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1.
Cell ; 151(5): 1042-54, 2012 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-23178123

RESUMO

The conserved transcriptional regulator heat shock factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC, and Hsf1 senses an RQC-mediated translation-stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.


Assuntos
Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico/genética , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina
2.
Nat Methods ; 17(10): 1040-1051, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32807956

RESUMO

The behavior and microscale processes associated with freely suspended organisms, along with sinking particles underlie key ecological processes in the ocean. Mechanistically studying such multiscale processes in the laboratory presents a considerable challenge for microscopy: how to measure single cells at microscale resolution, while allowing them to freely move hundreds of meters in the vertical direction? Here we present a solution in the form of a scale-free, vertical tracking microscope, based on a 'hydrodynamic treadmill' with no bounds for motion along the axis of gravity. Using this method to bridge spatial scales, we assembled a multiscale behavioral dataset of nonadherent planktonic cells and organisms. Furthermore, we demonstrate a 'virtual-reality system for single cells', wherein cell behavior directly controls its ambient environmental parameters, enabling quantitative behavioral assays. Our method and results exemplify a new paradigm of multiscale measurement, wherein one can observe and probe macroscale and ecologically relevant phenomena at microscale resolution. Beyond the marine context, we foresee that our method will allow biological measurements of cells and organisms in a suspended state by freeing them from the confines of the coverslip.


Assuntos
Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Microscopia/instrumentação , Microscopia/métodos , Animais , Invertebrados/classificação , Invertebrados/fisiologia , Larva/fisiologia , Movimento , Plâncton , Natação , Interface Usuário-Computador
3.
Nature ; 547(7662): 236-240, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28636604

RESUMO

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Animais , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA/metabolismo , Inativação Gênica , Heterocromatina/química , Heterocromatina/genética , Humanos , Ligantes , Camundongos , Células NIH 3T3 , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Fosforilação , Solubilidade , Fator de Transcrição TFIIB/metabolismo
4.
BMC Med Educ ; 22(1): 446, 2022 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-35681189

RESUMO

BACKGROUND: This study investigated the relevance of the revised 2-factor study process questionnaire (R-SPQ-2F) for exploring medical students' approaches to learning in Qatar and identify how factors like gender, age, educational attainment, and prior experience with health care influence students' adoption of deep approaches to learning. METHODS: The sample consisted of 108 medical students (44% male, 56% female) from all four years of medical school at Weill Cornell Medicine-Qatar (WCM-Q). Participants completed the 20-item R-SPQ-2F questionnaire to measure their learning approaches through a structural model contrasting deep and surface learning. Participants also completed a survey collecting demographic information. RESULTS: Statistical analysis revealed significant differences in deep learning approaches across year levels for both men and women. Additionally, educational attainment played a significant role in students' approaches to learning. CONCLUSIONS: Based on structural equation modeling, this cross-verification study supports the R-SPQ-2F instrument and offers additional evidence for its robustness and application to medical education. These findings may help educational and program leaders in Qatar better understand medical students' learning approaches to enhance their pedagogical practices.


Assuntos
Estudantes de Medicina , Currículo , Avaliação Educacional , Feminino , Humanos , Masculino , Catar , Inquéritos e Questionários
5.
Nature ; 496(7445): 377-81, 2013 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-23485968

