Chromosomal Events Research Articles

Somy evolution in the honey bee infecting trypanosomatid parasite, Lotmaria passim.

Lotmaria passim is a ubiquitous trypanosomatid parasite of honey bees nestled within the medically important subfamily Leishmaniinae. Although this parasite is associated with honey bee colony losses, the original draft genome-which was completed before its differentiation from the closely related Crithidia mellificae-has remained the reference for this species despite lacking improvements from newer methodologies. Here we report the updated sequencing, assembly, and annotation of the BRL type strain (ATCC PRA-422) of Lotmaria passim. The nuclear genome assembly has been resolved into 31 complete chromosomes and is paired with an assembled kinetoplast genome consisting of a maxicircle and 30 minicircle sequences. The assembly spans 33.7 Mb and contains very little repetitive content, from which our annotation of both the nuclear assembly and kinetoplast predicted 10,288 protein-coding genes. Analyses of the assembly revealed evidence of a recent chromosomal duplication event within chromosomes 5 and 6 and provides evidence for a high level of aneuploidy in this species, mirroring the genomic flexibility employed by other trypanosomatids as a means of adaptation to different environments. This high-quality reference can therefore provide insights into adaptations of trypanosomatids to the thermally regulated, acidic, and phytochemically rich honey bee hindgut niche, which offers parallels to the challenges faced by other Leishmaniinae during the challenges they undergo within insect vectors, during infection of mammals, and exposure to antiparasitic drugs throughout their multi-host life cycles. This reference will also facilitate investigations of strain-specific genomic polymorphisms, their role in pathogenicity, and the development of treatments for pollinator infection.

Unravelling the architecture of major histocompatibility complex class II haplotypes in rhesus macaques.

The regions in the genome that encode components of the immune system are often featured by polymorphism, copy number variation, and segmental duplications. There is a need to thoroughly characterize these complex regions to gain insight into the impact of genomic diversity on health and disease. Here we resolve the organization of complete major histocompatibility complex (MHC) class II regions in rhesus macaques by using a long-read sequencing strategy (Oxford Nanopore Technologies) in concert with adaptive sampling. In particular, the expansion and contraction of the primate DRB-region appear to be a dynamic process that involves the rearrangement of different cassettes of paralogous genes. These chromosomal recombination events are propagated by a conserved pseudogene, DRB6, which features the integration of two retroviral elements. In contrast, the DRA locus appears to be protected from rearrangements, which may be owing to the presence of an adjacently located truncated gene segment, DRB9 With our sequencing strategy, the annotation, evolutionary conservation, and potential function of pseudogenes can be reassessed, an aspect that was neglected by most genome studies in primates. Furthermore, our approach facilitates the characterization and refinement of an animal model essential to study human biology and disease.

Genome-Wide Identification and Functional Validation of Actin Depolymerizing Factor (ADF) Gene Family in Gossypium hirsutum L.

The Actin Depolymerizing Factor (ADF) protein, highly conserved among eukaryotes, is essential for plant growth, development, and stress responses. Cotton, a vital economic crop with applications spanning oilseed, textiles, and military sectors, has seen a limited exploration of its ADF gene family. This research has identified 118 unique ADF sequences across four principal cotton species: Gossypium hirsutum L., Gossypium barbadense Linn, Gossypium raimondii, and Asiatic cotton. The study found that the structural domains and physicochemical properties of these proteins are largely uniform across species. The ADF genes were classified into four subfamilies with a notable expansion in groups III and IV due to tandem and chromosomal duplication events. A thorough analysis revealed a high degree of conservation in gene structure, including exon counts and the lengths of introns and exons, with the majority of genes containing three exons, aligning with the characteristics of the ADF family. RNA-seq analysis uncovered a spectrum of responses by GhADFs to various abiotic stresses with GhADF19 showing the most significant reaction. Virus-induced gene silencing (VIGS) experiments were conducted to assess the role of GhADF19 in plant growth under abiotic stress. The results demonstrated that plants with silenced GhADF19 exhibited significantly slower growth rates and lower dry weights when subjected to cold, salt, and drought stress compared to the control group. This marked reduction in growth and dry weight under stress conditions highlights the potential importance of GhADF19 in stress tolerance mechanisms.

KaryoTap Enables Aneuploidy Detection in Thousands of Single Human Cells.

