cerebral hemorrhage using naoxueshu oral liquid: Protocol for a research study.

BACKGROUND: Naoxueshu oral liquid is the only approved drug for acute treatment of cerebral hemorrhage in China. It has been used widely for the treatment of acute ischemic stroke and acute hemorrhagic stroke. However, safety and efficacy data on the early use of Naoxueshu oral liquid are lacking. The main purpose of this study is to observe the benefit and safety of early use of Naoxueshu oral liquid (< 72 h of cerebral hemorrhage) and offer evidence into the potential superiority of Naoxueshu oral liquid in patients with hemorrhagic stroke, and its healthcare costs. METHODS: This registration study for the prevention and treatment of cerebral hemorrhage using Naoxueshu oral liquid will be a quantitative, prospective, multicenter, observational clinical registry study. We aim to register 2000 patients with cerebral hemorrhage within 7 days of disease onset. This study will be an observational study and not interfere with the medication regimen of participants. Hence, we will not allocate patients. The main observation indicators will be the hematoma volume and the proportion of reduction 14 days post-cerebral hemorrhage (or at hospital discharge), onset of new stroke (ischemic stroke, hemorrhagic stroke) within 12 months of disease onset, independence in everyday life activities (modified Rankin Scale score ≤ 2), total cost during hospitalization, and treatment costs. CONCLUSION: This registration study will offer strong evidence for the efficacy and safety of Naoxueshu oral liquid for the prevention and treatment of cerebral hemorrhage, particularly with regard to early use (72 h after onset). It will offer evidence into the potential advantages of Naoxueshu oral liquid in patients with hemorrhagic stroke, including healthcare costs.

CRISPR/Cas9 knockout of MTA1 enhanced RANKL-induced osteoclastogenesis in RAW264.7 cells partly via increasing ROS activities.

Metastasis-associated protein 1 (MTA1), belonging to metastasis-associated proteins (MTA) family, which are integral parts of nucleosome remodelling and histone deacetylation (NuRD) complexes. However, the effect of MTA1 on osteoclastogenesis is unknown. Currently, the regulation of MTA1 in osteoclastogenesis was reported for the first time. MTA1 knockout cells (KO) were established by CRISPR/Cas9 genome editing. RAW264.7 cells with WT and KO group were stimulated independently by RANKL to differentiate into mature osteoclasts. Further, western blotting and quantitative qRT-PCR were used to explore the effect of MTA1 on the expression of osteoclast-associated genes (including CTSK, MMP9, c-Fos and NFATc1) during osteoclastogenesis. Moreover, the effects of MTA1 on the expression of reactive oxygen species (ROS) in osteoclastogenesis was determined by 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Nuclear translocation of Nrf2 was assessed by immunofluorescence staining and western blotting. Our results indicated that the MTA1 deletion group could differentiate into osteoclasts with larger volume and more TRAP positive. In addition, compared with WT group, KO group cells generated more actin rings. Mechanistically, the loss of MTA1 increased the expression of osteoclast-specific markers, including c-Fos, NFATc1, CTSK and MMP-9. Furthermore, the results of qRT-PCR and western blotting showed that MTA1 deficiency reduced basal Nrf2 expression and inhibited Nrf2-mediated expression of related antioxidant enzymes. Immunofluorescence staining demonstrated that MTA1 deficiency inhibited Nrf2 nuclear translocation. Taken together, the above increased basal and RANKL-induced intracellular ROS levels, leading to enhanced osteoclast formation.

[Research progress in mechanism of puerarin in treating vascular dementia].

