Rapid screening of glomerular slit diaphragm integrity in larval zebrafish

2007 ◽  
Vol 293 (5) ◽  
pp. F1746-F1750 ◽  
Author(s):  
Dirk M. Hentschel ◽  
Michael Mengel ◽  
Lisa Boehme ◽  
Fabian Liebsch ◽  
Caroline Albertin ◽  
...  

Gene array-type experiments have identified large numbers of genes thought to be important for the integrity of the glomerular slit diaphragm. Confirmation of individual proteins has been limited by the expenses and time involved in generating transgenic or knockout mice for each candidate. We present a functional screening assay based on the clearance of a 70-kDa fluorescent dextran in another vertebrate system that is rapid and low in cost. In the pronephric glomerulus of larval zebrafish, we have demonstrated quantifiable loss of slit diaphragm integrity in a zebrafish model of puromycin aminonucleoside (PA) toxicity. In addition, after knockdown of CD2-associated protein (CD2AP) and podocin, two well-characterized genetic contributors to podocyte differentiation in mammals, we observed glomerular loss of serum macromolecules similar to that seen in mammalian kidneys with inborn mutations in these genes. Increased filtration of 70-kDa FITC-labeled dextran correlates with effacement of podocyte foot processes in ultrastructural analysis. These findings document the value of the zebrafish model in genomics and pharmacological screening applications.

1998 ◽  
Vol 36 (7) ◽  
pp. 2043-2045 ◽  
Author(s):  
David A. Sack ◽  
Albert J. Lastovica ◽  
Sunny H. Chang ◽  
Gary Pazzaglia

Campylobacter jejuni with Gm1 ganglioside in the core of its lipopolysaccharide has been associated with Guillain-Barrésyndrome. Since this epitope may be of considerable pathophysiologic importance and since this ganglioside binds cholera toxin, a rapid screening assay to detect bacteria that bind cholera toxin as an indication of Gm1 on their surfaces was developed. In the assay, bacterial lawns were grown on agar plates, harvested with phosphate-buffered saline, boiled, and incubated with a standard concentration of cholera B subunit. Preparations from strains with Gm1 were observed to inhibit the binding of cholera B subunit to Gm1 in a microtiter enzyme-linked immunosorbent assay. By using this assay with two groups of strains, 37 positive strains were detected among the 197 tested. Species with positive isolates included C. jejuni,Campylobacter coli, and Helicobacter pylori. The assay is capable of testing large numbers of isolates and should prove useful in future clinical and epidemiological studies of bacteria with this epitope.


2018 ◽  
Vol 314 (2) ◽  
pp. F280-F292 ◽  
Author(s):  
Ha Won Lee ◽  
Ehtesham Arif ◽  
Mehmet M. Altintas ◽  
Kevin Quick ◽  
Shrey Maheshwari ◽  
...  

Podocyte dysfunction and loss is an early event and a hallmark of proteinuric kidney diseases. A podocyte’s normal function is maintained via its unique cellular architecture that relies on an intracellular network of filaments, including filamentous actin (F-actin) and microtubules, that provides mechanical support. Damage to this filamentous network leads to changes in cellular morphology and results in podocyte injury, dysfunction, and death. Conversely, stabilization of this network protects podocytes and ameliorates proteinuria. This suggests that stabilization of podocyte architecture via its filamentous network could be a key therapeutic strategy for proteinuric kidney diseases. However, development of podocyte-directed therapeutics, especially those that target the cell’s filamentous network, is still lacking, partly because of unavailability of appropriate cellular assays for use in a drug discovery environment. Here, we describe a new high-content screening-based methodology and its implementation on podocytes to identify paullone derivatives as a novel group of podocyte-protective compounds. We find that three compounds, i.e., kenpaullone, 1-azakenpaullone, and alsterpaullone, dose dependently protect podocytes from puromycin aminonucleoside (PAN)-mediated injury in vitro by reducing PAN-induced changes in both the filamentous actin and microtubules, with alsterpaullone providing maximal protection. Mechanistic studies further show that alsterpaullone suppressed PAN-induced activation of signaling downstream of GSK3β and p38 mitogen-activated protein kinase. In vivo it reduced ADR-induced glomerular injury in a zebrafish model. Together, these results identify paullone derivatives as novel podocyte-protective agents for future therapeutic development.


2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Chelsea A. Weitekamp ◽  
Allison Kvasnicka ◽  
Scott P. Keely ◽  
Nichole E. Brinkman ◽  
Xia Meng Howey ◽  
...  

