Mouse Homologue Research Articles

The thiol methyltransferase activity of TMT1A (METTL7A) is conserved across species

Although few resistance mechanisms for histone deacetylase inhibitors (HDACis) have been described, we recently demonstrated that TMT1A (formerly METTL7A) and TMT1B (formerly METTL7B) can mediate resistance to HDACis with a thiol as the zinc-binding group by methylating and inactivating the drug. TMT1A and TMT1B are poorly characterized, and their normal physiological role has yet to be determined. As animal model systems are often used to determine the physiological function of proteins, we investigated whether the ability of these methyltransferases to methylate thiol-based HDACis is conserved across different species. We found that TMT1A was conserved across rats, mice, chickens, and zebrafish, displaying 85.7%, 84.8%, 60.7%, and 51.0% amino acid sequence identity, respectively, with human TMT1A. Because TMT1B was not found in the chicken or zebrafish, we focused our studies on the TMT1A homologs. HEK-293 cells were transfected to express mouse, rat, chicken, or zebrafish homologs of TMT1A and all conferred resistance to the thiol-based HDACIs NCH-51, KD-5170, and romidepsin compared to empty vector-transfected cells. Additionally, all homologs blunted the downstream effects of HDACi treatment such as increased p21 expression, increased acetylated histone H3, and cell cycle arrest. Increased levels of dimethylated romidepsin were also found in the culture medium of cells transfected to express any of the TMT1A homologs after a 24 h incubation with romidepsin compared to empty-vector transfected cells. Our results indicate that the ability of TMT1A to methylate molecules is conserved across species. Animal models may therefore be useful in elucidating the role of these enzymes in humans.

A mouse model of the protease-activated receptor 4 Pro310Leu variant has reduced platelet reactivity

BackgroundProtease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor. ObjectivesThe goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L). MethodsA point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbβ3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride–induced carotid artery injury model. ResultsPAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbβ3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis. ConclusionPAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts.

Translational toxicoepigenetic Meta-Analyses identify homologous gene DNA methylation reprogramming following developmental phthalate and lead exposure in mouse and human offspring

Although toxicology uses animal models to represent real-world human health scenarios, a critical translational gap between laboratory-based studies and epidemiology remains. In this study, we aimed to understand the toxicoepigenetic effects on DNA methylation after developmental exposure to two common toxicants, the phthalate di(2-ethylhexyl) phthalate (DEHP) and the metal lead (Pb), using a translational paradigm that selected candidate genes from a mouse study and assessed them in four human birth cohorts. Data from mouse offspring developmentally exposed to DEHP, Pb, or control were used to identify genes with sex-specific sites with differential DNA methylation at postnatal day 21. Associations of human infant DNA methylation in homologous mouse genes with prenatal DEHP or Pb were examined with a meta-analysis. Differential methylation was observed on 6 cytosines (adjusted-p < 0.05) and 90 regions (adjusted-p < 0.001). This translational approach offers a unique method that can detect conserved epigenetic differences that are developmentally susceptible to environmental toxicants.

Whole transcriptome screening for novel genes involved in meiosis and fertility in Drosophila melanogaster

Reproductive success requires the development of viable oocytes and the accurate segregation of chromosomes during meiosis. Failure to segregate chromosomes properly can lead to infertility, miscarriages, or developmental disorders. A variety of factors contribute to accurate chromosome segregation and oocyte development, such as spindle assembly and sister chromatid cohesion. However, many proteins required for meiosis remain unknown. In this study, we aimed to develop a screening pipeline for identifying novel meiotic and fertility genes using the genome of Drosophila melanogaster. To accomplish this goal, genes upregulated within meiotically active tissues were identified. More than 240 genes with no known function were silenced using RNA interference (RNAi) and the effects on meiosis and fertility were assessed. We identified 94 genes that when silenced caused infertility and/or high levels of chromosomal nondisjunction. The vast majority of these genes have human and mouse homologs that are also poorly studied. Through this screening process, we identified novel genes that are crucial for meiosis and oocyte development but have not been extensively studied in human or model organisms. Understanding the function of these genes will be an important step towards the understanding of their biological significance during reproduction.

