Similar Gene Research Articles

Isolation and genetic analysis of the chikungunya virus from Aedes aegypti and Aedes albopictus mosquitoes captured in Central America

Introduction. The habitat of mosquitoes belonging to the genera Aedes spp., Culex spp., Culiseta spp. is in South and Central America, including Nicaragua. Monitoring of the spread of mosquito vectors and assessment of the infection with arboviruses can provide information on possible occurrence of new diseases or an increase in the reported cases, changes in the infectivity of viruses for humans due to changes in pathogen transmitters.
 The purpose of this study was isolation and identification of arboviruses belonging to the Flavivirus and Alphavirus genera from A. albopictus, A. aegypti, Culiseta spp., Culex spp. mosquitoes captured in forests of Nicaragua.
 Materials and methods. A. albopictus, A. aegypti, Culiseta spp., Culex spp. mosquitoes were captured during the dry season in 2021 in forested areas of Nicaragua in four different locations. Mosquitoes were sorted into pools, each containing 5-8 mosquitoes (236 pools in total). Using the reverse transcription polymerase chain reaction, the pools were tested for the presence of chikungunya (CHIKV), dengue, Zika, and yellow fever viruses. Positive pools were inoculated into the C6/36 cell culture to obtain isolates and for their further sequencing.
 Results. The dengue virus was detected only in Aedes spp. mosquitoes: in 7 pools — A. aegypti, in 1 — A. albopictus. CHIKV was also detected only in Aedes spp. mosquitoes: in 3 pools — A. aegypti, in 1 — A. albopictus. The sequencing of nucleotide sequences of 6К, Е1, Е2, and NS1 genes of CHIKV isolated from A. albopictus mosquitoes showed that compared to the similar gene sequences from CHIKV isolates recovered from A. aegypti mosquitoes, the 6K gene region contained 4 nucleotide and 4 amino acid substitutions, while the E1 region contained 16 nucleotide substitutions, 10 of them led to amino acid substitutions; the E2 region contained 14 nucleotide and 11 amino acid substitutions; the NS1 region contained 33 nucleotide and 19 amino acid substitutions.

Genome-Wide Identification of AhMDHs and Analysis of Gene Expression under Manganese Toxicity Stress in Arachis hypogaea.

Malate dehydrogenase (MDH) is one kind of oxidation-reduction enzyme that catalyzes the reversible conversion of oxaloacetic acid to malic acid. It has vital functions in plant development, photosynthesis, abiotic stress responses, and so on. However, there are no reports on the genome-wide identification and gene expression of the MDH gene family in Arachis hypogaea. In this study, the MDH gene family of A. hypogaea was comprehensively analyzed for the first time, and 15 AhMDH sequences were identified. According to the phylogenetic tree analysis, AhMDHs are mainly separated into three subfamilies with similar gene structures. Based on previously reported transcriptome sequencing results, the AhMDH expression quantity of roots and leaves exposed to manganese (Mn) toxicity were explored in A. hypogaea. Results revealed that many AhMDHs were upregulated when exposed to Mn toxicity, suggesting that those AhMDHs might play an important regulatory role in A. hypogaea's response to Mn toxicity stress. This study lays foundations for the functional study of AhMDHs and further reveals the mechanism of the A. hypogaea signaling pathway responding to high Mn stress.

Intrinsic deletion at 10q23.31, including the PTEN gene locus, is aggravated upon CRISPR-Cas9-mediated genome engineering in HAP1 cells mimicking cancer profiles.

The CRISPR-Cas9 system is a powerful tool for studying gene functions and holds potential for disease treatment. However, precise genome editing requires thorough assessments to minimize unintended on- and off-target effects. Here, we report an unexpected 283-kb deletion on Chromosome 10 (10q23.31) in chronic myelogenous leukemia-derived HAP1 cells, which are frequently used in CRISPR screens. The deleted region encodes regulatory genes, including PAPSS2, ATAD1, KLLN, and PTEN We found that this deletion was not a direct consequence of CRISPR-Cas9 off-targeting but rather occurred frequently during the generation of CRISPR-Cas9-modified cells. The deletion was associated with global changes in histone acetylation and gene expression, affecting fundamental cellular processes such as cell cycle and DNA replication. We detected this deletion in cancer patient genomes. As in HAP1 cells, the deletion contributed to similar gene expression patterns among cancer patients despite interindividual differences. Our findings suggest that the unintended deletion of 10q23.31 can confound CRISPR-Cas9 studies and underscore the importance to assess unintended genomic changes in CRISPR-Cas9-modified cells, which could impact cancer research.

