Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.613
Filtrar
1.
J Ethnopharmacol ; 265: 113395, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32956757

RESUMO

ETHNOPHARMACOLOGY RELEVANCE: Armillaria mellea (Vahl) P. Kumm. (AM) is an edible mushroom that has been reported as treatment for several neurological disorders, such as dizziness and epilepsy in Asia. Importantly, AM shares a symbiotic relationship with Gastrodia elata Blume (GE), a medicinal herb with antidepressant-like properties. Researchers believe that AM may possess pharmacological properties similar to GE due to their symbiosis, however, few studies have investigated the pharmacological effect of AM. AIM OF THE STUDY: The aim of this study was to explore the potential of AM as an antidepressant in forced-swimming test (FST) and unpredictable chronic mild stress (UCMS) rodent models and investigate its possible underlying mechanism. MATERIALS AND METHODS: Rats were orally administrated with 250, 500, and 1000 mg/kg body weight (bw) water extract of AM (WAM) for 28 and 35 consecutive days prior to the FST and UCMS protocols, respectively. The cerebral serotonin (5-HT) and the metabolites in the frontal cortex of rats were measured. The brain was dissected and the blood was collected to investigate the levels of inflammatory-related signaling pathway. RESULTS: All doses of WAM reduced the immobility time in the FST without disturbing autonomic locomotion. All doses of WAM prevented stress-induced abnormal behaviors in the UCMS model, including decreased sucrose preference and hypoactivity. 500 and 1000 mg/kg bw WAM attenuated the stress-induced increases in IL-1ß and TNF-α in the serum and cerebrum. 1000 mg/kg bw WAM alleviated brain inflammation by reducing the protein expression of ionized calcium binding adaptor molecule 1. CONCLUSION: WAM exhibited acute and chronic antidepressant-like effects, and may result from the anti-inflammatory actions. Therefore, the development of AM as a dietary therapy or adjuvant for depression treatment should be considered.

2.
Chemosphere ; 262: 127905, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33182152

RESUMO

Pot experiments were conducted to study combined effects of Ca and Cd on contents of Cd and Ca, and membrane transporters activities (CC (calcium channel protein), ATPase and CAXs (cationic/H+ antiporter) of two-year old Panax notoginseng with application of different concentrations of Ca2+ (0, 180 and 360 mgkg-1, prepared by Ca(OH)2 and CaCl2, respectively) under Cd2+ (0, 0.6, 6.0, and 12.0 mgkg-1, prepared by CdCl2•2.5H2O) treatments. The results showed that soil available Cd contents decreased with Ca(OH)2 and CaCl2 application. Soil pH value increased with Ca(OH)2 application. The contents of Cd in all parts of P. notoginseng increased with the increase in Cd treatment concentrations. The Cd content of P. notoginseng decreased with Ca(OH)2 and CaCl2 treatments. The activities of CC and ATPase in the main root of P. notoginseng decreased with the increase in Cd treatment concentrations and application of CaCl2. The activities of CC and ATPase increased with Ca(OH)2application. The activity of CAXs in the main root of P. notoginseng increased with the increase of Cd treatment concentration. The results indicate that Ca and Cd should be both related to membrane transporters activities and activities of CC, ATPase and CAXs are promoted by cooperation of Ca2+and OH+, which suggest the Ca(OH)2 application should be better than application of CaCl2 for Cd detoxification.


Assuntos
Cádmio/toxicidade , Cálcio/metabolismo , Panax notoginseng/fisiologia , Poluentes do Solo/toxicidade , Adenosina Trifosfatases/análise , Cádmio/análise , Proteínas de Membrana Transportadoras/metabolismo , Panax notoginseng/química , Raízes de Plantas/metabolismo , Solo/química , Poluentes do Solo/análise
3.
Bioresour Technol ; 320(Pt A): 124359, 2020 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-33157448