RESUMO

A hallmark of histone H3 lysine 9 (H3K9)-methylated heterochromatin, conserved from the fission yeast Schizosaccharomyces pombe to humans, is its ability to spread to adjacent genomic regions. Central to heterochromatin spread is heterochromatin protein 1 (HP1), which recognizes H3K9-methylated chromatin, oligomerizes and forms a versatile platform that participates in diverse nuclear functions, ranging from gene silencing to chromosome segregation. How HP1 proteins assemble on methylated nucleosomal templates and how the HP1-nucleosome complex achieves functional versatility remain poorly understood. Here we show that binding of the key S. pombe HP1 protein, Swi6, to methylated nucleosomes drives a switch from an auto-inhibited state to a spreading-competent state. In the auto-inhibited state, a histone-mimic sequence in one Swi6 monomer blocks methyl-mark recognition by the chromodomain of another monomer. Auto-inhibition is relieved by recognition of two template features, the H3K9 methyl mark and nucleosomal DNA. Cryo-electron-microscopy-based reconstruction of the Swi6-nucleosome complex provides the overall architecture of the spreading-competent state in which two unbound chromodomain sticky ends appear exposed. Disruption of the switch between the auto-inhibited and spreading-competent states disrupts heterochromatin assembly and gene silencing in vivo. These findings are reminiscent of other conditionally activated polymerization processes, such as actin nucleation, and open up a new class of regulatory mechanisms that operate on chromatin in vivo.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Animais , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/ultraestrutura , Microscopia Crioeletrônica , Inativação Gênica , Heterocromatina/química , Heterocromatina/ultraestrutura , Histonas/química , Histonas/metabolismo , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Estrutura Terciária de Proteína , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/antagonistas & inibidores , Proteínas de Schizosaccharomyces pombe/ultraestrutura , Xenopus laevis
6.
Biochemistry ; 57(17): 2540-2548, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29644850

RESUMO

In eukaryotic cells, structures called heterochromatin play critical roles in nuclear processes ranging from gene repression to chromosome segregation. Biochemical and in vivo studies over the past several decades have implied that the diverse functions of heterochromatin rely on the ability of these structures to spread across large regions of the genome, to compact the underlying DNA, and to recruit different types of activities. Recent observations have suggested that heterochromatin may possess liquid droplet-like properties. Here, we discuss how these observations provide a new perspective on the mechanisms for the assembly, regulation, and functions of heterochromatin.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , DNA/genética , Heterocromatina/genética , Proteínas Cromossômicas não Histona/química , DNA/química , Regulação da Expressão Gênica/genética , Heterocromatina/química , Histonas/química , Histonas/genética , Nucleossomos/química , Nucleossomos/genética , Transição de Fase , Schizosaccharomyces/genética
7.
Int J Neuropsychopharmacol ; 21(12): 1079-1089, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30260415

RESUMO

Background: We hypothesized that propofol, a unique general anesthetic that engages N-methyl-D-aspartate and gamma-aminobutyric acid receptors, has antidepressant properties. This open-label trial was designed to collect preliminary data regarding the feasibility, tolerability, and efficacy of deep propofol anesthesia for treatment-resistant depression. Methods: Ten participants with moderate-to-severe medication-resistant depression (age 18-45 years and otherwise healthy) each received a series of 10 propofol infusions. Propofol was dosed to strongly suppress electroencephalographic activity for 15 minutes. The primary depression outcome was the 24-item Hamilton Depression Rating Scale. Self-rated depression scores were compared with a group of 20 patients who received electroconvulsive therapy. Results: Propofol treatments were well tolerated by all subjects. No serious adverse events occurred. Montreal Cognitive Assessment scores remained stable. Hamilton scores decreased by a mean of 20 points (range 0-45 points), corresponding to a mean 58% improvement from baseline (range 0-100%). Six of the 10 subjects met the criteria for response (>50% improvement). Self-rated depression improved similarly in the propofol group and electroconvulsive therapy group. Five of the 6 propofol responders remained well for at least 3 months. In posthoc analyses, electroencephalographic measures predicted clinical response to propofol. Conclusions: These findings demonstrate that high-dose propofol treatment is feasible and well tolerated by individuals with treatment-resistant depression who are otherwise healthy. Propofol may trigger rapid, durable antidepressant effects similar to electroconvulsive therapy but with fewer side effects. Controlled studies are warranted to further evaluate propofol's antidepressant efficacy and mechanisms of action. ClinicalTrials.gov: NCT02935647.


Assuntos
Anestésicos Intravenosos/farmacologia , Transtorno Depressivo Resistente a Tratamento/tratamento farmacológico , Eletroencefalografia/efeitos dos fármacos , Avaliação de Resultados em Cuidados de Saúde , Propofol/farmacologia , Adolescente , Adulto , Anestésicos Intravenosos/administração & dosagem , Anestésicos Intravenosos/efeitos adversos , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Propofol/administração & dosagem , Propofol/efeitos adversos , Adulto Jovem
8.
Mem Cognit ; 44(2): 207-19, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26450589

