High-level Gene Expression Research Articles

Genome-Wide Analysis of Trehalose-6-Phosphate Phosphatase Gene Family and Their Expression Profiles in Response to Abiotic Stress in Groundnut.

Trehalose-6-phosphate phosphatase (TPP) is a pivotal enzyme in trehalose biosynthesis which plays an essential role in plant development and in the abiotic stress response. However, little is currently known about TPPs in groundnut. In the present study, a total of 16 AhTPP genes were identified, and can be divided into three phylogenetic subgroups. AhTPP members within the same subgroups generally displayed similar exon-intron structures and conserved motifs. Gene collinearity analysis revealed that segmental duplication was the primary factor driving the expansion of the AhTPP family. An analysis of the upstream promoter region of AhTPPs revealed eight hormone- and four stress-related responsive cis-elements. Transcriptomic analysis indicated high expression levels of AhTPP genes in roots or flowers, while RT-qPCR analysis showed upregulation of the six tested genes under different abiotic stresses, suggesting that AhTPPs play roles in growth, development, and response to various abiotic stresses. Subcellular localization analysis showed that AhTPP1A and AhTPP5A were likely located in both the cytoplasm and the nucleus. To further confirm their functions, the genes AhTPP1A and AhTPP5A were individually integrated into yeast expression vectors. Subsequent experiments demonstrated that yeast cells overexpressing these genes displayed increased tolerance to osmotic and salt stress compared to the control group. This study will not only lay the foundation for further study of AhTPP gene functions, but will also provide valuable gene resources for improving abiotic stress tolerance in groundnut and other crops.

SOX11/PRDX2 axis modulates redox homeostasis and chemoresistance in aggressive mantle cell lymphoma

Mantle cell lymphoma (MCL) is an incurable B-cell neoplasm characterized by an aggressive behavior, short responses to conventional therapies and SOX11 overexpression, which is associated with aggressive disease features and inferior clinical outcome of patients. Oxidative stress is known to induce tumorigenesis and tumor progression, whereas high expression levels of antioxidant genes have been associated with chemoresistance in different cancers. However, the role of oxidative stress in MCL pathogenesis and the involvement of SOX11 regulating redox homeostasis in MCL cells are largely unknown. Here, by integrating gene set enrichment analysis of two independent series of MCL, we observed that SOX11+ MCL had higher reactive oxygen species (ROS) levels compared to SOX11− MCL primary tumors and increased expression of Peredoxine2 (PRDX2), which upregulation significantly correlated with SOX11 overexpression, higher ROS production and worse overall survival of patients. SOX11 knockout (SOX11KO) significantly reduced PRDX2 expression, and SOX11KO and PRDX2 knockdown (PRDX2KD) had increased ROS levels and ROS-mediated tumor cell death upon treatment with drugs, compared to control MCL cell lines. Our results suggest an aberrant redox homeostasis associated with chemoresistance in aggressive MCL through SOX11-mediated PRDX2 upregulation, highlighting PRDX2 as promising target for new therapeutic strategies to overcome chemoresistance in aggressive MCLs.

Involvement of BCR::ABL1 in laminin adhesion of Philadelphia chromosome-positive acute lymphoblastic leukemia through upregulation of integrin α6.

Adhesion of cancer cells to extracellular matrix laminin through the integrin superfamily reportedly induces drug resistance. Heterodimers of integrin α6 (CD49f) with integrin β1 (CD29) or β4 (CD104) are major functional receptors for laminin. Higher CD49f expression is reportedly associated with a poorer response to induction therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Moreover, a xenograft mouse model transplanted with primary BCP-ALL cells revealed that neutralized antibody against CD49f improved survival after chemotherapy. Considering the poor outcomes in Philadelphia chromosome (Ph)-positive ALL treated with conventional chemotherapy without tyrosine kinase inhibitors, we sought to investigate an involvement of the laminin adhesion. Ph-positive ALL cell lines expressed the highest levels of CD49f among the BCP-ALL cell lines with representative translocations, while CD29 and CD104 were ubiquitously expressed in BCP-ALL cell lines. The association of Ph-positive ALL with high levels of CD49f gene expression was also confirmed in two databases of childhood ALL cohorts. Ph-positive ALL cell lines attached to laminin and their laminin-binding properties were disrupted by blocking antibodies against CD49f and CD29 but not CD104. The cell surface expression of CD49f, but not CD29 and CD104, was downregulated by imatinib treatment in Ph-positive ALL cell lines, but not in their T315I-acquired sublines. Consistently, the laminin-binding properties were disrupted by the imatinib pre-treatment in the Ph-positive ALL cell line, but not in its T315I-acquired subline. BCR::ABL1 plays an essential role in the laminin adhesion of Ph-positive ALL cells through upregulation of CD49f.

