Journal of Visualized Experiments

Published by Journal of Visualized Experiments
Online ISSN: 1940-087X
Publications
0.8% agarose gel electrophoresis image of high molecular weight DNA collected from four depths in the subarctic Pacific Ocean (in duplicate), stained with the intercalating agent ethidium bromide (10 mg/ml). Gel was run at 15V for ~16hrs in 1X TBE gel running buffer. Sample bands are of good quality showing little evidence of mechanical shearing (shows single bands or smears as opposed to multiple bands) although the 10m extracts retain some RNA carry over (see smear in the 0.5 to 2.0 Kb range).  
Genomic DNA band illuminated by blue light after CsCl gradient centrifugation.  
Article
This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 microM Sterivex filters that have been treated with storage/lysis buffer and archived at -80 degrees C, and to purify this DNA using a cesium chloride density gradient. The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA. Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract. Part Five describes the optional purification via cesium chloride density gradient. It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.
 
General Heck cross-coupling reaction between an aryl bromide and an olefin. Click here to view larger image. 
Examples of industrially relevant organic compounds involving a palladium-catalyzed Heck cross-coupling reaction as key step in their synthesis. Click here to view larger image. 
Synthesis of dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium [(P(NC 5 H 10 ) 3 ) 2 Pd(Cl) 2 ] (1). Click here to view larger image. 
Gas chromatograms recorded from reaction mixtures of the Heck reaction of ethyl 4-bromobenzoate and styrene at 100 °C in DMF in the presence of ~10 mol% of tetrabutylammonium bromide and 0.05 mol% of catalyst, showing the time-dependent product formation. Note that the reaction time is slightly prolonged when compared to the data given in Table 1. This is due to periodical sampling. Click here to view larger image. 
The effect of ligand composition of dichloro{bis(aminophosphine)}palladium with the general formula of [(P{(NC 5 H 10 ) 3-n (C 6 H 11 ) n }) 2 Pd(Cl) 2 ] (where n = 0-2) on the complex stability and hence, on the ease of (water-induced) nanoparticle formation and hence, their catalytic performance under mild reaction conditions in the Heck cross-coupling reaction. Click here to view larger image. 
Article
Dichloro-bis(aminophosphine) complexes of palladium with the general formula of [(P{(NC5H10)3-n(C6H11)n})2Pd(Cl)2] (where n = 0-2), belong to a new family of easy accessible, very cheap, and air stable, but highly active and universally applicable C-C cross-coupling catalysts with an excellent functional group tolerance. Dichloro{bis[1,1',1''-(phosphinetriyl)tripiperidine]}palladium [(P(NC5H10)3)2Pd(Cl)2] (1), the least stable complex within this series towards protons; e.g. in the form of water, allows an eased nanoparticle formation and hence, proved to be the most active Heck catalyst within this series at 100 °C and is a very rare example of an effective and versatile catalyst system that efficiently operates under mild reaction conditions. Rapid and complete catalyst degradation under work-up conditions into phosphonates, piperidinium salts and other, palladium-containing decomposition products assure an easy separation of the coupling products from catalyst and ligands. The facile, cheap, and rapid synthesis of 1,1',1"-(phosphinetriyl)tripiperidine and 1 respectively, the simple and convenient use as well as its excellent catalytic performance in the Heck reaction at 100 °C make 1 to one of the most attractive and greenest Heck catalysts available. We provide here the visualized protocols for the ligand and catalyst syntheses as well as the reaction protocol for Heck reactions performed at 10 mmol scale at 100 °C and show that this catalyst is suitable for its use in organic syntheses.
 
Article
Unsymmetrical 1,2-diols are hardly accessible by reductive pinacol coupling processes. A successful execution of such a transformation is bound to a clear recognition and strict differentiation of two similar carbonyl compounds (aldehydes → secondary 1,2-diols or ketones → tertiary 1,2-diols). This fine-tuning is still a challenge and an unsolved problem for an organic chemist. There exist several reports on successful execution of this transformation but they cannot be generalized. Herein we describe a catalytic direct pinacol coupling process which proceeds via a retropinacol/cross-pinacol coupling sequence. Thus, unsymmetrical substituted 1,2-diols can be accessed with almost quantitative yields by means of an operationally simple performance under very mild conditions. Artificial techniques, such as syringe-pump techniques or delayed additions of reactants are not necessary. The procedure we describe provides a very rapid access to cross-pinacol products (1,2-diols, vicinal diols). A further extension of this new process, e.g. an enantioselective performance could provide a very useful tool for the synthesis of unsymmetrical chiral 1,2-diols.
 
Representative examples of green-positive E. faecalis (left), red positive E. faecium (middle), and negative (right) test results. 
Article
Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.
 
Sugar composition in the gut content of cotton leafworm. (A) Sugars in the gut content of larvae fed on cotton or (B) artificial diet by GC-MS characterization after aldononitrile acetate derivatization. The larval alimentary canal is divided into three regions: foregut, midgut and hindgut, shown in diagram (C). (D) Quantification of dominant glucose reveals a significant decrease in average content along the gut. However, cotton-feeding larvae exhibit higher amount of glucose in all gut regions. * and a indicate significant difference: P (* 1 ) = 0.0020, P (* 2 ) = 0.0366, P ( a ) = 0.0017. Error bars indicate standard errors. 1, Ribose; 2, Arabinose; 3, Mannose; 4, Glucose; 5, Galactose. doi:10.1371/journal.pone.0085948.g001 
Profiling of active bacteria. (A) For determining active bacteria, 13 C-labeled DNA is separated by density-gradient ultracentrifugation and subsequently retrieved from representative fractions, with darker color indicating labeled heavy DNA. All separated DNA samples (light, middle and heavy) were directly subjected to quantitative pyrosequencing for revealing species lineage and relative abundance. For each taxon X, metabolic activity is calculated as the difference in relative abundance between heavy fraction of the labeled sample and that of the control. (B) Isotopic ratio (d 13 C) of DNA samples. (C) Distribution of DNA content in gradient fractions of glucose treatments. Symbols: O, DNA extracted from the [ 12 C]-glucose control; &, DNA extracted from the [ 13 C]-glucose treatment for 24 h; m, DNA extracted from the [ 13 C]-glucose treatment for 48 h. Arrows indicate that considerable 13 C-labeled DNA shifted to heavy gradients, compared with the control. (D) Gradient fraction analysis by density after 40 h centrifugation of a 1.725 g ml 21 starting CsCl solution. Error bars indicate standard errors. doi:10.1371/journal.pone.0085948.g002 
Bacterial diversity and relative abundance in the gut microbiota of early-instar larvae. (A) Rarefaction curves of 16S rDNA sequences were obtained from representative SIP fractions of the control ([ 12 C]) and labeling treatment ([ 13 C]). (B) Relative abundance of bacterial taxa in different SIP fractions, represented in a relative area graph as revealed by pyrosequencing. Abbreviations: [ 12 C] Light, light fractions (fractions 9-11, Fig. 2A) of native-glucose amendment; [ 12 C] Middle, middle fraction (fraction 7) of that; [ 12 C] Heavy, heavy fractions (fractions 4-5) of that; [ 13 C] Light, light fractions of 13 C-glucose amendment; [ 13 C] Middle, middle fraction of that; [ 13 C] Heavy, heavy fractions of that. doi:10.1371/journal.pone.0085948.g003 
Bacterial diversity and relative abundance in the gut microbiota of late-instar larvae. (A) Rarefaction curves of 16S rDNA sequences were obtained from representative SIP fractions of early-instar and late-instar larvae. Abbreviations: E, representative fractions from earlyinstar larvae fed on 13 C-glucose; L, fractions from late-instar larvae fed on 13 C-glucose. (B) Relative abundance of bacterial taxa in different SIP fractions, represented in a relative area graph as revealed by pyrosequencing. Abbreviations are the same as in Fig. 3. doi:10.1371/journal.pone.0085948.g004 
Article
Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction(1), boosting the immune response(2), pheromone production(3), as well as nutrition, including the synthesis of essential amino acids(4,) among others. Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms. To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing (13)C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA(5). The incorporation of (13)C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled ((12)C) one. In the end, the (13)C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the (12)C-unlabeled similar one(6). Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species. Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, (13)C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.
 
