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 Intracellular colonization by Bd in zebrafish larvae Alerter l'administrateur Recommander à un ami Lien de l'article 

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A hallmark of chytrid infection in amphibians includes the presence of zoosporangia and rhizoid-like structures in the keratinized epidermal tissue layers of host skin28,30. To investigate symptoms of zebrafish larvae infection, Bd-infected larvae were labelled with calcofluor white (CFW). We first observed that CFW clearly labelled the chitinous cell wall of Bd sporangia in broth culture (Supplementary Fig. 2d). At 72 h.p.i., infected larvae showed fluorescent punctae (bright round spots) throughout the entire body and fin (Fig. 2c). Time-course imaging experiments showed that these phenotypes appeared within 48–72 h.p.i. (Fig. 2c), consistent with the appearance of fin erosion (Supplementary Fig. 2c). We quantified the number cable accessories of CFW-positive punctae on each larva and observed that larvae infected with a high dose of Bd zoospores show significantly more (4.7±0.6-fold) CFW-positive punctae compared to controls (Fig. 2d, Supplementary Fig. 2e). Larvae infected with a low dose of Bd zoospores show similar values to controls, highlighting the dose dependent nature of infection. Moreover, CFW-positive punctae colocalized with blister-like structures on eroded muscle of infected larvae (Fig. 2e), suggesting that both CFW-positive punctae and blistering of skin are symptoms of infection by Bd.

Consequence of Bd infection on zebrafish larvae host tissue
We used confocal microscopy to investigate the consequence of Bd infection on zebrafish larvae. Bd-infected larvae were fluorescently labelled with CFW and Evans Blue (EB) to detect tissue damage. CFW-positive punctae were observed on the fins and muscle of low dose infected larvae, which colocalize with EB-positive tissue damage (Fig. 3a, Supplementary Fig. 3a). Moreover, both CFW-positive punctae and EB-positive tissue damage were found to colocalize with fin erosion and blister-like structures on Bd-infected larvae (Supplementary Fig. 3b).

 

Intracellular colonization by Bd in zebrafish larvae
To determine how symptoms of infection in zebrafish larvae are related to the colonization of Bd on larvae skin, we labelled infected larvae at 72 h.p.i. with a novel Bd-specific monoclonal antibody, mAb 5C4. This antibody binds to a carbohydrate epitope on an extracellular antigen produced by Bd33, labelling both Bd zoospores and zoosporangia with minimal background labelling in vivo (Supplementary Fig. 4a,b). Infected larvae present areas of skin harbouring the Bd secreted antigen (Fig. 4a). Antibody labelling also colocalizes with blisters and actin reorganization on infected larvae, showing disruption of host tissue in response to Bd infection (Fig. 4a,b, Supplementary Fig. 4c). These results are consistent with the pattern of CFW-labelled punctae described above, indicating that mAb 5C4 is suitable for highly specific, in-depth analysis of the Bd infection process. Indeed, various stages of Bd infection can be visualized in infected larvae by 72 h.p.i. (Fig. 4c), including germ-tubes invading epidermal cells, encysting zoospores and intracellular zoosporangia amongst hyperplastic epithelial cell build-ups. These results show that zebrafish larvae undergo an infection process similar to that of amphibians, and that Bd infection of zebrafish can be powerful model system to study the invasion and proliferation of Bd in vivo.

  Aucun commentaire | Ecrire un nouveau commentaire Posté le 05-06-2017 à 08h46

 Our study shows that Bd is able to infect Alerter l'administrateur Recommander à un ami Lien de l'article 

Figure 4: Intracellular colonization by Bd in zebrafish larvae.
Figure 4
(a) Zebrafish larvae bath water was inoculated with high (>200 zsp per μl) dose Bd zoospores and incubated for 72 h.p.i., then fixed and labelled with Hoechst (for DNA; blue) and mAb 5C4 (for Bd; green) for visualization by confocal microscopy. Images taken at 63 X, maximum intensity projection of Z-stack shown here. Cartoon depicts imaged region. Representative images with arrows highlight colocalization between Bd and blisters on larvae skin. Scale bar, 50 μm. (b) Zebrafish larvae bath water was inoculated with high dose Bd zoospores and incubated for 72 h.p.i., then fixed and labelled with Hoechst (for DNA; blue), mAb 5C4 (for Bd; green) and phalloidin (for F-Actin; red) for visualization by confocal microscopy. Images taken at × 63, maximum intensity projection of Z-stack shown here. Cartoon depicts imaged region. Representative images with insets highlight Bd adjacent to host cell actin rearrangements. Scale bar, 50 μm. See also Supplementary Fig. 4c for host cell actin in control treated larvae. (c) Zebrafish larvae bath water was treated as in a. Images taken at × 40 or × 63, maximum intensity projection of Z-stack shown here. Images showing different stages of Bd invasion and infection on larvae, also depicted using cartoons in the right column. 1, rhizoid-like germ tube attached to encysting zoospore, 2, chytrid thallus growth on zebrafish larvae skin, 3, encysted sporangium amongst hyperplasic epithelial cells. Scale bars, 10 μm.
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Discussion
Our study shows that Bd is able to infect and multiply on zebrafish larvae treated with antibiotics in a dose dependent manner that mimics the process of infection seen in amphibians. This demonstration of a non-amphibian vertebrate host being infected by Bd widens the host range previously known to be exploited by this hypervirulent chytrid lineage. Using a Bd monoclonal antibody (mAb 5C4), we were able to image the different stages of Bd infection with unprecedented resolution in vivo. Collectively, these results validate zebrafish larvae as a powerful aquatic model system within which these host-Bd interactions can be more fully explored. Furthermore, our observations that treating zebrafish with antibiotics results in higher cable accessories burdens of infection highlight the use of probiotic bacteria to combat Bd infection34.

