The authors thank J. Priscu, A. Chiuchiolo and the McMurdo LTER limnology team for assistance in collection and preservation of the samples in Antarctica. We thank Ratheon Polar Services and PHI helicopters for logistical support. Light micrographs were generated in Miami's Center for Advanced Microscopy and Imaging Center. This work was supported by NSF Office of Polar Programs Grants 0631659 and 1056396.

1. Sample Acquisition
<ol>
	<li>Choose and prepare the sampling site one day prior to sampling the water column. This will allow the stratified layers of the water column to reform after disturbance due to drilling and ice-hole melting. Identify location of drill site by GPS.</li>
	<li>To access the water column, begin by drilling a hole through the ice with a Jiffy ice auger attached to a 4-inch Jiffy flight extension and cutting bit. To prevent freezing of the drill in the hole, try to avoid drilling into the liquid water by halting drilling approximately 4-6 inches above the top of the water column.</li>
	<li>The drill hole will need to be widened from a diameter of 15 to 60 cm prior to sampling. This is accomplished by use of a hole melter (Hotsy Model) which circulates heated polyethylene glycol through a copper pipe. Hole melting typically takes up to 18 hours.</li>
	<li>Prior to sampling the water column, ensure that all required equipment and sample storage vessels are assembled at the sampling site. Sampling materials include: Niskin bottle (5-10 L capacity), Winch with depth-calibrated cable, Messenger, sufficient sample bottles for each sampling depth and coolers for sample transport.</li>
	<li>Begin sampling early in the day (~5 am), as lake water filtering should be completed on the day of sampling. Collect water samples (1-5 L) from desired depths (for this study: 6-m, 15-m and 18-m sampling depths, measured from the piezometric water level in the ice hole) using a Niskin sampler attached to a calibrated hand winch. To prevent disturbance of the water column collect shallow depths before deeper ones.</li>
	<li>Store lake samples in coolers and transport from the sample site to the field lab using a sled attached to an ATV (all terrain vehicle). Samples should be processed or stabilized (for eg: flash freeze in liquid nitrogen) at the field laboratory immediately following sampling.</li>
</ol>
2. Development of Enrichment Cultures
<ol>
	<li>For cultivation of enrichment cultures,Concentrate 0.5-1 L of lake water onto 47 mm 0.45 &mu;m pore size polyethersulfone filters using gentle filtration (&lt; 0.3 atm). If the diversity of the natural community is desired, filter a second sample onto 47 mm 0.45 &mu;m pore size polyethersulfone filters and follow protocols according to Bielewicz et al. 4 for eukaryote phylogenetic diversity.</li>
	<li>Transfer filter to 50 mL sterile falcon tube containing ~20 mL of filtered lake water collected from the same sampling depth as the filter. Gently wash cells from the filter using a sterile pipette.</li>
	<li>Transfer cell suspension to a 25 cm2 sterile cell culture flask. Add filtered lake water collected from each sample depth to increase the culture volume to 45 mL. Set up multiple replicates for each sampling depth if enrichments on different culture media are desired.</li>
	<li>Supplement culture flasks with 50X sterile solutions to a final concentration of 1X of the following culture media: Bold's Basal Medium, BG11 and F/2. If cultivation of mixotrophic organisms is required, set-up two F/2-supplemented vessels and add 2-3 grains of sterile rice to one of the flasks.</li>
	<li>Incubate culture flasks at low temperature (4 &deg;C) and low irradiance (20 &mu;mol photons m-2 s-1) in a temperature-controlled growth incubator for at least 4 weeks in Antarctica prior to shipment to your U.S. laboratory. If isolates are desired, plate 250 &mu;L of enrichment culture on agar plates containing appropriate growth media after 2 weeks incubation.</li>
	<li>For shipment to the U.S., transfer enrichment cultures to sterile 50-mL falcon tubes. Request shipment requirement of "do not freeze" and "keep cool" on shipping request. While the shipping process from Antarctica is relatively fast (2 weeks by commercial air) and highly reliable, there is a possibility of loss of fastidious organisms during the transport process. Thus, investigators may want to preserve a subsample prior to shipping so that loss of certain species during the shipping process can be assessed.</li>
	<li>Upon arrival in the U.S., transfer 5 mL of enrichment culture into 45 mL of appropriate growth media in a 125-mL sterile Erlenmeyer flask capped with a sterile sponge. Enrichment cultures should be maintained as standing cultures in a temperature controlled environmental growth chamber (Diurnal Growth Chamber, VWR) at 4 &deg;C/20 &mu;mol photons m-2 s-1. Cultures should be transferred to fresh media every 30 days.</li>
</ol>
3. Cell Lysate Extraction from Filtered Enrichments
<ol>
	<li>Filter 5 mL of enrichment culture onto a 25 mm Whatman GF/F filter using gentle vacuum (&lt;10 psi). The filter may be stored for several weeks at -80 &deg;C prior to assaying RubisCO activity.</li>
	<li>Cut the filter into 4-5 sections with sterile steel scissors and transfer the pieces with sterile forceps to a 2 mL screw cap tube filled one fifth of the way full with 0.1 mm diameter zirconia/silica beads.</li>
	<li>Add 1.25 mL of filter extraction buffer (50 mM bicine, 10 mM MgCl2, 1 mM EDTA, 5 mg/mL BSA, and 0.1% triton X-100; add fresh: 10 mM NaHCO3, 10 mM DTT, 3.3 mM aminocaproic acid, 0.7 mM benzamidine, pH 7.8) 13 and disrupt cells using a Minibead beater three times for 30s on speed setting 48. To prevent heating of the sample, alternate beadbeating with 1 min ice incubation.</li>