Schematic diagram of the 40 pot capacity continuous multi-isotope labeling chamber from a bird's eye view . Dotted lines represent electrical wiring, while solid lines represent gas or water tubing. Click here to view larger image. 
A) Average CO 2 concentration (ppm) (+/- SE) over a twenty-four hour period for an entire growing season. B ) Average temperature (oC), open circles, and humidity (%), closed circles (+/- SE) over a twenty-four hour period for an entire growing season. Click here to view larger image. 
Article
Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or 2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration, and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.
 
Article
Gastric emptying studies in mice have been limited by the inability to follow gastric emptying changes in the same animal since the most commonly used techniques require killing of the animals and postmortem recovery of the meal(1,2). This approach prevents longitudinal studies to determine changes in gastric emptying with age and progression of disease. The commonly used [(13)C]-octanoic acid breath test for humans(3) has been modified for use in mice(4-6) and rats(7) and we previously showed that this test is reliable and responsive to changes in gastric emptying in response to drugs and during diabetic disease progression(8). In this video presentation the principle and practical implementation of this modified test is explained. As in the previous study, NOD LtJ mice are used, a model of type 1 diabetes(9). A proportion of these mice develop the symptoms of gastroparesis, a complication of diabetes characterized by delayed gastric emptying without mechanical obstruction of the stomach(10). This paper demonstrates how to train the mice for testing, how to prepare the test meal and obtain 4 hr gastric emptying data and how to analyze the obtained data. The carbon isotope analyzer used in the present study is suitable for the automatic sampling of the air samples from up to 12 mice at the same time. This technique allows the longitudinal follow-up of gastric emptying from larger groups of mice with diabetes or other long-standing diseases.
 
Article
Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive (13)C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive (13)C-methylation is a chemical reaction performed under mild conditions that modifies a protein's primary amino groups (lysine ε-amino groups and the N-terminal α-amino group) to (13)C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive (13)C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein (13)C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of (13)C-labeling at each primary amino group. One limitation of the method arises when the protein's N-terminal residue is a lysine because the α- and ε-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the (13)C-dimethylamines associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme, Lys1, are identified in (1)H-(13)C heteronuclear single-quantum coherence spectra.
 
The 13 C-assisted pathway analysis steps. 
Amino acids used for acquiring the labeling pattern of their metabolic precursors. ACoA, acetyl-CoA; AKG, α-Ketoglutarate; C5P, ribose 5-phosphate; CIT, citrate; E4P, erythrose 4-phosphate; G6P, glucose 6-phosphate; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PGA, 3-phosphoglycerate; PYR, pyruvate. 
GC peaks for 16 amino acids. TBDMS derivatized amino acids are cracked by MS into two fragments: (M-57) + , containing the entire amino acid, and (M-159) + , which lacks the α carboxyl group of the amino acid. For leucine and isoleucine, the (M-57)+ was overlapped by other mass peaks. We suggest using fragment (M-15)+ to analyze the entire amino acid labeling. The (f302)+ group is detected in most amino acids, which contains only the first (α-carboxyl group) and second carbons in an amino acid backbone. Because this MS peak often has high noise-to- signal ratios, (f302) + is not recommended for quantitatively analyzing the metabolic fluxes 7 . 
Article
Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is (13)C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a (13)C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps (2). First, we grow cells with (13)C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways(3, 4). Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active (2). Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways. (13)C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism(1). In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.
 
Isolation of protoplasts from Arabidopsis seedlings. A, Tissues from 14-day-old seedlings of Arabidopsis were collected and converted to protoplasts by a modified procedure of (Chen and Halkier, 2000). B , Protoplasts were purified by sucrose density gradient centrifugation and collected at the interface of enzyme solution and W5 uffer ( White arrows ). C , Bright-field microscopy of protoplasts. Microphotographs were collected using a cooled CCD camera interfaced with the Zeiss Axioscope 2 plus microscope. 
Article
Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi). Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants). We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 10(6)-10(7) protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.
 
Representative fluorescence image of Cryptosporidium and Giardia (oo)cysts. Cryptosporidium oocysts and Giardia cysts were stained with FITC labeled anti-Cryptosporidium/Giardia antibodies. Arrows, Giardia cysts; arrowheads, Cryptosporidium oocysts. A total of four Cryptosporidium oocysts and six Giardia cysts were found in the plane of focus. Samples observed under 200X magnification. 
Article
Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies (12). The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy. Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used(1,14). A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously(1-3,5-8,10,11). In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters(4). Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices(9,13) . This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.
 
Article
The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable cytokine-secreting cells. In order to label IL-17-secreting cells, a single cell suspension of mouse splenocytes is prepared and stimulated at 37 degrees C with PMA/ionomycin to induce cytokine secretion. To stop secretion cells are then placed on ice and are exposed to the IL-17 Catch Reagent a bi-specific antibody that binds to CD45 on the cell surface of leukocytes and to IL-17 as it is secreted and caught near the cell surface. Secretion is then re-started by increasing the temperature to 37 degrees C and IL-17 is trapped by the Catch Reagent. Secretion is then stopped again, by placing cells on ice. To detect the trapped IL-17, cells are incubated with a second IL-17-specific antibody conjugated to biotin and an Anti-Biotin-PE antibody. Cells can now be directly analyzed by flow cytometry or prepared for isolation and enrichment by subsequent labeling with Anti-PE conjugated MicroBeads.
 
