Correlation Of Gene Expression Research Articles

Degraded RNA from Human Anterior Cruciate Ligaments Yields Valid Gene Expression Profiles.

Correlating gene expression patterns with biomechanical properties of connective tissues provides insights into the molecular processes underlying the tissue growth and repair. Cadaveric specimens such as human knees are widely considered suitable for biomechanical studies, but their usefulness for gene expression experiments is potentially limited by the unavoidable, nuclease-mediated degradation of RNA. Here, we tested whether valid gene expression profiles can be obtained using degraded RNA from human anterior cruciate ligaments (ACLs). Human ACL RNA (N = 6) degraded in vitro by limited ribonuclease digestion resemble highly degraded RNA isolated from cadaveric tissue. PCR threshold cycle (Ct) values for 90 transcripts (84 extracellular matrix, 6 housekeeping) in degraded RNAs variably ranged higher than values obtained from their corresponding non-degraded RNAs, reflecting both the expected loss of target templates in the degraded preparations as well as differences in the extent of degradation. Relative Ct values obtained for mRNAs in degraded preparations strongly correlated with the corresponding levels in non-degraded RNA, both for each ACL as well as for the pooled results from all six ACLs. Nuclease-mediated degradation produced similar, strongly correlated losses of housekeeping and non-housekeeping gene mRNAs. RNA degraded in situ yielded comparable results, confirming that in vitro digestion effectively modeled degradation by endogenous ribonucleases in frozen and thawed ACL. We conclude that, contrary to conventional wisdom, PCR-based expression analyses can yield valid mRNA profiles even from RNA preparations that are more than 90% degraded, such as those obtained from connective tissues subjected to biomechanical studies. Furthermore, legitimate quantitative comparisons between variably degraded tissues can be made by normalizing data to appropriate housekeeping transcripts.

The contribution of larval zebrafish transcriptomics to chemical risk assessment

In Canada, the Canadian Environmental Protection Act (1999) requires human health and environmental risk assessments be conducted for new substances prior to their manufacture or import. While this toxicity data is historically obtained using rodents, in response to the international effort to eliminate animal testing, Health Canada is collaborating with the National Research Council (NRC) of Canada to develop a New Approach Method by refining existing NRC zebrafish models. The embryo/larval zebrafish model evaluates systemic (whole body) general toxicity which is currently unachievable with cell-based testing. The model is strengthened using behavioral, toxicokinetic and transcriptomic responses to assess non-visible indicators of toxicity following chemical exposure at sub-phenotypic concentrations. In this paper, the predictive power of zebrafish transcriptomics is demonstrated using two chemicals; Raloxifene and Resorcinol. Raloxifene exposure produced darkening of the liver and malformation of the nose/mandible, while Resorcinol exposure produced increased locomotor activity. Transcriptomic analysis correlated differentially expressed genes with the phenotypic effects and benchmark dose calculations determined that the transcriptomic Point of Departure (POD) occurred at subphenotypic concentrations. Correlating gene expression with apical (phenotypic) effects strengthens confidence in evaluation of chemical toxicity, thereby demonstrating the significant advancement that the larval zebrafish transcriptomics model represents in chemical risk assessment.

Identification of a survival associated gene trio in chemical induced breast cancer

Sporadic cases of breast cancer being more prevalent than the hereditary cases, can be largely attributed to environmental pollutants like polycyclic aromatic hydrocarbons (PAHs). The aim of the present study was to identify gene dysregulations and the associations in DMBA (a PAH) induced breast cancer. A breast cancer model was developed in Wistar rats (n = 40), using DMBA. Serum proteomics (2D electrophoresis and MALDI-TOF MS) followed by relative gene expression analysis in mammary glands were conducted to reach to the differential gene signatures. The candidate genes were subjected to survival analysis (by GEPIA2 and KM plotter) and infiltration analysis (by ImmuCellAI) for correlating gene expression with patient survival and immune cell infiltration respectively. Further, the regulatory network investigation (by Cytoscape) was performed to find out the transcription factors (TFs) and miRNAs of the concerned genes. A gene trio (ANXA5, MTG1, PPP2R5B), expressing differentially in early mammary carcinogenesis at 4 months (precancerous stage) till full-fledged cancerous stage (post 6 months) was identified. The altered gene expression was associated with less survival among breast cancer patients (n = 4019). The dysregulated expression also has a correlation with enhanced mammary infiltration of immune cells. Moreover, a regulatory network (comprising of 77 transcription factors and 50 miRNAs) involved in the regulation of candidate genes was also deciphered. The deregulated target genes can therefore be explored for reregulation via identified TFs and miRNAs, and survival thereby improved.

