Compared Gene Expression Research Articles

Mitral valve transcriptome analysis in thirty-four age-matched Cavalier King Charles Spaniels with or without congestive heart failure caused by myxomatous mitral valve disease.

We here report the results of a mitral valve transcriptome study designed to identify genes and molecular pathways involved in development of congestive heart failure (CHF) following myxomatous mitral valve disease (MMVD) in dogs. The study is focused on a cohort of elderly age-matched dogs (n = 34, age ~ 10years) from a single breed-Cavalier King Charles Spaniels (CKCS)-with a high incidence of MMVD. The cohort comprises 19 dogs (10♀, 9♂) without MMVD-associated CHF, and 15 dogs (6♀, 9♂) with CHF caused by MMVD; i.e., we compare gene expression in breed and age-matched groups of dogs, which only differ with respect to CHF status. We identify 56 genes, which are differentially expressed between the two groups. In this list of genes, we confirm an enrichment of genes related to the TNFβ-signaling pathway, extracellular matrix organization, vascular development, and endothelium damage, which also have been identified in previous studies. However, the genes with the greatest difference in expression between the two groups are CNTN3 and MYH1. Both genes encode proteins, which are predicted to have an effect on the contractile activity of myocardial cells, which in turn may have an effect on valvular performance and hemodynamics across the mitral valve. This may result in shear forces with impact on MMVD progression.

A combination of burn wound injury and Pseudomonas infection elicits unique gene expression that enhances bacterial pathogenicity.

The interaction between an underlying disease process and a specific pathogen may lead to the unique expression of genes that affect bacterial pathogenesis. These genes may not be observed during infection in the absence of, or with a different underlying process or infection during the underlying process with a different pathogen. To test this hypothesis, we used Nanostring technology to compare gene transcription in a murine-burned wound infected with P. aeruginosa. The Nanostring probeset allowed the simultaneous direct comparison of immune response gene expression in both multiple host tissues and P. aeruginosa in conditions of burn alone, infection alone, and burn with infection. While RNA-Seq is used to discover novel transcripts, NanoString could be a technique to monitor specific changes in transcriptomes between samples and bypass the additional adjustments for multispecies sample processing or the need for the additional steps of alignment and assembly required for RNASeq. Using Nanostring, we identified arginine and IL-10 as important contributors to the lethal outcome of burned mice infected with P. aeruginosa. While other examples of altered gene transcription are in the literature, our study suggests that a more systematic comparison of gene expression in various underlying diseases during infection with specific bacterial pathogens may lead to the identification of unique host-pathogen interactions and result in more precise therapeutic interventions.

PSXIII-8 Effects of Dietary Lactobacillus Reuteri on Immune Responses, Blood Profiles, Intestinal Morphology, and Ileal Gene Expression of Weaned Pigs

Abstract Weaning period is critical for gut health and immunity of pigs due to negative effect of weaning stress. Probiotics can be a potential alternative to antibiotics. However, there is limited information on potential mechanism of gut health and immunity of dietary Lactobacillus reuteri. This study was conducted to investigate the effects of dietary Lactobacillus reuteri on immune responses, blood profiles, intestinal morphology, and ileal gene expression of weaned pigs. A total of 48 weaned pigs [average initial body weight (BW) = 6.51 ± 0.87 kg; 21 days old] were allotted to 2 dietary treatments in a randomized complete block design (block: initial BW and sex). Dietary treatments had 6 replicate pens with 4 pigs per pen and fed for 4 weeks. Dietary treatments consisted of a basal weaner diet based on corn-soybean meal (CON) and CON supplemented with 0.02% Lactobacillus reuteri IDCC 3701 (LR). The LR was obtained from a commercial company (ILDONG Bioscience Co., Ltd., Pyeongtaek, Republic of Korea). Blood samples were collected from randomly selected one pig per pen on day 1, 7, 14, and 28. Serum was obtained from the blood sample to measure immune responses. On the last day of the experiment, the pigs were euthanized to collect small intestinal segments for intestinal morphology. Ileal mucosa were collected from ileal tissue for gene expression analysis. Measurements were serum cortisol, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β), and interleukin-6 (IL-6) using ELISA kits, blood profiles using an automated hematology analyzer, intestinal morphology using hematoxylin and eosin method, and ileal gene expression of tight junction proteins and inflammatory cytokines using qRT-PCR. Data were analyzed by using PROC GLM of SAS. Experimental unit was a pen. Statistical model for immune responses, blood profiles, and intestinal morphology included dietary treatments as a main effect and initial BW as a covariate. The t-test was used for the comparison of ileal gene expression. Pigs fed LR had drecreased (P < 0.05) serum TNF-α and IL-6 on day 7 and serum TGF-β1 on day 14 than those fed CON. In addition, pigs fed LR had greater (P < 0.05) hemoglobin concentration on day 7 than those fed CON, but had reduced (P < 0.05) hematocrit on day 14. Pigs fed LR had greater (P < 0.05) villus height to crypt depth ratio in duodenum than those fed CON. The LR upregulated (P < 0.05) claudin-1 gene compared with CON, but downregulated (P < 0.05) interleukin-8 and TNF-α genes. In conclusion, addition of dietary Lactobacillus reuteri in weaner diets improved intestinal morphology, modulated blood profiles and immune responses, and gene expression of inflammatory cytokines.

