Quantitative Transcriptome Research Articles

Defense strategies against sweet potato chlorotic stunt and pakakuy virus coinfection unraveled

AbstractThis study into the response of two Ipomoea batatas (L.) Lam cultivars, Melinda and Tio Joe, to coinfection with sweet potato chlorotic stunt virus (SPCSV) and sweet potato pakakuy virus (SPPV), employed a comprehensive approach encompassing symptomatology, real‐time quantitative polymerase chain reaction, metagenomics, and transcriptomics. SPCSV is a quarantine virus with synergistic effects, which decimate yields. SPPV is the most prevalent DNA virus in sweet potato germplasm, with a tendency to persist in meristems, posing a significant risk for germplasm transfer between territories. Graft inoculation experiments revealed that while Tio Joe remained asymptomatic for 12 weeks and suppressed virus replication, Melinda displayed symptoms early on and exhibited high virus titers. Metagenomic analyses corroborated these observations and confirmed that SPCSV and SPPV were responsible. Transcriptomic analysis unveiled disparities in gene expression between Melinda and Tio Joe. Differential gene expression was heightened and altered in Melinda as the viruses disrupted its gene expression. Its defense strategies, such as inducing abscisic acid signaling, were insufficient to overcome disruptive viral effects like oxidative stress, rendering it susceptible. In contrast, Tio Joe had relatively stable differential gene expression, indicating resistance to SPPV–SPCSV coinfection. Overexpressed genes such as sirtuin, rapid alkalization factor, and nuclear pore anchor triggered quantitative resistance, supported with enriched organelles such as mitochondrion and pathways such as proteasome and cutin, suberine, and wax biosynthesis. Tio Joe maintained its genome integrity and inhibited viral replication by tightly controlling gene expression and preventing reactive oxygen species accumulation.

The underlying mechanism of single and co-infection of DIV1 and WSSV in Litopenaeus vannamei

Viral diseases are one of the important factors restricting the shrimp industry. Decapod iridescent virus 1 (DIV1) and White spot syndrome virus (WSSV) are the two most harmful viruses to shrimp. DIV1 and WSSV were found to be carried and infected simultaneously in several samples of cultured and wild shrimps. In this study, the co-infection model of DIV1 and WSSV in Litopenaeus vannamei was constructed by TaqMan probe quantitative detection (qPCR), intestinal section, and transcriptome analysis. Our study also evaluated the virulence of the virus by calculating LC50 and detected the replication rates of DIV1 and WSSV at 7-time points and 2 tissues in L. vannamei by qPCR. As a result, DIV1 and WSSV could infect L. vannamei singly or co-infect L. vannamei. DIV1 replicated the fastest, at 12–16 h post-injection (hpi). The WSSV virus replicates the fastest 20–24 hpi. In different tissues, the amount of DIV1 carried by blood cells is higher than that in muscle, while WSSV is the opposite. In addition, the intestinal responses of DIV1 and WSSV single and co-infection of L. vannamei were explored using the RNA-Seq platform and bioinformatics analysis, and the differential expressed genes (DEGs) screened by comparison were as follows: there were significant differences in DEGs between the PBS group and the three virus infection groups, and there were differences in DEGs between the PBS group and single virus infection groups (the DIV1 group and the WSSV group), but there was no significant difference in the DEGs between the 2 single virus infection groups, and the Combine-infected group, especially, the DEGs between the WSSV group and the Combine-infected group were only 39. According to the above conclusions, it can be inferred that WSSV plays a major role in co-infection. In addition, pathways related to vitamin metabolism were significantly enriched in the viral infection group compared to the PBS group. Glycolysis/Gluconeogenesis, a hallmark of the Warburg effect, is also significantly enriched after viral infection. In this study, the co-infection phenomenon of DIV1 and WSSV was found for the first time in L. vannamei, which provided a new idea for developing and screening molecular markers for new strains resistant to complex viruses in L. vannamei.