Identification and bioinformatics analysis of MADS-box family genes containing K-box domain in maize

The MADS-box family genes are involved in the development of plant roots, leaves, flowers, and fruits, and play a crucial role in plant growth and development. Studying MADS-box genes with K-box domain is crucial to distinguish different types of MADS-box genes. This study systematically analysed the genomic structural information of maize MADS-box family members containing the K-box Domain at the genome-wide level using the maize (Zea mays) B73 genome as the reference sequence, and provided insight into the biological functions of the maize MADS-box family containing the K-box domain. According to the findings, 52 MADS-box family genes with K-box domain were identified and divided into 4 subgroups. The distribution of motif in the same subgroup was found to be relatively conservative, and all of them had MADS-box conserved domain and K-box domain. Gene structure analysis showed that the introns and exons of the same subgroup genes have similar gene structure, and different types of genes containing the K-box domain showed different exon/intron structure characteristics. Chromosome mapping showed that 52 genes containing the K-box domain were unevenly distributed on the 10 chromosomes of maize, most of which were distributed at both ends of the chromosome and a small number of genes were distributed near the centromere. Based on the analysis of cis-acting elements of it up-stream promoter, it was found that MADS-box family genes may be involved in light response, IAA, GA, ABA, and LTR signal pathways, indicating that they play a certain role in stress response and hormone signal transduction. The expression analysis of genes with the K-box domain in maize leaves treated with auxin and gibberellin revealed that MADS-box genes may have a regulatory effect on certain plant hormones. Through the identification and bioinformatics analysis of MADS-box family genes containing the K-box domain, it is helpful to further study the function and pathway of MADS-box family genes, and provide a theoretical basis for further re-search for the molecular mechanism of maize growth and development.

Bioinformatics and expression analysis of proline metabolism-related gene families in alfalfa under saline-alkali stress

Regulation of the proline metabolic pathway is essential for the accumulation of proline under abiotic stress and for the amelioration of plant stress resistance. Δ1-pyrroline-5-carboxylate synthase (P5CS), pyrroline-5-carboxylate reductase (P5CR), ornithine transaminase (δ-OAT), proline dehydrogenase (PDH), pyrroline-5-carboxylate dehydrogenase (P5CDH), and proline transporter (ProT) are the key enzymes in the proline metabolic pathway. However, the gene families responsible for proline metabolism have not yet been identified or reported in alfalfa. In this study, a total of 12 MsP5CSs, 4 MsP5CRs, 3 MsOATs, 6 MsPDHs, 2 MsP5CDHs, and 5 MsProTs were identified in the genome of alfalfa, and the members of the same subfamily had similar gene structures and conserved motifs. Analysis of cis-regulatory elements revealed the presence of light-responsive, hormone-regulated, and stress-responsive elements in the promoter regions of alfalfa proline metabolism-related genes. Following treatment with saline-alkali, the expression of MsP5CSs, MsP5CRs, MsOATs, and MsProTs was significantly upregulated, whereas the expression of MsPDH1.1, MsPDH1.3, and MsP5CDH was significantly downregulated. The proline content and enzyme activity of P5CS gradually increased, whereas the enzyme activity of PDH gradually decreased as the duration of stress increased. Root growth rates decreased upon MsP5CS1a suppression (MsP5CS1a-RNAi) in the hairy roots of alfalfa compared to the empty vector line under saline-alkali stress. These results show that proline metabolism-related genes play an important role in the saline-alkali stress tolerance of alfalfa and provide a theoretical basis for further research on the functions of proline metabolism-related genes in alfalfa in response to saline-alkali stress.

