Hox Genes Research Articles

Expression of HLA-DR by mesenchymal stromal cells in the platelet lysate era: an obsolete release criterion for MSCs?

BackgroundAccording to the definition of the International Society for Cell and Gene Therapy (ISCT), mesenchymal stromal cells (MSCs) do not express HLA-DR. This phenotypic marker as a release criterion for clinical use was established at a time when MSCs were expanded in fetal bovine serum (FBS)-containing media. Replacement of FBS with platelet lysate (PLs) as a medium supplement induced a significantly higher fraction of MSCs to express MHC class II antigens.MethodsAs this raised concerns that such MSCs may play the role of antigen-presenting cells for T cells, in the current study, we studied major factors that may induce HLA-DR on MSCs by means of flow cytometry and real-time polymerase chain reaction. The immunomodulatory potential of MSCs was assessed by a mixed lymphocyte reaction.ResultsOur results demonstrated that a very low percentage of generated and expanded MSCs in FBS express HLA-DR (median: 1.1%, range: 0.3–22%) compared to MSCs generated and expanded in PLs (median: 28.4%, range: 3.3–73.7%). Analysis of the cytokine composition of ten PLs showed a significant positive correlation between the levels of IL-1β, IL-4, IL-10, IL-17, bFGF and expression of HLA-DR, in contrast to no correlation with the age of MSC donors and HLA-DR (r = 0.21). Both MSCs expressing low and high levels of HLA-DR expressed class II transactivator (CIITA), a master gene coding for these molecules. Our results demonstrate for the first time that MSCs with constitutively high levels of HLA-DR also express moderate levels of indoleamine 2,3-dioxygenase (IDO). Treatment of MSCs with multiple doses of TGF-β1 at passage 0 (P0) and passage 1 (P1) completely abrogated HLA-DR and IDO expression. In contrast, treatment of MSCs with a single dose of TGF-β1 after P0 only partially reduced the expression of HLA-DR and CIITA. Remarkably, increased expression of HLA-DR on MSCs that constitutively express high levels of this antigen after overnight incubation with IFN-γ was rather unaffected by incubation with TGF-β1. However, treatment of MSCs with TGF-β1 for 24 h completely abrogated constitutive expression of IDO.ConclusionsIrrespective of HLA-DR expression at the population level, all MSC preparations significantly inhibited the proliferation of stimulated peripheral blood mononuclear cells, indicating that HLA-DR represents an obsolete release marker for the clinical use of MSCs.

The Impact of Early Genes and Gene Editing Technology Associated with Clustered Repeats on the Development of Human Body Organs

Early growth response 1 (EGR-1) and homeobox (Hox) proteins play crucial roles in regulating genes within the body, promoting cell renewal, and expediting the wound healing process. Conversely, Clustered Regularly Interspaced Palindromic Re-peats/Associated (CRISPR-Cas) technology is primarily involved in gene editing. However, significant gaps persist in understanding the mechanisms of key genes like EGR-1, CRISPR/Cas, and HOX genes in organ development. Unveiling their mechanisms is crucial for advancing organ development and discovering new therapeutic strategies. This study aims to investigate the roles of EGR-1, CRISPR/Cas, and HOX genes in organ development and growth. The study used CRISPR/Cas to investigate the impact of catalase mutations on organ regeneration. Early embryonic mutants were generated by injecting gRNAs and Cas9 protein into zygotes, followed by tail amputation in larvae. Additionally, the study explored the role of specific Hox genes in axon elongation and Wnt signaling regulation. EGR-1, induced by TGF-β1, enhanced collagen production, underscoring its importance in wound healing. Integration of EGR-1, HOX proteins, and CRISPR-Cas revealed a regulatory complex influencing organ development. The integration of EGR-1, HOX proteins, and CRISPR-Cas revealed a regulatory complex. EGR-1 aids wound healing, HOX proteins influence fetal development and organ formation, and CRISPR-Cas enables precise genome modifications.

