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Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.
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Encéfalo/fisiologia , Proteínas Ativadoras de GTPase/genética , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Optogenética/métodos , Ritmo Teta , Vigília , Potenciais de Ação , Animais , Encéfalo/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Ratos Sprague-Dawley , CorridaRESUMO
Structural maintenance of chromosome (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. SMC complexes form long rod-like structures, which can change to ring-like and elbow-bent conformations upon binding ATP, DNA, and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C-terminal half (aa230-505) is required for binding to its PpNSE6 partner, while the N-terminal half (aa1-230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head-proximal joint region and PpSMC5 hinge-proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Therefore, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow-bent conformation, the later model being consistent with the role of NSE5/NSE6 dimer as SMC5/6 loading factor to DNA lesions. In addition, we generated the P. patens Ppnse5KO1 mutant line with an N-terminally truncated version of PpNSE5, which exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the role of PpNSE5-PpSMC6 interaction in SMC5/6 loading to DNA lesions.
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Bryopsida , Proteínas de Plantas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Bryopsida/genética , Bryopsida/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Cromossomos de Plantas/genética , Ligação ProteicaRESUMO
With the advent of soft ionization techniques such as electrospray (ESI) and matrix-assisted laser desorption/ionization (MALDI) to produce intact gas-phase ions from nonvolatile macromolecules, mass spectrometry has become an essential technique in the field of polymeric materials. However, (co)polymers of very high molecular weight or with reticulated architectures still escape ESI or MALDI, mainly due to solubility issues. Strategies developed to tackle such an analytical challenge all rely on sample degradation to produce low-mass species amenable to existing ionization methods. Yet, chain degradation needs to be partial and controlled to generate sufficiently large species that still contain topological or architectural information. The present article reviews the different analytical degradation strategies implemented to perform mass spectrometry of these challenging synthetic polymers, covering thermal degradation approaches in sources developed in the 2000s, off-line sample pre-treatments for controlled chemical degradation of polymeric substrates, and most recent achievements employing reactive ionization modes to perform chemolysis on-line with MS.
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The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.
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Fatores de Ribosilação do ADP , Neoplasias , Humanos , Fatores de Ribosilação do ADP/metabolismo , Clorobenzenos , Pirazóis , Proteínas Ativadoras de GTPase/metabolismo , Fator 1 de Ribosilação do ADP/metabolismoRESUMO
Structural maintenance of chromosomes (SMC) complexes are molecular machines ensuring chromatin organization at higher levels. They play direct roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of long-armed SMC, kleisin, and kleisin-associated subunits. Additional factors, like NSE6 within SMC5/6, bind to SMC core complexes and regulate their activities. In the human HsNSE6/SLF2, we recently identified a new CANIN domain. Here we tracked down its sequence homology to lower plants, selected the bryophyte Physcomitrium patens, and analyzed PpNSE6 protein-protein interactions to explore its conservation in detail. We identified a previously unrecognized core sequence motif conserved from yeasts to humans within the NSE6 CANIN domain. This motif mediates the interaction between NSE6 and its NSE5 partner in yeasts and plants. In addition, the CANIN domain and its preceding PpNSE6 sequences bind both PpSMC5 and PpSMC6 arms. Interestingly, we mapped the PpNSE6-binding site at the PpSMC5 arm right next to the PpNSE2-binding surface. The position of NSE6 at SMC arms suggests its role in the regulation of SMC5/6 dynamics. Consistent with the regulatory role of NSE6 subunits, Ppnse6 mutant lines were viable and sensitive to the DNA-damaging drug bleomycin and lost a large portion of rDNA copies. These moss mutants also exhibited reduced growth and developmental aberrations. Altogether, our data showed the conserved function of the NSE6 subunit and architecture of the SMC5/6 complex across species.