EPCO-41. MICRORNA-MEDIATED DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMAS

Abstract Neuron-specific microRNAs miR-9/9* and miR-124 (miR-9/9*-124) are potent neurogenic molecules whose ectopic expression in non-neuronal somatic cells effectively induces direct cell fate conversion into a neuronal fate. Our recent work indicates that the fate transition occurs in distinct steps where miR-9/9*-124 first represses core gene regulatory networks to erase the original non-neuronal cell identity and subsequently activates the neuronal program to confer neuronal fate. Extending these findings, we reason that repression of similar gene regulatory networks by miR-9/9*-124 can also induce fate erasure in cancerous cells using glioblastoma as a paradigm. Glioblastoma multiforme (GBM) is a type of aggressive brain tumor with poor prognosis. Instead of having specific mutation profiles, GBMs present a global genetic network dysregulation which gives rise to a neural stem cell or progenitor cell-like state resembling embryonic neural development. Whereas miR-9/9*-124 plays a key role in inducing mitotic exit and orchestrating neurogenesis during development and neuronal reprogramming, we hypothesize that miR-9/9*-124 will instruct neuronal reprogramming of GBMs. Our results so far demonstrate that miR-9/9*-124 indeed exerts reprogramming activities in patient-derived GBM cell lines. Ectopic expression of miR-9/9*-124 in vitro readily induces GBM cells to exit cell cycle and promotes neuronal identity over time. In GBM intracerebral xenograft mouse model, mice receiving GBM cells overexpressing miR-9/9*-124 were able to survive longer than those with GBMs overexpressing non-specific microRNAs. We are currently carrying out transcriptomic and chromatin accessibility analyses to infer the epigenetic switch as GBM cells are reprogrammed to neurons. Based on these studies, we will further examine whether the GBM cells undergo irreversible fate erasure and are reprogrammed into a stable post-mitotic neuronal state. Our studies will provide insights into the plasticity of GBM fate that can be controlled by microRNAs and the key downstream regulatory nodes underlying fate transition of GBM to permanent post-mitotic state.

Prediction of lncRNA functions using deep neural networks based on multiple networks

BackgroundMore and more studies show that lncRNA is widely involved in various physiological processes of the organism. However, the functions of the vast majority of them continue to be unknown. In addition, data related to lncRNAs in biological databases are constantly increasing. Therefore, it is quite urgent to develop a computing method to make the utmost of these data.ResultsIn this paper, we propose a new computational method based on global heterogeneous networks to predict the functions of lncRNAs, called DNGRGO. DNGRGO first calculates the similarities among proteins, miRNAs, and lncRNAs, and annotates the functions of lncRNAs according to its similar protein-coding genes, which have been labeled with gene ontology (GO). To evaluate the performance of DNGRGO, we manually annotated GO terms to lncRNAs and implemented our method on these data. Compared with the existing methods, the results of DNGRGO show superior predictive performance of maximum F-measure and coverage.ConclusionsDNGRGO is able to annotate lncRNAs through capturing the low-dimensional features of the heterogeneous network. Moreover, the experimental results show that integrating miRNA data can help to improve the predictive performance of DNGRGO.

Inhibition of USP7 upregulates USP22 and activates its downstream cancer-related signaling pathways in human cancer cells

Deubiquitinases (DUBs) play important roles in various human cancers and targeting DUBs is considered as a novel anticancer therapeutic strategy. Overexpression of ubiquitin specific protease 7 and 22 (USP7 and USP22) are associated with malignancy, therapy resistance, and poor prognosis in many cancers. Although both DUBs are involved in the regulation of similar genes and signaling pathways, such as histone H2B monoubiquitination (H2Bub1), c-Myc, FOXP3, and p53, the interdependence of USP22 and USP7 expression has never been described. In the study, we found that targeting USP7 via either siRNA-mediated knockdown or pharmaceutical inhibitors dramatically upregulates USP22 in cancer cells. Mechanistically, the elevated USP22 occurs through a transcriptional pathway, possibly due to desuppression of the transcriptional activity of SP1 via promoting its degradation upon USP7 inhibition. Importantly, increased USP22 expression leads to significant activation of downstream signal pathways including H2Bub1 and c-Myc, which may potentially enhance cancer malignancy and counteract the anticancer efficacy of USP7 inhibition. Importantly, targeting USP7 further suppresses the in vitro proliferation of USP22-knockout (USP22-Ko) A549 and H1299 lung cancer cells and induces a stronger activation of p53 tumor suppressor signaling pathway. In addition, USP22-Ko cancer cells are more sensitive to a combination of cisplatin and USP7 inhibitor. USP7 inhibitor treatment further suppresses in vivo angiogenesis and tumor growth and induced more apoptosis in USP22-Ko cancer xenografts. Taken together, our findings demonstrate that USP7 inhibition can dramatically upregulate USP22 in cancer cells; and targeting USP7 and USP22 may represent a more effective approach for targeted cancer therapy, which warrants further study.51f9aZhqcv_kcNHk2RbtxAVideo

Functionally similar genes exhibit comparable/similar time-course expression kinetics in the UV-induced photoaged mouse model.