RESUMO

This study investigated the relative roles of visuospatial versus linguistic working memory (WM) systems in the online generation of bridging inferences while viewers comprehend visual narratives. We contrasted these relative roles in the visuospatial primacy hypothesis versus the shared (visuospatial & linguistic) systems hypothesis, and tested them in 3 experiments. Participants viewed picture stories containing multiple target episodes consisting of a beginning state, a bridging event, and an end state, respectively, and the presence of the bridging event was manipulated. When absent, viewers had to infer the bridging-event action to comprehend the end-state image. A pilot study showed that after viewing the end-state image, participants' think-aloud protocols contained more inferred actions when the bridging event was absent than when it was present. Likewise, Experiment 1 found longer viewing times for the end-state image when the bridging-event image was absent, consistent with viewing times revealing online inference generation processes. Experiment 2 showed that both linguistic and visuospatial WM loads attenuated the inference viewing time effect, consistent with the shared systems hypothesis. Importantly, however, Experiment 3 found that articulatory suppression did not attenuate the inference viewing time effect, indicating that (sub)vocalization did not support online inference generation during visual narrative comprehension. Thus, the results support a shared-systems hypothesis in which both visuospatial and linguistic WM systems support inference generation in visual narratives, with the linguistic WM system operating at a deeper level than (sub)vocalization.


Assuntos
Compreensão/fisiologia , Idioma , Memória de Curto Prazo/fisiologia , Percepção Visual/fisiologia , Adulto , Feminino , Humanos , Masculino , Adulto Jovem
9.
Proc Natl Acad Sci U S A ; 107(27): 12151-6, 2010 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-20566873

RESUMO

Microtubules are typically observed to buckle and loop during interphase in cultured cells by an unknown mechanism. We show that lateral microtubule movement and looping is a result of microtubules sliding against one another in interphase Drosophila S2 cells. RNAi of the kinesin-1 heavy chain (KHC), but not dynein or the kinesin-1 light chain, eliminates these movements. KHC-dependent microtubule sliding powers the formation of cellular processes filled with parallel microtubule bundles. The growth of these cellular processes is independent of the actin cytoskeleton. We further observe cytoplasmic microtubule sliding in Xenopus and Ptk2 cells, and show that antibody inhibition of KHC in mammalian cells prevents sliding. We therefore propose that, in addition to its well established role in organelle transport, an important universal function of kinesin-1 is to mediate cytoplasmic microtubule-microtubule sliding. This provides the cell with a dedicated mechanism to transport long and short microtubule filaments and drive changes in cell shape.


Assuntos
Forma Celular/fisiologia , Proteínas de Drosophila/fisiologia , Cinesinas/fisiologia , Microtúbulos/fisiologia , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Immunoblotting , Cinesinas/genética , Cinesinas/metabolismo , Microscopia Confocal , Simulação de Dinâmica Molecular , Interferência de RNA , Transfecção , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/fisiologia
10.
Front Syst Neurosci ; 17: 1172856, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37397237

RESUMO

Burst suppression is a brain state consisting of high-amplitude electrical activity alternating with periods of quieter suppression that can be brought about by disease or by certain anesthetics. Although burst suppression has been studied for decades, few studies have investigated the diverse manifestations of this state within and between human subjects. As part of a clinical trial examining the antidepressant effects of propofol, we gathered burst suppression electroencephalographic (EEG) data from 114 propofol infusions across 21 human subjects with treatment-resistant depression. This data was examined with the objective of describing and quantifying electrical signal diversity. We observed three types of EEG burst activity: canonical broadband bursts (as frequently described in the literature), spindles (narrow-band oscillations reminiscent of sleep spindles), and a new feature that we call low-frequency bursts (LFBs), which are brief deflections of mainly sub-3-Hz power. These three features were distinct in both the time and frequency domains and their occurrence differed significantly across subjects, with some subjects showing many LFBs or spindles and others showing very few. Spectral-power makeup of each feature was also significantly different across subjects. In a subset of nine participants with high-density EEG recordings, we noted that each feature had a unique spatial pattern of amplitude and polarity when measured across the scalp. Finally, we observed that the Bispectral Index Monitor, a commonly used clinical EEG monitor, does not account for the diversity of EEG features when processing the burst suppression state. Overall, this study describes and quantifies variation in the burst suppression EEG state across subjects and repeated infusions of propofol. These findings have implications for the understanding of brain activity under anesthesia and for individualized dosing of anesthetic drugs.