	<li>Centrifuge filter slurry for 2 min at 3000 x g at 4 &deg;C to form a loose pellet of GF/F filter fibers and cell wall debris.</li>
	<li>Remove a 200 &mu;L aliquot and immerse in 800 &mu;L of ice-cold 90% acetone. Measure extractable chlorophyll (chl) a in a spectrophotometer at wavelength values of 664 nm and 647 nm 14. Transfer the supernatant to a 1.5 mL microcentrifuge tube.</li>
	<li>Centrifuge remaining supernatant at 4 &deg;C for 2 min at 15,000 x g and transfer the supernatant (avoid the pellet of insoluble cellular membranes and remaining GF/F filter fibers) to a clean 1.5 mL microcentrifuge tube. This soluble cell lysate may now be used in the RubisCO carboxylase activity assay.</li>
	<li>Lysate may be stored at -80 &deg;C after addition of 12.5% glycerol and flash freezing in liquid nitrogen. A loss of activity may occur after multiple freeze/thaw cycles depending on the sensitivity of the enzymes in the lysate. Therefore, activity of fresh versus frozen lysate should be tested before large scale cell lysate extractions.</li>
</ol>
4. RubisCO Carboxylase Activity Filter Assay
<ol>
	<li>Transfer 125 &mu;L of soluble lysate to a 15 mL screw cap microcentrifuge tube (cap not needed) that has been chilled on ice. For negative control samples, add 125 &mu;L soluble lysate to 15 mL screw cap microcentrifuge tube, but do not add substrate (in step 4.7). Run duplicate reactions for all samples and negative controls.</li>
	<li>Prepare 10 mL of assay buffer fresh (50 mM Bicine-NaOH, pH 8.0, 50 mM NaHCO3, 25 mM MgCl2) and chill on ice. Aliquot enough of the assay buffer for all of the samples and negative controls (100 &mu;L per sample/control plus 100 &mu;L extra) to a 5 mL tube. Perform all subsequent steps under a fume hood.</li>
	<li>Add 40 &mu;L NaH14CO3 (500 &mu;Ci/mL) per mL of assay buffer to the aliquoted buffer and keep the buffer on ice.</li>
	<li>Remove 10 &mu;L of the assay buffer containing 14C and dilute it in 1 mL of assay buffer. After inverting to mix, add 100 &mu;L of the 1:100 diluted mixture to 3 mL of Bio-Safe II scintillation counting cocktail in a scintillation vial. Invert to mix. Scintillation counts (Beckman LS6500) should be over 18,000 cpm before continuing with the assay.</li>
	<li>When enough NaH14CO3 has been added to the assay buffer, move the reaction tubes to a dry bath preset to 25 &deg;C and incubate for 3-5 mins.</li>
	<li>Add 100 &mu;L assay buffer to the lysates and incubate for 5 mins at 25 &deg;C.</li>
	<li>Add 20 &mu;L of 15 mM Ribulose bisphosphate, to samples and allow the reaction to proceed for 5-10 mins at 25 &deg;C.</li>
	<li>Add 100 &mu;L propionic acid (100%) (Fisher) to stop the reaction. Centrifuge for 1 hour at 2,000 x g to exhaust unincorporated 14C.</li>
	<li>Transfer the total volume of the samples to scintillation vials containing 3 mL Bio-Safe II scintillation counting cocktail. Determine cpm of acid stable end products by scintillation counting (B. Witte, R. Tabita personal communication).</li>
	<li>Calculate RubisCO activity rates on a chl a basis 14.</li>
</ol>
5. Representative Results
We utilized enrichment culturing to isolate cold-adapted phototrophic and mixotrophic protists residing in the Antarctic Lake Bonney. To capture a greater diversity of organisms, we tested three growth media types: Bold's Basal Medium (BBM) 15, F/2-Si Marine Medium 16, 17 and BG11 Medium 18. Visual inspection of the enrichment cultures by light microscopy revealed that the cultures were dominated by phototrophic protists (indicated by the presence of chlorophyll pigment in most cells) and harbored a variety of cell morphologies depending on the sampling depth from which the inoculum was taken and the type of media used in the culture (Fig. 1).
We measured maximum rates of carboxylase activity catalyzed by the enzyme RubisCO as a proxy for carbon fixation potential. Chl a abundance was also monitored as an estimate of phototrophic protist biomass (Fig. 2). Despite cultivation of all cultures under the same temperature/light regime (ie. 4 &deg;C/20 &mu;mol m-2 s-1), carbon fixation potential and phototrophic biomass varied dramatically between the enrichment cultures. Maximum RubisCO activity was observed in enrichment cultures growing in either BBM (Enrichments 4 and 6) or BG11 (Enrichments 13 and 14) growth media, while cultures enriched on F/2 growth medium (Enrichments 19, 21, 23) exhibited a 4- to 34-fold lower maximum carboxylase activities. These differences were not due to lower biomass levels in the F/2 cultures, as carboxylase activity was expressed on a Chl a basis. Moreover, RubisCO activity did not correlate with chl a concentration (r = -0.023, p&gt;0.1; Fig. 2, inset). The BBM cultures inoculated with lake water from 6 m and 15 m (Enrichments 4 and 5) had the highest chl a levels, while all other cultures exhibited relatively low chl a, regardless of RubisCO enzyme activity (Fig. 2).
<img alt="Figure 1" src="/files/ftp_upload/3992/3992fig1.jpg" /> 
Figure 1. Representative microbial eukaryote enrichment cultures which have selected for the growth of different organisms. Identity of the culture is given in the lower left of the panels. All images represent light micrographs generated using oil immersion at 1000X magnification.
<img alt="Figure 2" src="/files/ftp_upload/3992/3992fig2.jpg" /> 
Figure 2. Potential carbon fixation capacity and extractable chlorophyll a levels in Antarctic microbial eukaryote enrichment cultures isolated from Lake Bonney and grown on various culture media. See Table 1 for culture details. Inset: Pearson correlation between chlorophyll a abundance and carbon fixation potential in Antarctic microbial eukaryote enrichment cultures.