Mutant p53 Drives the Development of Myelodysplastic Syndromes Via Dysregulating Pre-mRNA Splicing in Hematopoietic Stem and Progenitor Cells

Despite the clinical importance of TP53 mutations, to date there have been no studies on the functional impact of TP53 mutations on the initiation and progression of MDS. We found that heterozygous p53 mutant mice ( p53R248W/+) show extended survival and died within 15-20 months after birth. p53 mutant mice had hypercellular bone marrow and displayed cytopenia, thrombocytopenia, and anemia. Approximately 60% of p53R248W/+ mice developed MDS based upon pathological analysis of bone marrow (BM) and peripheral blood smears, while the remaining p53R248W/+ mice developed lymphoma and sarcoma. We further showed that mutant p53-driven MDS are transplantable. Thus, we have generated a mouse model of MDS that has some features of human MDS with TP53 mutations. To understand how mutant p53 drives MDS development, we performed RNA-seq studies to compare gene expression in hematopoietic stem and progenitor cells (HSPCs). GSEA analysis revealed that spliceosome genes were significantly downregulated in middle-aged p53 R248W/+ HSPCs compared to age-matched p53 +/+ HSPCs. We confirmed that the expression of splicing factors, such as Prpf3 and Prpf4, was significantly downregulated in middle-aged p53 R248W/+ HSPCs. We conducted alternative splicing analysis utilizing MISO (Mixture of Isoforms) algorithm to identify differences in exon inclusion ratio between WT and mutant HSPCs. We observed differential splicing of all classes of alternative splicing events, including Cassette Exons (CE), Competing 5′ and 3′ splice sites (A5' or A3'SS), Mutually Exclusive Exons (MXE), and Retained Introns (RI) in p53 R248W/+ cells compared to that of the WT HSPCs. Notably, we found that murine p53 mutant HSPCs exhibited aberrant splicing of key regulators of NFκBactivation, such as USP15. USP15 overexpression promotes NFκB expression through inhibiting its ubiquitination, whereas NFκB promotes USP15 expression. p53 mutant HSPCs tend to skip exon 7 and express high levels of short isoform of Usp15. The short isoform of Usp15, but not the long isoform of Usp15, activates NFκB in HSPCs, manifested by increased phosphorylation of p65. To determine the impact of mutant p53 on pre-mRNA splicing in human HSPCs, we ectopically expressed GFP or p53 R248W in human cord blood CD34 + cells and performed RNA-seq in transduced cells (GFP +). Human HSPCs expressing mutant p53 displayed alterations in pre-mRNA splicing compared to HSPCs expressing GFP. We found that human HSPCs expressing mutant p53 display aberrant splicing in key regulator of NFκB such as IKBKE (Inhibitor of nuclear factor kappa-B kinase subunit epsilon). Human HSPCs expressing mutant p53 tend to include exon 7 and express high levels of long isoform of IKBKE. The long isoform of IKBKE, but not the short isoform of IKBKE, activates NFκB in human HSPCs. Many proinflammatory cytokine genes are NFκB targets and we observed increased levels of pro-inflammatory cytokines, including IL-1β and IL-6, in the BM of middle-aged p53 mutant mice. Notably, we observed increased levels of IL-1β and IL-6 in the BM of MDS or AML patients with TP53 mutations. TP53 mutations co-occur with splicing factor mutations in MDS. To determine whether TP53 and SRSF2 mutations cooperate in pathogenesis of MDS, we have generated p53 R248W/+SRSF2 P95H/+ mice. We found that the survival of recipient mice repopulated with p53 R248W/+SRSF2 P95H/+ BM cells was significantly decreased compared to recipients repopulated with WT, p53 R248W/+, or SRSF2 P95H/+ BM cells. All recipient mice repopulated with p53 R248W/+SRSF2 P95H/+ BM cells died within 6 months and developed MDS or leukemia. Thus, we demonstrate that TP53 and SRSF2 mutations cooperate in MDS or leukemia development. In summary, we discovered that mutant p53 dysregulates pre-mRNA splicing in key regulators of inflammatory response during aging, thereby generating a chronic inflammatory microenvironment to drive MDS pathogenesis.

