Braunschweig, Germany
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Sikorski J.,Cell Cultures GmbH | Lapidus A.,U.S. Department of Energy | Copeland A.,U.S. Department of Energy | Glavina Del Rio T.,U.S. Department of Energy | And 44 more authors.
Standards in Genomic Sciences | Year: 2010

Sulfurospirillum deleyianum Schumacher et al. 1993 is the type species of the genus Sulfurospirillum. S. deleyianum is a model organism for studying sulfur reduction and dissimilatory nitrate reduction as an energy source for growth. Also, it is a prominent model organism for studying the structural and functional characteristics of cytochrome c nitrite reductase. Here, we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of the genus Sulfurospirillum. The 2,306,351 bp long genome with its 2,291 protein-coding and 52 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Gronow S.,Cell Cultures GmbH | Welnitz S.,Cell Cultures GmbH | Lapidus A.,U.S. Department of Energy | Nolan M.,U.S. Department of Energy | And 44 more authors.
Standards in Genomic Sciences | Year: 2010

Veillonella parvula (Veillon and Zuber 1898) Prévot 1933 is the type species of the genus Veillonella in the family Veillonellaceae within the order Clostridiales. The species V. parvula is of interest because it is frequently isolated from dental plaque in the human oral cavity and can cause opportunistic infections. The species is strictly anaerobic and grows as small cocci which usually occur in pairs. Veillonellae are characterized by their unusual metabolism which is centered on the activity of the enzyme methylmalonyl-CoA decarboxylase. Strain Te3 T, the type strain of the species, was isolated from the human intestinal tract. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the large clostridial family Veillonellaceae, and the 2,132,142 bp long single replicon genome with its 1,859 protein-coding and 61 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Lail K.,U.S. Department of Energy | Sikorski J.,Cell Cultures GmbH | Saunders E.,Los Alamos National Laboratory | Lapidus A.,U.S. Department of Energy | And 43 more authors.
Standards in Genomic Sciences | Year: 2010

Spirosoma linguale Migula 1894 is the type species of the genus. S. linguale is a free-living and non-pathogenic organism, known for its peculiar ringlike and horseshoe-shaped cell morphology. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is only the third completed genome sequence of a member of the family Cytophagaceae. The 8,491,258 bp long genome with its eight plasmids, 7,069 protein-coding and 60 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Pukall R.,Cell Cultures GmbH | Lapidus A.,U.S. Department of Energy | Glavina Del Rio T.,U.S. Department of Energy | Copeland A.,U.S. Department of Energy | And 43 more authors.
Standards in Genomic Sciences | Year: 2010

The genus Conexibacter (Monciardini et al. 2003) represents the type genus of the family Conexibacteraceae (Stackebrandt 2005, emend. Zhi et al. 2009) with Conexibacter woesei as the type species of the genus. C. woesei is a representative of a deep evolutionary line of des-cent within the class Actinobacteria. Strain ID131577 T was originally isolated from temperate forest soil in Gerenzano (Italy). Cells are small, short rods that are motile by peritrichous fla-gella. They may form aggregates after a longer period of growth and, then as a typical charac-teristic, an undulate structure is formed by self-aggregation of flagella with entangled bacteri-al cells. Here we describe the features of the organism, together with the complete sequence and annotation. The 6,359,369 bp long genome of C. woesei contains 5,950 protein-coding and 48 RNA genes and is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Ivanova N.,U.S. Department of Energy | Sikorski J.,Cell Cultures GmbH | Jando M.,Cell Cultures GmbH | Munk C.,Los Alamos National Laboratory | And 42 more authors.
Standards in Genomic Sciences | Year: 2010

Geodermatophilus obscurus Luedemann 1968 is the type species of the genus, which is the type genus of the family Geodermatophilaceae. G. obscurus is of interest as it has frequently been isolated from stressful environments such as rock varnish in deserts, and as it exhibits interesting phenotypes such as lytic capability of yeast cell walls, UV-C resistance, strong production of extracellular functional amyloid (FuBA) and manganese oxidation. This is the first completed genome sequence of the family Geodermatophilaceae. The 5,322,497 bp long genome with its 5,161 protein-coding and 58 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Tice H.,U.S. Department of Energy | Mayilraj S.,Cell Cultures GmbH | Mayilraj S.,Chandigarh Institute of Microbial Technology | Sims D.,Los Alamos National Laboratory | And 40 more authors.