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We have nothing to disclose.

Recent molecular studies have reported high diversity of single-celled eukaryotes across a range of environments 3, 19, 20; however, due to a lack of isolates across the full range of protist habitats the functional roles of these individual species in food webs are largely unknown. In this study, we have described methodologies to enrich for microbial eukaryote species exhibiting metabolic versatility from a relatively undersampled environment, a permanently ice-covered Antarctic lake. Cultures enriched from ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>BBM</td>
			<td>Sigma</td>
			<td>B5282</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BG11</td>
			<td>Sigma</td>
			<td>C3061</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>F/2</td>
			<td>Sigma</td>
			<td>G9903</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GF/F filter, 25 mm</td>
			<td>Fisher Scientific</td>
			<td>09-874-64</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GF/F filter, 47 mm</td>
			<td>Fisher Scientific</td>
			<td>09-874-71</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polyethersulfone filter, 0.45 &mu;m pore, 47 mm</td>
			<td>Pall Life Sciences</td>
			<td>61854</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile cell culture flask, 25 cm2</td>
			<td>Corning</td>
			<td>430639</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Diurnal growth chamber</td>
			<td>VWR</td>
			<td>35960-076</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zirconia/silica beads, 0.1 mm diamter</td>
			<td>BioSpec Products</td>
			<td>11079101z</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mini-Bead beater</td>
			<td>BioSpec Products</td>
			<td>3110BX</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Screw-cap microcentrifuge tube (1.5 &mu;L)</td>
			<td>USA Scientific</td>
			<td>1415-8700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaH14CO3</td>
			<td>ViTrax</td>
			<td>VC 194</td>
			<td>Keep in aliquots of 400 &mu;L at -20&deg;C</td>
		</tr>
		<tr>
			<td>RuBP</td>
			<td>Sigma</td>
			<td>R0878-100mg</td>
			<td>Dissolve in 10 mM Tris-propionic acid (pH 6.5)</td>
		</tr>
	</tbody>
</table>

establishment of microbial eukaryotic enrichment cultures from a chemically stratified antarctic lake and assessment of carbon fixation potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

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Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

