Clonal Selection Research Articles

Getting the Right Clones in an Automated Manner: An Alternative to Sophisticated Colony-Picking Robotics.

In recent years, the design-build-test-learn (DBTL) cycle has become a key concept in strain engineering. Modern biofoundries enable automated DBTL cycling using robotic devices. However, both highly automated facilities and semi-automated facilities encounter bottlenecks in clone selection and screening. While fully automated biofoundries can take advantage of expensive commercially available colony pickers, semi-automated facilities have to fall back on affordable alternatives. Therefore, our clone selection method is particularly well-suited for academic settings, requiring only the basic infrastructure of a biofoundry. The automated liquid clone selection (ALCS) method represents a straightforward approach for clone selection. Similar to sophisticated colony-picking robots, the ALCS approach aims to achieve high selectivity. Investigating the time analogue of five generations, the model-based set-up reached a selectivity of 98 ± 0.2% for correctly transformed cells. Moreover, the method is robust to variations in cell numbers at the start of ALCS. Beside Escherichia coli, promising chassis organisms, such as Pseudomonas putida and Corynebacterium glutamicum, were successfully applied. In all cases, ALCS enables the immediate use of the selected strains in follow-up applications. In essence, our ALCS approach provides a 'low-tech' method to be implemented in biofoundry settings without requiring additional devices.

KUFRI DAKSH: A NEW WATER-USE EFFICIENT TABLE POTATO VARIETY WITH CROSS TOLERANCE TO HIGH TEMPERATURE STRESS

Kufri (K) Daksh is a main season, medium maturing, high yielding, water-use-efficient table potato variety that can be grown in regions with limited water availability. It is a clonal selection from the cross between CP1748, having high water use efficiency and LT-1 tolerating high temperature stress. Its plants are tall and vigorous with field resistance to late blight. Its tubers are white-cream (light yellow), ovoid with shallow to medium eyes and cream flesh with good keeping quality. It is fertilizer responsive and can yield 30-35 t/ha under low water (≤ 20%) availability in middle Gangetic plains. K Daksh has better trade-off for high CO2 gain and low water loss through optimization in plant canopy leaf area and plant height components. K Daksh can be an excellent choice to tolerate high temperature and water stress regimes in changing climatic scenarios.

Artificial immune systems (GA-AIS) enabled power loss mitigation in distributed generation: X3PAIS optimization approaches

The distribution network is a crucial component of the power system, with industrialization driving increased energy demand. Traditional power-generating techniques, such as thermal and hydroelectric are not enough to meet this demand, leading to the development of Distributed Generation (DG). DG requires an extensive re-evaluation of the current power system, as it modifies energy losses and line flows. Inadequate integration of DG can cause power outages, disruption of protection coordination, and lead to islanding. AI can help overcome this issue by determining the best system architecture. Researchers have been interested in the Artificial Immune System (AIS) algorithm, which has room for development and lacks a fixed template. In order to improve AIS, X3PAIS, a hybridization strategy that combines clonal selection with a three-parent crossover has been developed within the scope of the study. X3PAIS was pre-tested using applications in a planetary gear train, a wastewater treatment facility, and mathematical calculations, showcasing its robustness and versatility. In the context of power distribution, X3PAIS is used in the multiple DG architecture of the power distribution system, reducing power losses by placing DG units in the best locations and sizing them to match load profiles. The four DGs' experiment results show that X3PAIS can minimize power losses by more than 89 %. To optimize power losses in the power distribution system, X3PAIS may be improved with a three-parent multiple-point crossover operation.