Detection of Occurrent Somatic Hypermutation and Associated Gene Expression Profile in Single B Cells

Introduction Somatic Hypermutation (SHM) is an endogenous mutator mechanism in B lymphocytes that preferentially targets the immunoglobulin V(D)J coding region and facilitates antibody affinity maturation. The exact frequency and impact of SHM in individual B cells remains elusive, as previous quantifications only considered cumulative outcomes at population level. We therefore employed single-cell RNA sequencing on primary follicular lymphoma (FL) cells, a B-cell malignancy retaining the germinal center (GC) B-cell phenotype, to identify and quantify individual SHM events. This approach allowed us to detect neovariants, defined as single nucleotide differences that originate from recent SHM events, and to explore the gene expression profiles of the B lymphoma cells with neovariants, particularly focusing on genes and pathways effectuating SHM. Methods We collected and processed samples from 12 follicular lymphoma (FL) and five chronic lymphocytic leukemia (CLL) patients as non-germinal center controls. Single cell whole cDNA libraries were prepared by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with semi-nested amplification using 3′ constant domain primers followed by 10X Genomics prep. Both single cell libraries were sequenced in paired-end mode on Illumina HiSeq 4000. We developed a customized method to preprocess scBCR data designed specifically for highly mutated FL BCR sequences. To ensure data quality and validity of putative neovariants, we applied a comprehensive filtering system, including stringent thresholds in terms of numbers of individual immunoglobulin (IG) mRNA molecules sequenced as defined by their UMI, and reads per position obtained from the median of the estimator's distribution, base quality scores, exclusion of cell doublets, position within the V(D)J region, and proportion of cell-level variants. Based on a reported IG mRNA half-life of 3 h and assuming a first-order decay, the filtering criteria implied that detected neovariants were created between 1 and 7 hours prior to the time of sampling. Results A median of 1112 cells (range: 215-16432) per case were analyzed. 1239 IG neovariants were detected in a total of 334 FL cells in 7 of the 12 FL cases. Neovariants were undetectable in control CLL cells. A remarkably high proportion (95,4%) of neovariants were found within known AID motifs (WRCY, WA, and RCG), suggesting active AID involvement in the hypermutation process. The observed fractions of FL cells carrying recent SHM events indicated initiation of FL pathophysiology at least 11,2 months prior to sampling. To further evaluate the role of AID, we compared gene expression profiles between FL cells with and without neovariants. The results demonstrated not only higher expression of AID but as well of pathways involved in SHM. Mismatch repair (hsa03430, 9 enriched genes with p.adjust < 0.001) and base excision repair (hsa03410, 17 enriched genes with p.adjust = 0.01), together with DNA replication (hsa03030, 14 enriched genes with p.adjust < 0.001), were the three most prominent pathways with upregulated genes. In cells with detectable neovariants, the MLH1/MSH2 genes, associated with the recognition of the deamination process, as well as POLH gene, which is involved in error-prone mutations in MMR, were highly expressed. Overall, we observed a more prominent expression of genes related to MMR than BER in cells with neovariants supported by a higher normalized enrichment score (NES) in MMR (MMR:1.93 and BER:1.6). This suggests not only an active SHM process but also potential interplay between AID and MMR in driving SHM in FL. Conclusions Here we report for the first time quantitative detection of ongoing somatic hypermutation at single cell level in FL. Based on the lifespan of IG mRNA, simultaneous detection of two IG mRNA transcripts differing by one (or a few) nucleotides only within a single cell permits to estimate the timing when this SHM event occurred. The detection of this phenomenon in 58% of FL samples and its association with AID expression suggests that AID-induced mutagenesis might be acting at a much higher rate than expected. Quantification of SHM at the single-cell level should facilitate the understanding of the cellular heterogeneity and dynamics in physiological GC reactions but also may offer novel insights into the AID-dependent shaping of the genomic landscape of germinal center B-cell neoplasms.

Combining Inotuzumab Ozogamicin (IO) with PARP Inhibitors, Olaparib and Talazoparib, Exerts Synergistic Cytotoxicity By Inhibiting the Repair of IO-Induced DNA Strand Breaks in IO-Resistant ALL Cells