Standards in Genomic Sciences | Year: 2010

Nakamurella multipartita (Yoshimi et al. 1996) Tao et al. 2004 is the type species of the mo-nospecific genus Nakamurella in the actinobacterial suborder Frankineae. The nonmotile, coccus-shaped strain was isolated from activated sludge acclimated with sugar-containing synthetic wastewater, and is capable of accumulating large amounts of polysaccharides in its cells. Here we describe the features of the organism, together with the complete genome se-quence and annotation. This is the first complete genome sequence of a member of the family Nakamurellaceae. The 6,060,298 bp long single replicon genome with its 5415 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


News Article | April 6, 2016
Site: www.nature.com

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were carried out using the TR146 buccal epithelial squamous cell carcinoma line32 obtained from the European Collection of Authenticated Cell Cultures (ECACC) and grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were routinely tested for mycoplasma contamination using mycoplasma-specific primers and were found to be negative. Prior to stimulation, confluent TR146 cells were serum-starved overnight, and all experiments were carried out in serum-free DMEM. C. albicans wild-type strains included the autotrophic strain BWP17 + CIp30 (ref. 33) and the parental strain SC5314 (ref. 34). Other C. albicans strains used and their sources are listed in Extended Data Tables 1 and 2. C. albicans cultures were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30 °C overnight. Cultures were washed in sterile PBS and adjusted to the required cell density. Antibodies to phospho-MKP1 and c-Fos were from Cell Signalling Technologies (New England Biolabs UK), mouse anti-human α-actin was from Millipore (UK), and goat anti-mouse and anti-rabbit horseradish peroxidase (HRP)-conjugated antibodies were from Jackson Immunologicals (Stratech Scientific, UK). Ece1p peptides were synthesized commercially (Proteogenix (France) or Peptide Synthetics (UK). ECE1 deletion was performed as previously described35. Deletion cassettes were generated by PCR36. Primers ECE1-FG and ECE1-RG were used to amplify pFA-HIS1 and pFA-ARG4 -based markers. C. albicans BWP17 (ref. 37), was sequentially transformed38 with the ECE1-HIS1 and ECE1-ARG4 deletion cassettes and then transformed with CIp10 (ref. 39), yielding the ece1∆/Δ deletion strain. For complementation, the ECE1 gene plus upstream and downstream intergenic regions were amplified with primers ECE1-RecF3k and ECE1-RecR and cloned into plasmid CIp10 at MluI and SalI sites. This plasmid was transformed into the uridine auxotrophic ece1Δ/Δ strain, yielding the ece1∆/Δ + ECE1 complemented strain. For generation of the ece1Δ/Δ + ECE1 strain, the CIp10-ECE1 was amplified with primers Pep3-F1 and Pep3-R1, digested with ClaI and re-ligated, yielding the CIp10 + ECE1 plasmid. This plasmid was transformed into the uridine auxotrophic ece1Δ/Δ strain, yielding the ece1Δ/Δ + ECE1 strain. All integrations were confirmed by PCR/sequencing and at least two independent isogenic transformants were created to confirm results. KEX1 deletion was performed exactly as the ECE1 deletion but using primers KEX1-FG and KEX1-RG for creating the deletion cassette. Fluorescent strains of ece1Δ/Δ and BWP17 were constructed as previously described40. Briefly, the ece1Δ/Δ and BWP17 strains were transformed with the pENO1-dTom-NATr plasmid. Primers used to clone and construct the ECE1 genes and intragenic regions are listed in Extended Data Table 4. Strains are listed in Extended Data Table 2. ECE1 promoter (primers 5′ECE1prom–NarI / 3′ECE1prom–XhoI) and terminator (5′ECE1term–SacII / 5′ECE1term–SacI) were amplified and cloned into pADH1-GFP. Resulting pSK-pECE1-GFP was verified by sequencing. C. albicans SC5314 was transformed with the pECE1-GFP transformation cassette38. Resistance to nourseothricin was used as selective marker and correct integration of GFP into the ECE1 locus was verified by PCR. Primers for cloning and validation are listed in Extended Data Table 4. Strains are listed in Extended Data Table 2. C. albicans cells grown on TR146 epithelial cells were collected into RNA pure (PeqLab), centrifuged and the pellet resuspended in 400 μl AE buffer (50 mM Na-acetate pH 5.3, 10 mM EDTA, 1% SDS). Samples were vortexed (30 s), and an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and incubated for 5 min (65 °C) before subjected to 2× freeze-thawing. Lysates were clarified by centrifugation and the RNA precipitated with isopropyl alcohol/0.3 M sodium acetate by incubating for 1 h at −20 °C. Precipitated pellets were washed (2× 1 ml 70% ice-cold ethanol), resuspended in DEPC-treated water and stored at −80 °C. RNA integrity and concentration was confirmed using a Bioanalyzer (Agilent). RNA (500 ng) was treated with DNase (Epicentre) and cDNA synthesized using Reverse Transcriptase Superscript III (Invitrogen). cDNA samples were used for qPCR with EVAgreen mix (Bio&Sell). Primers (ACT1-F and ACT1-R for actin, ECE1-F and ECE1-R for ECE1 Extended Data Table 4) were used at a final concentration of 500 nM. qPCR amplifications were performed using a Biorad CFX96 thermocycler. Data was evaluated using Bio-Rad CFX Manager 3.1 (Bio-Rad) with ACT1 as the reference gene and t as the control sample. TR146 cells were lysed using a modified RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease (Sigma-Aldrich) and phosphatase (Perbio Science) inhibitors41, left on ice (30 min) and then clarified (10 min) in a refrigerated microfuge. Lysate total protein content was determined using the BCA protein quantitation kit (Perbio Science). 