MiR-664a-5p promotes experimental membranous nephropathy progression through HIPK2/Calpain1/GSα-mediated autophagy inhibition.

We previously found that miR-664a-5p is specifically expressed in urinary exosomes of idiopathic membranous nephropathy (IMN) patients. Homeodomain-interacting protein kinase 2 (HIPK2), a nuclear serine/threonine kinase, plays an important role in nephropathy. But the function of these factors and their connection in MN are unclear. To investigate the function and mechanism of miR-664a-5p in MN, the miR-664a-5p expression in HK-2 cells, exosomes, podocytes and renal tissues were studied, as well as cell growth and apoptosis of these cells, the binding of miR-664a-5p to HIPK2 mRNA, the levels of relative proteins and autophagy. The MN progression in MN mice model was also studied. Albumin increased the miR-664a-5p content and apoptosis of HK-2 cells, which was blocked by miR-664a-5p antagomir. miR-664a-5p bound to the 3' UTR of HIPK2 mRNA, resulting in the up-regulation of Calpain1, GSα shear and the inhibition of autophagy level. Autophagy inhibitor CQ blocked the protective effect of miR-664a-5p antagomir, HIPK2 overexpression, Calpain inhibitor SJA6017 on albumin-mediated injury. MiR-664a-5p from albumin-treated HK-2 cells could be horizontally transported to podocytes through exosomes. Exosomes from albumin-treated HK-2 cells promoted progression of MN mice, AAV-Anti-miR-664-5p (mouse homology miRNA) could improve them. Albumin increases the miR-664a-5p level and causes changes of HIPK2/Calpain1/GSα pathway, which leads to autophagy inhibition and apoptosis up-regulation of renal tubular epithelial cells. miR-664a-5p can horizontally enter podocytes through exosomes resulting in podocytes injury. Targeted inhibition of miR-664a-5p can reduce the apoptosis of renal tubule cells and podocytes, and may improve the MN progression.

Molecular dynamic approach in the regulation of systemic XCT antiporter through biologically active molecules for ischemic stroke

ABSTRACT The heterodimeric amino acid transporter systemic XCT plays a crucial role in redox balance regulation by facilitating L-cystine and L-glutamate import/export in a 1:1 ratio. Translating findings from preclinical to clinical contexts poses challenges due to interspecies receptor variations. To understand these differences and achieve success during translating studies, in silico analysis of FDA-approved drugs was performed against three species for systemic XCT receptors with UniProt-ids D4ADU2 (Rattus norvegicus), Q9WTR6 (Mus musculus), and Q9UPY5 (Homo sapiens). Homology modelling produced validated 3D structures for D4ADU2 and Q9WTR6, achieving an excellent model quality score (∼84%). Virtual screening identified common hits in Rat and Mouse Homology Modelled Structures (RHMS and MHMS), including Avodart, Rolapitant, and Olmesartan. In Homo sapiens (PDBs 7P9U and 7P9 V), common hits Dihydroergotamine and Ergotamine showed binding affinities of −12.3 to −10.1 kcal/mol as compared to glutamate and cystine (−4.9 to −5.9 kcal/mol) within the same binding pocket. A 500 ns MD simulation confirmed structural stability, positive correlations and limited atomic displacements. Free energy calculations highlighted the top compounds highly favourable binding affinities across all receptors. These results shed light on structural and dynamical variations among the interspecies receptors-ligands interactions which provide valuable insights into transitioning from preclinical to clinical settings.

Bombesin receptor-activated protein homolog deficiency altered the pattern of pathological changes of psoriasis - like skin lesion in mice.