Vascular dementia(VD) is a condition of cognitive impairment due to acute and chronic cerebral hypoperfusion. The available therapies for VD mainly focus on mitigating cerebral ischemia, improving cognitive function, and controlling mental behavior. Achievements have been made in the basic and clinical research on the treatment of VD with traditional Chinese medicine(TCM) active components, including Ginkgo leaf extract, puerarin, epimedium, tanshinone, and ginsenoside. Most of these components have anti-inflammatory, anti-apoptotic, anti-oxidant, and neuroprotective effects, and puerarin demonstrates excellent performance in mitigating cholinergic nervous system disorders and improving synaptic plasticity. Puerarin, ginkgetin, and epimedium are all flavonoids, while tanshinone is a diterpenoid. Puerariae Lobatae Radix, pungent in nature, can induce clear Yang to reach the cerebral orifices and has the wind medicine functions of ascending, dispersing, moving, and scurrying. Puerariae Lobatae Radix entering collaterals will dredge blood vessels to promote blood flow, and that entering the sweat pore will open the mind, which is in line with the TCM pathogenesis characteristics of VD. This study reviews the progress in the mechanism of puerarin, the main active component of Puerariae Lobatae Radix, in treating VD. Puerarin can ameliorate cholinergic nervous system disorders, reduce excitotoxicity, anti-inflammation, inhibit apoptosis, alleviate oxidative stress injury, enhance synaptic plasticity, up-regulate neuroprotective factor expression, promote cerebral circulation metabolism, and mitigate Aß injury. The pathways of action include activating nuclear factor erythroid 2-related factor 2(Nrf2)/antioxidant response element(ARE), vascular endothelial growth factor(VEGF), extracellular regulated protein kinases(ERK), phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt), Janus-activating kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3), AMP-activated protein kinase(AMPK), as well as inhibiting the tumor necrosis factor α(TNF-α), transient receptor potential melastatin 2(TRPM2)/N-methyl-D-aspartate receptor(NMDAR), p38 mitogen-activated protein kinase(p38 MAPK), Toll-like receptor 4(TLR4)/nuclear factor-kappaB(NF-κB), early growth response 1(Egr-1), and matrix metalloproteinase 9(MMP-9). By reviewing the papers about the treatment of VD by puerarin published by CNKI, Wanfang, VIP, PubMed, and Web of Science in the last 10 years, this study aims to support the treatment and drug development for VD.

Cell cytoskeleton and proliferation study for the RANKL-induced RAW264.7 differentiation.

Although document studies (including ours) have been reported the achieved in vitro osteoclastic cellular model establishment from the RAW264.7 cell lineage, there was no study directly reported that American Type Culture Collection (ATCC) cell bank has various RAW264.7 cell lineages. Besides that, for our knowledge there was only one study compared the two different RAW264.7TIB-71 and RAW264.7CRL-2278 cell lineages for their osteoclastic differentiation, and they concluded that the RAW264.7CRL-2278 demonstrated to generate much osteoclast than RAW264.7TIB-71 . However, on the contrary to their results we noticed the fusion of RAW264.7TIB-71 in our previous studies was much compromising. Therefore, we try to explore the two cell lineages for their properties in osteoclastic differentiation with an in-depth cellular cytoskeletal study. Our current study has showed that comparing to the RAW264.7CRL-2278 , RAW264.7TIB-71 demonstrated a much higher efficacies for RANKL-stimulated osteoclastic differentiation. Besides that, in our depth cytoskeletal studies, we found that the RANKL-induced RAW264.7TIB-71 cells could finally differentiate into mature osteoclasts. However, regardless the various pre-treatment conditions, there was no mature osteoclast formed in RANKL-induced RAW264.7CRL-2278 cell lineage.

Genetic manipulation of primary human natural killer cells to investigate the functional and oncogenic roles of PRDM1.

Extra-nodal natural killer/T-cell lymphoma, nasal type (ENKTCL) is a highly aggressive lymphoma, where the tumor suppressor gene (TSG) PRDM1 is frequently lost/inactivated. We employed two different CRISPR/Cas9 approaches to generate PRDM1-/- primary NK cells to study its role in NK-cell homeostasis. PRDM1-/- NK cells showed a marked increase in cloning efficiency, higher proliferation rate and less apoptosis compared with their wild type counterparts. Gene expression profiling demonstrated a marked enrichment in pathways associated with proliferation, cell cycle, MYC, MYB and TCR/NK signaling in PRDM1-/- NK cells, but pathways associated with normal cellular functions including cytotoxic functions were down-regulated, suggesting that the loss of PRDM1 shifted NK cells toward proliferation and survival rather than the performance of its normal functions. We were also able to further modify a PRDM1 deleted clone to introduce heterozygous deletions of common TSG in ENKTCL such as TP53, DDX3X, or PTPN6. We have established an in vitro model to elucidate the major pathways through which PRDM1 mediates its homeostatic control of NK-cells. This approach can be applied to the study of other relevant genetic lesions and oncogenic collaborations in lymphoma pathogenesis.