Single-cell sequencing dissects the transcriptional identity of activated fibroblasts and identifies novel persistent distal tubular injury patterns in kidney fibrosis

Examining kidney fibrosis is crucial for mechanistic understanding and developing targeted strategies against chronic kidney disease (CKD). Persistent fibroblast activation and tubular epithelial cell (TEC) injury are key CKD contributors. However, cellular and transcriptional landscapes of CKD and specific activated kidney fibroblast clusters remain elusive. Here, we analyzed single cell transcriptomic profiles of two clinically relevant kidney fibrosis models which induced robust kidney parenchymal remodeling. We dissected the molecular and cellular landscapes of kidney stroma and newly identified three distinctive fibroblast clusters with “secretory”, “contractile” and “vascular” transcriptional enrichments. Also, both injuries generated failed repair TECs (frTECs) characterized by decline of mature epithelial markers and elevation of stromal and injury markers. Notably, frTECs shared transcriptional identity with distal nephron segments of the embryonic kidney. Moreover, we identified that both models exhibited robust and previously unrecognized distal spatial pattern of TEC injury, outlined by persistent elevation of renal TEC injury markers including Krt8 and Vcam1, while the surviving proximal tubules (PTs) showed restored transcriptional signature. We also found that long-term kidney injuries activated a prominent nephrogenic signature, including Sox4 and Hox gene elevation, which prevailed in the distal tubular segments. Our findings might advance understanding of and targeted intervention in fibrotic kidney disease.

BAF45D-binding to HOX genes was differentially targeted in H9-derived spinal cord neural stem cells

Chromatin accessibility has been used to define how cells adopt region-specific neural fates. BAF45D is one of the subunits of a specialised chromatin remodelling BAF complex. It has been reported that BAF45D is expressed in spinal cord neural stem cells (NSCs) and regulates their fate specification. Within the developing vertebrate spinal cord, HOX genes exhibit spatially restricted expression patterns. However, the chromatin accessibility of BAF45D binding HOX genes in spinal cord NSCs is unclear. In the present study, we found that in H9-derived spinal cord NSCs, BAF45D targets TBX6, a gene that regulates spinal cord neural mesodermal progenitors. Furthermore, BAF45D binding to the NES gene is much more enriched in H9-derived spinal cord NSCs chromatin compared to ESCs chromatin. In addition, BAF45D binding to anterior and trunk/central HOX genes, but not to lumbosacral HOX genes, was much more enriched in NSCs chromatin compared to ESCs chromatin. These results may shed new light on the role of BAF45D in regulating region-specific spinal cord NSCs by targeting HOX genes.

Hox genes and patterning the vertebrate body.

The diversity of vertebrate body plans is dizzying, yet stunning for the many things they have in common. Vertebrates have inhabited virtually every part of the earth from its coldest to warmest climates. They locomote by swimming, flying, walking, slithering, or climbing, or combinations of these behaviors. And they exist in many different sizes, from the smallest of frogs, fish and lizards to giraffes, elephants, and blue whales. Despite these differences, vertebrates follow a remarkably similar blueprint for the establishment of their body plan. Within the relatively small amount of time required to complete gastrulation, the process through which the three germ layers, ectoderm, mesoderm, and endoderm are created, the embryo also generates its body axis and is simultaneously patterned. For the length of this axis, the genes that distinguish the neck from the rib cage or the trunk from the sacrum are the Hox genes. In vertebrates, there was evolutionary pressure to maintain this set of genes in the organism. Over the past decades, much has been learned regarding the regulatory mechanisms that ensure the appropriate expression of these genes along the main body axes. Genetic functions continue to be explored though much has been learned. Much less has been discerned on the identity of co-factors used by Hox proteins for the specificity of transcriptional regulation or what downstream targets and pathways are critical for patterning events, though there are notable exceptions. Current work in the field is demonstrating that Hox genes continue to function in many organs long after directing early patterning events. It is hopeful continued research will shed light on remaining questions regarding mechanisms used by this important and conserved set of transcriptional regulators.

Synergistic effect of metformin and vitamin D3 on osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells under high d-glucose conditions