This study investigated the potential role of the mouse homolog of bombesin receptor-activated protein (BRAP) in imiquimod (IMQ) induced psoriasis - like skin inflammation. The expression of both human BRAP, encoded by C6orf89, and its mouse homolog, encoded by BC004004, has been found to be expressed abundantly in the keratinocytes. BC004004 knockout mice (BC004004-/-) were topically treated with IMQ daily for 7 days to test whether they were more vulnerable to psoriasis - like inflammation. We found that those mice exhibited an altered pattern of inflammation process compared to isogenic wild type control mice (BC004004+/+). BC004004-/- mice developed skin lesions with earlier and more acute onset, as well as a quicker remission. The cytokines related to pathogenesis of psoriasis also exhibited different expression patterns in IMQ treated BC004004-/- mice. On day 4 of IMQ treatment, BC004004-/- mice exhibited a higher expression level of IL-17A compared to BC004004+/+ mice, suggesting a more robust activation of Th17 cells in the knockout mice. The serum level of thymic stromal lymphopoietin (TSLP), one of the keratinocyte derived cytokines, was also increased in BC004004-/- mice and reached its peak on day 4. Knockdown of BRAP in cultured human keratinocyte-derived HaCaT cells by siRNA silencing led to increased release of TSLP. Our data suggest that the elevated of level of TSLP released from keratinocytes due to BRAP deficiency might mediate the crosstalk between the epidermal cells and immune cells and thereby contributing to the altered pathological changes observed in psoriasis - like skin lesion in knockout mice.

Computational prediction and experimental validation identify functionally conserved lncRNAs from zebrafish to human

Functional studies of long noncoding RNAs (lncRNAs) have been hindered by the lack of methods to assess their evolution. Here we present lncRNA Homology Explorer (lncHOME), a computational pipeline that identifies a unique class of long noncoding RNAs (lncRNAs) with conserved genomic locations and patterns of RNA-binding protein (RBP) binding sites (coPARSE-lncRNAs). Remarkably, several hundred human coPARSE-lncRNAs can be evolutionarily traced to zebrafish. Using CRISPR–Cas12a knockout and rescue assays, we found that knocking out many human coPARSE-lncRNAs led to cell proliferation defects, which were subsequently rescued by predicted zebrafish homologs. Knocking down coPARSE-lncRNAs in zebrafish embryos caused severe developmental delays that were rescued by human homologs. Furthermore, we verified that human, mouse and zebrafish coPARSE-lncRNA homologs tend to bind similar RBPs with their conserved functions relying on specific RBP-binding sites. Overall, our study demonstrates a comprehensive approach for studying the functional conservation of lncRNAs and implicates numerous lncRNAs in regulating vertebrate physiology.

Translatable plasma and CSF biomarkers for use in mouse models of Huntington's disease.

Huntington's disease is an inherited neurodegenerative disorder for which a wide range of disease-modifying therapies are in development and the availability of biomarkers to monitor treatment response is essential for the success of clinical trials. Baseline levels of neurofilament light chain in CSF and plasma have been shown to be effective in predicting clinical disease status, subsequent clinical progression and brain atrophy. The identification of further sensitive prognostic fluid biomarkers is an active research area, and total-Tau and YKL-40 levels have been shown to be increased in CSF from Huntington's disease mutation carriers. The use of readouts with clinical utility in the preclinical assessment of potential therapeutics should aid in the translation of new treatments. Here, we set out to determine how the concentrations of these three proteins change in plasma and CSF with disease progression in representative, well-established mouse models of Huntington's disease. Plasma and CSF were collected throughout disease progression from R6/2 transgenic mice with CAG repeats of 200 or 90 codons (R6/2:Q200 and R6/2:Q90), zQ175 knock-in mice and YAC128 transgenic mice, along with their respective wild-type littermates. Neurofilament light chain and total-Tau concentrations were quantified in CSF and plasma using ultrasensitive single-molecule array (Quanterix) assays, and a novel Quanterix assay was developed for breast regression protein 39 (mouse homologue of YKL-40) and used to quantify breast regression protein 39 levels in plasma. CSF levels of neurofilament light chain and plasma levels of neurofilament light chain and breast regression protein 39 increased in wild-type biofluids with age, whereas total-Tau remained constant. Neurofilament light chain and breast regression protein 39 were elevated in the plasma and CSF from Huntington's disease mouse models, as compared with wild-type littermates, at presymptomatic stages, whereas total-Tau was only increased at the latest disease stages analysed. Levels of biomarkers that had been measured in the same CSF or plasma samples taken at the latest stages of disease were correlated. The demonstration that breast regression protein 39 constitutes a robust plasma biomarker in Huntington's disease mouse models supports the further investigation of YKL-40 as a CSF biomarker for Huntington's disease mutation carriers. Neurofilament light chain and Tau are considered markers of neuronal damage, and breast regression protein 39 is a marker of inflammation; the similarities and differences in the levels of these proteins between mouse models may provide future insights into their underlying pathology. These data will facilitate the use of fluid biomarkers in the preclinical assessment of therapeutic agents for Huntington's disease, providing readouts with direct relevance to clinical trials.