20 μg of total protein was separated on 12% SDS–PAGE gels before transfer to nitrocellulose membranes (GE Healthcare). After probing with primary (1:1,000) and secondary (1:10,000) antibodies, membranes were developed using Immobilon chemiluminescent substrate (Millipore) and exposed to X-ray film (Fuji film). Human α-actin was used as a loading control. DNA binding activity of transcription factors was assessed using the TransAM transcription factor ELISA system (Active Motif) as previously described41, 42. Serum-starved TR146 epithelial cells were treated for 3 h before being differentially lysed to recover nuclear proteins using a nuclear protein extraction kit (Active Motif) according to the manufacturer’s protocol. Protein concentration was determined (BCA protein quantitation kit (Perbio Science)) and 5 μg of nuclear extract was assayed in the TransAM system according to the manufacturer’s protocol. Data was expressed as fold-change in A relative to resting cells. Cytokine levels in cell culture supernatants were determined using the Performance magnetic Fluorokine MAP cytokine multiplex kit (Bio-techne) and a Bioplex 200 machine. The data were analysed using Bioplex Manager 6.1 software to determine analyte concentrations. Following incubation, culture supernatant was collected and assayed for lactate dehydrogenase (LDH) activity using the Cytox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) according to the manufacturer’s instructions. Recombinant porcine LDH (Sigma-Aldrich) was used to generate a standard curve. Quantification of C. albicans adherence to TR146 epithelial cells was performed as described previously43. Briefly, TR146 cells were grown to confluence on glass coverslips for 48 h in tissue culture plates in DMEM medium. C. albicans yeast cells (2 × 105) were added into 1 ml serum-free DMEM, incubated for 60 min (37 °C/5% CO ) and non-adherent C. albicans cells removed by aspiration. Following washing (3× 1 ml PBS), cells were fixed with 4% paraformaldehyde (Roth) and adherent C. albicans cells stained with Calcofluor White and quantified using fluorescence microscopy. The number of adherent cells was determined by counting 100 high-magnification fields of 200 μm × 200 μm size. Exact total cell numbers were calculated based on the quantified areas and the total size of the cover slip. C. albicans invasion of epithelial cells was determined as described previously43. Briefly, TR146 epithelial cells were grown to confluence on glass coverslips for 48 h and then infected with C. albicans yeast cells (1 × 105), for 3 h in a humidified incubator (37 °C/5% CO ). Following washing (3× PBS), the cells were fixed with 4% paraformaldehyde. All surface adherent fungal cells were stained for 1 h with a rabbit anti-Candida antibody and subsequently with a goat anti-rabbit-Alexa Fluor 488 antibody. After rinsing with PBS, epithelial cells were permeabilized (0.1% Triton X-100 in PBS for 15 min) and fungal cells (invading and non-invading) were stained with Calcofluor White. Following rinsing with water, coverslips were visualized using fluorescence microscopy. The percentage of invading C. albicans cells was determined by dividing the number of (partially) internalized cells by the total number of adherent cells. At least 100 fungal cells were counted on each coverslip. TR146 cells (105 per ml) seeded on glass coverslips in DMEM/10% FBS were infected with C. albicans (2.5 × 104 cfu per ml) in DMEM and incubated for 6 h (37 °C/5% CO ). Cells were washed with PBS, fixed overnight (4 °C in 4% paraformaldehyde) and stained with Concanavalin A-Alexa Fluor 647 in PBS (10 μg ml−1) for 45 min at room temperature in the dark with gentle shaking (70 r.p.m.) to stain the fungal cell wall. Epithelial cells were permeabilized with 0.1% Triton X-100 for 15 min at 37 °C in the dark, then washed and stained with 10 μg ml−1 Calcofluor White (0.1 M Tris-HCl pH 9.5) for 20 min at room temperature in the dark with gentle shaking. Cells were rinsed in water and mounted on slides with 6 μl of ProLong Gold anti-fade reagent, before air drying for 2 h in the dark. Fluorescence microscopy was performed on a Zeiss Axio Observer Z1 microscope, and 5 phase images were taken per picture. For scanning electron microscopy (SEM) analysis, TR146 cells were grown to confluence on Transwell inserts (Greiner) and serum starved overnight in serum-free DMEM. After 5 h of C. albicans incubation on epithelial cells at an MOI of 0.01, cell media was removed and samples were fixed overnight at 4 °C with 2.5% (v/v) glutaraldehyde in 0.05 M HEPES buffer (pH 7.2) and post-fixed in 1% (w/v) osmium tetroxide for 1 h at room temperature. After washing, samples were dehydrated through a graded ethanol series before being critical point dried (Polaron E3000, Quorum Technologies). Dried samples were mounted using carbon double side sticky discs (TAAB) on aluminium pins (TAAB) and gold coated in an Emitech K550X sputter coater (Quorum Technologies Ltd). Samples were examined and images recorded using a FEI Quanta 200 field emission scanning electron microscope operated at 3.5 kV in high vacuum mode. Zebrafish infections were performed in accordance with NIH guidelines under Institutional Animal Care and Use Committee (IACUC) protocol A2009-11-01 at the University of Maine. To determine sample size, a power calculation was done for all experiments based on two-tailed t-tests in order to detect a minimum effect size of 0.8, with an alpha error probability of 0.05 and a power (1 – beta error probability) of 0.95. This gave a minimum number of 42 fish for each group. The fish selected for the experiments were randomly assigned to the different groups by picking them from a pool without bias and the groups were injected in different orders. No blinding was used to read the results. Ten to twenty zebrafish per group per experiment were maintained at 33 °C in E3 + PTU and used as previously described40. Briefly, 4 days post-fertilization (dpf) larvae were treated with 20 μg ml−1 dexamethasone dissolved in 0.1% DMSO 1 h before infection and thereafter. For tissue damage and neutrophil recruitment, individual AB or mpo:GFP fish (respectively) were injected into the swimbladder with 4 nl of PBS with or without 25–40 C. albicans yeast cells of ece1Δ/Δ-dTomato, ece1Δ/Δ + ECE1 + dTomato, ece1Δ/Δ + ECE1  + dTomato or BWP17-dTomato. For tissue damage, 1 nl of Sytox green (0.05 mM in 1% DMSO) was injected at 20 h post-infection into the swimbladder and fish were imaged by confocal microscopy at 24 h post-infection. For neutrophil recruitment, fish were imaged at 24 h post-injection. For synthetic peptide damage, AB or α-catenin:citrine44 fish were injected with 2 nl of