Comparative Physiological Analysis of Lignification, Anthocyanin Metabolism and Correlated Gene Expression in Red Toona sinensis Buds during Cold Storage

The characteristics of anthocyanin and lignin are important parameters in evaluating the quality of red Toona sinensis buds. Red T. sinensis buds are prone to senescence during postharvest storage, which subsequently affects their quality and sales. However, the mechanism of senescence in red T. sinensis buds under low-temperature conditions remains unclear. In this study, red T. sinensis buds were stored at 4 °C, and their anthocyanin and lignin contents as well as the enzyme activities of PAL (phenylalanine ammonia lyase), 4CL (4-coumarate-CoA ligase), CAD (cinnamyl alcohol dehydrogenase) and POD (peroxidase) were determined at 0, 1, 2 and 3 d after handing. Meanwhile, the cellular structure of postharvest red T. sinensis buds was observed by microscopy. The relative expression of lignin-related and anthocyanin-related genes was analyzed using qRT-PCR. The results show that the anthocyanin content of the leaves was higher than that of the petioles. After 3 d of storage, the anthocyanin content of the leaves was 4.66 times that of the petioles. Moreover, the lignin content of the red T. sinensis buds gradually increased. Compared with 0 d, the lignin content of the leaves and petioles increased by 331.8 and 94.14 mg·g−1, respectively. The enzyme activities of PAL, 4CL, CAD and POD increased during cold storage. The intercellular space and the arrangement of the palisade tissue and sponge tissue in the mesophyll of red T. sinensis buds became smaller and closer, respectively. The secondary cell wall of xylem cells thickened, the number of xylem cells increased, and the arrangement number of the xylem cells became closed in the leaf vein and petioles during red T. sinensis bud storage. The expression levels of anthocyanin-related (Except for TsCHS and TsANS) and lignin-related genes increased during red T. sinensis bud storage and are highly consistent with the accumulation patterns of anthocyanins and lignin. This study may serve as a reference for exploring the molecular mechanisms of senescence, regulating the quality and cultivating new varieties of red T. sinensis buds that have low lignin content but high anthocyanin content after harvest.

Proteasome Regulatory Atpases PSMC2 and PSMC6 Modulate Proteosome Inhibitor Sensitivity and Correlate with Survival in Multiple Myeloma

Background: Multiple myeloma (MM) cells synthesize and secrete massive amounts of immunoglobulins and thus are exquisitely sensitive to therapeutics that disrupt proteostasis. The 26S proteasome complex is a 2.5-MDa holoenzyme comprised of >32 individual subunits responsible for the bulk of intracellular protein degradation. 26S proteasomes are comprised of barrel-shaped proteolytic core complex, the 20S core particle (CP), capped at either or both ends by 19S regulatory particles (RPs), which confer ATPase activity and specificity for ubiquitinated proteins. Six distinct ATPases (PSMC1-6) form a heterohexameric ring-like structure that rests atop the 20S CP's α-subunits and is responsible for unfolding and shuttling target proteins for degradation within the 20S CP. The proteasome inhibitor (PI) bortezomib (BTZ) is a potent, selective, and reversible drug that dramatically improved the prognosis of multiple myeloma (MM) patients and remains the backbone of induction therapy for transplant-eligible new diagnosis MM (NDMM), but response to therapy remains highly variable. Here, we predicted that gene expression analysis of publically available datasets could identify components within the ubiquitin-proteasome system (UPS) that areprognostic in patients treated with BTZ, suggesting potential new targets in MM. Methods: We interrogated gene expression profiles (GEP) from the randomized, phase 3 Assessment of Proteasome Inhibition for Extending Remissions (APEX) study that compared BTZ with high-dose dexamethasone (DEX) in RRMM patients who had received 1-3 prior therapies. APEX datasets served as a test cohort to identify genes that influenced survival in patients treated with BTZ. Patients consented to genomic analyses of pretreatment tumor samples. CD138+ cells were isolated from BM aspirates and analyzed by RNA microarrays to generate GEP datasets. We correlated gene expression with response to BTZ or DEX and overall survival (OS). We then validated individual genes identified from the APEX analysis using the MM Research Foundation (MMRF) CoMMpass study, a longitudinal observational analysis of the clinical and molecular data for >1000 NDMM patients receiving various standard approved treatments. Finally, we used shRNA to knockdown PSMC2 in RPMI8226 and ARH77 cells and tested their sensitivity to BTZ and carfilzomib (CFZ). Results: Analysis of the APEX dataset revealed that increased expression of genes PSMC2 and PSMC6, members of the heterohexameric ATPase ring of the 19S RP, correlated with poorer OS (PSMC2- Figure 1A, PSMC6- Median OS 320 versus 620 days for upper and lower tertile PSMC6, respectively). Using the CoMMpass database, increased PSMC2 expression also correlated with a worse OS (Figure 1B), while increased PSMC6 correlated with worse OS as well as progression free survival (PFS) (OS- HR= 2.29 p=0.0014, PFS- HR=1.63 p=0.0109). PSMC6 displayed greater expression in 50 patients above the median RNA expression (Fragments per Kb transcript per million mapped reads (FPKM)= 1 to 100) while PSMC2 expression was elevated in 400 patients above the median (FPKM= 25 to 100). shRNA-mediated PSMC2 knockdown (KD) increased PI susceptibility in ARH77 and the RPMI8226 MM cells. In ARH77 cells, the BTZ LD50 decreased from 11 nM to 2.5 nM and the CFZ LD50 decreased from 11 to 9 nM in control and KD cells, respectively. In RPMI8226 cells, the BTZ LD50 decreased from 30 nM to 20 nM and the CFZ LD50 decreased from 28 to 17 nM in control and KD cells, respectively. Conclusion: Taken together, our results indicate that increased PSMC2 and PSMC6 correlates with OS in MM in both patients treated with BTZ in the APEX and with a variety of standard therapies in the CoMMpass database. Analysis in this validation cohort extends the clinical significance of PSMC2/PSMC6 beyond the landmark APEX trial and suggests they may inform survival in patients treated with modern triplet anti-myeloma therapies. In addition to the prognostic importance across our test and validation datasets, point mutations in each ATPase subunit have been associated with PI resistance. Consistent with these findings, PSMC2 KD increased sensitivity to PI therapy. Controlled inhibition or degradation of proteasome 19S ATPase subunits using small molecules, PROTACs or RNA-based modalities now provides an opportunity to therapeutically target proteostasis in MM and other malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts

G protein-coupled receptors (GPCRs) enable cells to sense environmental cues and are indispensable for coordinating vital processes including quorum sensing, proliferation, and sexual reproduction. GPCRs comprise the largest class of cell surface receptors in eukaryotes, and for more than three decades the pheromone-induced mating pathway in baker’s yeast Saccharomyces cerevisiae has served as a model for studying heterologous GPCRs (hGPCRs). Here we report transcriptome profiles following mating pathway activation in native and hGPCR-signaling yeast and use a model-guided approach to correlate gene expression to morphological changes. From this we demonstrate mating between haploid cells armed with hGPCRs and endogenous biosynthesis of their cognate ligands. Furthermore, we devise a ligand-free screening strategy for hGPCR compatibility with the yeast mating pathway and enable hGPCR-signaling in the probiotic yeast Saccharomyces boulardii. Combined, our findings enable new means to study mating, hGPCR-signaling, and cell-cell communication in a model eukaryote and yeast probiotics.

Time and age dependent regulation of neuroinflammation in a rat model of mesial temporal lobe epilepsy: Correlation with human data.

Acute brain insults trigger diverse cellular and signaling responses and often precipitate epilepsy. The cellular, molecular and signaling events relevant to the emergence of the epileptic brain, however, remain poorly understood. These multiplex structural and functional alterations tend also to be opposing - some homeostatic and reparative while others disruptive; some associated with growth and proliferation while others, with cell death. To differentiate pathological from protective consequences, we compared seizure-induced changes in gene expression hours and days following kainic acid (KA)-induced status epilepticus (SE) in postnatal day (P) 30 and P15 rats by capitalizing on age-dependent differential physiologic responses to KA-SE; only mature rats, not immature rats, have been shown to develop spontaneous recurrent seizures after KA-SE. To correlate gene expression profiles in epileptic rats with epilepsy patients and demonstrate the clinical relevance of our findings, we performed gene analysis on four patient samples obtained from temporal lobectomy and compared to four control brains from NICHD Brain Bank. Pro-inflammatory gene expressions were at higher magnitudes and more sustained in P30. The inflammatory response was driven by the cytokines IL-1β, IL-6, and IL-18 in the acute period up to 72 h and by IL-18 in the subacute period through the 10-day time point. In addition, a panoply of other immune system genes was upregulated, including chemokines, glia markers and adhesion molecules. Genes associated with the mitogen activated protein kinase (MAPK) pathways comprised the largest functional group identified. Through the integration of multiple ontological databases, we analyzed genes belonging to 13 separate pathways linked to Classical MAPK ERK, as well as stress activated protein kinases (SAPKs) p38 and JNK. Interestingly, genes belonging to the Classical MAPK pathways were mostly transiently activated within the first 24 h, while genes in the SAPK pathways had divergent time courses of expression, showing sustained activation only in P30. Genes in P30 also had different regulatory functions than in P15: P30 animals showed marked increases in positive regulators of transcription, of signaling pathways as well as of MAPKKK cascades. Many of the same inflammation-related genes as in epileptic rats were significantly upregulated in human hippocampus, higher than in lateral temporal neocortex. They included glia-associated genes, cytokines, chemokines and adhesion molecules and MAPK pathway genes. Uniquely expressed in human hippocampus were adaptive immune system genes including immune receptors CDs and MHC II HLAs. In the brain, many immune molecules have additional roles in synaptic plasticity and the promotion of neurite outgrowth. We propose that persistent changes in inflammatory gene expression after SE leads not only to structural damage but also to aberrant synaptogenesis that may lead to epileptogenesis. Furthermore, the sustained pattern of inflammatory genes upregulated in the epileptic mature brain was distinct from that of the immature brain that show transient changes and are resistant to cell death and neuropathologic changes. Our data suggest that the epileptogenic process may be a result of failed cellular signaling mechanisms, where insults overwhelm the system beyond a homeostatic threshold.