Identification and Quantification, Metabolism and Pharmacokinetics, Pharmacological Activities, and Botanical Preparations of Protopine: A Review.

Through pharmacological activity research, an increasing number of natural products and their derivatives are being recognized for their therapeutic value. In recent years, studies have been conducted on Corydalis yanhusuo W.T. Wang, a valuable medicinal herb listed in the Chinese Pharmacopoeia. Protopine, one of its components, has also become a research hotspot. To illustrate the identification, metabolism, and broad pharmacological activity of protopine and the botanical preparations containing it for further scientific studies and clinical applications, an in-depth and detailed review of protopine is required. We collected data on the identification and quantification, metabolism and pharmacokinetics, pharmacological activities, and botanical preparations of protopine from 1986 to 2021 from the PubMed database using "protopine" as a keyword. It has been shown that protopine as an active ingredient of many botanical preparations can be rapidly screened and quantified by a large number of methods (such as the LC-ESI-MS/MS and the TLC/GC-MS), and the possible metabolic pathways of protopine in vivo have been proposed. In addition, protopine possesses a wide range of pharmacological activities such as anti-inflammatory, anti-platelet aggregation, anti-cancer, analgesic, vasodilatory, anticholinesterase, anti-addictive, anticonvulsant, antipathogenic, antioxidant, hepatoprotective, neuroprotective, and cytotoxic and anti-proliferative activities. In this paper, the identification and quantification, metabolism and pharmacokinetics, pharmacological activities, and botanical preparations of protopine are reviewed in detail to lay a foundation for further scientific research and clinical applications of protopine.

[Screening of key genes and pathways of ischemic stroke and prediction of traditional Chinese medicines based on bioinformatics].

The aim of this paper was to explore the key genes and pathogenesis of ischemic stroke(IS) by bioinformatics, and predict the potential traditional Chinese medicines for IS. Based on the gene-chip raw data set of GSE22255 from National Center of Biotechnology Information(NCBI), the article enrolled in 20 patients with ischemic stroke and 20 sex-and age-matched controls, and differentially expressed genes(DEGs) were screened based on R language software. The DAVID tool and R language software were used to perform gene ontology(GO) biological process enrichment analysis and Kyoto encyclopedia of genes and gnomes(KEGG) pathway enrichment analysis. The DEGs were imported into STRING to construct a protein-protein interaction network, and the Molecular Complexity Module(MCODE) plug-in of Cytoscape software was used to visualize and analyze the key functional modules. Moreover, the core genes and the medical ontology information retrieval platform(Coremine Medical) were mapped to each other to screen the traditional Chinese medicines and construct drug-active ingredient-target network. Compared with healthy controls, 14 DEGs were obtained, of which 12 genes were up-regulated and 2 genes were down-regulated. DEGs were mainly involved in immune response, inflammatory process, signal transduction, and cell proliferation regulation. The interleukin-17(IL-17), nuclear factor kappaB(NF-κB), tumor necrosis factor(TNF), nucleotide binding oligomerization domain(NOD)-like receptor and other signaling pathways were involved in KEGG pathway enrichment analysis. The key modules of the DEGs-encoding protein interaction network mainly focused on 7 genes of TNF, JUN, recombinant immediate early response 3(IER3), recombinant early growth response protein 1(EGR1), prostaglandin-endoperoxide synthase 2(PTGS2), C-X-C motif chemokine ligand 8(CXCL8) and C-X-C motif chemokine ligand 2(CXCL2), which were involved in biological processes widely such as neuroinflammation and immunity. TNF and JUN were the key nodes in this module, which might become potential biological markers for diagnosis and prognosis evaluation of IS. The potential traditional Chinese medicines for the treatment of IS includes Salviae Miltiorrhizae Radix et Rhizoma, Croci Stigma, Scutellariae Radix, and Cannabis Fructus. The occurrence of stroke was the result of multiple factors. Dysregulation of genes and pathways related to immune regulation and inflammation may be the key link for the development of IS. This study provided research direction and theoretical basis for further exploring the mechanism of action of traditional Chinese medicine in the treatment of IS and searching for potential drug targets.

