We thank Fiona Foley for excellent lab support and zebrafish care. This work was supported an AHA predoctoral fellowship to G.W. (11PRE5730027) and NHLBI grants to H.J.Y. (R01HL66292) and J.D.A. (R01HL095690).

Overview of Stage-Specific Zebrafish Embryo Injections
Antisense morpholino oligonucleotides (MO), which bind to a targeted mRNA and disrupt protein expression from that transcript, are widely used in gene knockdown (loss-of-function) studies in zebrafish13,14. Gene Tools, LLC offers MOs that are tagged with either carboxyfluorescein (emits green fluorescence) or lissamine (emits red fluorescence) to detect MO in injected embryos using fluorescent microscopy. By injecting MO into the yolk cell at different stages of zebrafish development, it is possible to deliver the MO to specific compartments of the embryo (Figures 1A-F). MO injected into the yolk between the 1-4 cell stages (0-1 hpf) enters all embryonic cells (Figures 1A, D, D') via connections with the yolk that persist until the 32-cell stage15 to facilitate global knockdown. MO injected into the yolk during midblastula stages (2.5-3 hpf) can enter the progenitors of the DFCs (Figures 1B, E, E') likely through cytoplasmic bridges8 and knockdown gene function specifically in the DFC/KV cell lineage12, without entering most other embryonic cell lineages. As an important control to test whether gene function is required in DFC/KV or also in yolk12,16, it is also possible to restrict MO to the yolk cell by injecting between the dome-30% epiboly stages (~4.5 hpf) after all cytoplasmic bridges have closed (Figures 1C, F, F'). These injections have been used in combination to analyze gene function in DFC/KV cells17-24. To assess fluid flow in KV, fluorescent microbeads are injected into the KV lumen between the 6-10 somite stages (12-14 hpf) and then immediately imaged using videomicroscopy (Figures 1G-J). Microbeads are available that emit red or green fluorescence (or both), so it is possible to use different channels to image fluorescent microbeads and MO.
1. Stage-specific Injection of Morpholinos (MO)
Injection of MO into zebrafish embryos has been demonstrated previously25,26. Here, we briefly describe stage-specific MO injections. Following all injections, embryos are transferred to a Petri dish and incubated at 28.5 &deg;C.
<ol>
	<li>Global MO injection: Collect embryos immediately after fertilization and load them into an injection plate. Load fluorescent MO into a capillary needle and mount the needle onto a microinjector (e.g. Harvard Apparatus PLI-90 Pico-injector). Break the needle tip and adjust injection settings (pressure and/or time) to create an injection drop that has a volume of 1 nl as described25,26.Using a dissecting stereomicroscope, inject 1 nl of MO into the yolk (Figure 1A) of &ge;50 embryos per experiment. Work quickly to complete injections by the 4-cell stage.</li>
	<li>DFC-targeted MO injection: Collect embryos immediately after fertilization and incubate at 28.5 &deg;C. When the embryos have reached the 256-cell stage (~2.5 hpf), quickly load them into an injection plate and then inject 1 nl of fluorescent MO into the yolk of &ge;100 embryos between the 256-cell and 1,000-cell stages (Figure 1B).</li>
	<li>Yolk-targeted MO injection: Collect embryos immediately after fertilization and incubate at 28.5 &deg;C. At the dome stage (~4 hpf), load the embryos into an injection plate and then inject 1 nl of fluorescent MO in the yolk of &ge;100 embryos between the dome and 30% epiboly stages (Figure 1C).</li>
</ol>
2. Selecting Injected Embryos for Analysis
<ol>
	<li>When MO injected embryos reach the 75%-epiboly stage (8 hpf), remove unfertilized and dead embryos and then screen living embryos under a fluorescent dissecting microscope for distribution of the fluorescent MO. Then allow selected embryos to develop at 28.5 &deg;C.
	<ol>
		<li>For global MO injections, select embryos that have fluorescence evenly distributed in throughout all embryonic cells (Figure 1D; Figure 2A).</li>
		<li>For DFC-targeted MO injections, select embryos in which fluorescent MO has diffused throughout the yolk and appears concentrated at the dorsal blastoderm margin (DFCs) (Figure 1E; Figure 2B). At this stage, it can be difficult to visualize fluorescent DFCs due to bright fluorescence in the underlying yolk. Take care to exclude embryos in which the fluorescent MO has incorporated into embryonic cells other than DFCs or remains aggregated at the injection site (Figure 2D).</li>
		<li>For yolk-targeted MO injections, select embryos in which MO fluorescence is evenly distributed throughout the yolk and not observed in any embryonic cells (Figure 1F;Figure 2C). Again, remove embryos in which the fluorescent MO remains aggregated at the injection site.</li>
	</ol>
	</li>
	<li>Between the 2-4-somite stages (~11 hpf), screen embryos a second time under a fluorescent microscope using higher magnification. Then allow selected embryos to develop at 28.5 &deg;C.
	<ol>
		<li>For global MO injections, ensure selected embryos have MO fluorescence evenly distributed in KV and all embryonic cells (Figure 1D'; Figure 2E).</li>
		<li>For DFC-targeted MO injections, carefully select embryos that have MO fluorescence only in KV cells and the yolk (Figure 1E'). Transgenic embryos that express GFP in KV cells, such as Tg(Dusp6:d2EGFP)27, can aid in the identification of embryos in which MO has been successfully delivered to KV cells (Figure 2F). Discard embryos with MO fluorescence in embryonic cells other that KV cells.</li>