IntroductionVitamin D3 plays a vital role in bone health, with low levels of vitamin D3 being related to skeletal fragility, fractures, and metabolic disorders such as diabetes. Metformin is known as an antihyperglycemic agent for regulating blood sugar. A correlation between diabetes mellitus and osteoporosis is attracting considerable interest, and research to find the prevention and treatment is gradually being studied. In this study, we investigated the effect of metformin and vitamin D3 on osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (AT-MSCs) under high d-glucose concentrations and optimized by combining vitamin D3 and metformin in the process. MethodsROS production of AT-MSCs under high d-glucose conditions was measured by DCFH-DA assay. The differentiated AT-MSCs were analyzed by Alizarin Red S staining and optical density measurement. The investigation involved the examination of osteogenic master genes' expressions using quantitative reverse transcription polymerase chain reaction (qRT-PCR) techniques. ResultsInterestingly, the results have shown that human AT-MSCs will exhibit high ROS accumulation and low osteogenic differentiation capabilities, indicated by low calcium deposition, as well as low expression of indicative genes such as ALP, Runx-2 under high d-glucose conditions. The combination of vitamin D3 and metformin remarkedly accelerated the osteogenic differentiation of AT-MSCs under high d-glucose concentrations more effectively than the administration of either agent. ConclusionsThis study partially explains an aspect of an in vitro model for pre-clinical drug screening for osteoporosis-related diabetic pathological mechanisms, which can be applied for further research on the prevention or treatment of osteoporosis in diabetic patients.

Machine learning-based estimation of spatial gene expression pattern during ESC-derived retinal organoid development

Organoids, which can reproduce the complex tissue structures found in embryos, are revolutionizing basic research and regenerative medicine. In order to use organoids for research and medicine, it is necessary to assess the composition and arrangement of cell types within the organoid, i.e., spatial gene expression. However, current methods are invasive and require gene editing and immunostaining. In this study, we developed a non-invasive estimation method of spatial gene expression patterns using machine learning. A deep learning model with an encoder-decoder architecture was trained on paired datasets of phase-contrast and fluorescence images, and was applied to a retinal organoid derived from mouse embryonic stem cells, focusing on the master gene Rax (also called Rx), crucial for eye field development. This method successfully estimated spatially plausible fluorescent patterns with appropriate intensities, enabling the non-invasive, quantitative estimation of spatial gene expression patterns within each tissue. Thus, this method could lead to new avenues for evaluating spatial gene expression patterns across a wide range of biology and medicine fields.

Erwinia amylovora Type III Secretion System Inhibitors Reduce Fire Blight Infection Under Field Conditions.

Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.

Genetic modification of a Hox locus drives mimetic color pattern variation in a highly polymorphic bumble bee.

Müllerian mimicry provides natural replicates ideal for exploring mechanisms underlying adaptive phenotypic divergence and convergence, yet the genetic mechanisms underlying mimetic variation remains largely unknown. The current study investigates the genetic basis of mimetic color pattern variation in a highly polymorphic bumble bee, Bombus breviceps (Hymenoptera, Apidae). In South Asia, this species and multiple comimetic species converge onto local Müllerian mimicry patterns by shifting the abdominal setal color from orange to black. Genetic crossing between the orange and black phenotypes suggested the color dimorphism being controlled by a single Mendelian locus, with the orange allele being dominant over black. Genome-wide association suggests that a locus at the intergenic region between two abdominal fate-determining Hox genes, abd-A and Abd-B is associated with the color change. This locus is therefore in the same intergenic region but not the same exact locus as found to drive red-black mid-abdominal variation in a distantly related bumble bee species, B. melanopygus. Gene expression analysis and RNA interferences suggest that differential expression of an intergenic long non-coding RNA between abd-A and Abd-B at the onset setal color differentiation may drive the orange black color variation by causing a homeotic shift late in development. Analysis of this same color locus in comimetic species reveals no sequence association with the same color shift, suggesting that mimetic convergence is achieved through distinct genetic routes. Our study establishes Hox regions as genomic hotspots for color pattern evolution in bumble bees, and demonstrates how pleiotropic developmental loci can drive adaptive radiations in nature.

Molecular Profiling of Response and Resistance to Venetoclax-Decitabine Therapy in Acute Myeloid Leukemia