Integrin-associated molecules and signalling cross talking in osteoclast cytoskeleton regulation.

In the ageing skeleton, the balance of bone reconstruction could commonly be broken by the increasing of bone resorption and decreasing of bone formation. Consequently, the bone resorption gradually occupies a dominant status. During this imbalance process, osteoclast is unique cell linage act the bone resorptive biological activity, which is a highly differentiated ultimate cell derived from monocyte/macrophage. The erosive function of osteoclasts is that they have to adhere the bone matrix and migrate along it, in which adhesive cytoskeleton recombination of osteoclast is essential. In that, the podosome is a membrane binding microdomain organelle, based on dynamic actin, which forms a cytoskeleton superstructure connected with the plasma membrane. Otherwise, as the main adhesive protein, integrin regulates the formation of podosome and cytoskeleton, which collaborates with the various molecules including: c-Cbl, p130Cas , c-Src and Pyk2, through several signalling cascades cross talking, including: M-CSF and RANKL. In our current study, we discuss the role of integrin and associated molecules in osteoclastogenesis cytoskeletal, especially podosomes, regulation and relevant signalling cascades cross talking.

Cellular study of the LPS-induced osteoclastic multinucleated cell formation from RAW264.7 cells.

Despite the response to the receptor activator of nuclear factor κ-Β ligand (RANKL), a study has reported that lipopolysaccharide (LPS) could induce RAW264.7 linage osteoclastic differentiation. However, on the contrary, another study recently showed that the LPS-induced multinuclear cells from RAW264.7 did not express osteoclastic functions. Interestingly, in our previous study, we found that RAW264.7 cells pretreated with 10 ng LPS plus macrophage-colony stimulating factor did not show any effects for enhancing RANKL-induced osteoclastic cell differentiation. Therefore, in our current study, we aim to investigate the oteoclastogesis induction ability and efficacy of LPS in the RAW264.7 cell line and relevant molecular signaling. The osteoclastogenic activity of LPS-treated RAW264.7 linage was studied by bone resorption pits and fibrous actin study. Besides that, through polymerase chain reaction and western blot, we showed that the transcriptional factor c-Fos and Nfatc1 might be associated with LPS-induced osteoclastogenesis. Overall, the results of our current study showed positive proof for osteoclast generation from LPS-independent treatment, as well as established an optimal and efficient method for this process.

Double-walled hierarchical porous silica nanotubes loaded Au nanoparticles in the interlayer as a high-performance catalyst.

Double-walled hierarchical porous silica nanotubes (NTs) loaded Au nanoparticles (Au NPs) in the interlayer (SiO2@Au@SiO2 NTs) are synthesized by using tetraethoxysilane as silica source and hollow polydivinylbenzene (PDVB) nanowires as the sacrificial templates. The mesopores on the walls and the hollow structure of NTs (macropores) construct the hierarchical porous structure. The SiO2@Au@SiO2 NTs possess a high surface area of 405 m2 g-1 and an average pores size of 4.7 nm. The double-walled structure protects the Au NPs from environmental attacks, which shows an excellent catalytic activity even after reusing 10 times. Meanwhile, the hierarchical porous structure shows excellent catalytic ability and allows the catalytic reaction process to be completed within 5 min. This result indicates that double-walled silica NTs have vast potential in catalysis application due to the special structure.

Calibration, Compensation and Accuracy Analysis of Circular Grating Used in Single Gimbal Control Moment Gyroscope.