Affinity-independent memory B cell origin of the early antibody-secreting cell response in naive individuals upon SARS-CoV-2 vaccination

Memory B cells (MBCs) formed over the individual's lifetime constitute nearly half of the circulating B cell repertoire in humans. These pre-existing MBCs dominate recall responses to their cognate antigens, but how they respond to recognition of novel antigens is not well understood. Here, we tracked the origin and followed the differentiation paths of MBCs in the early anti-spike (S)response to mRNA vaccination in SARS-CoV-2-naive individuals on single-cell and monoclonal antibody levels. Pre-existing, highly mutated MBCs showed no signs of germinal center re-entry and rapidly developed into mature antibody-secreting cells (ASCs). By contrast, and despite similar levels of S reactivity, naive B cells showed strong signs of antibody affinity maturation before differentiating into MBCs and ASCs. Thus, pre-existing human MBCs differentiate into ASCs in response to novel antigens, but the quality of the humoral and cellular anti-S response improved through the clonal selection and affinity maturation of naive precursors.

Deciphering the importance of morphological aspects in drought tolerance in eucalyptus progenies

Water limitation is one of the factors that most impact plant growth and productivity, especially for Eucalyptus. For commercial eucalyptus plantations to develop properly, it is essential to select clones with drought-tolerant characteristics, especially in areas with low rainfall and well-defined dry periods. Accordingly, climate change, mainly related to increases in mean annual temperature, can lead to a more severe water deficit and ultimately harm plant development. Thus, the identification of indicators related to climatic variables enables the selection of clones with drought tolerance characteristics. Here, we used 27 families of eucalyptus progenies from crosses of plants with previously selected characteristics associated with drought tolerance, which were planted in a region characterized by high water deficiency. Sample harvesting was performed at 6, 18, and 30 months after planting, at the end of natural drought cycles. Meteorological data coupled with physiological analyses allow the early selection of Eucalyptus clones that are better able to tolerate recurrent drought events. Specific leaf area (SLA), leaf area (LA), length (L), width (W), and leaf water potential (ΨL) were analyzed. The parameters SLA, LA, and ΨL proved to be good bioindicators of Eucalyptus drought tolerance. Specific clones and progenies (e.g., GG2673, VM1, GG1923, GG1980, GG3389XGG4302, GG918XVS62, AEC2034xLR831, I3000XVM1, VM4XCAM, and VM1xVM4) were characterized by physiological adjustments and showed higher average annual volumetric increment (IMAvol) values and were assumed as genetic materials with potential drought tolerance.

Vascular injury derived apoptotic exosome-like vesicles trigger autoimmunity

According to a central tenet of classical immune theory, a healthy immune system must avoid self-reactive lymphocyte clones but we now know that B cells repertoire exhibit some level of autoreactivity. These autoreactive B cells are thought to rely on self-ligands for their clonal selection and survival. Here, we confirm that healthy mice exhibit self-reactive B cell clones that can be stimulated in vitro by agonists of toll-like receptor (TLR) 1/2, TLR4, TLR7 and TLR9 to secrete anti-LG3/perlecan. LG3/perlecan is an antigen packaged in exosome-like structures released by apoptotic endothelial cells (ApoExos) upon vascular injury. We demonstrate that the injection of ApoExos in healthy animals activates the IL-23/IL-17 pro-inflammatory and autoimmune axis, and produces several autoantibodies, including anti-LG3 autoantibodies and hallmark autoantibodies found in systemic lupus erythematosus. We also identify γδT cells as key mediators of the maturation of ApoExos-induced autoantibodies in healthy mice. Altogether we show that ApoExos released by apoptotic endothelial cells display immune-mediating functions that can stimulate the B cells in the normal repertoire to produce autoantibodies. Our work also identifies TLR activation and γδT cells as important modulators of the humoral autoimmune response induced by ApoExos.

Factor analytic selection tools and environmental feature-integration enable holistic decision-making in Eucalyptus breeding

Understanding the genotype-by-environment interaction (GEI) and considering it in the selection process is a sine qua non condition for the expansion of Brazilian eucalyptus silviculture. This study’s objective is to select high-performance and stable eucalyptus clones based on a novel selection index that considers the Factor Analytic Selection Tools (FAST) and the clone’s reliability. The investigation explores the nuances interplay of GEI and extends its insights by scrutinizing the relationship between latent factors and real environmental features. The analysis, conducted across seven trials in five Brazilian states involving 78 clones, employs FAST. The clonal selection was performed using an extended FAST index weighted by the clone’s reliability. Further insights about GEI emerge from the integration of factor loadings with 25 environmental features through a principal component analysis. Ten clones, distinguished by high performance, stability, and reliability, have been selected across the target population of environments. The environmental features most closely associated with factor loadings, encompassing air temperature, radiation, and soil characteristics, emerge as pivotal drivers of GEI within this dataset. This study contributes insights to eucalyptus breeders, equipping them to enhance decision-making by harnessing a holistic understanding-from the genotypes under evaluation to the diverse environments anticipated in commercial plantations.

