Positive Selection Method Research Articles

Method for selecting sainfoin biotypes

The article is devoted to the assessment of new criteria for the selection of sainfoin biotypes in the selection process at the initial stages of organogenesis. During the laying of the nursery of the source material, the summer sowing of sainfoin was carried out. The seeds of the samples obtained by the method of positive selection from the breeding collections of scientific institutions and wild-growing populations of sainfoin were sown. New criteria for the selection of sainfoin biotypes are based on the assessment of the intensity of root system development and the calculation of the coefficient of the negative effect of drought. The selection of sainfoin families with a powerful root system of plants (a volume of more than 6.5 cm³) and the calculation of the coefficient of the negative effect of drought at a level of no higher than 0.46 allows determining families with a high adaptive potential at the initial stages of plant development. The application of the selection of breeding material according to new criteria will solve the selection problems of forming the resistance of sainfoin plants to extreme hydrothermal conditions and insufficient moisture.

A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen.

Neutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil isolation and maintenance in culture. Here we compare four protocols based on density-gradient and immunomagnetic methods for isolation of murine neutrophils from bone marrow and spleen. Neutrophil isolation was performed using Ficoll 1.077/1.119 g/mL density gradient, Ficoll 1.083/1.090/1.110 g/mL density gradient and immunomagnetic method of negative and positive selection. The different protocols were compared with respect to sample purity, cell viability, yield, and cost. The functionality of isolated neutrophils was checked by NETosis analysis and neutrophil oxidative burst test. Obtained data revealed that given purity/yield/viability/cost ratio the protocol based on cell centrifugation on Ficoll 1.077/1.119 g/mL density gradient is recommended for isolation of neutrophils from bone marrow, whereas immunomagnetic method of positive selection using Dynabeads is recommended for isolation of splenic neutrophils.

CPSS-FAT: A consistent positive sample selection for object detection with full adaptive threshold

Recent CNN-based methods for object detection largely focus on training the backbone of the object detector, neglecting the key part of selecting positive/negative samples. Instead, based on our analyses about the limitations and inconsistency of existing positive sample selection, we propose a novel Consistent Positive Sample Selection (CPSS) method to select the positive samples automatically, which joints Box-IoU and normalized central distance of object-anchor by mutual weighting. Meanwhile, we employ a consistent approach to location quality evaluation for suppressing the false-positive predicted box when inferencing. Furthermore, an auxiliary Full Adaptive Threshold (FAT) post-processing is also adopted according to the objects’ occlusion level to improve the recall ratio. We implement the proposed CPSS-FAT detector using MS COCO 2017 and CityScapes datasets, the comparing results indicate that our approaches are effective and robust to the different objects in an open world. Especially, we achieve 52.2% AP and 43.1% ARS, outperforming most existing detectors.

Post‐translational modification and phenotype

Post‐translational modification and phenotype

Interleukin-33 Induces Neutrophil Extracellular Trap (NET) Formation and Macrophage Necroptosis via Enhancing Oxidative Stress and Secretion of Proatherogenic Factors in Advanced Atherosclerosis.

Interleukin-33 (IL-33) acts as an 'alarmin', and its role has been demonstrated in driving immune regulation and inflammation in many human diseases. However, the precise mechanism of action of IL-33 in regulating neutrophil and macrophage functioning is not defined in advanced atherosclerosis (aAT) patients. Further, the role of IL-33 in neutrophil extracellular trap (NET) formation in aAT and its consequent effect on macrophage function is not known. In the present study, we recruited n = 52 aAT patients and n = 52 control subjects. The neutrophils were isolated from both groups via ficoll/percoll-based density gradient centrifugation. The effect of IL-33 on the NET formation ability of the neutrophils was determined in both groups. Monocytes, isolated via a positive selection method, were used to differentiate them into macrophages from each of the study subjects and were challenged by IL-33-primed NETs, followed by the measurement of oxidative stress by calorimetric assay and the expression of the proinflammatory molecules by quantitative PCR (qPCR). Transcript and protein expression was determined by qPCR and immunofluorescence/ELISA, respectively. The increased expression of IL-33R (ST-2) was observed in the neutrophils, along with an increased serum concentration of IL-33 in aAT compared to the controls. IL-33 exacerbates NET formation via specifically upregulating CD16 expression in aAT. IL-33-primed NETs/neutrophils increased the cellular oxidative stress levels in the macrophages, leading to enhanced macrophage necroptosis and the release of atherogenic factors and matrix metalloproteinases (MMPs) in aAT compared to the controls. These findings suggested a pathogenic effect of the IL-33/ST-2 pathway in aAT patients by exacerbating NET formation and macrophage necroptosis, thereby facilitating the release of inflammatory factors and the release of MMPs that may be critical for the destabilization/rupture of atherosclerotic plaques in aAT. Targeting the IL-33/ST-2-NETs axis may be a promising therapeutic target for preventing plaque instability/rupture and its adverse complications in aAT.