[Introduction]Inotuzumab ozogamicin (IO) is highly effective in the treatment of relapsed/refractory CD22-positive B-acute lymphoblastic leukemia (B-ALL). IO exerts the antileukemic effect via DNA single- and double-strand breaks induced by calicheamicin. Although IO has been reported to show more than 80% response rate, the duration of remission is only 4.6 months, suggesting the early acquisition of drug resistance. Thus, it is crucial to elucidate the mechanism of resistance to IO and overcome it. Polyadenosine diphosphate-ribose polymerase (PARP) inhibitors, such as olaparib and talazoparib, show the antitumor effect by inhibiting DNA repair. This study was conducted to enhance the antitumor effect of IO by inhibiting IO-induced DNA damage repair using PARP inhibitors. The study attempted to overcome the resistance of B-ALL cell lines developed to be IO-resistant. [Methods]Ph-negative, CD22-positive B-ALL Reh cell line was used to determine the cytotoxicity of IO and PARP inhibitors. Inotuzumab ozogamicin was kindly provided by Pfizer. Six IO-resistant cell lines were newly established by long-term exposure of Reh cells to IO, followed by the limiting dilution. FACS analysis evaluated CD22 expression. WTS assay and Annexin-V positivity were used to determine the antileukemic effects. The Comet assay assessed the DNA repair function. The DNA microarray compared gene expression between Reh and IO-resistant Reh clones. [Results] The growth inhibition effect of IO was significantly augmented by the addition of the minimally toxic concentration of olaparib or talazoparib in Reh cells. The combination index values were 0.19 and 0.42, respectively, demonstrating a strong synergism. Combining IO with olaparib or talazoparib increased IO-induced apoptosis compared to single-agent IO. The apoptosis rates induced by 20 ng/mL IO with minimally toxic concentrations of olaparib or talazoparib were 51.7% and 66.1%, respectively, showing significantly higher apoptosis rates than IO alone (23.5%). Assessment of DNA strand breaks using the Comet assay showed that the recovery of IO-induced DNA damage was inhibited in the presence of PARP inhibitors, and DNA strand breaks persisted. Next, six IO-resistant Reh (Reh-IO-R) cell lines were established as described above. The expressions of CD22 were more than 60%. All 6 cell lines had more than 60-fold resistance to IO with the 50%-inhibitory concentration (IC 50) values 474.3～782.3 ng/mL, compared with that of Reh cells (2.1 ng/mL). The IC 50 values of olaparib or talazoparib were similar to Reh and Reh-IO-R cell lines. When IO was combined with olaparib or talazoparib, the growth of Reh-IO-R cells was inhibited, with median IC 50 values were 359.4[290.3-406.2] and 179.1[59.3-240.5] ng/mL, respectively. This combination also remarkably increased apoptosis induction in Reh-IO-R cells. The apoptosis rate of Reh cells at 100 ng/mL IO alone was 77%, whereas the apoptosis rate of 6 Reh-IO-R cell lines averaged 5.9% [5.7-6.6%]. The median percentage of apoptosis in Reh-IO-R cell lines was 12.0% by IO + olaparib and 17.1% by IO + talazoparib. Furthermore, synergism was obtained in Reh-IO-R cell lines with the combination of 1,000 ng/mL IO with olaparib or talazoparib. The median percentage of apoptotic cell death was 24.2% by IO 1,000 ng/mL alone and 45.3% by the IO+olaparib, and 56.8% by the IO+talazoparib. These results suggest that the potentiating effect of PARP inhibitors would be attributed to more DNA cleavage occurring. The expression of genes ( ATM, ATR, DNA-PK, CHK1/2, PARP, RAD, ERCC1, BRCA1/2 Ku70/80) related to DNA repair was similar between Reh and Reh-IO-R cells. Of note, ABCB1 (p-gp) was overexpressed in Reh-IO-R cells. These results suggest that the expression of p-gp was one of the mechanisms of the resistance to IO, and the combination of IO and PARP inhibitors was effective to a certain extent in the Reh-IO-R cells expressing p-gp. This would be because the PARP inhibitors were still effective in inhibiting the DNA repair in Reh-IO-R cells where the DNA repair function was not augmented. [Conclusion]The combination of IO with PARP inhibitors synergized the antileukemic effect of IO by inhibiting DNA strand break repair in CD22-positive ALL cell lines and IO-resistant ALL cells, suggesting that these results may contribute to overcoming the IO resistance mechanisms.

Multiple light signaling pathways control solar tracking in sunflowers.

Sunflowers are famous for their ability to track the sun throughout the day and then reorient at night to face east the following morning. This occurs by differential growth patterns, with the east sides of stems growing more during the day and the west sides of stems growing more at night. This process, termed heliotropism, is generally believed to be a specialized form of phototropism; however, the underlying mechanism is unknown. To better understand heliotropism, we compared gene expression patterns in plants undergoing phototropism in a controlled environment and in plants initiating and maintaining heliotropic growth in the field. We found the expected transcriptome signatures of phototropin-mediated phototropism in sunflower stems bending towards monochromatic blue light. Surprisingly, the expression patterns of these phototropism-regulated genes are quite different in heliotropic plants. Most genes rapidly induced during phototropism display only minor differences in expression across solar tracking stems. However, some genes that are both rapidly induced during phototropism and are implicated in growth responses to foliar shade are rapidly induced on the west sides of stems at the onset of heliotropism, suggesting a possible role for red light photoreceptors in solar tracking. To test the involvement of different photoreceptor signaling pathways in heliotropism, we modulated the light environment of plants initiating solar tracking. We found that depletion of either red and far-red light or blue light did not hinder the initiation or maintenance of heliotropism in the field. Together, our results suggest that the transcriptional regulation of heliotropism is distinct from phototropin-mediated phototropism and likely involves inputs from multiple light signaling pathways.

