This work was supported by the Wuhan Science and Technology Plan Project (2018201261638501).

1. Mosquito sampling and sorting
<ol>
	<li>Trap the adult mosquitoes through the light traps MXA-02 or carbon dioxide mosquito traps in the field.</li>
	<li>Kill the collected mosquitoes by dipping in liquid nitrogen10,11. Transport them to the laboratory by the cold-chain logistics system12. 
	NOTE: Dry ice was primarily used in the cold-chain logistics system.</li>
	<li>(Optional) If the sample sites were near the lab, directly send the alive mosquitoes in the net traps to the laboratory and euthanize them by freezing at &#8804;-20 &#176;C for 30 min4.</li>
	<li>Make a morphological identification of the collected mosquitoes through the tool book13 for classification and identification of mosquitoes. 
	NOTE: If necessary, DNA barcoding could be used to classify and identify mosquitoes14,15.&#160;</li>
	<li>Based on sampling dates and sites, assign them into different pools,&#160;and then store&#160;them in 2-5&#160;mL tubes.</li>
	<li>Draw a table to record each tube with mosquito species, number, sampling code, date, and sites.</li>
	<li>Print the labels with sampling codes and attach them to the tubes.</li>
	<li>Keep tubes with the mosquito samples at -80 &#176;C until further analysis.</li>
</ol>
2. Mosquito grinding
<ol>
	<li>Place 20-50 mosquitoes into each 2 mL sterile tube with 3 mm ceramic beads with 1.5 mL of Roswell Park Memorial Institute (RPMI) medium containing 2% Penicillin-Streptomycin-Amphotericin B Solution. 
	NOTE: Keep the tubes on ice oron anice tray.</li>
	<li>Homogenize the mosquitoes with a low-temperature tissue homogenizer by grinding for 30 s at 70 Hz at 4 &#176;C for three cycles16.</li>
	<li>Centrifuge the mixtures at 15,000 &#215; g for 30 min at 4 &#176;C and transfer the supernatants to new tubes.</li>
	<li>Centrifuge the supernatants again at 15,000 &#215; g for 10 min at 4 &#176;C to remove the mosquito debris12,17. 
	NOTE: If necessary, the supernatants can be filtered using 0.22 &#956;m filters.</li>
	<li>Aliquot the supernatants (P0) into 2 mL screw cap storage tubes (200 &#181;L per tube) and store them at -80 &#176;C.</li>
</ol>
3. Preparation of cells and cell culture maintenance medium
NOTE: Mosquito cell line C6/36 (Aedes albopictus RNAi-deficient) and vertebrate cell line BHK-21 (baby hamster kidney) were used for the virus amplification and isolation.
<ol>
	<li>Inoculate 1 &#215; 106 C6/36 cells into a 75 cm2 flask with 10 mL of RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) at 28 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Inoculate 1 &#215; 106 BHK-21 cells into a 75 cm2 flask with 10 mL of Dulbecco&#39;s modified Eagle medium (DMEM) supplemented with 10% FBS and 1% P/S at 37 &#176;C in a 5% CO2 humidified incubator. 
	NOTE: The cells were passaged two or three times per week17,18.</li>
	<li>Seed the cells cultured in a 75 cm2 flask (C6/36 or BHK-21 cells) into the 24-well plates (C6/36 per well: 1.4 &#215; 105; BHK-21 per well: 0.8 &#215; 105).</li>
	<li>Place the 24-well plates at 28 &#176;C or 37 &#176;C overnight.</li>
	<li>Observe the cells under the microscope and confirm whether cell confluency in each well is 80%-90%.</li>
	<li>Prepare the cell culture maintenance medium (500 mL). 
	&#8203;NOTE: For C6/36, the medium was RPMI medium supplemented with 2% FBS and 2% Penicillin-Streptomycin-Amphotericin B Solution. For BHK-21, the medium was DMEM medium supplemented with 2% FBS and 2% Penicillin-Streptomycin-Amphotericin B Solution.</li>
</ol>
4. Virus isolation
NOTE: All steps were carried out in a biosafety level 2 (BSL-2) laboratory. The safety level requirement of the biosafety laboratory was determined by the biosafety risk assessment based on the regulations of different countries and regions. The process must be performed in a biosafety cabinet.
<ol>