The hypomethylating agent decitabine and the BCL-2 inhibitor venetoclax combination (DEC-VEN) is now standard of care in AML, yet a subset of patients remains refractory, and factors that reduce efficacy remain to be fully elucidated. This study aimed to systematically identify molecular determinants of resistance via single-cell (sc)RNA-seq and scATAC-seq analysis of bone marrow (BM) samples from patients participating in a prospective clinical trial of DEC-VEN (NCT03404193). For scRNA-seq, 17 BM samples (total 39,607 cells) from 12 AML patients (median age 67; 8 responders; 4 non-responders; 5 pre- and post-treatment pairs) and 3 healthy BM controls were integrated and annotated into 23 transcriptional cell clusters. In the Uniform Manifold Approximation and Projection (UMAP) plot, C1 overlapped with healthy hematopoietic stem cells (HSCs), C2 appeared only in AML patients, and C3 overlapped with progenitor cells. C1 was characteristic of refractory AML and persisted after DEC-VEN treatment. (Figure 1) C1 cells showed significantly higher expression of FLT3, BCL2, MCL1 and transcription factors MYB, ETV6, RUNX1, LMO2, JUND compared to HSCs from healthy donors. DEC-VEN treatment markedly increased ribosomal protein ( RP ) genes and mitochondrial electron transfer COX family genes and increased expression of the leukemia stem cell (LSC) marker CD74 in C1 of non-responders. In C2 cells, high expression of LSC marker genes FLT3, PIM1, MYC and transforming master regulator genes MYB, ETV6, RUNX2, LMO2, STAT3, NOTCH1, BCL11A was observed. DEC-VEN significantly upregulated RP genes and CD74 in C2 in non-responders, as in C1. In responders, hemoglobin family genes were significantly increased in C2. C3 cells were characterized by highly expressed iron metabolism genes, which were further increased by DEC-VEN in responders. GSEA analysis demonstrated that DEC-VEN upregulated ATP metabolic processes and ribosomal biosynthesis and downregulated stem cell differentiation genes in refractory C1 and C2. In responders' AML clusters, DEC-VEN upregulated cellular oxidant detoxification and iron ion homeostasis and downregulated myeloid leukocyte activation. Next, we extended the AML transcriptome analysis by profiling accessible chromatin by scATAC-seq using BM from pre- and post- DEC -VEN treatment of 2 AML patients (1 refractory, 1 relapsed after remission with incomplete count recovery [CRi]), and 2 healthy controls (total 41,062 cells). Leukemia-like cluster (LLC) cells showed significantly higher chromatin accessibility of CD74, RUNX1, RUNX2, NOTCH1, LMO2, ETV6, JUND, STAT3, BCL2, BCL11A and enriched binding motifs of RUNX2, ETV6, MYB, JUN, JUND, STAT3 compared to other clusters, which was consistent with gene expression findings of C1 and C2 by scRNA-seq. DEC-VEN treatment induced further increased motif enrichment of STAT3 in LLC in CRi AML, and STAT3 RUNX2, IKZF1 in refractory AML. To interpret T cell kinetics in response to DEC-VEN in AML, T cells from scRNA-seq profiling were subclassified based on canonical gene expression. The results showed that the CD3+ and CD3+CD8+ cell signatures were significantly lower in non-responders compared to responders post-treatment (p<0.005). Notably, the ratio of exhausted CD8+ cell clusters was significantly higher in non-responders than in responders in DEC-VEN post-treatment (p<0.05), with downregulation of CD8+ effector marker GZMK, and regulator of CD8+ cytotoxic T cell development TOX. In summary, we identified a subpopulation of AML cells that overlap with HSCs in UMAP plots and are associated with DEC-VEN resistance. LSC-related genes were enriched in resistant AML clusters, while hemoglobin-related genes were increased in sensitive clusters. DEC-VEN induced mitochondrial metabolism genes in resistant cells and further enhanced erythrocytic differentiation in sensitive cells. In addition, scATAC-seq revealed LSC-specific chromatin accessibility patterns, and depletion of CD8+ T cells was observed in non-responders. These findings shed light on the molecular mechanisms of DEC-VEN resistance.

Nuclear Condensates Are a Therapeutic Vulnerability in NPM1-Mutant Acute Myeloid Leukemia