11.
medRxiv ; 2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37745479

RESUMO

Background: Anesthetic agents including ketamine and nitrous oxide have shown antidepressant properties when appropriately dosed. Our recent open-label trial of propofol, an intravenous anesthetic known to elicit transient positive mood effects, suggested that it may also produce robust and durable antidepressant effects when administered at a high dose that elicits an electroencephalographic (EEG) burst-suppression state. Here we report findings from a randomized controlled trial ( NCT03684447 ) that compared two doses of propofol. We hypothesized greater improvement with a high dose that evoked burst suppression versus a low dose that did not. Methods: Participants with moderate-to-severe, treatment-resistant depression were randomized to a series of 6 treatments at low versus high dose (n=12 per group). Propofol infusions were guided by real-time processed frontal EEG to achieve predetermined pharmacodynamic criteria. The primary and secondary depression outcome measures were the 24-item Hamilton Depression Rating Scale (HDRS-24) and the Patient Health Questionnaire (PHQ-9), respectively. Secondary scales measured suicidal ideation, anxiety, functional impairment, and quality of life. Results: Treatments were well tolerated and blinding procedures were effective. The mean [95%-CI] change in HDRS-24 score was -5.3 [-10.3, -0.2] for the low-dose group and -9.3 [-12.9, -5.6] for the high-dose group (17% versus 33% reduction). The between-group effect size (standardized mean difference) was -0.56 [-1.39, 0.28]. The group difference was not statistically significant (p=0.24, linear model). The mean change in PHQ-9 score was -2.0 [-3.9, -0.1] for the low dose and -4.8 [-7.7, -2.0] for the high dose. The between-group effect size was -0.73 [-1.59, 0.14] (p=0.09). Secondary outcomes favored the high dose (effect sizes magnitudes 0.1 - 0.9) but did not generally reach statistical significance (p>0.05). Conclusions: The medium-sized effects observed between doses in this small, controlled, clinical trial suggest that propofol may have dose-dependent antidepressant effects. The findings also provide guidance for subsequent trials. A larger sample size and additional treatments in series are likely to enhance the ability to detect dose-dependent effects. Future work is warranted to investigate potential antidepressant mechanisms and dose optimization.

12.
Pathogens ; 12(5)2023 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-37242341

RESUMO

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

13.
Biophys J ; 101(11): 2760-9, 2011 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-22261065

RESUMO

Eg5 is a homotetrameric kinesin-5 motor protein that generates outward force on the overlapping, antiparallel microtubules (MTs) of the mitotic spindle. Upon binding an MT, an Eg5 dimer releases one ADP molecule, undergoes a slow (∼0.5 s(-1)) isomerization, and finally releases a second ADP, adopting a tightly MT-bound, nucleotide-free (APO) conformation. This conformation precedes ATP binding and stepping. Here, we use mutagenesis, steady-state and pre-steady-state kinetics, motility assays, and electron paramagnetic resonance spectroscopy to examine Eg5 monomers and dimers as they bind MTs and initiate stepping. We demonstrate that a critical element of Eg5, loop 5 (L5), accelerates ADP release during the initial MT-binding event. Furthermore, our electron paramagnetic resonance data show that L5 mediates the slow isomerization by preventing Eg5 dimer heads from binding the MT until they release ADP. Finally, we find that Eg5 having a seven-residue deletion within L5 can still hydrolyze ATP and move along MTs, suggesting that L5 is not required to accelerate subsequent steps of the motor along the MT. Taken together, these properties of L5 explain the kinetic effects of L5-directed inhibition on Eg5 activity and may direct further interventions targeting Eg5 activity.