Skin photoaging induced by ultraviolet (UV) irradiation contributes to the formation of thick and coarse wrinkles. Humans are exposed to UV light throughout their lives. Therefore, it is crucial to determine the time-sequential effects of UV on the skin. In this study, we irradiated the mouse back skin with UV light for eight weeks and observed the changes in gene expressions via microarray analysis every week. There were more downregulated genes (514) than upregulated genes (123). The downregulated genes had more functional diversity than the upregulated genes. Additionally, the number of downregulated genes did not increase in a time-dependent manner. Instead, time-dependent kinetic patterns were observed. Interestingly, each kinetic cluster harbored functionally enriched gene sets. Since collagen changes in the dermis are considered to be a major cause of photoaging, we hypothesized that other gene sets contributing to photoaging would exhibit kinetics similar to those of the collagen-regulatory genes identified in this study. Accordingly, co-expression network analysis was conducted using 11 well-known collagen-regulatory seed genes to predict genes with similar kinetics. We ranked all downregulated genes from 1 to 504 based on their expression levels, and the top 50 genes were suggested to be involved in the photoaging process. Additionally, to validate and support our identified top 50 gene lists, we demonstrated that the genes (FN1, CCDC80, PRELP, and TGFBR3) we discovered are downregulated by UV irradiation in cultured human fibroblasts, leading to decreased collagen levels, which is indicative of photoaging processes. Overall, this study demonstrated the time-sequential genetic changes in chronically UV-irradiated skin and proposed 50 genes that are involved in the mechanisms of photoaging.

Abstract 15706: Common- and Rare-Variant Genetic Architecture of Heart Failure Across the Allele Frequency Spectrum

Background: Heart failure (HF) is a complex trait influenced by environmental and genetic factors. Genome-wide association studies (GWAS) have found common genetic variants associated with HF risk, but the extent that common and rare variants explain risk and implicate similar genes and pathways in HF remains less understood. Methods: For common variants, we meta-analyze existing GWAS of all-cause HF with data from the Million Veteran Program, including 207,346 individuals with HF and 2,151,210 without in diverse populations. We examine rare coding variants in the Penn Medicine BioBank (PMBB) using gene-collapsing association studies and burden heritability regression. We test for an interaction between a polygenic risk score (PRS) based on our GWAS meta-analysis and truncating variants in TTN (TTNtv). Results: GWAS meta-analysis identified 176 risk loci (146 previously unreported), implicating known risk factors for HF and genes involved in cardiac development. New signals at GIPR/ GLP1R highlight potential contributions of dysregulated glucose homeostasis and metabolism in HF. Gene collapsing studies identified associations between HF and rare loss-of-function variants in TTN (OR 2.94, 95% CI 2.43-3.56) and MYBPC3 (OR 7.71, 95% CI 3.97-14.97) at exome-wide significance. HF heritability mediated through common variants (4.5%) was significantly higher than for rare variants (1.8%), but rare variant heritability was further enriched in cardiomyopathy genes compared to common variants (27-fold vs. 3.4-fold). We found a significant interaction between a new multi-ancestry HF-associated PRS (AUC 0.72, 95% CI 0.72-0.73) and TTNtv carrier status (interaction OR 1.32, 95% CI 1.06-1.64). Conclusions: Common and rare variants contribute to HF heritability. Common variant HF heritability is highly polygenic, while rare variant burden heritability appears more concentrated in core cardiomyopathy genes. HF risk conferred by TTNtv carrier status is further increased by an individual’s polygenic contribution. Our results support the continued inclusion of cardiomyopathy genes in rare-variant genetic testing panels for HF but suggest a meaningful polygenic component to HF exists that is not currently captured by clinical genetic testing.

Diverse Bradyrhizobium spp. with Similar Symbiosis Genes Nodulate Peanut in Different Regions of China: Characterization of Symbiovar sv. Arachis.