	<li>Remove the cell culture medium from the 24-well plates and add 100 &#181;L of the cell culture maintenance medium and 100 &#181;L of the supernatant of the mosquito homogenate (P0) to each well.</li>
	<li>Incubate the plates at 28 &#176;C or 37 &#176;C for 60 min and gently shake them every 15 min to prevent cell drying.</li>
	<li>Remove the supernatant and rinse each well gently with 600 &#181;L of cell culture maintenance medium to remove the debris completely.</li>
	<li>Add 800 &#181;L of cell culture maintenance medium to each well and keep the plates in a 5% CO2 humidified incubator at 28 &#176;C or 37 &#176;C for 7 days.</li>
	<li>Monitor the cell state of each well under the microscope daily15.</li>
	<li>Collect the supernatants of cells (P1 supernatants) on the 7th day and store the supernatants at -80 &#176;C.</li>
	<li>Repeat steps 4.1-4.6 2x to get P2 and P3 supernatants. 
	NOTE: In step 4.3, rinsing each well gently with 600 &#181;L of cell culture maintenance medium was optional for the P2 and P3.</li>
	<li>Depending on which well shows the cytopathogenic effect (CPE) &#160;&#8212;&#160;cells die, pyknosis,&#160;and detach from the surface; fusion with adjacent cells to form syncytia; or the appearance of nuclear or cytoplasmic inclusion bodies &#8212; add 300-400 &#181;L of the supernatant of this CPE well into each well of the new 6-well plates containing cells (the cell confluency was 80%-90%) to greatly amplify the viruses.</li>
	<li>Collect the supernatants and aliquot them into 2 mL screw cap storage tubes (500 &#181;L per tube). Store them at -80 &#176;C. 
	&#8203;NOTE: After three generations of successive passaging in cells, the samples that did not induce CPE were discarded. If necessary, the generations of successive passaging in cells can be increased.</li>
</ol>
5. Detecting viral sequences by RT-PCR
<ol>
	<li>Extract total RNA from 200 &#181;L of the viral supernatant using a viral RNA mini kit16. 
	NOTE: Here, anautomated nucleic acid extraction system was used to extract total RNA following the instrument manufacturer&#39;s instructions.</li>
	<li>Convert the RNA to cDNA following the RT-PCR kit instructions using a PCR instrument.
	<ol>
		<li>Add 15 &#181;L of RNA mixed with 1 &#181;L of random primers into the PCR tube. Place the tube at 70 &#176;C for 5 min and then place it on ice for 5 min.</li>
		<li>Add 5 &#181;L of 5x buffer and 25 &#181;L of mixture (10 mM dNTP, 40 U/&#181;L RNase inhibitor, and 200 U/&#181;L M-MLV) into the tube and place the tube at 42 &#176;C for 60 min and 95 &#176;C for 10 min.</li>
		<li>Store the tube at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Use a PCR kit and universal primers (Table 1) to detect viruses by PCR.
	<ol>
		<li>For arboviruses, set up the PCR reaction system (total 30 &#181;L) with the following components: 3 &#181;L of 10x Taq buffer, 3 &#181;L of dNTP (1mM), 1 &#181;L of forward primer (10 &#181;m), 1 &#181;L of reverse primer (10 &#181;m), 0.3 &#181;L of Taq (10 U/&#181;L), 19.7 &#181;L of ddH2O, and 2 &#181;L of cDNA.</li>
		<li>For the detection of flaviviruses, set up the reaction process as: stage 1: 35 cycles of 94 &#176;C for 30 s, 52 &#176;C for 30 s, and 72 &#176;C for 30 s; stage 2: 72 &#176;C for 10 min.</li>
		<li>For the detection of alphaviruses, set up the reaction process as: stage 1: 35 cycles of 94 &#176;C for 30 s, 50 &#176;C for 30 s, and 72 &#176;C for 30 s; stage 2: 72 &#176;C for 10 min.</li>
		<li>For the detection of bunyaviruses, set up the reaction process as: stage 1: 35 cycles of 94 &#176;C for 30 s, 50 &#176;C for 30 s, and 72 &#176;C for 30 s; stage 2: 72 &#176;C for 10 min.</li>
	</ol>
	</li>
	<li>Detect the PCR amplification products by 1% agarose gel electrophoresis and send them for sequencing analysis. 
	NOTE: If conditions permit, the supernatants can be undergo next generation sequencing analysis to identfy new viruses and obtain the whole viral genome seqeunces.</li>
</ol>

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The authors have no conflicts of interest to disclose.