RESUMO

Bacterial, archaeal, and eukaryotic community composition and dynamics in leachate during solid waste decomposition were investigated using Illumina MiSeq sequencing. The functional enzyme-encoding genes of methanogenic pathways were also predicted via PICRUSt. Succession of bacterial, archaeal, and eukaryotic community composition in aerobic phase (AP), anaerobic acid phase (ACP), and methanogenic phase (MP) was observed. The main representatives of microbial phyla, genera, and species significantly (p < 0.05) differed at least two phases. Protist Ciliophora occurred at ACP and was prevalent in MP, suggesting a short food chain establishment in the methanogenesis. Bacterial, archaeal, fungi and eukaryotic community structure were all pH and biochemical oxygen demand (BOD5) dependent patter. Acetoclastic and hydrogenotrophic methanogenesis pathways with associated functional genes differed during solid waste decomposition and were inhibited in ACP.

4.
Adv Mater ; : e2005454, 2020 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-33169480

RESUMO

Metasurfaces present a potent platform to manipulate light by the spatial arrangement of sub-wavelength patterns with well-defined sizes and geometries, in thin films. Metasurfaces by definition are planar. However, it would be highly desirable to integrate metasurfaces with diverse, spatially programmed sub-wavelength features into a 3D monolith, to manipulate light within a compact 3D space. Here, a 3D photoengraving strategy is presented; that is, generation of such composite metasurfaces from a single microstructure via the irradiation of multiple interference laser beams onto different facets of the parent azopolymeric microstructure. Through "photofluidization," this technique enables independent inscription and erasing of metasurfaces onto and from individual facets of 3D monoliths with arbitrary shapes and dimensions, in a high-throughput fashion (over approximately a few cm2 at a time). By engraving discrete sub-wavelength 1D surface relief gratings of different pitches on different facets of an inverse pyramidal array, a multiplexing structure-color filter is demonstrated.

5.
Int J Clin Exp Pathol ; 13(10): 2586-2592, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33165433

RESUMO

Focal adhesion kinase is a non-receptor, tyrosine kinase of cells whose key functions are cell adhesion, migration, and invasion. Aberrant expression and regulation of FAK-mediated intracellular signaling pathways has been reported in several cancers and they are involved in cancer cell migration and apoptosis resistance. By RT-PCR, we found that cervical cancer cells showed a 4-fold increase of relative mRNA expression of FAK compared to control cells. In parallel, the FAK protein expression level was also elevated in cervical cancer cells. Interestingly, knockdown of FAK in cervical cancer cells showed attenuated cell proliferation and migration. Further, the FAK RNAi cells became more sensitive to chemotherapeutic drugs such as 5-FU and docetaxel and therefore the rate of cell survival is declined. The significant over-expression of FAK in cervical cancer cells might involve in cervical carcinogenesis and prolonged cell survival. This FAK overexpression might be a potential target for anti-cancer drugs to attenuate rapid cell proliferation and invasion by inducing apoptosis.

6.
J Proteome Res ; 2020 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-33175545

RESUMO

Tandem mass tag (TMT)-based mass spectrometry (MS) enables deep proteomic profiling of more than 10,000 proteins in complex biological samples but requires up to 100 µg protein in starting materials during a standard analysis. Here, we present a streamlined protocol to quantify more than 9000 proteins with 0.5 µg protein per sample by 16-plex TMT coupled with two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS). In this protocol, we optimized multiple conditions to reduce sample loss, including processing each sample in a single tube to minimize surface adsorption, increasing digestion enzymes to shorten proteolysis and function as carriers, eliminating a desalting step between digestion and TMT labeling, and developing miniaturized basic pH LC for prefractionation. By profiling 16 identical human brain tissue samples of Alzheimer's disease (AD), vascular dementia (VaD), and non-dementia controls, we directly compared this new microgram-scale protocol to the standard-scale protocol, quantifying 9116 and 10,869 proteins, respectively. Importantly, bioinformatics analysis indicated that the microgram-scale protocol had adequate sensitivity and reproducibility to detect differentially expressed proteins in disease-related pathways. Thus, this newly developed protocol is of general application for deep proteomics analysis of biological and clinical samples at sub-microgram levels.