Tumor microenvironmental signals reshape chromatin landscapes to limit the functional potential of exhausted T cells.

Response rates to immunotherapy in solid tumors remain low due in part to the elevated prevalence of terminally exhausted T cells, a hypofunctional differentiation state induced through persistent antigen and stress signaling. However, the mechanisms promoting progression to terminal exhaustion in the tumor remain undefined. Using the low-input chromatin immunoprecipitation sequencing method CUT&RUN, we profiled the histone modification landscape of tumor-infiltrating CD8+ T cells throughout differentiation. We found that terminally exhausted T cells had unexpected chromatin features that limit their transcriptional potential. Terminally exhausted T cells had a substantial fraction of active chromatin, including active enhancers enriched for bZIP/AP-1 transcription factor motifs that lacked correlated gene expression, which was restored by immunotherapeutic costimulatory signaling. Reduced transcriptional potential was also driven by an increase in histone bivalency, which we linked directly to hypoxia exposure. Enforced expression of the hypoxia-insensitive histone demethylase Kdm6b was sufficient to overcome hypoxia, increase function, and promote antitumor immunity. Our study reveals the specific epigenetic changes mediated by histone modifications during T cell differentiation that support exhaustion in cancer, highlighting that their altered function is driven by improper costimulatory signals and environmental factors. These data suggest that even terminally exhausted T cells may remain competent for transcription in settings of increased costimulatory signaling and reduced hypoxia.

Abstract 1337: Pan-cancer analysis of immune-associated genes and pathways dysregulated by tobacco reveals osteopontin as a key mediator of smoking-associated carcinogenesis

Abstract Background: Smoking remains a primary etiological agent of 12 different cancers. However, current understanding of the pathogenesis and progression of these smoking-induced cancers remains unclear. Though vaping has been proposed as a safer alternative to cigarette smoking, more research is needed to fully understand its potential to cause cancer. In particular, the chemicals in e-cigarette (e-cig) vapor have previously been demonstrated to cause immune dysregulation, similar to conventional cigarette smoke. In this study, we aimed to investigate the immune-associated (IA) genomic and transcriptomic landscapes of a panel of smoking-induced cancers in order to identify common and unique elements among these cancers and to determine whether certain immune dysregulations may be shared between smoking and vaping. Methods: Whole genome sequencing data from tumors and adjacent normal samples was obtained from The Cancer Genome Atlas (TCGA) to compare the IA landscapes of 5 different smoking-induced cancers: head and neck squamous cell carcinoma (HNSCC), esophageal carcinoma (ESCA), bladder urothelial carcinoma (BLCA), lung adenocarcinoma (LUAD), and lung squamous cell carcinoma (LUSC). Differentially expressed genes were identified and correlated to tumor stage and patient survival. Gene Set Enrichment Analysis and ReactomeFi were used to correlate IA gene expression to cancer pathway enrichment. The REVEALER algorithm was used to correlate gene expression to copy number variations and mutations. Results: We found that the genomic landscapes of smoking-induced cancers were largely unique. However, Osteopontin (OPN), an IA gene capable of regulating the EGFR and CD44 signaling cascades, was found to be upregulated along with its downstream targets in HNSCC, LUAD, LUSC, and ESCA smoking patients. Such upregulation was correlated to poorer prognosis for all 4 cancers. In vitro study indicated an upregulation of OPN and its downstream targets in cell lines exposed to both e-cig vapor and cigarette smoke. Conclusions: Despite few similarities among the IA landscapes of these smoking-induced cancers, we suggest the importance of OPN upregulation in HNSCC, LUSC, LUAD, and ESCA pathogenesis caused by smoking and, potentially, vaping. Citation Format: Jaideep Chakladar, Neil Shende, Wei Tse Li, Weg M. Ongkeko. Pan-cancer analysis of immune-associated genes and pathways dysregulated by tobacco reveals osteopontin as a key mediator of smoking-associated carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1337.