Abstract Background Across taxa, animals with depleted intestinal microbiomes show disrupted behavioral phenotypes. Axenic (i.e., microbe-free) mice, zebrafish, and fruit flies exhibit increased locomotor behavior, or hyperactivity. The mechanism through which bacteria interact with host cells to trigger normal neurobehavioral development in larval zebrafish is not well understood. Here, we monoassociated zebrafish with either one of six different zebrafish-associated bacteria, mixtures of these host-associates, or with an environmental bacterial isolate. Results As predicted, the axenic cohort was hyperactive. Monoassociation with three different host-associated bacterial species, as well as with the mixtures, resulted in control-like locomotor behavior. Monoassociation with one host-associate and the environmental isolate resulted in the hyperactive phenotype characteristic of axenic larvae, while monoassociation with two other host-associated bacteria partially blocked this phenotype. Furthermore, we found an inverse relationship between the total concentration of bacteria per larvae and locomotor behavior. Lastly, in the axenic and associated cohorts, but not in the larvae with complex communities, we detected unexpected bacteria, some of which may be present as facultative predators. Conclusions These data support a growing body of evidence that individual species of bacteria can have different effects on host behavior, potentially related to their success at intestinal colonization. Specific to the zebrafish model, our results suggest that differences in the composition of microbes in fish facilities could affect the results of behavioral assays within pharmacological and toxicological studies.


2001 ◽  
Vol 8 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Ulla Niewerth ◽  
Andreas Frey ◽  
Thomas Voss ◽  
Chantal Le Bouguénec ◽  
Georg Baljer ◽  
...  

ABSTRACT Pathogenic Escherichia coli strains are known to cause edema disease (ED) and postweaning diarrhea (PWD) in piglets. Although the exact mechanisms of pathogenicity that lead to ED-PWD remain to be elucidated, E. coli-borne Shiga-like toxin and adhesion-mediating virulence factors such as F18 adhesin or F4 fimbriae are believed to play a central role in ED-PWD. In light of these observations we investigated whether another E. coliadhesin, the plasmid-encoded AIDA (adhesin involved in diffuse adherence) might also be present in ED-PWD-causing E. coli isolates. For rapid screening for the AIDA system in large numbers of isolates, a multiplex PCR method along with a duplex Western blot procedure was developed. When screening 104 strains obtained from pigs with or without ED-PWD, we observed a high prevalence of the AIDA operon in porcine E. coli isolates, with over 25% of all strains being AIDA positive, and we could demonstrate a significant association of the intact AIDA gene (orfB) with ED-PWD, while defects in orfB were associated with the absence of disease. Although our data hint toward a contribution of AIDA to ED-PWD, further studies will be necessary since the presence of the AIDA genes was also associated with the presence of the Shiga-like toxin and F18 adhesin genes, two reported virulence factors for ED-PWD.


2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Warren Burggren ◽  
Regina Abramova ◽  
Naim Bautista ◽  
Regina Fritsche Danielson ◽  
Avi Gupta ◽  
...  