Intratumoral Cell Heterogeneity in Patient-Derived Glioblastoma Cell Lines Revealed by Single-Cell RNA-Sequencing.

Glioblastoma cell lines derived from different patients are widely used in tumor biology research and drug screening. A key feature of glioblastoma is the high level of inter- and intratumor heterogeneity that accounts for treatment resistance. Our aim was to investigate whether intratumor heterogeneity is maintained in cell models. Single-cell RNA sequencing was used to investigate the cellular composition of a tumor sample and six patient-derived glioblastoma cell lines. Three cell lines preserved the mutational profile of the original tumor, whereas three others differed from their precursors. Copy-number variation analysis showed significantly rearranged genomes in all the cell lines and in the tumor sample. The tumor had the most complex cell composition, including cancer cells and microenvironmental cells. Cell lines with a conserved genome had less diverse cellularity, and during cultivation, a relative increase in the stem-cell-derived progenitors was noticed. Cell lines with genomes different from those of the primary tumors mainly contained neural progenitor cells and microenvironmental cells. The establishment of cell lines without the driver mutations that are intrinsic to the original tumors may be related to the selection of clones or cell populations during cultivation. Thus, patient-derived glioblastoma cell lines differ substantially in their cellular profile, which should be taken into account in translational studies.

CHIP: a clonal odyssey of the bone marrow niche.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying somatic mutations. While CHIP is typically asymptomatic, it has garnered substantial attention due to its association with the pathogenesis of multiple disease conditions, including cardiovascular disease (CVD) and hematological malignancies. In this Review, we will discuss seminal and recent studies that have advanced our understanding of mechanisms that drive selection for mutant HSPCs in the BM niche. Next, we will address recent studies evaluating potential relationships between the clonal dynamics of CHIP and hematopoietic development across the lifespan. Next, we will examine the roles of systemic factors that can influence hematopoietic stem cell (HSC) fitness, including inflammation, and exposures to cytotoxic agents in driving selection for CHIP clones. Furthermore, we will consider how - through their impact on the BM niche - lifestyle factors, including diet, exercise, and psychosocial stressors, might contribute to the process of somatic evolution in the BM that culminates in CHIP. Finally, we will review the role of old age as a major driver of selection in CHIP.

Computational modeling of petroleum purification for removal of sulfur compounds: Process analysis for reduction of environmental impacts and material costs

In the course of this investigation, three machine learning models (Gaussian Process Regression, Decision Tree Regression, and Kernel Ridge Regression) were examined for determining the correlation between the input variables (x and y) which are spatial coordinates of model’s geometry, and the content of species in adsorption for sulfur capture. For the process modeling, mass transfer was analyzed, and the concentration distribution of sulfur compound was obtained via numerical solution of mass transfer equations, and then used for machine learning models. The machine learning models were trained using a dataset of 19,000 observations, and their performance was assessed through metrics including R2 score, MAE, and RMSE. Analysis of the results reveals that Decision Tree Regression surpassed the other two models in performance, with an R2 score of 0.9989, MAE of 6.64405E-01, and RMSE of 1.1277E+00. Gaussian Process Regression had an R2 score of 0.97106, MAE of 3.65541E+00, and RMSE of 5.6821E+00, while Kernel Ridge Regression had an R2 score of 0.86347, MAE of 8.26121E+00, and RMSE of 1.1330E+01. The Clonal Selection Algorithm was used for hyper-parameter optimization for all models. These findings demonstrate the potential of machine learning techniques for accurately and reliably predicting the concentration of chemical species and highlight the importance of considering the choice of model and hyper-parameter optimization for optimal performance.