Abstract 5331: Biological assessment of the virus-like drug conjugate AU-011 to specifically target a breadth of human cancer types

Abstract Introduction: AU-011 is a virus-like drug conjugate based on a human papillomavirus (HPV) derived virus-like particle (VLP) conjugated to a light-activated cytotoxic payload (IRDye700DX) currently in Phase II clinical trials for the treatment of primary choroidal melanoma. When activated by near-infrared light, AU-011 has a dual mechanism of action inducing rapid, tumor necrosis resulting in pro-immunogenic cell death and long-term anti-tumor immunity. HPV-derived VLPs bind a wide variety of tumor types via modified heparan sulfate proteoglycans (HSPG) found on the tumor surface. This study explores the breadth of tumor types that could benefit from a targeted treatment intervention with AU-011 and surveys genes and biological pathways that are positively and negatively associated with this targeting. Methods: AU-011 binding, cytotoxicity and HSPG specificity was surveyed in 127 tumor cell lines representing breast, cervical, CNS (glioblastoma, astrocytoma), colon, esophageal, gastric, hematopoietic, lung, liver, melanoma (cutaneous or choroidal), oropharyngeal, ovarian, pancreas, prostate, renal, urothelial and skin cancers, and sarcomas (osteosarcomas, mesotheliomas, retinoblastomas). Publicly available gene expression data for 101 of these cell lines was cross-referenced to identify gene signatures that correlated (either positively or negatively) with AU-011 binding and cytotoxicity. Results: AU-011 activity was observed for every tumor type examined, with some variation across several of the types. HSPG-specific binding was well-conserved across all tumor types tested except for most cells of lymphoid origin which are known to have HSPG deficiency. Collectively the tumor-derived cell lines exhibiting average binding EC50s < 100 pM were ocular cancers (choroidal melanomas and retinoblastomas) and solid tumors including urothelial, bone, breast, cervical, CNS, colon, cutaneous melanoma, esophageal, gastric, liver, lung, skin, oropharyngeal, ovarian, and renal. Urothelial, bone, breast, cervical, CNS, esophageal, gastric, liver, lung, melanoma, skin, ovarian, renal and retinoblastoma all exhibited AU-011 mediated cytotoxicity with an average potency of < 100pM. Correlative gene expression analysis demonstrated a strong association between AU-011 activity and genes involved in epithelial-to-mesenchymal transition, glycosaminoglycan biosynthesis/metabolism, and extracellular matrix interactions. Expression signatures for ribosomal activity and protein translation were negatively associated with AU-011 binding and activity. Conclusions: Collectively these data demonstrate the wide potential applicability of AU-011 to target a number of tumor types, particularly those derived from neural or epithelial lineages. Importantly, a large portion of these tumors are accessible making their AU-011 targeting clinically translatable. Citation Format: Rhonda C. Kines, Nathan R. Fons, Elisabet de los Pinos, John T. Schiller. Biological assessment of the virus-like drug conjugate AU-011 to specifically target a breadth of human cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5331.

Identification and classification of cis-regulatory elements in the amphipod crustacean Parhyale hawaiensis.

Emerging research organisms enable the study of biology that cannot be addressed using classical 'model' organisms. New data resources can accelerate research in such animals. Here, we present new functional genomic resources for the amphipod crustacean Parhyale hawaiensis, facilitating the exploration of gene regulatory evolution using this emerging research organism. We use Omni-ATAC-seq to identify accessible chromatin genome-wide across a broad time course of Parhyale embryonic development. This time course encompasses many major morphological events, including segmentation, body regionalization, gut morphogenesis and limb development. In addition, we use short- and long-read RNA-seq to generate an improved Parhyale genome annotation, enabling deeper classification of identified regulatory elements. We discover differential accessibility, predict nucleosome positioning, infer transcription factor binding, cluster peaks based on accessibility dynamics, classify biological functions and correlate gene expression with accessibility. Using a Minos transposase reporter system, we demonstrate the potential to identify novel regulatory elements using this approach. This work provides a platform for the identification of novel developmental regulatory elements in Parhyale, and offers a framework for performing such experiments in other emerging research organisms.