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11.3K Views.  Miami University. Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Video: Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Enrichment of NK Cells from Human Blood with the RosetteSep Kit from StemCell Technologies

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer and infections, but are also implicated in the early stages of pregnancy and transplant rejection. These cells are present in peripheral blood, from which they can be isolated. Cells can be isolated using either positive or negative selection. For positive selection we use antibodies directed to a surface marker present only on the cells of interest whereas for negative selection we use cocktails of antibodies targeted to surface markers present on all cells but the cells of interest. This latter technique presents the advantage of leaving the cells of interest free of antibodies, thereby reducing the risk of unwanted cell activation or differenciation. In this video-protocol we demonstrate how to separate NK cells from human blood by negative selection, using the RosetteSep kit from StemCell technologies. The procedure involves obtaining human peripheral blood (under an institutional review board-approved protocol to protect the human subjects) and mixing it with a cocktail of antibodies that will bind to markers absent on NK cells, but present on all other mononuclear cells present in peripheral blood (e.g., T lymphocytes, monocytes...). The antibodies present in the cocktail are conjugated to antibodies directed to glycophorin A on erythrocytes. All unwanted cells and red blood cells will therefore be trapped in complexes. The mix of blood and antibody cocktail is then diluted, overlayed on a Histopaque gradient, and centrifuged. NK cells (&gt;80% pure) can be collected at the interface between the Histopaque and the diluted plasma. Similar cocktails are available for enrichment of other cell populations, such as human T lymphocytes.

Natural killer cells are a small population of lymphocytes. Here we show how to isolate these cells from human blood by negative selection, using a kit from StemCell Technologies. The cells obtained are viable and untouched by antibodies, and therefore ready to be used for a number of procedures.

Natural killer (NK) cells are large granular cytotoxic lymphocytes that belong to the innate immune system and play major roles in fighting against cancer ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross-section (&#963;PSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq&#8242;/Fm&#8242;), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, the protocol describes how to measure PSII photophysiology of an epizoic alga Colacium sp.&#160;Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton), using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. M&#252;ller 1776, Cladocera, Daphniidae) on baseline fluorescence and &#963;PSII, Fv/Fm, Fq&#8242;/Fm&#8242;, and NPQNSV of planktonic Colacium sp. To validate this methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol could determine the effects of calcium (Ca) and manganese (Mn) on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II photophysiology and primary productivity. Here we describe a protocol to measure PSII photophysiology of epizoic alga, Colacium sp. on substrate zooplankton using cuvette-type FRRf.

Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

Collection, Fixation, and Mounting of Caudal Penduncle from Zebrafish after Cryoinjury

To begin, prepare two milliliters of 4%formalin or any other appropriate fixative. Also keep a petri dish, forceps, and surgical scissors ready before starting the procedure. Place the properly euthanized cryoinjured fish in a petri dish containing deionized water.Using scissors, perform a cut through the body, anterior and posterior to the caudal peduncle. Let the tissue bleed out in the deionized water inside the petri dish. Using forceps, collect and transfer the caudal peduncle into the prepared fixative solution in a microcentrifuge tube.Carefully invert the tube several times. Before mounting, wash the fixed tissue in PBS for 10 minutes on a rocker. Then transfer it into a two-milliliter microcentrifuge tube containing a solution of 30%sucrose in deionized water, pre-cooled to four degrees Celsius and gently invert the tube several times.Leave the tube upright at 4 degrees Celsius for at least 24 hours. Fill an embedding mold with a five-millimeter layer of OCT mounting medium. Using forceps, place the caudal peduncle at the bottom of the mold.Adjust its orientation for either transversal or coronal sections. Let the medium freeze in a box of dry ice. As soon as the tissue is stabilized in the desired position, fill up the rest of the mold before the OCT completely freezes over.Store the mold at 80 degrees Celsius for at least one hour before sectioning the tissue using a cryostat. The extent of cryoinjury at different days post-cryoinjury, or DPCI, was analyzed in the caudal peduncle sections using trichrome staining composed of Aniline Blue, Acid Fuschin, and Orange-G. The damaged areas were determined by the absence of orange staining.At 4 and 7 dpci, the transverse section displayed extensively degenerated skeletal muscle in the cryoinjured flank of the body. Immunofluorescence analysis showed that, at 4 DPCI, the injured side of the caudal peduncle contained abundant DAPI-positive cells but little to no F-actin and tropomyosin 1, indicating degenerated muscles. At 7 dpci, tropomyosin 1 and F-actin could be detected in the wound, close to the vertical body midline, indicating the start of new myofiber formation.At 30 dpci, both sides of the body displayed a similar distribution of F-actin staining, indicating efficient skeletal muscle restoration.