Electroretinography and Gene Expression Measures Implicate Phototransduction and Metabolic Shifts in Chick Myopia and Hyperopia Models

The Retinal Ion-Driven Fluid Efflux (RIDE) model theorizes that phototransduction-driven changes in trans-retinal ion and fluid transport underlie the development of myopia (short-sightedness). In support of this model, previous functional studies have identified the attenuation of outer retinal contributions to the global flash electroretinogram (gfERG) following weeks of myopia induction in chicks, while discovery-driven transcriptome studies have identified changes to the expression of ATP-driven ion transport and mitochondrial metabolism genes in the retina/RPE/choroid at the mid- to late-induction time-points. Less is known about the early time-points despite biometric analyses demonstrating changes in eye growth by 3 h in the chick lens defocus model. Thus, the present study compared gfERG and transcriptome profiles between 3 h and 3 days of negative lens-induced myopia and positive lens-induced hyperopia in chicks. Photoreceptor (a-wave and d-wave) and bipolar (b-wave and late-stage d-wave) cell responses were suppressed following negative lens-wear, particularly at the 3–4 h and 3-day time-points when active shifts in the rate of ocular growth were expected. Transcriptome measures revealed the up-regulation of oxidative phosphorylation genes following 6 h of negative lens-wear, concordant with previous reports at 2 days in this model. Signal transduction pathways, with core genes involved in glutamate and G-protein coupled receptor signalling, were down-regulated at 6 h. These findings contribute to a growing body of evidence for the dysregulation of phototransduction and mitochondrial metabolism in animal models of myopia.

Download Full-text

Exploring early time points of vimentin assembly in flow by fluorescence fluctuation spectroscopy

Lab on a Chip ◽

10.1039/d0lc00985g ◽

2021 ◽

Vol 21 (4) ◽

pp. 735-745

Author(s):

Eleonora Perego ◽

Sarah Köster

Keyword(s):

Reaction Kinetics ◽

Photon Counting ◽

Early Time ◽

Fluorescence Fluctuation Spectroscopy ◽

Microfluidic Mixing ◽

Time Points ◽

Photon Counting Histogram ◽

Kinetics Of

The combination of photon counting histogram and microfluidic mixing reveals early time points in reaction kinetics of biomolecule aggregation.

Download Full-text

A Pilot Study to Investigate the Efficacy of Nicotine Oral Soluble Film, Lozenge and Gum in Relief of Acute Smoking Cue-provoked Craving for Cigarette in Low Dependence Smokers

The Journal of Smoking Cessation ◽

10.1017/jsc.2014.5 ◽

2014 ◽

Vol 10 (2) ◽

pp. 87-95 ◽

Cited By ~ 1

Author(s):

Daniel Du ◽

James Borders ◽

Alex Selmani ◽

William Waverczak

Keyword(s):

Pilot Study ◽

Early Time ◽

Controlled Study ◽

Nicotine Gum ◽

Open Label ◽

Time Points ◽

Smoking Cues ◽

Significant Difference ◽

Nicotine Lozenge

Introduction: A new nicotine film that releases nicotine quickly may lead to faster craving relief.Aims: This study compares the efficacy of 2.5 mg nicotine film with 2 mg nicotine lozenge and 2 mg nicotine gum on relieving provoked craving in low dependence smokers.Methods: A randomised, open-label, active comparators controlled study was conducted in 120 subjects. Subjects were abstinent from smoking for 4 hours prior to being provoked with smoking cues. After post-provocation craving assessment, subjects were administered one dose of the 3 treatments: nicotine film, lozenge, or gum. Craving intensity was then assessed at 50 seconds, 3, 5, 7, 15, 20, 25 and 30 minutes after administration.Results/Findings: Three treatments reduced craving with similar maximum effects. The effect was maintained up to 30 minutes. Nicotine film significantly reduced more craving than lozenge at 50 seconds, 3 and 5 minutes. It also significantly reduced more craving than gum at 50 seconds and 3 minutes. There was no significant difference between lozenge and gum.Conclusions: Nicotine film, lozenge and gum have similar maximum craving relief. Nicotine film significantly reduced more craving than lozenge and gum at early time points. Nicotine film may be particularly useful to provide acute craving relief.

