Polyclonal Selection Research Articles

The adaptive roles of aneuploidy and polyclonality in Leishmania in response to environmental stress.

Aneuploidy is generally considered harmful, but in some microorganisms, it can act as an adaptive mechanism against environmental stress. Here, we use Leishmania-a protozoan parasite with remarkable genome plasticity-to study the early steps of aneuploidy evolution under high drug pressure (using antimony or miltefosine as stressors). By combining single-cell genomics, lineage tracing with cellular barcodes, and longitudinal genome characterization, we reveal that aneuploidy changes under antimony pressure result from polyclonal selection of pre-existing karyotypes, complemented by further and rapid de novo alterations in chromosome copy number along evolution. In the case of miltefosine, early parasite adaptation is associated with independent point mutations in a miltefosine transporter gene, while aneuploidy changes only emerge later, upon exposure to increased drug levels. Therefore, polyclonality and genome plasticity are hallmarks of parasite adaptation, but the scenario of aneuploidy dynamics depends on the nature and strength of the environmental stress as well as on the existence of other pre-adaptive mechanisms.

Polyclonal selection for abiotic stress tolerance in Arinto: Implications in yield and quality of the must

Polyclonal selection for abiotic stress tolerance in Arinto: Implications in yield and quality of the must

Fully and Partially Replicated Experimental Designs for Evaluating Intravarietal Variability in Grapevine

Background and Aims. In ancient grapevine varieties, the experimental design of field trials is crucial to providing a reliable evaluation of quantitative traits. The main purposes of this study are to demonstrate the benefits of the resolvable row-column design (RCD) for quantifying intravarietal variability and performing polyclonal selection and to compare the efficiency of fully and partially replicated designs for quantifying intravarietal variability to implement the latter designs for a preliminary analysis of that variability. Methods and Results. Linear mixed models were fitted to yield data obtained in field trials with fully and partially replicated designs. The results pointed out the importance of the RCD in controlling the spatial variability present in large field trials. Although less precise, a partially replicated design proved to be useful in evaluating intravarietal variability when the average of years was used. Conclusions. The results reinforced the importance of the RCD in increasing the efficiency of intravarietal variability quantification and polyclonal selection. The partially replicated design proved to be useful when the only objective was to perform a preliminary analysis of intravarietal variability. Significance of the Study. Understanding the role of experimental design in grapevine selection field trials will help grapevine breeders enhance their knowledge about variability within ancient varieties and implement more successful polyclonal selection.

Using CRISPR-X for Optimization of Antibodies Towards A30P α-synuclein Oligomers in Immunotherapy of Parkinson’s Disease

Introduction: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive cell death in the substantia nigra portion of the midbrain, which results in severe dopamine imbalance and a subsequent loss of refined motor control. Pathogenicity arises from mutations driving the misfolding of alpha synuclein (ɑ-syn), thus leading to oligomerization and formation of Lewy body aggregates in neurons. Previous research has identified the A30P mutated oligomers, which are connected to familial early-onset PD, as particularly toxic intermediates. However, there are insufficient methods of targeting them in isolation of other forms of the ɑ-syn protein. We aim to modify existing approaches to immunotherapy of PD, by use of CRISPR-X and phage display, to design antibodies that are highly specific to the A30P oligomer. Methods: We will use CRISPR-X to produce a diverse library of antibodies by introducing point mutations in the sequences encoding their variable light (VL) and heavy (VH) chains. Then, via insertion into bacteriophages for multiple rounds of phage display selection, we should obtain a polyclonal selection of antibodies that exclusively recognize epitopes on A30P ɑ-syn. Each variant then produces a distinct monoclonal phage colony to be incubated with relevant antigens for affinity testing via enzyme-linked immunosorbent assay (ELISA). Expected Results: We expect that measurements from ELISA would indicate low dissociation constants (KD) within the low micromolar (10-6) to nanomolar (10-7 to 10-9) range, when incubated with A30P oligomers. We predict that the resultant antibodies would have a higher affinity for the A30P oligomeric ɑ-syn than other forms of the protein. Furthermore, the novel antibodies are expected to have a higher specificity to this target antigen than the original antibodies that were modified. Discussion: By determining binding affinity, we verify the effectiveness of this method for improving specificity of in vitro antibody design. Upon further investigation using mice models, we would anticipate these novel antibodies could produce a specific reversal of the toxic effects of A30P in neuronal tissue. Conclusion: This study aims to establish a potential for CRISPR-X and phage selection to be used in antibody optimization for developing an immunotherapeutic treatment for PD.