		<li>For yolk-targeted MO injections, select embryos that have MO fluorescence exclusively in the yolk (Figure 1F': Figure 2G).</li>
	</ol>
	</li>
</ol>
3. Mounting Embryos to Analyze Fluid Flow in KV
<ol>
	<li>To analyze asymmetric fluid flow in KV of global MO, DFC-targeted MO or yolk-targeted MO injected embryos, carefully dechorionate ~20 embryos between 4-6 somite stages (~11-12 hpf) with sharp forceps.</li>
	<li>Prepare 50 ml of 1% low-melting point agarose and aliquot into 5 ml tubes to be maintained as a liquid in a dry bath at 42 &deg;C.</li>
	<li>Transfer one embryo to a glass depression slide (United Scientific Supplies, Inc.) and remove as much of the water as possible. Immobilize the embryo by adding enough warm 1% low-melting point agarose to just cover the embryo (too much agarose can interfere with subsequent imaging). As the agarose solidifies, use forceps to position the embryo such that KV is facing up (dorsal view) (Figure 1H). Mount 10 embryos with one embryo per slide. Work quickly to ensure all injections in the next step are completed by the 10 somite stage (14 hpf).</li>
</ol>
4. Injection of Fluorescent Microbeads into KV
<ol>
	<li>Dilute Fluoresbrite Multifuorescent 0.5 micron diameter Microspheres (Polysciences, Inc.) to 1:50 in sterile water in a 1.5 ml Eppendorf tube.</li>
	<li>Mix the 1:50 dilution of microbeads thoroughly by tapping the tube and load 3 &mu;l into a capillary needle. Using the same microinjector used for MO injections (e.g. Harvard Apparatus PLI-90 Pico-injector), break the needle tip with forceps and adjust the injection settings to generate the smallest possible (&lt;0.5 nl) injection drop.</li>
	<li>Under a dissecting microscope, align the needle with the KV lumen of the first mounted embryo. Insert the needle into the lumen and inject a small volume (&lt; 0.5 nl) of beads (Figure 1G). A small needle tip and small injection volume are critical to avoid damaging KV.</li>
	<li>Inject all mounted embryos. Once an embryo has been injected, a drop of sterile water can be added on top of the agarose to prevent it from drying out. During the course of the injections, the beads will often obstruct the needle opening. This requires re-breaking the needle tip and adjusting the injection volume by modulating the pressure or time setting of the microinjection apparatus. Replace the needle if it becomes blunt enough to cause significant damage during injection.</li>
	<li>Screen the injected embryos for successful delivery of beads under an upright fluorescent compound microscope (e.g. Zeiss AxioImager M1) using a 20X objective. First, determine whether KV structure is intact using brightfield illumination (Figure 1J), and then use fluorescence to observe the beads. Select embryos in which beads are present and moving inside the KV lumen. Discard embryos with KV damage or no floating beads in KV. It is easy to miss KV and deliver beads to nearby tissue or the underlying yolk. If unsuccessful, mount another 10 embryos and repeat the injection procedure.</li>
</ol>
5. Visualization and Analysis of KV Fluid Flow
<ol>
	<li>For each selected embryo with beads inside KV, add a drop of sterile water to cover the agarose and then observe KV under the upright fluorescent compound microscope using a 63X water dipping objective (Figure 1I). Alternatively, a coverslip can be applied for use with non-water dipping objectives and/or inverted microscopes.</li>
	<li>Use a high-speed camera mounted on the microscope (e.g. Zeiss AxioCamHSm) to record a 10 sec movie. Record both the beads using the fluorescent channel (approximately 70 frames/sec) and the KV lumen using a brightfield or differential interference contrast (DIC) channel.</li>
	<li>To visualize all bead movements over time, import the movie of fluorescent beads into ImageJ software (free download at <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>) and create a maximum projection of all fluorescent signals in the movie. To perform the maximum projection in ImageJ, click "Image&rarr;Stacks&rarr; Z project." In the "Z project" window, project all slices with projection type of "Max intensity". This image can be superimposed on a DIC image of the KV in which the beads were imaged (Figures 3A-B).</li>
	<li>The movements of individual beads can be tracked using ImageJ. A plugin ("Manual Tracking") can be downloaded at <a href="http://rsbweb.nih.gov/ij/plugins/track/track.html" target="_blank">http://rsbweb.nih.gov/ij/plugins/track/track.html</a>. Open the fluorescent beads movie in ImageJ and run the Manual Tracking function. In the window of Manual Tracking, check "show parameters" and input parameters for"time intervals" and "x/y calibration". Manually select beads that remain in the focal plane of the movie for &ge;50 frames and then track &ge;5 beads per embryo. The path and velocity of each bead is generated by the software (Figures 3 C-D).</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+function+for+embryonic+nodal+cilia.">Conserved function for embryonic nodal cilia.</a> Nature. 418 (6893), 37-38 (2002).</li><li>Kramer-Zucker, A. 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No conflicts of interest declared.