Stylized graphs presenting 17β-estradiol concentrations in rats (A; n=15) 4 and mice (B; n=10) 6 as a consequence of daily hormone administration in Nutella. The peroral Nutella method renders daily peaks, however not by far as high as produced by subcutaneous or intravenous methods. This mimics the pharmacokinetics of human hormone replacement therapy, and the peroral treatment can proceed for as long as needed, even if the example above only present an initial 96 hours. The shaded areas correspond to physiological 17β-estradiol serum concentrations in intact female rats and mice respectively. Even though not visually demonstrated in the graphs, there is inter-individual variability in the 17β-estradiol concentrations, rendering coefficients of variation from 40 to 132 % in rats and from 13 to 219 % in mice with the Nutella method (very low mean concentrations often generate high coefficients of variation).  
Article
Estrogens are a family of female sexual hormones with an exceptionally wide spectrum of effects. When rats and mice are used in estrogen research they are commonly ovariectomized in order to ablate the rapidly cycling hormone production, replacing the 17β-estradiol exogenously. There is, however, lack of consensus regarding how the hormone should be administered to obtain physiological serum concentrations. This is crucial since the 17β-estradiol level/administration method profoundly influences the experimental results. We have in a series of studies characterized the different modes of 17β-estradiol administration, finding that subcutaneous silastic capsules and per-oral nut-cream Nutella are superior to commercially available slow-release pellets (produced by the company Innovative Research of America) and daily injections in terms of producing physiological serum concentrations of 17β-estradiol. Amongst the advantages of the nut-cream method, that previously has been used for buprenorphine administration, is that when used for estrogen administration it resembles peroral hormone replacement therapy and is non-invasive. The subcutaneous silastic capsules are convenient and produce the most stable serum concentrations. This video article contains step-by-step demonstrations of ovariectomy and 17β-estradiol hormone replacement by silastic capsules and peroral Nutella in rats and mice, followed by a discussion of important aspects of the administration procedures.
 
Article
The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function (1). At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models. Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR (2). We herein present in addition small animal positron emission tomography (PET) using (18)F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation (3). Further, this method can be applied for follow-up to improve monitoring of transplant rejection (4).
 
Article
Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC) and carotid intimal medial thickness (C-IMT) provide information about the burden of disease. However, despite multiple validation studies of CAC, and C-IMT, these modalities do not accurately assess plaque characteristics, and the composition and inflammatory state of the plaque determine its stability and, therefore, the risk of clinical events. [(18)F]-2-fluoro-2-deoxy-D-glucose (FDG) imaging using positron-emission tomography (PET)/computed tomography (CT) has been extensively studied in oncologic metabolism. Studies using animal models and immunohistochemistry in humans show that FDG-PET/CT is exquisitely sensitive for detecting macrophage activity, an important source of cellular inflammation in vessel walls. More recently, we and others have shown that FDG-PET/CT enables highly precise, novel measurements of inflammatory activity of activity of atherosclerotic plaques in large and medium-sized arteries. FDG-PET/CT studies have many advantages over other imaging modalities: 1) high contrast resolution; 2) quantification of plaque volume and metabolic activity allowing for multi-modal atherosclerotic plaque quantification; 3) dynamic, real-time, in vivo imaging; 4) minimal operator dependence. Finally, vascular inflammation detected by FDG-PET/CT has been shown to predict cardiovascular (CV) events independent of traditional risk factors and is also highly associated with overall burden of atherosclerosis. Plaque activity by FDG-PET/CT is modulated by known beneficial CV interventions such as short term (12 week) statin therapy as well as longer term therapeutic lifestyle changes (16 months). The current methodology for quantification of FDG uptake in atherosclerotic plaque involves measurement of the standardized uptake value (SUV) of an artery of interest and of the venous blood pool in order to calculate a target to background ratio (TBR), which is calculated by dividing the arterial SUV by the venous blood pool SUV. This method has shown to represent a stable, reproducible phenotype over time, has a high sensitivity for detection of vascular inflammation, and also has high inter-and intra-reader reliability. Here we present our methodology for patient preparation, image acquisition, and quantification of atherosclerotic plaque activity and vascular inflammation using SUV, TBR, and a global parameter called the metabolic volumetric product (MVP). These approaches may be applied to assess vascular inflammation in various study samples of interest in a consistent fashion as we have shown in several prior publications.
 
Article
Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults (1). BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via β adrenergic receptor (2, 3). Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters (4-6). BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance (6-8). While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT (9-13), there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of (18)F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of β-adrenoceptor antagonist propranolol (14, 15), or the enhanced BAT activation by β3 agonist BRL37344 (16). The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.
 
Article
Stable isotopes are essential tools in biological mass spectrometry. Historically, O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao, Miyagi and Rao and Ye et al.). O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques. Depending on the protease utilized, O-labeling can result in the incorporation of up to two O-atoms in the C-terminal carboxyl group of the cleavage product. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction. In our PALeO (protease-assisted labeling employing O-enriched water) adaptation of enzymatic O-labeling, we utilized 50% O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases and monitor proteolytic reactions. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second O-atom. Such "double-labeling" enzymes can be used for postdigestion O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing O-enriched water beyond enzymes and uses acidic pH conditions to introduce O-stable isotope signatures into peptides.
 
Article
Swiss microbial geneticist, Werner Arber shared the 1978 Nobel Prize in Physiology or Medicine with Hamilton Smith and Daniel Nathans for their discovery of restriction endonucleases. Werner Arber was born in Granichen, Switzerland in 1929. Following a public school education, he entered the Swiss Polytechnical School in Zurich in 1949, working toward a diploma in natural sciences. There, his first research experience involved isolating and characterizing an isomer of chlorine. Following graduation in 1953, Arber joined a graduate program at the University of Geneva, taking on an assistanceship in electron microscopy (EM), in which he studied gene transfer in the bacterial virus (bacteriophage) lambda. Eventually encountering limitations with EM as a tool, he began using microbial genetics as a methodology for his studies. The study of microbial genetics had been possible for a relatively short time: DNA had been discovered to carry genetic information only a decade before he d entered the field. After earning his Ph.D. in 1958, Arber continued to develop skills in microbial genetics, working with colleagues in the United States for a short time before returning to Geneva at beginning of 1960. There, he continued working on lambda transduction in E. coli, but found that the virus would not efficiently propagate. Recalling research done seven years earlier by Joe Bertani and Jean Weigle on "host-controlled restriction-modification", he realized there must be a host-controlled modification of the invading DNA, and sought to identify the mechanism. Based on Grete Kallengerger s work that demonstrated degradation of both irradiated and non-irradiated phage lambda following injection in a host, Arber and his graduate student, Daisy Dussoix further investigated the fate of DNA, and found that restriction and modification (later determined to be postreplicative nuclotide methylation) directly affected DNA, but did not cause mutations. They also found that theses were properties of the bacterial strains, and that both viral and cellular DNA were degraded. Together, Arber and Dussoix reported their findings to scientific community in 1961 at the First International Biophysics Congress in Stockholm. Aber also presented the research to the Science Faculty of University of Geneva in 1962, earning the Plantamour-Prevost prize. Based on his work and the work of others, he hypothesized that an enzyme in the host bacterium cut DNA into smaller pieces at specific sites, and methylase modified the host DNA to protect it from the digestive enzyme. These theories were later confirmed by Urs Kuhnlein, who found that mutation of specific sites rendered the phage resistant to cleavage; Hamilton smith, who identified Type II endonuclease HindII; and Daniel Nathans, who used HindII to break the SV40 virus into 11 fragments, allowing him to determine its method of replication. Since the discovery of restriction endonucleases, researchers have used them as tools to study the functions of genes of all types of organisms. Restriction enzymes have also facilitated the study of gene functions and enabled production of substances of medical and nutritional importance. Arber feels that in the next few decades we will learn much from the study of epigentics --factors that can affect the phenotype of an organism without changing the genetic information--. He is proud that, in that studying restriction degradation and DNA methylation in the 1960s, he was among the first in studying epigenetic phenomenon.
 