Cytopathic Effect Detection and Clonal Selection using Deep Learning.

In biotechnology, microscopic cell imaging is often used to identify and analyze cell morphology and cell state for a variety of applications. For example, microscopy can be used to detect the presence of cytopathic effects (CPE) in cell culture samples to determine virus contamination. Another application of microscopy is to verify clonality during cell line development. Conventionally, inspection of these microscopy images is performed manually by human analysts. This is both tedious and time consuming. In this paper, we propose using supervised deep learning algorithms to automate the cell detection processes mentioned above. The proposed algorithms utilize image processing techniques and convolutional neural networks (CNN) to detect the presence of CPE and to verify the clonality in cell line development. We train and test the algorithms on image data which have been collected and labeled by domain experts. Our experiments have shown promising results in terms of both accuracy and speed. Deep learning algorithms achieve high accuracy (more than 95%) on both CPE detection and clonal selection applications, resulting in a highly efficient and cost-effective automation process.

Decoding Clonal Hematopoiesis: Emerging Themes and Novel Mechanistic Insights.

Clonal hematopoiesis (CH), the relative expansion of mutant clones, is derived from hematopoietic stem cells (HSCs) with acquired somatic or cytogenetic alterations that improve cellular fitness. Individuals with CH have a higher risk for hematological and non-hematological diseases, such as cardiovascular disease, and have an overall higher mortality rate. Originally thought to be restricted to a small fraction of elderly people, recent advances in single-cell sequencing and bioinformatics have revealed that CH with multiple expanded mutant clones is universal in the elderly population. Just a few years ago, phylogenetic reconstruction across the human lifespan and novel sensitive sequencing techniques showed that CH can start earlier in life, decades before it was thought possible. These studies also suggest that environmental factors acting through aberrant inflammation might be a common theme promoting clonal expansion and disease progression. However, numerous aspects of this phenomenon remain to be elucidated and the precise mechanisms, context-specific drivers, and pathways of clonal expansion remain to be established. Here, we review our current understanding of the cellular mechanisms driving CH and specifically focus on how pro-inflammatory factors affect normal and mutant HSC fates to promote clonal selection.

Recombinant Antibody-Producing Stable CHOK1 Pool Stability Study.

Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements.

Modern breeding methods in forestry aimed at preserving genetic diversity

This research is devoted to analysing the impact of modern breeding measures in forestry on the level of genetic diversity of forest tree species. It has been found that the main source of improved seed material for the genetic restoration of forests is base forest seed orchards. Aspects of the influence of determining factors – background pollination and the number of clones – on the indicators of genetic variability of progeny have been analysed in detail. The potential of background pollination in forest seed orchards in the context of a significant decrease in the effectiveness of breeding measures and a parallel increase in the level of genetic variability of progeny has been studied. An analysis of data on the minimum number of clones in seed orchards has been carried out based on the practical experience of other developed countries. The dynamics of clone variability in terms of fertility have been investigated. The potential impact of clonal selection on the genetic diversity of tree species, particularly in reducing it, has been identified. It has been confirmed that integrating the concept of family forestry, which involves the use of vegetative propagation techniques, into the breeding strategy significantly increases the level of genetic variability in progeny. The study has demonstrated that the multiple-population breeding system provides the optimal preconditions for synergising the process of long-term intensive breeding and preserving the gene pool of tree species. It has been determined that there is no negative impact on genetic diversity from implementing a complex of optimally planned breeding programs. At the same time, the potential for intensifying the quality of gene pool conservation in the process of forest ecosystem restoration through the use of improved seed material and clones in artificial orchards has been established. Special attention is given to the maintenance of ex situ – valuable genetic material in forest seed production facilities, including forest seed orchards, trial cultures, and clone archives of plus trees. Research has shown that the implementation of modern innovative solutions and scientific recommendations can minimise the loss of genetic diversity in forest tree species. The results can be applied in contemporary forestry breeding programs

Theories of immune recognition: Is anybody right?