Dynamics of HER3 and its correlated gene expression profile in EGFR-mutated NSCLC tumor treated with EGFR-TKI toward enhancing effectiveness of patritumab deruxtecan (HER3-DXd; U3-1402).

e21175 Background: Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a standard first-line therapy for activated EGFR-mutated non-small-cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance and is hence a promising target for anticancer treatment. Patritumab deruxtecan (HER3-DXd) is an antibody drug conjugate comprised of a fully human anti-HER3 IgG1 monoclonal antibody, covalently linked to a topoisomerase 1 inhibitor payload, an exatecan derivative, via a tetrapeptide-based cleavable linker. Clinical trials of theHER3-DXd demonstrated its anticancer activity in EGFR-mutated NSCLC. However, the genomic background that regulates HER3 expression is unknown. This study was conducted with the aim to clarify the genomic background for HER3 regulation in EGFR-mutated NSCLC tumors and to explore the strategy for enhancing the anticancer activity of HER3-DXd. Methods: 48 paired samples were obtained before EGFR-TKI treatment and after the acquisition of EGFR-TKI resistance from patients with EGFR-mutated NSCLC. HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pre-treatment to post-EGFR-TKI resistance acquisition. For statistical tests, linear regression analysis was performed to investigate whether a factor, including EGFR-TKI administration, affected HER3 expression. Pearson correlation coefficients were calculated to explore the relationships between HER3 expression level and other genomic expression in the tumors. Results: We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared to paired pretreatment samples (mean H-score 100.8 vs. 155.9, paired t-test p = 0.0007). Although genomic alterations including EGFR secondary T790M mutation did not correlate with HER3 augmentation, RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation (GSEA; normalized enrichment score -1.78, p = 0.004). Multivariable regression analysis of HER3 augmentation revealed that re-biopsy under continuing EGFR-TKI treatment independently effected HER3 augmentation. Indeed, an in-vitro study also showed that EGFR-TKI increased HER3 expression via repressed AKT phosphorylation in multiple EGFR-mutated cancers, and it finally enhanced the anticancer activity of HER3-DXd. Conclusions: Our findings highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC that is currently being evaluated in a clinical trial for patients with EGFR-mutated NSCLC tumors, not only with regard to an acquired resistance to EGFR-TKI but also in EGFR-TKI-naïve tumors (NCT04676477).

Genotype-phenotype correlation of T-cell subtypes reveals senescent and cytotoxic genes in Alzheimer's disease.

Recent studies identifying expression quantitative trait loci (eQTLs) in immune cells have uncovered important links between disease risk alleles and gene expression trends in monocytes, T cells and other cell types. However, these studies are generally done with young, healthy subjects, limiting the utility of their findings for age-related conditions such as Alzheimer's disease (AD). We have performed RNA sequencing on four T-cell subsets in genome-wide genotyped and well-characterized AD subjects and age- and sex-matched controls from the Religious Orders Study/Memory and Aging Project. We correlated gene expression data with AD neuropathological traits and with single-nucleotide polymorphisms to detect eQTLs. We identified several significant genes involved in T-cell senescence and cytotoxicity, consistent with T-cell RNA sequencing studies in aged/AD cohorts. We identified unexpected eQTLs previously associated with neuropsychiatric disease traits. Finally, we discovered that pathways related to axon guidance and synaptic function were enriched among trans-eQTLs in coding regions of the genome. Our data strengthen the potential link between T-cell senescence and age-related neurodegenerative disease. In addition, our eQTL data suggest that T-cell phenotypes may influence neuropsychiatric disorders and can be influenced by genes involved in neurodevelopmental processes.

Single-cell transcriptomics provides insights into hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic heart disease that is characterized by unexplained segmental hypertrophy that is usually most pronounced in the septum. While sarcomeric gene mutations are often the genetic basis for HCM, the mechanistic origin for the heterogeneous remodeling remains largely unknown. A better understanding of the gene networks driving the cardiomyocyte (CM) hypertrophy is required to improve therapeutic strategies. Patients suffering from HCM often receive a septal myectomy surgery to relieve outflow tract obstruction due to hypertrophy. Using single-cell RNA sequencing (scRNA-seq) on septal myectomy samples from patients with HCM, we identify functional links between genes, transcription factors, and cell size relevant for HCM. The data show the utility of using scRNA-seq on the human hypertrophic heart, highlight CM heterogeneity, and provide a wealth of insights into molecular events involved in HCM that can eventually contribute to the development of enhanced therapies.