Vascularized fascial flap for reconstruction of combined oral mucosa-mandibular defects: The multi-modal biological assessment

ObjectiveThe reconstruction of composite defects in the oral and maxillofacial region using vascularized fascial flaps, such as the fibular, iliac, and temporal fascial flaps, has gained increasing attention among surgeons. However, there remains uncertainty regarding the suitability of fascial flaps as transplants, as well as their healing processes and outcomes, due to their non-mucosal nature. This study aims to comprehensively assess the biological aspects of vascularized fascial flaps at clinical, histological, and genetic levels, with the goal of providing essential biological references for their clinical application. Study DesignThis study enrolled three patients who underwent reconstruction of combined oral mucosa-mandibular defects using fibular vascularized fascial flaps between 2020 and 2023. Data regarding changes in the appearance of the fascial flaps, bulk-RNA sequencing, and histological slices of initial fascia, initial gingiva, and transformed fascia were collected and analyzed. ResultsWithin three months, the fascial flaps exhibited rapid epithelial coverage and displayed distinct characteristics resembling mucosa. High-throughput RNA sequencing analyses and histological slices revealed that the transformed fascia exhibited tissue structures similar to mucosa and demonstrated unique advantages in promoting blood vessel formation and reducing scarring through the high-level expression of relevant genes. ConclusionThese findings emphasize the potential and feasibility of utilizing vascularized fascial flaps for oral mucosa reconstruction, establishing their unique advantage as transplant materials, and providing significant biological information and references for their selection and clinical application.

APLF facilitates interstrand DNA crosslink repair and replication fork protection to confer cisplatin resistance.

Replication stress converts the stalled forks into reversed forks, which is an important protection mechanism to prevent fork degradation and collapse into poisonous DNA double-strand breaks (DSBs). Paradoxically, the mechanism also acts in cancer cells to contribute to chemoresistance against various DNA-damaging agents. PARP1 binds to and is activated by stalled forks to facilitate fork reversal. Aprataxin and polynucleotide kinase/phosphatase-like factor (APLF) binds to PARP1 through the poly(ADP-ribose) zinc finger (PBZ) domain and is known to be involved in non-homologous end joining (NHEJ). Here, we identify a novel function of APLF involved in interstrand DNA crosslink (ICL) repair and fork protection. We demonstrate that PARP1 activity facilitates the APLF recruitment to stalled forks, enabling the FANCD2 recruitment to stalled forks. The depletion of APLF sensitizes cells to cisplatin, impairs ICL repair, reduces the FANCD2 recruitment to stalled forks, and results in nascent DNA degradation by MRE11 nucleases. Additionally, cisplatin-resistant cancer cells show high levels of APLF and homologous recombination-related gene expression. The depletion of APLF sensitizes cells to cisplatin and results in fork instability. Our results reveal the novel function of APLF to facilitate ICL repair and fork protection, thereby contributing to cisplatin-resistant phenotypes of cancer cells.