The development of targeted therapies against drivers of malignancy requires a robust mechanistic understanding of the pathogenic axis underlying disease. Several subtypes of acute myeloid leukemias (AML), such as those driven by mutations in IDH1/2, FLT3, and KMT2A, have benefitted from the development of novel therapeutics that disrupt their transcriptional program. In contrast, there are no approved therapies that target the mutant version of Nucleophosmin (NPM1), which is the most common driver in adult AML. Mutations in NPM1 result in export of the mutant protein (NPM1c) to the cytoplasm, prompting the suggestion that aberrant localization leads to neomorphic and pathogenic functions of NPM1c in the cytoplasm. In parallel, recent data from several groups has suggested that NPM1c directly regulates gene expression within the nucleus. Despite this progress, a well-defined mechanism for NPM1c-mediated gene regulation is lacking, hindering the development of NPM1c-targeted therapies. To investigate the function of NPM1c, we generated over a dozen AML cell lines expressing unique NPM1c truncations and characterized their impact on the leukemic transcriptome and cell morphology. To our surprise, we found that although the majority of these truncated proteins reside in the cytoplasm, most were unable to recapitulate the transcriptional program driven by full-length NPM1c, and cells subsequently underwent differentiation and growth arrest. We further explored the chromatin binding ability of these truncated proteins to key genomic regions such as the HOXA cluster and MEIS1 through CUT&RUN analysis. Next, we identified a region of the NPM1c protein critical for active nuclear import (NLS). Strikingly, disruption of this region enforces cytoplasmic sequestration of the truncated NPM1c protein. However, cells harboring this truncation fail to maintain leukemic gene expression and differentiate. Together these results suggest that aberrant localization and/or shuttling of NPM1c between cellular compartments is insufficient to maintain leukemic gene expression and cell-stemness. We next considered whether NPM1c had a de-novo function that may explain its role in gene regulation. We have recently shown that NPM1c forms phase-separated condensates in the nucleus that regulate HOX gene expression and are necessary for stem-ness. These NPM1c condensates recruit multiple gene regulatory factors including XPO1, NUP98, and KMT2A (Figure 1). Through selective degradation of NPM1c, or pharmacological inhibition of XPO1, we found that disruption of nuclear condensates abrogates recruitment of these protein co-factors. We further explore the effect of drug compounds known to inhibit NPM1-mutant AML on condensate behavior and chromatin binding. This condensate behavior is a defining feature of heterotypic phase separation, a process in which two or more biomolecules form multivalent interactions to create a condensate. Importantly, we found that significant overexpression of NPM1c causes an imbalance in the ratio between NPM1c and its binding partners, disrupting normal condensate formation, and causing subsequent differentiation and growth arrest. Collectively, these results suggest that therapeutic targeting of NPM1c, or a host of interacting partners, may disrupt condensate formation and the leukemic transcriptional program, providing an exciting new therapeutic vulnerability in NPM1-mutant AML.

Genomic and transcriptomic survey of bryozoan Hox and ParaHox genes with emphasis on phylactolaemate bryozoans

BackgroundBryozoans are mostly sessile aquatic colonial invertebrates belonging to the clade Lophotrochozoa, which unites many protostome bilaterian phyla such as molluscs, annelids and brachiopods. While Hox and ParaHox genes have been extensively studied in various lophotrochozoan lineages, investigations on Hox and ParaHox gene complements in bryozoans are scarce.ResultsHerein, we present the most comprehensive survey of Hox and ParaHox gene complements in bryozoans using four genomes and 35 transcriptomes representing all bryozoan clades: Cheilostomata, Ctenostomata, Cyclostomata and Phylactolaemata. Using similarity searches, phylogenetic analyses and detailed manual curation, we have identified five Hox genes in bryozoans (pb, Dfd, Lox5, Lox4 and Post2) and one ParaHox gene (Cdx). Interestingly, we observed lineage-specific duplication of certain Hox and ParaHox genes (Dfd, Lox5 and Cdx) in some bryozoan lineages.ConclusionsThe bryozoan Hox cluster does not retain the ancestral lophotrochozoan condition but appears relatively simple (includes only five genes) and broken into two genomic regions, characterized by the loss and duplication of serval genes. Importantly, bryozoans share the lack of two Hox genes (Post1 and Scr) with their proposed sister-taxon, Phoronida, which suggests that those genes were missing in the most common ancestor of bryozoans and phoronids.