Proteome-wide copy-number estimation from transcriptomics

Protein copy numbers constrain systems-level properties of regulatory networks, but proportional proteomic data remain scarce compared to RNA-seq. We related mRNA to protein statistically using best-available data from quantitative proteomics and transcriptomics for 4366 genes in 369 cell lines. The approach starts with a protein’s median copy number and hierarchically appends mRNA–protein and mRNA–mRNA dependencies to define an optimal gene-specific model linking mRNAs to protein. For dozens of cell lines and primary samples, these protein inferences from mRNA outmatch stringent null models, a count-based protein-abundance repository, empirical mRNA-to-protein ratios, and a proteogenomic DREAM challenge winner. The optimal mRNA-to-protein relationships capture biological processes along with hundreds of known protein-protein complexes, suggesting mechanistic relationships. We use the method to identify a viral-receptor abundance threshold for coxsackievirus B3 susceptibility from 1489 systems-biology infection models parameterized by protein inference. When applied to 796 RNA-seq profiles of breast cancer, inferred copy-number estimates collectively re-classify 26–29% of luminal tumors. By adopting a gene-centered perspective of mRNA–protein covariation across different biological contexts, we achieve accuracies comparable to the technical reproducibility of contemporary proteomics.

MDia2 is an important mediator of MRTF-A-dependent regulation of breast cancer cell migration.

Dysregulated actin cytoskeleton gives rise to aberrant cell motility and metastatic spread of tumor cells. This study evaluates the effect of overexpression of wild-type versus functional mutants of MRTF-A on migration and invasion of breast cancer (BC) cells. Our studies indicate that SRF's interaction is critical for MRTF-A-induced promotion of both two-dimensional and three-dimensional cell migration, while the SAP-domain function is important selectively for three-dimensional cell migration. Increased MRTF-A activity is associated with more effective membrane protrusion, a phenotype that is attributed predominantly to SRF's interaction with MRTF. We demonstrate formin-family protein mDia2 as an important mediator of MRTF-stimulated actin polymerization at the leading edge and cell migration. Multiplexed quantitative immunohistochemistry and transcriptome analyses of clinical BC specimens further demonstrate a positive correlation between nuclear localization of MRTF with malignant traits of cancer cells and enrichment of MRTF-SRF gene signature in pair-matched distant metastases versus primary tumors. In conclusion, this study establishes a novel mechanism of MRTF-dependent regulation of cell migration and provides evidence for the association between MRTF activity and increased malignancy in human BC, justifying future development of specific small molecule inhibitors of the MRTF-SRF transcriptional complex as potential therapeutic agents in BC.

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR

BackgroundReal-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results.ResultsWe developed a software named “Gene Selector for Validation” (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully.ConclusionThe GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data.

Effects of Refrigerated Storage on Restarted Morphological Development of Dictyophora indusiata Fruiting Bodies

Mature Dictyophora indusiata fruiting bodies are brittle and broken easily during storage. Peach-shaped Dictyophora indusiata before maturity respond well to refrigerated maintenance, but some cannot resume their development after storage. This study analyzed the effects of refrigerated storage and transportation on the restarted development of Dictyophora indusiata fruiting bodies using quantitative transcriptome analysis. The refrigerated (4 °C, 3 d) peach-shaped Dictyophora indusiata (CK) was used as the control. After induction treatment for 3 d (26 ± 2 °C, 95 ± 3% RH), 81.25% of CK could achieve restarted development and mature (D-M), whereas 18.75% failed (D-P) to restart. Quantitative transcriptome analysis revealed that 1389 and 4451 differentially expressed genes (DEGs) were identified in the D-P and D-M groups when compared with the results for the CK group, respectively. DEG annotation and functional analysis revealed that D-P did not initiate energy and nutrient metabolism. Most DEGs involving the phosphatidylinositol signaling pathway and the MAPK signaling pathway were significantly downregulated or unchanged in the D-P and significantly upregulated in the D-M groups. These results suggested that the phosphatidylinositol signaling pathway may play a crucial role in transmitting environmental signals and initiating the morphogenesis of CK, and that the downstream MAPK signaling pathway may be responsible for signal transmission, thereby regulating cellular activities. This study provides a theoretical basis for regulating the growth and development of postharvest Dictyophora indusiata fruiting bodies.