Article
Robert Huber and his colleagues, Johann Deisenhofer and Hartmut Michel, elucidated the three-dimensional structure of the Rhodopseudomonas viridis photosynthetic reaction center. This membrane protein complex is a basic component of photosynthesis - a process fundamental to life on Earth - and for their work, Huber and his colleagues received the 1988 Nobel Prize in Chemistry. Because structural information is central to understanding virtually any biological process, Huber likens their discovery to "switching on the light" for scientists trying to understand photosynthesis. Huber marvels at the growth of structural biology since the time he entered the field, when crystallographers worked with hand-made instruments and primitive computers, and only "a handful" of crystallographers would meet annually in the Bavarian Alps. In the "explosion" of structural biology since his early days of research, Huber looks to the rising generation of scientists to solve the remaining mysteries in the field - such as the mechanisms that underlie protein folding. A strong proponent of science mentorship, Huber delights in meeting young researchers at the annual Nobel Laureate Meetings in Lindau, Germany. He hopes that among these young scientists is an "Einstein of biology" who, he says with a twinkle in his eye, "doesn't know it yet." The interview was conducted by JoVE co-founder Klaus J. Korak at the Lindau Nobel Laureate Meeting 2008 in Lindau, Germany.
 
Article
At extremely low temperatures, certain materials conduct electrical currents without resistance - a phenomenon called superconductivity. An analogous state occurs in extremely low-temperature fluids, in which molecules flow without internal friction or resistance. Such a state, termed superfluidity, was predicted for helium-3 shortly after the Bardeen, Cooper, and Schrieffer (BCS) theory of 1957 explained the superconductivity of metals. The search for helium-3 superfluidity commenced; it continued for about ten years without success, and was all but abandoned by the time Douglas D. Osheroff began his graduate studies at Cornell University. "The general wisdom was that the superfluidity of helium-3 was a pipe dream of the theorists," says Osheroff. "But in fact, it did occur." In 1972, Osheroff - together with David M. Lee and Robert C. Richardson - demonstrated the transition of helium-3 to a superfluid state at temperatures near absolute zero (-273 degrees C). Twenty-four years after their discovery, Osheroff and colleagues won the Nobel Prize in Physics. Osheroff's scientifically inquisitive nature developed long before his landmark discovery. "A lot of people did crazy things when they were young" says Osheroff, whose wild experimentation including building a 100,000-volt X-ray machine as a teenager. Now as a faculty member at Stanford University, Osheroff stresses the importance of mentoring undergraduates to get them excited about science, and to guide them on what to do next. As for graduate school, Osheroff says that "it doesn't matter what is it that you study as a graduate student - the important thing is learning how to study."
 
Article
English Chemist Harold Kroto shared the 1996 Nobel Prize in Chemistry with Robert Curl and Richard Smalley for their discovery of Fullerenes (C(60;)), molecules composed completely of carbon (C(60;)) that form hollow spheres (also known as Buckyballs), tubes, or ellipsoids. These structures hold the potential for use in future technologies ranging from drug development and antimicrobial agents, to armor and superconductors. Harold Kroto was born in Wisbech, Cambridgeshire in 1939 and grew up in Bolton. Educated at Bolton School, he entered Sheffield University in 1958 to study Chemistry. During his time there he played tennis for the university team, illustrated the university's magazine covers, and played folk music with other students. Enjoying his time at Sheffield very much, he chose to stay on and complete a Ph.D. in Chemistry under Richard Dixon. Following graduation in 1964, Kroto went on to post doc at the National Research Council (NRC) in Ottowa, Canada where microwave spectroscopy became his specialty. After two years of study at the NRC he spent a year at Bell Laboratories. He then accepted a position as a tutorial fellow at the University of Sussex, where he was soon offered a permanent position. There, he applied his expertise in microwave spectroscopy to the field of astronomy and spent several fruitful years detecting long carbon chains in the interstellar medium. Upon hearing of the work of Richard Smalley at Rice, who developed a laser that could vaporize graphite, Kroto thought they could use Smalley's instrument to see carbon chains similar to those they had observed in interstellar matter. He suggested his idea for an experiment to Bob Curl, also at Rice. In 1985 he traveled to Rice to perform the experiment (and also to visit a half-price bookstore he'd heard about in Houston). Although he felt certain that the apparatus would create the carbon chains, the experiment revealed a totally unexpected result: the spontaneous formation of spherical shapes, which they called Buckminster Fullerenes in honor of the architect who popularized the geodesic dome. Though he is pleased to have received the Nobel Prize, Kroto does not believe in prizes or competition as a motivator for scientific (or athletic) progress. Rather, he believes that the pursuit of science or athletics should be simply for the enjoyment or interest in the subject matter, and he prefers to investigate subjects that other people aren't working on. Kroto has mixed feelings about the effect the prize has had on his life. On the one hand, he would like to be able to spend more time pursuing graphic design, something he has always deeply enjoyed. On the other hand, he now enjoys a sense of responsibility for supporting the scientific community. As an atheist, Kroto feels that science is, in itself, atheistic. He doesn't accept anything without evidence. Kroto expresses concern about people holding positions of power who do not use evidence as a basis for decision-making. "When they are prepared to accept one of 20-30 stories from thousands of years ago, I wonder what else they are prepared to accept when it comes to decisions which affect me?" Kroto is particularly worried about the effect of policies that require the teaching of non-scientific ideas, to the detriment of evidence-based scientific education. He points to the forced teaching of creationism in public schools and the existence of a "creation museum" in the United States as sources of misinformation that have given rise to "a whole generation of school children who've been abused."
 
Article
Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine ((19)F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by (19)F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound (19)F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by (19)F MR spectroscopy.
 
Article
In vivo (19)F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.
 
Article
Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy. NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.
 
Article
It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology. Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism. In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by (1)H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis). The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described. Hepatic metabolic profiling is measured directly from an intact liver biopsy using (1)H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism. This method can also be applied to any tissue biopsy.
 