Transcriptome-based comparison reveals key genes regulating allometry growth of forelimb and hindlimb bone in duck embryos

Allometric growth of the forelimb and hindlimb is a widespread phenomenon observed in vertebrates. As a typical precocial bird, ducks exhibit more advanced development of their hindlimbs compared to their forelimbs, enabling them to walk shortly after hatching. This phenomenon is closely associated with the development of long bones in the embryonic stage. However, the molecular mechanism governing the allometric growth of duck forelimb and hindlimb bones is remains elusive. In this study, we employed phenotypic, histological, and gene expression analyses to investigate developmental differences between the humerus (forelimb bone) and tibia/femur (hindlimb bones) in duck embryos. Our results revealed a gradual increase in weight and length disparity between the tibia and humerus from E12 to E28 (embryo age). At E12, endochondral ossification was observed solely in the tibia but not in the humerus. The number of differentially expressed genes (DEGs) gradually increased at H12 vs. T12, H20 vs. T20, and H28 vs. T28 stages consistent with phenotypic variations. A total of 38 DEGs were found across all 3 stages. Protein-protein interaction network analysis demonstrated strong interactions among members of HOXD gene family (HOXD3/8/9/10/11/12), HOXB gene family (HOXB8/9), TBX gene family (TBX4/5/20), HOXA11, SHOX2, and MEIS2. Gene expression profiling indicated higher expression levels for all HOXD genes in the humerus compared to tibia while opposite trends were observed for HOXA/HOXB genes with low or no expression detected in the humerus. These findings suggest distinct roles played by different clusters within HOX gene family during skeletal development regulation of duck embryo's forelimbs versus hind limbs. Notably, TBX4 exhibited high expression levels specifically in tibia whereas TBX5 showed similar patterns exclusively within humerus as seen previously across other species' studies. In summary, this study identified key regulatory genes involved in allometric growth of duck forelimb and hindlimb bones during embryonic development. Skeletal development is a complex physiological process, and further research is needed to elucidate the regulatory role of candidate genes in endochondral ossification.

Morphological, histological and gene-expression analyses on stolonization in the Japanese Green Syllid, Megasyllis nipponica (Annelida, Syllidae)

Benthic annelids belonging to the family Syllidae (Annelida, Errantia, Phyllodocida) exhibit a unique reproduction mode called “schizogamy” or “stolonization”, in which the posterior body part filled with gametes detaches from the original body, as a reproductive unit (stolon) that autonomously swims and spawns. In this study, morphological and histological observations on the developmental processes during stolonization were carried out in Megasyllis nipponica. Results suggest that the stolon formation started with maturation of gonads, followed by the formation of a head ganglion in the anteriormost segment of the developing stolon. Then, the detailed stolon-specific structures such as stolon eyes and notochaetae were formed. Furthermore, expression profiles of genes involved in the anterior–posterior identity (Hox genes), head determination, germ-line, and hormone regulation were compared between anterior and posterior body parts during the stolonization process. The results reveal that, in the posterior body part, genes for gonadal development were up-regulated, followed by hormone-related genes and head-determination genes. Unexpectedly, Hox genes known to identify body parts along the anterior–posterior axis showed no significant temporal expression changes. These findings suggest that during stolonization, gonad development induces the head formation of a stolon, without up-regulation of anterior Hox genes.

Marked intestinal trans-differentiation by autoimmune gastritis along with ectopic pancreatic and pulmonary trans-differentiation

BackgroundAutoimmune gastritis (AIG) is a prevalent chronic inflammatory disease with oncogenic potential that causes destruction of parietal cells and severe mucosal atrophy. We aimed to explore the distinctive gene expression profiles, activated signaling pathways, and their underlying mechanisms.MethodsA comprehensive gene expression analysis was conducted using biopsy specimens from AIG, Helicobacter pylori-associated gastritis (HPG), and non-inflammatory normal stomachs. Gastric cancer cell lines were cultured under acidic (pH 6.5) conditions to evaluate changes in gene expression.ResultsGastric mucosa with AIG had a unique gene expression profile compared with that with HPG and normal mucosa, such as extensively low expression of ATP4A and high expression of GAST and PAPPA2, which are involved in neuroendocrine tumorigenesis. Additionally, the mucosa with AIG and HPG showed the downregulation of stomach-specific genes and upregulation of small intestine-specific genes; however, intestinal trans-differentiation was much more prominent in AIG samples, likely in a CDX-dependent manner. Furthermore, AIG induced ectopic expression of pancreatic digestion-related genes, PNLIP, CEL, CTRB1, and CTRC; and a master regulator gene of the lung, NKX2-1/TTF1 with alveolar fluid secretion-related genes, SFTPB and SFTPC. Mechanistically, acidic conditions led to the downregulation of master regulator and stemness control genes of small intestine, suggesting that increased environmental pH may cause abnormal intestinal differentiation in the stomach.ConclusionsAIG induces diverse trans-differentiation in the gastric mucosa, characterized by the transactivation of genes specific to the small intestine, pancreas, and lung. Increased environmental pH owing to AIG may cause abnormal differentiation of the gastric mucosa.

