Later Stages Of Development Research Articles

Elucidating the genetics of post-harvest shelf-life of cucumber fruits and identification of associated QTLs and candidate genes

Cucumber is a perishable vegetable crop with limited storability, and development of genotypes with extended shelf-life will help in reducing post-harvest loss. One natural variant, DC-48 with extended shelf-life, was used to understand the genetics of the associated traits and identify the genomic regions and associated candidate genes. Scanning electron microscopy revealed differences in the structure of the epicarp in the genotypes. Programmed cell death (PCD) and appearance of senescence-related dark spots (SRDS) in the epicarp of the cucumber fruit with low shelf-life were also detected. Retention of green colour, absence of any shrinkage, retention of fruit firmness and slow change in the pH of the endocarp were essential traits associated with extended shelf-life. Single recessive gene govern retention of green colour while the remaining traits were polygenic. One QTL detected through QTL-seq on chromosome 4 was associated with retention of green colour spanning over a region of 5.01 Mb. Conventional mapping in F2 progenies with PCR-based markers also identified one QTL within the same region. RNA-seq and RT-PCR analysis at two different developmental stages revealed two genes in the QTL region namely, Csa_4G000860 with unknown function and Csa_4G016490 with acyl-transferase activity in the early and late developmental stages of the fruit, respectively associated with retention of green colour. Two PCR-based markers, InDel 861 and InDel 016, closely linked with retention of green colour can be used in marker-assisted back-cross introgression. This study provides greater insight into the molecular network associated with extended shelf-life in cucumber.

(Invited) Improving Certainty in Battery Safety Profiles through Early-Stage Evaluation

There is currently a great desire to reduce carbon emissions through electrification of all transportation sectors by utilizing battery energy storage. However, existing technology is unable to meet the performance target demands of certain transportation sectors (e.g., aviation). As a result, and due to a growing demand for conventional Li-ion battery materials, there is significant motivation to generate new battery technologies. During early-stage of development, the primary focus is demonstrating the achievement of performance metrics for the anticipated application. Once performance metrics have been demonstrated, the focus turns to scale-up. It isn’t until the later stages of development, perhaps even during the push for large scale manufacturing, that safety is more closely analyzed. At that stage, a significant amount of research dollars has been invested into that technology. To complicate matters, it is widely understood that the safety profile of large format cells (e.g., multi-layer pouch cells) can be significantly different than the safety profile of small format cells (e.g., coin cells) of the exact same chemistry. The work presented here examines the gap that exists in the understanding of a commercial-scale full cell's safety characteristics through a combination of characterization, experiments, and modeling using only information gained at the lab-scale, early-stage development (i.e., materials and small format cells). Characterization and experiment will be developed in such a way as to determine and quantify key metrics that will be utilized as modeling inputs. A modeling framework will utilize those key metrics to predict the large format safety profile of a specific chemistry. Experimental work, where cells are exposed to abusive conditions, will allow for the refinement of input parameters and refinement of the modeling framework to increase certainty in large format safety profile. This talk will highlight the planned approach to utilize a battery technology’s materials and small format cells to develop a picture of what may be expected in the safety profile of a large format cell.Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia LLC, a wholly owned subsidiary of Honeywell International Inc. for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525.

Investigating the Applicability of Rotating Disk Electrode Measurements for Determining Catalyst Activity in Fuel Cells

Currently, the mass production of fuel cells is mainly limited by the availability and the price of platinum, which is typically used as the catalyst for the hydrogen oxidation reaction (HOR) as well as for the oxygen reduction reaction (ORR) in proton exchange membrane fuel cells (PEMFC). To eliminate or at least reduce this limitation, it is necessary to develop new catalytic systems, which are based on other, cheaper active components or at least require less platinum. For the characterisation of catalysts, many different methods are in use for the physical and the chemical part. For example, the rotating disk electrode (RDE) is a widely used method for characterizing catalysts in electrochemistry. However, RDE measurement does not reflect the same reaction conditions as exist in a fuel cell. Nevertheless, characterisation by RDE tests is often used for pre-selection at an early stage of catalyst development. In later stages of development, catalyst investigations are carried out by full-cell or half-cell tests.In this work, we will compare the oxygen reduction reaction (ORR) properties of Pt/C catalysts from RDE measurements with those from CCM measurements in half-cell and full-cell configurations to determine what correlations can be made between the different type of investigations.

Floral structure and development in Nandina domestica and implications for systematic affinities in Berberidaceae and perianth evolution in Ranunculales

Abstract In Berberidaceae, the phylogenetic position and systematic affinities of the monotypic genus Nandina have long been debated and remain unclear. In addition, previous comparative studies have provided conflicting reports on the origin and development of its petals. Using scanning electron microscopy and microtome sections, we studied the structure and development of the flower and perianth in Nandina domestica to re-evaluate its affinities and homology of its petals. Our results confirm that N. domestica essentially shares the same Bauplan (organization) with other Berberidaceae and its petals are neither delayed in development nor much differentiated from sepals. However, we sometimes also observed the initiation of additional primordia that become confluent with the base of stamens of the outer whorl, but do not develop further and are no longer visible in later stages of development. Our study thus not only reconciles previous conflicting interpretations on the origin and development of petals in Nandina, but also confirms that it has a unique suite of vegetative and reproductive features supporting its position as a distinct member of the family. In addition, it suggests that the most recent common ancestors of both Berberidaceae and Ranunculales probably had two types of petaloid organs with distinct origin and development but still sharing a deep common genetic identity.

An Advanced Early-Stage Production Forecasting Model for Middle-High Rank Coal Development

Reasonable production prediction of coalbed methane (CBM) is of great significance for improving the economic benefit of CBM reservoirs. Current prediction methods for CBM production focus on the later stages of development, with few studies on early production forecasting. The objective of this work is to provide a reliable new idea for the early production prediction of CBM through various analyses and demonstrations. First, the CBM development modes are classified according to the production characteristics of the Panhe demonstration block of Shaanxi Province, China. Second, an efficient and feasible early production prediction model is established based on the geological potential and development potential. Finally, using the proposed model, different modes’ production characteristics and optimization strategies are analyzed. The research shows that the gas production profiles can be divided into two modes: single-peak mode (SPM) and multipeak mode (MPM). The peak production and average EUR of the SPM are 49.6% and 32.4% higher than those of the MPM, but the stable production period is only 0.2~1 year. In terms of the geological potential of CBM wells, the gas content, critical desorption pressure, and formation coefficient of the SPM are 6.7%, 13.3%, and 37.8% higher than those of the MPM, and the gas wells are mainly located in the high part of the coal seam (the average height difference is about 20 m). Besides, the concept of quasidesorption degree Pdq is innovatively introduced to characterize the development potential of gas well. The Pdq has an exponential relationship with CBM production, and the coefficient of the exponential term in SPM is approximately 22% larger than that in MPM. Moreover, the production of gas wells is greatly affected by the continuity of production. In the process of gas production, the influence of factors such as equipment shutdown should be minimized. To examine the applicability of the proposed method, the model is applied to an actual CBM well in Panhe, and the prediction accuracy is higher than 85%.

IMPROVING THE EFFICIENCY OF HYDROCARBON DEVELOPMENT BY IMPROVING THE METHODS OF CONTROL AND REGULATION OF PROCESSES

Identification of the regularity of pressure in the oil reservoir, considering the regularity of improving the method of control and reservoir development. The study used theoretical and experimental analyzes of the results with the use of software. The results of the study will improve the accuracy of the design and development of oil and gas fields and can be used to improve the production technology of oil fields at their later stages of development. Taking into account the angle, the pressure distributions in the reservoir are changed in a circle, a new model is proposed in which the pressure dynamics is changed, it is solved by a two-dimensional problem taking known solutions. According to the known distribution of pressure in the reservoir, obtained experimentally, the characteristics of this parameter along the axis of the segment of the oil reservoir are fixed under changing boundary conditions. A mathematical model is proposed that considers the distribution of pressure along the angle of inclination of wells, will improve the prediction of pressure distribution in producing wells, which leads to the improvement of methods for monitoring and regulating development in oil fields.

SetDB1 and Su(var)3-9 are essential for late stages of larval development of Drosophila melanogaster.

Methylation of H3K9 histone residue is a marker of gene silencing in eukaryotes. Three enzymes responsible for adding this modification - G9a, SetDB1/Egg, and Su(var)3-9 - are known in Drosophila. To understand how simultaneous mutations of SetDB1 and Su(var)3-9 may affect the fly development, appropriate combinations were obtained. Double mutants egg; Su(var)3-9 displayed pronounced embryonic lethality, slower larval growth and died before or during metamorphosis. Analysis of transcription in larval salivary glands and wing imaginal disks indicated that the effect of double mutation is tissue-specific. In salivary gland chromosomes, affected genes display low H3K9me2 enrichment and are rarely bound by SetDB1 or Su(var)3-9. We suppose that each of these enzymes directly or indirectly controls its own set of gene targets in different organs, and double mutation results in an imbalanced developmental program. This also indicates that SetDB1 and Su(var)3-9 may affect transcription via H3K9-independent mechanisms. Unexpectedly, in double and triple mutants, amount of di- and tri-methylated H3K9 is drastically reduced, but not completely absent. We hypothesize that this residual methylation implies the existence of additional H3K9-specific methyltransferase in Drosophila.

Morphological features of microglial cells in a 5xFAD mouse model of Alzheimer's disease

BACKGROUND: Aging is an inevitable and irreversible process associated with increased risk of developing various neurodegenerative diseases, one of which is Alzheimer's disease. Currently, the role of glial cells, in particular microglia, in the pathogenesis of Alzheimer's disease is being actively studied. However, only a few studies have correlated the morphological features of microglia and their spatial arrangement in relation to β-amyloid plaques. AIM: Describe the main morphological parameters of microglia in the 5xFAD mouse model of Alzheimer's disease at a late stage of pathology development. METHODS: As the studied object, mice were chosen by the age of 15–16 months of the 5xFAD line, as a model of acceleid amyloidosis. The immunohistochemical staining of the study of the morphological diversity of microglia was carried out on the cuts of the cortex of the mouse brain. The obtained confocal images performed an immunogystological analysis of the cuts of the cerebral cortex when analyzed using the Imagej application using the plugins of Skeleton, AnalyzeSkeleton (2D/3D) and FracLac. RESULTS: During the study, 5xFAD mice were divided into two groups (n=3 each). Carriers of the app and psen1 transgenes were assigned to the “FAD” group, and wild-type mice were assigned to the “Wt” group (control). We analyzed 3–4 sagittal sections (50 µm) of the brain from each mouse. The results showed that microglial cells from mice with signs of Alzheimer's disease have smaller fractal dimension, lacunarity and branching. CONCLUSION: The presence of β-amyloid plaques contributes to the migration of microglia to the focus of inflammation, its proliferation and transition to the phagocytic and dystrophic subtype. According to fractal analysis, there is a significant (p ≤0.05) decrease in the average branching of microglial processes, a decrease in fractal dimension and lacunarity.

Exploring symptom-level associations between anxiety and depression across developmental stages of adolescence: a network analysis approach

BackgroundAnxiety and depression often co-occur during adolescence, but the associations between symptoms of these two disorders in this developmental period are not yet fully understood. Network analysis provides a valuable approach to uncover meaningful associations among symptoms and offers insights for prevention and intervention strategies. This study aimed to investigate symptom-level associations between anxiety and depression using network analysis and to identify core symptoms, bridge symptoms, and differences in network structure across different stages of adolescence.MethodsThe cross-sectional study was conducted in March 2022 in Shenzhen, China. Participants completed the Generalized Anxiety Disorder Scale-7 and Patient Health Questionnaire Depression Scale, along with demographic questionnaires assessing age and gender. Chinese adolescents aged 10 to 17 who were in Grades 5 or 6 of elementary school, Grades 1 or 2 of middle school, or Grades 1 or 2 of high school, and who could comprehensively understand and read Chinese were recruited as participants. Students in Grade 3 of middle and high schools were excluded due to their upcoming high school or college entrance examinations. Based on age, participants were categorized into early, middle, and late developmental stages of adolescence.Results“Loss of control” was among the most central symptoms in the comorbidity network throughout all three developmental stages; “excessive worry” and “anhedonia” emerged as the core symptoms in early adolescence, and “restlessness” as the core symptom in late adolescence. “Anhedonia,” “sad mood,” and “fatigue” were identified as bridge symptoms between anxiety and depression across all three developmental stages of adolescence. The global strength of the network in middle adolescence was significantly higher compared to the other two stages.ConclusionThese findings highlight the core and bridge symptoms that require special attention and intervention at each stage of adolescence. Moreover, significantly higher network connectivity in middle adolescence suggests this is a critical period for intervention to prevent the development of comorbid mental disorders.

An experimental study on horizontal well waterflooding in the Cretaceous porous carbonate reservoir of Oman

Porous carbonate reservoirs, prevalent in the Middle East, are lithologically dominated by bioclastic limestones, exhibiting high porosity, low permeability, intricate pore structure, and strong heterogeneity. Waterflooding through horizontal wells is commonly used for exploiting these reservoirs. However, challenges persist, such as significant uncertainty and complex operational procedures regarding adjustment effects during the exploitation. The USH reservoir of the Cretaceous D oilfield, Oman exemplifies typical porous carbonate reservoirs. It initially underwent depletion drive using vertical wells, followed by horizontal well waterflooding in the late stage. Currently, the oilfield is confronted with substantial developmental challenges, involving the understanding of residual oil distribution, effective water cut control, and sustaining oil production since it has entered the late development stage. Employing a microscopic visualization displacement system equipped with electrodes, this study elucidated the waterflooding mechanisms and residual oil distribution in the late-stage development of the USH reservoir. The results reveal that the reservoir's vertical stacking patterns act as the main factor affecting the horizontal well waterflooding efficacy. Distinct water flow channels emerge under varying reservoir stacking patterns, with post-waterflooding residual oil predominantly distributed at the reservoir's top and bottom. The oil recovery can be enhanced by adjusting the waterflooding's flow line and intensity. The findings of this study will provide theoretical insights of waterflooding mechanisms and injection-production adjustments for exploiting other porous carbonate reservoirs in the Middle East through horizontal wells.

Distinct Roles for COMPASS Core Subunits Set1, Trx, and Trr in the Epigenetic Regulation of Drosophila Heart Development.

Highly evolutionarily conserved multiprotein complexes termed Complex of Proteins Associated with Set1 (COMPASS) are required for histone 3 lysine 4 (H3K4) methylation. Drosophila Set1, Trx, and Trr form the core subunits of these complexes. We show that flies deficient in any of these three subunits demonstrated high lethality at eclosion (emergence of adult flies from their pupal cases) and significantly shortened lifespans for the adults that did emerge. Silencing Set1, trx, or trr in the heart led to a reduction in H3K4 monomethylation (H3K4me1) and dimethylation (H3K4me2), reflecting their distinct roles in H3K4 methylation. Furthermore, we studied the gene expression patterns regulated by Set1, Trx, and Trr. Each of the COMPASS core subunits controls the methylation of different sets of genes, with many metabolic pathways active early in development and throughout, while muscle and heart differentiation processes were methylated during later stages of development. Taken together, our findings demonstrate the roles of COMPASS series complex core subunits Set1, Trx, and Trr in regulating histone methylation during heart development and, given their implication in congenital heart diseases, inform research on heart disease.

Hard-to-recover reserves of gas, liquid petroleum hydrocarbons, bituminous components, rare, rare-earth and noble metals of the gas-saturated part of oil and gas condensate and gas condensate fields at the late stage of development

Many oil and gas condensate and gas condensate fields discovered in the ‘60s and ‘70s of the last century are now at the late stage of development. Research of the Laboratory for Hard-to-Recover Hydrocarbon Reserves of the Oil and Gas Research Institute of the Russian Academy of Sciences has been devoted to justify an increase in the resource potential and attain a maximum of recovery efficiency for long-developed fields using the evidence from the Orenburg and the Vuktyl oil and gas condensate fields. The involvement of the unique reserves of hard-to-recover oil will help to enhance declining production in old oil and gas producing regions.

On the issue of injection of CO2 into a gas-condensate deposit within the framework of the technology of extraction of oil and retrograde condensate at concentrations below the filtration threshold from gas-saturated oil and gas source deposits of oil and gas condensate fields, at the late stage of development

The study was carried out as part of the work on the hydrodynamic justification of the technology for extracting oil and retrograde condensate at concentrations below the filtration threshold from gas-saturated oil and gas source deposits of oil and gas condensate fields, at the late stages of development. On the basis of a test multicomponent model using the key parameters of the Vuktyl oil and gas condensate field, it is shown that the injection of carbon dioxide, for the early replacement of the “dry” gas injected at the previous stage, entails risks of reducing the liquid hydrocarbon recovery factor and the risks of precipitation of asphaltene-paraffin deposits.

Developmental stages and episode-specific regulatory genes in andromonoecious melon flower development.

Given the lack of specific studies on floral development in melon (Cucumis melo L.), we carried out an extensive study involving morphological and transcriptomic analyses to characterize floral development in this species. Using an andromonoecious line, we analysed the development of floral buds in male and hermaphrodite flowers with both light microscopy and scanning electron microscopy. Based on flower lengths, we established a correlation between the developmental stages and four main episodes of floral development and conducted an extensive RNA sequencing analysis of these episodes. We identified 12 stages of floral development, from the appearance of the floral meristems to anthesis. The main structural differences between male and hermaphrodite flowers appeared between stages 6 and 7; later stages of development leading to the formation of organs and structures in both types of flowers were also described. We analysed the gene expression patterns of the four episodes in flower development to find the genes that were specific to each given episode. Among others, we identified genes that defined the passage from one episode to the next according to the ABCDE model of floral development. This work combines a detailed morphological analysis and a comprehensive transcriptomic study to enable characterization of the structural and molecular mechanisms that determine the floral development of an andromonoecious genotype in melon. Taken together, our results provide a first insight into gene regulation networks in melon floral development that are crucial for flowering and pollen formation, highlighting potential targets for genetic manipulation to improve crop yield of melon in the future.

Disease Activity Indices in Systemic Lupus Erythematosus: What Is New?

Systemic lupus erythematosus (SLE) is a complicated multisystem autoimmune disease that carries substantial mortality and morbidity. The development of new therapies is partly hindered by the lack of a flawless instrument to gauge disease activity in different organs for the primary efficacy outcome. Until a global consensus is reached regarding the most preferred assessment tool, application of any of the existing disease activity indices is acceptable in clinical practice and research. However, one should have a more thorough understanding of the strength and limitations of individual disease activity instruments. This article updates the SLE disease activity indices commonly used in clinical trials and their latest stage of development.

Abstract B039: T cell recruitment dynamics at different stages of primary and metastatic colorectal cancer

Abstract Colorectal cancer (CRC) is the fourth deadliest cancer worldwide, primarily due to metastasis. The current standard therapy for patients with CRC includes neoadjuvant therapies combined with surgery, often resulting in severe complications that affect patients’ lifestyles. Hence, there is a critical need to improve our understanding of the tumor microenvironment to improve the current therapeutics for metastatic CRC. We characterized T cell dynamics in response to tumors at early (1-2 weeks) versus late stages (6-7 weeks) of CRC development in different colon compartments (tumor, epithelium, lamina propria), as well as in colon-draining lymph nodes and the liver as potential metastasis sites. To achieve this, we injected by colonoscopy tumor AKP mouse organoids (mutations in APC, Kras, and P53 genes) or metastatic AKP mouse organoids in the colon walls of a novel fate-mapping strain (iSell-tomato). This model allows for the labelling of peripheral T cells that are recruited to the tissue or tumor sites. Our initial data showed an enhanced recruitment of CD4 and CD8ab T cells to the colon epithelium in early stage AKP (non-metastatic) tumor models compared to late-stage AKP and metastatic-AKP models at both stages. These recruited cells exhibited an enhanced anti-tumorigenic profile characterized by the production of IFNg and reduced frequency of FoxP3+ CD4 regulatory T cells (Tregs). In contrast, T cells recruited to early stage metastatic-AKP tumors and late-stage tumors of the colon and liver exhibited enhanced exhaustion characterized by an increased frequency of PD1+ CD4 and CD8ab T cells. These data suggest a time- and aggressiveness-dependent effect of tumors on immune cell infiltrates that, in turn, affect tumor progression and metastasis. Citation Format: Marwa A Saad, Angelina Bilate, Daniel Mucida. T cell recruitment dynamics at different stages of primary and metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B039.

Unraveling the Role of Sex in Endothelial Cell Dysfunction: Evidence From Lineage Tracing Mice and Cultured Cells.

Biological sex differences play a vital role in cardiovascular diseases, including atherosclerosis. The endothelium is a critical contributor to cardiovascular pathologies since endothelial cells (ECs) regulate vascular tone, redox balance, and inflammatory reactions. Although EC activation and dysfunction play an essential role in the early and late stages of atherosclerosis development, little is known about sex-dependent differences in EC. We used human and mouse aortic EC as well as EC-lineage tracing (Cdh5-CreERT2 Rosa-YFP [yellow fluorescence protein]) atherosclerotic Apoe-/- mice to investigate the biological sexual dimorphism of the EC functions in vitro and in vivo. Bioinformatics analyses were performed on male and female mouse aortic EC and human lung and aortic EC. In vitro, female human and mouse aortic ECs showed more apoptosis and higher cellular reactive oxygen species levels than male EC. In addition, female mouse aortic EC had lower mitochondrial membrane potential (ΔΨm), lower TFAM (mitochondrial transcription factor A) levels, and decreased angiogenic potential (tube formation, cell viability, and proliferation) compared with male mouse aortic EC. In vivo, female mice had significantly higher lipid accumulation within the aortas, impaired glucose tolerance, and lower endothelial-mediated vasorelaxation than males. Using the EC-lineage tracing approach, we found that female lesions had significantly lower rates of intraplaque neovascularization and endothelial-to-mesenchymal transition within advanced atherosclerotic lesions but higher incidents of missing EC lumen coverage and higher levels of oxidative products and apoptosis. RNA-seq analyses revealed that both mouse and human female EC had higher expression of genes associated with inflammation and apoptosis and lower expression of genes related to angiogenesis and oxidative phosphorylation than male EC. Our study delineates critical sex-specific differences in EC relevant to proinflammatory, pro-oxidant, and angiogenic characteristics, which are entirely consistent with a vulnerable phenotype in females. Our results provide a biological basis for sex-specific proatherosclerotic mechanisms.

Loss of Aspm causes increased apoptosis of developing neural cells during mouse cerebral corticogenesis.

Abnormal spindle-like microcephaly associated (ASPM) is a causative gene of primary autosomal recessive microcephaly. Microcephaly is considered to be a consequence of a small brain, but the associated molecular mechanisms are not fully understood. In this study, we generated brain-specific Aspm knockout mice to evaluate the fetal brain phenotype and observed cortical reduction in the late stage of murine cortical development. It has been reported that the total number of neurons is regulated by the number of neural stem and progenitor cells. In the Aspm knockout mice, no apparent change was shown in the neural progenitor cell proliferation and there was no obvious effect on the number of newly generated neurons in the developing cortex. On the other hand, the knockout mice showed a constant increase in apoptosis in the cerebral cortex from the early through the late stages of cortical development. Furthermore, apoptosis occurred in the neural progenitor cells associated with DNA damage. Overall, these results suggest that apoptosis of the neural progenitor cells is involved in the thinning of the mouse cerebral cortex, due to the loss of the Aspm gene in neocortical development.

Immunoglobulin Light Chains Control Permissiveness to Malignant B-Cell Transformation By RAS-Pathway Lesions

Background: RAS-pathway activating mutations (e.g. NRAS, KRAS, BRAF) cause constitutive ERK-activation which can lead to divergent outcomes. ERK-activation not only drives early stages of pre-B cell differentiation (Yasuda et al., 2008) but also induces negative selection and cell death to eliminate aberrantly activated B-cells at later stages of development (Limnander et al., 2011). Germline mutations in this pathway cause lymphoproliferative disorder and autoimmunity but not leukemia. Furthermore, activating lesions are very common in B-cell malignancies derived from the earliest (e.g. B-ALL) and the latest (e.g. multiple myeloma) stages of B-cell development, but they are rarely found in B-cell receptor (BCR)-expressing mature B-cell derived tumors ( Figure 1). These findings are consistent with previous observations that transgenic mouse models for RAS-oncogene were able to transform BCR-negative B-cell precursors to develop B-cell acute lymphoblastic leukemia (Chan et al., 2020). In contrast, transgenic expression of these oncogenes fails to transform BCR-positive mature B-cells (Mullins et al., 2013; Chung et al., 2014). Signals from the BCR, consisting of immunoglobulin (Ig) heavy chains (μ HC) and conventional light chains (kappa LC or lambda LC), and its precursor the pre-BCR, consisting of μ HC and surrogate light chains (SLC; λ5 and VpreB), regulate the development and function of B-cells. Here, we investigated whether structural elements of the (pre-) BCR determine the outcome of oncogenic RAS activation and how oncogenic RAS in B-ALL avoids cell death to promote leukemogenesis. Results: We first studied the impact of oncogenic RAS-activation in BCR-positive mantle cell lymphoma cells. Expression of NRAS G12D in BCR-positive mantle cell lymphoma (JeKo-1) cells transiently reduced the surface expression of μ HC and eventually resulted in depletion of cells from culture in growth competition assays. We then examined whether CRISPR/Cas9-based genetic ablation of BCR components will enable permissiveness to oncogenic RAS signaling. Despite loss of μ HC function, oncogenic RAS caused depletion of JeKo-1 cells from culture. In contrast, this effect was partially reversed by genetic ablation of the constant region of immunoglobulin kappa LC ( IGKC). We next examined whether reconstitution of surrogate light chain (SLC) components of the pre-BCR will confer permissiveness to oncogenic RAS signaling in JeKo-1 cells. We engineered JeKo-1 cells to express Dox-inducible expression of λ5 (Igll1) and VpreB. In the presence of kappa LC, overexpression of SLC alone did not reverse the deleterious impact of oncogenic RAS expression. Notably, reconstitution of SLC components following genetic deletion of IGKC amplified the effect of IGKC-deletion alone and largely rescued cells from oncogenic RAS-induced survival disadvantage. Taken together, our results showed that immunoglobulin light chains control the consequences of oncogenic RAS activation in B-cells - while SLC of the pre-BCR plays a productive role in enabling oncogenic RAS-signaling, conventional light chain of the BCR plays a counterproductive role. Supporting this scenario, we found that SLC enabled oncogenic RAS to massively induce the proto-oncogene Bcl6 at the expense of the tumor suppressor Prdm1 to avoid cell death, promoting RAS-driven transformation of B-cell precursors. Upon genetic ablation of λ5, upregulation of Bcl6 was abrogated while Prdm1 was de-repressed. Consequently, RAS-driven malignant transformation of B-cell precursors was subverted. Reflecting the critical role of SLC in enabling RAS-BCL6 signaling, mature B-cells, which express BCR and lack SLC, failed to induce Bcl6 upon oncogenic RAS-activation, leading to upregulation of Prdm1. This observation may explain why persistent activation of the RAS-pathway in mature B-cells can lead to negative selection and cell death (Limnander et al., 2011). Conclusions: Our results demonstrated that immunoglobulin light chains determine permissiveness to RAS-driven malignant transformation of B-cells. Expression of SLC enables RAS-BCL6 activation, allowing B-cell precursors to avoid PRDM1-mediated tumor suppression to promote leukemogenesis. In contrast, conventional LC opposes oncogenic RAS signaling, providing a mechanistic basis of the low frequency of RAS-pathway activating mutations in BCR-expressing mature B-cell lymphomas.

Transgenic Mouse Model for Translational Immunotherapy Studies on Multiple Myeloma

Background Genetically engineered mouse models that recapitulate key immunological features of human multiple myeloma may lend themselves to preclinical design and testing of novel immune therapies that target members of the tumor necrosis factor (TNF) receptor superfamily in addition to BCMA. One model of this sort are double-transgenic IL6Myc mice that spontaneously develop myeloma-like plasma cell tumors (PCTs) with full genetic penetrance (100% tumor incidence) and relatively short latency (4-5 months) in presence of an intact innate and adaptive immune system (Rutsch et. al. Blood 2010). IL6Myc mice exhibit serum M-spikes, respond to proteasome inhibition, and develop osteolytic lesions mimicking myeloma bone disease (Duncan K et. al. Blood Cancer J 2013, Sun F et. al. Haematologica 2020). Here, we examined changes in the immune tumor microenvironment during PCT progression in IL6Myc mice. We found that neoplastic plasma cell development was accompanied by accumulation of inhibitory T cells, T cell exhaustion, and elevated serum levels of MIP-3α and MCP-5. A subset of aberrant plasma cells expressed the TNF receptors, TACI and BAFF-R, which, together with their ligands, APRIL and BAFF, may serve as immunotherapeutic targets in myeloma not responding to BCMA-targeted treatment (Wong DP et. al. Nat Commun 2022, Braunstein M et. al. Expert Rev Hematol 2021). Materials and methods Double-transgenic IL6Myc mice were bred by intercrossing homozygous transgenic C.iMyc ΔEμ (Duncan K et. al. Blood Cancer J 2013) and heterozygous transgenic C.IL6 mice (Kovalchuk AL. et. al. Proc Natl Acad Sci USA 2002). Age-matched IL6Myc mice were investigated at an early (5-7 weeks), intermediate (10-12 weeks) and late (14-17 weeks) stage of PCT development using flow cytometry (Cytek Aurora) and serum cytokine levels (Bio-Plex) as main research tools. Results Bone marrow (BM) plasma cells, defined as CD45-gated B220 -CD138 + cells, increased during PCT development, as one might have expected: 10.5 ± 5.8% and 26.8 ± 6.4% ( p< 0.05) in early- and late-stage mice, respectively. The proportion of B220 +CD19 + B cells fell accordingly: 29.6 ± 4.1% vs 5.5 ± 4.9% ( p< 0.0001). Consistent with the notion that the BM microenvironment of PCT-bearing mice is immunosuppressive, there was a 2-fold elevation of TGF-β expressing T cells and a 4-fold elevation of IL-10 expressing T cells in the BM when late-stage mice were compared to early-stage mice. A sizeable fraction of aberrant plasma cells expressed TACI and BAFF-R in addition to BCMA ( Figure 1, left). Spleens of IL6Myc mice undergoing tumor development contained increasing numbers of FoxP3 + regulatory T cells (8.6 ± 2.1% at early stage vs 14.0 ± 4.6% at late stage, p< 0.05) and CD11b +Ly-6G/C + myeloid-derived suppressor cells (21.2 ± 9.2% vs 51.6 ± 10.1%, p< 0.01). Exhausted T cells, detected via expression of PD-1, TIM-3, LAG-3 and TIGIT, increased in sync with PCT progression ( Figure 1, right). Elevated serum levels of inflammatory cytokines, including MIP-3α (95.3 ± 41.9 pg/ml in early-stage mice vs 514 ± 279 pg/ml in late-stage mice, p< 0.01) and MCP-5 (121 ± 43.9 pg/ml vs 284 ± 70.5 pg/ml, p< 0.05), agreed with flow-based tissue data. Conclusion Neoplastic plasma cell development in IL6Myc mice takes place in an immune suppressed tumor microenvironment similar to that promoting human myeloma. IL6Myc-driven PCTs may provide a good experimental model system for novel immunotherapeutic approaches that target TACI and BAFF-R or their ligands, APRIL and BAFF. Previous work to that end holds great promise but has been challenging thus far (Goodman DB et. al. Sci Transl Med 2022, Lee L et. al. J Immunother Cancer 2023).