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Proteínas Cromossômicas não Histona , Reparo do DNA , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos , Domínios Proteicos , Proteínas de Ciclo Celular/metabolismoRESUMO
The Arabidopsis splicing factor serine/arginine-rich 45 (SR45) contributes to several biological processes. The sr45-1 loss-of-function mutant exhibits delayed root development, late flowering, unusual numbers of floral organs, shorter siliques with decreased seed sets, narrower leaves and petals, and altered metal distribution. SR45 bears a unique RNA recognition motif (RRM) flanked by one serine/arginine-rich (RS) domain on both sides. Here, we studied the function of each SR45 domains by examining their involvement in: (i) the spatial distribution of SR45; (ii) the establishment of a protein-protein interaction network including spliceosomal and exon-exon junction complex (EJC) components; and (iii) the RNA binding specificity. We report that the endogenous SR45 promoter is active during vegetative and reproductive growth, and that the SR45 protein localizes in the nucleus. We demonstrate that the C-terminal arginine/serine-rich domain is a determinant of nuclear localization. We show that the SR45 RRM domain specifically binds purine-rich RNA motifs via three residues (H101, H141, and Y143), and is also involved in protein-protein interactions. We further show that SR45 bridges both mRNA splicing and surveillance machineries as a partner of EJC core components and peripheral factors, which requires phosphoresidues probably phosphorylated by kinases from both the CLK and SRPK families. Our findings provide insights into the contribution of each SR45 domain to both spliceosome and EJC assemblies.
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Proteínas de Arabidopsis , Arabidopsis , Éxons , Fatores de Processamento de RNA , Splicing de RNA , Humanos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Splicing de RNA/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Inclusions comprised of microtubule-associated protein tau (tau) are implicated in a group of neurodegenerative diseases, collectively known as tauopathies, that include Alzheimer's disease (AD). The spreading of misfolded tau "seeds" along neuronal networks is thought to play a crucial role in the progression of tau pathology. Consequently, restricting the release or uptake of tau seeds may inhibit the spread of tau pathology and potentially halt the advancement of the disease. Previous studies have demonstrated that the Mammalian Suppressor of Tauopathy 2 (MSUT2), an RNA binding protein, modulates tau pathogenesis in a transgenic mouse model. In this study, we investigated the impact of MSUT2 on tau pathogenesis using tau seeding models. Our findings indicate that the loss of MSUT2 mitigates human tau seed-induced pathology in neuron cultures and mouse models. In addition, MSUT2 regulates many gene transcripts, including the Adenosine Receptor 1 (A1AR), and we show that down regulation or inhibition of A1AR modulates the activity of the "ArfGAP with SH3 Domain, Ankyrin Repeat, and PH Domain 1 protein" (ASAP1), thereby influencing the internalization of pathogenic tau seeds into neurons resulting in reduction of tau pathology.
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Doença de Alzheimer , Tauopatias , Camundongos , Humanos , Animais , Encéfalo/patologia , Proteínas tau/metabolismo , Tauopatias/patologia , Doença de Alzheimer/patologia , Neurônios/patologia , Camundongos Transgênicos , Mamíferos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
The opium poppy (Papaver somniferum) is a global commercial crop that has been historically valued for both medicinal and culinary purposes. Naturally occurring opium alkaloids including morphine, codeine, thebaine, noscapine, and papaverine are found primarily in the latex produced by the plant. If the plant is allowed to fully mature, poppy seeds that do not contain the opium alkaloids will form within the pods and may be used in the food industry. It is possible for the seeds to become contaminated with alkaloids by the latex during harvesting, posing a potential health risk for consumers. In the USA, there have been more than 600 reported adverse events including 19 fatalities that may be linked to the consumption of a contaminated poppy-containing product such as home-brewed poppy seed tea. Unwashed poppy seeds and pods may be purchased over the Internet and shipped worldwide. The Forensic Chemistry Center, US Food and Drug Administration (FDA) has evaluated several mass spectrometers (MS) capable of rapid screening to be used for high-throughput analysis of samples such as poppy seeds. These include a direct analysis in real-time (DART) ambient ionization source coupled to a single-quadrupole MS, an atmospheric solids analysis probe (ASAP) ionization source coupled to the same MS, and ion mobility spectrometers (IMS). These instruments have been used to analyze 17 poppy seed samples for the presence of alkaloids, and the results were compared to data obtained using liquid chromatography with mass spectral detection (LC-MS/MS). Results from the 17 poppy seed samples indicate that the DART-MS, ASAP-MS, and IMS devices detect many of the same alkaloids confirmed during the LC-MS/MS analyses, although both the false-positive and false-negative rates are higher, possibly due to the non-homogeneity of the samples and the lack of chromatographic separation.