Abstract C047: Comprehensive curation of label-free proteomics data for 186 patient-derived xenograft (PDX) models in HuBase™

Abstract Introduction. HuBase™, a Crown Bioscience Inc. database, hosts pharmacological and genomic data from over 2500 patient-derived xenograft (PDX) models. It is widely used in pharmacology for model selection and biomarker analysis. Recognizing the pivotal role of proteomics data in unraveling intricate molecular mechanisms underlying biological processes and diseases, we acknowledge the challenge posed by the significant sequence similarity between mouse and human homologs in PDX samples, which hampers the accurate characterization of proteomics data using traditional run-then-separate protocols. In this study, building upon our previously published separate-then-run protocol1, we established a label-free proteomics quantification pipeline to accurately determine the protein expression level of 186 PDX models in a systematic manner. The resulting proteomics data have been seamlessly integrated into HuBase™, constituting the first comprehensive collection of PDX proteomics data derived from mouse cell-depleted samples. Methods. Mouse stromal cells were removed from the 186 PDX models with Mouse Cell Depletion Kit. The mouse depleted samples were analyzed using the 4D Data-Independent Acquisition (4D-DIA) technology. Quality control (QC) samples were measured at every 15 MS runs to assess technical variability. Raw MS data was processed with Spectronaut® 17 to generate label-free quantification (LFQ), which were log-transformed for subsequent analysis. To address batch effects, the Combat method in the sva R package was used on 2 different batches. Additionally, a subset of 10 models without mouse cell removal, underwent the same proteomics quantification pipeline for comparative analysis with their mouse depleted counterparts. Results. We quantified the expression levels of over 12,000 proteins per sample, with a significant number of proteins showing positive correlations with mRNA expression in all PDX samples. After the batch effect correction, we observed no systematic discrepancies between the two batches, and the median intensity of QC samples remained comparable. Through a comparative analysis of 10 pairs of PDX models with and without mouse stromal cell removal, we found differentially expressed proteins were enriched in the extracellular matrix for non-mouse depleted samples, which could be attributed to the upregulation of these proteins in mouse stromal cells. The significant presence of some mouse proteins underscores the importance of removing mouse stromal cells prior to proteomic characterization to avoid adversely confounding subsequent analyses leading to misinterpretations. Conclusion. Powered by state-of-the-art 4D-DIA technology, we have curated the first comprehensive PDX proteomics dataset derived from mouse-depleted samples, offering additional value for PDX model selection and exploration of potential biomarkers. References. 1. Shi, Z., Mao, B., Chen, X., Hao, P. &amp; Guo, S. Mouse Stromal Cells Confound Proteomic Characterization and Quantification of Xenograft Models. Cancer Research Communications 3, 202–214 (2023). Citation Format: Hengyuan Liu, Xiaobo Chen, Sheng Guo, Binchen Mao, Rekha Pal. Comprehensive curation of label-free proteomics data for 186 patient-derived xenograft (PDX) models in HuBase™ [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C047.