Although the specific host factors necessary for Bd infection remain to be discovered, Bd is commonly found to parasitize the keratinized tissue of both amphibian and non-amphibian hosts8,9,35,36. Consistent with these observations, we found Bd parasitizing zebrafish larvae structures known to express high levels of keratin, such as the edges of the caudal fin32, where we observed fin erosion, tissue damage and apoptotic cells. These observations are in agreement with studies showing widespread apoptosis of amphibian skin cells in response to Bd infection6,31. However, in common with many macroparasite disease systems37, not all larvae became infected and mortality is heterogeneous among experiments (Supplementary Table 1), suggesting that there are unknown factors underlying the susceptibility of zebrafish larvae to Bd.

  Aucun commentaire | Ecrire un nouveau commentaire Posté le 05-06-2017 à 08h48

 The advent of optogenetics provided researchers Alerter l'administrateur Recommander à un ami Lien de l'article 


The advent of optogenetics provided researchers with powerful tools that manipulate living systems with exquisite spatiotemporal precision using non-invasive visible light. The plethora of optical reporters to probe changes in cellular structure, second messenger levels, post-translational modifications and even conformation of individual proteins was until recently matched by few optical actuators, mostly chemically caged compounds released by ultraviolet light. Synthetic biology has now provided numerous designer channels and proteins that can be activated in different cells and distinct subcellular regions for precisely defined periods or using temporally patterned inputs. Nevertheless, the design of an optogenetic regulator for a new target is often a strenuous multidisciplinary task guided by combinations of crystallography, directed evolution, library screening, molecular modelling and/or mutational interweaving strategies6,7,8,9,10. It therefore remains challenging for a typical research lab to implement new optogenetic designs for new proteins and pathways.

Here we develop and evaluate a simple protocol for developing optogenetic regulators of new targets, specifically MAP kinases JNK and p38, for which the currently available tools are limited in spatiotemporal precision. We demonstrate that a LOV2-based regulator of JNK, OptoJNKi, can be applied as an optopharmacological tool without the need for gene transfer, and provide detailed protocols to successfully produce and transfer recombinant OptoJNKi into neurons. To target MAP kinases, we made use of known D-domain interactions55,56. We found AsLOV2Jα sterically constrained JNK inhibitor peptides, showing considerable dark/lit-state JNK-binding ratios for the optimal peptide (~30-fold, Fig. 1f,g). This was surprising considering reported difficulties achieving steric hindrance-based light regulation of either proteins or short peptides6,8,9. Molecular dynamic simulation suggested that phenylalanine at or near the C terminus could be ‘caged’ by a novel non-polar LOV2 domain pocket formed between residues of the Iβ-sheet and the Aβ/Bβ loop (Fig. 9). This distal pocket might cooperate with the dark-state α-helical form of the Jα sequence to constrain and thereby generate steric hindrance of fused inhibitor peptide that can be released by light, leading to LED Tube China regulation of JNK that we observed. In support of this, a point mutation in this pocket releases the JIP sequence (without disrupting protein stability as judged by soluble expression level) so that dark-state binding to JNK became indistinguishable from lit-state binding (Fig. 9d).

These structural considerations suggested a simple protocol

  Aucun commentaire | Ecrire un nouveau commentaire Posté le 06-06-2017 à 08h28

 GAL4-cJun luciferase reporter assays Alerter l'administrateur Recommander à un ami Lien de l'article 