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Alcaloides , Papaver , Papaver/química , Ópio/análise , Cromatografia Líquida , Espectrometria de Mobilidade Iônica , Látex/análise , Espectrometria de Massas em Tandem , Morfina , Alcaloides/análise , Sementes/químicaRESUMO
Allergic asthma is a chronic inflammatory disease of airways involving complex mechanisms, including MAS-related GPR family member X2 (MRGPRX2) and its orthologue MRGPRB2 on mast cells (MCs). Although miRNAs have been previously shown to related to allergic asthma, the role of miR-212/132 in this process has not been studied. In this study, the predicted pairing of miRNAs and MRGPRX2 (MRGPRB2) mRNAs was carried out by online databases and the function was verify using in vivo and in vitro experiments. Database prediction showed that miR-212/132 interact with MRGPRX2 and MRGPRB2. miR-212/132 mimics alleviated MRGPRB2 mRNA expression as well as pathology changes in lungs and AHR of mice with airway inflammation in vivo. The expression level of MRGPRB2 in the mice lungs after inhaled OVA was also decreased by miR-212/132 mimics. Meanwhile, miR-212/132 inhibited MCs degranulation and cytokines release triggered by C48/80 in vitro. Further, ASAP1 (ARF GTPase-Activating Protein 1) was selected from the junction related pathways using RNAseq and KEGG enrichment. ASAP1 mRNA level was upregulated in airway inflammation and MCs activation and decreased by miR-212/132 mimics. miR-212/132 attenuated OVA-induced airway inflammation by inhibiting MCs activation through MRGPRX2 and ASAP1.
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Asma , Mastócitos , MicroRNAs , Animais , Camundongos , Asma/induzido quimicamente , Asma/genética , Asma/metabolismo , Degranulação Celular , Inflamação/induzido quimicamente , Inflamação/metabolismo , Mastócitos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Ovalbumina/efeitos adversos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , RNA Mensageiro/metabolismo , HumanosRESUMO
In this paper, we report two Accelerated Stability Assessment Program (ASAP) studies for a pediatric drug product. Whereas the first study using a generic design failed to establish a predictive model, the second one was successful after troubleshooting the first study and customizing the study conditions. This work highlighted important lessons learned from designing an ASAP study for formulations containing excipients that could undergo phase change at high humidity levels. The stability predictions by the second ASAP model were consistent with available long-term stability data of the drug product under various storage conditions in two different packaging configurations. The ASAP model was part of the justifications accepted by the health authority to submit a stability package with reduced long-term stability data from the primary stability batches for a Supplemental New Drug Application (sNDA).
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Química Farmacêutica , Estabilidade de Medicamentos , Excipientes , Excipientes/química , Química Farmacêutica/métodos , Umidade , Armazenamento de Medicamentos , Embalagem de Medicamentos/métodos , Embalagem de Medicamentos/normas , Composição de Medicamentos/métodos , Humanos , Criança , Preparações Farmacêuticas/química , Pediatria/métodosRESUMO
PURPOSE: The summary presented herein covers recommendations on the early detection of prostate cancer and provides a framework to facilitate clinical decision-making in the implementation of prostate cancer screening, biopsy, and follow-up. This is Part II of a two-part series focusing on initial and repeat biopsies, and biopsy technique. Please refer to Part I for discussion of initial prostate cancer screening recommendations. MATERIALS AND METHODS: The systematic review utilized to inform this guideline was conducted by an independent methodological consultant. The systematic review was based on searches in Ovid MEDLINE and Embase and Cochrane Database of Systematic Reviews (January 1, 2000-November 21, 2022). Searches were supplemented by reviewing reference lists of relevant articles. RESULTS: The Early Detection of Prostate Cancer Panel developed evidence- and consensus-based guideline statements to provide guidance in prostate cancer screening, initial and repeat biopsies, and biopsy technique. CONCLUSIONS: The evaluation of prostate cancer risk should be focused on the detection of clinically significant prostate cancer (Grade Group 2 or higher [GG2+]). The use of laboratory biomarkers, prostate MRI, and biopsy techniques described herein may improve detection and safety when a prostate biopsy is deemed necessary following prostate cancer screening.