Investigating chromosomal instability and aneuploidy within tumors is essential for understanding tumorigenesis and developing diagnostic and therapeutic strategies. Single-cell DNA sequencing technologies have enabled such analyses, revealing aneuploidies specific to individual cells within the same tumor. However, it has been difficult to scale the throughput of these methods to detect rare aneuploidies while maintaining high sensitivity. To overcome this deficit, we developed KaryoTap, a method combining custom targeted DNA sequencing panels for the Tapestri platform with a computational framework to enable detection of chromosome- and chromosome arm-scale aneuploidy (gains or losses) and copy number neutral loss of heterozygosity in all human chromosomes across thousands of single cells simultaneously. KaryoTap allows detecting gains and losses with an average accuracy of 83% for arm events and 91% for chromosome events. Importantly, together with chromosomal copy number, our system allows us to detect barcodes and gRNAs integrated into the cells' genome, thus enabling pooled CRISPR- or ORF-based functional screens in single cells. As a proof of principle, we performed a small screen to expand the chromosomes that can be targeted by our recently described CRISPR-based KaryoCreate system for engineering aneuploidy in human cells. KaryoTap will prove a powerful and flexible approach for the study of aneuploidy and chromosomal instability in both tumors and normal tissues.

Expanding the Molecular Spectrum of Carcinoma Ex Pleomorphic Adenoma: An Analysis of 84 Cases With a Novel HMGA2::LINC02389 Fusion.

Carcinoma ex pleomorphic adenoma (CXPA) is an aggressive epithelial and/or myoepithelial neoplasm that arises in association with a pleomorphic adenoma (PA). Its etiopathogenesis remains poorly understood, but it is believed that the development of this tumor is due to the accumulation of genetic, protein, metabolic, and epigenetic alterations in a PA. A retrospective review of the Salivary Gland Tumor Registry in Pilsen yielded 84 CXPA, namely 25/84 salivary duct carcinoma (SDC), 15/84 myoepithelial carcinoma (MC), 1/84 epithelial-myoepithelial carcinoma (EMC), and 1/84 adenoid cystic carcinoma (AdCC). All 84 CXPA cases were analyzed by next-generation sequencing (NGS) and/or fluorescence in situ hybridization (FISH). Forty-three tumors originally diagnosed as CXPA (43/84, 51.2%) showed some molecular alteration. Fusion transcripts were identified in 12/16 (75%) CXPA, including LIFR::PLAG1, CTNNB1::PLAG1, FGFR1::PLAG1, and a novel fusion, HMGA2::LINC02389. Most of the fusions were confirmed by FISH using PLAG1 (6/11) and HMGA2 (1/1) gene break probes. Split signals indicating gene break were identified by FISH for PLAG1 (12/17), HMGA2 (3/4), EWSR1 (7/22), and MYB (2/7). Concerning pathogenic mutations, only CXPA with epithelial differentiation (SDC) presented these alterations, including HRAS mutation (2/4), TP53 (1/4), PTEN (1/4), and ATK1 (1/4). In addition, amplifications in ERBB2 (17/35), MDM2 (1/4), and EWSR1 (1/7) were detected. A novel finding was the discovery of an HMGA2::LINC02389 fusion in 1 patient with EMC ex-PA. The present results indicate that molecular profiling of CXPA with myoepithelial differentiation (MC) tends to reveal chromosomal fusion events, whereas CXPA with epithelial differentiation (SDC) tends to have a higher frequency of pathogenic mutations and gene amplifications.

Revolutionizing soybean genomics: How CRISPR and advanced sequencing are unlocking new potential.

Soybean Glycine max L., paleopolyploid genome, poses challenges to its genetic improvement. However, the development of reference genome assemblies and genome sequencing has completely changed the field of soybean genomics, allowing for more accurate and successful breeding techniques as well as research. During the single-cell revolution, one of the most advanced sequencing tools for examining the transcriptome landscape is single-cell RNA sequencing (scRNA-seq). Comprehensive resources for genetic improvement of soybeans may be found in the SoyBase and other genomics databases. CRISPR-Cas9 genome editing technology provides promising prospects for precise genetic modifications in soybean. This method has enhanced several soybean traits, including as yield, nutritional value, and resistance to both biotic and abiotic stresses. With base editing techniques that allow for precise DNA modifications, the use of CRISPR-Cas9 is further increased. With the availability of the reference genome for soybeans and the following assembly of wild and cultivated soybeans, significant chromosomal rearrangements and gene duplication events have been identified, offering new perspectives on the complex genomic structure of soybeans. Furthermore, major single nucleotide polymorphisms (SNPs) linked to stachyose and sucrose content have been found through genome-wide association studies (GWAS), providing important tools for enhancing soybean carbohydrate profiles. In order to open up new avenues for soybean genetic improvement, future research approaches include investigating transcriptional divergence processes, enhancing genetic resources, and incorporating CRISPR-Cas9 technologies.