Download Full-text

F-actin serves as a template for cytokeratin organization in cell free extracts

Journal of Cell Science ◽

10.1242/jcs.115.7.1373 ◽

2002 ◽

Vol 115 (7) ◽

pp. 1373-1382 ◽

Cited By ~ 2

Author(s):

Kari L. Weber ◽

William M. Bement

Keyword(s):

Time Course ◽

Early Time ◽

Dynamic Imaging ◽

Actin Network ◽

Intact Cells ◽

Time Points ◽

Egg Extracts ◽

Actin Cables

The microtubule, F-actin, and intermediate filament systems are often studied as isolated systems, yet the three display mutual interdependence in living cells. To overcome limitations inherent in analysis of polymer-polymer interactions in intact cells, associations between these systems were assessed in Xenopus egg extracts. In both fixed and unfixed extract preparations, cytokeratin associated with F-actin cables that spontaneously assembled in the extracts. Time-course experiments revealed that at early time points cytokeratin cables were invariably associated with F-actin cables,while at later time points they could be found without associated F-actin. In extract samples where F-actin assembly was prevented, cytokeratin formed unorganized aggregates rather than cables. Dynamic imaging revealed transport of cytokeratin by moving F-actin as well as examples of cytokeratin release from F-actin. Experimental alteration of F-actin network organization by addition of α-actinin resulted in a corresponding change in the organization of the cytokeratin network. Finally, pharmacological disruption of the F-actin network in intact, activated eggs disrupted the normal pattern of cytokeratin assembly. These results provide direct evidence for an association between F-actin and cytokeratin in vitro and in vivo, and indicate that this interaction is necessary for proper cytokeratin assembly after transition into the first mitotic interphase of Xenopus.

Download Full-text

An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1883-1893.1991 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1883-1893

Author(s):

Y C Li ◽

J Ross ◽

J A Scheppler ◽

B R Franza

Keyword(s):

Human Immunodeficiency Virus ◽

Protein Synthesis ◽

Early Time ◽

In Vitro Transcription ◽

Jurkat Cells ◽

Time Points ◽

Immunodeficiency Virus ◽

Hiv 1

In this report we introduce a simple, fast, and reliable method to prepare whole cell or nuclear extracts from small numbers of cells. These extracts were used to study transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in vitro. Our results revealed that the time courses of activation of extracts derived from cells stimulated with the mitogenic lectin phytohemagglutinin (PHA) or with the tumor promoter phorbol 12-myristate 13-acetate (PMA) are different. PMA induces a rapid onset of increased in vitro transcription from the HIV-1 LTR, while PHA causes a slow and sustained response. The biochemical relevance of protein synthesis inhibition by cycloheximide treatment of cells was investigated. In these studies, PMA induction of a change in in vitro transcriptional activity is not dependent on protein synthesis. Cycloheximide alone is insufficient to induce activation. Oligonucleotide-mediated site-directed mutagenesis demonstrated that mutation of the TATA box in the LTR ablated initiation of both basal-level transcription and activation by extracts from cells stimulated with PMA. Surprisingly, mutation of both kappa B sites in the LTR reduced but did not eliminate the in vitro response to extracts prepared at early time points after PHA or PMA stimulation of Jurkat cells. The reduction was greater in extracts derived from cells treated with PMA. Deletion analysis of the HIV-1 LTR revealed at least one region (-464 to -252) capable of suppressing in vitro transcription in extracts from Jurkat cells stimulated by PMA. This result is consistent with early studies of the HIV-1 LTR in transient transfection assays. We therefore have been able to observe distinct regulatory events at early time points after cells are exposed to agents known to induce transcription of both the HIV-1 LTR reporter gene constructs and the HIV-1 provirus itself.

Download Full-text

Abstract 88: Sphingolipid Profiling Identifies Large Vessel Occlusions at Early Time Points: Results of the ASPIRE-Stroke Study

Stroke ◽

10.1161/str.49.suppl_1.88 ◽

2018 ◽

Vol 49 (Suppl_1) ◽

Author(s):

Sunil A Sheth ◽

Songmi Lee ◽

Anthony T Iavarone ◽

Gregory J Wong ◽

Raymond Liou ◽

...

