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The pathological hallmarks of Parkinson's disease (PD) are degeneration of dopamine (DA) neurons of the substantia nigra (SN) and the presence of alpha-synuclein (α-syn)-rich Lewy bodies in DA cells that remain. To model these aspects of the disease, we previously showed that high titer (5.1×10exp12 gp/ml) AAV1/2 driven expression of A53T α-syn in the SN of rats caused nigrostriatal pathology including a loss of DA neurons, but also with toxicity in the GFP control group. In the current study, we evaluate the effects of two lower titers by dilution of the vector (1∶3 [1.7×10exp12] and 1∶10 [5.1×10exp11]) to define a concentration that produced pathology specific for α-syn. In GFP and empty vector groups there were no behavioural or post-mortem changes at 3 or 6 weeks post-administration at either vector dose. Dilution of the AAV1/2 A53T α-syn (1:3) produced significant paw use asymmetry, reductions in striatal tyrosine hydroxylase (TH), and increases in DA turnover at 3 weeks in the absence of overt pathology. By 6 weeks greater evidence of pathology was observed and included, reductions in SN DA neurons, striatal DA, TH and DA-transporter, along with a sustained behavioural deficit. In contrast, the 1:10 AAV1/2 A53T α-syn treated animals showed normalization between 3 and 6 weeks in paw use asymmetry, reductions in striatal TH, and increased DA turnover. Progression of dopaminergic deficits using the 1:3 titer of AAV1/2 A53Tα-syn provides a platform for evaluating treatments directed at preventing and/or reversing synucleinopathy. Use of the 1:10 titer of AAV1/2 A53T α-syn provides an opportunity to study mechanisms of endogenous compensation. Furthermore, these data highlight the need to characterize the titer of vector being utilized, when using AAV to express pathogenic proteins and model disease process, to avoid producing non-specific effects. 相似文献
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Objectives
To develop a site-specific integration strategy for CAR-T engineering by using a non-viral vector dependent on adeno-associated viral (AAV) genome, which tends to be integrated into AAVS1 site with the help of its Rep proteins.Results
AAV-dependent vectors were produced in Sf9 cells. Structural analyses revealed the vector as covalently close-ended, linear duplex molecules, which was termed “CELiD” DNA. A plasmid CMV-Rep was constructed to express the integrases Rep78 and Rep68. Jurkat cells were co-electroporated with “CELiD” DNA and plasmid CMV-Rep in order to specifically integrate CAR gene into AAVS1 site. We examined 71 stably transfected Jurkat clones by nested PCR, sequencing and southern blotting, of which 30 clones bore CAR gene within AAVS1 site. The site-specific integration efficiency was nearly 42.2 %.Conclusions
The AAV-dependent vector preferentially integrated CAR into AAVS1 site, which could be further used in human T cell modification and enhance the security of CAR-T therapy.4.
Anika?M.?Helferich Wolfgang?P.?Ruf Veselin?Grozdanov Axel?Freischmidt Marisa?S.?Feiler Lisa?Zondler Albert?C.?Ludolph Pamela?J.?McLean Jochen?H.?Weishaupt Karin?M.?Danzer
Background
Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) are both neurodegenerative diseases leading to impaired execution of movement. α-Synuclein plays a central role in the pathogenesis of PD whereas Cu, Zn superoxide dismutase (SOD1) is a key player in a subset of familial ALS cases. Under pathological conditions both α-synuclein and SOD1 form oligomers and fibrils. In this study we investigated the possible molecular interaction of α-synuclein and SOD1 and its functional and pathological relevance.Results
Using a protein-fragment complementation approach and co-IP, we found that α-synuclein and SOD1 physically interact in living cells, human erythrocytes and mouse brain tissue. Additionally, our data show that disease related mutations in α-synuclein (A30P, A53T) and SOD1 (G85R, G93A) modify the binding of α-synuclein to SOD1. Notably, α-synuclein accelerates SOD1 oligomerization independent of SOD1 activity.Conclusion
This study provides evidence for a novel interaction of α-synuclein and SOD1 that might be relevant for neurodegenerative diseases.5.