Activation of Wnt/β-catenin signaling is critical for the tumorigenesis of choroid plexus.

Choroid plexus (ChP) is the secretory epithelial structure located in brain ventricles. Choroid plexus tumors (CPTs) are rare neoplasms predominantly occurring in young patients with intensified malignancy in children. CPT treatment is hindered by insufficient knowledge of the tumor pathology and limited availability of valid models. Genomic and transcriptomic data from CPT patients were analyzed to identify the putative pathological pathway. Cellular and molecular techniques were employed to validate bioinformatic results in CPT patient samples. Pharmacologic inhibition of Wnt/β-catenin signaling was assessed in CPT cells. Cell-based assays of ChP cell lines were performed following CRISPR-Cas9-derived knockout and over-expression of Wnt/β-catenin pathway genes. 3D CPT model was generated through CRISPR-Cas9-derived knockout of APC. We discovered that Wnt/β-catenin signaling is activated in human CPTs, likely as a consequence of large-scale chromosomal instability events of the CPT genomes. We demonstrated that CPT-derived cells depend on autocrine Wnt/β-catenin signaling for survival. Constitutive Wnt/β-catenin pathway activation, either through knock-out of the negative regulator APC or overexpression of the ligand WNT3A, induced tumorigenic properties in ChP 2D in vitro models. Increased activation of Wnt/β-catenin pathway in ChP organoids, through treatment with a potent GSK3β inhibitor, reduced the differentiation of mature ChP epithelia cells. Remarkably, the depletion of APC was sufficient to induce the oncogenic transformation of ChP organoids. Our research identifies Wnt/β-catenin signaling as a critical driver of CPT tumorigenesis and provides the first 3D in vitro model for future pathological and therapeutic studies of CPT.

A familial, telomere-to-telomere reference for human de novo mutation and recombination from a four-generation pedigree.

Using five complementary short- and long-read sequencing technologies, we phased and assembled >95% of each diploid human genome in a four-generation, 28-member family (CEPH 1463) allowing us to systematically assess de novo mutations (DNMs) and recombination. From this family, we estimate an average of 192 DNMs per generation, including 75.5 de novo single-nucleotide variants (SNVs), 7.4 non-tandem repeat indels, 79.6 de novo indels or structural variants (SVs) originating from tandem repeats, 7.7 centromeric de novo SVs and SNVs, and 12.4 de novo Y chromosome events per generation. STRs and VNTRs are the most mutable with 32 loci exhibiting recurrent mutation through the generations. We accurately assemble 288 centromeres and six Y chromosomes across the generations, documenting de novo SVs, and demonstrate that the DNM rate varies by an order of magnitude depending on repeat content, length, and sequence identity. We show a strong paternal bias (75-81%) for all forms of germline DNM, yet we estimate that 17% of de novo SNVs are postzygotic in origin with no paternal bias. We place all this variation in the context of a high-resolution recombination map (~3.5 kbp breakpoint resolution). We observe a strong maternal recombination bias (1.36 maternal:paternal ratio) with a consistent reduction in the number of crossovers with increasing paternal (r=0.85) and maternal (r=0.65) age. However, we observe no correlation between meiotic crossover locations and de novo SVs, arguing against non-allelic homologous recombination as a predominant mechanism. The use of multiple orthogonal technologies, near-telomere-to-telomere phased genome assemblies, and a multi-generation family to assess transmission has created the most comprehensive, publicly available "truth set" of all classes of genomic variants. The resource can be used to test and benchmark new algorithms and technologies to understand the most fundamental processes underlying human genetic variation.

Abstract Mo049: Loss of Y chromosome and cardiovascular events in chronic kidney disease