Effect of selection methods on seed potato quality in the following season

Most potato farmers in Indonesia select the small tubers at harvest for planting in the following season, the so-called farmers’ practice (FP). This propagation method is cheap, but the small tubers may come from less healthy plants, which increases the build-up of diseases with accelerated yield decreases over the seasons. Alternatively, farmers may identify healthy plants within the growing season and select those for propagation, the so-called positive plant selection method (PPSM). An experiment was carried out to evaluate the effects of PPSM compared to FP on yields in the following season in the two main potato growing areas of West Java, i.e., Pangalengan and Garut. Generations G2 and G3 of cv. Granola and one generation of the imported cv. Atlantic were used. Selected seeds using PPSM and FP were planted in the second season in a randomized complete block design. Results show that yields of seeds selected through PPSM were significantly higher compared to seeds selected through FP, over both locations, on average, 7.4, 5.5 and 1.2 ton ha−1 for Granola G2 and G3, and the Atlantic, respectively. These yield increases represent an increase in the gross revenue of 30.8 to 51.8 million IDR ha−1 for Granola and 1.9 to 7.8 million IDR ha−1 for Atlantic at a farm gate price of 7,000 and 6,500 IDR kg−1, respectively. This study confirms that PPSM is superior to FP and improves the quality of the farms saved seeds.

In vitro expansion of fetal liver hematopoietic stem cells

Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34+) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.

Human Leukemia T-Cell Lines as Alternatives to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type B.

Staphylococcal enterotoxin type B (SEB) is associated with food poisoning. Current methods for the detection of biologically active SEB rely upon its ability to cause emesis when administered to live kittens or monkeys. This technique suffers from poor reproducibility and low sensitivity and is ethically disfavored over concerns for the welfare of laboratory animals. The data presented here show the first successful implementation of an alternative method to live animal testing that utilizes SEB super-antigenic activity to induce cytokine production for specific novel cell-based assays for quantifiable detection of active SEB. Rather than using or sacrificing live animals, we found that SEB can bind to the major histocompatibility complex (MHC) class II molecules on Raji B-cells. We presented this SEB–MHC class II complex to specific Vβ5.3 regions of the human T-cell line HPB-ALL, which led to a dose-dependent secretion of IL-2 that is capable of being quantified and can further detect 10 pg/mL of SEB. This new assay is 100,000 times more sensitive than the ex vivo murine splenocyte method that achieved a detection limit of 1 µg/mL. The data presented here also demonstrate that SEB induced proliferation in a dose-dependent manner for cells obtained by three different selection methods: by splenocyte cells containing 22% of CD4+ T-cells, by CD4+ T-cells enriched to >90% purity by negative selection methods, and by CD4+ T-cells enriched to >95% purity by positive selection methods. The highly enriched and positively isolated CD4+ T-cells with the lowest concentration of antigen-presenting cells (APC) (below 5%) provided higher cell proliferation than the splenocyte cells containing the highest concentration of APC cells.

Construction of a highly error-prone DNA polymerase for developing organelle mutation systems.

A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3′–5′ exonuclease function resulted in a modest 5–8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.

СИСТЕМЫ СЕЛЕКЦИИ ТОПОЛЕЙ В СВЯЗИ С СИСТЕМАМИ ИХ РАЗМНОЖЕНИЯ

We consider poplar selection systems in connection with their reproduction systems. Poplar is a model object on which it is possible to test all known systems for the selection of woody plants. Positive selection and synthetic selection methods (hybridization, mutagenesis, polyploidy, genetic engineering, biotechnology) effectively “work” on poplar. Poplar is propagated by seed, vegetative and in vitro biotechnology. Amphimixis or seed propagation of poplar is used to create hybrid seed plantations, for example white poplar × aspen. In natural poplar populations, spontaneous hybridization occurs, leading to the emergence of new species. The ability to vegetatively propagate poplar successfully solves the problem of plus breeding: selected best biotypes are propagated by in vitro grafting, cuttings or regenerants, and plantation cultures are created. Synthetic breeding methods: hybridization is widely used on poplar; dozens of poplar hybrids for forest crops and landscaping have been obtained; Induced mutants and polyploids are obtained. Breeders get their first genetically modified forms using genetic engineering. By microcloning, economically valuable individuals are propagated and regenerants are used to create forest crops and landscaping.