Assuntos
Cinesinas/química , Cinesinas/metabolismo , Multimerização Proteica , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Cinética , Microtúbulos/metabolismo , Modelos Moleculares , Sondas Moleculares/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Nucleotídeos/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Soluções , Relação Estrutura-Atividade , ortoaminobenzoatos/metabolismo
14.
J Theor Biol ; 289: 107-15, 2011 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-21872609

RESUMO

Loop 5 (L5) is a conserved loop that projects from the α2-helix adjacent to the nucleotide site of all kinesin-family motors. L5 is critical to the function of the mitotic kinesin-5 family motors and is the binding site for several kinesin-5 inhibitors that are currently in clinical trials. Its conformational dynamics and its role in motor function are not fully understood. Our previous work using EPR spectroscopy suggested that L5 alters the nucleotide pocket conformation of the kinesin-5 motor Eg5 (Larson et al., 2010). EPR spectra of a spin-labeled nucleotide analog bound at the nucleotide site of Eg5 display a highly immobilized component that is absent if L5 is shortened or if the inhibitor STLC is added (Larson et al., 2010), which X-ray structures suggest stabilizes an L5 conformation pointing away from the nucleotide site. These data, coupled with the proximity of L5 to the nucleotide site suggest L5 could interact with a bound nucleotide, modulating function. Here we use molecular dynamics (MD) simulations of Eg5 to explore the interaction of L5 with the nucleotide site in greater detail. We performed MD simulations in which the L5-domain of the Eg5·ADP X-ray structure was manually deformed via backbone bond rotations. The L5-domain of Eg5 was sufficiently lengthy that portions of L5 could be located in proximity to bound ADP. The MD simulations evolved to thermodynamically stable structures at 300 K showing that L5 can interact directly with bound nucleotide with significant impingement on the ribose hydroxyls, consistent with the EPR spectroscopy results. Taken together, these data provide support for the hypothesis that L5 modulates Eg5 function via interaction with the nucleotide-binding site.


Assuntos
Cinesinas/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Cinesinas/genética , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética
15.
Iperception ; 12(2): 2041669521994150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35145614

RESUMO

Visual crowding, the impairment of object recognition in peripheral vision due to flanking objects, has generally been studied using simple stimuli on blank backgrounds. While crowding is widely assumed to occur in natural scenes, it has not been shown rigorously yet. Given that scene contexts can facilitate object recognition, crowding effects may be dampened in real-world scenes. Therefore, this study investigated crowding using objects in computer-generated real-world scenes. In two experiments, target objects were presented with four flanker objects placed uniformly around the target. Previous research indicates that crowding occurs when the distance between the target and flanker is approximately less than half the retinal eccentricity of the target. In each image, the spacing between the target and flanker objects was varied considerably above or below the standard (0.5) threshold to either suppress or facilitate the crowding effect. Experiment 1 cued the target location and then briefly flashed the scene image before participants could move their eyes. Participants then selected the target object's category from a 15-alternative forced choice response set (including all objects shown in the scene). Experiment 2 used eye tracking to ensure participants were centrally fixating at the beginning of each trial and showed the image for the duration of the participant's fixation. Both experiments found object recognition accuracy decreased with smaller spacing between targets and flanker objects. Thus, this study rigorously shows crowding of objects in semantically consistent real-world scenes.

16.
medRxiv ; 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34013298

RESUMO

Serology has provided valuable diagnostic and epidemiological data on antibody responses to SARS-CoV-2 in diverse patient cohorts. Deployment of high content, multiplex serology platforms across the world, including in low and medium income countries, can accelerate longitudinal epidemiological surveys. Here we report multiSero, an open platform to enable multiplex serology with up to 48 antigens in a 96-well format. The platform consists of three components: ELISA-array of printed proteins, a commercial or home-built plate reader, and modular python software for automated analysis (pysero). We validate the platform by comparing antibody titers against the SARS-CoV-2 Spike, receptor binding domain (RBD), and nucleocapsid (N) in 114 sera from COVID-19 positive individuals and 87 pre-pandemic COVID-19 negative sera. We report data with both a commercial plate reader and an inexpensive, open plate reader (nautilus). Receiver operating characteristic (ROC) analysis of classification with single antigens shows that Spike and RBD classify positive and negative sera with the highest sensitivity at a given specificity. The platform distinguished positive sera from negative sera when the reactivity of the sera was equivalent to the binding of 1 ng mL âˆ'1 RBD-specific monoclonal antibody. We developed normalization and classification methods to pool antibody responses from multiple antigens and multiple experiments. Our results demonstrate a performant and accessible pipeline for multiplexed ELISA ready for multiple applications, including serosurveillance, identification of viral proteins that elicit antibody responses, differential diagnosis of circulating pathogens, and immune responses to vaccines.