Background: Chronic kidney disease represents one of the strongest risk factors for cardiovascular diseases, and particularly for heart failure. Despite improved pharmaceutical treatments mortality remains high. Recently, experimental studies demonstrated that mosaic loss of Y chromosome (LOY) associates with cardiac fibrosis in mice. Since diffuse cardiac fibrosis is the common denominator for progression of all forms of heart failure, we determined the association of LOY on mortality and cardiovascular disease outcomes in patients with chronic kidney disease. Methods: LOY was quantified in patients with stable CKD (CARE for HOMe study, derivation cohort, N=279) and dialysis patients (4D study, validation cohort, N=544). The association between LOY and mortality, combined cardiovascular and heart failure-specific endpoints, and echocardiographic measures was assessed. Results: In CARE for HOMe, mortality was higher in men as compared to women across the entire range of impaired renal function. The frequency of LOY increased with age. LOY was associated with increased mortality (HR 2.58, 95%CI 1.33-5.03) and risk for cardiac decompensation or death (HR 2.30, 95%CI 1.23-4.27). Patients with LOY >17 % showed a significant decline of ejection fraction and an increase of E/E’ within five years. Consistently, validation in the 4D study revealed that LOY was associated with increased mortality (HR 3.13, 95%CI 1.97-4.99), higher risk of death due to heart failure and sudden cardiac death (HR 4.14, 95%CI 2.00-8.57), but not atherosclerotic events (see figure). Patients with LOY >17 % showed significantly higher plasma levels of soluble ST2, a biomarker for myocardial fibrosis. Mechanistically, intermediate monocytes from patients with LOY >17 % showed significantly higher CCR2 expression and higher plasma levels of the CCR2 ligand CCL2, which may have contributed to increased heart failure events. Conclusions: LOY identifies male patients with chronic kidney disease at high risk for mortality and heart failure events.

Loss of Y Chromosome and Cardiovascular Events in Chronic Kidney Disease.

Chronic kidney disease represents one of the strongest risk factors for cardiovascular diseases, and particularly for heart failure. Despite improved pharmaceutical treatments, mortality remains high. Recently, experimental studies demonstrated that mosaic loss of Y chromosome (LOY) associates with cardiac fibrosis in male mice. Since diffuse cardiac fibrosis is the common denominator for progression of all forms of heart failure, we determined the association of LOY on mortality and cardiovascular disease outcomes in patients with chronic kidney disease. LOY was quantified in men with stable chronic kidney disease (CARE for HOMe study, n=279) and dialysis patients (4D study, n=544). The association between LOY and mortality, combined cardiovascular and heart failure-specific end points, and echocardiographic measures was assessed. In CARE for HOMe, the frequency of LOY increased with age. LOY >17% was associated with increased mortality (heart rate, 2.58 [95% CI, 1.33-5.03]) and risk for cardiac decompensation or death (heart rate, 2.30 [95% CI, 1.23-4.27]). Patients with LOY >17% showed a significant decline of ejection fraction and an increase of E/E' within 5 years. Consistently, in the 4D study, LOY >17% was significantly associated with increased mortality (heart rate, 2.76 [95% CI, 1.83-4.16]), higher risk of death due to heart failure and sudden cardiac death (heart rate, 4.11 [95% CI, 2.09-8.08]), but not atherosclerotic events. Patients with LOY >17% showed significantly higher plasma levels of soluble interleukin 1 receptor-like 1, a biomarker for myocardial fibrosis. Mechanistically, intermediate monocytes from patients with LOY >17% showed significantly higher C-C chemokine receptor type 2 expression and higher plasma levels of the C-C chemokine receptor type 2 chemokine (C-C motif) ligand 2, which may have contributed to increased heart failure events. LOY identifies male patients with chronic kidney disease at high risk for mortality and heart failure events.

P-520 Analyses of mosaic embryos reveal critical regions on human chromosome 11 that involve embryo implantation