Genetic gains of selection in ancient grapevine cultivars

The genetic diversity accumulated over centuries within ancient grapevine cultivars is the raw material for any genetic selection. The greater the intravarietal variability, the greater will be the gains obtainable from the selection and the capacity of the cultivar to respond to present and future challenges of a viticulture in which the growing of ancient cultivars is valued. Therefore, it should be efficiently evaluated, which involves the establishment of field trials with powerful experimental designs and the fitting of appropriate models for data analysis. The key point is to perform well-planned and accurate phenotyping, because it is a requisite for any efficient genetic selection in grapevine. For quantitative genetics analyses focused on selection, linear mixed models are fitted to large data sets using residual maximum-likelihood estimation. In this work, those models were fitted to yield and must quality traits data from initial field trials of 11 ancient cultivars. The objective was to show that good and accurate phenotyping allows the implementation of efficient polyclonal selection (selection of a superior group of genotypes within one cultivar). The results obtained showed that, for all traits and cultivars studied, it was possible to perform polyclonal selection with high and precise genetic gains of selection, reinforcing the importance of this type of selection for the vine and wine industry.

Methods for conservation of intra-varietal genetic variability in ancient grapevine varieties

Conservation of intra-varietal genetic variability is a crucial strategy to preserve traditional viticulture and to face future challenges. Conservation can be performed through two processes: (1) conservation without other immediate objectives; (2) conservation which simultaneously ensures the evaluation of important quantitative traits (such as yield, must quality traits, tolerance to abiotic stresses). The second strategy permits the quantification of intra-varietal genetic variability for important quantitative traits and the selection of a superior group of genotypes (polyclonal selection). Based on the results obtained from the conservation of ancient Portuguese varieties, the abovementioned methods for conservation of intra-varietal diversity are compared, and the advantages and disadvantages of each one are discussed.

Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani

The parasite Leishmania donovani causes a fatal disease termed visceral leishmaniasis. The process through which the parasite adapts to environmental change remains largely unknown. Here we show that aneuploidy is integral for parasite adaptation and that karyotypic fluctuations allow for selection of beneficial haplotypes, which impact transcriptomic output and correlate with phenotypic variations in proliferation and infectivity. To avoid loss of diversity following karyotype and haplotype selection, L. donovani utilizes two mechanisms: polyclonal selection of beneficial haplotypes to create coexisting subpopulations that preserve the original diversity, and generation of new diversity as aneuploidy-prone chromosomes tolerate higher mutation rates. Our results reveal high aneuploidy turnover and haplotype selection as a unique evolutionary adaptation mechanism that L. donovani uses to preserve genetic diversity under strong selection. This unexplored process may function in other human diseases, including fungal infection and cancer, and stimulate innovative treatment options.

Optimization Design of Crane Box Girder Based on the Improved Polyclonal Selection Algorithm

On the basis of the polyclonal selection algorithm, an improved polyclonal selection algorithm based on negative selection is put forward in this paper. Inspired by the diversity and auto-tolerance of the negative selection, the population diversity is increased by the clone deletion and clone supply to improve the polyclonal selection algorithm. Finally, the optimization design of the crane box girder is realized based on the improved polyclonal selection algorithm.

Immune Optimization of PID Control Parameters for DC Motor

In order to select the optimal PID control parameters for the brushless DC(Direct Current) motor of the two wheeled self balancing electric vehicle, a bacterial foraging algorithm-based polyclonal selection algorithm (BFA-PSA) is proposed. The bacterial foraging algorithm is introduced into the polyclonal selection algorithm (PSA) to solve the convergence phenomenon which often occurs in the basic PSA at the late of the optimization and causes the algorithm to fall into local minima. The optimization results of the functions show that the searching ability and searching efficiency of the proposed BFA-PSA are improved. Finally, on the basis of the transform function model of the brushless DC motor, the proposed algorithm is used in the optimization of the PID control parameters, and the system response results show the validity of the BFA-PSA.

Comprehensive Analysis of Yeast Surface Displayed cDNA Library Selection Outputs by Exon Microarray to Identify Novel Protein-Ligand Interactions