17.
Biophys J ; 98(11): 2619-27, 2010 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-20513406

RESUMO

Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, L5, and the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of S-trityl-l-cysteine (STLC), an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small-molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of the nucleotide state.


Assuntos
Cinesinas/química , Regulação Alostérica , Cisteína/análogos & derivados , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Microtúbulos/química , Modelos Moleculares , Movimento (Física) , Conformação Proteica
18.
Top Cogn Sci ; 12(1): 311-351, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31486277

RESUMO

Understanding how people comprehend visual narratives (including picture stories, comics, and film) requires the combination of traditionally separate theories that span the initial sensory and perceptual processing of complex visual scenes, the perception of events over time, and comprehension of narratives. Existing piecemeal approaches fail to capture the interplay between these levels of processing. Here, we propose the Scene Perception & Event Comprehension Theory (SPECT), as applied to visual narratives, which distinguishes between front-end and back-end cognitive processes. Front-end processes occur during single eye fixations and are comprised of attentional selection and information extraction. Back-end processes occur across multiple fixations and support the construction of event models, which reflect understanding of what is happening now in a narrative (stored in working memory) and over the course of the entire narrative (stored in long-term episodic memory). We describe relationships between front- and back-end processes, and medium-specific differences that likely produce variation in front-end and back-end processes across media (e.g., picture stories vs. film). We describe several novel research questions derived from SPECT that we have explored. By addressing these questions, we provide greater insight into how attention, information extraction, and event model processes are dynamically coordinated to perceive and understand complex naturalistic visual events in narratives and the real world.


Assuntos
Atenção/fisiologia , Desenhos Animados como Assunto , Compreensão/fisiologia , Movimentos Oculares/fisiologia , Filmes Cinematográficos , Narração , Reconhecimento Visual de Modelos/fisiologia , Teoria Psicológica , Humanos
19.
J Biomed Opt ; 14(3): 034048, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19566340

RESUMO

The Nikon C1 confocal laser scanning microscope is a relatively inexpensive and user-friendly instrument. We describe a straightforward method to convert the C1 for multiphoton microscopy utilizing direct coupling of a femtosecond near-infrared laser into the scan head and fiber optic transmission of emission light to the three-channel detector box. Our adapted system can be rapidly switched between confocal and multiphoton mode, requires no modification to the original system, and uses only a few custom-made parts. The entire system, including scan mirrors and detector box, remain under the control of the user-friendly Nikon EZ-C1 software without modification.


Assuntos
Microscopia Confocal/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Animais , Desenho de Equipamento , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Camundongos , Camundongos Transgênicos , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios , Fibras Ópticas , Pólen/ultraestrutura , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Retina/citologia , Retina/metabolismo , Rodopsina/biossíntese , Rodopsina/química , Rodopsina/genética
20.
J Vis ; 9(10): 6.1-16, 2009 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-19810787

RESUMO

Which region of the visual field is most useful for recognizing scene gist, central vision (the fovea and parafovea) based on its higher visual resolution and importance for object recognition, or the periphery, based on resolving lower spatial frequencies useful for scene gist recognition, and its large extent? Scenes were presented in two experimental conditions: a "Window," a circular region showing the central portion of a scene, and blocking peripheral information, or a "Scotoma," which blocks out the central portion of a scene and shows only the periphery. Results indicated the periphery was more useful than central vision for maximal performance (i.e., equal to seeing the entire image). Nevertheless, central vision was more efficient for scene gist recognition than the periphery on a per-pixel basis. A critical radius of 7.4 degrees was found where the Window and Scotoma performance curves crossed, producing equal performance. This value was compared to predicted critical radii from cortical magnification functions on the assumption that equal V1 activation would produce equal performance. However, these predictions were systematically smaller than the empirical critical radius, suggesting that the utility of central vision for gist recognition is less than predicted by V1 cortical magnification.


Assuntos
Reconhecimento Visual de Modelos/fisiologia , Campos Visuais/fisiologia , Adolescente , Humanos , Modelos Neurológicos , Estimulação Luminosa/métodos , Percepção Espacial/fisiologia , Córtex Visual/fisiologia , Adulto Jovem
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