Background
Secondary pallidonigral transneuronal degeneration after a remote primary cerebral infarct can mimic recurrent stroke at clinical presentation. We describe a patient with secondary pallidonigral degeneration following a previous putaminal infarct, which was diagnosed through diffusion-weighted (DWI) and T2-weighted imaging (T2WI).Case presentation
A 64-year-old man complained of an acute relapse of right-lower-limb weakness following a cerebral infarction 2 months before presentation. Recurrent cerebral stroke was initially diagnosed in the emergency room. DWI of the brain revealed a subacute to chronic infarct in the left putamen and new acute cytotoxic edema in the left substantia nigra (SN) and globus pallidus while T2WI also showed hyperintensity in the same regions. The SN was outside the aforementioned middle cerebral arterial territory, which includes the putamen. These findings are compatible with the diagnosis of acute pallidonigral injury secondary to striatal infarction. The patient had fully recovered from his right-lower-limb weakness after 1 month.Conclusions
Secondary pallidonigral degeneration may mimic recurrent stroke. DWI along with T2WI facilitates elucidation of this clinicopathological entity, and thus unnecessary treatment can be avoided.6.
Santhanasabapathy Rajasekaran Jayashree Thatte Jayaprakash Periasamy Alok Javali Manjunath Jayaram Dwaipayan Sen Akshaya Krishnagopal Giridhara R. Jayandharan Ramkumar Sambasivan 《BMC biotechnology》2018,18(1):70
Background
Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection.Results
We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells.Conclusions
In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.7.
Wojciech Bobela Sameer Nazeeruddin Graham Knott Patrick Aebischer Bernard L. Schneider 《Molecular neurodegeneration》2017,12(1):80
Background
Metabolic perturbations and slower renewal of cellular components associated with aging increase the risk of Parkinson’s disease (PD). Declining activity of AMPK, a critical cellular energy sensor, may therefore contribute to neurodegeneration.Methods
Here, we overexpress various genetic variants of the catalytic AMPKα subunit to determine how AMPK activity affects the survival and function of neurons overexpressing human α-synuclein in vivo.Results
Both AMPKα1 and α2 subunits have neuroprotective effects against human α-synuclein toxicity in nigral dopaminergic neurons. Remarkably, a modified variant of AMPKα1 (T172Dα1) with constitutive low activity most effectively prevents the loss of dopamine neurons, as well as the motor impairments caused by α-synuclein accumulation. In the striatum, T172Dα1 decreases the formation of dystrophic axons, which contain aggregated α-synuclein. In primary cortical neurons, overexpression of human α-synuclein perturbs mitochondrial and lysosomal activities. Co-expressing AMPKα with α-synuclein induces compensatory changes, which limit the accumulation of lysosomal material and increase the mitochondrial mass.Conclusions
Together, these results indicate that modulating AMPK activity can mitigate α-synuclein toxicity in nigral dopamine neurons, which may have implications for the development of neuroprotective treatments against PD.8.
Xiaoying Zhang Wei Chen Ruth Guillermo Gudiseva Chandrasekher Radhey S Kaushik Alan Young Hesham Fahmy Chandradhar Dwivedi 《BMC research notes》2010,3(1):220
Background
α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action.Methods
MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells.Results
α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G2/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G2/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells.Conclusions
This study for the first time identifies effects of α-santalol in G2/M phase arrest and describes detailed mechanisms of G2/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models.9.
Monte S. Willis Amro Ilaiwy Megan D. Montgomery Paul C. Simpson Brian C. Jensen 《Metabolomics : Official journal of the Metabolomic Society》2016,12(10):155
Introduction
Alpha-1-adrenergic receptors (α1-ARs) are G-protein coupled receptors (GPCRs) with three highly homologous subtypes (α1A, α1B, and α1D). Of these three subtypes, only the α1A and α1B are expressed in the heart. Multiple pre-clinical models of heart injury demonstrate cardioprotective roles for the α1A. Non-selective α1-AR activation promotes glycolysis in the heart, but the functional α1-AR subtype and broader metabolic effects have not been studied.Objectives
Given the high metabolic demands of the heart and previous evidence indicating benefit from α1A activation, we chose to investigate the effects of α1A activation on the cardiac metabolome in vivo.Methods
Mice were treated for 1 week with a low, subpressor dose of A61603, a highly selective and potent α1A agonist. Cardiac tissue and serum were analyzed using a non-targeted metabolomics approach.Results
We identified previously unrecognized metabolic responses to α1A activation, most notably broad reduction in the abundance of polyunsaturated fatty acids (PUFAs) and endocannabinoids (ECs).Conclusion
Given the well characterized roles of PUFAs and ECs in inflammatory pathways, these findings suggest a possible role for cardiac α1A-ARs in the regulation of inflammation and may offer novel insight into the mechanisms underlying the cardioprotective benefit of selective pharmacologic α1A activation.10.