The accuracy of the circular grating is the key point for control precision of the single gimbal control moment gyroscope servo system used in civilian micro-agile satellites. Instead of using the multi reading heads to eliminate eccentricity errors, an algorithm compensation method based on a calibration experiment using a single reading head was proposed to realize a low-cost and high accuracy angular position measurement. Moreover, the traditional hardware compensation method using double reading heads was also developed for comparison. Firstly, the single gimbal control moment gyroscope system of satellites was introduced. Then, the errors caused by the installation of the reading head were studied and the mathematic models of these errors were developed. In order to construct the compensation function, a calibration experiment using the autocollimator and 24-sided prism was performed. Comparison of angle error compensation using the algorithm and hardware method was presented, and results showed that the algorithm compensation method proposed by this paper achieved the same accuracy level as the hardware method. Finally, the proposed method was further verified through a control system simulation.

Overview of RAW264.7 for osteoclastogensis study: Phenotype and stimuli.

Bone homeostasis is preserved by the balance of maintaining between the activity of osteogenesis and osteoclastogenesis. However, investigations for the osteoclastogenesis were hampered by considerable difficulties associated with isolating and culturing osteoclast in vivo. As the alternative, stimuli-induced osteoclasts formation from RAW264.7 cells (RAW-OCs) have gain its importance for extensively osteoclastogenic study of bone diseases, such as rheumatoid arthritis, osteoporosis, osteolysis and periodontitis. However, considering the RAW-OCs have not yet been well-characterized and RAW264.7 cells are polymorphic because of a diverse phenotype of the individual cells comprising this cell linage, and different fate associated with various stimuli contributions. Thus, in present study, we provide an overview for current knowledge of the phenotype of RAW264.7 cells, as well as the current understanding of the complicated interactions between various stimuli and RAW-OCs in the light of the recent progress.

Regulation effects of melatonin on bone marrow mesenchymal stem cell differentiation.

Melatonin's therapeutic potential has been highly underestimated because its biological functional roles are diverse and relevant mechanisms are complicated. Among the numerous biological activities of melatonin, its regulatory effects on pluripotent mesenchymal stem cells (MSCs), which are found in bone marrow stem cells (BMSCs) and adipose tissue (AD-MSC), have been recently proposed, which has received increasingly more attention in recent studies. Moreover, receptor-dependent and receptor-independent responses to melatonin are identified to occur in these cells by regulating signaling pathways, which drive the commitment and differentiation of MSCs into osteogenic, chondrogenic, or adipogenic lineages. Therefore, the aim of our current review is to summarize the evidence related to the utility of melatonin as a regulatory agent by focusing on its relationship with the differentiation of MSCs. In particular, we aimed to review its roles in promoting osteogenic and chondrogenic differentiation and the relevant signaling cascades involved. Also, the roles that melatonin and, particularly, its receptors play in these processes are highlighted.

Evaluation of efficacy on RANKL induced osteoclast from RAW264.7 cells.

Established RAW264.7 cell lines for osteoclastic differentiation has been widely engaged in bone homeostasis research, however, the efficacy of RANKL independently stimulating has rarely been defined, because protocols were usually developed and modified by various laboratories. Otherwise, problematic issues are also lie in the cell's seeding density, RANKL stimulating time point, and distinguishing osteoclastogenesis ability of RANKL-treated RAW264.7 cells. Therefore, in the current study, we examined the efficacy of various concentrations of RANKL-treated RAW264.7 for its osteoclastic differentiation with or without pretreated other costimulators such as: LPS and/or M-CSF. The oteoclastogenesis ability of RANKL-treated RAW264.7 cells was demonstrated by bone resorption pit, F-actin, and osteoclastogenesis specific marker studies. Besides that, through tartrate-resistant acid phosphatase (TRAP) staining, we clarified to start the treatment with 30 ng/ml RANKL at 12 hr after seeded RAW264.7 with the density of 6.25 × 10 3 cells/cm 2 manifested an significantly increased number of multinucleated osteoclastic cells. Overall, our results establishing an optimal method for RANKL independently inducing RAW 264.7 cell osteoclastic differentiation, which could efficiently generate osteoclasts in vitro for significant advances in our understanding of bone biology.

Inhibition effects of a natural inhibitor on RANKL downstream cellular signalling cascades cross-talking.