CSIG-08. GAIN-OF-FUNCTION MUTANT P53 REGULATES LONG-NONCODING RNAS TO CONTROL HIF1Α TRANSCRIPTION IN GLIOBLASTOMA

Abstract P53 is mutated in a large subset of glioblastoma (GBM). Most hot-spot p53 mutants are gain-of-function (with only partially understood mechanisms). To investigate the role of gain-of-function mutant p53 (GOF-Mut-p53), we performed ChIP-seq analysis of GOF-Mut-p53 R273H, the most common p53 mutation in GBM. We identified and verified numerous GOF-Mut-p53 targets that differed from those of wildtype p53. Among the 2,830 targets of GOF-Mut-p53 were 535 long non-coding RNAs (lncRNAs), the majority of which we also found enriched in Mut-p53 human tumors and deregulated in the TCGA database. LncRNAs play important regulatory roles in cancer. One of the most highly enriched lncRNAs was LINC00643. We first performed ChIP confirmation of GOF-Mut-p53 binding to the promoter of LINC00643. We then showed that GOF-Mut-p53 inhibits LINC00643 expression. Furthermore, we found that LINC00643 is deregulated in human GBM and LGG and correlated with patient survival. Overexpression of LINC00643 led to a significant inhibition of GBM cell proliferation, migration, invasion and in vivo xenograft growth. Co-expression of human LINC00643 or its mouse homologue with PDGF and Cre in RCAS transgenic mice (N/tv-a; Ink4a-arf-/-; Pten-fl/fl) reduced tumor growth and improved animal survival. These data show that GOF-Mut-p53 exerts oncogenic effects in GBM by downregulating LINC00643. To elucidate the mechanisms of action of LINC00643, we performed Chromatin Isolation by RNA purification high-throughput sequencing (CHIRP-seq) to identify its binding targets. We found that LINC00643 binds to the HIF1α enhancer region. Overexpression of LINC00643 and knockdown of GOF-Mut-p53 in GBM cells under hypoxia growth conditions reduced HIF1α mRNA and protein expression. LINC00643 also regulated the expression of numerous hypoxia-regulating genes downstream of HIF1α. Our study shows for the first time that GOF-Mut-p53 transcriptionally regulates a subset of lncRNAs and that GOF-Mut-p53-regulated LINC00643 regulates hypoxia in GBM.

MODL-32. EGFRVIII OVEREXPRESSION AND LOSS OF MOUSE SPECIFIC CDKN2A IN GLIAL CELLS LEADS TO GLIOMAGENESIS IN A NOVEL MOUSE MODEL

Abstract A new mouse model for the classical subtype of human glioblastoma has been generated using Cre-mediated EGFRvIII overexpression and homozygous p19-ARF deletion (the mouse homolog of human p14-ARF/CDKN2A) in GFAP expressing cells. Transgenic mice develop intraparchenymal and/or leptomeningeal brain lesions with some spinal cord invasion as early as 1 month old and 95% of mice die by 6 months due to hydrocephalus and/or paralysis. Mice with high grade tumors have worse survival and similar features to human classical glioblastoma such as necrosis, high levels of mitosis, and infiltration of tumor cells into normal brain. Immunohistochemical analysis confirms EGFRvIII overexpression and p19-ARF loss in tumor cells, along with patchy positive GFAP and positivity for Olig2, S100β and NeuN, suggesting that tumor cells arise from a progenitor glial cell type. Adherent and neurosphere primary culture of dissociated tumors indicate that tumor cells maintain EGFRvIII expression in culture and are able to generate xenograft tumors by 3 weeks after intracranial injections into NODSCID mice. Xenograft tumors are reminiscent of the primary tumor, with similar histopathological features and immunohistochemical staining. This novel mouse model can be used to study diffuse glioma with a leptomeningeal component.

Depth recordings of the mouse homologue of the Reward Positivity.

We recently advanced a rodent homologue for the reward-specific, event-related potential component observed in humans known as the Reward Positivity. We sought to determine the cortical source of this signal in mice to further test the nature of this homology. While similar reward-related cortical signals have been identified in rats, these recordings were all performed in cingulate gyrus. Given the value-dependent nature of this event, we hypothesized that more ventral prelimbic and infralimbic areas also contribute important variance to this signal. Depth probes assessed local field activity in 29 mice (15 males) while they completed multiple sessions of a probabilistic reinforcement learning task. Using a priori regions of interest, we demonstrated that the depth of recording in the cortical midline significantly correlated with the size of reward-evoked delta band spectral activity as well as the single trial correlation between delta power and reward prediction error. These findings provide important verification of the validity of this translational biomarker of reward responsiveness, learning, and valuation.

Bioinformatic Analysis of Recurrent Genomic Alterations and Corresponding Pathway Alterations in Ewing Sarcoma.