Abstract Study question Why some mosaic embryos can be implanted, whereas others cannot? What are the secrets hidden in the human genome that control embryo implantation? Summary answer By data-mining genomic information from PGT-A results of 109 mosaic embryos, we have defined the critical regions in chr11 that play essential roles in implantation. What is known already Embryo implantation is usually a bottleneck step for a successful IVF treatment. What factors control the embryo implantation process has been pursued for many years, and the answers remain largely unknown. PGT-A is a widely applied procedure to identify embryos with normal diploid chromosomes to increase the clinical pregnancy rate per embryo transfer. However, some poor responder patients may only have mosaic embryos that can be used for transfer. We thus investigate the critical regions of the human chromosomes that are important for controlling embryo implantation. Study design, size, duration One hundred and nine mosaic blastocysts were analyzed based on PGT-A assay from 94 patients (ages ranging from 30 to 49 years old, mean age = 38.9 years) undergoing IVF between November 2020 and March 2022. The impact of chromosomal regions on implantation was explored by analyzing the sequencing results together with the pregnancy outcome of mosaic embryos. Additional published outcome data of 1000 mosaic embryo transfers was used to confirm our findings. Participants/materials, setting, methods Pregnant outcomes were followed, including hCG examination, sac formation, and live birth. Whole genome data analysis was performed using individual PGT-A results. Different types of chromosome abnormalities were calculated, and heatmap and clustering analysis were used to define the critical chromosome regions for embryo implantation. In addition, gene set enrichment and pathway analyses were conducted to reveal the functions of genes within the critical regions. Main results and the role of chance Among the transferred 109 mosaic embryos, the clinical outcomes of hCG positive, sac formation, and live birth rates were 45.8%, 42.2%, and 32.1%, respectively. There were 74 (67.8%) and 35 (32.1%) embryos carrying single and multiple mosaic chromosomes, respectively, resulting in 182 chromosomal mosaicism events. The trisomy mosaicism (133/182, 73.0%) occurred with higher frequency than monosomy mosaicism (49/182, 26.9%). Using the heatmap and clustering analysis, our data showed clustered monosomy mosaicism in non-pregnant embryos, especially for chr2, chr4, chr11, chr19, and chr22. Further refinement using segmental mosaic embryos has identified the critical regions, ranging from 10M to 40M in size on different chromosomes that are involved in hundreds of protein-coding genes and known dosage-sensitive genes. Importantly, functional analyses of the genes within the critical regions of chromosome 11 revealed enrichments in embryo invasion and angiogenesis, and suggest that loss of the chromosomal copy of these regions may result in implantation failure. Limitations, reasons for caution It’s still preliminary. More data is required to further confirm the conclusion. Wider implications of the findings Our study provides insights into the chromosomal regions involved in human embryo implantation and will improve the embryo prioritizing strategy in future IVF clinics. Importantly it is the first time the genomics information and gene-mapping strategy have been applied in mosaic embryos to uncover mechanisms underlying human embryo implantation. Trial registration number not applicable

Aurora Kinases: Their Role in Cancer and Cellular Processes

Aurora kinases, belonging to a highly conserved family of serine/threonine kinases with critical roles in the regulation of the cell cycle, comprise three members: Aurora kinase A, B, and C, which serve as key mitotic regulators essential for maintaining chromosome stability. Aurora kinases play crucial roles in multiple events in mitotic such as the coordination of chromosomal and cytoskeletal events, regulation of the spindle assembly checkpoint pathway and cytokinesis to ensure the smooth progression of the cell cycle. Besides their mitotic functions, Aurora kinases are also involved in the regulation of meiosis. Gene amplification/mutation and overexpression of Aurora kinases have been detected in various solid and haematological cancers. In human tumours, Aurora kinases exhibit oncogenic roles associated with their mitotic roles, which drive the cancer cell proliferation and survival. Deregulation of Aurora kinase activity causes failure in centrosome function, spindle assembly, chromosomal alignment, and cytokinesis, eventually resulting in the mitotic abnormalities and genetic instability. These findings emphasize the crucial functions of Aurora kinases in cancer, prompting their recognition as valuable targets for cancer therapy. This review provides an overview of the structures and functions of Aurora kinases and sheds light on their oncogenic roles in cancer.

Chromosome segregation during spermatogenesis occurs through a unique center-kinetic mechanism in holocentric moth species.

Precise regulation of chromosome dynamics in the germline is essential for reproductive success across species. Yet, the mechanisms underlying meiotic chromosomal events such as homolog pairing and chromosome segregation are not fully understood in many species. Here, we employ Oligopaint DNA FISH to investigate mechanisms of meiotic homolog pairing and chromosome segregation in the holocentric pantry moth, Plodia interpunctella, and compare our findings to new and previous studies in the silkworm moth, Bombyx mori, which diverged from P. interpunctella over 100 million years ago. We find that pairing in both Bombyx and Plodia spermatogenesis is initiated at gene-rich chromosome ends. Additionally, both species form rod shaped cruciform-like bivalents at metaphase I. However, unlike the telomere-oriented chromosome segregation mechanism observed in Bombyx, Plodia can orient bivalents in multiple different ways at metaphase I. Surprisingly, in both species we find that kinetochores consistently assemble at non-telomeric loci toward the center of chromosomes regardless of where chromosome centers are located in the bivalent. Additionally, sister kinetochores do not seem to be paired in these species. Instead, four distinct kinetochores are easily observed at metaphase I. Despite this, we find clear end-on microtubule attachments and not lateral microtubule attachments co-orienting these separated kinetochores. These findings challenge the classical view of segregation where paired, poleward-facing kinetochores are required for accurate homolog separation in meiosis I. Our studies here highlight the importance of exploring fundamental processes in non-model systems, as employing novel organisms can lead to the discovery of novel biology.