The objective of this method was to offer a practical way for isolating mosquito-associated viruses using various cell lines. It is critical to add the Antibiotic-Antimycotic (Penicillin-Streptomycin-Amphotericin) to the supernatants of the mosquito homogenates to avoid contamination by bacteria or fungi. Mosquitoes and viral supernatants obtained in the field must be refrigerated at -80 &#176;C to avoid repeated freeze-thaw cycles.
Another critical step in the protocol was grinding. Mosquito ...

Mosquitoes are a group of important pathogenic arthropod vectors. There are approximately 3,500 species of mosquitoes in the family Culicidae1,2. The development of high-throughput sequencing technologies has led to the discovery of many novel, virus-like sequences in mosquitoes from different parts of the world3. Generally, these mosquito-associated viruses can be classified into two main groups: MBVs and MSVs.
MBVs are a group of diverse viruses that are causative agents of many human or animal illnesses, such as yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), and Rift Valley fever virus (RVFV)4. They have seriously threatened public health by causing severe morbidity and mortality in both humans and animals across the world. MBVs naturally sustain a life cycle between diverse hosts through transmission from an infected mosquito to a na&#239;ve host, as well as from a virus-infected host and to a feeding mosquito5. Therefore, these viruses can infect both mosquito cell lines and vertebrate cell lines in the lab1.
MSVs, which include Yichang virus (YCN), Culex flavivirus (CxFV), and Chaoyang virus (CHAOV), are a subgroup of insect-specific viruses1,6,7. In recent years, there has been a rise in the discovery of novel MSVs, and some of these MSVs have been found to have an impact on the transmission of MBVs. For example, CxFV, which can be a persistent infection in Culex pipiens, could suppress WNV replication at an early stage8. Another insect-specific flavivirus, cell-fusing agent virus (CFAV), has been found to inhibit the propagation of DENV and Zika virus (ZIKV) in Aedes aegypti mosquitoes9. Thus, this protocol is a useful approach for isolating mosquito-associated viruses and can help in further research into the distribution of pathogens related to mosquitoes and the control of mosquito-borne diseases.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m membrane filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td>Polymer films with specific pore ratings.To remove cell debris and bacteria.</td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>CORNING</td>
			<td>3524</td>
			<td>Containers for cell</td>
		</tr>
		<tr>
			<td>75 cm2 flasks</td>
			<td>CORNING</td>
			<td>430641</td>
			<td>Containers for cell</td>
		</tr>
		<tr>
			<td>a sterile 2 mL tube with 3 mm ceramic beads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>Antibiotic in the medium to prevent contamination from bacteria and fungi</td>
		</tr>
		<tr>
			<td>Automated nucleic acid extraction system</td>
			<td>NanoMagBio</td>
			<td>S-48</td>
			<td></td>
		</tr>
		<tr>
			<td>BHK-21 cells</td>
			<td>National Virus Resource Center, Wuhan Institute of Virology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C6/36 cells</td>
			<td>National Virus Resource Center, Wuhan Institute of Virology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal machine</td>
			<td>Himac</td>
			<td>CF16RN</td>
			<td>Instrument for centrifugation of mosquito samples</td>
		</tr>
		<tr>
			<td>CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s minimal essential medium (DMEM)</td>
			<td>Gibco</td>
			<td>C11995500BT</td>
			<td>medium for vertebrate cell lines</td>
		</tr>
		<tr>
			<td>Ebinur Lake virus</td>
			<td></td>
			<td></td>
			<td>Cu20-XJ isolation</td>
		</tr>
		<tr>
			<td>Feta Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10099141C</td>
			<td> 
			 