Abstract 5365: Profiling circulating tumor cell RNA from a large blood screening volume: A pilot study using diagnostic leukapheresis followed by the NanoString low RNA input nCounter assay

Abstract Introduction and objective: The presence of circulating tumor cells (CTC) is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. The aim of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by gene profiling using the NanoString low RNA input nCounter assay to achieve reliable CTC gene profiling. Methods: Blood samples were collected from 4 mPCa patients and 3 healthy donors. In the LP procedure, different blood elements were separated based on density. The mononuclear cell layer (presumed to contain any CTCs) was collected. The Alere™ CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method for isolation of EpCAM+ CTCs, followed by RT-qPCR for PSA, PSMA, and HOXB13 was used to detect CTCs from LP. The Ct value thresholds for positive CTC signal by RT-qPCR were set based on healthy donor LP subjected to AdnaTest as a negative control (PSA 36.3, PSMA 36.82, HOXB13 40). The cDNA of cells captured by EpCAM+ selection (including putative CTCs) and of the EpCAM+ depleted white blood cells (WBC) were assayed by the nCounter PanCancer Progression Panel to determine expression of 770 selected mRNAs. The NanoString nCounter Low RNA Input Kit with the multiplex 770-gene primer pool was used for the pre-amplification of cDNA and overnight hybridization with the PanCancer Progression panel. To validate the assay, healthy donor LPs were spiked with known numbers of PCa cell line LN95 cells. Results: For mPCa patients, the LP procedure took 136.2±10.6 min, with a screening volume of 10L of peripheral blood. The mean volume of mPCa LP product was 123.64±12.23 ml. The WBC concentration was 69.75±28.2×109/L. All samples were subjected to AdnaTest in triplicate and CTCs were identified in 3 out of 4 patients. The positive Ct values of PSA were 33.86±0.81, 32.14±0.31 and 33.09±0.34; PSMA were 35.55±1.02, 33.55±0.83 and 35.30±0.65; HOXB13 were 36.25±0.34, 37.15±0.22 and 37.09±0.33. To assess profiling of CTCs isolated from LP using the nCounter platform, 100, 500, and 1000 LN95 cells were spiked in healthy LPs and were analyzed with the AdnaTest. Within each spiking group, pre-spiking WBC, captured CTC, post-capture WBC, and LN95 cells in PBS alone were subjected to the Nanostring assay. Eisen Cluster analysis separated pre- and post- WBCs, captured CTCs, LN95 cells into three groups. The gene expression of captured CTCs correlated with those of LN95 cells, and the correlation increased with the spiking cancer cell number (R2=0.45, 0.57, and 0.70, respectively). The gene expression of pre-spiked WBCs were highly correlated with those of post-capture WBCs regardless of spiking cancer cell number (R2=0.83, 0.84 and 0.85). Conclusions: Analyzing CTCs in LP samples by AdnaTest is feasible and offers a promising method for liquid biopsy of mPCa patients. The NanoString low RNA input nCounter assay can provide reliable gene expression profiling of CTC. Citation Format: Liang Dong, Chung-Ying Huang, Changxue Lu, Diane Reyes, Sarah R. Amend, Jun Luo, Kenneth Pienta. Profiling circulating tumor cell RNA from a large blood screening volume: A pilot study using diagnostic leukapheresis followed by the NanoString low RNA input nCounter assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5365.