2000 ◽  
Vol 113 (23) ◽  
pp. 4141-4142
Author(s):  
B.E. Maden

Illuminating Life: Selected Papers from Cold Spring Harbor (1903–1969) by J. Witkowski Cold Spring Harbor Laboratory Press (2000) pp. 383 + xvi. ISBN 0–87969-566-8 $25.00 If you are anywhere on the spectrum from frequent Cold Spring Harbor visitor to someone who barely knows that Symposia of that name were until recently published in maroon covers, and if you want to learn more of the history of this remarkable research centre, then this book is for you. At first sight, Illuminating Life looks like a coffee table book, but it is much more than that. Jan Witkowski has assembled a history of the Cold Spring Harbor Laboratories from their inception in 1890 through to 1968, illustrated by a selection of research papers from 1903 to 1969. Each one or two papers is preceded by an interpretative essay and a biographical note on the principal author(s), and the whole is introduced by an informative historical preface. At the end are three obituaries from the literature summarizing the lives of three key players, Davenport, Harris and Demerec. For a book of this size and compass, the essentials can be assimilated remarkably quickly, and at $25 the book is exceptional value for money. First read the preface. Then the essays. These are gems, and at two or three pages each there is no need to postpone them until later! Then dip into a few research papers. Then re-read the preface. Then you will know a lot about Cold Spring Harbor. If you read the obituaries you will know even more. Here are just a few impressions. On p. 364 there is a photograph of one of the early buildings, the James Laboratory. The laboratory was constructed for $12,000 in 1928 for biophysics research (p117). It looks tiny, but in the early years of the Symposia, which were then on biophysical topics, it housed a galaxy of summer visitors including Curtis and Cole (electrophysiology), J. Z. Young (nerve conduction), Davison and Danielli (need one say more?) and many others. If biophysics under Reginald Harris (1924–36) was what made Cold Spring Harbor Quantitative, then the quest for the genetic material and its properties is what has made it most widely famed. The book brings out the seminal contributions of Demerec, both as scientist and as director (1941–60) and of McClintock, Hershey, Cairns (director 1963–8) and others. The chosen research papers include many that are landmarks in science, from maize to bacteria and phage, and generally they are easy to read. They are largely devoid of the ponderous throat-clearing and innumerable citations that are so much a part of scientific literature today. Many examples could be given of such ease of style and freedom from excess verbage, but one will suffice here. ‘Aggregation of DNA is often suspected but seldom studied. In phage λ we found a DNA that can form characteristic and stable complexes. A first account of them is given here’. That is the entire introduction in Hershey, A. D., Burgi, E. and Ingraham, L. (1963), Cohesion of DNA molecules isolated from phage λ. Proc. Nat. Acad. Sci. USA 49, 748–755. The paper by de Lucia and Cairns (1969) on ‘Isolation of an E. coli strain with a mutation affecting DNA polymerase’ is a fitting choice with which to conclude the compilation. In the late 1960s something seemed not quite right about the Kornberg enzyme as the putative engine of replication. Several suspicious inconsistencies were accumulating. How to test these suspicions? Random mutagenesis, a precise and rapid screening assay applicable to thousands of isolates. The rest is history. What of the last thirty years? The spine of the cover says, rather enigmatically, ‘Volume 1’; the reviewer could find no statement elsewhere in the book that more is to follow. Perhaps we can look forward to Volume 2. Surely that volume will contain, among many other landmark papers, one called ‘An amazing sequence arrangement at the 5 ends of Adenovirus 2 messenger RNA’.


2020 ◽  
Vol 23 (4) ◽  
pp. 285-295
Author(s):  
Fang Wang ◽  
Ping Jing ◽  
Peiyan Zhan ◽  
Hongyi Zhang

Introduction The objective of this study is to investigate the role of thyroid hormone (TH) in the pathogenesis of intestinal dysganglionosis (ID). Methods A zebrafish model of congenital hypothyroidism (CH) was created by exposing the larvae to the 6-propyl-2-thiouracil (PTU). The enteric neurons were labeled with anti-HuC/D antibodies. The number of enteric neurons was counted. The larval intestine was dissociated and stained with anti-p75 and anti-α4 integrin antibodies. Mitosis and apoptosis of the p75+ α4 integrin+ enteric neural crest cells (ENCCs) were studied using flow cytometry. Intestinal motility was studied by analyzing the transit of fluorescent tracers. Results PTU (25 mg/L) significantly reduced TH production at 6- and 9-days post fertilization without changing the body length, body weight, and intestinal length of the larvae. Furthermore, PTU inhibited mitosis of ENCCs and reduced the number of enteric neurons throughout the larval zebrafish intestine. Importantly, PTU inhibited intestinal transit of fluorescent tracers. Finally, thyroxine supplementation restored ENCC mitosis, increased the number of enteric neurons, and recovered intestinal motility in the PTU-treated larvae. Conclusions PTU inhibited TH production, reduced the number of enteric neurons, impaired intestinal motility, and impeded ENCC mitosis in zebrafish, suggesting a possible role of CH in the pathogenesis of ID.


1976 ◽  
Vol 59 (4) ◽  
pp. 766-769
Author(s):  
Albert B Karasz ◽  
John J Maxstadt ◽  
Juergen Reher ◽  
Frank Decocco

Abstract A procedure is described for the examination of large numbers of ground beef samples for the 4 most commonly used preservatives. A single sample solution is prepared and aliquots are taken for the various tests. Sulfite is determined colorimetrically with acid-bleached p-rosaniline. Ascorbic acid is titrated with 2,6-dichloroindophenol. Benzoic and sorbic acids are measured by ultraviolet absorption. Negative samples are rapidly sorted out in this procedure and positive samples are completed for quantitative determination of the preservative. Recoveries are 95% for sodium sulfite, 103% for sodium benzoate, 90% for potassium sorbate, and 81 % expected recovery for sodium ascorbate. The limit of detection is 0.005% for potassium sorbate, and 0.01% for the 3 other preservatives.


2012 ◽  
Vol 84 ◽  
pp. 84-91 ◽  
Author(s):  
Jesús Pablo García-Cambero ◽  
Myriam Catalá ◽  
Yolanda Valcárcel

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