			Provide nutrition for cells</td>
		</tr>
		<tr>
			<td>high-speed low-temperature tissue homogenizer</td>
			<td>servicebio</td>
			<td>KZ-III-F</td>
			<td>Instrument for grinding</td>
		</tr>
		<tr>
			<td>incubator (28 &#176;C)</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td>Instrument for cell culture</td>
		</tr>
		<tr>
			<td>incubator (37 &#176;C)</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td>Instrument for cell culture</td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15410-122</td>
			<td>Antibiotic in the medium to prevent contamination from bacteria</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Amphotericin B Solution</td>
			<td>Gibco</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator (-80 &#176;C)</td>
			<td>sanyo</td>
			<td>MDF-U54V</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute&#160; medium (RPMI)</td>
			<td>Gibco</td>
			<td>C11875500BT</td>
			<td>medium for mosiquto cell lines</td>
		</tr>
		<tr>
			<td>Screw cap storage tubes (2 mL)</td>
			<td>biofil</td>
			<td>&#160;FCT010005</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile pestles</td>
			<td>Tiangen</td>
			<td>OSE-Y004</td>
			<td>Consumables&#160; for grinding</td>
		</tr>
		<tr>
			<td>TGrinder OSE-Y30 electric tissue grinder</td>
			<td>Tiangen</td>
			<td>OSE-Y30</td>
			<td>Instrument for grinding</td>
		</tr>
		<tr>
			<td>The dissecting microscope</td>
			<td>ZEISS</td>
			<td>stemi508</td>
			<td></td>
		</tr>
		<tr>
			<td>the light traps MXA-02</td>
			<td>Maxttrac</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The mosquito absorbing machine</td>
			<td>Ningbo Bangning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>The pipette tips</td>
			<td>Axygen</td>
			<td>TF</td>
			<td></td>
		</tr>
		<tr>
			<td>The QIAamp viral RNA mini kit</td>
			<td>QIAGEN</td>
			<td>52906</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td>0203-5-PO</td>
			<td></td>
		</tr>
	</tbody>
</table>

mosquito-associated virus isolation from field-collected mosquitoes

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two main categories of these new mosquito-associated viruses are &#34;mosquito-borne viruses (MBVs)&#34; and &#34;mosquito-specific viruses (MSVs)&#34;. These novel viruses might be pathogenic to both vertebrates and mosquitoes, or they could just be symbiotic with mosquitoes. Entity viruses are essential to confirm the biological characters of these viruses. Thus, a detailed protocol has been described here for virus isolation and amplification from field-collected mosquitoes. First, the mosquito samples were prepared as supernatants of mosquito homogenates. After centrifugation twice, the supernatants were then inoculated into either mosquito cell line C6/36 or vertebrate cell line BHK-21 for virus amplification. After 7 days, the supernatants were collected as the P1 supernatants and stored at -80 &#176;C. Next, P1 supernatants were passaged twice more in C6/36 or BHK-21 cells while the cell status was being checked daily. When cytopathogenic effect (CPE) on the cells was discovered, these supernatants were collected and used to identify viruses. This protocol serves as the foundation for future research on mosquito-associated viruses, including MBVs and MSVs.

After inoculation with the supernatants of the mosquito homogenates (P0), the C6/36 cells exhibited a wide intercellular space, and exfoliated cells were observed at 120 h (Figure 1A) compared to the uninoculated cells (control) at the same time (Figure 1B). After incubating the BHK-21 cells with the P3 supernatants, visible CPE was observed in the BHK-21 cells at 48 h (Figure 1C) in contrast to the control cells (<strong class="xfi...

Watch this Scientific Journal Video about Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes at JoVE.com

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

Numerous novel virus-like sequences have been found in mosquitoes due to the extensive use of sequencing technologies. We provide an effective procedure for isolating and amplifying viruses using vertebrate and mosquito cell lines, which might serve as the basis for future studies on mosquito-associated viruses, including mosquito-borne and mosquito-specific viruses.

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two ...

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Research

JoVE Journal

Biology

1.7K Views.  Chinese Academy of Sciences. Numerous novel virus-like sequences have been found in mosquitoes due to the extensive use of sequencing technologies. We provide an effective procedure for isolating and amplifying viruses using vertebrate and mosquito cell lines, which might serve as the basis for future studies on mosquito-associated viruses, including mosquito-borne and mosquito-specific viruses.

Video: Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...