Isolation and characterization of circulating tumor cells using a novel workflow combining the CellSearch® system and the CellCelector™

Circulating tumor cells (CTC) are rare cells which have left the primary tumor to enter the blood stream. Although only a small CTC subgroup is capable of extravasating, the presence of CTCs is associated with an increased risk of metastasis and a shorter overall survival. Understanding the heterogeneous CTC biology will optimize treatment decisions and will thereby improve patient outcome. For this, robust workflows for detection and isolation of CTCs are urgently required. Here, we present a workflow to characterize CTCs by combining the advantages of both the CellSearch^® and the CellCelector? micromanipulation system. CTCs were isolated from CellSearch^® cartridges using the CellCelector? system and were deposited into PCR tubes for subsequent molecular analysis (whole genome amplification (WGA) and massive parallel multigene sequencing). By a CellCelector? screen we reidentified 97% of CellSearch^® SKBR‐3 cells. Furthermore, we isolated 97% of CellSearch^®‐proven patient CTCs using the CellCelector? system. Therein, we found an almost perfect correlation of R² = 0.98 (Spearman's rho correlation, n = 20, p < 0.00001) between the CellSearch^® CTC count (n = 271) and the CellCelector? detected CTCs (n = 252). Isolated CTCs were analyzed by WGA and massive parallel multigene sequencing. In total, single nucleotide polymorphisms (SNPs) could be detected in 50 genes in seven CTCs, 12 MCF‐7, and 3 T47D cells, respectively. Taken together, CTC quantification via the CellCelector? system ensures a comprehensive detection of CTCs preidentified by the CellSearch^® system. Moreover, the isolation of CTCs after CellSearch^® using the CellCelector? system guarantees for CTC enrichment without any contaminants enabling subsequent high throughput genomic analyses on single cell level. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:125–132, 2017     

Genetical and physical assignments of equine microsatellites—first integration of anchored markers in horse genome mapping

Twenty equine microsatellites were isolated from a genomic phage library, and their genetical and physical localization was sought by linkage mapping and fluorescent in situ hybridization (FISH). Nineteen of the markers were found to be polymorphic with, in most cases, heterozygosities exceeding 50%. The markers were mapped in a Swedish reference family for gene mapping, comprising eight half-sib families from Standardbred and Icelandic horse sires. Segregation was analyzed against a set of 35 other markers typed in the pedigree. Thirteen of the microsatellites showed linkage to at least one other marker, with a total of 21 markers being involved in these linkages. In parallel, 18 of the microsatellites could be assigned to their chromosomal region by FISH. These assignments involved eight equine autosomes: ECA1, 2, 4, 6, 9, 10, 15, and 16. The genetical and physical mappings revealed by this study represent a significant extension of the current knowledge of the equine genome map. Received: 24 September 1996 / Accepted: 1 December 1996     

β-Carotene synthesis in isolated chromoplasts from Narcissus pseudonarcissus

A system has been established from isolated intact chromoplasts of Narcissus pseudonarcissus flowers that synthesizes geranylgeraniol, an unknown polyprenoid alcohol, phytoene, and -carotene from [1-¹⁴C]isopentenyl pyrophosphate in a good yeild. Long chain pyrophosphates are not accumulated. San 6706 inhibits the dehydrogenation of phytoene, whereas nicotine does not lead to an accumulation of lycopene. Separation and identification of polyprenoid lipids was performed by HPLC. The properties and advantages of the chromoplast system are discussed.Abbreviations IPP isopentenyl pyrophosphate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - GC gas chromatography - Sau 6706 4-chloro-5-(dimethylamino)-2-,,-(trifluoro-m-tolyl)3(2H)-pyridazinone     

N-Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure

A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.     

MultiRTA: A simple yet reliable method for predicting peptide binding affinities for multiple class II MHC allotypes

Background

The binding of peptide fragments of antigens to class II MHC is a crucial step in initiating a helper T cell immune response. The identification of such peptide epitopes has potential applications in vaccine design and in better understanding autoimmune diseases and allergies. However, comprehensive experimental determination of peptide-MHC binding affinities is infeasible due to MHC diversity and the large number of possible peptide sequences. Computational methods trained on the limited experimental binding data can address this challenge. We present the MultiRTA method, an extension of our previous single-type RTA prediction method, which allows the prediction of peptide binding affinities for multiple MHC allotypes not used to train the model. Thus predictions can be made for many MHC allotypes for which experimental binding data is unavailable.

Results

We fit MultiRTA models for both HLA-DR and HLA-DP using large experimental binding data sets. The performance in predicting binding affinities for novel MHC allotypes, not in the training set, was tested in two different ways. First, we performed leave-one-allele-out cross-validation, in which predictions are made for one allotype using a model fit to binding data for the remaining MHC allotypes. Comparison of the HLA-DR results with those of two other prediction methods applied to the same data sets showed that MultiRTA achieved performance comparable to NetMHCIIpan and better than the earlier TEPITOPE method. We also directly tested model transferability by making leave-one-allele-out predictions for additional experimentally characterized sets of overlapping peptide epitopes binding to multiple MHC allotypes. In addition, we determined the applicability of prediction methods like MultiRTA to other MHC allotypes by examining the degree of MHC variation accounted for in the training set. An examination of predictions for the promiscuous binding CLIP peptide revealed variations in binding affinity among alleles as well as potentially distinct binding registers for HLA-DR and HLA-DP. Finally, we analyzed the optimal MultiRTA parameters to discover the most important peptide residues for promiscuous and allele-specific binding to HLA-DR and HLA-DP allotypes.