peptide (9 ng or 1.25 ng per fish) or vehicle (40% DMSO or 5% DMSO) + SytoxGreen (0.05 mM in 1% DMSO) or SytoxOrange (0.5 mM in 10% DMSO) and the fish imaged by confocal microscopy 4 h later. Numbers of neutrophils and damaged cells observed were counted and tabulated for each fish. Live zebrafish imaging was carried out as previously described40. Briefly, fish were anaesthetized in Tris-buffered Tricaine (200 μg ml−1, Western Chemicals) and further immobilized in a solution of 0.4% low-melting-point agarose (LMA, Lonza) in E3 + Tricaine in a 96-well plate glass-bottom imaging dish (Greiner Bio-On). Confocal imaging was carried out using an Olympus IX-81 inverted microscope with an FV-1000 laser scanning confocal system (Olympus). Images were collected and processed using Fluoview (Olympus) and Photoshop (Adobe Systems). Panels are either a single slice for the differential interference contrast channel (DIC) with maximum projection overlays of fluorescence image channels (red-green), or maximum projection overlays of fluorescence channels. The number of slices for each maximum projection is specified in the legend of individual figures. Murine infections were performed under UK Home Office Project Licence PPL 70/7598 in dedicated animal facilities at King’s College London. No statistical method was used to pre-determine sample size. No method of randomization was used to allocate animals to experimental groups. Mice in the same cage were part of the same treatment. The investigators were not blinded during outcome assessment. A previously described murine model of oropharyngeal candidiasis using female BALB/c mice45 was modified to use for investigating early infection events. Briefly, mice were treated subcutaneously with 3 mg per mouse (in 200 μl PBS with 0.5% Tween 80) of cortisone acetate on days −1 and +1 post-infection. On day 0, mice were sedated for ~75 min with an intra-peritoneal injection of 110 mg per kg ketamine and 8 mg per kg xylazine, and a swab soaked in a 107 cfu per ml of C. albicans yeast culture in sterile saline was placed sublingually for 75 min. After 2 days, mice were euthanized, the tongue excised and divided longitudinally in half. One half was weighed, homogenized and cultured to derive quantitative Candida counts. The other half was processed for histopathology and immunohistochemistry. C. albicans-infected murine tongues were fixed in 10% (v/v) formal-saline before being embedded and processed in paraffin wax using standard protocols. For each tongue, 5-μm sections were prepared using a Leica RM2055 microtome and silane coated slides. Sections were dewaxed using xylene, before C. albicans and infiltrating inflammatory cells were visualized by staining using Periodic Acid-Schiff (PAS) stain and counterstaining with haematoxylin. Sections were then examined by light microscopy. Histological quantification of infection was undertaken by measuring the area of infected epithelium and expressed as a percentage relative to the entire epithelial area. TR146 epithelial cells were grown in 35-mm Petri dishes (Nunc) for 48 h before recordings at low cell density (10–30% confluence). Cells were superfused with a modified Krebs solution (120 mM NaCl, 3 mM KCl, 2.5 mM CaCl , 1.2 mM MgCl , 22.6 mM NaHCO , 11.1 mM glucose, 5 mM HEPES pH 7.4). Isolated cells were recorded at room temperature (21–23 °C) in whole cell mode using microelectrodes (5–7 MΩ) containing 90 mM potassium acetate, 20 mM KCl, 40 mM HEPES, 3 mM EGTA, 3 mM MgCl , 1 mM CaCl (free Ca2+ 40 nM), pH 7.4. Cells were voltage clamped at −60 mV using an Axopatch 200A amplifier (Axon Instruments) and current/voltage curves were generated by 1 s steps between −100 to +50 mV. Treatments were applied to the superfusate to produce the final required concentration, with vehicle controls similarly applied. Data was recorded using Clampex software (PClamp 6, Axon Instrument) and analysed with Clampfit 10. TR146 cells were grown in a 96-well plate overnight until confluent. The medium was removed and 50 μl of a Fura-2 solution (5 μl Fura-2 (Life Technologies) (2.5 mM in 50% Pluronic F-127 (Life Technologies):50% DMSO), 5 μl probenecid (Sigma) in 5 ml saline solution (NaCl (140 mM), KCl (5 mM), MgCl (1 mM), CaCl (2 mM), glucose (10 mM) and HEPES (10 mM), adjusted to pH 7.4)) was added and the plate incubated for 1 h at 37 °C/5% CO . The Fura-2 solution was replaced with 50 μl saline solution and baseline fluorescence readings (excitation 340 nm/emission 520 nm) taken for 10 min using a FlexStation 3 (Molecular Devices). Ece1 peptides were added at different concentrations and readings immediately taken for up to 3 h. The data was analysed using Softmax Pro software to determine calcium present in the cell cytosol and expressed as the ratio between excitation and emission spectra. tBLMs with 10% tethering lipids and 90% spacer lipids (T10 slides) were formed using the solvent exchange technique46, 47 according to the manufacturer’s instructions (SDx Tethered Membranes Pty Ltd, Sydney, Australia). Briefly, 8 μl of 3 mM lipid solutions in ethanol were added, incubated for 2 min and then 93.4 μl buffer (100 mM KCl, 5 mM HEPES, pH 7.0) was added. After rinsing 3× with 100 μl buffer the conductance and capacitance of the membranes were measured for 20 min before injection of Ece1 peptides at different concentrations. All experiments were performed at room temperature. Signals were measured using the tethaPod (SDx Tethered Membranes Pty, Sydney, Australia). Intercalation of Ece1 peptides into phospholipid liposomes was determined by FRET spectroscopy applied as a probe-dilution assay48. Phospholipids mixed with each 1% (mol/mol) of the donor dye NBD-phosphatidylethanolamine (NBD-PE) and of the acceptor dye rhodamine-PE, were dissolved in chloroform, dried, solubilized in 1 ml buffer (100 mM KCl, 5 mM HEPES, pH 7.0) by vortexing, sonicated with a titan tip (30 W, Branson sonifier, cell disruptor B15), and subjected to three cycles of heating to 60 °C and cooling down to 4 °C, each for 30 min. Lipid samples were stored at 4 °C for at least 12 h before use. Ece1 peptide was added to liposomes and intercalation was monitored as the increase of the quotient between the donor