Sepideh Shahbazi Nooshin Haghighipour Sepehr Soleymani Seyed Alireza Nadji Azam Bolhassani 《Biotechnology letters》2018,40(6):923-931
Objective
In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry.Results
The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p?<?0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (~?83.94 and ~?77.01% for HEK-293T), and cancer cells (~?71.69 and ~?67.19% for TC-1, and ~?73.86 and ~?67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by +36 GFP reached ~?60–80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (~?63.66%) compared to TurboFect (~?32.95%); however, the efficiency level of hPP10 was only ~?17.51 and ~?16.36% in TC-1 and A549 cells.Conclusions
Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.11.
Background
We propose, model, and implement a novel system of population-level intervention against a virus. One context is a treatment against a chronic infection such as HIV. The underlying principle is a form of virus ‘wars’ in which a benign, transmissible agent is engineered to protect against infection by and spread of a lethal virus. In our specific case, the protective agent consists of two entities, a benign virus and a gene therapy vector mobilized by the benign virus.Results
Numerical analysis of a mathematical model identified parameter ranges in which adequate, population-wide protection is achieved. The protective system was implemented and tested using E. coli, bacteriophage M13 and a phagemid vector mobilized by M13 to block infection by the lethal phage T5. Engineering of M13 profoundly improved its dynamical properties for facilitating spread of the gene therapy vector. However, the gene therapy vector converts the host cell to resist T5 too slowly for protection on a time scale appropriate for T5.Conclusions
Overall, there is a reasonable marriage between the mathematical model and the empirical system, suggesting that such models can be useful guides to the design of such systems even before the models incorporate most of the relevant biological details.12.
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Baninia Habchi Sandra Alves Delphine Jouan-Rimbaud Bouveresse Bilel Moslah Alain Paris Yannick Lécluse Pascal Gauduchon Pierre Lebailly Douglas N. Rutledge Estelle Rathahao-Paris 《Metabolomics : Official journal of the Metabolomic Society》2017,13(4):45
Introduction
To perform large scale metabolomic analyses, high throughput approaches are required. The direct introduction mass spectrometry (DIMS) approach appears to be very attractive to achieve this goal. However, processing DIMS data is still very challenging due to the large number of samples and the intrinsic complexity of the mass spectra.Objectives
The objective of this study is to develop a computational procedure, based on an innovative chemometric method, i.e. Independent component–discriminant analysis (IC–DA), for processing DIMS data.Method
Metabolomic fingerprints were obtained by direct introduction high resolution mass spectrometry (DI-HRMS) analysis of urine samples of subjects that had been professionally exposed to pesticides. Spectral data were processed using the developed IC–DA procedure. Results obtained from this method were compared to those obtained by the conventional Partial least squares–discriminant analysis (PLS–DA). For both the IC–DA and PLS–DA methods, a validation was performed based on a permutation test.Result
IC–DA results enabled a good detection of discriminant variables and a clear discrimination of control samples and exposure classes whereas a less striking discrimination was obtained with PLS–DA. Putative annotation of these variables was performed using metabolomic databases. Targeted correlation analysis was used for the detection of ions associated with the most discriminant variables, consolidating their identity assignment.Conclusion
This study demonstrated the efficiency of IC–DA to discriminate the different exposure groups. As well the improvement of high throughput metabolomic studies was provided by combining DI–HRMS with this new chemometric tool.14.
Background
The aim of this study is to explore the expression of alpha-synuclein (α-synuclein) in benign, atypical, and anaplastic meningiomas and determine its role in the malignant progression of meningiomas.Methods
Expression of α-synuclein was measured in 44 meningioma samples by real-time PCR analysis. The effects of overexpression or knockdown of α-synuclein on meningioma cell growth, invasiveness, and tumorigenicity were determined.Results
Atypical and anaplastic meningiomas displayed significantly greater levels of α-synuclein mRNA, relative to benign tumors. Depletion of α-synuclein decreased cell proliferation and colony formation and promoted apoptosis in IOMM-Lee meningioma cells, whereas overexpression of α-synuclein facilitated cell proliferation and colony formation in CH-157MN meningioma cells. Silencing of α-synuclein attenuated IOMM-Lee cell migration and invasion. In contrast, ectopic expression of α-synuclein increased the invasiveness of CH-157MN cells. In vivo studies further demonstrated that downregulation of α-synuclein significantly retarded meningioma growth in nude mice. At the molecular level, the phosphorylation levels of Akt, mTOR, p70S6K and 4EBP were significantly decreased in α-synuclein-depleted IOMM-Lee cells.Conclusions
In conclusion, α-synuclein upregulation contributes to aggressive phenotypes of meningiomas via the Akt/mTOR pathway and thus represents a potential therapeutic target for malignant meningiomas.15.