Conclusions

The MultiRTA method yields competitive performance but with a significantly simpler and physically interpretable model compared with previous prediction methods. A MultiRTA prediction webserver is available at http://bordnerlab.org/MultiRTA.

     

Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus

Summary A system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.Abbreviations GUS ß-glucuronidase - PPV Plum Pox Virus - BA 6-benzylaminopurine - NPTII neomycin phosphotransferase II - CP coat protein - CaMV Cauliflower Mosaic Virus - P35S 35S promoter - MS Murashige and Skoog - PCR polymerase chain reaction - P/C/I phenol/chloroform/isoamylalcohol - RNase ribonuclease - dNTP deoxyribonucleosidetriphosphate - DMSO dimethyl sulfoxide     

The influence of savanna trees on nutrient,water and light availability and the understorey vegetation

In an East African savanna herbaceous layer productivity and species composition were studied around Acacia tortilis trees of three different age classes, as well as around dead trees and in open grassland patches. The effects of trees on nutrient, light and water availability were measured to obtain an insight into which resources determine changes in productivity and composition of the herbaceous layer. Soil nutrient availability increased with tree age and size and was lowest in open grassland and highest under dead trees. The lower N:P ratios of grasses from open grassland compared to grasses from under trees suggested that productivity in open grassland was limited by nitrogen, while under trees the limiting nutrient was probably P. N:P ratios of grasses growing under bushes and small trees were intermediate between large trees and open grassland indicating that the understorey of Acacia trees seemed to change gradually from a N-limited to a P-limited vegetation. Soil moisture contents were lower under than those outside of canopies of large Acacia trees suggesting that water competition between trees and grasses was important. Species composition of the herbaceous layer under Acacia trees was completely different from the vegetation in open grassland. Also the vegetation under bushes of Acacia tortilis was different from both open grassland and the understorey of large trees. The main factor causing differences in species composition was probably nutrient availability because species compositions were similar for stands of similar soil nutrient concentrations even when light and water availability was different. Changes in species composition did not result in differences in above-ground biomass, which was remarkably similar under different sized trees and in open grassland. The only exception was around dead trees where herbaceous plant production was 60% higher than under living trees. The results suggest that herbaceous layer productivity did not increase under trees by a higher soil nutrient availability, probably because grass production was limited by competition for water. This was consistent with the high plant production around dead trees because when trees die, water competition disappears but the high soil nutrient availability remains. Hence, in addition to tree soil nutrient enrichment, below-ground competition for water appears to be an important process regulating tree-grass interactions in semi-arid savanna.     

3-D Structure of multilaminar lysosomes in antigen presenting cells reveals trapping of MHC II on the internal membranes

In late endosomes and lysosomes of antigen presenting cells major histocompatibility complex class II (MHC II) molecules bind peptides from degraded internalized pathogens. These compartments are called MHC class II compartments (MIICs), and from here peptide-loaded MHC II is transported to the cell surface for presentation to helper T-lymphocytes to generate an immune response. Recent studies from our group in mouse dendritic cells indicate that the MHC class II on internal vesicles of multivesicular late endosomes or multivesicular bodies is the main source of MHC II at the plasma membrane. We showed that dendritic cell activation triggers a back fusion mechanism whereby MHC II from the inner membranes is delivered to the multivesicular bodies' outer membrane. Another type of MIIC in B-lymphocytes and dendritic cells is more related to lysosomes and often appears as a multilaminar organelle with abundant MHC II-enriched internal membrane sheets. These multilaminar lysosomes have a functioning peptide-loading machinery, but to date it is not clear whether peptide-loaded MHC II molecules from the internal membranes can make their way to the cell surface and contribute to T cell activation. To obtain detailed information on the membrane organization of multilaminar lysosomes and investigate possible escape routes from the lumen of this organelle, we performed electron tomography on cryo-immobilized B-lymphocytes and dendritic cells. Our high-resolution 3-D reconstructions of multilaminar lysosomes indicate that their membranes are organized in such a way that MHC class II may be trapped on the inner membranes, without the possibility to escape to the cell surface.     

Structural protein requirements in equine arteritis virus assembly

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order Nidovirales. EAV particles contain seven structural proteins: the nucleocapsid protein N, the unglycosylated envelope proteins M and E, and the N-glycosylated membrane proteins GP(2b) (previously named G(S)), GP(3), GP(4), and GP(5) (previously named G(L)). Proteins N, M, and GP(5) are major virion components, E occurs in virus particles in intermediate amounts, and GP(4), GP(3), and GP(2b) are minor structural proteins. The M and GP(5) proteins occur in virus particles as disulfide-linked heterodimers while the GP(4), GP(3), and GP(2b) proteins are incorporated into virions as a heterotrimeric complex. Here, we studied the effect on virus assembly of inactivating the structural protein genes one by one in the context of a (full-length) EAV cDNA clone. It appeared that the three major structural proteins are essential for particle formation, while the other four virion proteins are dispensable. When one of the GP(2b), GP(3), or GP(4) proteins was missing, the incorporation of the remaining two minor envelope glycoproteins was completely blocked while that of the E protein was greatly reduced. The absence of E entirely prevented the incorporation of the GP(2b), GP(3), and GP(4) proteins into viral particles. EAV particles lacking GP(2b), GP(3), GP(4), and E did not markedly differ from wild-type virions in buoyant density, major structural protein composition, electron microscopic appearance, and genomic RNA content. On the basis of these results, we propose a model for the EAV particle in which the GP(2b)/GP(3)/GP(4) heterotrimers are positioned, in association with a defined number of E molecules, above the vertices of the putatively icosahedral nucleocapsid.