7.
Aging Cell ; 19(11): e13252, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33040455

RESUMO

The risk of colitis and colorectal cancer increases markedly throughout adult life, endangering the health and lives of elderly individuals. Previous studies have proposed that bacterial translocation and infection are the main risk factors for these diseases. Therefore, in the present study, we aimed to identify the underlying mechanism by focusing on the mucus barrier function and mucin-type O-glycosylation. We evaluated alterations in the colon mucus layer in 2-, 16-, and 24-month-old mice and aged humans. Aged colons showed defective intestinal mucosal barrier and changed mucus properties. The miR-124-3p expression level was significantly increased in the aged distal colonic mucosa, which was accompanied by an increase in pathogens and bacterial translocation. Meanwhile, T-synthase, the rate-limiting enzyme in O-glycosylation, displayed an age-related decline in protein expression. Further experiments indicated that miR-124-3p modulated O-glycosylation by directly targeting T-synthase. Moreover, young mice overexpressing miR-124-3p exhibited abnormal glycosylation, early-onset, and more severe colitis. These data suggest that miR-124-3p predisposes to senile colitis by reducing T-synthase, and the miR-124-3p/T-synthase/O-glycans axis plays an essential role in maintaining the physiochemical properties of colonic mucus and intestinal homeostasis.

8.
Nanomedicine (Lond) ; 15(26): 2543-2561, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33103961

RESUMO

Aim: Cells with CD133 overexpression, a theoretical cancer stem cells (CSCs) marker, have been shown to induce colorectal cancer (CRC) initiation and relapse. Therefore, the detection and treatment of CSCs are the most important factors in overcoming CRC. Materials & methods: Herein, we developed a magnetite-based nanomedicine (superparamagnetic iron oxide@poly(sodium styrene sulfonate)/irinotecan/human serum albumin-anti-CD133 nanoparticle) using loco-regional hyperthermia combined with chemotherapy for CRC- and CSC-specific targeting treatment. Results: The designed nanoparticles were highly biocompatible and exhibited a higher temperature increase rate under radiofrequency generator irradiation. The nanoparticles could be used as a T2-weighted magnetic resonance imaging contrast media, and also applied during hyperthermia and chemotherapy to display a synergistic anticancer effect. Conclusion: Therefore, the superparamagnetic iron oxide@poly(sodium styrene sulfonate)/irinotecan/human serum albumin-anti-CD133 nanoparticles are a powerful candidate for future antitumor strategies.

9.
Nat Commun ; 11(1): 5253, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067459

RESUMO

Enterovirus 71 (EV71)-neutralizing antibodies correlate with protection and have potential as therapeutic agents. We isolate and characterize a panel of plasmablast-derived monoclonal antibodies from an infected child whose antibody response focuses on the plateau epitope near the icosahedral 3-fold axes. Eight of a total of 19 antibodies target this epitope and three of these potently neutralize the virus. Representative neutralizing antibodies 38-1-10A and 38-3-11A both confer effective protection against lethal EV71 challenge in hSCARB2-transgenic mice. The cryo-electron microscopy structures of the EV71 virion in complex with Fab fragments of these potent and protective antibodies reveal the details of a conserved epitope formed by residues in the BC and HI loops of VP2 and the BC and HI loops of VP3 spanning the region around the 3-fold axis. Remarkably, the two antibodies interact with the epitope in quite distinct ways. These plateau-binding antibodies provide templates for promising candidate therapeutics.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterovirus Humano A/química , Enterovirus Humano A/genética , Infecções por Enterovirus/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização
10.
Artigo em Inglês | MEDLINE | ID: mdl-33125207

RESUMO

Fibers that can reversibly and passively change colors along with body temperatures are highly desired for potential applications including temperature sensors, smart wearables, and photonic devices. Here, we develop a facile strategy to fabricate thermochromic photonic fibers, which could exhibit tunable structural colors as a function of temperatures. The thermochromic fibers are prepared by aligning superparamagnetic, carbon-encapsulated Fe3O4 colloidal nanocrystal clusters (Fe3O4@C CNCs) in a thermoresponsive hydrogel, poly(N-isopropylacrylamide) (PNIPAM), forming chain-like structures under an external magnetic field before gelation. When the fiber is transferred from air to water at room temperature, it changes color from dark green to red as it swells. The red color can be reversibly changed back to green as the temperature is raised to 36 °C, while the fiber shrinks and the reflection peak shifts from 642 to 494 nm. The swelling of the fiber is anisotropic: by 60% in the diameter direction but 45% in the length direction. Therefore, the fiber can act as a thermochromic actuator.