The clonal selection theory (CST) is the centrepiece of the current paradigm used to explain immune recognition and memory. Throughout the past decades, the original CST had been expanded and modified to explain new experimental evidences since its original publication by Burnet. This gave origin to new paradigms that govern experimental immunology nowadays, such as the associative recognition of antigen model and the stranger/danger signal model. However, these new theories also do not fully explain experimental findings such as natural autoimmune immunoglobulins, idiotypic networks, low and high dose tolerance, and dual-receptor T and B cells. To make sense of these empirical data, some authors have been trying to change the paradigm of immune cognition using a systemic approach, analogies with brain processing and concepts from second-order cybernetics. In the present paper, we review the CST and some of the theories/hypotheses derived from it, focusing on immune recognition. We point out their main weaknesses and highlight arguments made by their opponents and believers. We conclude that, until now, none of the proposed theories can fully explain the totality of immune phenomena and that a theory of everything is needed in immunology.

Фенотипическая оценка клонов плюсовых деревьев сосны обыкновенной (Pinus sylvestris L.) в лесостепной зоне Башкирского Предуралья

An assessment of the main morphometric features of the ramets of clones of Scots pine plus trees in a comparative aspect has been carried out. The vegetative progeny of plus trees of the species are represented as part of a clonal plantation created in 2005 in the Dyurtyulinskiy forestry of the forest-steppe zone of the Republic of Bashkortostan in an area with the С2 type of forest growing conditions. Compliance with the breeding and genetic principle of phenotypic assessment of intraand intergroup differences in Scots pine clones and ramets, as well as methodical and methodological requirements for conducting the field stage of the study, has been ensured. Taxation indicators (the height and diameter of the stem, the diameter and length of the crown, the increment of the central shoot in height) have been taken into account for 100 clones at a continuous count. A varying nature of the distribution of the average values of the analyzed stem indicators in the vegetative progeny of plus trees has been revealed. The best growth in stem diameter (25.5±0.73 cm) and height (11.2±0.16 m), as well as central shoot increment (0.45±0.02 m), are typical for the progeny of clones of the plus tree 29/17. Clones 263/40, 264/41, 262/39 with taxation indicators above the average for the entire set of trees in the clone archive are of high breeding value, which indicates the specificity of the genotypes of these specimens. The levels of significance of the differences and the intensity of selection of the best clones of plus trees based on morphometric features have been determined, which indicates the different nature of the individual non-identity of each of the plus trees relative to the rest of the trees studied. The valuable genotypes of the progeny of Scots pine plus trees identified in the clone archive can be recommended for further use in forest seed breeding when creating highly productive and sustainable artificial plantations of this valuable tree species in the region.

Retraction Note: A study on car flow organization in the loading end of heavy haul railway based on immune clonal selection algorithm

Retraction Note: A study on car flow organization in the loading end of heavy haul railway based on immune clonal selection algorithm

Clearance of residual genome editing components used for ex vivo genome-editing of allogeneic cell therapy products