Using stage-specific injections to target MO to the DFC/KV cell lineage is a useful approach to study cell-autonomy of gene function and avoid pleiotropic phenotypes caused by global gene knockdown. However, these injections can be technically challenging. Injection of MO between the 256-cell and 1,000-cell stages can result in three possible outcomes: 1) the MO remains aggregated at the injection site, 2) the MO diffuses throughout the yolk and enters DFC/KV cells or 3) the MO diffuses throughout the yolk and enters DFC...

<table border="1">
<tbody>
	<tr>
 	<td>Name of Reagent/Material</td>
 <td>	Company</td>
 <td>	Catalogue Number</td>
 </tr>
 <tr>
 	<td>Standard Control oligo-Lissamine tagged	</td>
 <td>Gene Tools, LLC</td>
 <td></td>
 </tr>
 <tr>
 	<td>Custom Rock2b morpholino oligo</td>
 <td>Gene Tools, LLC</td>
 <td></td>
 </tr>
 <tr>
 	<td>Fluoresbrite Multifluorescent 0.5 micron Microspheres</td>
 <td>Polysciences, Inc.</td>
 <td>24054</td>
 </tr>
</tbody>
</table>

analysis of gene function and visualization of cilia-generated fluid flow in kupffer's vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Stage-specific MO injections provide a useful approach to analyze gene function in specific compartments of the embryo. Figure 1 presents a flow chart of the injection strategies used to test gene function in DFC/KV cells and how to introduce fluorescent beads to visualize fluid flow in KV. The distribution of fluorescent MO in successful stage-specific injected embryos is shown schematically in Figures 1D-F and in live embryos in Figure 2. An unsuccessful MO injection, ...

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Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

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14.8K Views.  Upstate Medical University. Cilia-generated fluid flow in Kupffer’s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

Video: Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In situ Hybridization for Sipunculus nudus Coelomic Fluid

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

In situ Hybridization for Sipunculus nudus Coelomic Fluid

This protocol describes an effective in situ hybridization approach to detect the mRNA expression levels and spatial patterns of target genes in Sipunculus nudus coelomic fluid.

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture method to obtain a sufficient number of LG stem cells (LGSCs) is one obstacle for further research and application. The three-dimensional (3D), serum-free culture method for adult mouse LGSCs is well established and shown here. The LGSCs could be continuously passaged and induced to differentiate to acinar or ductal-like cells.
For the LGSC primary culture, the LGs from 6-8-week-old mice were digested with dispase, collagenase I, and trypsin-EDTA. A total of 1 &#215; 104 single cells were seeded into 80 &#181;L of matrix gel-lacrimal gland stem cell medium (LGSCM) matrix in each well of a 24-well plate, precoated with 20 &#181;L of matrix gel-LGSCM matrix. The mix was solidified after incubation for 20 min at 37 &#176;C, and 600 &#181;L of LGSCM added.
For LGSC maintenance, LGSCs cultured for 7 days were disaggregated into single cells by dispase and trypsin-EDTA. The single cells were implanted and cultured according to the method used in the LGSC primary culture. LGSCs could be passaged over 40 times and continuously express stem/progenitor cell markers Krt14, Krt5, P63, and nestin. LGSCs cultured in LGSCM have self-renewal capacity and can differentiate into acinar or ductal-like cells in vitro and in vivo.

The three-dimensional, serum-free culture method for adult lacrimal gland (LG) stem cells is well established for the induction of LG organoid formation and differentiation into acinar or ductal-like cells.

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...