198 Differential Gene Expression and Metabolic Pathway Comparisons of Liver and Duodenum from Suckling Piglets Given One or Two Iron Dextran Injections

Abstract Six female littermate pairs were used in an experiment to evaluate the mRNA expression in tissues from piglets given one or two 1 mL injections of iron dextran (200 mg Fe/mL, Uniferon, Pharmacosmos Inc. Watchung, NJ). All pigs in the litter were administered the first 1 mL injection < 24 hours after birth and on d 7, pigs were paired by body weight (BW; 1.72 ± 0.13 kg) and one pig from each pair was randomly selected as control (CON) and the other received a second injection (+Fe). At weaning on d 22, each piglet was anesthetized, and samples of liver and duodenum were taken from the anesthetized piglets and placed in DNA/RNA Shield (Zymo, Irvine, CA) for preservation until mRNA extraction. RNA-Seq libraries were sequenced to a depth of at least 30 million read pairs (150 bp paired-end sequencing) per sample. Differential Gene Expression data were analyzed with GeneSpring (Agilent, Santa Clara, CA) with a fold-change (FC) of |1.2| P < 0.05 (Table 1). Pathway analysis was conducted with Ingenuity Pathway Analysis (IPA QIAGEN, Redwood City, CA) software with Z-score cutoff of P < 0.05. In the duodenum 435 genes were significantly changed with a FC ≥|1.2| P < 0.05. Most notably, Claudin 1 and Claudin 2 were inversely affected by the second dose of iron. Claudin 1 (CLDN1, FC = 4.48, P = 0.0423) is responsible for cell-to-cell adhesion in the epithelial cell sheets and serves as a barrier to prevent water and solutes from passing through the paracellular space. Claudin 2 (CLDN2, FC = -1.41, P = 0.0097) was down regulated because it is expressed in cation leaky epithelia. In the liver 362 genes were expressed with a FC ≥|1.2| P < 0.05. The gene most affected by a second dose of 200 mg Fe was HAMP (Hepcidin Antimicrobial Peptide) with an FC of 40.8. HAMP is a liver-produced hormone that is the main circulating regulator of Fe absorption and distribution across tissues. It also controls the major flows of Fe into plasma by promoting endocytosis and degradation of ferroportin (SLC4A1). This leads to the retention of Fe in Fe-exporting cells and decreased flow of Fe into plasma. Metabolic pathway changes in the duodenum and liver provide evidence for the improved feed conversion and growth rates in pigs given two iron injections pre-weaning. In the duodenum, there is a down regulation of gluconeogenesis (P < 0.05). Concurrently, there is a decrease in the production of urea in the liver (P < 0.05). These observations show that there is less need for gluconeogenesis, thus less urea production from deaminated amino acids. The genomic and pathway analyses provided empirical evidence linking gene expression with phenotypic observations of piglet health and growth improvements.

Ceramide synthase 4 overexpression exerts oncogenic properties in breast cancer

BackgroundCeramide, a bioactive signaling sphingolipid, has long been implicated in cancer. Members of the ceramide synthase (CerS) family determine the acyl chain lengths of ceramides, with ceramide synthase 4 (CerS4) primarily generating C18–C20-ceramide. Although CerS4 is known to be overexpressed in breast cancer, its role in breast cancer pathogenesis is not well established.MethodsTo investigate the role of CerS4 in breast cancer, public datasets, including The Cancer Genome Atlas (TCGA) and two Gene Expression Omnibus (GEO) datasets (GSE115577 and GSE96058) were analyzed. Furthermore, MCF-7 cells stably overexpressing CerS4 (MCF-7/CerS4) as a model for luminal subtype A (LumA) breast cancer were produced, and doxorubicin (also known as Adriamycin [AD])-resistant MCF-7/ADR cells were generated after prolonged treatment of MCF-7 cells with doxorubicin. Kaplan–Meier survival analysis assessed the clinical significance of CERS4 expression, while Student’s t-tests or Analysis of Variance (ANOVA) compared gene expression and cell viability in different MCF-7 cell lines.ResultsAnalysis of the public datasets revealed elevated CERS4 expression in breast cancer, especially in the most common breast cancer subtype, LumA. Persistent CerS4 overexpression in MCF-7 cells activated multiple cancer-associated pathways, including pathways involving sterol regulatory element–binding protein, nuclear factor kappa B (NF-κB), Akt/mammalian target of rapamycin (mTOR), and β-catenin. Furthermore, MCF-7/CerS4 cells acquired doxorubicin, paclitaxel, and tamoxifen resistance, with concomitant upregulation of ATP-binding cassette (ABC) transporter genes, such as ABCB1, ABCC1, ABCC2, ABCC4, and ABCG2. MCF-7/CerS4 cells were characterized by increased cell migration and epithelial–mesenchymal transition (EMT). Finally, CERS4 knockdown in doxorubicin-resistant MCF-7/ADR cells resulted in reduced activation of cancer-associated pathways (NF-κB, Akt/mTOR, β-catenin, and EMT) and diminished chemoresistance, accompanied by ABCB1 and ABCC1 downregulation.ConclusionsChronic CerS4 overexpression may exert oncogenic effects in breast cancer via alterations in signaling, EMT, and chemoresistance. Therefore, CerS4 may represent an attractive target for anticancer therapy, especially in LumA breast cancer.

Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

Hydroxamic acids: New players in the multifactorial mechanisms of maize resistance to Striga hermonthica

Striga hermonthica is the most widespread and destructive plant parasite infesting maize and other major crops in sub-Saharan Africa where it causes severe yield losses and threatens food security. Several tolerant maize lines supporting reduced S. hermonthica emergence have been deployed. However, the molecular bases of such resistance are yet poorly understood. Based on a time course comparative gene expression analysis between susceptible and resistant maize lines we have confirmed resistance mechanisms known to be activated upon plant parasite infestation and identified potential novel players worth further investigation e.g. iron homeostasis and mitochondrial respiration-related genes. Most intriguingly, we show a previously unknown strategy of maize post-attachment resistance based on DIMBOA accumulation in S. hermonthica-infested maize roots. S. hermonthica infestation triggers positive regulation of gene expression in the hydroxamic acid (HA) pathway culminating with an accumulation of benzoxazinoids (BX), known for their antifeedant, insecticidal, antimicrobial, and allelopathic activities. We demonstrate that HA root content is positively correlated with S. hermonthica resistance in the resistant parent and its progenies and in unrelated maize lines. Downregulation of HA genes causes increased susceptibility to S. hermonthica infestation in loss-of-function maize mutants. While the mechanism of BX action in parasitic plant resistance is yet to be uncovered, the potential of this discovery for developing effective control and breeding strategies is enormous.

DNA methylation and transcriptome analysis reveal epigenomic differences among three macaque species.

Macaques (genus Macaca) are the most widely distributed non-human primates, and their evolutionary history, gene expression profiles, and genetic differences have been extensively studied. However, the DNA methylomes of macaque species are not available in public databases, which hampers understanding of epigenetic differences among macaque species. Epigenetic modifications can potentially affect development, physiology, behavior, and evolution. Here, we investigated the methylation patterns of the Tibetan macaque (M. thibetana; TM), Chinese rhesus macaque (M. mulatta lasiota; CR), and crab-eating macaque (M. fascicularis; CE) through whole-genome bisulfite sequencing from peripheral blood. We compared genome-wide methylation site information for the three species. We identified 12,128 (CR vs. CE), 59,165 (CR vs. TM), and 39,751 (CE vs. TM) differentially methylated regions (DMRs) in the three macaques. Furthermore, we obtained the differentially expressed genes (DEGs) among the three macaque species. The differences between CR and CE were smaller at both the methylome and transcriptome levels than compared with TM (CR vs. TM and CE vs. TM). We also found a change in the density of single nucleotide mutations in DMRs relative to their flanking regions, indicating a potential mechanism through which genomic alterations may modulate methylation landscapes, thereby influencing the transcriptome. Functional enrichment analyses showed the DMR-related genes were enriched in developmental processes and neurological functions, such as the growth hormone-related pathway, insulin secretion pathway, thyroid hormone synthesis pathway, morphine addiction, and GABAergic synapses. These differences may be associated with variations in physiology and habitat among the macaques. Our study provides one of the first genome-wide comparisons of genetic, gene expression, and epigenetic variations across different macaques. Our results should facilitate further research on comparative genomic and genetic differences in macaque species.

Single Cell Transcriptomics Identifies Distinct Choroid Cell Populations Involved in Visually Guided Eye Growth.

Postnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. Many studies suggest that the ocular choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refractive development. To elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations during conditions in which the eye is undergoing emmetropization. UMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of significant gene expression changes were relatively small (< 2 fold). The highest changes in gene expression were identified in a rare cell population (0.11% - 0.49% of total choroidal cells). This cell population expressed high levels of neuron-specific genes as well as several opsin genes suggestive of a rare neuronal cell population that is potentially light sensitive. Our results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization as well as insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.

Depletion of IQ motif-containing GTPase activating protein 2 (IQGAP2) reduces hepatic glycogen and impairs insulin signaling

The liver is critical in maintaining metabolic homeostasis, regulating both anabolic and catabolic processes. Scaffold protein IQ motif-containing GTPase activating protein 2 (IQGAP2) is highly expressed in the liver and implicated in fatty acid uptake. However, its role in coordinating either fed or fasted responses is not well understood. Here we report that IQGAP2 is widely expressed in the liver that is pronounced in the pericentral region. Although control and IQGAP2 knockout mouse model showed comparable hepatic gene expression in the fasted state, we found significant defects in fed state responses. Glycogen levels were reduced in the periportal region when IQGAP2 was deleted. Consistently, we observed a decrease in phosphorylated glycogen synthase kinase 3α and total glycogen synthase protein in the fed IQGAP2 knockout mice which suggest inadequate glycogen synthesis. Moreover, immunoprecipitation of IQGAP2 revealed its interaction with GSK3 and GYS. Furthermore, our study demonstrated that knocking down IQGAP2 invitro significantly decreased the phosphorylation of AKT and forkhead box O3 proteins downstream of insulin signaling. These findings suggest that IQGAP2 contributes to liver fed state metabolism by interacting with glycogen synthesis regulators and affecting the phosphorylation of insulin pathway components. Our results suggest that IQGAP2 plays a role in regulating fed state metabolism.