Phosphatidylinositides are important signaling molecules that interact with a myriad of cellular proteins, many of which remain unidentified. We previously screened a yeast surface displayed human proteome library to identify protein fragments with affinity for the phosphatidylinositides, phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate. Much of the diversity in the screened selection outputs was represented by clones present at low frequencies, suggesting that a significant number of additional phosphatidylinositide-binding protein fragments might be present in the selection outputs. In the studies described in this report, we developed a novel cDNA library analysis method and comprehensively analyzed the polyclonal selection outputs from the phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate selections using a high-density exon microarray. In addition to the nine previously reported phosphatidylinositide-binding protein fragments, we identified 37 new phosphatidylinositide-binding candidates. Nine of 37 contain known phosphatidylinositide-binding domains, whereas the remaining 28 contain no known phosphatidylinositide-binding domain. We cloned and confirmed phosphatidylinositide binding by fluorescence-activated cell sorting for 17 of these novel candidate protein fragments. Our experiments suggest that phosphatidylinositide binding by these 17 novel protein fragments is dependent on both the inositol phosphate "headgroup" and the lipid "tail." This is in contrast with the PH domain containing fragments we tested, for which the inositol phosphate headgroup was sufficient for binding. The novel PtdIns-binding fragments come from a wide variety of proteins, including splicing factors, transcription factors, a kinase, and a polymerase. Intriguingly, 11 of the phosphatidylinositide-binding protein fragments are from nuclear proteins, including four containing homeobox domains. We found that phosphatidylinositides and double-stranded DNA oligonucleotides derived from homeobox domain target sequences compete for binding to homeobox domain-containing protein fragments, suggesting a possible mechanism for phospholipid-dependent transcriptional regulation. FACS enrichment of target-binding clones in yeast human cDNA display libraries coupled with comprehensive analysis of the selection output by DNA microarray analysis is an effective method for investigating common as well as rare protein interactions. In particular, this method is well suited for the study of small molecule/protein and drug/protein interactions.

Accelerated Proliferative Senescence of Rat Embryo Fibroblasts after Stable Transfection of Multiple Copies of the c-Myc DNA-Binding Sequence

The protooncogene c-myc positively regulates cellular proliferation whereas it exhibits negative effects on both cellular senescence and differentiation. Ectopic overexpression of c-myc in transfection experiments or titration of the c-myc mRNA by antisense oligonucleotides has demonstrated that small changes of the concentration of cellular c-myc mRNA or protein levels can be crucial for these processes. In view of the role of c-Myc as a transcription factor, most of these effects may be mediated via its binding to specific DNA sequences. Here we studied the cellular reactions after manipulating the cellular concentration of c-Myc DNA-binding sites. Multiple copies of the c-Myc-binding sequence GACCACGTGGTC or, alternatively, the control sequence GACCAGCTGGTC that displays only a poor affinity for c-Myc were stably introduced into the genome of rat embryo fibroblasts. Transfection with the c-Myc-binding sequence yielded much lower clone numbers and sizes than transfection with the control sequence. After polyclonal selection and further subcultivation cells transfected with c-Myc-binding sequence exhibited a reduced growth rate and achieved less than two-thirds of the cumulative population doublings before becoming senescent and irreversibly growth arrested compared to the controls. Southern blot analysis demonstrated that 30 binding sequences on average were integrated into the cellular genome. Our results can be interpreted as competition of the ectopically introduced c-Myc-binding sequences with the functional genomic ones and assume that a fairly low number of the latter exist in the normal cellular genome. Hence, only a low copy number of introduced c-Myc-binding sequences is sufficient to cause signs of accelerated proliferative senescence.

Mechanism of antigenic variation in an individual epitope on influenza virus N9 neuraminidase

Monoclonal antibodies which inhibit influenza virus neuraminidase (NA) and which therefore indirectly neutralize virus infectivity bind to epitopes located on the rim of the active-site crater. The three-dimensional structure of one of these epitopes, recognized by monoclonal antibody NC41, has previously been determined (W. R. Tulip, J. N. Varghese, R. G. Webster, G. M. Air, W. G. Laver, and P. M. Colman, Cold Spring Harbor Symp. Quant. Biol. 54:257-263, 1989). Nineteen escape mutants of influenza virus A/tern/Australia/G70c/75 (N9) NA selected with NC41 were sequenced. A surprising restriction was seen in the sequence changes involved. Ten mutants had a Ser-to-Phe change at amino acid 372, and six others had mutations at position 367. No escape mutants with changes at 369 or 370 were found, although these mutations were selected with other antibodies and rendered the epitope unrecognizable by antibody NC41. Another N9 NA, from A/ruddy turnstone/NJ/85, which differs by 14 amino acids from the tern virus NA, still bound antibody NC41. Epitope mapping by selecting multiple escape mutants with antibody NC41 thus identified only three of the five polypeptide loops on NA that contact the antibody. Escape mutants selected sequentially with three different monoclonal antibodies showed three sequence changes in two loops of the NC41 epitope. The multiple mutants were indistinguishable from wild-type virus by using polyclonal rabbit antiserum in double immunodiffusion tests, but NA inhibition titers were fourfold lower. The results suggest that although the NC41 epitope contains 22 amino acids, only a few of these are so critical to the interaction with antibody that a single sequence change allows selection of an escape mutant. In that case, the variety of amino acid sequence changes which can lead to polyclonal selection of new epidemic viruses during antigenic drift might be very limited.