A Selected Stenotaphrum secundatum as Superior Shade Tolerant Forage Resource

<p class="awabstrak2">The obstacle in planting and developing forage plants is limited land. One solution to anticipate this is to utilize land in plantations by introducing forage shade-tolerant among the main crops. The area of oil palm plantations in Indonesia reaches 14,677,560 ha potentially used in the integration system. This article outlines the superiority of <em>Stenotaphrum secundatum</em> from the selection results so that the reader gets comprehensive information about this grass, both in terms of production, nutritional quality and digestibility. <em>Stenotaphrum secundatum</em> selection’s variety grass is a forage shade tolerant that had derived by positive mass selection method which had had been tried it’s adaptability at two different elevation in North Sumatra. This grass is very suitable to be integrated in plantation land. The average fresh yield of <em>S. secundatum</em> at 55 and 75% shading level reached 2,386 and 2,001 g/m2/harvest, respectively. The digestibility of Steno grass selection’s result on growing Boerka goat shows a fairly good value, which ranges from 60.7% to 72.8%. Palatability of <em>S. secundatum</em> grass in goats is very good with consumption reaching 3.6% of body weight. <em>S. secundatum</em> grass, besides being tolerant of shade, is also resistant to pests, as long as it is maintained with good management. The crossing of polyploid Steno grass with diploid has produced drought tolerant varieties. This grass also has advantages in terms of dry matter production, nutrient content and crude protein production compared to other grass species and can be planted in wider plantation areas with 55-75% shade.</p>

A Self-Supervised Learning Framework for Road Centerline Extraction From High-Resolution Remote Sensing Images

Road extraction from the high-resolution remote sensing image is significant for the land planning, vehicle navigation, etc. The existing road extraction methods normally need many preprocessing and subsequent optimization steps. Therefore, an automatic road centerline extraction method based on the self-supervised learning framework for high-resolution remote sensing image is proposed. This proposed method does not need to manually select training samples and other optimization steps, such as the nonroad area removing. First, the positive sample selection method combining the spectral and shape features is proposed to extract the road sample. Then, the one-class classifier framework is introduced and the random forest positive unlabeled learning classifier is constructed to get the posterior probability of the pixel belonging to road. The shape feature and the posterior probability are combined to form the final road network in the object-oriented way. Finally, the road centerline is obtained through the tensor voting algorithm. In order to verify the effectiveness of the proposed algorithm, high-resolution remote sensing images and benchmark datasets are used to do experiments. The indexes of the completeness ratio, the correctness ratio, and the detection quality are used for the quantitative accuracy evaluation. Compared with the supervised, the unsupervised, and the one-class classification road extraction algorithms, this proposed algorithm achieves high accuracy and efficiency. For the deep learning method comparison, the deep learning method performs well in most cases especially in the complex urban area. However, the deep learning method needs a large number of samples and a long training time, and our self-supervised learning framework does not need the training samples.

Abstract 1383: Isolating circulating tumor cells from a large screening blood volume: A pilot study using diagnostic leukapheresis

Abstract Purpose: The presence of circulating tumor cells (CTC) in blood is associated with poor prognosis in metastatic prostate cancer (mPCa) patients. For non-metastatic PCa patients and patients with biochemical recurrence (BCR), it is extremely difficult to find CTCs in 5-10 ml of peripheral blood (PB). The purpose of this study is to evaluate the feasibility of using diagnostic leukapheresis (LP) followed by other enrichment technologies to increase the CTC yield by using a large screening blood volume. Methods: Blood samples were collected from one healthy donor and one mPCa patient. During LP procedure, different blood elements were separated to different layers based on their densities. The mononuclear cell layer (that also contains CTCs) was collected. To assess recovery rate of different CTC isolation and detection methods, the healthy donor LP was spiked with a known number of PCa cell line LNCaP cells. Three CTC isolation and detection methods were used, including FAST (Clinomics, Ulsan, South Korea), a size-selection platform followed by immunofluorescence (IF); the AccuCyte-CyteFinder system (RareCyte, Inc., Seattle, WA), a selection-free method that utilizes IF; the AlereTM CTC AdnaTest (AdnaGen, Langenhagen, Germany), an EpCAM positive selection method followed by RT-qPCR. The criteria for defining a CTC are DAPI+, CK/EpCAM+, and CD45-. The Ct value thresholds for positive CTC signal by RT-qPCR were EpCAM 36.4, PSA 36.3, HOXB13 40, AR 35.4. Results: For the healthy donor LP with spiked LNCaP cells, the CTC recovery rate from FAST was 53.7%~67.2%. For Adna test, 0, 44, 95 and 1000 LNCaP cells were spiked in 5 ml of LP respectively. The Ct values of EpCAM were 36.28, 29.32, 28.22 and 24.76. For the mPCa patient, two tubes of 7.5 ml of PB were collected for FAST and RareCyte. The FAST found 3 CTCs while the RareCyte found 1 CTC. An LP sample was also collected from the same mPCa patient. The LP sample volume was 162 ml (WBC concentration was 83×109/L). Triplicates of 5 ml of samples were sent through AdnaTest and CTCs were identified in all samples (Ct: EpCAM 34.21±0.77, PSA 33.86±1.81, HOXB13 36.25±0.34, AR 35.29±0.22). Then the remainder of the mPCa LP was subjected to Ficoll-Paque density centrifugation to further concentrate mononuclear cells to a 90 ml volume. 1 ml samples were then applied to FAST in triplicate. The CTC numbers were 22 and 15. The density of cells on one of the filters was too high to be successfully scanned. Conclusion: Finding CTCs in LP samples by size-based technology or AdnaTest is feasible and offers a promising method to do liquid biopsy for mPCa patients. Screening a larger volume of PB by LP has significantly increased the CTC yield compared to using the same capture technology on 7.5 ml of PB. Citation Format: Liang Dong, Zhongyuan Zhang, Changxue Lu, Diane Reyes, Amber de Groot, Sarah R. Amend, Jun Luo, Kenneth J. Pienta. Isolating circulating tumor cells from a large screening blood volume: A pilot study using diagnostic leukapheresis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1383.