THU396 Digital Spatial Profiling Of Human Parathyroid Tumors Reveals Distinct Cellular And Molecular Alterations Linked To Vitamin D Deficiency

Abstract Disclosure: C. Tu: None. W. Chang: None. J.A. Sosa: Advisory Board Member; Self; Novo Nordisk, AstraZeneca, Eli Lilly &amp; Company. Grant Recipient; Self; Exelixis, Inc., Eli Lilly &amp; Company. J. Koh: None. Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder characterized by disrupted calcium homeostasis secondary to inappropriately elevated parathyroid hormone (PTH) secretion. Low levels of serum 25-hydroxyvitamin D (25OHD) are significantly more prevalent in PHPT patients than in the general population, but the pathophysiological basis for this association remains unclear. We employed a spatially defined in situ whole-transcriptomics and selective proteomics profiling approach to compare gene expression patterns and cellular composition in parathyroid adenomas from 9 vitamin D-deficient or 34 vitamin D-replete PHPT patients with 12 eucalcemic cadaveric donor parathyroid glands as normal tissue controls. Parathyroid oxyphil cell content is markedly higher in tumors from vitamin D-deficient PHPT patients (Def-Ts) relative to tumors from vitamin D-replete PHPT patients (Rep-Ts) (47.8% vs 17.8%, respectively; p &amp;lt;0.0001) and normal donor parathyroid glands (7.7%). Def-Ts display increased expression of genes encoding electron transport chain (corr. coefficient=0.546), proteasomal catabolism (corr. coefficient=0.587), and oxidative phosphorylation (corr. coefficient=0.982) pathway components. In contrast, Rep-Ts are characterized by upregulation of the ras (corr. coefficient=0.632) and myc (corr. coefficient=0.811) signaling pathways, suggestive of an oncogene-driven etiology. Parathyroid oxyphil cells are comparable to chief cells at the transcriptional level, and vitamin D-status is reflected in the transcriptional profiles of both cell types in a similar manner. Among PHPT patients who did not present as vitamin D deficient, three distinct tumor transcriptional profile clusters showed a dose-dependent relationship to pre-operative 25OHD levels (p &amp;lt;0.0001 by ANOVA). Relative to normal tissue, the abundance of calcium sensing receptor (CaSR) protein is reduced in Def-Ts (mean protein count 253.6 vs 492.3, respectively; p=0.039 for difference), while the type B γ-aminobutyric acid receptor 1 (GABAB1R) is increased (mean protein count 114.5 vs 64.65, respectively; p=0.0099 for difference). These expression changes resulted in a vitamin D dose-dependent reduction in the ratio of CaSR to GABAB1R receptors in Def-T tumor cells (r2 = 0.218, p&amp;lt;0.0001), favoring the formation of calcium sensing-attenuated CaSR:GABAB1R heteromers. The abundance of the amyloid precursor protein (APP) peptide derivative Aβ1-42, a putative GABAB1R ligand, is preferentially increased in Def-Ts compared to normal tissue (mean protein count 5797 vs 4500, respectively; p=0.0318). Collectively, these findings suggest a novel mechanism where chronic vitamin D deficiency can contribute to tonic PTH hypersecretion in PHPT patients by increasing GABAB1R-mediated antagonism of CaSR activity in parathyroid adenomas. Presentation: Thursday, June 15, 2023

Characterization of 3D organotypic epithelial tissues reveals tonsil-specific differences in tonic interferon signaling.

Three-dimensional (3D) culturing techniques can recapitulate the stratified nature of multicellular epithelial tissues. Organotypic 3D epithelial tissue culture methods have several applications, including the study of tissue development and function, drug discovery and toxicity testing, host-pathogen interactions, and the development of tissue-engineered constructs for use in regenerative medicine. We grew 3D organotypic epithelial tissues from foreskin, cervix, and tonsil-derived primary cells and characterized the transcriptome of these in vitro tissue equivalents. Using the same 3D culturing method, all three tissues yielded stratified squamous epithelium, validated histologically using basal and superficial epithelial cell markers. The goal of this study was to use RNA-seq to compare gene expression patterns in these three types of epithelial tissues to gain a better understanding of the molecular mechanisms underlying their function and identify potential therapeutic targets for various diseases. Functional profiling by over-representation and gene set enrichment analysis revealed tissue-specific differences: i.e., cutaneous homeostasis and lipid metabolism in foreskin, extracellular matrix remodeling in cervix, and baseline innate immune differences in tonsil. Specifically, tonsillar epithelia may play an active role in shaping the immune microenvironment of the tonsil balancing inflammation and immune responses in the face of constant exposure to microbial insults. Overall, these data serve as a resource, with gene sets made available for the research community to explore, and as a foundation for understanding the epithelial heterogeneity and how it may impact their in vitro use. An online resource is available to investigate these data (https://viz.datascience.arizona.edu/3DEpiEx/).