Impact of polyploidization on genome evolution and phenotypic diversity in oil-tea Camellia

Oil-tea Camellia (OTC) is a valuable oil crop with diverse applications across the food, cosmetics, and medicine sectors. However, understanding the impact of chromosome polyploidization on genome evolution and phenotypic diversity in OTC remains challenging. This study addressed this knowledge gap by focusing on the origins and impacts of OTC polyploidization. The chromosomal karyotypes of 10 representative samples were examined, and their relationships were elucidated through hierarchical clustering analysis of karyotype parameters. Based on the phylogeny of the genus Camellia, the ancestral 1 C-value and 1 Cx-value were reconstructed, revealing an increase in the 1 C-value and a reduction in the 1 Cx-value associated with polyploidization. Notable positive correlations between ploidy level, genome size, and fruit size were highlighted by phylogenetic generalized least squares analysis. The phenotypic characteristics and chromosomal evolution of OTC were reconstructed. It was inferred that the genus Camellia, which was originally diploid, underwent 11 chromosomal duplication events. With the emergence of polyploidization, phenotypic traits transitioned from small fruits, autumn–winter flowering, and white flowers to larger fruits, spring flowering, and red flowers. Biogeographically, it is suggested that OTC first appeared in the Lingnan region, and subsequent species dispersion and hybridization significantly influenced the emergence of polyploid species. In conclusion, evidence has shown that OTC polyploidization increases genome size and enhances phenotypic diversity. This research enhances our understanding of the complex genetic mechanisms that drive species evolution, influence genome changes, and contribute to the domestication of Camellia plants. Furthermore, these findings offer insights that can inform future breeding and cultivation strategies for OTC.

Chromosome-level genome assemblies of 2 hemichordates provide new insights into deuterostome origin and chromosome evolution.

Deuterostomes are a monophyletic group of animals that includes Hemichordata, Echinodermata (together called Ambulacraria), and Chordata. The diversity of deuterostome body plans has made it challenging to reconstruct their ancestral condition and to decipher the genetic changes that drove the diversification of deuterostome lineages. Here, we generate chromosome-level genome assemblies of 2 hemichordate species, Ptychodera flava and Schizocardium californicum, and use comparative genomic approaches to infer the chromosomal architecture of the deuterostome common ancestor and delineate lineage-specific chromosomal modifications. We show that hemichordate chromosomes (1N = 23) exhibit remarkable chromosome-scale macrosynteny when compared to other deuterostomes and can be derived from 24 deuterostome ancestral linkage groups (ALGs). These deuterostome ALGs in turn match previously inferred bilaterian ALGs, consistent with a relatively short transition from the last common bilaterian ancestor to the origin of deuterostomes. Based on this deuterostome ALG complement, we deduced chromosomal rearrangement events that occurred in different lineages. For example, a fusion-with-mixing event produced an Ambulacraria-specific ALG that subsequently split into 2 chromosomes in extant hemichordates, while this homologous ALG further fused with another chromosome in sea urchins. Orthologous genes distributed in these rearranged chromosomes are enriched for functions in various developmental processes. We found that the deeply conserved Hox clusters are located in highly rearranged chromosomes and that maintenance of the clusters are likely due to lower densities of transposable elements within the clusters. We also provide evidence that the deuterostome-specific pharyngeal gene cluster was established via the combination of 3 pre-assembled microsyntenic blocks. We suggest that since chromosomal rearrangement events and formation of new gene clusters may change the regulatory controls of developmental genes, these events may have contributed to the evolution of diverse body plans among deuterostomes.

Non-clinical evaluation of NT-175, an autologous T cell product engineered to express an HLA-A*02:01-restricted TCR targeting TP53 R175H and resistant to TGF-b inhibition.