Article
Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition(1). Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce(2). Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration(3)Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study(4). Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.
 
Representative image of AO staining for nucleated white blood cells used in order to determine cell concentration in diluted whole blood samples. 
IL-1β production measured in whole blood supernatants from a healthy donor upon stimulation with LPS at increasing cell concentrations. Samples were assayed in triplicate. 
Representative results of IL-1β secretion measured in whole blood supernatants from two healthy normal controls as well as two patients presenting with IL-1β-associated autoinflammatory manifestations. 
Article
Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1β(1, 2, 3). Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1β upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system (4, 5, 6, 7). A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1β to its mature, bioactive form (4, 5, 6, 8, 9, 10). The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture (2). The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.
 
Normal Endothelial Vasodilator Function. Representative recording of an individual with normal endothelial vasodilator function, characterized by an increase in the signal amplitude after cuff release relative to baseline.  
Endothelial Vasodilator Dysfunction. Representative recording of an individual with endothelial vasodilator dysfunction.
Article
The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction. In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT (peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response. In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.
 
Article
Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 microL. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.
 
Article
Peter Agre, born in 1949 in Northfield Minnesota, shared the 2003 Nobel Prize in Chemistry with Roderick MacKinnon for his discovery of aquaporins, the channel proteins that allow water to cross the cell membrane. Agre's interest medicine was inspired by the humanitarian efforts of the Medical Missionary program run by the Norwegians of his home community in Minnesota. Hoping to provide new treatments for diseases affecting the poor, he joined a cholera laboratory during medical school at Johns Hopkins. He found that he enjoyed biomedical research, and continued his laboratory studies for an additional year after medical school. Agre completed his clinical training at Case Western Hospitals of Cleveland and the University of North Carolina, and returned to Johns Hopkins in 1981. There, his serendipitous discovery of aquaporins was made while pursuing the identity of the Rhesus (Rh) antigen. For a century, physiologists and biophysicists had been trying to understand the mechanism by which fluid passed across the cell's plasma membrane. Biophysical evidence indicated a limit to passive diffusion of water, suggesting the existence of another mechanism for water transport across the membrane. The putative "water channel," however, could not be identified. In 1988, while attempting to purify the 30kDa Rh protein, Agre and colleagues began investigating a 28 kDa contaminant that they believed to be a proteolytic fragment of the Rh protein. Subsequent studies over the next 3-4 years revealed that the contaminant was a membrane-spanning oligomeric protein, unrelated to the Rh antigen, and that it was highly abundant in renal tubules and red blood cells. Still, they could not assign a function to it. The breakthrough came following a visit with his friend and former mentor John Parker. After Agre described the properties of the mysterious 28 kDa protein, Parker suggested that it might be the long-sought-after water channel. Agre and colleagues tested this idea by expressing the protein in Xenopus oocytes, which typically have low water permeability. When the test oocytes were placed in a hypotonic solution, they swelled and exploded, thus revealing the function of the unknown protein as a water channel, which they named aquaporin. The Nobel Prize enabled Agre to take his research and scientific interests in new directions. He felt that over the years his work had continually taken him further from his original interests in third-world diseases, so he shifted his focus back in that direction. He now serves as the director of the Malaria institute at Johns Hopkins where he has applied his knowledge to the study of the malarial parasite and the Anopheles mosquito, which both express aquaporins. In addition, since winning the Nobel Prize, he has enjoyed increased opportunities for bringing science to the public and for "encouraging young people to go into science."
 
Article
Aaron Ciechanover was born in Haifa, Israel in October 1947. He shared the Nobel Prize in Chemistry in 2004 with Avram Hershko and Irwin Rose for their discovery of ubiquitin-mediated protein degradation. When Ciechanover began his work on proteolysis, the field was outside the realm of scientific mainstream as many thought that the fundamental secrets relating to sequence specificity were relevant to the synthetic side, or code side. The notion that specific sequences could selectively guide a destructive process did not naturally occur to scientists including Ciechanover himself. The emergence of controversial evidence demonstrating a requirement for metabollic energy in intracellular protein degradation, refuted the idea that cellular proteolysis was an entirely exergonic process occuring in the lysosme and prompted Ciechanover, Hershko, and Rose to "launch an attack" on the system, in order to uncover true pathway. Later findings of Ciechanover and subsequent groups showed that not only was the process energy-dependent, but that 8% of the human genome is remarkabley one large ubiquitin system. Following the recapitulation and reflection of his work, Ciechanover shares insights into his principal and philosophical approach to science and life alltogether. The life and work of Aaron Ciechanover are deeply rooted and influenced by Judaism and Israel and it is therefore that with only brief intermission, Ciechanover spent his scientific career in Israel as he is--through his presence and work--able to contribute and shape presence and future of the State of Israel.
 
Article
The field of quantum optics rests on the work of Roy Glauber, who helped elucidate the nature of light as both particles and waves. According to Glauber, quantum optics allowed "all sorts of experiments...that never could have been done before." He suggests that it was not his "small revelation" that the Nobel Committee awarded, but rather the decades of research that followed his own. Nonetheless, Glauber received one-half of the 2005 Nobel Prize in Physics "for his contribution to the quantum theory of optical coherence" while the other half was shared by John Hall and Theodor Hänsch for their work on laser-based precision spectroscopy. Glauber admits that the behavior of light seems strange and unintuitive--yet the phenomena that Einstein called "spooky action at a distance" may have many practical applications. In this candid interview, Glauber shares his thoughts about working at Los Alamos National Laboratory--his shock to learn that he was helping to build The Bomb, and his dismay about how it was used. At Los Alamos, Glauber met two of his major influences: Julian Schwinger, who was Glauber's thesis advisor at Harvard, and Los Alamos scientific director Robert Oppenheimer, who facilitated his early post-doctoral research. Glauber also tells a poignant account of how his marriage fell victim to the social upheaval of the 1960's, and how he was left to raise two children alone. Despite the difficulties of reconciling academia with family, Glauber is amused to find himself revered by women as "someone who has raised children and nonetheless had a successful academic career."
 