Abstract 7057: Validation of OXPHOS gene amplification as a driver of treatment resistance in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer deaths in the U.S. and worldwide. Up to 90% of all lung cancers are non-small cell lung cancer (NSCLC). NSCLC is a complex and highly aggressive disease typical for poor prognosis and high rates of treatment resistance. Clinical management of NSCLC varies according to the stage. High-dose stereotactic body radiation therapy (SBRT) is the standard of care for localized non-resectable NSCLC. However, tumor hypoxia is a significant barrier to effective radiation therapy. In the last decade, first line NSCLC treatment has been substantially reinforced with the introduction of immunotherapy targeting immune checkpoints such as programmed cell death 1 (PD-1), yet long-term disease control occurs in less than 25% of NSCLC patients. Therefore, understanding the mechanisms of treatment resistance is essential to address the dire need of introducing novel synergistic therapies to enhance anti-cancer treatment response in refractory NSCLC tumors. The basis of tumor hypoxia has traditionally been attributed to the oxygen supply deficit as malformed tumor vasculature fails to meet the high demand of the rapidly proliferating tumor mass. However, our preliminary analysis of NSCLC patient datasets in the Cancer Genome Atlas (TCGA) PanCancer dataset revealed a significant correlation between high-level expression of nuclear genes encoding mitochondrial subunits essential for oxidative phosphorylation (OXPHOS), and high-level expression of hypoxia-regulated genes (Buffa hypoxia score). Furthermore, we have observed a direct correlation between copy number amplification (CNA) of several OXPHOS genes and hypoxia levels in patient samples. Because mitochondrial function consumes up to 90% of available cellular oxygen, its activity may be indirectly regulating oxygen availability in the tumor microenvironment by rapidly consuming oxygen upon its delivery to the tumor. Moreover, we have identified that almost 40% of NSCLC patients harbor CNA of these essential OXPHOS genes. To provide mechanistic insight into these clinical observations, we have generated mouse syngeneic NSCLC cell lines with modified mRNA expression levels of OXPHOS genes (representing deletion, diploid and amplified number of copies) identified in our screen and observed a direct effect on the rates of mitochondrial oxygen consumption in these cells. Our study investigates the previously unknown role of OXPHOS gene copy number in driving mitochondrial oxygen demand and thus contributing to tumor hypoxia and treatment resistance in NSCLC. Citation Format: Martin Benej, Katarina Benejova, Rebecca Hoyd, Caroline Wheeler, Daniel Spakowicz, Nicholas C. Denko. Validation of OXPHOS gene amplification as a driver of treatment resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7057.

Abstract 4253: High expression of specific gene profiles in organoids from African American pancreatic cancer tumors

Abstract Pancreatic cancer remains one of the leading causes of cancer-related deaths in the United States with limited therapies for advanced stages of this disease. African Americans have the highest morbidity and mortality rates of pancreatic cancer compared to other racial groups. Many factors are known to contribute to this disparity such as, socioeconomic and lifestyle factors. One of the major factors contributing to this disparity is the lack of African Americans with pancreatic cancer in clinical trials or genomic databases. In order to gain insight into the biology or genetic differences that may exist in signals driving the aggressiveness of this cancer in this population more studies are needed. Innovative in vitro models are needed to investigate possible differences in molecular changes that may exist in African Americans. Studies have shown that organoids developed from metastatic and primary tumors can give sights into genetic and epigenetic changes detected in several type of cancers. Using a Human Pancreatic Adenocarcinoma TagMan Array 96 (Applied Biosystems), which contained 76 genes known to be expressed in pancreatic cancer, pancreatic organoids from African American metastatic tumors revealed high expression levels of specific genes. Usually K-ras is highly expressed in pancreatic tumors but it was not significantly expressed in the organoids from African Americans metastatic tumors. However, preliminary results from organoids, compared to pancreatic cancer cell line spheroids, showed significantly increased gene expression of HSP90AA1 (13-fold), MMP1(36-fold), BRACA2 (20-fold), MDM2 (19-fold), RB1 (14-fold), Rel (12-fold), STAT1 (15-fold), STAT5B (5-fold), and TGFBR1 (1- fold). High expression of HSP90AA1 is related to poor prognosis, constitutively expression of STAT5B confers gemcitabine chemoresistance and promote invasiveness, and mutations in BRCA2 can increase pancreatic cancer risk. Further studies are being done to validate these findings in African American pancreatic tumors and to ascertain their relevance to the metastatic progression phenotype. Citation Format: Beverly D. Lyn-Cook, Fatemeh Nouri Emamzaden, Beverly Word, George Hammons. High expression of specific gene profiles in organoids from African American pancreatic cancer tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4253.