SMAD1 Is Dispensable for CDX2 Induction but Required for the Repression of Ectopic Small-Intestinal Gene Expression in Human-Pluripotent-Stem-Cell-Derived Colonic Organoids

The generation of gastrointestinal tissues from human pluripotent stem cells has provided unprecedented insight into the molecular mechanisms that drive the patterning of the primitive gut tube. Previous work has identified bone-morphogenetic-protein (BMP) signaling as an important mediator of mid/hindgut versus foregut and hindgut versus midgut cell fate choice. Inhibition of BMP signaling during gut tube morphogenesis inhibits the expression of the pan-intestinal transcription factor CDX2. Treatment of CDX2+ mid/hindgut cultures with BMP patterns them into hindgut, which gives rise to colonic organoids (HCOs). While the role for BMP signaling is clear, the molecular mechanisms through which BMP signaling patterns the mid/hindgut and colon remain unclear. BMPs bind to BMP receptors, activating a signaling cascade that results in the activation of SMADs, which function as transcription factors. We hypothesized that one of these factors, SMAD1, would be necessary for establishing the CDX2 domain and the colon domain. Unexpectedly, endoderm derived from SMAD1-deficient induced pluripotent stem cells was capable of inducing CDX2 in response to WNT and FGF signaling. In addition, CDX2+ gut tube cultures could activate posterior HOX genes in response to BMP. However, examination of HCOs following cytodifferentiation revealed that SMAD1-deficient HCOs ectopically expressed small-intestinal markers despite expressing posterior HOX genes. These results indicate that there is redundancy of SMADs during early hindgut patterning but that SMAD1 is required for the inhibition of small-intestinal gene expression in HCOs.

PATH-41. INTEGRATED MOLECULAR ANALYSIS REVEALS HYPERMETHYLATION AND OVEREXPRESSION OF HOX GENES TO BE POOR PROGNOSTICATORS IN IDH MUTANT GLIOMA

Abstract BACKGROUND Diffuse gliomas represent over 80% of malignant brain tumors ranging from low-grade to aggressive high-grade lesions. Within IDH-mutant gliomas there is a high variability in survival and a need to more accurately predict outcome. METHODS To identify and characterize a predictive signature of outcome in gliomas, we utilized an integrative molecular analysis (using methylation, mRNA, copy number variation (CNV) and mutation data), analyzing a total of 729 IDH-mutant samples including a test set of 99 from University Health Network (UHN) and two validation cohorts including the German Cancer Research Center (DKFZ) and The Cancer Genome Atlas (TCGA). RESULTS Cox regression analysis of methylation data from the UHN cohort identified CpG-based signatures that split the glioma cohort into two prognostic groups strongly predicting survival that were validated using two independent cohorts from TCGA and DKFZ (all p-values< 0.0001). The methylation signatures that predicted poor outcome also exhibited high CNV instability and hypermethylation of HOX gene probes. Integrated multi-platform analyses using mRNA and methylation (iRM) showed that parallel HOX gene overexpression and simultaneous hypermethylation were significantly associated with increased mutational load, high aneuploidy and worse survival (p-value< 0.0001). A 7-HOX gene signature was developed and validated using the most significantly associated HOX genes with patient outcome in both 1p/19q codeleted and non-codeleted IDHmut gliomas. CONCLUSIONS HOX gene methylation and expression provide important prognostic information in IDH-mutant gliomas that are not captured by current molecular diagnostics. A 7-HOX gene signature of outcome shows significant survival differences in both 1p/19q codeleted and non-codeleted IDH-mutant gliomas.