Ewing Sarcoma (ES) is an aggressive, mesenchymal malignancy associated with a poor prognosis in the recurrent or metastatic setting with an estimated overall survival (OS) of <30% at 5 years. ES is characterized by a balanced, reciprocal chromosomal translocation involving the EWSR1 RNA-binding protein and ETS transcription factor gene (EWS-FLI being the most common). Interestingly, murine ES models have failed to produce tumors phenotypically representative of ES. Genomic alterations (GA) in ES are infrequent and may work synergistically with EWS-ETS translocations to promote oncogenesis. Aberrations in fibroblast growth factor receptor (FGFR4), a receptor tyrosine kinase (RTK) have been shown to contribute to carcinogenesis. Mouse embryonic fibroblasts (MEFs) derived from knock-in strain of homologous Fgfr4G385R mice display a transformed phenotype with enhanced TGF-induced mammary carcinogenesis. The association between the FGFRG388R SNV in high-grade soft tissue sarcomas has previously been demonstrated conferring a statistically significant association with poorer OS. How the FGFR4G388R SNV specifically relates to ES has not previously been delineated. To further define the genomic landscape and corresponding pathway alterations in ES, comprehensive genomic profiling (CGP) was performed on the tumors of 189 ES patients. The FGFR4G388R SNV was identified in a significant proportion of the evaluable cases (n = 97, 51%). In line with previous analyses, TP53 (n = 36, 19%), CDK2NA/B (n = 33, 17%), and STAG2 (n = 22, 11.6%) represented the most frequent alterations in our cohort. Co-occurrence of CDK2NA and STAG2 alterations was observed (n = 5, 3%). Notably, we identified a higher proportion of TP53 mutations than previously observed. The most frequent pathway alterations affected MAPK (n = 89, 24% of pathological samples), HRR (n = 75, 25%), Notch1 (n = 69, 23%), Histone/Chromatin remodeling (n = 57, 24%), and PI3K (n = 64, 20%). These findings help to further elucidate the genomic landscape of ES with a novel investigation of the FGFR4G388R SNV revealing frequent aberration.

Signature morphoelectric properties of diverse GABAergic interneurons in the human neocortex.

Human cortex transcriptomic studies have revealed a hierarchical organization of γ-aminobutyric acid-producing (GABAergic) neurons from subclasses to a high diversity of more granular types. Rapid GABAergic neuron viral genetic labeling plus Patch-seq (patch-clamp electrophysiology plus single-cell RNA sequencing) sampling in human brain slices was used to reliably target and analyze GABAergic neuron subclasses and individual transcriptomic types. This characterization elucidated transitions between PVALB and SST subclasses, revealed morphological heterogeneity within an abundant transcriptomic type, identified multiple spatially distinct types of the primate-specialized double bouquet cells (DBCs), and shed light on cellular differences between homologous mouse and human neocortical GABAergic neuron types. These results highlight the importance of multimodal phenotypic characterization for refinement of emerging transcriptomic cell type taxonomies and for understanding conserved and specialized cellular properties of human brain cell types.