2560 Background: Adoptive transfer of T-cell receptor (TCR)-engineered T-cells to target shared cancer neoantigens is a promising new immunotherapy approach for patients harboring mutations in tumor suppressor genes such as TP53. TP53 is the most commonly mutated gene across all cancer types, with the R175H mutation being the most prevalent across different indications. NT-175 is an autologous engineered T-cell product expressing an HLA-A*02:01-restricted TCR that specifically targets the TP53 R175H mutation. NT-175 is additionally engineered to lack TGF-β receptor II (TGFBR2) expression, rendering T-cells insensitive to TGF-b-mediated inhibition in the tumor microenvironment. Methods: For manufacturing of NT-175, CD4 and CD8 T-cells are enriched from leukapheresis material, activated in vitro and gene-edited to knock-out both endogenous TGFBR2 and TCR β constant 1/2 (TRBC1/2) and knock-in an HLA-A*02:01-restricted TP53 R175H neoantigen-specific TCR at the TCR α constant (TRAC) locus using CRISPR-Cas9 technology. NT-175 product functionality was evaluated in vivo models and in vitro. T-cell reactivity was assessed by measuring cytotoxicity, proliferation, and cytokine production. The benefit of TGFBR2 knock-out (KO) in the presence of TGF-b was evaluated by measuring phosphorylation of SMAD2/3 proteins, the impact on cell viability and serial killing of target cells. For determination of NT-175 product safety, cross-reactivity and HLA-specificity assessments were carried out. A comprehensive analysis of potential off-target editing by CRISPR/Cas9 was performed to assess potential risk of genotoxicity in clinical grade NT-175. Results: High reactivity of the NT-175 TCR against TP53 R175H and HLA-A*02:01 expressing target cells was observed. T-cell activation and functionality was highly specific, as demonstrated by a lack of reactivity against TP53 WT, minimal cross-reactivity against antigens with up to 4 mismatches to the minimal TP53 R175H encoding epitope recognized by the NT-175 TCR, and lack of T-cell activation in the absence of HLA-A*02:01. In the presence of TGF-b, TGFBR2 KO TCR-edited T-cells displayed inhibition of SMAD2/3 phosphorylation, increased cell viability and increased cytotoxicity and proliferation in serial stimulation assays. In vivo, NT-175 T-cells were able to induce tumor clearance in two independent models. Low frequency chromosomal translocation events (<0.1%) between on-target and off-target Cas9 cleavage sites were detectable in NT-175 T-cells. However importantly, these did not result in autonomous cytokine-independent growth. Conclusions: Non-clinical studies revealed a favorable safety and efficacy profile for NT-175 and supported further clinical development of a TGF-β-resistant TCR-edited T-cell product for mutant TP53-targeted cancer immunotherapy.

Non-clinical evaluation of NT-112, an autologous T cell product engineered to express an HLA-C*08:02-restricted TCR targeting KRAS G12D and resistant to TGF-b inhibition.

e14533 Background: Adoptive transfer of T-cell receptor (TCR)-engineered T-cells to target shared cancer neoantigens is a promising new immunotherapy approach for patients harboring mutations in oncogenes such as KRAS. Activating mutations in KRAS are among the most prevalent oncogenic driver mutations in human cancers, with KRAS G12D being the most frequent KRAS mutation in colorectal carcinoma and pancreatic cancer. NT-112 is an autologous engineered T-cell product expressing an HLA-C*08:02-restricted TCR that specifically targets the KRAS G12D mutation. NT-112 is additionally engineered to lack TGF-β receptor II (TGFBR2) expression, rendering T-cells insensitive to TGF-b-mediated inhibition in the tumor microenvironment. Methods: For manufacturing of NT-112, CD4 and CD8 T-cells are enriched from leukapheresis material, activated in vitro and gene-edited to knock-out both endogenous TGFBR2 and TCR β constant 1/2 (TRBC1/2) and knock-in an HLA-C*08:02-restricted KRS G12D neoantigen-specific TCR at the TCR α constant (TRAC) locus using CRISPR-Cas9 technology. NT-112 product functionality was evaluated in both in vivo models and in vitro co-culture experiments with a panel of cell lines including those endogenously expressing the KRAS G12D mutation. T-cell reactivity was assessed by measuring cytotoxicity, proliferation, and cytokine production. For determination of NT-112 product safety, cross-reactivity and allo-reactivity assessments were carried out. A comprehensive analysis of potential off-target editing by CRISPR/Cas9 was performed to assess potential risk of genotoxicity in clinical grade NT-112. Results: High reactivity of the NT-112 TCR against KRAS G12D and HLA-C*08:02-expressing target cells was observed. T-cell activation and functionality was highly specific, as demonstrated by a lack of reactivity against KRAS WT, minimal cross-reactivity in Alanine and Glycine scans, and lack of T-cell activation in the absence of HLA-C*08:02. In vivo, NT-112 T-cells were able to induce tumor clearance in two independent models. Low frequency chromosomal translocation events (<0.1%) between on-target and off-target Cas9 cleavage sites were detectable in NT-112 T-cells. However importantly, these did not result in autonomous cytokine-independent growth. Conclusions: Non-clinical studies revealed a favorable safety and efficacy profile for NT-112 and supported further clinical development of a TGF-β-resistant TCR-edited T-cell product for mutant KRAS-targeted cancer immunotherapy.

Genome-wide identification of bZIP transcription factors and their expression analysis in Platycodon grandiflorus under abiotic stress.