fluorescence intensity I at 531 nm and the acceptor intensity I at 593 nm (FRET signal) independent of time. CD measurements were performed using a Jasco J-720 spectropolarimeter (Japan Spectroscopic Co., Japan), calibrated as described previously49. CD spectra represent the average of four scans obtained by collecting data at 1 nm intervals with a bandwidth of 2 nm. The measurements were performed in 100 mM KCl, 5 mM HEPES, pH 7.0 at 25 °C and 40 °C in a 1.0 mm quartz cuvette. The Ece1-III concentration was 15 μM. Planar lipid bilayers were prepared using the Montal-Mueller technique50 as described previously51. All measurements were performed in 5 mM HEPES, 100 mM KCl, pH 7.0 (specific electrical conductivity 17.2 mS per cm) at 37 °C. Candida strains were cultured for 18 h in hyphae inducing conditions (YNB medium containing 2% sucrose, 75 mM MOPSO buffer pH 7.2, 5 mM N-acetyl-d-glucosamine, 37 °C). Hyphal supernatants were collected by filtering through a 0.2 μm PES filter, and peptides were enriched by solid phase extraction (SPE) using first C4 and subsequently C18 columns on the C4 flowthrough. After drying in a vacuum centrifuge, samples were resolubilized in loading solution (0.2% formic acid in 71:27:2 ACN/H O/DMSO (v/v/v)) and filtered through a 10 kDa MWCO filter. The filtrate was transferred into HPLC vials and injected into the LC-MS/MS system. LC-MS/MS analysis was carried out on an Ultimate 3000 nano RSLC system coupled to a QExactive Plus mass spectrometer (ThermoFisher Scientific). Peptide separation was performed based on a direct injection setup without peptide trapping using an Accucore C4 column as stationary phase and a column oven temperature of 50 °C. The binary mobile phase consisting of A) 0.2% (v/v) formic acid in 95:5 H O/DMSO (v/v) and B) 0.2% (v/v) formic acid in 85:10:5 ACN/H O/DMSO (v/v/v) was applied for a 60 min gradient elution: 0–1.5 min at 60% B, 35–45 min at 96% B, 45.1–60 min at 60% B. The Nanospray Flex Ion Source (ThermoFisher Scientific) provided with a stainless steel emitter was used to generate positively charged ions at 2.2 kV spray voltage. Precursor ions were measured in full scan mode within a mass range of m/z 300–1600 at a resolution of 70k FWHM using a maximum injection time of 120 ms and an automatic gain control target of 1e6. For data-dependent acquisition, up to 10 most abundant precursor ions per scan cycle with an assigned charge state of z = 2–6 were selected in the quadrupole for further fragmentation using an isolation width of m/z 2.0. Fragment ions were generated in the HCD cell at a normalized collision energy of 30 V using nitrogen gas. Dynamic exclusion of precursor ions was set to 20 s. Fragment ions were monitored at a resolution of 17.5k (FWHM) using a maximum injection time of 120 ms and an AGC target of 2e5. Thermo raw files were processed by the Proteome Discoverer (PD) software v1.4.0.288 (Thermo). Tandem mass spectra were searched against the Candida Genome Database (http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly22/current/C_albicans_SC5314_A22_current_orf_trans_all.fasta.gz; status: 2015/05/03) using the Sequest HT search algorithm. Mass spectra were searched for both unspecific cleavages (no enzyme) and tryptic peptides with up to 4 missed cleavages. The precursor mass tolerance was set to 10 p.p.m. and the fragment mass tolerance to 0.02 Da. Target Decoy PSM Validator node and a reverse decoy database was used for (q value) validation of the peptide spectral matches (PSMs) using a strict target false discovery (FDR) rate of <1%. Furthermore, we used the score versus charge state function of the Sequest engine to filter out insignificant peptide hits (xcorr of 2.0 for z = 2, 2.25 for z = 3, 2.5 for z = 4, 2.75 for z = 5, 3.0 for z = 6). At least two unique peptides per protein were required for positive protein hits. TransAM and patch clamp data were analysed using a paired t-test while cytokines, LDH and calcium influx data were analysed using one-way ANOVA with all compared groups passing an equal variance test. Murine in vivo data was analysed using the Mann–Whitney test. Zebrafish data was analysed using the Kruskal–Wallis test with Dunn’s multiple comparison correction. In all cases, P < 0.05 was taken to be significant.


No statistical methods were used to predetermine sample size. The experiments were not randomized, and the investigators were not blinded to allocation during experiments and outcome assessment. Wild-type and mutant versions of human DDB1 (Q16531), human CRBN (Q96SW2), human CK1α (P48729) and human IKZF1 (Q13422) were subcloned into pAC-derived vectors29 and recombinant proteins expressed as N-terminal His , StrepII or StrepII-Avi fusions in Trichoplusia ni High-Five insect cells using the baculovirus expression system (Invitrogen). For purification of His -DDB1-His -CRBN, His -DDB1∆BPB-StrepII-CRBN∆1–40 and His -DDB1∆BPB-His -CRBN, cells were resuspended in buffer containing 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH 8.0, 200 mM NaCl, 0.25 mM tris(2-carboxyethyl)phosphine (TCEP), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1× protease inhibitor cocktail (Sigma-Aldrich) and lysed by sonication. Cells expressing StrepII-Avi-CK1α or truncated versions of StrepII-Avi-IKZF1 (Δ256–519, Δ197–238/Δ256–519 and Δ1–82/Δ197–238/Δ256–519; full-length IKZF1 forms aggregates during purification) were lysed in the presence of 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.25 mM TCEP, 1 mM PMSF and 1× protease inhibitor cocktail (Sigma-Aldrich). Following ultracentrifugation, the soluble fraction was passed over Strep-Tactin Sepharose (IBA) or His-Select nickel affinity resin (Sigma-Aldrich) and following elution, affinity tags were removed from CK1α and IKZF1 by overnight TEV protease treatment as indicated. The affinity-purified protein was further purified via ion exchange chromatography (Poros 50HQ and 50HS) and subjected to size-exclusion chromatography in 50 mM HEPES pH 7.4, 200 mM NaCl and 0.25 mM TCEP. The protein-containing fractions were concentrated using ultrafiltration (Millipore) and flash frozen (DDB1–CRBN constructs at 40–120 μM, TEV cleaved CK1α at ~280 μM, StrepII-Avi-CK1α at ~50 μM, TEV