SAT034 Exploring The Role Of Zfp664 In Hepatic Glucose Regulation

Abstract Disclosure: A. Tsay: None. J. Wang: None. Genome wide association studies (GWAS) identify the association between variants at genomic loci and common diseases. These variants consist of single nucleotide polymorphisms (SNPs), which can be localized within coding or non-coding regions. The former could affect the functions of proteins encoded by these genes. The latter could modulate the expression of genes contained or localized nearby these SNPs. Several SNPs associated with metabolic disease such as diabetes, obesity, and dyslipidemia are found in and nearby the human ZNF664 gene, which encodes a zinc finger protein that is predicted to be a transcriptional regulator. The mouse homolog of ZNF664 is Zfp664, whose amino acid sequence is identical to ZNF664. To learn whether Zfp664 contains a transcriptional activation or repression function, the expression plasmid harboring the cDNA of full length or deletion mutants of mouse Zfp664 fused to yeast GAL4 DNA binding domain (DBD) was transfected into HEK293T cells along with a luciferase reporter plasmid containing 5 copies of the GAL4 binding sites. We saw decreased luciferase activity in cells overexpressing the GAL4 DBD and full length Zfp664 fusion protein. Thus, Zfp664 acts as a transcriptional repressor. The deletion analysis identified that amino acid 7 to 95 of Zfp664 was required for its transcriptional repressive activity. To test the metabolic function of hepatic Zfp664, 8 weeks old male C57Bl/6J mice were infected with adeno-associated virus (AAV) expressing either scramble small hairpin RNA (shRNA, as a control) or shRNA targeting Zfp664 and then fed a normal chow diet or western diet (42% kcal from fat, 0.2% total cholesterol) for 12 weeks. We found that mice infected with AAV expressing Zfp664 shRNA had a lower expression of Zfp664 in the liver but not in epididymal white adipose tissue and gastrocnemius muscle. Under chow diet feeding, not only did hepatic Zfp664 knockdown not affect glucose tolerance and insulin sensitivity, but it also did not affect fasting glucose, insulin, triglyceride, and total cholesterol levels. In contrast, in mice fed a western diet, hepatic Zfp664 knockdown worsened glucose tolerance and elevated fasting insulin levels. Moreover, the expression of hepatic gluconeogenic genes, such as phosphoenolpyruvate carboxykinase (Pck1) and the catalytic subunit of glucose-6-phosphatase (G6pc), was increased. The fasting plasma triglyceride and total cholesterol levels were not affected by hepatic Zfp664 knockdown in western diet fed mice. Overall, these results discover the novel role of Zfp664 in the regulation of hepatic glucose homeostasis. Presentation: Saturday, June 17, 2023

Knockout of the BRAP homolog in mice leads to abnormal tracheal cilia.

Both bombesin receptor-activated protein (BRAP) and its mouse homolog have been found to be expressed in bronchial epithelia but with unclear functions. Using electron microscopy combined with histological assays, we found that BRAP homolog deficiency in mice led to abnormal tracheal cilia. Rab-3A-interacting protein (Rabin8), a protein that might play a role in cilia development, was screened by yeast two-hybrid and further verified to have interaction with human BRAP by co-immunoprecipitation and pulldown assays. The expression levels of Rabin8, together with acetylated α-tubulin, a marker of cilia, were either downregulated by knockdown of BRAP or upregulated by overexpression of BRAP in cultured immortalized human bronchial epithelial cells. These results reveal a role for BRAP in airway cilia formation.

Liver Metabolomics and Inflammatory Profiles in Mouse Model of Fentanyl Overdose Treated with Beta-Lactams.

Fentanyl is a highly potent opioid analgesic that is approved medically to treat acute and chronic pain. There is a high potential for overdose-induced organ toxicities, including liver toxicity, and this might be due to the increase of recreational use of opioids. Several preclinical studies have demonstrated the efficacy of beta-lactams in modulating the expression of glutamate transporter-1 (GLT-1) in different body organs, including the liver. The upregulation of GLT-1 by beta-lactams is associated with the attenuation of hyperglutamatergic state, which is a characteristic feature of opioid use disorders. A novel experimental beta-lactam compound with no antimicrobial properties, MC-100093, has been developed to attenuate dysregulation of glutamate transport, in part by normalizing GLT-1 expression. A previous study showed that MC-100093 modulated hepatic GLT-1 expression with subsequent attenuation of alcohol-increased fat droplet content in the liver. In this study, we investigated the effects of fentanyl overdose on liver metabolites, and determined the effects of MC-100093 and ceftriaxone in the liver of a fentanyl overdose mouse model. Liver samples from control, fentanyl overdose, and fentanyl overdose ceftriaxone- or MC-100093-treated mice were analyzed for metabolomics using gas chromatography-mass spectrometry. Heatmap analysis revealed that both MC-100093 and ceftriaxone attenuated the effects of fentanyl overdose on several metabolites, and MC-100093 showed superior effects. Statistical analysis showed that MC-100093 reversed the effects of fentanyl overdose in some metabolites. Moreover, enrichment analysis revealed that the altered metabolites were strongly linked to the glucose-alanine cycle, the Warburg effect, gluconeogenesis, glutamate metabolism, lactose degradation, and ketone body metabolism. The changes in liver metabolites induced by fentanyl overdose were associated with liver inflammation, an effect attenuated with ceftriaxone pre-treatments. Ceftriaxone normalized fentanyl-overdose-induced changes in liver interleukin-6 and cytochrome CYP3A11 (mouse homolog of human CYP3A4) expression. Our data indicate that fentanyl overdose impaired liver metabolites, and MC-100093 restored certain metabolites.