A total of 219 rhizobial strains isolated from peanut grown in soils from six peanut croplands in Zhengyang county, Henan Province, were typed by PCR-RFLP of IGS sequences. Their phylogenetic relationships were refined on representative strains using sequence analyses of 16S rRNA genes, housekeeping genes (atpD, recA, glnII) and symbiosis genes (nodA, nodC and nifH). The 219 rhizobial isolates were classified into 13 IGS types, and twenty representatives were defined within eight Bradyrhizobium genospecies: B. guangdongense covering 5 IGS types (75.2% of total isolates), B. guangzhouense (2 IGS types, 2.7% total isolates), B. zhengyangense (1 IGS type, 11.3% total isolates) and five novel genospecies (5 IGS types, 0.9 to 3.2% total isolates). All representative strains had identical nodA, nodC and nifH sequences except for one nifH sequence. With this one exception, these sequences were identical to those of the type strains of Bradyrhizobium species and several Bradyrhizobium genospecies isolated from peanut in different regions of China. The nodC sequences of all strains showed < 67% similarity to the closest strains on the Genbank database indicating that they are representative of a novel Bradyrhiobium symbiovar. This study has shown that (1) diverse Bradyrhizobium spp. with similar symbiosis genes nodulate peanut in different regions of China. (2) Horizontal transfer of genes involved in nodulating peanut is common between Bradyrhizobium species in soils used to grow the crop in China. (3) The strains studied here are representative of a novel Bradyrhizobium symbiovar that nodulates peanut in China. We propose the name sv. arachis for this novel symbiovar indicating that the strains were isolated from Arachis hypogaea. Results here have practical implications in relation to the selection of rhizobial inoculants for peanut in China.

Genome-Wide Identification and Characterization of CLAVATA3/EMBRYO SURROUNDING REGION (CLE) Gene Family in Foxtail Millet (Setaria italica L.).

The CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) genes encode signaling peptides that play important roles in various developmental and physiological processes. However, the systematic identification and characterization of CLE genes in foxtail millet (Setaria italica L.) remain limited. In this study, we identified and characterized 41 SiCLE genes in the foxtail millet genome. These genes were distributed across nine chromosomes and classified into four groups, with five pairs resulting from gene duplication events. SiCLE genes within the same phylogenetic group shared similar gene structure and motif patterns, while 34 genes were found to be single-exon genes. All SiCLE peptides harbored the conserved C-terminal CLE domain, with highly conserved positions in the CLE core sequences shared among foxtail millet, Arabidopsis, rice, and maize. The SiCLE genes contained various cis-elements, including five plant hormone-responsive elements. Notably, 34 SiCLE genes possessed more than three types of phytohormone-responsive elements on their promoters. Comparative analysis revealed higher collinearity between CLE genes in maize and foxtail millet, which may be because they are both C4 plants. Tissue-specific expression patterns were observed, with genes within the same group exhibiting similar and specific expression profiles. SiCLE32 and SiCLE41, classified in Group D, displayed relatively high expression levels in all tissues except panicles. Most SiCLE genes exhibited low expression levels in young panicles, while SiCLE6, SiCLE24, SiCLE25, and SiCLE34 showed higher expression in young panicles, with SiCLE24 down-regulated during later panicle development. Greater numbers of SiCLE genes exhibited higher expression in roots, with SiCLE7, SiCLE22, and SiCLE36 showing the highest levels and SiCLE36 significantly down-regulated after abscisic acid (ABA) treatment. Following treatments with ABA, 6-benzylaminopurine (6-BA), and gibberellic acid 3 (GA3), most SiCLE genes displayed down-regulation followed by subsequent recovery, while jasmonic acid (JA) and indole-3-acetic acid (IAA) treatments led to upregulation at 30 min in leaves. Moreover, identical hormone treatments elicited different expression patterns of the same genes in leaves and stems. This comprehensive study enhances our understanding of the SiCLE gene family and provides a foundation for further investigations into the functions and evolution of SiCLE genes in foxtail millet.

Unusual Oligomeric Laccase-like Oxidases from Ascomycete Curvularia geniculata VKM F-3561 Polymerizing Phenylpropanoids and Phenolic Compounds under Neutral Environmental Conditions.