Exosomes derived from human urine–derived stem cells ameliorate IL-1β-induced intervertebral disk degeneration

BackgroundHuman intervertebral disk degeneration (IVDD) is a sophisticated degenerative pathological process. A key cause of IVDD progression is nucleus pulposus cell (NPC) degeneration, which contributes to excessive endoplasmic reticulum stress in the intervertebral disk. However, the mechanisms underlying IVDD and NPC degeneration remain unclear.MethodsWe used interleukin (IL)-1β stimulation to establish an NPC-degenerated IVDD model and investigated whether human urine–derived stem cell (USC) exosomes could prevent IL-1β-induced NPC degeneration using western blotting, quantitative real-time polymerase chain reaction, flow cytometry, and transcriptome sequencing techniques.ResultsWe successfully extracted and identified USCs and exosomes from human urine. IL-1β substantially downregulated NPC viability and induced NPC degeneration while modulating the expression of SOX-9, collagen II, and aggrecan. Exosomes from USCs could rescue IL-1β-induced NPC degeneration and restore the expression levels of SOX-9, collagen II, and aggrecan.ConclusionsUSC-derived exosomes can prevent NPCs from degeneration following IL-1β stimulation. This finding can aid the development of a potential treatment strategy for IVDD.

Identification and expression analysis of nuclear factor Y transcription factor genes under drought, cold and Eldana infestation in sugarcane (Saccharum spp. hybrid)

BackgroundThe Nuclear Factor Y (NF-Y) transcription factor (TF) gene family plays a crucial role in plant development and response to stress. Limited information is available on this gene family in sugarcane.ObjectivesTo identify sugarcane NF-Y genes through bioinformatic analysis and phylogenetic association and investigate the expression of these genes in response to abiotic and biotic stress.MethodsSugarcane NF-Y genes were identified using comparative genomics from functionally annotated Poaceae and Arabidopsis species. Quantitative PCR and transcriptome analysis assigned preliminary functional roles to these genes in response to water deficit, cold and African sugarcane borer (Eldana saccharina) infestation.ResultsWe identify 21 NF-Y genes in sugarcane. Phylogenetic analysis revealed three main branches representing the subunits with potential discrepancies present in the assignment of numerical names of some NF-Y putative orthologs across the different species. Gene expression analysis indicated that three genes, ShNF-YA1, A3 and B3 were upregulated and two genes, NF-YA4 and A7 were downregulated, while three genes were upregulated, ShNF-YB2, B3 and C4, in the plants exposed to water deficit and cold stress, respectively. Functional involvement of NF-Y genes in the biotic stress response were also detected where three genes, ShNF-YA6, A3 and A7 were downregulated in the early resistant (cv. N33) response to Eldana infestation whilst only ShNF-YA6 was downregulated in the susceptible (cv. N11) early response.ConclusionsOur research findings establish a foundation for investigating the function of ShNF-Ys and offer candidate genes for stress-resistant breeding and improvement in sugarcane.

Nkx1.2 deletion decreases fat production in zebrafish.

This study aimed to investigate the role of Nkx1-2, a transcription factor with the NK homeobox domain, in the regulation of fat production. Gene expression was analyzed using quantitative real-time polymerase chain reaction or transcriptome sequencing. CRISPR/Cas9 technology was employed to generate nkx1.2 knockout zebrafish and nkx1.2-deleted 3T3-L1 cells. Lipid droplet production in zebrafish larvae was visually quantified using Nile red staining, whereas lipid droplets in 3T3-L1 cells were stained with Oil red O. The binding of Nkx1-2 to the promoter was verified through an electrophoretic mobility shift assay experiment. Nkx1-2 plays crucial roles in the regulation of fat production in zebrafish. Knockout of nkx1.2 in zebrafish leads to weight loss, accompanied by significantly reduced lipid droplet production and decreased visceral and liver fat content. Furthermore, genes related to lipid biosynthesis are significantly downregulated. In 3T3-L1 preadipocytes, Nkx1-2 induces differentiation into mature adipocytes by binding to the cebpa promoter, thereby activating its transcription. Additionally, the expression of nkx1.2 is regulated by the p38 MAPK, JNK, or Smad2/3 signaling pathways in 3T3-L1 cells. Our findings suggest that Nkx1-2 functions as a positive regulator of fat production, playing a critical role in adipocyte differentiation and lipid biosynthesis.