Article
American biochemist Roger Tsien shared the 2008 Nobel Prize in Chemistry with Martin Chalfie and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Tsien, who was born in New York in 1952 and grew up in Livingston New Jersey, began to experiment in the basement of the family home at a young age. From growing silica gardens of colorful crystallized metal salts to attempting to synthesize aspirin, these early experiments fueled what would become Tsien's lifelong interest in chemistry and colors. Tsien's first official laboratory experience was an NSF-supported summer research program in which he used infrared spectroscopy to examine how metals bind to thiocyanate, for which he was awarded a $10,000 scholarship in the Westinghouse Science Talent Search. Following graduation from Harvard in 1972, Tsien attended Cambridge University in England under a Marshall Scholarship. There he learned organic chemistry --a subject he'd hated as an undergraduate-- and looked for a way to synthesize dyes for imaging neuronal activity, generating BAPTA based optical calcium indicator dyes. Following the completion of his postdoctoral training at Cambridge in 1982, Tsien accepted a faculty position at the University of California, Berkeley. There he and colleagues developed and improved numerous small molecule indicators, including indicators fura-2 and indo-1. In 1989, Tsien moved his laboratory to the University of California at San Diego, where he and his colleagues developed the enhanced mutant of GFP as a way to devise a cyclic AMP (cAMP) sensor for use in live cells. They initially engineered molecules to take advantage of the conformational change that occurs when cAMP binds to protein kinase A (PKA). By labeling one part of PKA with fluoroscein and another with a rhodamine, they hoped to detect Fluorescence Resonance Energy Transfer (FRET), which would occur when the two molecules were in close proximity. The initial experiments presented numerous difficulties due to the challenges of expressing PKA subunits in E. coli, labeling the protein without destroying its function, and delivering the protein to cells via microinjection. Eventually, Tsien sought a more elegant approach, hoping to use and modify a naturally fluorescent protein that could be expressed in the cell. GFP originally described by Davenport in 1955, extracted and purified by Shimomura in 1965, and cloned by Prasher in 1992 was an appealing candidate. To make the protein more useful for their FRET studies, Tsien and colleagues modified the amino acid structure of the protein (S65T). The improved protein had an excitation peak near that of fluoroscein, and was photostable. Tsien and colleagues also solved the protein's crystal structure, enabling them to generate additional colors with spectral properties suitable for FRET. However, when they attempted to use the GFP proteins in the detection of cAMP, they experienced further difficulties with PKA. Instead, their first successful use of GFP derivatives for FRET was in the detection of intracellular calcium using their engineered calmodulin-based calcium indicator, Cameleon. In a short time, Tsien's work has led to further technological developments and important scientific findings. GFP and its derivatives have been used in a wide range of biological applications, from the study of protein localization to understanding how HIV spreads from cell to cell. The need for such probes is highlighted by the abundance of research conducted using these fluorescent proteins, as well as the continued development of similar fluorescent proteins, such as the coral-derived dsRED. Tsien is currently developing genetically encoded Infrared Fluorescent Proteins (IFPs), which with their long emission wavelengths of >700 nm, have the ability to pass through living tissue and improve imaging in living organisms. He is also building synthetic molecules for use in humans. He cites team effort and the contributions of students and post-docs as key components of progress and success: "Even if I had the time, I couldn't have done the experiments, because I don't know how. It's very much a team effort."
 
Article
American Biologist Martin Chalfie shared the 2008 Nobel Prize in Chemistry with Roger Tsien and Osamu Shimomura for their discovery and development of the Green Fluorescent Protein (GFP). Martin Chalfie was born in Chicago in 1947 and grew up in Skokie Illinois. Although he had an interest in science from a young age-- learning the names of the planets and reading books about dinosaurs-- his journey to a career in biological science was circuitous. In high school, Chalfie enjoyed his AP Chemistry course, but his other science courses did not make much of an impression on him, and he began his undergraduate studies at Harvard uncertain of what he wanted to study. Eventually he did choose to major in Biochemistry, and during the summer between his sophomore and junior years, he joined Klaus Weber's lab and began his first real research project, studying the active site of the enzyme aspartate transcarbamylase. Unfortunately, none of the experiments he performed in Weber's lab worked, and Chalfie came to the conclusion that research was not for him. Following graduation in 1969, he was hired as a teacher Hamden Hall Country Day School in Connecticut where he taught high school chemistry, algebra, and social sciences for 2 years. After his first year of teaching, he decided to give research another try. He took a summer job in Jose Zadunaisky's lab at Yale, studying chloride transport in the frog retina. Chalfie enjoyed this experience a great deal, and having gained confidence in his own scientific abilities, he applied to graduate school at Harvard, where he joined the Physiology department in 1972 and studied norepinephrine synthesis and secretion under Bob Pearlman. His interest in working on C. elegans led him to post doc with Sydney Brenner, at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England. In 1982 he was offered position at Columbia University. When Chalfie first heard about GFP at a research seminar given by Paul Brehm in 1989, his lab was studying genes involved in the development and function of touch-sensitive cells in C. elegans. He immediately became very excited about the idea of expressing the fluorescent protein in the nematode, hoping to figure out where the genes were expressed in the live organism. At the time, all methods of examining localization, such as antibody staining or in situ hybridization, required fixation of the tissue or cells, revealing the location of proteins only at fixed points in time. In September 1992, after obtaining GFP DNA from Douglas Prasher, Chalfie asked his rotation student, Ghia Euskirchen to express GFP in E. coli, unaware that several other labs were also trying to express the protein, without success. Chalfie and Euskirchen used PCR to amplify only the coding sequence of GFP, which they placed in an expression vector and expressed in E.coli. Because of her engineering background, Euskirchen knew that the microscope in the Chalfie lab was not good enough to use for this type of experiment, so she captured images of green bacteria using the microscope from her former engineering lab. This work demonstrated that GFP fluorescence requires no component other than GFP itself. In fact, the difficulty that other labs had encountered stemmed from their use of restriction enzyme digestions for subcloning, which brought along an extra sequence that prevented GFP's fluorescent expression. Following Euskirchen's successful expression in E. coli, Chalfie's technician Yuan Tu went on to express GFP in C. elegans, and Chalfie published the findings in Science in 1994. Through the study of C. elegans and GFP, Chalfie feels there is an important lesson to be learned about the importance basic research. Though there has been a recent push for clinically-relevant or patent-producing (translational) research, Chalfie warns that taking this approach alone is a mistake, given how "woefully little" we know about biology. He points out the vast expanse of the unknowns in biology, noting that important discoveries such as GFP are very frequently made through basic research using a diverse set of model organisms. Indeed, the study of GFP bioluminescence did not originally have a direct application to human health. Our understanding of it, however, has led to a wide array of clinically-relevant discoveries and developments. Chalfie believes we should not limit ourselves: "We should be a little freer and investigate things in different directions, and be a little bit awed by what we're going to find."
 