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Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/patologia , Próstata/diagnóstico por imagem , Próstata/patologia , Detecção Precoce de Câncer , Antígeno Prostático Específico , Revisões Sistemáticas como Assunto , Biópsia , Imageamento por Ressonância Magnética , Biópsia Guiada por Imagem/métodosRESUMO
Several automated molecular methods have emerged for distinguishing eukaryote species based on DNA sequence data. However, there are knowledge gaps around which of these single-locus methods is more accurate for the identification of microalgal species, such as the highly diverse and ecologically relevant diatoms. We applied genetic divergence, Automatic Barcode Gap Discovery for primary species delimitation (ABGD), Assemble Species by Automatic Partitioning (ASAP), Statistical Parsimony Network Analysis (SPNA), Generalized Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP) using partial cox1, rbcL, 5.8S + ITS2, ITS1 + 5.8S + ITS2 markers to delineate species and compare to published polyphasic identification data (morphological features, phylogeny and sexual reproductive isolation) to test the resolution of these methods. ASAP, ABGD, SPNA and PTP models resolved species of Eunotia, Seminavis, Nitzschia, Sellaphora and Pseudo-nitzschia corresponding to previous polyphasic identification, including reproductive isolation studies. In most cases, these models identified diatom species in similar ways, regardless of sequence fragment length. GMYC model presented smallest number of results that agreed with previous published identification. Following the recommendations for proper use of each model presented in the present study, these models can be useful tools to identify cryptic or closely related species of diatoms, even when the datasets have relatively few sequences.
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Diatomáceas , Diatomáceas/genética , DNA , Código de Barras de DNA Taxonômico , FilogeniaRESUMO
INTRODUCTION AND OBJECTIVES: Hepatocellular carcinoma (HCC) may be diagnosed using the GAAP and ASAP models; our goal was to verify and evaluate their diagnostic effectiveness compared to alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin (DCP), and AFP & DCP for both HCC and HCC caused by the hepatitis B virus (HBV). PATIENTS AND METHODS: GAAP and ASAP models were validated and compared using a retrospective investigation of 938 patients from our hospital between July 2020 and July 2021. RESULTS: Both the GAAP and ASAP models had better diagnostic efficacy than AFP, DCP, AFP & DCP. The GAAP model achieved better performance in section A for the detection of HCC and in section C for the detection of HBV-HCC than the ASAP model. The Hosmer-Lemeshow test showed that the GAAP and ASAP models were well-calibrated for the diagnoses of these two groups. To be more specific, the area under curve (AUC) of the GAAP model for HCC detection in section A was 0.862 [95% confidence interval (CI): 0.838-0.883], and that of the ASAP model was 0.850 [95% CI: 0.826-0.872]. The AUC of the GAAP model for HBV-HCC detection in section C was 0.897 [95% CI: 0.872-0.918], and that of the ASAP model was 0.878 [95% CI: 0.852-0.902]. CONCLUSIONS: The GAAP model was more accurate and reliable than the AFP, DCP, AFP and DCP, as well as the ASAP model in section A for the detection of HCC and in section C for the detection of HBV-HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , alfa-Fetoproteínas , Estudos Retrospectivos , Neoplasias Hepáticas/patologia , Biomarcadores Tumorais , Biomarcadores , Precursores de Proteínas , Protrombina , Vírus da Hepatite BRESUMO
BACKGROUND: Diagnostic panels based on multiple biomarkers and clinical characteristics are considered more favorable than individual biomarker to diagnose hepatocellular carcinoma (HCC). Based on age, sex, alpha-fetoprotein (AFP), and protein induced by vitamin K absence II (PIVKA-II) with/without AFP-L3, ASAP and GALAD models are potential diagnostic panels. The diagnostic performances of these two panels were compared relative to HCC detection among patients with various etiologies of chronic liver diseases (CLDs). METHODS: A multicenter case-control study recruited CLDs patients with and without HCC from 14 Chinese hospitals. The etiologies of CLDs included hepatitis B virus (HBV), hepatitis C virus (HCV), alcoholic liver disease (ALD), and nonalcoholic fatty liver disease (NAFLD). Using area under the receiver operating characteristic curve (AUC) values, the diagnostic performances of ASAP and GALAD models were compared to detect HCC among patients with various etiologies of CLDs. RESULTS: Among 248 HCC patients and 722 CLD controls, the ASAP model demonstrated the highest AUC (0.886) to detect HCC at any stage, outperforming the GALAD model (0.853, P = 0.001), as well as any individual biomarker (0.687-0.799, all P < 0.001). In the subgroup analysis of various CLDs etiologies, the ASAP model outperformed the GALAD model to HCC independent of CLDs etiology. In addition, the ASAP model performed better in detecting early-stage (BCLC stage 0/A) HCC versus the GALAD model. CONCLUSIONS: Despite using one less laboratory variable (AFP-L3), the ASAP model demonstrated better diagnostic performance than the GALAD model to detect all-stage HCC among patients with various etiologies of CLDs-related HCC.