The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

Species-wide quantitative transcriptomes and proteomes reveal distinct genetic control of gene expression variation in yeast

Gene expression varies between individuals and corresponds to a key step linking genotypes to phenotypes. However, our knowledge regarding the species-wide genetic control of protein abundance, including its dependency on transcript levels, is very limited. Here, we have determined quantitative proteomes of a large population of 942 diverse natural Saccharomyces cerevisiae yeast isolates. We found that mRNA and protein abundances are weakly correlated at the population gene level. While the protein coexpression network recapitulates major biological functions, differential expression patterns reveal proteomic signatures related to specific populations. Comprehensive genetic association analyses highlight that genetic variants associated with variation in protein (pQTL) and transcript (eQTL) levels poorly overlap (3%). Our results demonstrate that transcriptome and proteome are governed by distinct genetic bases, likely explained by protein turnover. It also highlights the importance of integrating these different levels of gene expression to better understand the genotype-phenotype relationship.

Overexpression of RBM15 modulated the effect of trophoblast cells by promoting the binding ability between YTHDF2 and the CD82 3′UTR to decrease the expression of CD82

Pre-eclampsia (PE) is a syndrome with no specific pathological mechanism and is specific to pregnancy. The combined analysis of proteomics and transcriptomics possesses many benefits for treating this disease. m6A modification plays a major role in PE; however, mechanism have not been studied clearly. This study investigated the potential mechanism underlying the role of m6A in PE. Mass spectrometry-based label-free quantitative proteomics and transcriptomics experiments were conducted on the placenta of patients with pre-eclampsia and normal pregnancies, and the two omics were followed by joint analysis. Total m6A modification in placental tissues, HTR8/SVneo cells, and JEG-3 cells was measured by dot blot. The levels of RBM15 and CD82 in tissues and cells were detected using qPCR. The protein levels of G3BP1, RBM15, MMP-2, YTHDF2, and MMP-9 were measured by western blotting. The function, migration, and invasion characteristics of HTR8/SVneo and JEG-3 cells were measured using Transwell assays. SRAMP predicted the m6A modification site in the CD82 mRNA 3'UTR, and this was confirmed using luciferase activity and YTHDF2-RIP. m6A modification was promoted in the PE group, and the RBM15 abundance was increased. Overexpression of RBM15 increased m6A modification. However, overexpression of RBM15 suppressed the expression of MMP-2 and MMP-9 and also the migratory and invasive capabilities of HTR8/SVneo and JEG-3 cells. CD82 expression levels were decreased in PE, and CD82 expression was confirmed via qPCR, western blotting and immunofluorescence. Furthermore, RBM15 overexpression reduced CD82 mRNA and protein levels. Luciferase activity and YTHDF2-RIP results verified that overexpression of RBM15 promoted the binding ability between YTHDF2 and the CD82 3'UTR, thereby decreasing CD82 expression. Finally, CD82 overexpression reversed the effect of RBM15 overexpression on the expression of MMP-2 and MMP-9 and on the migratory and invasive capabilities of the cells. Overexpression of RBM15 hindered the migratory and invasive capabilities of trophoblasts, while concurrently enhancing m6A modification. The potential mechanism was that overexpression of RBM15 promoted the binding capability between YTHDF2 and CD82 3'UTR and decrease the expression of CD82. Thus, this study provides a theoretical basis for the treatment of PE.

Proteomic and Transcriptomic Analyses Provide New Insights into the Mechanism Underlying Lipid Deterioration in Pecan Kernels during Storage.

Pecan nuts are rich in lipids that tend to deteriorate during storage. Tandem mass-tag-based quantitative proteomics and transcriptomics were used to investigate the changes in the protein and gene profiles of stored pecan kernels for the first time. Our previous lipidomic data were jointly analyzed to elucidate the coordinated changes in lipid molecules and related proteins/genes. The mechanism underlying lipid deterioration in pecan kernels during storage was revealed by multiomics analyses. Lipid metabolism-related pathways were activated during pecan storage. Phospholipases, triacylglycerol lipases, lipoxygenases, and oil body-related proteins/genes were highly expressed during storage, revealing their involvement in lipid deterioration. These data provide rich information and will be valuable for future genetic or chemical research to alleviate lipid deterioration in pecans.