LCM of Drosophila peripheral neurons. (a) Age matched third instar larvae are selected, washed and (b) embedded in a cryomold with OCT and frozen at -80°C, (c) 8μm serial tissue sections are created using a standard cryostat and (d) placed uniformly on clean glass slides. The glass slides with the affixed tissue sections are stored at -80°C prior to LCM processing. (e,f) Immediately preceding LCM, the tissue sections are thawed and briefly fixed in 70% ethanol followed by brief rinse in ddH2O. (g) The slides are briefly incubated with trypsin and rinsed with ddH2O to remove cuticles (black) from the larval sections. (h,i) tissue sections without larval cuticle are then further dehydrated in an ethanol gradient and finally cleared in xylene. (j) The LCM cap with a thermolabile polymer is placed on the tissue section and the laser is pulsed on the selected fluorescent cell body. The laser pulse melts the polymer and engulfs the selected cell body. The polymer cap, along with the captured cell body is lifted, and (k) RNA extraction buffer is added to the cell. The captured cell, along with the RNA extraction buffer is incubated at 42°C for 30 minutes and can be either stored at -80°C, or directly processed for RNA purification.  
LCM facilitates high precision capture of da neurons. (a) Representative image of a dehydrated and trypsin treated 8 micron tissue section prior to performing LCM, showing two class-IV da neurons labeled with GFP by the ppk-GAL4,UASmCD8::GFP reporter strain. Note, one of the neurons is highlighted (arrowhead) for capture. (b) Cell body of the highlighted neuron (arrowhead) is cleanly micro-dissected with high specificity from the tissue section. (c) End-on view of a single class-IV da neuron captured on the LCM cap. (d) Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) electropherogram of total RNA isolated from LCM-derived da neurons, showing excellent RNA quality as indicated by the presence of sharp 5.8S, 18S, and 28S rRNA peaks.  
qRT-PCR reveals substantial enrichment of neuronal marker gene expression in LCM captured da neurons relative to whole animal. qRT-PCR analyses of neuron-specific marker gene expression (elav) in LCM captured da neurons (21-7-GAL4,UAS-mCD8::GFP) and whole animals in age matched third instar larvae was performed in triplicate. Relative levels of elav expression in LCM captured da neurons were normalized to the endogenous control (rp49) and relative to whole larvae using the ΔΔCt method 23 . A 2.5 fold enrichment in the relative levels of elav in LCM captured da neuron samples was observed relative to whole animals.  
Article
The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification, Fluorescent Activated Cell Sorting (FACS), and RNA binding protein based strategies, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells, including da neurons at the third instar larval stage of development. Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP transgene under the control of either the class IV da neuron specific ppk-GAL4 driver or the pan-da neuron specific 21-7-GAL4 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.
 
Article
The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (> or = 20 L) filtration of microbial biomass, ranging between 0.22 microm and 2.7 microm in diameter, from the water column. Two 20 L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.
 
Arrangement of chromosome combinations on the OctoChrome device and expected chromosomal painting results. 
Positioning of sample slide over the OctoChrome device. 
A. A normal cell with chromosomes 8, 12, 21 painted in Square 2. 
B. Representative abnormal cells with leukemia-specific chromosomal translocation and aneuploidy in Square 2. 
Article
Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei(1). The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis(2). Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene(3-6), indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application(7). Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)(8). The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)(9). Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers(8,10).
 
Representative gel stained after loading samples of a protein purification (*: molecular weight marker). 
CBB stained gel that has not been washed long enough before CBB staining. The protein bands appear weaker (note that the marker lane * contains the same amount of protein as in Fig. 1). 
Article
In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80 mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staining of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compounds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.
 
Sensitivity of colloidal CBB G-250 in protein in-gel staining. A sample mixture of fi ve proteins (BSA/66 kDa, ovalbu- min/45 kDa, casein/25 kDa, myoglobin/17 kDa, and cytochrome C/12 kDa) was separated in one-dimensional SDS gels and visualized using the colloidal staining protocols according to Neuhoff ( a ), Candiano ( b ), Anderson ( c ), and Kang ( d ). Lane 1 , molecular weight marker; Lanes 2 – 8 , proteins loaded in decreasing amount per lane (200, 100, 50, 20, 10, 5, and 2 ng). Without destaining of the gels, a detection limit of about 5 ng is achieved. 
Article
Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches. Several improvements of the original Coomassie protocol(1) have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution(2), and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol(3). Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol(4). The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins. Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.
 
Schematic of the modified pcDNA 3.1 expression vector. The plasmid was digested with EcoRV and EcoRI to include the affinitytag and TEV cleavage site on the amino terminus of Sin3A. To clone untagged versions of HDAC1 and SDS3, the vector was digested with KpnI and EcoRI. 
SDS-PAGE showing the first step of the purification. The "bound protein" lane shows the complex bound to the affinity resin. Following TEV digestion, the tag present on Sin3A-HID is cleaved off and the complex is eluted from the resin as shown in the second and third lanes of the gel. 
Chromatogram of protein-complex purified using size exclusion chromatography. Note that the pure complex eluted in the void volume because it forms a dimer in solution. 
SDS-PAGE showing the fractions of the gel filtration shown in Figure 3. 
Article
The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.
 
Article
Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFRalpha, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80 degrees C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.
 
Article
The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28°C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28°C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37°C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28°C for further incubation. We find that the initial incubation at 28°C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.
 
NPCs show directed migration in EFs. PCs showed highly directed migration towards the cathode when exposed to EFs, red lines and blue arrows represent trajectories and direction of cell movement (A). B shows migration paths of NPCs. Bar: 50 μm.  
Transplanted NPCs show directed migration towards the cathode in the organotypic spinal cord slice. (A) NPCs labeled with Hoechst 33342 were transplanted into the organotypic spinal cord slice at the starting point of the EF treatment. NPCs migrated directionally towards the cathode for 2.5 hours, at which point the EF polarity was reversed (B). Altering EF polarity triggered a sharp reversal of electrotaxis towards the new cathode (C). (D) Image of transplanted NPCs within the spinal cord slice at the end of the time-lapse recording. (E) A 3D reconstruction of transplanted NPCs within the spinal cord slice. 3D scanning sections were 300 μm in thickness, starting from the middle and ending at the bottom of the slice. Dotted lines indicate the relative positions of the same population of transplanted cells at the beginning, reversal, and end points of the EF treatment (A-C, respectively). Arrow heads indicate the same population of Hoechst 33342 labelled NPCs. Bar: 50 μm.
Article
Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system(1,2). These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans(3,4). In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types(5,6), including neural progenitor cells (NPCs)(7,8). Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously(5,11). Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures(9,10). Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.
 
Photographs of phase-contrast microscopy of primary culture of macrophages. Example of morphology of M1 (A, B) and M2 (C, D) macrophages. M1 macrophages were obtained by lipopolysaccharide treatment for 4 hr at day 12. M2 macrophages were obtained by IL-4 treatment starting from day 6 until day 12. Phase-contrast microscopy allows clear identification of both subtypes of macrophages. Photographs at low (10X) (A, C) and high (40X) (B, D) magnification were performed at day 12 of culture. Scale bar: 50 μm. Please click here to view a larger version of this figure.
Bioinformatic analysis of a representative 2D DIGE gel from macrophage proteins with Progenesis SameSpots software. Twenty areas were detected to perform the differential analysis between M1 and M2 macrophages. The bottom of the figure indicates the color code for the fold-change of volume spots. Please click here to view a larger version of this figure. 
Article
The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.
 
Article
Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.
 