Keyword(s):

Early Time ◽

Large Vessel ◽

Time Points ◽

Stroke Study

Download Full-text

ASSOCIATION OF LIPID WTTH THE CYTOSKELETON (CS) OF AGGREGATING PLATELETS PRELABELED WITH 3H-PALMITTC ACID

10.1055/s-0038-1643639 ◽

1987 ◽

Author(s):

A E Livne ◽

A M Packham ◽

A M Guccione ◽

F J Mustard

Keyword(s):

Electron Microscopy ◽

Membrane Proteins ◽

Palmitic Acid ◽

Protease Activity ◽

Lipid Extraction ◽

Early Time ◽

Human Platelets ◽

Dnase I ◽

Time Points ◽

Triton X

To investigate the association of lipid with the CS of platelets during aggregation, rabbit and human platelets were isolated and labeled with 3H-palmitic acid; lipid extraction showed about 80% in phospholipid. Limited aggregation was induced with ADP (undpr conditions in which release of granule contents did not occur) or thrombin, and CS isolated after lysis with 1% Triton X-1DD, 5 mM EGTA. CS from unactivated platelets had approx. 0.03% of the total platelet label, but after aggregation with ADP (2 µM) or thrombin (0.l U/ml) for 20-30 sec, 3 to 5% of the label was with the CS. Stirring was necessary and added fibrinogen enhanced aggregation and association of label with the CS; incorporation of label increased exponentially as aggregation proceeded and decreased exponentially during deaggregation. The amount of label with the CS was related more directly to the number of sites of contact than to the numbor of platelets in the aggregates. To determine how much label was bound to CS, some experiments were done in which the CS was disrupted and loss of label was assessed. In these experiments, DNase I and Ca2+ were added to the Triton X-100 lysis medium to cause actin depolymerisation, under conditions in which the Ca2+-dependent protease activity was inhibited. This treatment greatly reduced the association of label with the CS in samples taken from early time points on the aggregation curves: when aggregation proceeded further, a large proportion of label was not dissociated by this treatment. These findings, electron microscopy, and enrichment of the CS of aggregated platelets with only some membrane proteins labeled by the 125lactopproxidase method, indicated that under the conditions of limited aggregation used, the 3H-labeled lipid was mainly associated with the CS and not with trapped membrane fragments resulting from incomplete lysis. Si nee the pattern of CS 1abeli ng (3H-palmit at el and selective association of some of the membrane proteins with the cytoskeleton/1ipid complex was the same with ADP and thrombin, the reactions must be dependent on aggregation and not on events associated with the release of granule contents.

Download Full-text

A new treatment for unresectable liver tumours: long-term studies of electrolytic lesions in the pig liver

Clinical Science ◽

10.1042/cs0980561 ◽

2000 ◽

Vol 98 (5) ◽

pp. 561-567 ◽

Cited By ~ 9

Author(s):

Simon A. WEMYSS-HOLDEN ◽

Gavin S. M. ROBERTSON ◽

Ashley R. DENNISON ◽

Paula S. VANDERZON ◽

Pauline de la M. HALL ◽

...

Keyword(s):

Animal Model ◽

Early Time ◽

Hepatic Necrosis ◽

Large Animal Model ◽

Large Animal ◽

Large Area ◽

Liver Tumours ◽

Pig Liver ◽

Time Points ◽

Electrolytic Lesions

The majority of liver tumours are inoperable and an alternative treatment to surgical resection is urgently needed. Electrolysis has been investigated in a rat model and the procedure is safe, with accurate and predictable effects. The necrosis produced has also been shown to cause destruction of tumour deposits in the rat liver. A similar evaluation in a large animal model was necessary before clinical trials could commence. Using platinum electrodes connected to a d.c. generator, areas of hepatic necrosis were created in the pig liver. Animals were killed at various time points after treatment to assess the extent of healing. Treatment was uneventful and all animals made a full recovery. No animal died from the treatment or had to be killed prematurely. After 2 days of treatment, healing was minimal but at successive time points there was progressive evidence of healing, such that after 4 months, the original electrolytic lesion was greatly reduced in size and the large area of necrosis seen at the early time points was largely replaced by a fibrous scar with only small islands of necrotic tissue. In a large animal model, electrolysis is a safe method for creating areas of hepatic necrosis. The lesions heal with time and are associated with minimal morbidity. The results support a trial of electrolysis in patients with unresectable liver tumours.