TIGAR exacerbates obesity by triggering LRRK2-mediated defects in macroautophagy and chaperone-mediated autophagy in adipocytes

ABSTRACT Obesity is one of the most common metabolic diseases around the world, which is distinguished by the abnormal buildup of triglycerides within adipose cells. Recent research has revealed that autophagy regulates lipid mobilization to maintain energy balance. TIGAR (Trp53 induced glycolysis regulatory phosphatase) has been identified as a glycolysis inhibitor, whether it plays a role in the metabolism of lipids is unknown. Here, we found that TIGAR transgenic (TIGAR+/+) mice exhibited increased fat mass and trended to obesity phenotype. Non-target metabolomics showed that TIGAR caused the dysregulation of the metabolism profile. The quantitative transcriptome sequencing identified an increased levels of LRRK2 and RAB7B in the adipose tissue of TIGAR+/+ mice. It was confirmed in vitro that TIGAR overexpression increased the levels of LRRK2 by inhibiting polyubiquitination degradation, thereby suppressing macroautophagy and chaperone-mediated autophagy (CMA) while increasing lipid accumulation which were reversed by the LRRK2 inhibitor DNL201. Furthermore, TIGAR drove LRRK2 to interact with RAB7B for suppressing lysosomal degradation of lipid droplets, while the increased lipid droplets in adipocytes were blocked by the RAB7B inhibitor ML282. Additionally, fat-specific TIGAR knockdown of TIGAR+/+ mice alleviated the symptoms of obesity, and adipose tissues-targeting superiority DNL201 nano-emulsion counteracted the obesity phenotype in TIGAR+/+ mice. In summary, the current results indicated that TIGAR performed a vital function in the lipid metabolism through LRRK2-mediated negative regulation of macroautophagy and CMA in adipocyte. The findings suggest that TIGAR has the potential to serve as a viable therapeutic target for treating obesity and its associated metabolic dysfunction.

Identification and characterization of the critical genes encoding Cd-induced enhancement of SOD isozymes activities in Zhe-Maidong (Ophiopogon japonicus).

Superoxide dismutase (SOD) protects plants from abiotic stress-induced reactive oxygen species (ROS) damage. Here, the effects of cadmium (Cd) exposure on ROS accumulation and SOD isozymes, as well as the identification of significant SOD isozyme genes, were investigated under different Cd stress treatments to Zhe-Maidong (Ophiopogon japonicus). The exposure to Cd stress resulted in a notable elevation in the SOD activity in roots. Cu/ZnSODa and Cu/ZnSODb were the most critical SOD isozymes in response to Cd stress, as indicated by the detection results for SOD isozymes. A total of 22 OjSOD genes were identified and classified into three subgroups, including 10 OjCu/ZnSODs, 6 OjMnSODs, and 6 OjFeSODs, based on the analysis of conserved motif and phylogenetic tree. Cu/ZnSOD-15, Cu/ZnSOD-18, Cu/ZnSOD-20, and Cu/ZnSOD-22 were the main genes that control the increase in SOD activity under Cd stress, as revealed via quantitative PCR and transcriptome analysis. Additionally, under various heavy metal stress (Cu2+, Fe2+, Zn2+, Mn2+), Cu/ZnSOD-15, Cu/ZnSOD-18, and Cu/ZnSOD-22 gene expression were significantly upregulated, indicating that these three genes play a critical part in resisting heavy metal stress. The molecular docking experiments performed on the interaction between oxygen ion (O2•-) and OjSOD protein have revealed that the critical amino acid residues involved in the binding of Cu/ZnSOD-22 to the substrate were Pro135, Ile136, Ile140, and Arg144. Our findings provide a solid foundation for additional functional investigations on the OjSOD genes, as well as suggestions for improving genetic breeding and agricultural management strategies to increase Cd resistance in O. japonicus.

P090 Prevotella copri promotes colitis in mice by reducing expression of ATF4 and disturbing the gut microbiota

Abstract Background Colitis is a disease that carries a significant global disease burden, with a growing prevalence. Studies in humans and mice have explored the role of the intestinal microbiota that contribute to inflammatory bowel disease (IBD). Prevotella copri is a common human gut microbe that has been linked to inflammatory disorders. Methods C57BL/6 mice were divided into four groups: normal control group, P. copri group, DSS model group, DSS model+ P. copri group. Mice were gavaged with P. copri or PBS for 14 consecutive days, starting from the first day. Experimental colitis was induced by 2% DSS treatment from day 8. Mouse colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, immunohistochemistry, and transcriptome. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography-tandem mass spectrometry. The analysis of the microbiome was done through the 16S ribosomal DNA sequencing. Results In the present study, our experiment confirmed that P. copri was significantly associated with the exacerbation of DSS-induced colitis by reducing expression of ATF4 and altering the gut microbiota composition. Leucine concentration of serum and IEC were increased in P. copri-gavaged mice, which clearly suppresses ATF4 expression. The dysbiosis of the intestinal microbiota and impaired α-defensin production by Paneth cells are also the leading causes of why P. copri-gavaged mice are more susceptible to DSS-induced colitis. Conclusion Taken together, the results proposed that P. copri aggravated the severity of colitis in the mice with DSS-induced colitis. This was done by reducing the expression of ATF4 and disturbing the gut microbiota. Thus, targeting P. copri may be an effective strategy in the treatment of IBD.