High-Throughput IgG Reformatting and Expression.

We have recently described a one-step zero-background IgG reformatting method that enables the rapid reformatting of phage-displayed antibody fragments into a single-mammalian cell expression vector for full IgG expression (Chen et al. Nucleic Acids Res 42:e26, 2014). The strategy utilizes our unique positive selection method, referred to as insert-tagged (InTag) positive selection, where a positive selection marker (e.g. chloramphenicol-resistance gene) is cloned together with the antibody inserts into the expression vector. The recombinant clones containing the InTag adaptor are then positively selected without cloning background, thus bypassing the need to plate out cultures and screen colonies. This IgG reformatting method is rapid and can be automated and performed in a high-throughput (HTP) format. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated. We have further optimized the protocol for IgG reformatting since the initial publication of this method (Chen et al. Nucleic Acids Res 42:e26, 2014) and also updated the transient transfection protocol using Expi293F cells, which are described herein.

Preservation of Antigen-Specific Functions of αβ T Cells and B Cells Removed from Hematopoietic Stem Cell Transplants Suggests Their Use As an Alternative Cell Source for Advanced Manipulation and Adoptive Immunotherapy.

Hematopoietic stem cell transplantation is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αβ T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αβ T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αβ T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing, and presentation were fully preserved. Therefore, we propose that separated αβ T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification.

The Response of White Yam (Dioscorea rotundata Poir) Tuber Portions to Positive Selection for Quality Seed Yam Production

The production of yams is constrained by high cost and unavailability of clean planting materials, pests and diseases. Vegetative propagation has also caused a build of up diseases, reported to cause up to 80% yield reduction due to scarcity of quality declared seeds. Planting of disease-free material has been found to be effective in reducing disease problems in plants. This study was conducted to produce clean seed yam by reducing yam diseases through positive selection method. In this method, apparently healthy yam plants were identified, tagged (positive selection), assessed for yam mosaic virus incidence and severity using the scale 0 or 1 and 1-5 respectively. The harvested clean tubers from tagged plants were planted the following season so as to determine the rate of response of each genotype and tuber portion to positive selection. At the end of two cycles of positive selection, analysis of variance for percentage number of positive yam plants, YMV incidence, YMV severity and tuber yield shows significant difference (p ≤ 0.05) between positive selection and no selection for both field and screen house plants. It was observed that positively selected plants performed significantly (p ≤ 0.05) better than no selection plants. For field experiment, Number of positively selected plants was highest in positive TDr89/02665 with mean value of 75.00% while no selection Ogoja had the least number of positive plants (17.8%). A yield increase of 1.80 t/ha was recorded due to the application of positive selection method in two cycles. For screen house experiment, result followed a similar trend; however with a reduction in YMV incidence and severity. It is worthy to note that tail portions of yam were lest infected with YMV, hence more healthy plants were selected from the tail portion.

A Positive Selection for Nucleoside Kinases in E. coli.