Background aimsClustered Regularly Interspaced Short Palindromic Repeats (CRISPR)–associated genome editing (GE) components (e.g., nucleases, guide RNAs (gRNAs), and plasmids) are used to genetically modify cells during development of ex vivo genome-edited cell therapies. Prolonged presence of GE components may increase the risk of unintended genome modifications (e.g., off-target editing and chromosomal rearrangements). This risk is a function of the stability of the GE components, culture conditions (i.e., culture length, media changes, etc.), and the nature of the GE component (i.e., only plasmids can be integrated into a cell's genome). Testing for residual GE components on ex vivo genetically edited drug products is generally recommended in current regulatory guidance (CBER 2024).For allogenic cell therapies derived from induced pluripotent stem cells (iPSC), cells typically undergo clonal selection and extensive culturing following completion of genome editing. This post-engineering clonal selection substantially reduces the amount of residual GE components while the long-duration cell culture significantly reduces the presence of active residual GE components. Here we present a case in which the need for testing of the drug product for residual GE components has been eliminated. MethodsIn silico modeling was used to estimate clearance mechanisms across a variety of relevant assumptions, including disposition of extracellular GE components via media changes and dilution of intracellular GE components via cell expansion. Determining the ability of each GE component—alone or in complex with other GE components—to modify genomic material was assessed by a series of both in vitro and ex vivo (i.e., engineering cells) studies. For the in vitro studies, a DNA cutting assay was developed to assess the ability of the component to cut a representative DNA strand. For the ex vivo modification of cells, an assessment of the knock-out of the relevant gene was completed by flow cytometry specifically assessing the presence or absence of protein expression on the modified cells. The persistence and stability of GE components were examined under cell-mimicking conditions and in ex vivo modified cells. The components were stressed under multiple conditions mimicking a range of culture conditions and tested in the aforementioned DNA cutting assay. The presence of residual gRNA was directly assessed in the ex vivo modified cells via a gRNA-specific digital droplet polymerase chain reaction (ddPCR) assay. ResultsSimulations estimating genome editing residual clearance via dilution for extracellular residuals (via media changes) or intracellular residuals (via cell doubling) demonstrate clearance of measurable residuals within 28 days of cell culture. Studies simulating the stability of genome editing residuals estimate less than 7 days for the nuclease, gRNA and ribonucleoprotein (RNP) complex. gRNA was undetectable by 8 days post-engineering under actual engineering conditions. Additionally, without gRNA present, CRISPR Cas12a nucleases did not demonstrate evidence of cutting.In contrast, plasmid DNA can be randomly integrated into the genome and free plasmid is highly stable under cell culture-like conditions (50+ days). Additionally, plasmid DNA integrated in cells will propagate during mitosis, leading to the additional risk of expansion of an unintentional integration event. ConclusionsBoth the gRNA and nuclease in the RNP complex are required for DNA cutting. Neither individual component nor the complex are stable beyond 7 days in culture-mimicking conditions. These findings suggest that the risk of unplanned genomic modification resulting from residual gRNA or nuclease is minimal for processes in which extensive culture is performed after the completion of genome editing and clonal selection. However, the risk of residual plasmid DNA integration is significantly higher regardless of the manufacturing process. The residual plasmid itself is quite stable (at least 50 days) and the risk of random, off-target integration is present. By establishing the stability of these components, we have demonstrated that testing for residual gRNA or nuclease is not warranted for clonally derived allogeneic cell therapies.

Characteristics of large granular lymphocyte leukemia associated with variable common immunodeficiency disorders: A study of 12 cases.

Common Variable Immunodeficiency Disorders (CVID) and Large Granular Lymphocytes leukemia (LGLL) exhibit diverse clinical manifestations including infections, dysimmunity, and lymphoproliferation. Recent decades have seen the discovery of new genes in the lymphopoiesis pathway, such as JAK-STAT. This case series supplemented by a literature review aims to describe clinical and biological characteristics of patients with both CIVD and LGLL. Patients were included through a call for comments to French and Belgian centers and through a literature review via PubMed. Clinical characteristics were compared to two large French cohort involving CVID and LGLL patients. Twelve patients were included. In all cases, CVID precedes LLGL (median diagnosis delay for LLGL was 7 years). Most cases presented with splenomegaly and autoimmune cytopenia. Ten out of 12 patients underwent splenectomy during follow up. Patients with LGLL and CVID differ from patients without immune deficiency in term of clinical presentation and prognosis. We suggest CVID may act as a trigger of LGL lymphocytosis, due to endogenous and exogenous antigenic pressure leading to the selection of a dominant LGL clone and stimulation of the JAK-STAT pathway. The role of splenomegaly and splenectomy in LGLL onset warrant further investigation in future studies.

Cost-effective clonal selection and AIS-based load balancing in cloud computing environment

Cost-effective clonal selection and AIS-based load balancing in cloud computing environment