Abstract A089: Quantitative MRI metrics and single cell transcriptome reveal effects of stroma-directed drug and chemotherapy in a GEM model of pancreatic cancer

Abstract Hypothesis: The dense stroma present in pancreatic ductal adenocarcinoma (PDA) harbors a unique tumor microenvironment (TME) that is immune suppressive and underlies the chemo-resistance of PDA. We have evaluated synthetic vitamin D combined with chemoimmunotherapy in a pilot clinical study (NCT03519308). To elucidate the underlying mechanisms of this treatment, we designed a matched study in a genetically engineered mouse model of PDA (KPC mice) to test the hypothesis that synthetic vitamin D enhances the effect of chemotherapy with or without immune checkpoint inhibition. To detect changes in TME, we have integrated diffusion weighted (DW) and dynamic contrast enhanced (DCE) MRI with immunohistochemistry (IHC) and single cell RNA sequencing (scRNAseq). Methods: KPC mice were randomly assigned to one of four groups: 1) Control (untreated); 2) Nab-paclitaxel + gemcitabine + cisplatin (Chemo); 3) Chemo + synthetic vitamin D (calcipotriol, Cal); 4) Chemo + Cal + PD-L1. The treatment lasted for 14 days followed by euthanasia and tumor collection for IHC and scRNAseq. Respiratory motion-robust DW- and DCE-MRI were performed on treatment days 0, 7 and 14. Results: Compared to consistent tumor growth (measured by MRI) in the Control group, both progression and regression were observed in groups 2) - 4), suggesting that the KPC model captured the varied treatment responses observed in the clinic. Capillary perfusion/permeability of the tumor measured by Ktrans (a DCE-MRI metric) was reduced at day 7 in the Chemo group (P<0.05), whereas Chemo+Cal reversed the reduction of Ktrans. Chemo-induced Ktrans reduction was corroborated with a significant reduction of microvascular density by IHC of CD31 compared to the Control. Addition of PD-L1 did not appear to enhance the tumor growth delay by Chemo+Cal. scRNAseq analysis revealed that while Chemo significantly delayed the tumor growth compared to the Control, it increased the portion of mesenchymal cells in the tumor and upregulated epithelial-to-mesenchymal transition (EMT) genes known to resist chemotherapy. In contrast, Chemo+Cal increased the fraction of epithelial cells and downregulated EMT genes compared to Chemo. Trasncriptome analysis of the tumor cells demonstrated that Chemo treatment increased the expression of Fibronectin 1 (FN1), a gene associated with tumor invasiveness, EMT, and metastasis, while lowering the expression of Trefoil Factor Family1 (Tff1), which is related to tumor-suppression and chemosensitivity. Chemo+Cal treatment reversed the expression of both FN1 and Tff1 gene compared to Chemo treatment. Conclusion: Single cell transcriptome analysis provided valuable insights into treatment-induced molecular subtype changes, which may predict future resistance and invasiveness. Our study revealed important mechanisms of calcipotriol that enhance the efficacy of chemotherapy by improving microvascular perfusion and permeability and by reducing chemo-resistance by enriching epithelial cells in PDA tumors. Citation Format: Hoon Choi, Mamta Gupta, Thomas Karasic, Miguel Joaquim, Emma Furth, Stephen Pickup, Cynthia Clendenin, Hee Kwon Song, Yong Fan, Jeffrey Duda, James Gee, Mark Rosen, Peter O’Dwyer, Rong Zhou. Quantitative MRI metrics and single cell transcriptome reveal effects of stroma-directed drug and chemotherapy in a GEM model of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A089.