Myricitrin is a natural occurring flavonoid glycoside that possesses effects on inhibiting nitric oxide (NO) transmission and preventing inflammatory reaction. Although previous study showed the myricitrin possesses antibone loss effects via reducing the expression of IL-6 and partially suppressing reactive oxygen species (ROS) production. However, the effects of myricitrin on nuclear factor-kappaB ligand (RANKL)-stimulated osteoclastogenesis have not yet been further investigated. The current study was aimed to demonstrating the inhibitory effects of myricitrin on RANKL-stimulated osteoclastogenesis and relevant mechanisms. We found myricitrin significantly suppressed osteoclastogenesis suggesting that it may acts on RANKL/RANK induced downstream signal cross cascading in osteoclast precursors. In that, our Western blotting results showed myricitrin significantly attenuated RNAKL/MAPKs (phosphorylation of p38, ERK, JNK) and AKT signal cascading. Complementing previous study, our results suggesting as a natural inhibitor, myricitrin possesses the potential therapeutic effects on inflammatory osteolysis.

Picroside II Inhibits RANKL-Mediated Osteoclastogenesis by Attenuating the NF-κB and MAPKs Signaling Pathway In Vitro and Prevents Bone Loss in Lipopolysaccharide Treatment Mice.

Picroside II, one of the major components isolated from the seed of natural plant picrorhiza, is widely used in traditional Chinese medicine. The present study was performed to define effects of picroside II on nuclear factor-kappaB ligand (RANKL)-stimulated osteoclast differentiation in vitro and on lipopolysaccharide (LPS)-induced bone loss in vivo. The bone marrow cells (BMMs) were harvested and induced with RANKL followed by treatment with picroside II at several doses, and the differentiation of osteoclasts from these cells was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and resorption pit formation assay. The effects of picroside II on osteoclastogenesis were studied by examining RANKL-induced osteoclast F-actin ring formation and osteoclast bone resorption. Moreover, we explored the mechanisms of these downregulation effects by performed Western blotting and quantitative RT-PCR examination. Results demonstrated picroside II strongly inhibited RANKL-induced osteoclast formation when added during the early stage of BMMs cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Moreover, picroside II markedly decreased the phosphorylation of p38, ERK, JNK, p65, and I-κB degradation, and significantly suppressed c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), both the key transcription factors during osteoclastogenesis. Furthermore, in vivo studies verified the bone protection effects of picroside II. These results collectively suggested that picroside II acted as an anti-resorption agent by blocking osteoclast activation. J. Cell. Biochem. 118: 4479-4486, 2017. © 2017 Wiley Periodicals, Inc.

Picrasidine I from Picrasma Quassioides Suppresses Osteoclastogenesis via Inhibition of RANKL Induced Signaling Pathways and Attenuation of ROS Production.

BACKGROUND/AIMS: Osteoporosis is a metabolic bone disorder that tortures about millions of people worldwide. Recent study demonstrated agents derived from picrasma quassioides is a promising drug for targets multiple signaling pathways. However its potential in treatment of bone loss has not been fully understood. METHODS: The bone marrow macrophages (BMMs) were cultured and induced with M-CSF and RANKL followed by picrasidine I (PI) treatment. Then the effects of PI on osteoclast formation were evaluated by counting tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Moreover, effects of PI on bone resorption activity of mature osteoclast were studied through bone resorption pit counting and actin ring structure analysis. Further, the involved potential signaling pathways cross-talking were investigated by performed Western blotting and quantitative real-time PCR examination. RESULTS: Results demonstrated PI strongly inhibited RANKL induced osteoclast formation from its precursors. Mechanistically, the inhibitory effect of PI on osteoclast differentiation was due to the suppression of osteoclastogenic transcription factors, c-Fos and NFATc1. Moreover, PI markedly blocked the RANKL-induced osteoclastogenesis by attenuating MAPKs and NF-κB signaling pathways. In addition, PI decreased the ROS generation in osteoclast and osteoblast. CONCLUSION: Taken together our data demonstrate that PI has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-κB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors.