11.
Chin Med J (Engl) ; 2020 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-33074843

RESUMO

BACKGROUND: Resting-state functional magnetic resonance imaging (rs-fMRI) is a promising method for the study of brain function. Typically, rs-fMRI is performed on anesthetized animals. Although different functional connectivity (FC) in various anesthetics on whole brain have been studied, few studies have focused on different FC in the aged brain. Here, we measured FC under three commonly used anesthesia methods and analyzed data to determine if the FC in whole brain analysis were similar among groups. METHODS: Twenty-four male aged Wistar rats were randomly divided into three groups (n = 8 in each group). Anesthesia was performed under either isoflurane (ISO), combined ISO + dexmedetomidine (DEX) or α-chloralose (AC) according to the groups. Data of rs-fMRI was analyzed by FC in a voxel-wise way. Differences in the FC maps between the groups were analyzed by one-way analysis of variance and post hoc two-sample t tests. RESULTS: Compared with ISO + DEX anesthesia, ISO anesthesia caused increased FC in posterior brain and decreased FC in the middle brain of the aged rat. AC anesthesia caused global suppression as no increase in FC was observed. CONCLUSION: ISO could be used as a substitute for ISO + DEX in rat default mode network studies if the left temporal association cortex is not considered important.

12.
Adv Mater ; : e2004270, 2020 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33043501

RESUMO

Liquid crystal elastomers (LCEs) are of interest for applications such as soft robotics and shape-morphing devices. Among the different actuation mechanisms, light offers advantages such as spatial and local control of actuation via the photothermal effect. However, the unwanted aggregation of the light-absorbing nanoparticles in the LCE matrix will limit the photothermal response speed, actuation performance, and repeatability. Herein, a near-infrared-responsive LCE composite consisting of up to 0.20 wt% poly(ethylene glycol)-modified gold nanorods (AuNRs) without apparent aggregation is demonstrated. The high Young's modulus, 20.3 MPa, and excellent photothermal performance render repeated and fast actuation of the films (actuation within 5 s and recovery in 2 s) when exposed to 800 nm light at an average output power of ≈1.0 W cm-2 , while maintaining a large actuation strain (56%). Further, it is shown that the same sheet of AuNR/LCE film (100 µm thick) can be morphed into different shapes simply by varying the motifs of the photomasks.

13.
Nanoscale Res Lett ; 15(1): 197, 2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-33052458

RESUMO

In this study, self-catalyzed ß-FeSi2 nanowires, having been wanted but seldom achieved in a furnace, were synthesized via chemical vapor deposition method where the fabrication of ß-FeSi2 nanowires occurred on Si (100) substrates through the decomposition of the single-source precursor of anhydrous FeCl3 powders at 750-950 °C. We carefully varied temperatures, duration time, and the flow rates of carrier gases to control and investigate the growth of the nanowires. The morphology of the ß-FeSi2 nanowires was observed with scanning electron microscopy (SEM), while the structure of them was analyzed with X-ray diffraction (XRD) and transmission electron microscopy (TEM). The growth mechanism has been proposed and the physical properties of the iron disilicide nanowires were measured as well. In terms of the magnetization of ß-FeSi2, nanowires were found to be different from bulk and thin film; additionally, longer ß-FeSi2 nanowires possessed better magnetic properties, showing the room-temperature ferromagnetic behavior. Field emission measurements demonstrate that ß-FeSi2 nanowires can be applied in field emitters.