The unique oligomeric alkaliphilic laccase-like oxidases of the ascomycete C. geniculata VKM F-3561 (with molecular masses about 1035 and 870 kDa) were purified and characterized for the first time. The ability of the enzymes to oxidize phenylpropanoids and phenolic compounds under neutral environmental conditions with the formation of previously unknown di-, tri-, and tetrameric products of transformation was shown. The possibility to obtain industrially valuable compounds (dihydroxybenzyl alcohol and hydroxytyrosol) from caffeic acid using laccase-like oxidases of C. geniculata VKM F-3561 has been shown. Complete nucleotide sequence of the laccase gene, which is expressed at the peak of alkaliphilic laccase activity of the fungus, and its promoter region were determined. Based on the phylogenetic analysis of the nucleotide sequence, the nearest relationship of the isolated laccase gene with similar genes of fungi of the genera Alternaria, Bipolaris, and Cochliobolus was shown. Homologous model of the laccase structure was predicted and a proton channel was found, which was presumably responsible for the accumulation and transport of protons to T2/T3-copper center in the alkaliphilic laccase molecule and providing the functional activity of the enzyme in the neutral alkaline environment conditions.

Allergy and autoimmunity in children: non-mutually exclusive diseases. A narrative review.

In last decades a simultaneous increase in the prevalence of atopic and autoimmune disorders in pediatric population has been observed. Despite the Th1-Th2 paradigm, supporting the polarization of the immune system with Th1 response involved in autoimmune diseases and Th2 response leading to hypersensitivity reactions, recent evidence suggests a possible coexistence of common pathogenic pathways as result of shared immune dysregulation. Similar genes and other mechanisms such as epithelial barrier damage, gut microbiota dysbiosis and reduced number of T regs and IL-10 contribute to the onset of allergy and autoimmunity. IgA deficiency is also hypothesized to be the crosslink between celiac disease and allergy by lowering gut mucous membrane protection from antigens and allergens. The present narrative review aims to give an overview of the co-occurrence of allergic and autoimmune disorders (celiac disease, inflammatory bowel diseases, type 1 diabetes mellitus, thyroid disease, juvenile idiopathic arthritis) in pediatric population, based on the available evidence. We also highlighted the common pathogenic pathways that may underpin both. Our findings confirm that allergic and autoimmune diseases are commonly associated, and clinicians should therefore be aware of the possible coexistence of these conditions in order to ameliorate disease management and patient care. Particular attention should be paid to the association between atopic dermatitis or asthma and celiac disease or type 1 diabetes and vice versa, for therapeutic interventions. Further studies are needed to better clarify mechanisms involved in the pathogenesis and eventually identify new therapeutic strategies.

Finding information about uncharacterized Drosophila melanogaster genes.

Genes that have been identified in the genome but remain uncharacterized with regards to function offer an opportunity to uncover novel biological information. Novelty is exciting but can also be a barrier. If nothing is known, how does one start planning and executing experiments? Here, we provide a recommended information-mining workflow and a corresponding guide to accessing information about uncharacterized Drosophila melanogaster genes, such as those assigned only a systematic coding gene identifier. The available information can provide insights into where and when the gene is expressed, what the function of the gene might be, whether there are similar genes in other species, whether there are known relationships to other genes, and whether any other features have already been determined. In addition, available information about relevant reagents can inspire and facilitate experimental studies. Altogether, mining available information can help prioritize genes for further study, as well as provide starting points for experimental assays and other analyses.

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4+ T-cells for the treatment of Hyper IgM1.

Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4+ T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

Heterogeneous Gene Fusion Transcripts Found in t(7;12)(q36;p13) Acute Myeloid Leukemia but with Similar Gene Expression Profile