Quantitative trait locus (QTL) mapping and transcriptome profiling identify QTLs and candidate genes associated with heat stress response during reproductive development in Camelina sativa

AbstractCamelina sativa (L.) Crantz is a low‐input oilseed crop that has great potential in providing sustainable feedstock for biofuels and bioproducts. Climate change is threatening production of camelina with rising global temperatures. Elucidating the genetic response to high temperatures is essential for successful breeding of heat‐tolerant camelina varieties. Here, we report a combinatorial approach to identifying candidate genes associated with heat stress by quantitative trait locus (QTL) mapping and comparative transcriptome profiling. A population of recombinant inbred lines (RILs) was grown in a controlled growth chamber under the high‐temperature regimes for 14 days beginning at the onset of the reproductive stage. Several traits related to seed production were evaluated at maturity. The QTL analysis identified several regions with co‐located traits on chromosomes 8, 10, and 12. Two RILs with contrasting phenotypic responses to heat stress were chosen for gene expression profiling via RNA sequencing. Multiple pathways and genes were found to be strongly affected by heat stress, and many genes expressed differently between the two RILs. Several genes identified within the QTL regions were considered strong candidates that may control heat tolerance during reproduction in camelina. These studies provide resources for future studies that may assist in improving the heat tolerance of camelina.

A universal protocol for high-quality DNA and RNA isolation from diverse plant species

Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (βME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15–100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis.

Ubiquitin-Mediated Degradation of MORF4L1 By FBXO11 Suppresses Cholesterol Biosynthesis in T Cell Acute Lymphoblastic Leukemia

The substrate receptor of SKP1-Cullin1-FBOX complex, FBXO11, is frequently mutated in both diffuse large B cell lymphoma and acute myeloid leukemia, and typically functions as a tumor suppressor by targeting substrates for ubiquitin-mediated degradation. Although FBXO11 is also mutated in human T-cell acute lymphoblastic leukemia (T-ALL) cell lines, the underlying mechanism in T-ALL development remains to be established. Here, we identified that both endogenous activation and exogenous overexpression of FBXO11 could suppress the growth of T-ALL cell in vitro and in vivo. While conversely, loss of FBXO11 accelerates the T-ALL cell proliferation. Transcriptomic and total quantitative proteomic analyses in FBXO11-depleted T-ALL cells reveal activation of the cholesterol biosynthesis pathway with accumulation of intracellular cholesterol levels. Importantly, we identified a chromatin regulator MORF4L1, mortality factor 4 like protein 1, as a functional substrate of FBXO11 in T-ALL cells. We first observed that FBXO11 exhibited frequent copy number alterations in T-ALL cell lines and in patient cohorts from public datasets. Moreover, by CRISPR SAM (Synergistic Activation Mediator) endogenous activation and lentiviral mediated gene overexpression, we validated that FBXO11 played a role in inhibiting tumor cell proliferation in T-ALL cells both in vitro and in vivo. Conversely, loss of FBXO11 accelerated T-ALL cell proliferation. Taken together, our results suggest that FBXO11 functions as a tumor suppressor in T-ALL. Mechanistically, we uncovered that the absence of FBXO11 could activate the cholesterol synthesis signaling pathway from both the RNA-seq and the TMT proteomic analysis in FBXO11-depleted MOLT4 cells compared to control cells. Concordantly, loss of FBO11 resulted in the accumulation of intracellular cholesterol level, suggesting that FBXO11 may suppress cholesterol biosynthesis in T-ALL cells. To understand how FBXO11 controls cholesterol biosynthesis, we further identified the potential substrates from the combined quantitative transcriptome and proteome analysis. MORF4L1, a chromatin modifier involved in histone acetylation, was identified as a key substrate of FBXO11 based on its upregulation at the protein level in FBXO11-depleted cells. Moreover, our data suggest MORF4L1 is a regulator of cholesterol biosynthesis in T-ALL. The inhibition of MORF4L1 suppressed the cellular growth of T-ALL cell lines, and led to the downregulation of the expression of cholesterol-related genes, such as gene HMGCR and HMGCS1. Of note, suppression of MORF4L1 and its cholesterol biosynthesis target genes such as HMGCR, HMGCS1 could rescue the accelerated growth effect after loss of FBXO11, indicating a functional role for MORF4L1 in T-ALL development. In summary, our data support a model wherein the degradation of MORF4L1 by FBXO11 suppresses leukemia cell growth by controlling cholesterol biosynthesis. The identification of this regulatory mechanism is required for T-ALL and provides valuable insights into the metabolic vulnerabilities of leukemia.