cleaved IKZF1 at ~350 μM and StrepII-Avi-IKZF1 at ~150 μM). Proteins were stored at −80 °C. Attempts to crystallize CK1α, lenalidomide and CRBN with the full-length DDB1 adaptor protein were unsuccessful. Because the WD40 β-propeller B (BPB) of DDB1 is not involved in CRBN binding, we generated a human DDB1 construct in which a GNGNSG-linker replaced the BPB domain (residues 396–705; DDB1∆BPB). For crystallization of the DDB1∆BPB–CRBN–lenalidomide–CK1α complex, His DDB1∆BPB–StrepII–CRBN∆1–40 at 70 μM was mixed with lenalidomide at 80 μM before the addition of TEV cleaved full-length CK1α at 80 μM. The mixture was incubated on ice for 1 h and subsequently centrifuged at 20,000g for 30 min at 4 °C. Crystallization plates were set up and stored at room temperature. Crystals appeared within 3 days after mixing the protein solution 1:1 with the reservoir containing 70 mM Tris pH 7.0, 140 mM MgCl and 7% (w/v) PEG 8000 and continued growing until day 13 in MRC 2 Well Crystallization format vapour diffusion plates (Swissci). Crystals were cryo-protected in reservoir solution supplemented with 20% ethylene glycol and flash-cooled in liquid nitrogen. Diffraction data were collected at the Swiss Light Source (beamline PXII) with a Pilatus 6M detector at the wavelength of the Zn-edge (1.28162 Å) and a temperature of 100 K. Data were indexed and integrated using XDS30 and scaled using AIMLESS supported by other programs of the CCP4 suite31. The optimal high-resolution cut-off (2.45 Å) was determined based on the half-set correlation criterion32 (CC  = 0.45 for the highest resolution shell). Data processing statistics, refinement statistics and model quality parameters are provided in Extended Data Table 1. The DDB1∆BPB–CRBN–lenalidomide–CK1α quaternary complex crystallized in space group P1 with two complexes in the unit cell. PHASER33 was used to determine the structure by molecular replacement using a crystallographic model of DDB1 omitting the BPB domain, a CK1α homology model generated with Modeller34 based on a CK1δ crystal structure (PDB entry 4TWC), and human CRBN (PDB entry 4TZ4) as search models. The initial model was iteratively improved with COOT and refined using PHENIX.REFINE35 and autoBUSTER36, with ligand restraints generated by Grade server (Global Phasing). Figures were generated with PyMOL (The PyMOL Molecular Graphics System, Version 1.7.2.2 Schrödinger, LLC) and model quality was assessed with MOLPROBITY37. Interaction surfaces were determined with PISA38. The IKZF1 homology model was calculated using Modeller based on a multiple sequence alignment with the experimental zinc-finger structures of Aart, YY1 and Kasio (PDB entries 2I13, 1UBD, 2LT7). Purified human NEDD8(M1C) (NEDD8) was incubated with DTT (8 mM) at 4 °C for 1 h. DTT was removed using a S200 16/60 gel filtration column in a buffer containing 50 mM Tris pH 7.3 and 150 mM NaCl. Alexa-488–C5-maleimide (Invitrogen) was dissolved in 100% DMSO and mixed with NEDD8 to achieve fourfold molar excess of Alexa-488–C5-maleimide. NEDD8 labelling was carried out at room temperature for 3 h in a vacuum desiccator and stored overnight at 4 °C. Labelled NEDD8 was purified on a S200 16/60 gel filtration column in 50 mM Tris pH 7.5, 150 mM NaCl, 0.25 mM TCEP and 10% (v/v) glycerol, concentrated by ultrafiltration (Millipore), flash frozen (~40–80 μM) in liquid nitrogen and stored at −80 °C. In vitro CRL4CRBN reconstitution and CUL4A neddylation was performed as described4, 19, 21. His –CUL4A–His –RBX1 at 3.5 μM was incubated with His –DDB1–His –CRBN at 1.5 μM (wild-type or mutant forms) in a reaction mixture containing 3.8 μM Alexa-488–NEDD8, 50 nM NAE1/UBA3 (E1), 150 nM UBC12 (E2), 1 mM ATP, 50 mM Tris pH 7.5, 100 mM NaCl, 2.5 mM MgCl , 0.5 mM DTT and 5% (v/v) glycerol for 2 h at room temperature. Gel filtration purified neddylated CRL4CRBN ( CRL4CRBN) was concentrated to 2–4 μM, flash frozen and stored at −80 °C. Alexa-488–NEDD8 labelling efficiency of different CRL4CRBN mutant complexes was determined by measuring total fluorescent intensity at equal concentration (excitation at 490 nm, emission at 540 nm) using a Safire2 microplate reader from Tecan (Extended Data Fig. 4f). Purified StrepII-Avi-tagged CK1α or IKZF1 were biotinylated in vitro at a concentration of 25–50 μM by incubation with final concentrations of 2.5 μM BirA enzyme and 0.2 mM D-Biotin in 50 mM HEPES pH 7.4, 200 mM NaCl, 10 mM MgCl , 0.25 mM TCEP and 20 mM ATP. The reaction was incubated for 1 h at room temperature and stored at 4 °C for 14–16 h. Biotinylated proteins were purified by gel filtration chromatography and stored at −80 °C (StrepII-Avi-CK1α at ~25 μM, StrepII-Avi-IKZF1 at ~40 μM). Increasing concentrations of Alexa-488–NEDD8-labelled CRL4CRBN ( CRL4CRBN) were added to pre-mixed biotinylated IKZF1 at 80 nM or CK1α at 100 nM, terbium-coupled streptavidin at 4 nM (Invitrogen) and IMiDs or 2′-deoxyuridine at 5 μM (final concentrations) in 384-well microplates (Greiner, 784076) in a buffer-containing 50 mM Tris pH 7.5, 100 mM NaCl, 0.1% pluronic acid and 1% DMSO (see also figure legends). Before TR-FRET measurements were conducted, the reactions were incubated for 15 min at room temperature. After excitation of terbium (Tb) fluorescence at 337 nm, emission at 490 nm (Tb) and 520 nm (Alexa 488) were recorded with a 70 μs delay to reduce background fluorescence and the reaction was followed over 1 h by recording 60 technical replicates of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio. Data were analysed with GraphPad Prism 6 assuming equimolar binding of the probe to the receptor ( CRL4CRBN) using the following equations: The concentration of the receptor in the bound state, [C ], can be calculated for that setting by the law of mass action: K is the equilibrium constant for the dissociation, and [C ] and [C ] are the total concentrations of the probe and the receptor, respectively. The K value can be calculated from the change in the fluorescence intensity, FI, observed by a titration of the receptor at constant probe concentrations according to: FI is the observed fluorescence intensity, and FI and FI the fluorescence intensities of the probe in its free and its bound states, respectively. The