14.
Artigo em Inglês | MEDLINE | ID: mdl-33097426

RESUMO

BACKGROUND: Chlorhexidine is one of the most essential ingredients in infection control applications. Except qacA, the effects of other various efflux-medicated biocide genes (including qacB, qacC, qacEΔ1, qacH or norA) on biguanides resistance are still controversial. In addition, most of the studies have discussed the effect of qacA/B on clinical S. aureus isolates but not that qacA or qacB individually. METHODS: In total, 254 methicillin-resistant Staphylococcus aureus (MRSA), selected 30 methicillin-susceptible S. aureus (MSSA) clinical isolates from different patients during 2014-2015 and 15 S. aureus quality control strains (including Mu3 and Mu50) were included in the study. Various biocide genes, including qacA/B, qacC, qacH, qacEΔ1, and different types of norA, were determined through conventional PCR. S. aureus isolates with qacA/B (+) were analyzed using high-resolution melting curve (HRM) to differentiate qacA from qacB. The chlorhexidine MIC was determined using the agar dilution method. Univariate and multivariate statistics were analyzed to see which biocide resistant genes had effects on chlorhexidine MIC. RESULTS: Results of all HRM analyses (n = 22) were consistent with those of Sanger sequencing for differentiation of qacA from qacB. None of the isolates harbored qacH and only one MRSA harbored qacEΔ1. The harboring rate of qacA, qacB, and qacC among MRSA/MSSA isolates was 7.1% (n = 18)/0%, 38.2% (n = 97)/0%, and 7.5% (n = 19)/3.3% (n = 1), respectively. The most type of norA was norAI (n = 158), followed by norAIII (n = 87) and norAII (n = 9) among MRSA isolates. Based on the results of multivariate logistic regression analyses, only qacA and qacB would increase chlorhexidine MIC from ≤ 1 ug/ml to ≥ 2 ug/ml in MRSA isolates (P < 0.001) but not qacC or norA types (P=0.976 and 0.633 or 0.933, respectively). In addition, only qacA but not qacB was contributed to elevate chlorhexidine from ≤ 1 ug/ml to 4 ug/ml in MRSA isolates (P < 0.001 and 0.017, respectively). CONCLUSION: HRM analysis can be a great method to differentiate qacA from qacB. The biocide gene with the most effect on chlorhexidine MIC in S. aureus isolates was qacA, followed by qacB, but qacC and different types of norA did not have any effect on chlorhexidine susceptibility. Further investigation on the influence of qacB, qacC and types of norA on chlorhexidine susceptibility is necessary.

15.
J Comp Eff Res ; 9(14): 985-1002, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33025800

RESUMO

Aim: Breast cancer is a leading cause of cancer among women. Because guidelines on screening for breast cancer for certain ages are controversial, many experts advocate the use of shared decision making (SDM) using validated decision aids (DAs). Recent studies have concluded that DAs are beneficial; however, the results have great heterogeneity. Therefore, further studies are needed to improve understanding of these tools. Objective: This systematic review and meta-analysis aimed to investigate the impact of using web-based DAs in women aged 50 years and below facing the decision to be screened for breast cancer in comparison with usual care. Methods: PubMed, Web of Science, Embase and the Cochrane CENTRAL databases were searched up to February 2020 for studies assessing web-based DAs for women making a breast cancer screening decision and reported quality of decision-making outcomes. Using a random-effects model or a fixed-effects model, meta-analyses were conducted pooling results using mean differences (MD), standardized mean differences (SMD) and relative risks (RR). Results: Of 1097 unique citations, three randomized controlled trials and two before-after studies met the study eligibility criteria. Compared with usual care, web-based DAs increased knowledge (SMD = 0.69; 95% CI: 0.57-0.80; p < 0.00001), reduced decision conflict and increased the proportion of women who made an informed choice (RR = 1.86; 95% CI: 1.38 to 2.50; p < 0.0001), but did not change the intention of women deciding to be screened or affect decision regret. Conclusion: This analysis showed the positive effect of web-based DAs on patient-centered outcomes in breast cancer screening. In the future, more internet devices and free or larger discount WI-FI should be established to ensure more women can benefit from this effective tool.