GAL4-cJun luciferase reporter assays in cerebellar granule neurons
Cerebellar granule neurons were transfected with a firefly luciferase reporter plasmid driven by five GAL4 elements in tandem, pGL3-G5E4Δ38 (12.5% of total DNA), pcDNA3-GAL4cJun plasmid encoding the transactivation domain of c-Jun (residues 5–105) fused to the DNA-binding domain of GAL4 (residues 1–147; 12.5% of total DNA), and pRL-CMV (12.5% of total DNA) expressing sea pansy luciferase as an internal standard against which signals were normalized24. CMV was selected for normalization because pcDNA3-GAL4cJun is also driven by CMV promoter. To activate the JNK pathway, either pEBG-ΔMEKK1 (10% of total DNA), a plasmid encoding MAPK-kinase kinase 1 (MEKK1) kinase domain (amino acids 1,174–1,493) was co-transfected, or cells were subjected to 4?h of WTS. Differentially targeted mCherry-OptoJNKi constructs or empty vector (mCherry-C1) were co-transfected as indicated. For WTS experiments, all OptoJNKi constructs were transfected at 40% of total DNA. For the specific case of ΔMEKK1-co-expression experiments, mCherry-3xNLS-OptoJNKi was transfected at 40% of total DNA but pilot titration evaluations indicated light-independent artefacts at high levels of the NES-targeted form; by transfecting at only 2% of total DNA, we avoided effects in dark conditions, as can be seen in the figures. We did not find a suitable percentage level for use of H2B-OptoJNKi in ΔMEKK1-transfected cells, suggesting a possible unexpected interaction between these two constructs, so we were unable to use H2B-OptoJNKi in this specific JNK-activation model, even though there was no evidence of dark effects of H2B-OptoJNKi in the WTS model. This emphasizes the need for careful titration of constructs and inclusions of appropriate controls in every model. But this is not unique to OptoJNKi as the risks of overexpression are well known, potentially affecting use of ΔMEKK1 and most other constructs. It should be noted that, in cerebellar neuron cultures as used here, the 10% level of ΔMEKK1 plasmid used had little impact on overall expression as judged by Renilla luciferase internal control signal, but it substantially increased the GAL4-cJun-driven firefly luciferase response. For the ΔMEKK1 plates, 2?h after transfection, wells were individually illuminated as indicated. Illumination was carried out using blue LEDs (peak 465–467?nm) with 500?Hz pulsed-width modulation to achieve 0.6?mW?cm?2 incident power on the well bottom (duty cycle modulation was set according to measured LED Tube China at 100% duty cycle using an Ex-Cite XR2100 power meter from Lumen Dynamics/LDGI, Mississauga, Ontario, Canada). The illumination was delivered in pulses of 1.5?s length or as indicated. Pulses were repeated with periodicity and for time windows as indicated in the figures. For ΔMEKK1 experiments, dual luciferase reporter assay was carried out on samples lysed 18–20?h after transfection. For the WTS plates, conditioned cell culture medium was replaced by minimal essential medium (cat# 11700) 20?h after transfection and wells were maintained in a cell culture incubator (Cytomat 2C, Thermo) for a further 4?h before lysis, under the illumination conditions described. Dual luciferase reagent (Promega) was used to assay firefly (reporter) and Renilla (internal standard) luciferase activities in samples lysed in passive-lysis buffer (Promega) according to the manufacturer’s instructions24.

  Aucun commentaire | Ecrire un nouveau commentaire Posté le 06-06-2017 à 08h37

 Does the Cable TV Alerter l'administrateur Recommander à un ami Lien de l'article 

Additional Fees: Does the Cable TV,  Package Price include hidden costs or any extra costs? Read the fine print. Then compare the Cable TV,  plan based against the new cost plus the new fees.  Many providers provide connection of a home network for a fee. Often you can connect all your computers without this fee if you have a router. So you can analyze the fee for connection against the cost of the router. with a $9 a month provider fee, and a $40 router, and a 2 year contract, the provider fee will cost you $216. You could save $180 by having a router installed.

Cable TV, Plan Offers: Each Cable TV, plan will have a list of Offers to it. Compare these Components and you may find that one set of Offers is unimportant to you, but that another set is more important. Having faster broadband speeds if you don't need that much bandwidth may be a waste of money.
Internet Speed: Most internet networks provide high speeds, you need to contrast the speed plans. Always read the terms of service to ensure if the providerthe network throttles their bandwidth or blocks high speed users. Will your usage be affected if you use Netflix or other video sites or if you do a lot of file transfer or online game playing?
Equipment: Cable TV contracts all include additional equipment. Most Cable TV providers include the cost of the equipment in the contract. Your choices of equipment will vary from provider to provider. Some choices that you may consider are DVR, High Definition and room to room viewing via wireless.
High Definition: Many Security alarm cable TV deals sport HD channels as a ruse to get you in. Bear in mind, not all shows are simulcast in HD. So keep in mind that just because the provider is calling it an HD channel that muchof the content on that channel won't be in HD all the time.
Cable TV Sports Packages: Many Cable TV channel lineups lean heavily toward sports. If you are a sports fan, verify that the Cable TV package carries the teams that you want to watch most. Often, there is some give and take on having  more sports channels versus just those channels which really matter to you. You may find that a lower priced package may have a better mix of channels for your needs.
How to Compare Cable TV, Internet Specials in City State

  Aucun commentaire | Ecrire un nouveau commentaire Posté le 07-06-2017 à 04h18


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