Assessment of Ecotropic Viral Integration Site 2B (EVI2B) Gene in Juvenile Myelomonocytic Leukemia and Neurofibromatosis Type 1 NF1 Tumors.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that affects the development and growth of various tissues. NF1 is a major risk factor for the development of malignancies, particularly malignant peripheral nerve sheath tumors, optic gliomas, and leukemia. NF1 encodes a neurofibromin. Three genes, EVI2A, EVI2B, and OMGP, are embedded within intron 27b of NF1. However, the function of these genes remains unclear. EVI2A and EVI2B encode for putative transmembrane proteins. Mouse homologs are associated with viral insertions involved in leukemia in mice. Mouse Evi2b has been identified as a direct target gene of C/EBPα, a transcription factor critical for myeloid differentiation. Also possible is that these genes are related to the leukemia observed in patients with NF1. These genes might act as modifiers of NF1 phenotypic variations. Therefore, we investigated the EVI2B gene in leukemia and NF1 tumors. We analyzed DNA from 10, 20, and 3 patients with NF1, leukemia, and NF1-leukemia, respectively, and six NF1 tumor tissues. DNA sequencing analysis was used to identify the viral integration sequence, and the protein amounts and EVI2B gene expression were analyzed by flow cytometry and quantitative real-time PCR techniques. The EVI2B gene expression was increased in cutaneous neurofibroma compared with the control both at the level of protein and mRNA. However, its expression in plexiform neurofibroma was decreased significantly at protein level and increased at mRNA level compare to control. Moreover, integration of 455 bases near the 3' end of the exon was detected. When this integrated sequence was blasted into the NCBI retroviral genome database, an 87% match with the HIV-1 virus envelope gene was obtained. These preliminary results show that EVI2B might be important in NF1 tumorigenesis and leukemia.

Restriction of SARS-CoV-2 replication by receptor transporter protein 4 (RTP4).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is subject to restriction by several interferon-inducible host proteins. To identify novel factors that limit replication of the virus, we tested a panel of genes that we found were induced by interferon treatment of primary human monocytes by RNA sequencing. Further analysis showed that one of the several candidates genes tested, receptor transporter protein 4 (RTP4), that had previously been shown to restrict flavivirus replication, prevented the replication of the human coronavirus HCoV-OC43. Human RTP4 blocked the replication of SARS-CoV-2 in susceptible ACE2.CHME3 cells and was active against SARS-CoV-2 Omicron variants. The protein prevented the synthesis of viral RNA, resulting in the absence of detectable viral protein synthesis. RTP4 bound the viral genomic RNA and the binding was dependent on the conserved zinc fingers in the amino-terminal domain. Expression of the protein was strongly induced in SARS-CoV-2-infected mice although the mouse homolog was inactive against the virus, suggesting that the protein is active against another virus that remains to be identified. IMPORTANCE The rapid spread of a pathogen of human coronavirus (HCoV) family member, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), around the world has led to a coronavirus disease 2019 (COVID-19) pandemic. The COVID-19 pandemic spread highlights the need for rapid identification of new broad-spectrum anti-coronavirus drugs and screening of antiviral host factors capable of inhibiting coronavirus infection. In the present work, we identify and characterize receptor transporter protein 4 (RTP4) as a host restriction factor that restricts coronavirus infection. We examined the antiviral role of hRTP4 toward the coronavirus family members including HCoV-OC43, SARS-CoV-2, Omicron BA.1, and BA.2. Molecular and biochemical analysis showed that hRTP4 binds to the viral RNA and targets the replication phase of viral infection and is associated with reduction of nucleocapsid protein. Significant higher levels of ISGs were observed in SARS-CoV-2 mouse model, suggesting the role of RTP4 in innate immune regulation in coronavirus infection. The identification of RTP4 reveals a potential target for therapy against coronavirus infection.