Developing a recombinant expression system for human serine protease PRSS23

The PRSS23 gene, encoding putative serine protease 23, has been associated with several diseases by correlative gene expression studies and genetic‐based comparative models. Our group has recently developed data implicating the expression of this gene as a potential driver of ovarian cancer malignancy. However, there have been no studies defining PRSS23 protein structure or demonstrating its hypothesized proteolytic function. We aim to elucidate PRSS23 protein structure and enzymatic activity. The first step to achieve this goal is to develop a robust expression system and purification protocol for recombinant PRSS23 protein. We hypothesize that developing a methodology to produce PRSS23 recombinant protein will enable us to determine its crystal structure, evaluate its biological and enzyme function, and ultimately target the enzyme therapeutically. We strategized to maximize chances of producing PRSS23 in its native form and with high yield by testing multiple systems and host species: human, yeast, and bacterial. We anticipate, based on the protein sequence, that PRSS23 is endogenously secreted and may be activated in the secretory pathway by furin, releasing the C‐terminal catalytic domain from a smaller N‐terminal domain. The protein is also predicted to be glycosylated. We generated expression plasmids for full‐length PRSS23, codon‐optimized for expression in human and Pichia pastoris expression systems, incorporating a His‐tag for detection and purification. In the human HEK 293F and HEK 293T cell systems, despite optimizing transfection protocols and testing multiple expression constructs, secretion signals, and promoters, the expression level remained very low and was judged unlikely to produce sufficient protein for structural and functional studies. Integration of codon‐optimized PRSS23 into the genome of P. pastoris produced integrants showing a spectrum of expression levels from very low to moderate. Experiments are ongoing to optimize expression conditions of the best clone and develop a purification workflow involving ammonium sulfate precipitation, immobilized nickel ion chromatography, and additional chromatographic steps as needed. In parallel, we cloned the catalytic domain of PRSS23, appropriately codon‐optimized and also containing a C‐terminal His‐tag, into an expression vector for Escherichia coli. High levels of protein were produced as insoluble inclusion bodies, and a protocol was developed for nickel ion affinity purification under denaturing conditions. Experiments are ongoing to identify suitable conditions for in vitro refolding of the PRSS23 catalytic domain. Additionally, we are evaluating a maltose binding protein fusion with the PRSS23 catalytic domain as an alternative strategy to improve soluble protein production in E. coli. Significant quantities of pure protein will be needed to enable crystal structure determination and enzymatic activity elucidation, to unravel the biological mechanism of action and identify endogenous substrates, and potentially lead to new therapeutic strategies targeting PRSS23. The expression systems and purification workflows developed here will be a step forward toward these long‐term goals.

215 Impact of Maternal Diabetes on Neonatal Body Composition, Energy Homeostasis and Hypothalamic Salivary Gene Expression

OBJECTIVES/GOALS: Infants of diabetic mothers (IDMs) may exhibit decreased oral intake, requiring nasogastric feedings and prolonged hospitalization. We hypothesize that increased insulin exposure and resulting overgrowth in utero disrupts hypothalamic regulation of food intake, correlates to body composition and impacts feeding in IDMs. METHODS/STUDY POPULATION: Infants born at ≥ 35 weeks gestation to mothers with gestational or type II diabetes (IDM cohort), and normoglycemic mothers (control cohort) were recruited. Infants born to mothers with Type I DM or preeclampsia and with a history of intrauterine growth restriction, opioid exposure, or major congenital anomalies were excluded. Salivary expression of known hunger signaling genes 5AMP-activated protein kinase (AMPK), Neuropeptide Y receptor Y2 (NPY2R), leptin (LEP), ghrelin (GHRL), proopiomelanocortin (POMC), and adiponectin (ADIPOQ) were quantified using RT-qPCR. Body composition was assessed via skinfold measurements and compared and correlated between cohorts. Feeding outcomes were recorded. RESULTS/ANTICIPATED RESULTS: 23 infants were recruited in each cohort. POMC and AMPK were expressed by 71% and 88% of infants respectively in both cohorts. NPY2R was expressed by 79% and 83% of the diabetic cohort and normoglycemia cohort respectively, while GHRL was expressed by 75% and 79% of the diabetic cohort and normoglycemia cohort, respectively. LEP and ADIPOQ were not reliably expressed in either cohort. Infants with a higher body fat percentage were less likely to express NPY2R (OR= 0.76). There was no significant association between body fat percentage and expression of AMPK, POMC, or GHRL. Only 3 IDMs were noted by providers to exhibit poor oral intake, limiting our ability to correlate gene expression and body composition with feeding outcomes. DISCUSSION/SIGNIFICANCE: Non-invasive assessment of hunger signaling gene expression is possible through salivary analysis of AMPK, POMC, NPY2R, and GHRL. Given the paucity of IDMs with poor feeding in our study, future studies should target IDMs requiring feeding support to understand mechanisms driving aberrant feeding behavior.

Differential Expression of Calycosin-7-O-β-D-glucoside Biosynthesis Genes and Accumulation of Related Metabolites in Different Organs of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao Under Drought Stress.

Calycosin-7-O-β-D-glycoside (CG), as a flavonoid, plays an important role in the abiotic stress response of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). CG is also an active ingredient in A. mongholicus with high medicinal value. However, the response mechanism of the CG biosynthetic pathway of drought stress is not clear. In this research, drought stress was inflicted upon A. mongholicus, and the variations in flavonoid metabolites and the correlating gene expression in CG biosynthesis were studied in roots, stems, and leaves of A. mongholicus by UHPLC-MRM-MS/MS and qRT-PCR. Drought stress reduced the dry weight and increased the content of malondialdehyde (MDA) and proline. Drought was beneficial to the accumulation of L-phenylalanine and 4-coumaric acid in leaves and promoted the accumulation of all target compounds in the roots, except calycosin. Overexpression of AmIOMT was observed in the leaves, but the content of formononetin which is the product of isoflavone O-methyltransferase (IOMT) catalysis was higher in stems than in leaves. This research aims to further understand the acclimation of abiotic stress and the regulation mechanism of flavonoid accumulation in A. mongholicus.

RNA in situ hybridisation as a molecular diagnostic technique targeting IBA‐1 and CD204 in canine histiocytic sarcoma

BackgroundCanine histiocytic sarcoma (HS) is an aggressive cancer with morphologically variable features; therefore, obtaining a definitive diagnosis can be challenging. Two proteins, IBA‐1, ionised calcium‐binding adapter molecule 1, and CD204, a macrophage scavenger receptor, have been shown to be specific immunohistochemical markers helpful in distinguishing HS from other tumour types with similar morphological features.ObjectivesThis study was performed to demonstrate the use of RNA in situ hybridisation (ISH) technology allowing single‐molecule RNA visualisation in formalin‐fixed paraffin‐embedded (FFPE) tissues as a molecular tool for the diagnosis of canine HS.MethodsReverse transcription polymerase chain reaction (RT‐PCR) and western blot analysis for IBA‐1 and CD204 were performed to correlate gene expression and protein expression of these two markers in the histiocytic sarcoma DH82 cell line. RNA‐ISH for IBA‐1 and CD204 was performed on the DH82 cell line to validate the RNA‐ISH probes. RNA‐ISH and immunohistochemistry (IHC) were performed in clinical HS FFPE samples to demonstrate mRNA and protein expression of IBA‐1 and CD204. FFPE archived samples of canine round cell tumours, melanoma and anaplastic sarcoma were used as negative controls.ResultsRNA‐ISH and IHC showed moderate to strong expression for IBA‐1 and CD204 in the neoplastic cells in both the canine DH82 cell line and the archived canine HS samples. RNA‐ISH and IHC showed scattered positive staining in the control tumours samples, consistent with macrophagic infiltration.ConclusionRNA‐ISH for CD204 and IBA‐1 appeared to have a high specificity and sensitivity in our samples and may be an additional valuable diagnostic technique in identifying HS.

The chromatin accessibility landscape in the dental pulp of mouse molars and incisors.

The dental pulp is a promising source of progenitor cells for regenerative medicine. The natural function of dental pulp is to produce odontoblasts to generate reparative dentin. Stem cells within the pulp tissue originate from the migrating neural crest cells and possess mesenchymal stem cell properties with the ability to differentiate into multiple lineages. To elucidate the transcriptional control mechanisms underlying cell fate determination, we compared the transcriptome and chromatin accessibility in primary dental pulp tissue derived from 5-6-day-old mice. Using RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq), we correlated gene expression with chromatin accessibility. We found that the majority of ATAC-seq peaks were concentrated at genes associated with development and cell differentiation. Most of these genes were highly expressed in the mouse dental pulp. Surprisingly, we uncovered a group of genes encoding master transcription factors that were not expressed in the dental pulp but retained open chromatin states. Within this group, we identified key developmental genes important for specification of the neural crest, adipocyte, neural, myoblast, osteoblast and hepatocyte lineages. Collectively, our results uncover a complex relationship between gene expression and the chromatin accessibility landscape in the mouse dental pulp.

Lighting from Top and Side Enhances Photosynthesis and Plant Performance by Improving Light Usage Efficiency.

Light is a critical environmental factor that influences plant growth and development, ranging from seed germination to flowering and fruiting. This study was carried out to explore how the optimal combination of various lighting directions increases the light usage efficiency and influences the plant morphophysiology, by investigating the plant growth parameters, leaf anatomy, epidermal morphology, stomatal properties, chlorophyll content, key physiological changes, and correlated gene expressions. In closed-type plant growth chambers, rooted cuttings of two chrysanthemum (Chrysanthemum morifolium Ramat.) cultivars, “Pearl Egg” and “Gaya Glory”, were subjected to a 10-h photoperiod with 600 μmol∙m−2·s−1 photosynthetic photon flux density (PPFD) provided by light-emitting diodes (LEDs) in each light-direction combination (top (1/1) (T), top (1/2) + side (1/2) (TS), top (1/2) + bottom (1/2) (TB), side (1/2) + bottom (1/2) (SB), and top (1/3) + side (1/3) + bottom (1/3) (TSB)). The TS lighting significantly enhanced the morphophysiological performance, compared to the other lighting direction combinations. Notably, the excellent branch formation and earlier flowering were induced by the TS lighting in both “Pearl Egg” and “Gaya Glory” plants.