Novel LRRK2 GTP-binding inhibitors reduced degeneration in Parkinson's disease cell and mouse models.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson's disease (PD) and contribute to sporadic PD. LRRK2 contains Guanosine-5'-triphosphate (GTP) binding, GTPase and kinase activities that have been implicated in the neuronal degeneration of PD pathogenesis, making LRRK2, a potential drug target. To date, there is no disease-modifying drug to slow the neuronal degeneration of PD and no published LRRK2 GTP domain inhibitor. Here, the biological functions of two novel GTP-binding inhibitors of LRRK2 were examined in PD cell and mouse models. Through a combination of computer-aided drug design (CADD) and LRRK2 bio-functional screens, two novel compounds, 68: and 70: , were shown to reduce LRRK2 GTP binding and to inhibit LRRK2 kinase activity in vitro and in cultured cell assays. Moreover, these two compounds attenuated neuronal degeneration in human SH-SY5Y neuroblastoma cells and mouse primary neurons expressing mutant LRRK2 variants. Although both compounds inhibited LRRK2 kinase activity and reduced neuronal degeneration, solubility problems with 70: prevented further testing in mice. Thus, only 68: was tested in a LRRK2-based lipopolysaccharide (LPS)-induced pre-inflammatory mouse model. 68: reduced LRRK2 GTP-binding activity and kinase activity in brains of LRRK2 transgenic mice after intraperitoneal injection. Moreover, LPS induced LRRK2 upregulation and microglia activation in mouse brains. These findings suggest that disruption of GTP binding to LRRK2 represents a potential novel therapeutic approach for PD intervention and that these novel GTP-binding inhibitors provide both tools and lead compounds for future drug development.

Fast Confocal Raman Imaging Using a 2-D Multifocal Array for Parallel Hyperspectral Detection.

We present the development of a novel confocal hyperspectral Raman microscope capable of imaging at speeds up to 100 times faster than conventional point-scan Raman microscopy under high noise conditions. The microscope utilizes scanning galvomirrors to generate a two-dimensional (2-D) multifocal array at the sample plane, generating Raman signals simultaneously at each focus of the array pattern. The signals are combined into a single beam and delivered through a confocal pinhole before being focused through the slit of a spectrometer. To separate the signals from each row of the array, a synchronized scan mirror placed in front of the spectrometer slit positions the Raman signals onto different pixel rows of the detector. We devised an approach to deconvolve the superimposed signals and retrieve the individual spectra at each focal position within a given row. The galvomirrors were programmed to scan different focal arrays following Hadamard encoding patterns. A key feature of the Hadamard detection is the reconstruction of individual spectra with improved signal-to-noise ratio. Using polystyrene beads as test samples, we demonstrated not only that our system images faster than a conventional point-scan method but that it is especially advantageous under noisy conditions, such as when the CCD detector operates at fast read-out rates and high temperatures. This is the first demonstration of multifocal confocal Raman imaging in which parallel spectral detection is implemented along both axes of the CCD detector chip. We envision this novel 2-D multifocal spectral detection technique can be used to develop faster imaging spontaneous Raman microscopes with lower cost detectors.

A nanotweezer system for evanescent wave excited surface enhanced Raman spectroscopy (SERS) of single nanoparticles.

We experimentally demonstrate the integration of near-field optical tweezers with surface enhanced Raman scattering (SERS) spectroscopy by using the optical evanescent wave from a silicon nitride waveguide to trap single shell-isolated metallic nanoparticles (NPs) and simultaneously excite SERS signals of Raman reporter molecules adsorbed on the surface of the trapped metallic NPs. Both evanescent wave excited Stokes and anti-Stokes SERS spectra of waveguide trapped single silver (Ag) NPs were acquired, which were compared to their far-field SERS spectra. We investigated the trapping of bare and shell-isolated metallic NPs and determined that the addition of a shell to the metallic NPs minimized particle-induced laser damage to the waveguide, which allowed for the stable acquisition of the SERS spectra. This work realizes a new nanophotonic approach, which we refer to as near-field light scattering Raman (NLS-Raman), for simultaneous near-field optical trapping and SERS characterization of single metallic NPs.