CSIG-24. LINEAR GRADING COEFFICIENT REVEALS EMBRYONIC DEVELOPMENT GENE SIGNATURE AS TREATMENT-INDEPENDENT PROGNOSTIC FACTOR IN IDH-MUTANT ASTROCYTOMA

Abstract BACKGROUND Since there is variation in prognosis within grade II/III and grade IV IDH-mutant astrocytomas, we sought to identify features that may aid further tumor grading. METHODS RNAseq was performed on IDH-mutant astrocytomas from the CATNON trial and overlaid with matching genome-wide methylation data. This dataset was extended by multi-omics data of IDH-mutant astrocytomas from the TCGA and GLASS-NL studies. We defined a linear grading coefficient (LGC) based on calibrated DNA methylation-based probability scores and performed differential gene expression (DGE) regression analysis on the LGC. Statistical analyses were intersected across the CATNON and TCGA cohorts ("discovery set") and validated on GLASS-NL. Single-nucleus RNA-sequencing (snRNAseq) data of high-grade IDH-mutant astrocytomas were utilized to explore the source of expression signals. Immunofluorescence was used to confirm findings on protein level. RESULTS DGE regression analysis revealed that higher-grade IDH-mutant tumors (high LGC) exhibited mRNA upregulation of key embryonic developmental transcription factors (HOX[A/C/D], NKX, PAX, and TBX). Although the vast majority of CpG sites in LGC high tumors were hypomethylated, homeobox genes were strongly hypermethylated in our discovery set. HOX gene clusters showed gene-ordered (differential) expression where e.g. HOXD10 >D9 >D8 >D4 >D1 and HOXA3 >A7 >A9 >A13 suggesting derepression of enhancer sequences up/downstream of the gene cluster. Our expression-based embryonic development signature was positively correlated with, but not restricted to, the G-CIMP-low subtype and homozygous CDKN2AB deletion. The signature was prognostic (log-rank test) on both the discovery (CATNON: p< 0.0005, TCGA: p< 0.0005) and validation set (GLASS-NL: p< 0.005). As our signature was developed on molecular data from primary resections, its prognostic value is treatment-independent. SnRNAseq on high-grade IDH-mutant astrocytomas indicated expression of embryonic development genes from tumor cells, not restricted to the stemness program. CONCLUSION Our study highlights upregulation of embryonic development genes as potential molecular mechanism associated with malignancy of IDH-mutant astrocytomas.

Identification and functional characterization of the putative members of the CTDK-1 kinase complex as regulators of growth and development in Aspergillus nidulans and Aspergillus fumigatus.

Aspergillus fumigatus has been included by the World Health Organization in the priority list of fungal pathogens because (i) it causes 90% of invasive aspergillosis cases, with a high mortality rate, and (ii) infections are becoming increasingly resistant to azole antifungals. A. nidulans is an opportunistic pathogen and a saprotroph which has served during the last 80 years as a reference system for filamentous fungi. Here, we characterized the role in morphogenesis and development of the putative transcriptional cyclin/kinase complex CTDK-1 in both aspergilli. The null mutants of the corresponding genes showed delayed germination, aberrant conidiophore development, and inhibition of cleistothecia production. While in higher eukaryotes this complex is formed only by a cyclin and a kinase, the fungal complex would incorporate a fungal-specific third component, FlpB, which would enable the interaction between the kinase (Stk47) and the cyclin (FlpA) and may be used as a target for antifungals.

A protein chimera for dsRNA binding and delivery into oocytes for large-scale gene silencing in crustacean aquaculture

RNA interference (RNAi) is one of the most intensively used gene silencing techniques for gene function studies and biotechnology applications. However, the gonads, specifically the ovary, of arthropods appear to be intractable to efficient gene silencing. In this article, we report the development of a tool for specific binding and delivery of double-stranded (ds) RNA to the crustacean oocyte for gene silencing. The tool, an oocyte-specific silencing protein chimera, which we designated OSSCot, is composed of a dsRNA-binding domain retrieved from a specific Macrobrachium rosenbergii dsRNA binding protein and 24-amino acid peptide derived from the yolk protein, vitellogenin. The results of the in vitro and in vivo examinations carried out in this study indicate that an OSSCot-piggybacked dsRNA specifically enters the oocytes. The results also indicate that the OSSCot protein protects the dsRNA from degradation by endonucleases in the ovary. When OSSCot was linked to the specific PAX6-like dsRNA of M. rosenbergii (where PAX6 is the master gene for eye development) and injected into the hemolymph of vitellogenic females, the expression of the RNAi enzyme machinery was enhanced, and eye development was impaired in the embryos of the treated females. These findings validate the concept of exploiting OSSCot to piggyback a functional dsRNA into crustacean oocytes for gene silencing. The newly developed tool will find application for oocyte-specific gene silencing in other crustacean species and for research and biotechnology applications.