Comparative transcriptome analysis between long- and short-term survival after pig-to-monkey cardiac xenotransplantation reveals differential heart failure development

ABSTRACT Cardiac xenotransplantation is the potential treatment for end-stage heart failure, but the allogenic organ supply needs to catch up to clinical demand. Therefore, genetically-modified porcine heart xenotransplantation could be a potential alternative. So far, pig-to-monkey heart xenografts have been studied using multi-transgenic pigs, indicating various survival periods. However, functional mechanisms based on survival period-related gene expression are unclear. This study aimed to identify the differential mechanisms between pig-to-monkey post-xenotransplantation long- and short-term survivals. Heterotopic abdominal transplantation was performed using a donor CD46-expressing GTKO pig and a recipient cynomolgus monkey. RNA-seq was performed using samples from POD60 XH from monkey and NH from age-matched pigs, D35 and D95. Gene-annotated DEGs for POD60 XH were compared with those for POD9 XH (Park et al. 2021). DEGs were identified by comparing gene expression levels in POD60 XH versus either D35 or D95 NH. 1,804 and 1,655 DEGs were identified in POD60 XH versus D35 NH and POD60 XH versus D95 NH, respectively. Overlapped 1,148 DEGs were annotated and compared with 1,348 DEGs for POD9 XH. Transcriptomic features for heart failure and inhibition of T cell activation were observed in both long (POD60)- and short (POD9)-term survived monkeys. Only short-term survived monkey showed heart remodeling and regeneration features, while long-term survived monkey indicated multi-organ failure by neural and hormonal signaling as well as suppression of B cell activation. Our results reveal differential heart failure development and survival at the transcriptome level and suggest candidate genes for specific signals to control adverse cardiac xenotransplantation effects.

High and Low Levels of ABCB1 Expression Are Associated with Two Distinct Gene Signatures in Lung Tissue of Pulmonary TB Patients with High Inflammation Activity.

P-glycoprotein (encoded by the ABCB1 gene) has a dual role in regulating inflammation and reducing chemotherapy efficacy in various diseases, but there are few studies focused on pulmonary TB patients. In this study, our objective was to identify a list of genes that correlate with high and low levels of ABCB1 gene expression in the lungs of pulmonary TB patients with different activity of chronic granulomatous inflammation. We compared gene expression in two groups of samples (with moderate and high activity of tuberculomas) to identify their characteristic gene signatures. Gene expression levels were determined using quantitative PCR in samples of perifocal area of granulomas, which were obtained from 65 patients after surgical intervention. Subsequently, two distinct gene signatures associated with high inflammation activity were identified. The first signature demonstrated increased expression of HIF1a, TGM2, IL6, SOCS3, and STAT3, which correlated with high ABCB1 expression. The second signature was characterized by high expression of TNFa and CD163 and low expression of ABCB1. These results provide insight into various inflammatory mechanisms and association with P-gp gene expression in lung tissue of pulmonary TB patients and will be useful in the development of a host-directed therapy approach to improving the effectiveness of anti-TB treatment.

Incorporating Primer Amplification Efficiencies in Quantitative Reverse Transcription Polymerase Chain Reaction Experiments; Considerations for Differential Gene Expression Analyses in Zebrafish.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is commonly used to measure the mRNA expression of target genes in zebrafish. Gene expression values from RT-qPCR are typically reported as relative fold-changes, and relative quantification of RT-qPCR data incorporates primer amplification efficiency values for each target gene. We describe the influence of the primer amplification efficiency analysis method on RT-qPCR gene expression fold-change calculations. This report describes (1) a sample analysis demonstrating incorporation of primer amplification efficiency into RT-qPCR analysis for comparing gene expression of a gene of interest between two groups when normalized to multiple reference genes, (2) the influence of differences in primer amplification efficiencies between measured genes on gene expression differences calculated from theoretical delta-Cq (dCq) values, and (3) an empirical comparison of the influence of three methods of defining primer amplification efficiency in gene expression analyses (delta-delta-Cq [ddCq], standard curve, LinRegPCR) using mRNA measurements of a set of genes in zebrafish embryonic development. Given the need to account for the influence of primer amplification efficiency along with the simplicity of using software programs (LinRegPCR) to measure primer amplification efficiency from RT-qPCR data, we encourage using empirical measurements of primer amplification efficiency for RT-qPCR analysis of differential gene expression in zebrafish.

1190P Comparative gene expression and mutational profiling of neuroendocrine neoplasm in relation to clinical outcome

1190P Comparative gene expression and mutational profiling of neuroendocrine neoplasm in relation to clinical outcome