16.
Complement Ther Clin Pract ; 41: 101240, 2020 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-32977216

RESUMO

BACKGROUND AND PURPOSE: and purpose: Massage has gained increasing attention for reducing peri-operative anxiety. We aimed to investigate the effectiveness of massage for peri-operative anxiety in adults. METHODS: Six English electronic databases were comprehensively searched from their inception to February 2020. Subgroup analysis, quality assessment, sensitivity analysis, meta-regression and publication bias assessment were performed. RESULTS: Twenty-five controlled trials comprising 2494 participants were included. The meta-analysis indicated that massage could significantly reduce peri-operative anxiety for most types of surgical patients. Specifically, it was effective for pre-, intra- and post-operative anxiety. Acupoint or specific body reflex area massage showed a larger effect than general massage did. Massage delivered by professionals and non-professionals were both effective. Massage lasting 10-20 min per session was the most worthy of recommendation. Massage was concomitant with the improvement of peri-operative vital signs and post-operative pain. CONCLUSION: Massage is a promising complementary therapy for ameliorating peri-operative anxiety in adults.

17.
J Med Internet Res ; 22(8): e19056, 2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32865500

RESUMO

BACKGROUND: The mass media have been condemned for encouraging young people to take dietary supplements (DS). Media literacy, which includes authors and audiences (AA), messages and meanings (MM), and representation and reality (RR) domains, is a new approach to teaching young adults to make better informed health decisions. However, it is not clear which domains are the most important for media literacy education. OBJECTIVE: The purpose of this study is to investigate the associations among individual factors, media literacy, and DS use. METHODS: The survey instrument included demographic items, the DS Media Literacy Scale (DSMLS), and DS use items (users or nonusers, types of DS, current use of DS, and intention to use DS in the future). The DSMLS is an 11-item instrument designed to assess college students' AA, MM, and RR media literacy in relation to DS. A total of 467 Taiwanese college students participated in the study. Descriptive statistical analysis, logistic regression analysis, and multiple regression analysis were conducted. RESULTS: A total of 338/467 (72.4%) participants reported using DS, and 176/467 (37.7%) consumed 3 or more supplements. Moreover, the MM media literacy domain was associated with having been a DS user (odds ratio 0.63, P=.002), current DS use (ß=-.10, P=.02), and intention to use DS in the future (ß=-.12, P=.011). Finally, perceived importance of health was positively related to current DS use (ß=.18, P=.001) and intention to use DS in the future (ß=.18, P=.001). CONCLUSIONS: This study showed that the majority of Taiwanese college students were DS users and used multiple types of supplements. Moreover, students with lower MM media literacy were more likely to be DS users, to take DS more frequently, and to have higher intentions for future frequent DS use. Finally, those who placed extreme importance on health were more likely to take DS frequently and have higher intentions for future frequent DS use.

18.
Mol Neurodegener ; 15(1): 51, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32907630

RESUMO

BACKGROUND: Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response. METHODS: We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or H2O2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage. RESULTS: We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm. CONCLUSIONS: This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.

19.
Cells ; 9(9)2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32887382

RESUMO

The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer's disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.

20.
Dev Cell ; 55(3): 272-288.e5, 2020 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898476

RESUMO

The dysregulation of the metabolic regulator TOR complex I (TORC1) contributes to a wide array of human pathologies. Tuberous sclerosis complex (TSC) is a potent inhibitor of TORC1. Here, we demonstrate that the Rag GTPase acts in both the amino-acid-sensing and growth factor signaling pathways to control TORC1 activity through the regulation of TSC dynamics in HeLa cells and Drosophila. We find that TSC lysosomal-cytosolic exchange increases in response to both amino acid and growth factor restriction. Moreover, the rate of exchange mirrors TSC function, with depletions of the Rag GTPase blocking TSC lysosomal mobility and rescuing TORC1 activity. Finally, we show that the GATOR2 complex controls the phosphorylation of TSC2, which is essential for TSC exchange. Our data support the model that the amino acid and growth factor signaling pathways converge on the Rag GTPase to inhibit TORC1 activity through the regulation of TSC dynamics.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA