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Journal articles on the topic 'Gene editing germinale'
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1
Sutton, Agneta. "Editing della linea germinale: quali sono i rischi sociali e morali? / Germ-line gene editing: What are the social and moral risks?" Medicina e Morale 65, no. 2 (September 21, 2016): 123–30. http://dx.doi.org/10.4081/mem.2016.430.
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Dovremmo accogliere tutti i possibili sviluppi dell’editing genetico? L’editing genetico delle cellule somatiche potrebbe essere considerato alla pari delle terapie convenzionali volte a trattare particolari patologie o ad alleviarne i sintomi. Tale intervento interesserebbe esclusivamente il singolo paziente trattato. Esso potrebbe quindi essere ben accolto come un nuovo tipo di trattamento per i tumori e le malattie del sangue, come ad esempio la beta-talassemia. Diversamente, l’editing della linea germinale avrebbe effetti ereditari. Ciò solleva preoccupazioni particolari riguardo al rischio medico. I rischi medici non sono, tuttavia, gli unici tipi di rischi che possono derivare dalla modificazione genetica della linea germinale. Nel contributo non vengono discussi i rischi medici, ma quelli sociali e morali correlati alla manipolazione genetica della linea-germinale. ---------- Should we welcome all developments in gene editing? Somatic cell gene editing would be on a par with conventional therapies aimed at treating particular conditions or alleviating symptoms. It would solely affect the individual patient treated. It could thus serve as a welcome new kind of treatment for cancers and blood diseases such as ß-thalassaemia. Germ-line gene editing, on the other hand, would have hereditary effects. This raises special concerns about medical mishaps. Medical risks are, however, not the only kinds of risks in the case of germline gene editing. Discussed here are not the medical risks, but the social and moral risks of germ-line-gene editing.
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Ben Shlush, Ilan Ben, Aviva Samach, Cathy Melamed-Bessudo, Daniela Ben-Tov, Tal Dahan-Meir, Shdema Filler-Hayut, and Avraham A. Levy. "CRISPR/Cas9 Induced Somatic Recombination at the CRTISO Locus in Tomato." Genes 12, no. 1 (December 31, 2020): 59. http://dx.doi.org/10.3390/genes12010059.
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Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F1 hybrids from divergent parental lines, each with a different mutation at the Carotenoid isomerase (CRTISO) locus. IHSR events, namely crossover or gene conversion (GC), between the two CRTISO mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F2 seeds were grown to test for germinal transmission of HR events. Two out of five F1 plants showing red sectors had their IHSR events germinally transmitted to F2, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.
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Henderson, Sam W., Steven T. Henderson, Marc Goetz, and Anna M. G. Koltunow. "Efficient CRISPR/Cas9-Mediated Knockout of an Endogenous PHYTOENE DESATURASE Gene in T1 Progeny of Apomictic Hieracium Enables New Strategies for Apomixis Gene Identification." Genes 11, no. 9 (September 10, 2020): 1064. http://dx.doi.org/10.3390/genes11091064.
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Most Hieracium subgenus Pilosella species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic Hieracium by targeting mutations in the endogenous PHYTOENE DESATURASE (PDS) gene using Agrobacterium-mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of PDS knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify PDS gene edits. Up to 86% of seeds produced albino seedlings with complete PDS knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing Cas9 segregation from the PDS target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic Hieracium, enabled development of the discussed strategies for future identification of causal apomixis genes.
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Yan, Yi, and Betty Diamond. "A crucial role of IL-6 in B cell tolerance induction after antigen activation (167.6)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 167.6. http://dx.doi.org/10.4049/jimmunol.186.supp.167.6.
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Abstract Receptor editing, clonal deletion and anergy are the key components of the central tolerance. It is well characterized that receptor editing plays the most important role among the three mechanisms. We recently identified a post germinal center tolerance check point, where receptor editing is re-induced to diminish the autoreactive B cells generated by class switching and somatic hypermutation. Here we demonstrate that this de-novo re-induction of the recombinase RAG gene in antigen-activated B cells is reduced by neutralization of IL-6. Serum level of autoreactive anti-dsDNA antibodies is increased in mice immunized with a peptide mimetope of dsDNA if they are given antibodies to IL-6 to reduce receptor editing. The same effect is observed in mice haploid for IL-6 when compared to WT mice. This dependence on IL-6 is B cell intrinsic, as demonstrated in adoptive transfer experiments when B cells from either WT mice or IL-6+/- mice were reconstituted in uMT mice. Our data reveal a novel but crucial role of IL-6 in regulation of the B cell autoimmunity, and show that Il-6 serves a regulatory function as well as being proinflammatory.
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Hikida, Masaki, Yasunori Nakayama, Yumi Yamashita, Yoshio Kumazawa, Shin-Ichi Nishikawa, and Hitoshi Ohmori. "Expression of Recombination Activating Genes in Germinal Center B Cells: Involvement of Interleukin 7 (IL-7) and the IL-7 Receptor." Journal of Experimental Medicine 188, no. 2 (July 20, 1998): 365–72. http://dx.doi.org/10.1084/jem.188.2.365.
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Mouse germinal center (GC) B cells have been shown to undergo secondary V(D)J (V, variable; D, diversity; J, joining) recombination (receptor editing) mediated by the reexpressed products of recombination activating gene (RAG)-1 and RAG-2. We show here that interleukin (IL)-7 as well as IL-4 was effective in inducing functional RAG products in mouse IgD+ B cells activated via CD40 in vitro. Blocking of the IL-7 receptor (IL-7R) by injecting an anti– IL-7R monoclonal antibody resulted in a marked suppression of the reexpression of RAG-2 and subsequent V(D)J recombination in the draining lymph node of immunized mice, whereas RAG-2 expression was not impaired in immunized IL-4–deficient mice. Further, these peripheral B cells activated in vitro or in vivo were found to express IL-7R. These findings indicate a novel role for IL-7 and IL-7R in inducing receptor editing in GC B cells.
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Hikida, Masaki, and Hitoshi Ohmori. "Rearrangement of λ Light Chain Genes in Mature B Cells In Vitro and In Vivo. Function of Reexpressed Recombination-activating Gene (RAG) Products." Journal of Experimental Medicine 187, no. 5 (March 2, 1998): 795–99. http://dx.doi.org/10.1084/jem.187.5.795.
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V(D)J (V, variable; D, diversity; J, joining) combination of immunoglobulin (Ig) genes established in premature B cells has been thought to be conserved throughout differentiation at mature stages. However, germinal center (GC) B cells have been shown to reexpress recombination-activating gene (RAG)-1 and RAG-2 proteins in immunized mice. Here, we present several lines of evidence indicating that RAG proteins thus induced are functional as the V(D)J recombinase. DNA excision product reflecting Vλ1 to Jλ1 rearrangement was generated in parallel with the expression of RAG genes in mature mouse B cells that were activated in vitro with LPS and IL-4. Similar λ chain gene rearrangement was observed in the draining lymph node of immunized mice. Further, B cells that underwent λ gene rearrangement were shown by in situ PCR to be localized within GCs. Thus, secondary rearrangement of Ig genes (receptor editing) can occur in mature B cells.
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Vavassori, Valentina, Elisabetta Mercuri, Genni Marcovecchio, Maria Carmina Castiello, Daniele Canarutto, Claudia Asperti, Aurelien Jacob, et al. "Towards Clinical Translation of Hematopoietic Cell Gene Editing for Treating Hyper-IgM Type 1." Blood 138, Supplement 1 (November 5, 2021): 3978. http://dx.doi.org/10.1182/blood-2021-148572.
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Abstract Hyper-IgM Type 1 (HIGM1) is caused by mutations of CD40L, whose absence in CD4 T cells impairs signaling for B cell activation and Ig class-switching. Since unregulated CD40L expression leads to lymphoproliferations/lymphomas in the mouse model of the disease, gene correction must preserve the physiological regulation of the gene. Gene editing of either autologous T cells or hematopoietic stem cells (HSC) held promise for treating HIGM1. We developed a "one size fits all" editing strategy to insert a 5'-truncated corrective CD40L cDNA in the first intron of the native human gene, effectively making expression conditional to targeted insertion in the intended locus. By exploiting a protocol that preserves T stem memory cells (TSCM), we reproducibly obtained ~40% of editing efficiency in healthy donor and patients derived T cells, restoring regulated, although partial, CD40L surface expression. The reconstituted level of expression, however, was sufficient to fully restore helper function to B cells. In order to select, track and potentially deplete edited T cells, we coupled the corrective cDNA with a clinically compatible selector gene and confirmed that enriched T cells preserved their engraftment capacity in NSG mice. Unexpectedly, the presence of an IRES-linked downstream coding frame counteracted the shorter half-life of transcript from the edited locus, allowing replenishment of intracellular stores and surface translocation of physiological amounts of CD40L upon activation. We also tailored the CD40L editing strategy to human HSC, reaching up to 15-30% editing in HSC long term engrafting NSG mice, depending on the HSC source. We then modelled the therapeutic potential of both T cell and HSC gene therapy by infusing increasing proportions of WT murine cells, as surrogates of edited cells, in HIGM1 mice. Administration of functional T cells at clinically relevant doses in HIGM1 mice, preconditioned or not with different lymphodepleting regimens, achieved long term stable T cell engraftment and partial rescue of antigen specific IgG response and germinal center formation in splenic follicles after vaccination with a thymus dependent antigen. Remarkably, infusion of T cells from mice pre-exposed to the antigen, mimicking treatment of chronically infected patients, was effective even in absence of conditioning and protected the mice from a disease relevant infection induced by the opportunistic pathogen Pneumocystis murina. Transplantation of functional T cells admixed with an equal number of HIGM1 T cells resulted in lower vaccination response, indicating competition between WT and HIGM1 cells and implying that increasing the fraction of corrected cells in the graft by selection would improve immune reconstitution. Concerning HSC gene therapy, transplanting 25% WT cells along with HIGM1 ones in HIGM1 mice - mirroring the editing efficiencies achieved in human HSC - rescued antigen specific IgG response and established protection from pathogen comparably to T cell therapy. These findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T cell as competitive strategy to HSC gene therapy, because of more straightforward translation, lower safety challenges and potentially comparable clinical benefits. We thus embarked in assessing GMP compliant reagents and protocols for T cell activation, culture and editing and developed a scalable manufacturing process. Optimization of clinical grade culture conditions allowed further increasing editing efficiency, total cellular yield and maintenance of TSCM thus paving the way to the design of a clinical trial. Disclosures Naldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.
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Santini, Paul A., Bing He, April Chiu, Susan C. Ball, Kang Chen, Lawrence A. Kingsley, Charles R. Rinaldo, et al. "HIV-1 induces targeted down-regulation of the Ig gene-diversifying enzyme AID in the germinal center of infected lymphoid follicles (45.1)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S57. http://dx.doi.org/10.4049/jimmunol.178.supp.45.1.
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Abstract Class switching from IgM to IgG and IgA is essential for antiviral immunity and requires activation-induced cytidine deaminase (AID), an APOBEC family member with DNA-editing activity. Germinal center (GC) B cells express AID upon activation by CD4+ T cells through CD40 ligand (CD40L) and IL-4. HIV-1 is thought to impair IgG and IgA responses to viral antigens, opportunistic pathogens and vaccines by causing progressive loss of CD4+ T cells and by rendering B cells poorly responsive to CD4+ T cell help. It remains unknown whether HIV-1 targets AID to hamper protective IgG and IgA responses. We found that infected GCs contained less AID, but normal APOBEC3G, an AID-related RNA-editing protein. AID down-regulation was not associated with local loss of CD4+ T cells and CD40L, but rather correlated with decreased activation of AID-inducing transcription factors, such as NF-κB and STAT6, and with increased expression of feedback inhibitors of NF-κB and STAT6, including IκBα, SOCS1 and SOCS3. AID down-regulation also correlated with accumulation of the viral protein Nef in the GC and with trafficking of Nef within membrane channels connecting infected myeloid cells to B cells. Together with our recent in vitro studies showing that Nef penetrates B cells and inhibits class, the present in vivo data suggest that HIV-1 evades protective IgG and IgA responses by targeting the class switch recombinase machinery.
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Ghia, Emanuela M., Sonia Jain, George F. Widhopf, Laura Z. Rassenti, Michael J. Keating, William G. Wierda, John G. Gribben, et al. "Use of IGHV3–21 in chronic lymphocytic leukemia is associated with high-risk disease and reflects antigen-driven, post–germinal center leukemogenic selection." Blood 111, no. 10 (May 15, 2008): 5101–8. http://dx.doi.org/10.1182/blood-2007-12-130229.
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Abstract We examined the chronic lymphocytic leukemia (CLL) cells of 2457 patients evaluated by the CLL Research Consortium (CRC) and found that 63 (2.6%) expressed immunoglobulin (Ig) encoded by the Ig heavy-chain-variable-region gene (IGHV), IGHV3-21. We identified the amino acid sequence DANGMDV (motif-1) or DPSFYSSSWTLFDY (motif-2) in the Ig heavy-chain (IgH) third complementarity-determining region (HCDR3) of IgH, respectively, used by 25 or 3 cases. The IgH with HCDR3 motif-1 or motif-2, respectively, was paired with Ig light chains (IgL) encoded by IGLV3-21 or IGKV3-20, suggesting that these Ig had been selected for binding to conventional antigen(s). Cases that had HCDR3 motif-1 had a median time from diagnosis to initial therapy comparable with that of cases without a defined HCDR3 motif, as did cases that used mutated IGHV3-21 (n = 27) versus unmutated IGHV3-21 (n = 30). Of 7 examined cases that used Ig encoded by IGHV3-21/IGLV3-21, we found that 5 had a functionally rearranged IGKV allele that apparently had incurred antigendriven somatic mutations and subsequent rearrangement with KDE. This study reveals that CLL cells expressing IGHV3-21/IGLV3-21 most likely were derived from B cells that had experienced somatic mutation and germinal-center maturation in an apparent antigen-driven immune response before undergoing Ig-receptor editing and after germinal-center leukemogenic selection.
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Jing, Changshuang, Min Wei, Peng Fang, Rentao Song, and Weiwei Qi. "Pollen-Specific CRISPR/Cas9 System to Increase Heritable Gene Mutations in Maize." Agriculture 11, no. 8 (August 7, 2021): 751. http://dx.doi.org/10.3390/agriculture11080751.
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The CRISPR/Cas9 system has been widely utilized in plant biotechnology as a gene editing tool. However, a conventional design with ubiquitously expressed CRISPR/Cas9 was observed to cause large numbers of somatic mutations that complicated the identification of heritable mutations. We constructed a pollen-specific CRISPR/Cas9 (PSC) system using pollen-specific promoters of maize Profilin 1 and Profilin 3 (pZmPRO1 and pZmPRO3) to drive Cas9 expression, and the bZIP transcription factor Opaque2 (O2) was employed as the target gene. The maize ubiquitin promoter (pZmUbi)-driven CRISPR/Cas9 (UC) system was employed as a control. We generated transgenic plants for the PSC and UC systems and analyzed three independent events for each system. We found that the pZmPRO1 PSC system generated no target gene mutations in the T0 generation but successfully generated 0–90% target gene mutations in the T1 generation. A total of 31 of 33 mutations in the T1 generation could be inherited in the T2 generation. In addition, 88.9–97.3% of T2 mutations were from the T1 generation. The UC system generated mutations in the T0 generation, and 0%, 50% and 92.9% of T1 mutations were from the T0 generation. Our results demonstrate that the PSC system provided stable, heritable mutants in the next generation, and this approach might also be applied in other crops using germinal cell-specific CRISPR/Cas9 systems to facilitate plant breeding.
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Pasqualucci, Laura, Mara Compagno, Tongwei Mo, Paula Smith, Herbert C. Morse, V. V. V. S. Murty, and Riccardo Dalla-Favera. "Activation Induced Cytidine Deaminase (AID) Is Required for Germinal-Center Derived Lymphomagenesis." Blood 108, no. 11 (November 16, 2006): 223. http://dx.doi.org/10.1182/blood.v108.11.223.223.
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Abstract Most B cell non-Hodgkin’s lymphomas (B-NHL) derive from germinal center (GC) B cells and their pathogenesis is associated with the accumulation of distinct genetic lesions, including chromosomal translocations and a more recently identified mechanism of genomic instability, termed aberrant somatic hypermutation. These alterations are thought to be due to mistakes occurring during two GC-associated immunoglobulin (Ig) genes remodeling processes: class switch recombination (CSR) and somatic hypermutation (SHM). However, this model has never been formally proven. To conclusively investigate the role of CSR and SHM in the pathogenesis of B-NHL, we examined whether lymphoma development in mice requires the function of activation induced cytidine deaminase (AID), a DNA editing enzyme expressed specifically in GC and activated B cells and essential for both processes. Three transgenic mouse models were generated by crossing lymphoma-prone mice (λMYC, λMYC/IμHABCL6 and IμHABCL6) with mice (AID−/−) that are unable to undergo both SHM and CSR. The λMYC mice develop a diffusely infiltrating monoclonal proliferation of pre-GC origin, with unmutated IgV genes and lack of BCL6 expression, and therefore presumably independent from AID-associated DNA remodeling events. Conversely, lymphomas in λMYC/IμHABCL6 and IμHABCL6 mice recapitulate GC/post GC-derived malignancies, in that the former display somatically mutated IgV genes and upregulation of post-GC markers (CD138) in most of the cases, while the latter develop a splenic lymphoproliferative syndrome that culminates, past 12 months of age, in clonal B cell lymphomas with DLBCL morphology and somatically mutated IgV genes (~70% of the animals) (Cattoretti et al., Cancer Cell 7:445–455, 2005). Mice were monitored for tumor incidence and survival, and a combination of histologic, immunophenotypic and gene expression profiling analysis was used for tumor characterization. As expected, no significant differences in event-free survival and lymphoma type were observed between AID-proficient and AID-deficient λMYC mice, in agreement with their pre-GC derivation. Conversely, a phenotypic shift of the tumor was observed in λMYC/IμHABCL6 mice when bred into an AID−/− background, with >80% of the cases (N=21/26) reverting to a pre-GC phenotype (loss of GC/post GC markers) undistinguishable from that of the λMYC and λMYC/AID−/− mice. Gene expression profile analysis on representative cases (N=10 λMYC/IμHABCL6 and 5 each for λMYC, λMYC/AIDKO, λMYC/IμHABCL6/AIDKO) confirmed significant phenotypic similarities between pre-GC derived λMYC lymphomas and the λMYC/IμHABCL6/AID −/− lymphomas, which co-segregated in a separate cluster from λMYC/IμHABCL6 tumors. Analogously, a significant reduction in DLBCL frequency was observed in the IμHABCL6/AIDKO cohort as compared to IμHABCL6 mice (N= 4/19, 21% vs 8/14, 57%; p=0.03). Taken together, these results indicate that GC-derived lymphomas cannot develop in the absence of AID, thereby providing direct support to the notion that AID-mediated mistakes in antigen receptor gene modification events (CSR and SHM) represent major contributors to B-NHL pathogenesis.
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Hoang, Doan, and Simon Hoang. "Deep learning - cancer genetics and application of deep learning to cancer oncology." Vietnam Journal of Science and Technology 60, no. 6 (December 30, 2022): 885–928. http://dx.doi.org/10.15625/2525-2518/17256.
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Arguably the human body has been one of the most sophisticated systems we encounter but until now we are still far from understanding its complexity. We have been trying to replicate human intelligence by way of artificial intelligence but with limited success. We have discovered the molecular structure in terms of genetics, performed gene editing to change an organism’s DNA and much more, but their translatability into the field of oncology has remained limited. Conventional machine learning methods achieved some degree of success in solving problems that we do not have an explicit algorithm. However, they are basically shallow learning methods, not rich enough to discover and extract intricate features that represent patterns in the real environment. Deep learning has exceeded human performance in pattern recognition as well as strategic games and are powerful for dealing with many complex problems. High-throughput sequencing and microarray techniques have generated vast amounts of data and allowed the comprehensive study of gene expression in tumor cells. The application of deep learning with molecular data enables applications in oncology with information not available from clinical diagnosis. This paper provides fundamental concepts of deep learning, an essential knowledge of cancer genetics, and a review of applications of deep learning to cancer oncology. Importantly, it provides an insightful knowledge of deep learning and an extensive discussion on its challenges. The ultimate purpose is to germinate ideas and facilitate collaborations between cancer biologists and deep learning researchers to address challenging oncological problems using advanced deep learning technologies.
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Zhang, Zhaohan, Wanpeng Wang, Shahid Ali, Xiao Luo, and Linan Xie. "CRISPR/Cas9-Mediated Multiple Knockouts in Abscisic Acid Receptor Genes Reduced the Sensitivity to ABA during Soybean Seed Germination." International Journal of Molecular Sciences 23, no. 24 (December 18, 2022): 16173. http://dx.doi.org/10.3390/ijms232416173.
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Abscisic acid (ABA) is an important plant hormone that regulates numerous functions in plant growth, development, and stress responses. Several proteins regulate the ABA signal transduction mechanism in response to environmental stress. Among them, the PYR1/PYL/RCAR family act as ABA receptors. This study used the CRISPR/Cas9 gene-editing system with a single gRNA to knock out three soybean PYL genes: GmPYL17, GmPYL18, and GmPYL19. The gRNA may efficiently cause varying degrees of deletion of GmPYL17, GmPYL18, and GmPYL19 gene target sequences, according to the genotyping results of T0 plants. A subset of induced alleles was successfully transferred to progeny. In the T2 generation, we obtained double and triple mutant genotypes. At the seed germination stage, CRISPR/Cas9-created GmPYL gene knockout mutants, particularly gmpyl17/19 double mutants, are less susceptible to ABA than the wild type. RNA-Seq was used to investigate the differentially expressed genes related to the ABA response from germinated seedlings under diverse treatments using three biological replicates. The gmpyl17/19-1 double mutant was less susceptible to ABA during seed germination, and mutant plant height and branch number were higher than the wild type. Under ABA stress, the GO enrichment analysis showed that certain positive germination regulators were activated, which reduced ABA sensitivity and enhanced seed germination. This research gives a theoretical basis for a better understanding of the ABA signaling pathway and the participation of the key component at their molecular level, which helps enhance soybean abiotic stress tolerance. Furthermore, this research will aid breeders in regulating and improving soybean production and quality under various stress conditions.
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Tsubata, Takeshi. "B-cell tolerance and autoimmunity." F1000Research 6 (March 29, 2017): 391. http://dx.doi.org/10.12688/f1000research.10583.1.
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Self-reactive B cells are tolerized at various stages of B-cell development and differentiation, including the immature B-cell stage (central tolerance) and the germinal center (GC) B-cell stage, and B-cell tolerance involves various mechanisms such as deletion, anergy, and receptor editing. Self-reactive B cells generated by random immunoglobulin variable gene rearrangements are tolerized by central tolerance and anergy in the periphery, and these processes involve apoptosis regulated by Bim, a pro-apoptotic member of the Bcl-2 family, and regulation of B-cell signaling by various phosphatases, including SHIP-1 and SHP-1. Self-reactive B cells generated by somatic mutations during GC reaction are also eliminated. Fas is not directly involved in this process but prevents persistence of GC reaction that allows generation of less stringently regulated B cells, including self-reactive B cells. Defects in self-tolerance preferentially cause lupus-like disease with production of anti-nuclear antibodies, probably due to the presence of a large potential B-cell repertoire reactive to nucleic acids and the presence of nucleic acid-induced activation mechanisms in various immune cells, including B cells and dendritic cells. A feed-forward loop composed of anti-nuclear antibodies produced by B cells and type 1 interferons secreted from nucleic acid-activated dendritic cells plays a crucial role in the development of systemic lupus erythematosus.
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Kuraoka, Masayuki, T. Holl, Dongmei Liao, Mandy Womble, and Garnett Kelsoe. "Aicda mediates central tolerance in B cells (167.24)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 167.24. http://dx.doi.org/10.4049/jimmunol.186.supp.167.24.
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Abstract Activation-induced cytidine deaminase (AID), the product of Aicda gene, is required for somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin genes. Although crucial for humoral immunity, these processes exact genomic damage that often results in cell death, and occasionally oncogenesis. In mice, AID expression, SHM and CSR are thought to be restricted to mature B cells in germinal centers. We reported that developmentally immature B cells in mice and humans express low levels of AID, which support limited SHM and CSR. However, the physiological significance of AID expression and activity in these B-cell compartments is controversial. Here we show that low levels of AID in immature mouse bone marrow B cells suppress the development of autoreactivity. Aicda deficient mice exhibit significantly increased serum autoantibody and reduced capacity to purge autoreactive immature/transitional B cells; the clonal deletion and heavy chain receptor editing characteristics of autoimmune 3H9 VDJ knock-in mice is significantly impaired in the absence of AID expression. In vitro, AID deficient immature/transitional B cells are more resistant to anti-IgM induced apoptosis than their normal counterparts. Thus, early AID expression in immature B cells plays a fundamental and unanticipated role in purging self-reactive B cells during their development in the bone marrow. We are currently investigating signals that induce AID expression in immature B cells in vitro.
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Ten Hacken, Elisa, Shanye Yin, Kendell Clement, Robert A. Redd, Maria Hernandez-Sanchez, Shuqiang Li, Michaela Gruber, et al. "Interrogation of Individual CLL Loss-of-Function Lesions By CRISPR In Vivo Editing Reveals Common and Unique Pathway Alterations." Blood 134, Supplement_1 (November 13, 2019): 684. http://dx.doi.org/10.1182/blood-2019-127673.
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Mouse models represent invaluable tools for the systematic evaluation of cancer drivers, yet models that address the impact of putative genetic drivers of chronic lymphocytic leukemia (CLL) on B cell development and function are largely lacking. To study recurrent loss-of-function (LOF) mutations observed in human CLL, we established a transplant model that can rapidly evaluate genetic lesions. First, we crossed mice carrying B-cell restricted Cre expression (Cd19-cre) with mice carrying conditional Cas9-GFP, to generate a strain expressing B cell-restricted Cas9 (Cd19-Cas9). Next, we optimized methods for in vitro engineering of early stem and progenitor cells (Lin- Sca-1+ c-kit+ [LSK]) from Cd19-Cas9 mice using lentivirus expressing sgRNAs (mCherry+)targeting Atm, Trp53, Chd2, Birc3, Mga, or Samhd1. We chose LSKs because of their high transducibility and long-term repopulating potential. Last, we transplanted the single sgRNA-expressing LSKs into sub-lethally irradiated CD45.1 recipient mice, and then confirmed presence of ~45-85% gene-edited sequences (>70% carrying frameshift mutations) in edited B cells (GFP+mCherry+) at 2 months post-transplant, by PCR-based targeted deep sequencing and CRISPResso software analysis. We also verified presence of gene alterations (and putative off-target lesions) at the single cell DNA level (targeted sequencing by Tapestri, Mission Bio). We first asked whether presence of the 6 LOFs could impact B cell developmental trajectories in marrow, spleen and peritoneum at 4 months post-transplant, a time point by which B cells are considered to achieve optimal host reconstitution (n=5/group, including a non-targeting control group). No marked changes were observed in mice with Atmindel, Trp53indel, Chd2indel, Birc3indel or Samhd1indel, as analyzed by flow cytometry. Of interest, however, Mgaindel mice were detected to have increased germinal center (B220+CD95+CD38-) and marginal zone (B220+CD21highCD23-) splenic B cells, and also showed increased B1a (CD5+ B220low CD23- CD43+) and decreased B1b (CD5- B220low CD23- CD43+) cells in the peritoneum (p<0.05, ANOVA). These results indicate that the likely negative regulatory role that Mga exerts on MYC networks may directly impact germinal center formation and cell fate determination in B cells. The overall abundance of edited B cells in spleen and blood of each group was higher (overall median: 17.0%; 90%CI 6.7-58.8%) than the non-targeting control (8.4%; 90%CI 1.6-14.2%) at 4 months post-transplant (n=8/group, p<0.05, ANOVA), and abundance of edited cells increased in peripheral bleeds at 4 vs. 2 months (n=8/group, p<0.05, Wilcoxon signed rank test). This suggests that presence of individual alterations can alter pro-survival pathways in mature B cells, through mechanisms that may, at least partly, be shared across LOFs. To address this question, we analyzed the transcriptional profiles of edited B cell splenocytes (n=3/group), and compared them to their non-edited counterparts (GFP+mCherry- splenocytes from the same animal), identifying a total of ~3900 differentially expressed genes among the 6 groups (p<0.05, paired Student's t test). Notably, changes in gene expression were highly concordant across 5 of the 6 groups (Spearman r >0.37 for each of the 10 pairs of 5 groups), with the exception of Mgaindel, consistent with its unique phenotype, observed in developmental studies. Gene ontology analyses using Enrichr confirmed commonalities in pathway dysregulations across the 5 similar groups of mice (p<0.05), such as modulation of Notch signaling in Chd2indel, Samhd1indel, and Birc3indel, serine/glycine metabolism in Atmindel, Trp53indel, and Chd2indel, and oxidative phosphorylation in Atmindel and Samhd1indel. Unique to Mgaindel, we saw enrichment of the GOs for transcriptional mis-regulation in cancer and cellular senescence, both relevant for tumorigenesis and B cell development. In conclusion, we demonstrate that common LOFs typical of patients with CLL lead to increased cellular fitness in B-cell restricted mouse models, while dysregulating pro-survival pathways relevant to B cell development, CLL pathogenesis and more broadly to tumorigenesis. We are currently exploring phenotypic similarities and differences through tailored functional assays, while addressing the relative contribution of each alteration to CLL development in multiplexed edited mouse lines. Disclosures Wang: Mission Bio Inc.: Employment. Jacob:Mission Bio Inc.: Employment. Flynn:Mission Bio Inc.: Employment. Ruff:Mission Bio Inc.: Employment. Jones:Mission Bio Inc.: Employment. Neuberg:Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.
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Nagahara, Shiori, Tetsuya Higashiyama, and Yoko Mizuta. "Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube." Plant Reproduction 34, no. 3 (June 19, 2021): 191–205. http://dx.doi.org/10.1007/s00497-021-00418-z.
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Abstract Key message Biolistic delivery into pollen. Abstract In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.
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Wu, Xiaosheng, Renee C. Tschumper, Albert Gutierrez, Stephen A. Mihalcik, Grzegorz S. Nowakowski, Daniela Capello, and Diane F. Jelinek. "Somatic Hyperrepair: A Novel Tumor Suppression Mechanism for Germinal Center B Cells." Blood 114, no. 22 (November 20, 2009): 92. http://dx.doi.org/10.1182/blood.v114.22.92.92.
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Abstract Abstract 92 BACKGROUND: All somatic cell types, including all B lineage cells, constitutively express a number of DNA repair proteins to maintain genomic stability and thwart tumorigenesis in the face of ongoing constitutive levels of DNA damage. Compromises in expression of these essential DNA repair factors have been shown to readily induce the development of various cancers including B cell lymphomas, suggesting expression levels are tightly regulated and limited in quantity. In germinal center (GC) B cells, however, additional DNA repair capacity is likely required to counterbalance the heightened level of mutagenic activity owing to the induced expression of activation-induced cytidine deaminase (AID) during somatic hypermutation (SHM). AID is a DNA editing enzyme which introduces somatic mutations into immunoglobulin (Ig) V regions at an estimated rate which is almost a million-fold higher than the spontaneous rate in somatic cells. HYPOTHESIS: We hypothesize that to maintain the genomic wellness of GC B cells which are undergoing SHM, there is a need for induction of an accompanying robust DNA repair system. We further hypothesize that inefficient induction of these repair genes may predispose to malignant transformation. METHODS: Using tonsillar tissue sections and purified tonsillar B cell subpopulations, we compared expression levels of various DNA repair genes and related these levels to AID expression across the subsets using immunohistochemistry, real-time RT-PCR, and Western blot analysis. To characterize the nature of signals capable of inducing expression levels of AID and/or DNA repair proteins, peripheral blood B cells were activated in vitro using a panel of stimuli, including coculture with activated CD4 T cells. As a surrogate measure of mismatch repair (MMR) activity in the relative absence of T cell help, we quantitated the number of somatic hypermutations at A/T sites in the Ig heavy chain variable (IGHV) region genes in a collection of IGHV sequences obtained from normal B cells and HIV-related lymphoma cells. RESULTS: Using immunohistochemistry, we observed that, similar to the expression of AID, DNA MMR genes are significantly induced in tonsillar GC B cells. These results were further validated using a more sensitive real-time RT-PCR assay and analysis by Western blotting. By expanding our DNA repair gene panel, we observed that proteins of homologous recombination, base excision repair and DNA single strand break signalling pathway are also similarly induced in GC B cells at RNA, protein, and functional levels compared to their expression in naïve and memory B cells. By contrast, expression of non-homologous end joining and DNA double strand break signalling molecules are unchanged. We have termed this selective induction of repair mechanisms in GC B cells as somatic hyperrepair (SHR). To identify pathways that lead to the activation of AID and SHR, we used an in vitro system and a variety of stimuli and we discovered that multiple B cell stimuli including CpG, CD40L, and anti-BCR could each independently induce the expression of AID while SHR induction strictly required the engagement of CD4+ T cells. This provocative observation suggests a novel role for CD4+ T cells in mitigating tumorigenesis of post-GC B cells through their ability to induce the SHR pathway in cells that have been induced to undergo SHM. To demonstrate the possible role of SHR in lymphomagenesis, we analysed the mutation pattern of IGHV genes from a panel of B cell lymphomas obtained from HIV infected (CD4+ T cell suppressed) patients. We found that HIV-related lymphoma cells displayed a significantly lower frequency of SHM at A/T positions relative to normal memory B cells, indicative of compromised MMR of their precursor cells during GC transit. Our findings resolve the lingering paradox that B cell malignancies are overwhelmingly prevalent under T cell suppression conditions such as HIV infection, post-organ transplant, and aging. Finally, our results also suggest for the first time that mounting efficient tumor suppression for some cells may depend on signals transmitted by neighboring cells and the specific microenvironment. Disclosures: No relevant conflicts of interest to declare.
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El Hussien, Marwa Ali, Chao-Yuan Tsai, Yuhkoh Satouh, Daisuke Motooka, Daisuke Okuzaki, Masahito Ikawa, Hitoshi Kikutani, and Shuhei Sakakibara. "Multiple tolerance checkpoints restrain affinity maturation of B cells expressing the germline precursor of a lupus patient-derived anti-dsDNA antibody in knock-in mice." International Immunology 34, no. 4 (December 1, 2021): 207–23. http://dx.doi.org/10.1093/intimm/dxab111.
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Abstract Anti-dsDNA antibodies are a hallmark of systemic lupus erythematosus and are highly associated with its exacerbation. Cumulative evidence has suggested that somatic hypermutation contributes to the high-affinity reactivity of anti-dsDNA antibodies. Our previous study demonstrated that these antibodies are generated from germline precursors with low-affinity ssDNA reactivity through affinity maturation and clonal expansion in patients with acute lupus. This raised the question of whether such precursors could be subjected to immune tolerance. To address this, we generated a site-directed knock-in (KI) mouse line, G9gl, which carries germline-reverted sequences of the VH–DH–JH and Vκ–Jκ regions of patient-derived, high-affinity anti-dsDNA antibodies. G9gl heterozygous mice had a reduced number of peripheral B cells, only 27% of which expressed G9gl B-cell receptor (BCR). The remaining B cells harbored non-KI allele-derived immunoglobulin heavy (IgH) chains or fusion products of upstream mouse VH and the KI gene, suggesting that receptor editing through VH replacement occurred in a large proportion of B cells in the KI mice. G9gl BCR-expressing B cells responded to ssDNA but not dsDNA, and exhibited several anergic phenotypes, including reduced surface BCR and shortened life span. Furthermore, G9gl B cells were excluded from germinal centers (GCs) induced by several conditions. In particular, following immunization with methylated bovine serum albumin-conjugated bacterial DNA, G9gl B cells occurred at a high frequency in memory B cells but not GC B cells or plasmablasts. Collectively, multiple tolerance checkpoints prevented low-affinity precursors of pathogenic anti-dsDNA B cells from undergoing clonal expansion and affinity maturation in GCs.
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Brisou, Gabriel, Laurent Jallades, Alexandra Traverse-Glehen, Francoise Berger, Aurélie Verney, Gilles Salles, and Thomas Wenner. "Expression of a Dysfunctional Activation Induced Cytidine Deaminase Is Correlated with Disease Progression in Splenic Marginal Zone Lymphoma." Blood 120, no. 21 (November 16, 2012): 2397. http://dx.doi.org/10.1182/blood.v120.21.2397.2397.
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Abstract Abstract 2397 B cells can undergo at least two differentiation pathways, dependent of T cells or not, starting from follicular or marginal zone B cells respectively. The T-independent response, less understood than the germinal center reaction, is triggered by specific antigens and arises from marginal zone B cells. During this development, some B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR), triggered by the same DNA editing enzyme called Activation Induced Cytidine Deaminase (AID). The splenic marginal zone lymphoma (SMZL) is a rare lymphoproliferative disorder characterized by a clonal expansion of B cells in the marginal zone of the spleen. These B-cells underwent SHM in roughly 60% of the cases but nearly none underwent CSR. These observations suggest that tumor clones originate from a particular activated B cell subset not transiting through the germinal center. In order to confirm this hypothesis, we focused our work on the status and impact of AID in this disease and worked on purified B cells extracted from spleen of well-characterized SMZL cases. We determined AID status by quantitative RT-PCR analysis on 27 SMZL samples and compared it with 5 controls. In the SMZL group the relative level of expression of AID is heterogeneous but two subgroups could be distinguished: one considered as expressing AID (14 cases out of the 27 analyzed), the remaining considered as not expressing AID. When we compared AID expression rate with occurrence of SHM and CSR, no clear correlation between AID expression and presence of SHM or CSR could be observed suggesting that AID, when expressed, is dysfunctional. To address this hypothesis, we first analyzed AID protein by immunohistochemistry and a good correlation between IHC signal and AID mRNA expression level has been observed. As AID gene was not mutated, we next focused our work on AID mRNA splicing variants as these variants exhibit different functions according to the domain of the protein they contain in a murine model. We found that SMZL B cells express various splicing variants of AID mRNA, some of those variants corresponding to the full length isoform (n = 6/17), and other variants corresponding to AID-ΔE4a (n = 2/17) or AID-ΔE4 (n = 7/17) isoforms known to be expressed in normal germinal center B cells as well as in Chronic Lymphocytic and Acute Lymphoblastic Leukemia. These findings indicate that although expressed at the mRNA and protein levels, AID may not be fully functional in SMZL cases. Finally we addressed the potential clinical significance of AID expression. We identified for that purpose a group of “progressive SMZL” patients that had received immuno-chemotherapy after splenectomy because of a significant risk of progression or transformation into aggressive large B cell lymphoma (n = 8/27) pre-empting outcome differences. We found a higher proportion of AID expressing patients in the defined “progressive SMZL” group (n = 7/8) as compared to the proportion found in the “indolent SMZL” group (n = 5/14, p = 0,03). Altogether, this data suggest that the B cell clone leading to SMZL originate from the marginal zone and support the hypothesis of a lymphoproliferative disorder affecting the T-independent response. AID expression in SMZL may reflect an advanced stage of the disease and could be correlated with the evolution of the lymphoma into a more clinically or pathologically aggressive form. Disclosures: No relevant conflicts of interest to declare.
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Ghia, Emanuela M., Laura Z. Rassenti, George F. Widhopf, Gregg J. Silverman, Andrew W. Greaves, and Thomas J. Kipps. "KDE Analysis in Chronic Lymphocytic Leukemia Patients with B Cells Expressing IGHV3-21 and Discordant Mutational Status between Heavy and Light Chain." Blood 110, no. 11 (November 16, 2007): 1127. http://dx.doi.org/10.1182/blood.v110.11.1127.1127.
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Abstract Patients with chronic lymphocytic leukemia (CLL) cells that use IGHV3-21 typically have aggressive disease similar to that of patients with CLL cells that use unmutated IGHV. However, the IGHV3-21 genes expressed in CLL often have less than 98% sequence homology with the known germline IGHV3-21 and hence are considered mutated. However, many “mutated” IGHV3-21 in CLL have higher homology with the germline IGHV3-21 (e.g. >97%) than do most other IGHV in CLL that also are considered mutated (mean percent homology = 93 ± 4% (SD), N= 1,260). As such, some argue that 97% be used as the threshold for defining IGHV mutation status. To test whether this definition is valid for IGHV3-21 we examined the mutation status of the rearranged and/or expressed Ig light chain V genes. We examined 63 (2.6%) IgVH3-21 cases from a large cohort (N=2,457) of CLL patients evaluated by the CRC. Forty of 63 (63.5%) IGHV3-21 cases used ? and 23 (36.5%) used ? light chains. While the ?-expressing cases had perfect correlation in mutational status between IgH and IgL, 12 of 40 (30%) ?-expressing cases were discordant in that they had IGHV3-21 with 94–97.6% germline sequence homology and IGLV that were all unmutated. Nine of these 12 (75%) also displayed a strikingly homologous and short IgH third complementary determining region (CDR) motif (DANGMDV) and light chains encoded by IGLV3-21. To study the differentiation history of such cases, we examined for productively-rearranged, and formerly-expressed, IGKV alleles and non-functional IGKV alleles that had undergone rearrangement with the kappa deleting element (KDE). Analysis for IGKV-KDE rearrangements was performed on 7 of the 9 IGHV3-21/IGLV3-21 cases. Two cases had two non-functional IGKV alleles and five (71%) had one non-functional IGKV allele and one productively-rearranged IGKV allele that apparently was aborted via receptor editing. All 5 productively rearranged IGKV alleles had somatic mutations with germline-homology ranging from only 93.6% to 97.6% (median 96.3%). Non-conservative mutations were clustered in the CDRs, arguing that they were selected in an apparent antigen-driven response. All 9 non-functional IGKV alleles had 100% homology with known germline IGKV genes. For comparison, we examined for IGKV-KDE rearrangements in 4 cases of ?-expressing CLL cells that used the unmutated IGHV1-69 gene and IGLV3-09. All such cases each had two non-functional IGKV alleles that had 100% homology with known IGKV rearranged with KDE, indicating that none of these cases experienced IGKV receptor editing, a significantly lower incidence than that noted for cases using IGHV3-21/IGLV3-21 (P<0.01). These results indicate that despite sharing association with more aggressive disease, most CLL cases that use IGHV3-21/IGLV3-21 are distinctive from cases that use unmutated IGHV1-69 in their B cell differentiation history in that they are derived from B cells that formerly expressed ? light chains that had undergone somatic mutation and selection in an antigen-driven immune response prior to undergoing IGKV receptor editing. Despite expressing IGHV with >97% homology to the known germline IGHV3-21 and IGLV with >98% homology to known germline IGLV, these cases have experienced somatic mutation and apparent germinal center maturation prior to leukemogenesis.
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Bertolo, Cristina, Raquel Malumbres, Ainara Sagardoy, Eloy F. Robles, Jose I. Martinez-Ferrandis, Sergio Roa, Izidore S. Lossos, et al. "LITAF, a BCL6 Target Gene, Regulates Autophagia in B Cells and Is Essential for T-Cell Dependent Humoral Responses." Blood 118, no. 21 (November 18, 2011): 1391. http://dx.doi.org/10.1182/blood.v118.21.1391.1391.
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Abstract Abstract 1391 LITAF was discovered as a p53-induced transcript that promoted TNFa secretion in monocytes in response to LPS. We previously reported that LITAF is inactivated by deletion or promoter hypermethylation in germinal center-derived B-cell lymphomas. However, the function of LITAF in B lymphocytes is unknown. Using gene expression analysis of isolated B-cell subpopulation and immunohistochemical studies of tonsil lymphoid follicles we found that LITAF is expressed in naïve B lymphocytes and is repressed within the germinal centers (GCs). Thus, LITAF showed an opposite expression to BCL6, an essential regulator of GC development and function. Likewise, expression of LITAF and BCL6 were inversely correlated in cell lines and biopsies from patients with B-cell lymphoma, further suggesting a link between LITAF and BCL6. ChIP-on-chip and ChIP-sequencing analyses of B cells coupled with luciferase reporter assays revealed that BCL6 repressed LITAF expression by binding to its promoter. Accordingly, BCL6 silencing with siRNAs or after exposure to a BCL6-inhibitor peptide increased LITAF expression, indicating that LITAF is transcriptionally repressed by BCL6 in GC B lymphocytes and in B-cell lymphoma cells. To initially elucidate the function of LITAF in B cells, gain-and-loss of function experiments were performed in different cellular models. LITAF expression was not related to TNFa secretion after LPS exposure, nor modulated cell proliferation or apoptosis in B cells. However, sustained expression of LITAF in B-cell lymphoma cells increased cell size, lysosome content and mitochondrial mass. Gene expression microarray studies defined a LITAF-related transcriptional signature containing genes involved in the regulation of endomembranes, vesicle trafficking and protein transport. Accordingly, immunofluorescence analysis co-localized LITAF with lysosomes and with autophagosomes expressing LC3, the mammalian homolog of yeast autophagy-related protein (Atg8), as well as with the lysosomal sorting-associated proteins NEDD4 and TSG101, both in normal CD19+ B lymphocytes and in B-cell lymphoma cells. In addition, LITAF expression induced autophagic activity in B cells, shown by an increase in the FL1/FL3 ratio after acridine orange staining and by converting LC3-I to LC3-II, which were more evident upon cell starvation. Together, these data suggest that LITAF may play a role in the processing of proteins in autophagosomes through regulating autophagy. To investigate LITAF function in vivo, we generated mice with targeted deletion of the Litaf gene in B lymphocytes by using the Cre-loxP system. Litaf -mb1-Cre (Litaf−/− ) mice developed healthy and showed normal distribution of hematopoietic cell subpopulations. However, Litaf−/− mice were unable to develop full T-cell dependent immune responses, presenting PNA-stained, Litaf-negative GCs that were absent or had marked reduction in size and number. Accordingly, reduced amounts of IgM, IgG1 and IgG3 antibodies as a consequence of abnormal class switch recombination (CSR) were detected in immunized mice. However, in experiments testing CSR in vitro, in which B cells are artificially activated in the absence of T cells, the amounts of IgM/IgG1/IgG3 did not differ between knock-out and control groups. Similarly, mouse immunization with a T-cell independent antigen did not induce differences in immunoglobulin production. Further studies of GCs in T-cell immunized Litaf−/− mice using an antibody for the Class II-associated invariant chain peptide (CLIP) revealed that the atrophic GCs in Litaf−/− mice showed strong CLIP expression in comparison to wild-type littermates. In normal immune responses, CLIP peptides bind to MHC class II molecules in endolysosomes, until they are displaced by the antigen, then releasing CLIP and allowing MHC II-antigen complexes to be transported to the cell membrane for T-cell presentation. The failure to develop appropriate immune responses together with the accumulation of CLIP peptides in Litaf -deficient mice indicate that Litaf is essential for adequate T-cell dependent immune responses in GC B lymphocytes, possibly through facilitating the presentation of the antigens to MHC II molecules in the endolysosomes. Once this process is assembled and the T-cell activated B lymphocytes enter the GCs, BCL6 represses LITAF to prevent additional interactions between B and T cells during BCR editing. Disclosures: No relevant conflicts of interest to declare.
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Navarro, M., C. Bluguermann, M. Von Meyeren, V. Bariani, C. Osycka, and A. Mutto. "2 Role of histone H3 lysine 9 trimethylation during bovine pre-implantation embryonic development." Reproduction, Fertility and Development 31, no. 1 (2019): 126. http://dx.doi.org/10.1071/rdv31n1ab2.
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Histones play an important role in DNA’s compaction and organisation into the cellular nucleus. Depending on which histone modification occurs, chromatin can take a conformation of heterochromatin or euchromatin, which are associated with gene repression or expression, respectively. Histone H3 lysine 9 (H3K9) trimethylation (H3K9me3) is associated with gene silencing. At least 3 methyltransferases are able to change the methylation status of H3K9: SUV39H1, SUV39H2, and SETDB1. In several mammalian species, modulation of H3K9 methylation status has been demonstrated to be necessary to achieve a successful pre-implantation embryonic development after IVF or somatic cell NT. The aim of this work was to study the role of H3K9me3 in IVF pre-implantation bovine embryos. For this purpose, immunostaining of H3K9me3 at different pre-implantation stages of development was performed. Further, the relative abundances of the methyltransferases SUV39H1 and SUV39H2 were measured by real-time PCR using luciferase transcript as an exogenous gene for normalization. Finally, to evaluate H3K9me3 involvement during pre-implantation embryonic development, we generated SUV39H1 or SUV39H2 knockout embryos by the CRISPR/Cas9 system. We designed guide RNA targeting SUV39H1 or SUV39H2 and co-injected the presumptive zygote’s cytoplasm 18h post-fertilization with Cas9 protein. At Day 8 post-fertilization, the number of blastocysts was assessed and embryos were immunostained to evaluate H3K9me3. Results were analysed using Student’s t-test or ANOVA with the post-hoc Tukey test depending on data set (P ≤ 0.05) and reported as means±standard errors of the mean. Oocytes at germinal vesicle stage and metaphase II as well as embryos at different stages of pre-implantation development (2, 4, and 8 cells, morula, and blastocyst; n=6) were immunoreactive for H3K9me3. Expression of SUV39H1 and SUV39H2 mRNA decreased significantly as embryonic development progressed, reaching undetectable levels at stages where genome activation had already occurred (morula and blastocyst; P<0.0001, n=3). When zygotes were co-injected with the guide RNA targeting SUV39H1/Cas9, embryonic production showed a significant increase compared with the control [42.26%±5.03 (28/65) v. 23.86%±3.99 (21/88), respectively; P=0.034, n=4], and H3K9me3 immunostaining was reduced in treated embryos. Editing efficiency was estimated at 66%. In contrast, no statistical differences were found in embryonic production or H3K9me3 immunostaining in embryos co-injected with the guide RNA targeting SUV39H2/Cas9 (P=0.57, n=3). In conclusion, we were able to characterise H3K9me3 and determine transcript levels of methyltransferases SUV39H1 and SUV39H2 in oocytes and different stages of pre-implantation embryonic development. We also demonstrated that SUV39H1 deletion led to an increased embryonic production, suggesting that H3K9me3 removal would allow a greater relaxation of the heterochromatin and consequently a successful activation of embryonic genes. This highlights the essential role of H3K9me3 during bovine pre-implantation embryonic development.
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Pasqualucci, Laura, Mara Compagno, Wei Keat Lim, Adina Grunn, Subhadra V. Nandula, Marta Scandurra, Francesco Bertoni, et al. "Mutations in Multiple Genes Cause Deregulation of the NFkB Pathway in Diffuse Large B-Cell Lymphoma." Blood 112, no. 11 (November 16, 2008): 801. http://dx.doi.org/10.1182/blood.v112.11.801.801.
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Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease comprising multiple biologically and clinically distinct subgroups, including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Gene expression profile studies have shown that a key feature of its most aggressive subtype, ABC-DLBCL, is the constitutive activation of the NF-kB transcription complex. However, except for a small fraction of cases (Lenz et al., Science 2008), it remains unclear whether NF-kB activation in these tumors reflects an intrinsic program of the cell of origin or represents a primary pathogenetic event. To address this question, we first characterized 165 DLBCL samples (18 cell lines and 147 primary biopsies, including 26 ABC, 28 GCB, 10 unclassified and 83 not profiled) for the presence of active, nuclear NF-kB complexes by using immunohistochemical/immunofluorescence staining of NFKB1 p105/p50 (as a readout for the canonical pathway) and NFKB2 p100/p52 (as a readout for the non-canonical pathway). Nuclear localization of NF-kB, indicative of constitutive activity, was observed in 14/26 (54%) ABC-DLBCL and 8/28 (28%) GCB-DLBCL primary biopsies, as well as in 3/10 (30%) unclassified and 48/83 (58%) non-profiled cases, and correlated with significant enrichment in expression of NF-kB target genes, as assessed by gene set enrichment analysis (GSEA) of transcriptionally profiled cases. In addition, the more sensitive GSEA approach detected a gene expression signature of NF-kB activity in >90% ABC-DLBCLs and 53% GCB-DLBCLs, indicating that constitutive activation of this key signaling pathway is a common feature of ABC-DLBCL but can also be observed in a smaller fraction of GCB-DLBCL. To investigate whether NF-kB activity represents a primary pathogenetic event, we then screened for mutations the complete coding sequences of 31 genes encoding for NF-kB pathway components in a panel of 14 ABC-DLBCLs, which was expanded to 48 samples (12 ABC, 26 GCB and 10 not profiled) for validation of the mutated genes. The results showed that >50% of ABC-DLBCL (n=15/26) and a smaller fraction of GCB-DLBCL (n=8/26, 31%) carry somatic mutations in multiple genes, including negative (TNFAIP3/A20) and positive (CARD11, TRAF2, TRAF5, MAP3K7/TAK1 and TNFRSF11A/RANK) regulators of NF-kB. Of these, the A20 gene, which encodes for a ubiquitin-editing enzyme involved in termination of NF-kB responses, is the most commonly affected, with ~27% ABC-DLBCLs and 25% (8/32) immunohistochemically classified non-GC DLBCL displaying biallelic A20 inactivation by somatic mutations and/or deletions. Sequence changes include premature nonsense mutations, frameshift deletions/insertions and splice site mutations, leading to severely truncated proteins that lack functionally relevant domains and have thus lost their enzymatic activity. In virtually all mutated cases, FISH analysis revealed loss of the second allele, while 4 additional samples showed biallelic deletion of the gene. Thus, A20 is inactivated by a classic “two-hit” mechanism, suggesting a tumor suppressor role. Less frequently, missense mutations of CARD11 (10%) and TRAF2 (4%) produce molecules with significantly enhanced ability to activate NF-kB in transient transfection/reporter gene assays. Our results demonstrate that NF-kB activation in DLBCL is caused by genetic lesions affecting multiple genes, whose loss or activation may promote lymphomagenesis by leading to abnormally prolonged NF-kB responses. These findings provide the rationale and the assays for the identification of patients amenable to NF-kB targeted therapeutic intervention.
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Li, Michael Y., Lauren C. Chong, Elizabeth Chavez, Bruce W. Woolcock, Adele Telenius, Vivian Lam, Gerald Krystal, et al. "TRAF3 Loss Drives Alternative NF-κB Pathway Activation in Diffuse Large B-Cell Lymphoma." Blood 136, Supplement 1 (November 5, 2020): 22–23. http://dx.doi.org/10.1182/blood-2020-141126.
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Introduction: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor family that regulates gene expression programs contributing to inflammation and cell survival. NF-κB signaling occurs via two branches: classical and alternative, and is often enriched in somatic mutations of key pathway members in several lymphoid malignancies. Here, we reveal deregulation and constitutive activation of the alternative NF-κB pathway in a subset of DLBCL patients with recurrent genomic loss of the gene encoding tumor necrosis factor receptor-associated factor 3 (TRAF3), a regulator of the NF-κB signaling pathway. Methods and Results: To uncover novel driver mutations of DLBCL pathogenesis and tumor maintenance, we performed Affymetrix SNP6.0 copy number analysis on 347 de novo DLBCL samples from patients uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). We observed frequent, focal genomic loss of chr:14q32.31-32 which included TRAF3 and RCOR1 (7%, 22/313) in the minimally deleted region and an enrichment of activated B-cell-like (ABC) subtype cases over germinal center B-cell-like (GCB) subtype cases, confirming previously published data (Chan et al, Blood 2014). RNAseq of these DLBCL samples revealed a significant reduction of TRAF3 mRNA in chr:14q32.31-32 deleted cases compared to copy number neutral cases (p=0.002). Next, we focused on characterizing the phenotypic consequences of TRAF3 loss in DLBCL. We used CRISPR/Cas9 gene editing to knock out TRAF3 in 2 GCB-DLBCL (DOHH2, OCI-LY1) and 2 ABC-DLBCL (HBL1, OCI-LY3) cell lines. We performed immunoblotting analysis of NF-κB pathway members on cell fractionated samples of TRAF3 knockout cells and found increased levels of the NF-κB inducing kinase NIK (a direct target of TRAF3-mediated ubiquitin-proteasome degradation) and a concomitant increased nuclear translocation of NF-κB transcription factor complex subunits RelB and p52. Proteasome blockade restored RelB cytoplasmic localization and reduced processed p52 protein in TRAF3 knockout GCB-DLBCL lines only, indicating other factors may contribute to alternative NF-κB activation in ABC-DLBCL. Moreover, classical NF-κB activation remained unaffected, highlighting the specific role of TRAF3 regulation on the alternative NF-κB pathway in DLBCL. Consistent with these findings, TRAF3 knockout cells exhibited NF-κB-dependent transcriptional upregulation by luciferase reporter activity and elevated pro-inflammatory cytokine production (IL-6, TNF-β) by Luminex and ELISA. To study transcriptome changes as a result of TRAF3 loss-of-function, we performed RNAseq and differential gene expression analysis on wildtype and TRAF3 knockout DLBCL cell lines as well as primary DLBCL samples (N=347). We found enrichment of NIK and NF-κB associated pathways in TRAF3 deficient DLBCL and uncovered additional enriched gene sets including those involved in cell cycle regulation, cell division and metabolism, suggesting a potential proliferative and survival advantage. Conclusion: Our findings link TRAF3 loss-of-function to clinical and gene expression phenotypes in DLBCL and highlight alternative NF-κB activation as a pathogenically important pathway in both GCB and ABC subtypes. Future studies will be directed towards comprehensive evaluation of NF-κB inhibitors for effective blockade of constitutive alternative NF-κB activation in DLBCL. Disclosures Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; AbbVie: Consultancy.
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Takata, Katsuyoshi, Daisuke Ennishi, Ali Bashashati, Saeed Saberi, Elena Viganò, Shannon Healy, Julie S. Nielsen, et al. "Somatic PRAME Deletions Are Associated with Decreased Immunogenicity, Apoptosis Resistance and Poor Outcomes in Diffuse Large B-Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 667. http://dx.doi.org/10.1182/blood-2018-99-113516.
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Abstract Background: The current standard of care in diffuse large B-cell lymphoma (DLBCL) consists of chemotherapy and therapeutic monoclonal antibodies that have significantly improved patient outcomes over the past 15 years. However, a large proportion of patients suffer from refractory or relapsed disease. Therefore, the development of new therapeutic strategies for this subgroup of patients, who are threatened by a high chance of disease-related death, represents an important unmet clinical need. Methods: We enrolled into our study 347 de novo DLBCL patients uniformly treated with R-CHOP from the BC Cancer population-based cohort between September 2000 and January 2012. RNAseq and high-resolution copy number analysis were performed and correlated with clinical outcome data and tumor microenvironment composition. We also performed functional studies to investigate PRAME-mediated memory T-cell responses and gene expression changes. Results: We discovered novel, highly focal deletions of 22q11.22, including the PRAME gene in 13% (44/338) of the cases. The deletions cluster in a narrow chromosomal region that includes a very small number of genes (VpreB1, ZNF280A/B, PRAME, GGTLC2, miR-650). Of clinical importance, 22q11.22 deletions were found significantly more frequently in germinal centre B-cell-like (GCB) type DLBCL (17% (31/180) vs. activated B-cell-like (ABC) type: 8% (8/98), P < 0.01), and were also significantly associated with worse outcome, which was specifically observed in GCB-DLBCL (5-year disease specific survival, non-PRAME-deleted: 84.5% vs. PRAME-deleted: 67.2%, P = 0.026). Homozygous deletions were more strongly associated with poor outcome than heterozygous deletions. Interestingly, 90% of PRAME-deleted cases were Ig-lambda restricted (P < 0.001). PRAME is a prominent member of the cancer testis antigen (CTA) family of proteins that are expressed in various types of cancers, but not in normal tissues, including normal mature B-cells, apart from male germinal cells. Due to the cancer-specific expression of CTAs, these molecules are considered promising targets for cancer immunotherapy using cytotoxic T-cells and tumor vaccination approaches. To determine the association with tumor microenvironment composition, we analyzed CD4/CD8 flow cytometry data from DLBCL patient samples. The numbers of CD4 and CD8-positive T cells were significantly lower in PRAME-deleted cases compared to wild type (CD4: P < 0.001, CD8: P = 0.013). Notably, RNAseq analysis revealed that the HLA-A*0201 genotype was seen significantly more often in PRAME deleted cases (PRAME wt: 2.5% vs. PRAME deleted: 10.8%, P = 0.005). In order to functionally characterize its interaction with the immune microenvironment, we utilized enzyme-linked immunoSpot (ELISPOT) assays to investigate memory T-cell reactions of patient-derived T cells to PRAME antigens using patient-derived peripheral blood mononuclear cells (PBMC) and measured IFN-g production (7 control healthy donors, 4 PRAME-deleted and 4-wild type patients). While T cells from PRAME-replete patients had no reaction to PRAME antigens, PRAME-deleted patient-derived T-cells had significant reactions to 4 independent PRAME peptides. These data suggest that PRAME-deleted tumor cells can escape from cytotoxic T-cell attack to gain growth advantage. Next, we performed PRAME knock-out (KO) experiments using CRISPR/Cas9 genome editing to clarify the cell autonomous effects of PRAME deletions. Using 2 different cell lines (Karpas422 and SUDHL-4), we found TNFSF10 (TRAIL) expression was significantly down-regulated in homozygous PRAME-KO cell lines compared to wild type. The soluble form of TRAIL (sTRAIL) was also reduced, as measured with enzyme-linked immunosorbent assays. These results suggest that PRAME downregulated cells may contribute to cell survival via TRAIL and sTRAIL reduction. Conclusion: We identified recurrent PRAME deletions and characterized their clinical and functional role in DLBCL. Our findings contribute to the understanding of cell-autonomous and extrinsic roles of PRAME deletions in lymphomagenesis and may lead to the discovery of new therapeutic avenues to simultaneously treat the tumor and the host. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Janssen: Research Funding; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Tioma: Research Funding; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.
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Chu, Charles C., Piers E. M. Patten, Thomas MacCarthy, Chaohui Yuan, Xiao-Jie Yan, Jacqueline C. Barrientos, Jonathan E. Kolitz, Steven L. Allen, Kanti R. Rai, and Nicholas Chiorazzi. "IGHV-D-J Ultra-Deep Sequencing Reveals APOBEC and AID Targeted Mutations during Clonal Evolution of CLL in a Xenograft Mouse Model." Blood 124, no. 21 (December 6, 2014): 300. http://dx.doi.org/10.1182/blood.v124.21.300.300.
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Abstract Targeted ultra-deep sequencing of chronic lymphocytic leukemia (CLL) cells has enabled the assessment of subclone development based on mutations in the IGHV-D-J signature sequence in the dominant CLL clone. We have utilized the Roche 454 FLX pyrosequencing system, which can generate long sequencing reads containing both the immunoglobulin variable region (IGHV-D-J) and part of the immunoglobulin μ constant region (IGHM) in a single sequence, to analyze the mutational characteristics of newly evolved subclones to determine if they derive from AID/APOBEC activity. APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) is a family of cytidine deaminases that includes AID (activation-induced cytidine deaminase). AID is required for somatic hypermutation in germinal center B lymphocytes. CLL cells, like most non-germinal center B lymphocytes, generally do not express AID. However, AID expression in a small fraction of the CLL clone correlates with worse patient outcome. This observation has led to the hypothesis that abnormal AID expression promotes new off-target non-immunoglobulin mutations and DNA deletions and rearrangements leading to the development of more aggressive disease. CLL is not alone in this hypothesis, as AID is involved in the evolution of other leukemias/lymphomas and reportedly in other types of tumors such as breast and gastrointestinal cancers. Large scale cataloguing of somatic mutations by ultra-deep sequencing of a wide array of cancers has revealed an AID/APOBEC mutational signature in many cancers, including CLL (Alexandrov et al. 2013 Nature). Thus, AID/APOBEC family members may be involved in somatic mutations leading to the evolution of aggressive cancers. To test if AID/APOBEC proteins could be mutationally active in CLL, we analyzed the characteristics of new mutations found in IGHV-D-J-M in CLL cells that were activated in vivo after adoptive transfer into alymphoid NOD/Shi-scid,γcnull (NSG) mice. This CLL xenograft model activates a large fraction of CLL cells, which become AID+ and facilitates the detection of new subclones expressing mutated IGHV-D-Js by ultra-deep sequencing. Four unmutated IGHV CLL (U-CLL) and 3 mutated IGHV (>2% compared to germline) CLL (M-CLL) samples were each adoptively transferred into individual NSG mice. After expansion of CLL, the mice were sacrificed and the specific CLL clone IGHV-D-J-M was amplified from xenograft mouse spleen cDNA. Pre-transfer CLL cell cDNA was also amplified to establish a baseline comparison. Ultra-deep sequencing of these samples resulted in 2,318,800 sequence reads, which were subsequently trimmed according to the Roche 454 algorithm and further processed by custom R scripts. The sequence reads were aligned to the dominant CLL clone IGHV-D-J-M sequence to remove insertions, deletions, and poor quality (<20) nucleotides, resulting in 1,700,839 blocks of sequences of the same length. The dominant CLL clones represented 92.3% (1,569,059 blocks) of these sequences and were excluded. Mutated subclone sequences that occurred at least twice were extracted. Xenograft subclones not found in the pre-transfer sample were selected for analyses of the characteristics of newly generated CLL mutations. New xenograft CLL subclones could be identified in all samples (3.2 – 12.3 new subclones / read bp *106). AID mutational characteristics in new subclones were assessed using the SHMTool (http://scb.aecom.yu.edu/shmtool) algorithms to calculate mutation frequencies in IGHV-D-J relative to IGHM and at AID mutation hotspots and coldspots. Other APOBEC family members have a different mutation hotspot site, which we analyzed by custom R scripts. All CLL samples showed evidence of AID mutational activity: higher IGHV-D-J versus IGHM mutations in all cases, increased AID hotspot mutation frequency in five cases, and decreased AID coldspot mutation frequency in five cases. Increased APOBEC hotspot mutational activity was seen in four cases. This APOBEC mutational activity is consistent with increased APOBEC3 gene expression and the genomic somatic mutation pattern observed recently in CLL (Rebhandl et al. 2014 Leukemia). Thus, both U-CLL and M-CLL are capable of producing mutations characteristic of AID or APOBEC activity. These data are consistent with the hypothesis that AID/APOBEC may promote new mutations leading to the evolution of aggressive CLL. Disclosures No relevant conflicts of interest to declare.
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Takata, Katsuyoshi, Lauren C. Chong, Daisuke Ennishi, Avinash Thakur, Shannon Healy, Elena Viganò, Tao Dao, et al. "The Tumor Associated Antigen PRAME Exhibits Dualistic Functions That Are Targetable in Diffuse Large B-Cell Lymphoma." Blood 136, Supplement 1 (November 5, 2020): 34. http://dx.doi.org/10.1182/blood-2020-141168.
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Background: With the goal of translating biological discovery into clinical actionability, deciphering crosstalk in the cellular ecosystem of the tumor microenvironment (TME) has emerged as a research focus. Although comparatively little is known about the immune biology of diffuse large B-cell lymphoma (DLBCL), as reflected in clonal selection of specific somatic gene mutations in response to immune system pressure and the specific composition of the TME, PRAME has emerged as a prominent member of the cancer germline antigen/ tumor associated antigen (TAA) family of proteins. It is expressed in various types of cancers, but generally not in normal tissues, apart from male germinal cells, and triggers autologous T-cell mediated immune responses. PRAME is highlighted as a new cancer therapeutic target of T-cell or antibody-based immunotherapies with promising anti-tumor responses in early phase clinical trials and pre-clinical models for several types of cancers. In the context of developing new immunotherapies, targeting TAAs that are presented by major histocompatibility complexes on tumor cells is a promising therapeutic strategy for patients that experience treatment failure. Material and methods: We performed integrative genomic analysis of whole-transcriptome RNAseq, targeted genomic sequencing, and high-resolution copy number analysis in 347 de novo DLBCL tumors from patients uniformly treated with R-CHOP. Findings were correlated with pathological and clinical parameters, as well as TME composition. Using DLBCL-derived cell lines (7 EZH2 mutated and 5 wt) and CRISPR-Cas9 genome editing, we performed in vitro functional studies to characterize cell-intrinsic effects of PRAME knockout. Moreover, we studied EZH2 inhibition in a murine model of Ezh2 mutant lymphoma with a focus on mechanistic links between EZH2 activity and PRAME expression, as well as TME composition (cell-extrinsic effects). Results: We discovered recurrent, and highly focal deletions of 22q11.22 including the PRAME gene, which were associated with poor treatment outcome, independent of pathological and clinical risk factors. To explore PRAME-deletion-associated phenotypes and interaction with the tumor microenvironment (TME), we analyzed corresponding RNAseq, immunohistochemistry (IHC), and flow-cytometry data from our DLBCL cohort and utilized enzyme-linked immunospot (ELISPOT) assays using patient-derived peripheral blood mononuclear cells. PRAME deletions contributed to an immunologically "cold" TME, representing a somatically acquired mechanism to evade anti-tumor T-cell response (cell-extrinsic effect). Using PRAME knock out by CRISPR-Cas9 in vitro, TRAIL-mediated apoptotic signaling was impaired. In addition, PRAME down-modulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. Using proximity ligation assays and co-IP, we demonstrated that PRAME directly interacted with EZH2 as a negative regulator, establishing a link between PRAME deletions and EZH2 mutations with anti-apoptotic signaling in DLBCL (cell-intrinsic effect). An in vivo murine model of Ezh2 mutant lymphoma showed decreased T-cell infiltration in the TME and Ezh2 inhibition induced PRAME restoration as compared to vehicle controls. IHC for CD3, CD4, FOXP3, and GZMB revealed significant increases in various T-cell populations of the TME, including Tregs and cytotoxic T cells. EZH2 inhibition with EPZ-6438 abrogated these dualistic effects leading to increased immune cell infiltration in the tumor microenvironment and acceleration of apoptosis via PRAME restoration. Moreover, restoration of PRAME antigen presentation by EZH2 inhibition resulted in enhancement of PRAME binding using a T-cell receptor mimic PRAME antibody (Pr20), suggesting immunotherapeutic potential. Conclusion: Our findings highlight multiple functions of PRAME during lymphomagenesis. PRAME restoration by EZH2 inhibition provides a preclinical rationale for synergistic therapies combining epigenetic re-programming with PRAME-targeted therapies. Disclosures Dao: Eureka Therapeutics: Consultancy. Melnick:Jubilant: Consultancy; Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding. Scheinberg:Lantheus: Current equity holder in private company; Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Enscyse: Current equity holder in private company; Arvenas: Current equity holder in private company; Pfizer: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Oncopep: Consultancy. Scott:Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding. Steidl:Curis Inc: Consultancy; Roche: Consultancy; AbbVie: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy.
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Insanguine Mingarro, Ferdinando A. "Apuntes de sociología biojurídica a partir de la edición genética germinal." Anales de la Cátedra Francisco Suárez 55 (June 17, 2020). http://dx.doi.org/10.30827/acfs.v55i0.15319.
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Objetivo del presente artículo es poner de relieve las contradicciones de la relación entre bioética y bioderecho que, lejos de ser un problema meramente teórico, tiene un impacto real en las reglamentaciones de las innovaciones en el campo de la biomedicina. Con el ánimo de demostrar esa fuerte conexión de la cuestión aparentemente teórica con la praxis, el ensayo, dotado de una metodología inductiva, moverá sus primeros pasos a partir de las técnicas de edición genética germinal. En la primera parte del trabajo se aclararán matices científicos de dichas técnicas y se presentarán los primeros avances de la reglamentación jurídica que tienen su origen en el Consejo de Europa. Finalmente, a través de los límites y contradicciones de la reglamentación supuestamente biojurídica de la edición genética germinal se pondrá en evidencia la asimetría que domina en la relación entre bioética y bioderecho. ---The goal of this article is to stand out contradictions in the relationship among bioethics and biolaw that, far from being merely a theoretical quandary, has a real impact in the regulations of innovations in the biomedical field. Aiming to show that strong connection between the apparently theoretical question with the praxis, the essay, with an inductive methodology, goes straight showing the first steps of regulation of the germ-line gene editing. In the first part of the article, after a summary about the scientific highlights of germ-line gene editing, we undertake an investigation on the juridical regulation of germline-gene editing, based on the Council of Europe’s provisions. Finally, showing the limitations and contradictions of these supposedly biojuridical regulations, we highlight the dominant asymmetry in the relationship among bioethics and biolaw.
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Lu, Qing Shi Mimmie, and Lining Tian. "An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase genes." BMC Biotechnology 22, no. 1 (February 15, 2022). http://dx.doi.org/10.1186/s12896-022-00737-7.
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Abstract Background Genome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean (Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regeneration is still not widespread, largely due to difficulties with transformation or low mutation efficiencies. Results We sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seedlings, we used ‘half-seed’ explants derived from imbibed seeds for Agrobacterium-mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75–100% mutation efficiencies. Indel frequencies varied widely among plants (3–100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27–100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were transmitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants. Conclusions Using simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement.
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Corinaldesi, Clarissa, Antony B. Holmes, Qiong Shen, Eli Grunstein, Laura Pasqualucci, Riccardo Dalla-Favera, and Katia Basso. "Tracking Immunoglobulin Repertoire and Transcriptomic Changes in Germinal Center B Cells by Single-Cell Analysis." Frontiers in Immunology 12 (January 12, 2022). http://dx.doi.org/10.3389/fimmu.2021.818758.
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In response to T-cell-dependent antigens, mature B cells in the secondary lymphoid organs are stimulated to form germinal centers (GCs), which are histological structures deputed to antibody affinity maturation, a process associated with immunoglobulin gene editing by somatic hypermutation (SHM) and class switch recombination (CSR). GC B cells are heterogeneous and transition across multiple stages before being eliminated by apoptosis or committing to post-GC differentiation as memory B cells or plasma cells. In order to explore the dynamics of SHM and CSR during the GC reaction, we identified GC subpopulations by single-cell (sc) transcriptomics and analyzed the load of immunoglobulin variable (V) region mutations as well as the isotype class distribution in each subpopulation. The results showed that the large majority of GC B cells display a quantitatively similar mutational load in the V regions and analogous IGH isotype class distribution, except for the precursors of memory B cells (PreM) and plasma cells (PBL). PreM showed a bimodal pattern with about half of the cells displaying high V region germline identity and enrichment for unswitched IGH, while the rest of the cells carried a mutational load similar to the bulk of GC B cells and showed a switched isotype. PBL displayed a bias toward expression of IGHG and higher V region germline identity compared to the bulk of GC B cells. Genes implicated in SHM and CSR were significantly induced in specific GC subpopulations, consistent with the occurrence of SHM in dark zone cells and suggesting that CSR can occur within the GC.
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Meshcheryakova, Anastasia, Peter Pietschmann, Philip Zimmermann, Igor B. Rogozin, and Diana Mechtcheriakova. "AID and APOBECs as Multifaceted Intrinsic Virus-Restricting Factors: Emerging Concepts in the Light of COVID-19." Frontiers in Immunology 12 (July 1, 2021). http://dx.doi.org/10.3389/fimmu.2021.690416.
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The AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme catalytic subunit) family with its multifaceted mode of action emerges as potent intrinsic host antiviral system that acts against a variety of DNA and RNA viruses including coronaviruses. All family members are cytosine-to-uracil deaminases that either have a profound role in driving a strong and specific humoral immune response (AID) or restricting the virus itself by a plethora of mechanisms (APOBECs). In this article, we highlight some of the key aspects apparently linking the AID/APOBECs and SARS-CoV-2. Among those is our discovery that APOBEC4 shows high expression in cell types and anatomical parts targeted by SARS-CoV-2. Additional focus is given by us to the lymphoid structures and AID as the master regulator of germinal center reactions, which result in antibody production by plasma and memory B cells. We propose the dissection of the AID/APOBECs gene signature towards decisive determinants of the patient-specific and/or the patient group-specific antiviral response. Finally, the patient-specific mapping of the AID/APOBEC polymorphisms should be considered in the light of COVID-19.
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Poddar, Snigdha, Jaclyn Tanaka, Jamie H. D. Cate, Brian Staskawicz, and Myeong-Je Cho. "Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays." Plant Methods 16, no. 1 (November 12, 2020). http://dx.doi.org/10.1186/s13007-020-00692-4.
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Abstract Background An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Results Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. Conclusion The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.
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You, Hong, Malcolm K. Jones, Deanne J. Whitworth, and Donald P. McManus. "Innovations and Advances in Schistosome Stem Cell Research." Frontiers in Immunology 12 (March 5, 2021). http://dx.doi.org/10.3389/fimmu.2021.599014.
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Schistosomes infect about 250 million people globally causing the devastating and persistent disease of schistosomiasis. These blood flukes have a complicated life cycle involving alternating infection of freshwater snail intermediate and definitive mammalian hosts. To survive and flourish in these diverse environments, schistosomes transition through a number of distinct life-cycle stages as a result of which they change their body plan in order to quickly adapt to each new environment. Current research suggests that stem cells, present in adults and larvae, are key in aiding schistosomes to facilitate these changes. Given the recent advances in our understanding of schistosome stem cell biology, we review the key roles that two major classes of cells play in the different life cycle stages during intramolluscan and intramammalian development; these include the germinal cells of sporocysts involved in asexual reproduction in molluscan hosts and the neoblasts of adult worms involved in sexual reproduction in human and other mammalian hosts. These studies shed considerable new light in revealing the stem cell heterogeneity driving the propagation of the schistosome life cycle. We also consider the possibility and value of establishing stem cell lines in schistosomes to advance schistosomiasis research. The availability of such self-renewable resources will provide new platforms to study stem cell behavior and regulation, and to address fundamental aspects of schistosome biology, reproductive development and survival. In turn, such studies will create new avenues to unravel individual gene function and to optimize genome-editing processes in blood flukes, which may lead to the design of novel intervention strategies for schistosomiasis.
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Li, Quanlin, Wenxue Zhai, Jiaping Wei, and Yanfeng Jia. "Rice lipid transfer protein, OsLTPL23, controls seed germination by regulating starch-sugar conversion and ABA homeostasis." Frontiers in Genetics 14 (January 16, 2023). http://dx.doi.org/10.3389/fgene.2023.1111318.
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Seed germination is vital for ensuring the continuity of life in spermatophyte. High-quality seed germination usually represents good seedling establishment and plant production. Here, we identified OsLTPL23, a putative rice non-specific lipid transport protein, as an important regulator responsible for seed germination. Subcellular localization analysis confirmed that OsLTPL23 is present in the plasma membrane and nucleus. The knockout mutants of OsLTPL23 were generated by CRISPR/Cas9-mediated genome editing, and osltpl23 lines significantly germinated slower and lower than the Nipponbare (NIP). Starch and soluble sugar contents measurement showed that OsLTPL23 may have alpha-amylase inhibitor activity, and high soluble sugar content may be a causal agent for the delayed seed germination of osltpl23 mutants. Transcript profiles in the germinating seeds exhibited that the abscisic acid (ABA)-responsive genes, OsABI3 and OsABI5, and biosynthesis genes, OsNCED1, OsNCED2, OsNCED3 and OsNCED4, are obviously upregulated in the osltpl23 mutants compared to NIP plants, conversely, ABA metabolism genes OsABA8ox1, OsABA8ox2 and OsABA8ox3 are stepwise decreased. Further investigations found that osltpl23 mutants displays weakened early seedling growth, with elevated gene expresssion of ABA catabolism genes and repressive transcription response of defence-related genes OsWRKY45, OsEiN3, OsPR1a, OsPR1b and OsNPR1. Integrated analysis indicated that OsLTPL23 may exert an favorable effect on rice seed germination and early seedling growth via modulating endogenous ABA homeostasis. Collectively, our study provides important insights into the roles of OsLTPL23-mediated carbohydrate conversion and endogenous ABA pathway on seed germination and early seedling growth, which contributes to high-vigor seed production in rice breeding.
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Bernardo-Álvarez, Mª Ángela. "El derecho a la información veraz y la libertad de investigación ante los nuevos avances de la biotecnología: el caso de CRISPR-Cas de edición genómica." Memorias de la Real Sociedad Española de Historia Natural, February 18, 2019, 95–107. http://dx.doi.org/10.29077/bol/113/e02_bernardo.
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Resumen La biotecnología asiste a una nueva revolución gracias a CRISPR-Cas, una herramienta para editar el genoma de forma precisa, rápida, sencilla y económica. Este sistema, que sirve como mecanismo de inmunidad adaptativa en células procariotas contra el ataque de virus, ha permitido identificar dianas farmacológicas, estudiar el origen de determinadas afecciones o crear modelos animales de enfermedades. CRISPR-Cas cuenta además con potenciales aplicaciones sanitarias, como su posible uso como terapia génica en células somáticas y en la línea germinal. Sin embargo, la edición genómica también puede entrañar algunos riesgos. El debate sobre el balance entre los beneficios y los problemas de la tecnología, que puede limitar el desarrollo de ciertas investigaciones, debe involucrar a la sociedad de forma inclusiva, anticipatoria e informada. Para ello es importante analizar las implicaciones jurídicas, éticas y sociales del sistema CRISPR-Cas desde la perspectiva constitucional del derecho a la libertad de investigación y a la libertad de información, dos elementos clave de los países democráticos. Abstract Biotechnology is witnessing a new revolution thanks to CRISPR-Cas, a tool to edit the genome accurately, quickly, easily and economically. This system, which works as a mechanism of adaptive immunity in prokaryotic cells against the attack of viruses, has made it possible to identify pharmacological targets, study the origin of certain conditions or develop animal models of diseases. CRISPR-Cas also has potential health applications, such as its possible use as gene therapy in somatic cells and in the germline. However, genome editing can also involve some risks. The debate about the balance between the benefits and problems of this technology, which can limit the development of certain research activities, should involve society in an inclusive, anticipatory and informed manner. For this it is important to analyse the legal, ethical and social implications of the CRISPR-Cas system from the constitutional perspective of the right to freedom of research and freedom of information, two key elements of democratic countries.
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Zamò, Alberto, Elena Gerhard-Hartmann, German Ott, Ioannis Anagnostopoulos, David W. Scott, Andreas Rosenwald, and Hilka Rauert-Wunderlich. "Routine application of the Lymph2Cx assay for the subclassification of aggressive B-cell lymphoma: report of a prospective real-world series." Virchows Archiv, October 11, 2022. http://dx.doi.org/10.1007/s00428-022-03420-6.
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AbstractThe subclassification of diffuse large B-cell lymphoma (DLBCL) into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes has become mandatory in the 2017 update of the WHO classification of lymphoid neoplasms and will continue to be used in the WHO 5th edition. The RNA-based Lymph2Cx assay has been validated as a reliable surrogate of high-throughput gene expression profiling assays for distinguishing between GCB and ABC DLBCL and provides reliable results from formalin-fixed, paraffin-embedded (FFPE) material. This test has been previously used in clinical trials, but experience from real-world routine application is rare. We routinely applied the Lymph2Cx assay to day-to-day diagnostics on a series of 147 aggressive B-cell lymphoma cases and correlated our results with the immunohistochemical subclassification using the Hans algorithm and fluorescence in situ hybridization findings using break-apart probes for MYC, BCL2, and BCL6. The routine use of the Lymph2Cx assay had a high technical success rate (94.6%) with a low rate of failure due to poor material and/or RNA quality. The Lymph2Cx assay was discordant with the Hans algorithm in 18% (23 of 128 cases). Discordant cases were mainly classified as GCB by the Hans algorithm and as ABC by Lymph2Cx (n = 11, 8.6%). Only 5 cases (3.9%) were classified as non-GCB by the Hans algorithm and as GCB by Lymph2Cx. Additionally, 5.5% of cases (n = 7) were left unclassified by Lymph2Cx, whereas they were defined as GCB (n = 4) or non-GCB (n = 3) by the Hans algorithm. Our data support the routine applicability of the Lymph2Cx assay.
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Hu, Shan, Lijuan Peng, Haixia Ding, Wei Di Mo, and Zhi Cheng Zhou. "First Report of Colletotrichum fioriniae Causing Anthracnose on Rhododendron delavayi in China." Plant Disease, March 29, 2022. http://dx.doi.org/10.1094/pdis-01-22-0002-pdn.
Full textAbstract:
Rhododendron delavayi Franch, a member of Ericaceae family, is globally famous for its garden flowers with significant ornamental value (Liu et al., 2020). In July 2020 and 2021, a disease survey of R. delavayi groves was conducted in Baili Azalea Forest Area (N27°10'-27°20', E 105°04'-106°04'). We arbitrarily selected an area with around 280 R. delavayi trees covering 2.5 hectares in R. delavayi grove where 20-35% of leaves showed symptoms of anthracnose. Typical symptoms included elliptical to irregularly shaped brown lesions on leaves and masses of black dots clustered on it. About 30 pieces of leaves with anthracnose lesions were collected. A few black dots were picked from the lesions with a sterilized needle, plated on water agar and incubated at 25℃ for 12 h to observe spore germination (Fang, 2007). Then the germinated spores were transferred onto PDA medium for further purification and morphological observation. Fourteen single-spore isolates with similar morphology were obtained. The surface of the colony was white or gray and spongy; the edge was smooth; and the back side was pinkish brown after 7 days of growth on PDA. Conidia were spindle-shaped, transparent, 11.1-16.6×3.6-4.9 μm (n=50). Appressorium from conidia was nearly ovate or proximate, brown or dark brown in color, 4.3-10.3 ×3.2-7.6 μm (n=50). These characteristics are consistent with Colletotrichum fioriniae reported by Shivas and Tan (2009). DNA was extracted from a representative isolate MYDJ12. The internal transcribed spacer region (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB), actin (ACT), and chitin synthase 1 (CHS-1) genes were amplified using primer pairs described by Damm et al. (2012). The sequences were deposited in GenBank with accession number MW692854 (ITS), MW727518 (GAPDH), MW727519 (TUB2), MW727520 (ACT), and MW727521 (CHS-1). BLASTN searches of the ITS, GADPH, TUB2, ACT and CHS-1 genes revealed 100% (540/540 nucleotides), 100% (254/254 nucleotides), 99.38% (4488/491 nucleotides), 98.77% (242/245 nucleotides) and 100% (282/282 nucleotides) homology with those of C. fioriniae CBS:128517T in GenBank (NR_111747, JQ948622, JQ949943, JQ949613 and JQ948953 respectively). The phylogenetic tree showed the isolate MYDJ12 to cluster with C. fioriniae CBS:128517T. Finally, two-year old R. delavayi plants (n=5) were inoculated by wounding with a syringe needle and placing 10 μL of spore suspension (106 spores per mL) of the isolate MYDJ12 on three leaves per plant. Control leaves were inoculated with sterile water. The experiment was conducted twice. Inoculated leaves were wrapped in parafilm tape and then the plants were placed in a greenhouse at 25°C with high relative humidity (90 to 95%). Seven days after incubation, brown lesions appeared, similar to those observed in the grove. Black dots clustered on the lesions after 15 days. Re-isolation was conducted 20 days after inoculation. From all the five inoculated plants, similar symptoms were observed, and the same pathogen was re-isolated. One of the isolates was selected for morphological observation and multi-gene (ITS, GAPDH, ACT, TUB2 and CHS-1) analysis indicated the reisolated fungus to be C. fioriniae. No fungal pathogens were isolated from mock inoculated plants. This study can provide effective management and useful information for the control of this disease on R. delavayi in Baili Azalea Forest Area. References: Damm, U., et al. 2012. Stud Mycol 73: 37. Fang, Z. D. 2007. Research Methods of Plant Diseases (Third edition). China Agriculture Press. Liu, J., et al. 2020. Mitochondrial DNA B 5:37. Shivas, R, G; Tan, Y, P. 2009. Fungal Divers 39:111.