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SH3 domains are common protein binding modules. The target sequence of SH3 domains is usually a proline-rich motif (PRM) containing a minimal "PxxP" sequence. The mechanism of how different SH3 domains specifically choose their targets from vast PxxP-containing sequences is still not very clear, as many reported SH3/PRM interactions are weak and promiscuous. Here, we identified the binding of the SH3 domain of ASAP1 to the PRM of MICAL1 with a sub-µM binding affinity, and determined the crystal structure of ASAP1-SH3 and MICAL1-PRM complex. Our structural and biochemical analyses revealed that the target-binding pocket of ASAP1-SH3 contains two negatively charged patches to recognize the "xPx + Px+" sequence in MICAL1-PRM and consequently strengthen the interaction, differing from the typical SH3/PRM interaction. This unique PRM-binding pocket is also found in the SH3 domains of GTPase Regulator associated with focal adhesion kinase (GRAF) and Src kinase associated phosphoprotein 1 (SKAP1), which we named SH3AGS. In addition, we searched the Swiss-Prot database and found ~130 proteins with the SH3AGS-binding PRM in silico. Finally, gene ontology analysis suggests that the strong interaction between the SH3AGS-containing proteins and their targets may play roles in actin cytoskeleton regulation and vesicle trafficking.
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Prolina , Domínios de Homologia de src , Sítios de Ligação , Sequência de Aminoácidos , Prolina/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: RNA-binding protein with serine-rich domain 1 (RNPS1) is a member of a splicing-dependent mega Dalton protein complex or exon junction complex (EJC). During splicing, RNPS1 acts as a protector of global transcriptome integrity by suppressing the usage of cryptic splice sites. Additionally, RNPS1 functions in almost all stages of mRNA metabolism, including constitutive splicing, alternative splicing, translation and nonsense-mediated mRNA decay (NMD). The aim of the present study was to generate a highly specific polyclonal antibody against human RNPS1. METHODS AND RESULTS: A plasmid, pHis-TEV-RNPS1, has been constructed to overexpress recombinant RNPS1 (22-305 amino acids) by cloning the nucleotide sequence downstream of an N-terminal His-tag in the parent plasmid pHis-TEV. The recombinant plasmid was then transformed into Rosetta and expression was induced using IPTG. The His-tagged RNPS1 protein was purified using Ni-NTA affinity chromatography. The rabbit antiserum was then obtained by immunizing rabbits with the purified recombinant RNPS1 protein. The antiserum was further purified by antigen-immunoaffinity chromatography. The sensitivity and the specificity of the polyclonal antibody were assessed by enzyme-linked immunosorbent assay (ELISA) and knockdown assay. ELISA demonstrated that the antibody has a high binding affinity for RNPS1 and the usable titre is 1:2000. CONCLUSION: The antibody detected RNPS1 in human, mouse cell lines and rat tissue in Western blot. Importantly, the antibody efficiently detected the decrease in RNPS1 expression in siRNA induced knockdown assay, indicating the specificity of the antibody. The polyclonal antibody against RNPS1 will be a useful tool for performing further functional studies on RNPS1.
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Splicing de RNA , Proteínas de Ligação a RNA , Animais , Anticorpos , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Sítios de Splice de RNA , Proteínas de Ligação a RNA/genética , Coelhos , Ratos , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
Biopolymers based on polylactic acid (PLA) and starch have numerous advantages, such as coming from renewable sources or being compostable, though they can have deficiencies in mechanical properties, and for this reason, polyester resins are occasionally added to them in order to improve their properties. In this work, migration from a PLA sample and from another starch-based biopolymer to three different food simulants was studied. Attention was focused on the determination of oligomers. The analysis was first performed by ultraperformance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS), which allowed the identification of the oligomers present in migration. Then, the samples were analyzed by two ambient desorption/ionization techniques directly coupled to mass spectrometry (ADI), direct analysis in real-time coupled to standardized voltage and pressure (DART-MS) and atmospheric pressure solids analysis probe (ASAP-MS). These methodologies were able to detect simultaneously the main oligomers migrants and their adducts in a very rapid and effective way. Nineteen different polyester oligomers, fourteen linear and five cyclic, composed of different combinations of adipic acid [AA], propylene glycol [PG], dipropylene glycol [DPG], 2,2-dibutyl-1,3-propanediol [DBPG], or isobutanol [i-BuOH] were detected in migration samples from PLA. In migration samples from starch-based biopolymer, fourteen oligomers from poly(butylene adipate co-terephthalate) polyester (PBAT) were identified, twelve cyclic and two linear. The results from ADI techniques showed that they are a very promising alternative tool to assess the safety and legal compliance of food packaging materials.