Engineering heterologous nucleoside kinases inside E. coli is a difficult process due to the integral role nucleosides play in cell division and transcription. Nucleoside analogs are used in many kinase screens that depend on cellular metabolization of the analogs. However, metabolic activation of these analogs can be toxic through disruptions of DNA replication and transcription because of the analogs’ structural similarities to native nucleosides. Furthermore, the activity of engineered kinases can be masked by endogenous kinases in the cytoplasm, which leads to more difficulties in assessing target activity. A positive selection method that can discern a heterologous kinases’ enzymatic activity without significantly influencing the cell’s normal metabolic systems would be beneficial. We have developed a means to select for a nucleoside kinase’s activity by transporting the kinase to the periplasmic space of an E. coli strain that has its PhoA alkaline phosphatase knocked out. Our proof-of-principle studies demonstrate that the herpes simplex virus thymidine kinase (HSV-TK) can be transported to the periplasmic space in functional form by attaching a tat-signal sequence to the N-terminus of the protein. HSV-TK phosphorylates the toxic nucleoside analog 3’-azido-3’-deoxythymidine (AZT), and this charged, monophosphate form of AZT cannot cross the inner membrane. The translocation of HSV-TK provides significant resistance to AZT when compared to bacteria lacking a periplasmic HSV-TK. However, resistance decreased dramatically above 40 μg/ml AZT. We propose that this threshold can be used to select for higher activity variants of HSV-TK and other nucleoside kinases in a manner that overcomes the efficiency and localization issues of previous selection schemes. Furthermore, our selection strategy should be a general strategy to select or evaluate nucleoside kinases that phosphorylate nucleosides such as prodrugs that would otherwise be toxic to E. coli.

Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis.

Patient-derived xenograft (PDX) mouse models are increasingly used for preclinical therapeutic testing of human cancer. A limitation in molecular and genetic characterization of PDX tumors is the presence of integral murine stroma. This is particularly problematic for genomic sequencing of PDX models. Rapid and dependable approaches for quantitating stromal content and purifying the malignant human component of these tumors are needed. We used a recently developed technique exploiting species-specific polymerase chain reaction (PCR) amplicon length (ssPAL) differences to define the fractional composition of murine and human DNA, which was proportional to the fractional composition of cells in a series of lung cancer PDX lines. We compared four methods of human cancer cell isolation: fluorescence-activated cell sorting (FACS), an immunomagnetic mouse cell depletion (MCD) approach, and two distinct EpCAM-based immunomagnetic positive selection methods. We further analyzed DNA extracted from the resulting enriched human cancer cells by targeted sequencing using a clinically validated multi-gene panel. Stromal content varied widely among tumors of similar histology, but appeared stable over multiple serial tumor passages of an individual model. FACS and MCD were superior to either positive selection approach, especially in cases of high stromal content, and consistently allowed high quality human-specific genomic profiling. ssPAL is a dependable approach to quantitation of murine stromal content, and MCD is a simple, efficient, and high yield approach to human cancer cell isolation for genomic analysis of PDX tumors.

Abstract 4975: Detection and characterization of circulating and disseminated tumor cells in patients with bladder and prostate cancer

Abstract Introduction: Increasing evidence suggests that cancer cells display dynamic molecular changes in response to systemic therapy. Circulating tumor cells (CTCs) in the peripheral blood and disseminated tumor cells (DTCs) in bone marrow represent a readily available source of cancer cells with which to measure this dynamic process. To date, many strategies to isolate and characterize these cells have been described. These techniques, however, have been limited by the requirement for positive or negative selection thus resulting in cell losses. Herein we report a method to detect and characterize CTCs and DTCs in matched peripheral blood and bone marrow samples without need for a selection step. Methods: Following blood or bone marrow aspirate collection, nucleated cells were extracted using a modified density-media centrifugation technique. Nucleated cells were spread on positively charged slides and stained with epithelial markers (Pan-cytokeratin and EpCAM) and a leukocyte marker CD45. High throughput scanning microscopy was performed followed by image processing and subsequent ranking for probability of being a cancer cell. Candidate cancer cells were defined as events which were positive for PanCK and/or EpCAM but negative for CD45. Candidate cells were independently confirmed with high resolution microscopy. Results: Cytokeratin-positive, EpCAM-positive DTCs and CTCs were readily detectable in patients with clinically-localized bladder and prostate cancer without need for positive or negative selection. Conclusions: Future work aims to incorporate disease specific makers to confirm that the observed cells truly represent cells of either bladder or prostate origin. This method offers the flexibility to preserve samples for downstream molecular assays including single cell isolation and sequencing. All told, this assay provides a novel means to detect and characterize CTCs and DTCs without positive or negative selection methods. Citation Format: James E. Verdone, Emma E. van der Toom, Kenneth J. Pienta, Michael A. Gorin. Detection and characterization of circulating and disseminated tumor cells in patients with bladder and prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4975.