The Basic Leucine Zipper (bZIP) transcription factors (TFs) family is among of the largest and most diverse gene families found in plant species, and members of the bZIP TFs family perform important functions in plant developmental processes and stress response. To date, bZIP genes in Platycodon grandiflorus have not been characterized. In this work, a number of 47 PgbZIP genes were identified from the genome of P. grandiflorus, divided into 11 subfamilies. The distribution of these PgbZIP genes on the chromosome and gene replication events were analyzed. The motif, gene structure, cis-elements, and collinearity relationships of the PgbZIP genes were simultaneously analyzed. In addition, gene expression pattern analysis identified ten candidate genes involved in the developmental process of different tissue parts of P. grandiflorus. Among them, Four genes (PgbZIP5, PgbZIP21, PgbZIP25 and PgbZIP28) responded to drought and salt stress, which may have potential biological roles in P. grandiflorus development under salt and drought stress. Four hub genes (PgbZIP13, PgbZIP30, PgbZIP32 and PgbZIP45) mined in correlation network analysis, suggesting that these PgbZIP genes may form a regulatory network with other transcription factors to participate in regulating the growth and development of P. grandiflorus. This study provides new insights regarding the understanding of the comprehensive characterization of the PgbZIP TFs for further exploration of the functions of growth and developmental regulation in P. grandiflorus and the mechanisms for coping with abiotic stress response.

In-Depth Characterization of bZIP Genes in the Context of Endoplasmic Reticulum (ER) Stress in Brassica campestris ssp. chinensis.

Numerous studies have been conducted to investigate the genomic characterization of bZIP genes and their involvement in the cellular response to endoplasmic reticulum (ER) stress. These studies have provided valuable insights into the coordinated cellular response to ER stress, which is mediated by bZIP transcription factors (TFs). However, a comprehensive and systematic investigations regarding the role of bZIP genes and their involvement in ER stress response in pak choi is currently lacking in the existing literature. To address this knowledge gap, the current study was initiated to elucidate the genomic characteristics of bZIP genes, gain insight into their expression patterns during ER stress in pak choi, and investigate the protein-to-protein interaction of bZIP genes with the ER chaperone BiP. In total, 112 members of the BcbZIP genes were identified through a comprehensive genome-wide analysis. Based on an analysis of sequence similarity, gene structure, conserved domains, and responsive motifs, the identified BcbZIP genes were categorized into 10 distinct subfamilies through phylogenetic analysis. Chromosomal location and duplication events provided insight into their genomic context and evolutionary history. Divergence analysis estimated their evolutionary history with a predicted divergence time ranging from 0.73 to 80.71 million years ago (MYA). Promoter regions of the BcbZIP genes were discovered to exhibit a wide variety of cis-elements, including light, hormone, and stress-responsive elements. GO enrichment analysis further confirmed their roles in the ER unfolded protein response (UPR), while co-expression network analysis showed a strong relationship of BcbZIP genes with ER-stress-responsive genes. Moreover, gene expression profiles and protein-protein interaction with ER chaperone BiP further confirmed their roles and capacity to respond to ER stress in pak choi.

Comparative Genomics Reveal Phylogenetic Relationship and Chromosomal Evolutionary Events of Eight Cervidae Species.

Cervidae represents a family that is not only rich in species diversity but also exhibits a wide range of karyotypes. The controversies regarding the phylogeny and classification of Cervidae still persist. The flourishing development of the genomic era has made it possible to address these issues at the genomic level. Here, the genomes of nine species were used to explore the phylogeny and chromosomal evolutionary events of Cervidae. By conducting whole-genome comparisons, we identified single-copy orthologous genes across the nine species and constructed a phylogenetic tree based on the single-copy orthologous genes sequences, providing new insights into the phylogeny of Cervidae, particularly the phylogenetic relationship among sika deer, red deer, wapiti and Tarim red deer. Gene family analysis revealed contractions in the olfactory receptor gene family and expansions in the histone gene family across eight Cervidae species. Furthermore, synteny analysis was used to explore the chromosomal evolutionary events of Cervidae species, revealing six chromosomal fissions during the evolutionary process from Bovidae to Cervidae. Notably, specific chromosomal fusion events were found in four species of Cervus, and a unique chromosomal fusion event was identified in Muntiacus reevesi. Our study further completed the phylogenetic relationship within the Cervidae and demonstrated the feasibility of inferring species phylogeny at the whole-genome level. Additionally, our findings on gene family evolution and the chromosomal evolutionary events in eight Cervidae species lay a foundation for comprehensive research of the evolution of Cervidae.