Cytogenetic aberrations are often involved in acute myeloid leukemia (AML) and can serve as diagnostic markers, prognosis predictors and impact the choice of therapy. A translocation t(7;12)(q36;p13) is reported in up to 20-30% of AML patients typically diagnosed before 24 months of age (Beverloo, Panagopoulos et al. 2001, Cancer Res 61(14): 5374-5377). However, in a retrospective survey of the Nordic Organization of Pediatric Hematology and Oncology (NOPHO) registry, only 4% of reported AML diagnosed before 24 months of age had t(7;12) (Espersen, Noren-Nystrom et al. 2018, Genes Chromosomes Cancer 57(7): 359-365). The t(7;12) is difficult to identify with conventional karyotyping, why the true frequency of t(7;12) may be underestimated. Similarly, the reported prognosis for t(7;12) patients has varied ranging from good (Espersen, Noren-Nystrom et al. 2018) to dismal (von Bergh, van Drunen et al. 2006, Genes Chromosomes Cancer 45(8): 731-739). The translocation has been reported to give rise to an in-frame fusion transcript, MNX1::ETV6. However, detection of this fusion is reported only in 50% of cases in contrast to high expression of MNX1 in 100% (von Bergh, van Drunen et al. 2006). The aim of this study was to determine the frequency of the t(7;12) in pediatric AML patients in the NOPHO AML-2004/2012 protocols, their event-free and overall survival and to investigate the presence of additional genetic alterations. Patients in Sweden, Denmark, or Iceland between 2004 to 2020 diagnosed with AML before 2 years of age that had not been reported with a recurrent genetic aberration plus all patients diagnosed with t(7;12) AML were identified. Bone marrow, or peripheral blood samples from patients were retrieved from the NOPHO biobank. This cohort constituted 86% (31 out of 36) of all patients that fulfilled these criteria. In total 89 AML patients were diagnosed before 2 years of age. The t(7;12) AML cases were identified by screening for high expression of MNX1 and presence of fusion transcripts using whole transcriptome sequencing (WTS). Whole genome sequencing (WGS) was done to identify chromosomal rearrangements, and translocation breakpoints. At diagnosis, four patients were diagnosed with t(7;12). A central karyotype review of all patients reported two additional cases. No further cases were identified using WTS. Thus, the frequency of t(7;12) AML was 7% (6 out of 89) and for patients diagnosed before 12 months of age 11% (5 out of 47). In total 12 cases with t(7;12) were identified in the complete NOPHO registry but only 11 patients received treatment according to NOPHO protocol. The relapse rate was 54% (6 out of 11). All relapse cases underwent allogeneic hematopoietic stem cell (HSC) transplantation in complete remission two (CR2), and their overall survival (OS) was 83% (5 out of 6). Hence OS for all t(7;12) patients treated according to the NOPHO2004 and NOPHO2012 protocols was 91% (10 out of 11). Analysis of WTS data showed similar gene expression profile between our t(7;12) cases and t(7;12) cases retrieved from the TARGET database. Genes that were typically overexpressed in all t(7;12) AML were, MNX1, MNX1-AS1, MNX1-AS2, LIN28B, BAMBI, MAF, CRISP3, EDIL3, CTTNBP2, KRT72, and AGR2, where MNX1 seems to be uniquely expressed in this type of leukemia (Nilsson et al. 2022, Int J Cancer 151(5): 770-782). Analysis of fusion transcripts showed presence of MNX1::ETV6 in just one case. The remaining cases instead showed presence of other fusion transcripts involving ETV6, NOM1::ETV6, ETV6::NOM1, ETV6::LMBR1 and ETV6::BMAL2. WGS verified a fusion between NOM1 and ETV6 in 3 cases. The patient with ETV6::BMAL2 showed that 16 Mb of chromosome 12 was inserted into chromosome 7 placing a large part of ETV6 near MNX1. The fusion transcript ETV6::BMAL2 is the product of what remains on chromosome 12. In conclusion, frequency of t(7;12) AML in the NOPHO treatment cohort was approximately 7%, with 54% relapse rate, but most patients were salvaged by HSCT in CR2. Heterogeneous gene fusion transcripts were identified in this subgroup of AML where ETV6::NOM1 was most frequent. However, all cases showed similar gene expression signatures which underlines that the leukemia driving event is ectopic expression of MNX1 (Waraky, A., 2023, Haematologica, DOI: 10.3324/haematol.2022.282255) and should therefore be the defining classifying criteria of this type of AML.

Lipid Nanoparticle-Mediated Hit-and-Run Approaches Yield Efficient and Safe In Situ Gene Editing in Human Skin.

Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for in situ gene editing. We observed distinct LNP composition and cell-specific effects such as an extended presence of RNP in slow-cycling epithelial cells for up to 72 h. While obtaining similar gene editing rates using Cas9 RNP and mRNA with MC3-based LNPs (10-16%), mRNA-loaded LNPs proved to be more cytotoxic. Interestingly, ionizable lipids with a pKa ∼ 7.1 yielded superior gene editing rates (55%-72%) in two-dimensional (2D) epithelial cells while no single guide RNA-dependent off-target effects were detectable. Unexpectedly, these high 2D editing efficacies did not translate to actual skin tissue where overall gene editing rates between 5%-12% were achieved after a single application and irrespective of the LNP composition. Finally, we successfully base-corrected a disease-causing mutation with an efficacy of ∼5% in autosomal recessive congenital ichthyosis patient cells, showcasing the potential of this strategy for the treatment of monogenic skin diseases. Taken together, this study demonstrates the feasibility of an in situ correction of disease-causing mutations in the skin that could provide effective treatment and potentially even a cure for rare, monogenic, and common skin diseases.

Tertiary lymphoid structures as local perpetuators of organ-specific immune injury: implication for lupus nephritis.

In response to inflammatory stimuli in conditions such as autoimmune disorders, infections and cancers, immune cells organize in nonlymphoid tissues, which resemble secondary lymphoid organs. Such immune cell clusters are called tertiary lymphoid structures (TLS). Here, we describe the potential role of TLS in the pathogenesis of autoimmune disease, focusing on lupus nephritis, a condition that incurs major morbidity and mortality. In the kidneys of patients and animals with lupus nephritis, the presence of immune cell aggregates with similar cell composition, structure, and gene signature as lymph nodes and of lymphoid tissue-inducer and -organizer cells, along with evidence of communication between stromal and immune cells are indicative of the formation of TLS. TLS formation in kidneys affected by lupus may be instigated by local increases in lymphorganogenic chemokines such as CXCL13, and in molecules associated with leukocyte migration and vascularization. Importantly, the presence of TLS in kidneys is associated with severe tubulointerstitial inflammation, higher disease activity and chronicity indices, and poor response to treatment in patients with lupus nephritis. TLS may contribute to the pathogenesis of lupus nephritis by increasing local IFN-I production, facilitating the recruitment and supporting survival of autoreactive B cells, maintaining local production of systemic autoantibodies such as anti-dsDNA and anti-Sm/RNP autoantibodies, and initiating epitope spreading to local autoantigens. Resolution of TLS, along with improvement in lupus, by treating animals with soluble BAFF receptor, docosahexaenoic acid, complement inhibitor C4BP(β-), S1P1 receptor modulator Cenerimod, dexamethasone, and anti-CXCL13 further emphasizes a role of TLS in the pathogenesis of lupus. However, the mechanisms underlying TLS formation and their roles in the pathogenesis of lupus nephritis are not fully comprehended. Furthermore, the lack of non-invasive methods to visualize/quantify TLS in kidneys is also a major hurdle; however, recent success in visualizing TLS in lupus-prone mice by photon emission computed tomography provides hope for early detection and manipulation of TLS.

Ratiometric Inclusion of Fibroblasts Promotes Both Castration-Resistant and Androgen-Dependent Tumorigenic Progression in Engineered Prostate Cancer Tissues.

To investigate the ratiometric role of fibroblasts in prostate cancer (PCa) progression, this work establishes a matrix-inclusive, 3D engineered prostate cancer tissue (EPCaT) model that enables direct coculture of neuroendocrine-variant castration-resistant (CPRC-ne) or androgen-dependent (ADPC) PCa cells with tumor-supporting stromal cell types. Results show that the inclusion of fibroblasts within CRPC-ne and ADPC EPCaTs drives PCa aggression through significant matrix remodeling and increased proliferative cell populations. Interestingly, this is observed to a much greater degree in EPCaTs formed with a small number of fibroblasts relative to the number of PCa cells. Fibroblast coculture also results in ADPC behavior more similar to the aggressive CRPC-ne condition, suggesting fibroblasts play a role in elevating PCa disease state and may contribute to the ADPC to CRPC-ne switch. Bulk transcriptomic analyses additionally elucidate fibroblast-driven enrichment of hallmark gene sets associated with tumorigenic progression. Finally, the EPCaT model clinical relevancy is probed through a comparison to the Cancer Genome Atlas (TCGA) PCa patient cohort; notably, similar gene set enrichment is observed between EPCaT models and the patient primary tumor transcriptome. Taken together, study results demonstrate the potential of the EPCaT model to serve as a PCa-mimetic tool in future therapeutic development efforts.