Schematic representation of the experimental procedures toward the preparation of chromosome paints. Click here to view larger image. 
The major steps in chromosome microdissection. A) Laser-assisted cutting of the chromosomal region of interest through the membrane. B) The membrane with a hole after the catapulting is performed. C) The view of the catapulted piece of the membrane with a chromosomal segment in it attached to the adhesive cap. The arrow indicates the heterochromatin of the X chromosome that remained on the slide. The asterisk shows a piece of another chromosome that remained on the slide. Click here to view larger image. 
Agarose gel images showing DNA after WGA. A) Low molecular weight (200-500 bp) DNA from arm 3R after using the WGA4 and WGA3 GenomePlex kits. B) High molecular weight (10-20 kb) DNA from arm 2L after REPLI-g amplification. The 100 bp ladder is shown in the left lanes. The tables below gel images show DNA concentrations measured by Nanodrop. Click here to view larger image. 
Painting of interphase (A), prophase (B), prometaphase (C) and metaphase (D) chromosomes from larval imaginal discs of the An. gambiae Mopti strain using three probes generated from microdissected material labeled by WGA3. The 2R arm is labeled in green (fluorescein); the 2L arm is unlabeled; the 3R arm is in pink, a mixture of red (Cy-3) and orange (Cy-5); the 3L arm is labeled in orange (Cy-5). The X chromosome has a red label corresponding to the 18S rDNA probe. Chromatin is stained in blue (DAPI). Brightly stained regions of chromosomes correspond to the heterochromatin. Click here to view larger image. Video 1. The process of LCM of the An. gambiae polytene chromosomes. Click here to view Video 1. Video 2. Whole mount 3D FISH performed on An. gambiae ovarian nurse cells. The probe is labeled in Cy-3 (depicted in blue) and was made from a microdissected 2R chromosome arm. Chromatin was stained with DAPI and is depicted by cyan pseudo-coloring (light blue). Click here to view Video 2. 
Article
Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.
 
Workflow of leukemic cell purification from PB. Blood from CLL patients is transferred into a 15mL tube (1), Human B-cell enrichment cocktail is added to the sample (2) and incubated for 20 minutes. Blood is then diluted with PBS in a proportion of 1:1 and laid on the top of the density gradient (3). Subsequent sample centrifugation (4) allows the formation of multiple layers: a) plasma, b) B lymphocytes, c) Ficoll, d) red cells and unwanted cells. Purified B lymphocytes (b layer) are then collected (5), washed and purity of cell preparation is analyzed by flow cytometry (6). Please click here to view a larger version of this figure.
HS1 phosphorylation status influences cytoskeletal functionality in CLL primary samples. (1) Migration on transwell of primary samples in the presence or absence of SDF-1. In the graph are displayed the means SEM of the number of cells acquired in 1 minute at the flow cytometer (n = 7 of 1 spot HS1 vs n = 12 of 2 spot HS1). (2) Spontaneous adhesion was measured after cell labeling and 1 hr incubation in 96-well plates. Displayed are the means SEM for primary samples (n = 7 of 1 spot HS1 vs n = 12 of 2 spot HS1). (3) F-actin polymerization capability of primary samples. Displayed are the means SEM of the relative F-actin content of CLL cells after stimulation with SDF-1 and staining with FITC-labeled phalloidin (n = 5 of 1 spot HS1 vs n = 5 of 2 spot HS1). MFI: Mean Fluorescence Intensity. The histogram represent as example of sample acquisition. Please click here to view a larger version of this figure. 
Article
The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease's biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as "spots" on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.
 
Representative TCS UV-Vis absorbance spectrum. TCS has a robust peak at 280 nm, allowing easy determination of A 280 , as well as affording the ability to use the molar extinction coefficient of 4,200 L/mol/cm 12 to determine the actual concentration of TCS dissolved in tyrodes buffer. The yellow line indicates the peak at 280 nm. In this example, the absorbance value at 280 nm is 0.11876, which indicates a TCS concentration of 28.28 µM. Click here to view larger image.  
A representative degranulation response of IgE-sensitized RBL mast cells exposed to 0.0004 µg/ml DNP-BSA antigen and TCS (0-20 μM). A spontaneous release value (no antigen present) is depicted for reference. Values represent mean ± standard deviation of triplicate samples. As presented, data were normalized to control (0 µM TCS), and significant differences were determined in Prism software with a one-way ANOVA followed by a Tukey's post hoc test (comparisons made to 0.001 µM TCS average response). Significance is represented by ***p<0.001. Click here to view larger image.  
A representative degranulation response of RBL mast cells stimulated with 180 nM A23187 calcium Ionophore in the presence of TCS (0-20 μM). A spontaneous release sample (no ionophore present) is depicted for reference. Values represent mean ± standard deviation of triplicate samples. As presented, data are normalized to control (0 µM TCS), and significant differences were determined in Prism software with a one-way ANOVA followed by a Tukey's post hoc test (comparisons made to 0.001 µM TCS average response). Significance is represented by ***p<0.001; **p<0.01. Click here to view larger image.  
Article
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells(3-5). Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells(6), can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies(1). Originally published by Naal et al.(1), we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here. Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease(7-11), although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function(2). In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280 = 4,200 L/M/cm)(12). This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
 
Article
The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro. Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.
 
Article
Spring-like materials are ubiquitous in nature and of interest in nanotechnology for energy harvesting, hydrogen storage, and biological sensing applications. For predictive simulations, it has become increasingly important to be able to model the structure of nanohelices accurately. To study the effect of local structure on the properties of these complex geometries one must develop realistic models. To date, software packages are rather limited in creating atomistic helical models. This work focuses on producing atomistic models of silica glass (SiO2) nanoribbons and nanosprings for molecular dynamics (MD) simulations. Using an MD model of "bulk" silica glass, two computational procedures to precisely create the shape of nanoribbons and nanosprings are presented. The first method employs the AWK programming language and open-source software to effectively carve various shapes of silica nanoribbons from the initial bulk model, using desired dimensions and parametric equations to define a helix. With this method, accurate atomistic silica nanoribbons can be generated for a range of pitch values and dimensions. The second method involves a more robust code which allows flexibility in modeling nanohelical structures. This approach utilizes a C++ code particularly written to implement pre-screening methods as well as the mathematical equations for a helix, resulting in greater precision and efficiency when creating nanospring models. Using these codes, well-defined and scalable nanoribbons and nanosprings suited for atomistic simulations can be effectively created. An added value in both open-source codes is that they can be adapted to reproduce different helical structures, independent of material. In addition, a MATLAB graphical user interface (GUI) is used to enhance learning through visualization and interaction for a general user with the atomistic helical structures. One application of these methods is the recent study of nanohelices via MD simulations for mechanical energy harvesting purposes.
 
Top-cited authors
Tsuyoshi Miyakawa
  • Fujita Health University
Michael Seibenhener
  • Auburn University
Sean C Piantadosi
  • University of Pittsburgh
Keizo Takao
  • University of Toyama
Marom Bikson
  • City College of New York