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Embalagem de Alimentos , Poliésteres/análise , Amido/análise , Adipatos/análise , Cromatografia Líquida de Alta Pressão , Contaminação de Alimentos/análise , Espectrometria de MassasRESUMO
Monosaccharides and disaccharides are important dietary components, but if insufficiently metabolized by some population subgroups, they are also linked to disease patterns. Thus, the correct analytical identification, quantification, and labeling of these food components are crucial to inform and potentially protect consumers. Enzymatic assays and high-performance anion-exchange chromatography with pulsed amperometric detection are established methods for the quantification of monosaccharides and disaccharides that, however, require long measuring times (60-180 min). Accelerated methods for the identification and quantification of the nutritionally relevant monosaccharides and disaccharides d-glucose, d-galactose, d-fructose, sucrose, lactose, and maltose were therefore developed. To realize this goal, the NMR experiments HSQC (heteronuclear single quantum coherence) and acceleration by sharing adjacent polarization (ASAP)-HSQC were applied. Measurement times were reduced to 27 and 6 min, respectively, by optimizing the interscan delay and applying non-uniform sampling. The optimized methods were used to quantify d-glucose, d-galactose, d-fructose, sucrose, and lactose in various dairy products. Results of the HSQC and ASAP-HSQC methods are equivalent to the results of the reference methods in terms of both precision and accuracy, demonstrating that these methods can be used to correctly analyze nutritionally relevant monosaccharides and disaccharides in short times.
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Dissacarídeos , Monossacarídeos , Laticínios , Dissacarídeos/metabolismo , Frutose , Galactose , Glucose , Lactose , Monossacarídeos/metabolismo , SacaroseRESUMO
An accelerated stability model approach was demonstrated to accurately predict the long-term shelf life of example drug substances and drug products (indigo carmine tablets and L-ascorbic acid powder) where appearance changes were shelf life-limiting. The products were exposed outside of packaging to conditions from 50 to 90 °C and 0-80% relative humidity for up to one month to accelerate appearance changes. The appearance changes of stressed samples were quantitated using the CIELAB color scale (calculated ΔE*), where a visual assessment of appearance changes likely to be noticeable was used to assign a ΔE* specification limit. ASAPprime® software was employed to create an isoconversion paradigm, modeled in packaging by the moisture-modified Arrhenius equation, that predicted the color changes of the products within the error bars of the model to nine months at 25 °C/60% RH, 30 °C/65% RH, and 40 °C/75% RH. Overall, these case studies indicate that the ASAPprime® approach for accelerated stability studies are a fast, accurate approach to modeling appearance changes.
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Ácido Ascórbico , Índigo Carmim , Estabilidade de Medicamentos , Umidade , Pós , Comprimidos , TemperaturaRESUMO
The Arf GTPase-activating protein (Arf GAP) with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) establishes a connection between the cell membrane and the cortical actin cytoskeleton. The formation, maintenance, and turnover of actin filaments and bundles in the actin cortex are important for cell adhesion, invasion, and migration. Here, using actin cosedimentation, polymerization, and depolymerization assays, along with total internal reflection fluorescence (TIRF), confocal, and EM analyses, we show that the N-terminal N-BAR domain of ASAP1 directly binds to F-actin. We found that ASAP1 homodimerization aligns F-actin in predominantly unipolar bundles and stabilizes them against depolymerization. Furthermore, the ASAP1 N-BAR domain moderately reduced the spontaneous polymerization of G-actin. The overexpression of the ASAP1 BAR-PH tandem domain in fibroblasts induced the formation of actin-filled projections more effectively than did full-length ASAP1. An ASAP1 construct that lacked the N-BAR domain failed to induce cellular projections. Our results suggest that ASAP1 regulates the dynamics and the formation of higher-order actin structures, possibly through direct binding to F-actin via its N-BAR domain. We propose that ASAP1 is a hub protein for dynamic protein-protein interactions in mechanosensitive structures, such as focal adhesions, invadopodia, and podosomes, that are directly implicated in oncogenic events. The effect of ASAP1 on actin dynamics puts a spotlight on its function as a central signaling molecule that regulates the dynamics of the actin cytoskeleton by transmitting signals from the plasma membrane.