assay window is described by the overall change in the fluorescence intensity, (FI  − FI ). Counter titrations with unlabelled proteins were carried out by mixing CRL4CRBN at 0.5–1 μM with 200 nM biotinylated CK1α or 160 nM biotinylated IKZF1 in the presence of 8 nM terbium-coupled streptavidin and IMiDs at 10–20 μM. After 15 min incubation on ice, increasing amounts of unlabelled DDB1∆BPB–CRBN (0.04–40 μM) or IKZF1 (0.04–20 μM; wild-type or mutant forms or pre-incubated with consensus or control DNA as stated in the EMSA section) were added to the pre-assembled CRL4CRBN–CK1α/IKZF1 complexes in a 1:1 volume ratio and incubated for 5 min at room temperature. IMiD titrations were carried out by premixing CRL4CRBN, CK1α/IKZF1 and 8 nM terbium-coupled streptavidin before addition of increasing concentrations of each IMiD (0.005–5 μM) in a 1:1 volume ratio (see figure legends for final concentrations). The 520/490 nm ratios were plotted to calculate the half maximal effective concentrations (EC ) or half maximal inhibitory concentrations (IC ) assuming a single binding site using GraphPad Prism 6. IC values were converted to the respective K as described39. Three biological replicates were carried out per experiment. The standard deviation was derived from the sum of the mean absolute error of 10 technical replicates (per data point and replicate) and the standard deviation of the biological replicates. Cy5-conjugated thalidomide4 (10 nM) was mixed with increasing concentrations of either wild-type or mutant forms of purified DDB1–CRBN (0.004–2 μM) in a 384-well microplate (Greiner, 784076) and incubated for 30 min at room temperature. CRBN–IMiD interactions were measured in 50 mM Tris pH 7.5, 100 mM NaCl, 0.1% pluronic acid and 1% DMSO by change in fluorescence polarization using a PHERAstar FS microplate reader (BMG Labtech). The Cy5-thalidomide bound fraction was calculated as described40. Data were plotted and analysed using GraphPad Prism 6 assuming a single IMiD binding site on CRBN. Hct116 cells were purchased from The European Collection of Cell Cultures (ECACC, Sigma-Aldrich), immediately used for experiments and regularly tested for mycoplasma contamination. Cells were cultured in L-lysine and L-arginine free DMEM supplemented with unlabelled L-lysine, L-arginine, 10% FBS and 2 mM L-glutamine. Cells were grown to approximately 50% confluency and the medium was exchanged for DMEM supplemented with 13C,15N-labelled L-lysine (Lys8) and 13C,15N-labelled L-arginine (Arg10) containing lenalidomide at 30 μM or equivalent amounts of DMSO as control. Cells were incubated for 16 h and harvested for mass spectrometry analysis in 0.5 M Tris-HCl pH 8.6, 6 M guanidine hydrochloride, reduced in 16 mM TCEP for 30 min, and alkylated in 35 mM iodoacetamide for 30 min in the dark. The proteins were digested at 37 °C with lysyl endopeptidase (Wako) after dilution to ~2 M guanidine hydrochloride (with 50 mM Tris-HCl pH 7.3, 5 mM CaCl buffer) for 6 h, and after dilution to <1 M guanidine hydrochloride with trypsin (Promega) at 37 °C overnight. The resulting peptides were desalted using C solid state extraction cartridges and offline fractionated into 36 fractions by basic reverse phase chromatography. The 36 fractions were recombined to a final of 12 samples. Generated peptides were separated on an EASY n-LC 1000 liquid chromatography system equipped with a C EASY-Spray column coupled to an Orbitrap Fusion mass spectrometer (all from Thermo Scientific). Maxquant41 was used for .RAW file processing and controlling peptide and protein level false-discovery rates, assembling proteins from peptides, and protein quantification from peptides. Peptides were searched against a human Uniprot database with both forward and reverse sequences. For analysis, we did not utilize the SILAC component of the experiment, but instead used the Maxquant LFQ algorithm to quantify the relative abundance of casein kinase isoforms across three independent replicates. In vitro ubiquitination was performed by mixing wild-type or mutant CRL4CRBN at 70 nM with a reaction mixture containing IMiDs at 350 nM or 10 μM, CK1α at 500 nM, E1 (UBA1, BostonBiochem) at 40 nM, E2 (UBCH5a, BostonBiochem) at 1 μM, wild-type (20 μM) or lysine-free (10 μM) ubiquitin as indicated. Reactions were carried out in 50 mM Tris pH 7.5, 30 mM NaCl, 5 mM MgCl2, 0.2 mM CaCl , 1 mM ATP, 0.1% Triton X-100 and 0.1 mg ml−1 BSA, incubated for 15–30 min at 30 °C and analysed by western blot using anti-CK1α (abcam, ab108296, 1:20,000) and anti-rabbit IRDye 800CW antibodies (LI-COR, 926-32211, 1:10,000). Blots were scanned on a LI-COR Odyssey infrared imaging system. To identify target lysines following in vitro ubiquitination of CK1α in the presence or absence of lenalidomide, samples were precipitated with 20% (v/v) trichloroacetic acid followed by several acetone washes. The precipitated protein was dissolved in 10 μl of 500 mM Tris pH 8.6, 6 M guanidine hydrochloride and 8 mM TCEP and incubated with 18 mM iodoacetamide for 30 min at room temperature in the dark. After addition of 50 μl 50 mM Tris pH 7.4, 5 mM CaCl , samples were incubated with 200 ng trypsin at 37 °C for 12–14 h. Mass spec was carried out on a FUSION Orbitrap, the data was searched with MASCOT, site probabilities were calculated with ASCOR and peak integration for relative quantification was performed using ProgenesisLC. Equimolar ratios of complementary DNA single strands at 200 mM were mixed in 10 mM Tris pH 8.0, 50 mM NaCl and 1 mM MgCl , incubated at 95 °C for 2 min and annealed by slowly lowing temperature using a thermal cycler. IKZF1 was mixed with duplex DNA (consensus DNA: 5′-TCAGAAAAAGGGAATTCCGTCAC-3′; control DNA: 5′-TCAGACACTTTTGGTACTGTCAC-3′) in 50 mM HEPES pH 7.4, 200 mM NaCl, 0.25 mM TCEP and 10% (v/v) glycerol and incubated on ice for 15 min before the addition of DDB1–CRBN and pomalidomide, as indicated. Binding reactions were incubated for 30 min at room temperature, applied to a 4–16% NativePAGE Novex Bis-Tris gel (Invitrogen) and separated in 1× NativePAGE buffer at 150 V for 75 min at room temperature. Gels were stained with 1 μg ml−1 ethidium bromide in 1× NativePAGE buffer followed by Coomassie staining.