Download Full-text

Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments

Stem Cells International ◽

10.1155/2018/4809673 ◽

2018 ◽

Vol 2018 ◽

pp. 1-15

Author(s):

Quyen A. Tran ◽

Visar Ajeti ◽

Brian T. Freeman ◽

Paul J. Campagnola ◽

Brenda M. Ogle

Keyword(s):

Stem Cell ◽

Cell Differentiation ◽

Human Mesenchymal Stem Cells ◽

Early Time ◽

Stem Cell Differentiation ◽

Model Systems ◽

Ecm Remodeling ◽

Time Points ◽

3D Microenvironment

Developmental studies and 3D in vitro model systems show that the production and engagement of extracellular matrix (ECM) often precede stem cell differentiation. Yet, unclear is how the ECM triggers signaling events in sequence to accommodate multistep process characteristic of differentiation. Here, we employ transcriptome profiling and advanced imaging to delineate the specificity of ECM engagement to particular differentiation pathways and to determine whether specificity in this context is a function of long-term ECM remodeling. To this end, human mesenchymal stem cells (hMSCs) were cultured in 3D bioprinted prisms created from ECM proteins and associated controls. We found that exogenous ECM provided in 3D microenvironments at early time points impacts on the composition of microenvironments at later time points and that each evolving 3D microenvironment is uniquely poised to promote stem cell differentiation. Moreover, 2D cultures undergo minimal ECM remodeling and are ill-equipped to stimulate pathways associated with development.

Download Full-text

The role of microRNA-3085 in chondrocyte function

Scientific Reports ◽

10.1038/s41598-020-78606-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Linh Le ◽

Lingzi Niu ◽

Matthew J. Barter ◽

David A. Young ◽

Tamas Dalmay ◽

...

Keyword(s):

Feedback Inhibition ◽

Interleukin 1 ◽

Early Time ◽

Null Cell ◽

Time Points ◽

Vitro Analysis ◽

In Vitro Analysis ◽

Cultured Chondrocytes

AbstractMicroRNAs have been shown to play a role in cartilage development, homeostasis and breakdown during osteoarthritis. We previously identified miR-3085 in humans as a chondrocyte-selective microRNA, however it could not be detected by Northern blot. The aim of the current study was to prove that miR-3085 is a microRNA and to investigate the function of miR-3085 in signaling pathways relevant to cartilage homeostasis and osteoarthritis. Here, we confirm that miR-3085 is a microRNA and not another class of small RNA using (1) a pre-miR hairpin maturation assay, (2) expression levels in a Dicer null cell line, and (3) Ago2 pulldown. MicroRNA-3085-3p is expressed more highly in micromass than monolayer cultured chondrocytes. Transfection of miR-3085-3p into chondrocytes decreases expression of COL2A1 and ACAN, both of which are validated as direct targets of miR-3085-3p. Interleukin-1 induces the expression of miR-3085-3p, at least in part via NFκB. In a feed-forward mechanism, miR-3085-3p then potentiates NFκB signaling. However, at early time points after transfection, its action appears to be inhibitory. MyD88 has been shown to be a direct target of miR-3085-3p and may be responsible for the early inhibition of NFκB signaling. However, at later time points, MyD88 knockdown remains inhibitory and so other functions of miR-3085-3p are clearly dominant. TGFβ1 also induces the expression of miR-3085-3p, but in this instance, it exerts a feedback inhibition on signaling with SMAD3 and SMAD4 shown to be direct targets. This in vitro analysis shows that miR-3085-3p functions in chondrocytes to induce IL-1-signaling, reduce TGFβ1 signaling, and inhibit expression of matrix genes. These data suggest that miR-3085-3p has a role in chondrocyte function and could contribute to the process of osteoarthritis.

Download Full-text