Expression and purification of classical swine fever virus E2 protein from Sf9 cells using a modified vector 总被引:1,自引:0,他引:1
Objective
To develop a simple method for efficient expression of classical swine fever virus (CSFV) E2 protein.Results
The pFastBac HT B vector (pFastHTB-M1) was modified by adding a melittin signal peptide sequence. The E2 gene fragment without the transmembrane region was cloned into pFastHTB-M1. The modified vector has clear advantage over the original one, as evidenced by the purified recombinant E2 protein that was detected significantly by SDS-PAGE.Conclusions
The modified vector has the potential for large-scale production and easy purification of the CSFV E2 protein or other proteins of interests.16.
Andrelisse Arruda Viviane Castelo Branco Reis Vinícius Daniel Ferreira Batista Bruno Sahim Daher Luiza Cesca Piva Janice Lisboa De Marco Lidia Maria Pepe de Moraes Fernando Araripe Gonçalves Torres 《Biotechnology letters》2016,38(3):509-517
Objectives
To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.Results
P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.Conclusions
A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.17.
Benjamin H Natelson Roxann Intriligator Neil S Cherniack Helena K Chandler Julian M Stewart 《Dynamic medicine : DM》2007,6(1):2
Context
Patients with chronic fatigue syndrome and those with orthostatic intolerance share many symptoms, yet questions exist as to whether CFS patients have physiological evidence of orthostatic intolerance.Objective
To determine if some CFS patients have increased rates of orthostatic hypotension, hypertension, tachycardia, or hypocapnia relative to age-matched controls.Design
Assess blood pressure, heart rate, respiratory rate, end tidal CO2 and visual analog scales for orthostatic symptoms when supine and when standing for 8 minutes without moving legs.Setting
Referral practice and research center.Participants
60 women and 15 men with CFS and 36 women and 4 men serving as age matched controls with analyses confined to 62 patients and 35 controls showing either normal orthostatic testing or a physiological abnormal test.Main outcome measures
Orthostatic tachycardia; orthostatic hypotension; orthostatic hypertension; orthostatic hypocapnia or combinations thereof.Results
CFS patients had higher rates of abnormal tests than controls (53% vs 20%, p < .002), but rates of orthostatic tachycardia, orthostatic hypotension, and orthostatic hypertension did not differ significantly between patients and controls (11.3% vs 5.7%, 6.5% vs 2.9%, 19.4% vs 11.4%, respectively). In contrast, rates of orthostatic hypocapnia were significantly higher in CFS than in controls (20.6% vs 2.9%, p < .02). This CFS group reported significantly more feelings of illness and shortness of breath than either controls or CFS patients with normal physiological tests.Conclusion
A substantial number of CFS patients have orthostatic intolerance in the form of orthostatic hypocapnia. This allows subgrouping of patients with CFS and thus reduces patient pool heterogeneity engendered by use of a clinical case definition.18.
Background
Gestational trophoblastic disease (GTD) is a heterogeneous group of disorders characterized by abnormal trophoblast tissue. Molar and non-molar hydropic placental changes are the most common forms of GTD. Differential diagnosis of GTD is sometimes problematic. Recently, p53 expression was identified as a good marker for distinguishing GTD types.Aims
Comparison of p53 expression in partial hydatidiform mole (PHM) and hydropic abortion.Methods
In this prospective cross-sectional study, molar and non-molar hydropic pregnancy specimens were collected. Immunohistochemical staining, based on the Labeled Streptavidin Biotin (LSAB) technique, was carried out on multiple 4 mm paraffin block sections prepared from formalin-fixed trophoblastic tissues. Polymer-based Envision was used to assess p53 tumor suppressor protein immunoreactivity. p53 expression was then compared between both groups.Results
In the study, 40 patients were included: 20 with confirmed PHM and 20 with hydropic pregnancy. p53 protein was positive in 60% of patients with PHM and 25% of patients with hydropic pregnancy. The p53 positive rate was significantly higher in patients with PHM (p = 0.027). Moreover, patients with PHM had a significantly high grade of staining (p<0.001).Conclusion
Our findings indicate that immunohistochemical analysis of p53 protein can be used to distinguish PHM and hydropic pregnancy.19.
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