WiseGuyReports.Com Publish a New Market Research Report On – “3D Cell Cultures Market 2016 World Technology,Development,Trends and Opportunities Market Research Report to 2021”. This report studies 3D Cell Cultures in Global market, especially in North America, Europe, China, Japan, Southeast Asia and India, focuses on top manufacturers in global market, with Production, price, revenue and market share for each manufacturer, covering  Sigma  Lonza  3D Biomatrix  Ams Bio  Life Technologise  Microtissues  Labome  Tecan  Lena Bio  3D Biotek  Scivax Life Sciences  Corning Incorporated For more information or any query mail at [email protected] Market Segment by Regions, this report splits Global into several key Regions, with production, consumption, revenue, market share and growth rate of 3D Cell Cultures in these regions, from 2011 to 2021 (forecast), like  North America  Europe  China  Japan  Southeast Asia  India Split by product type, with production, revenue, price, market share and growth rate of each type, can be divided into  Membrane Type  Foam / gel Type  Microcarriers Type Split by application, this report focuses on consumption, market share and growth rate of 3D Cell Cultures in each application, can be divided into  Tissue Engineering  Tumor Model  Stem Cell Research  Drug Discovery Global 3D Cell Cultures Market Research Report 2016  1 3D Cell Cultures Market Overview  1.1 Product Overview and Scope of 3D Cell Cultures  1.2 3D Cell Cultures Segment by Type  1.2.1 Global Production Market Share of 3D Cell Cultures by Type in 2015  1.2.2 Membrane Type  1.2.3 Foam / gel Type  1.2.4 Microcarriers Type  1.3 3D Cell Cultures Segment by Application  1.3.1 3D Cell Cultures Consumption Market Share by Application in 2015  1.3.2 Tissue Engineering  1.3.3 Tumor Model  1.3.4 Stem Cell Research  1.3.5 Drug Discovery  1.4 3D Cell Cultures Market by Region  1.4.1 North America Status and Prospect (2011-2021)  1.4.2 Europe Status and Prospect (2011-2021)  1.4.3 China Status and Prospect (2011-2021)  1.4.4 Japan Status and Prospect (2011-2021)  1.4.5 Southeast Asia Status and Prospect (2011-2021)  1.4.6 India Status and Prospect (2011-2021)  1.5 Global Market Size (Value) of 3D Cell Cultures (2011-2021) 2 Global 3D Cell Cultures Market Competition by Manufacturers  2.1 Global 3D Cell Cultures Production and Share by Manufacturers (2015 and 2016)  2.2 Global 3D Cell Cultures Revenue and Share by Manufacturers (2015 and 2016)  2.3 Global 3D Cell Cultures Average Price by Manufacturers (2015 and 2016)  2.4 Manufacturers 3D Cell Cultures Manufacturing Base Distribution, Sales Area and Product Type  2.5 3D Cell Cultures Market Competitive Situation and Trends  2.5.1 3D Cell Cultures Market Concentration Rate  2.5.2 3D Cell Cultures Market Share of Top 3 and Top 5 Manufacturers  2.5.3 Mergers & Acquisitions, Expansion 7 Global 3D Cell Cultures Manufacturers Profiles/Analysis  7.1 Sigma  7.1.1 Company Basic Information, Manufacturing Base and Its Competitors  7.1.2 3D Cell Cultures Product Type, Application and Specification  7.1.2.1 Type I  7.1.2.2 Type II  7.1.3 Sigma 3D Cell Cultures Production, Revenue, Price and Gross Margin (2015 and 2016)  7.1.4 Main Business/Business Overview  7.2 Lonza  7.2.1 Company Basic Information, Manufacturing Base and Its Competitors  7.2.2 3D Cell Cultures Product Type, Application and Specification  7.2.2.1 Type I  7.2.2.2 Type II  7.2.3 Lonza 3D Cell Cultures Production, Revenue, Price and Gross Margin (2015 and 2016)  7.2.4 Main Business/Business Overview  7.3 3D Biomatrix  7.3.1 Company Basic Information, Manufacturing Base and Its Competitors  7.3.2 3D Cell Cultures Product Type, Application and Specification  7.3.2.1 Type I  7.3.2.2 Type II  7.3.3 3D Biomatrix 3D Cell Cultures Production, Revenue, Price and Gross Margin (2015 and 2016)  7.3.4 Main Business/Business Overview  7.4 Ams Bio  7.4.1 Company Basic Information, Manufacturing Base and Its Competitors  7.4.2 3D Cell Cultures Product Type, Application and Specification  7.4.2.1 Type I  7.4.2.2 Type II  7.4.3 Ams Bio 3D Cell Cultures Production, Revenue, Price and Gross Margin (2015 and 2016)  7.4.4 Main Business/Business Overview  7.5 Life Technologise  7.5.1 Company Basic Information, Manufacturing Base and Its Competitors  7.5.2 3D Cell Cultures Product Type, Application and Specification  7.5.2.1 Type I  7.5.2.2 Type II  7.5.3 Life Technologise 3D Cell Cultures Production, Revenue, Price and Gross Margin (2015 and 2016)  7.5.4 Main Business/Business Overview  7.6 Microtissues  7.6.1 Company Basic Information, Manufacturing Base and Its Competitors  7.6.2 3D Cell Cultures Product Type, Application and Specification  7.6.2.1 Type I  7.6.2.2 Type II  7.6.3 Microtissues 3D Cell Cultures Production, Revenue, Price and Gross Margin (2015 and 2016)  7.6.4 Main Business/Business Overview  7.7 Labome  7.7.1 Company Basic Information, Manufacturing Base and Its Competitors  7.7.2 3D Cell Cultures Product Type, Application and Specification  7.7.2.1 Type I  7.7.2.2 Type II  7.7.3 Labome 3D Cell Cultures Production, Revenue, Price and Gross Margin (2015 and 2016)  7.7.4 Main Business/Business Overview  7.8 Tecan  7.8.1 Company Basic Information, Manufacturing Base and Its Competitors  7.8.2 3D Cell Cultures Product Type, Application and Specification  7.8.2.1 Type I  7.8.2.2 Type II For more information or any query mail at [email protected] Wise Guy Reports is part of the Wise Guy Consultants Pvt. Ltd. and offers premium progressive statistical surveying, market research reports, analysis & forecast data for industries and governments around the globe. Wise Guy Reports features an exhaustive list of market research reports from hundreds of publishers worldwide. We boast a database spanning virtually every market category and an even more comprehensive collection of market research reports under these categories and sub-categories.

Loading Cell Cultures GmbH collaborators
Loading Cell Cultures GmbH collaborators