Abstract 2706: Identification and characterization of cancer-associated polymorphonuclear cell subsets in mice

Abstract Background: Neutrophils are the main type of polymorphonuclear cells (PMN) and comprise most immune cells in the body. PMN cells are upregulated in cancer and accumulate in the tumors. Tumor-associated PMNs comprise a heterogeneous population consisting of immunologically active mature neutrophils and immature polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). The phenotype of these cells evolves with disease progression and is implicated in modulating immune response. However, there is yet to be a consensus to define PMN populations in tumors and blood. Methods: Here, we used single-cell RNA-seq, bulk RNA sequencing, flow cytometry, and immunohistochemistry (IHC) analysis to characterize cancer-associated PMN-MDSCs in tumor-bearing mice. Results: Single-cell RNA-seq data from lung tumors and healthy individuals revealed five metaclusters (MC): MCA, MCB, MCC, MCD, and MCE. MCA and MCB made up 34.4 and 65.1% of PMNs in healthy tissues, respectively. MCA, MCB, MCC, MCD, and MCE comprised 3.8%, 23.5% 3.3% 14.6%, and 54.7% of PMNs in tumors, respectively. Genes enriched in MCA and MCE were associated with MDSCs and myeloid progenitor cells. MCB and MCC were associated with inflammatory responses mediated by TNF-alpha signaling and neutrophil degranulation. MCD was associated with a CD33 myeloid cell phenotype and had high expression levels of CD274, Pdcd1lg2, Havcr2, and Tigit genes. Cells from MCE were associated with protein translation signatures and had elevated levels of Nt5e. Gene expression signatures from single-cell RNA-seq data were compared with bulk transcriptomic data from healthy mouse prostates and Pten-null prostate tumors characterized by high PMN cell infiltration. Gene set enrichment analysis showed enrichments after androgen deprivation therapy and Cluster MCE was associated with castration-resistant tumors. Flow cytometric and IHC analysis confirmed the enrichment of PMN cells expressing elevated levels of proteins identified from gene expression analysis including CD73, PD-L1, and PD-L2. Conclusion: This study offers further insights into the distinct subsets of PMN-MDSCs present in tumors. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Takashi Kikuchi, Yasunori Mori, Takafumi Minami, Kazuhiro Yoshimura, Kazutoshi Fujita, Kazuto Nishio, Hirotsugu Uemura. Identification and characterization of cancer-associated polymorphonuclear cell subsets in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2706.

Abstract 3668: Plasma cell-free DNA profiling for deciphering cellular origins and immune competence in cancer patients

Abstract Plasma cell-free DNA (cfDNA) originates from apoptotic cells in the blood, providing a real-time insight into the biology of various cell types. The fragment coverage pattern of cfDNA across the genome is known to reflect the distinct chromatin status of originating cells. While extensive research has focused on fragments derived from cancer, known as circulating tumor DNA (ctDNA), for inference about cancer biology, cfDNA fragments from healthy cells have received little attention. Our study explores the potential of cfDNA to decipher the relative abundance of distinct blood cell types, particularly immune system cell types. We collected a cohort of 273 bladder (BC) and colorectal cancer (CRC) patients and 45 healthy controls for plasma whole genome sequencing and assessed the fragment coverage profile at transcription start sites (TSS) of protein coding genes. A significant negative correlation was observed between TSS coverage and whole blood gene expression (Spearman’s rho = -0.65). Furthermore, TSS coverage revealed a bimodal distribution, characterized by modes corresponding to high and low gene expression levels in blood cells. This may suggest the cfDNA coverage profile as a binary expression status classifier to distinguish between expressed and unexpressed genes in blood cells. We further evaluated TSS with differential coverage between age groups of healthy individuals and relapse status of cancer patients. Notably, out of the TSS that significantly differentiated middle-aged (aged 40-65) from older individuals (aged 65+), 31 were also able to distinguish relapse from non-relapse patients. Among these TSS were CD36, SLAMF7, TABBP and PDCD1, known to be involved in immune related pathways. Finally, we evaluated coverage at cell-type specific TSS and found dendritic cells (P-value: CRC = 8e-06, BC = 5.1e-3), monocytes (CRC = 1.6e-04, BC = 2.5e-3), and NK cells (CRC = 8.5e-04, BC = 0.026) to exhibit a significant increase in coverage in the cancer cohorts compared to healthy controls. Conversely, T-cells showed a decrease in coverage (CRC = 0.041, BC = 0.062). In conclusion, cfDNA coverage profile may identify TSS with differential coverage between patient groups and characterize a shift in blood cell type composition as a response to cancer. Future work may show if fragment coverage quantifies the relative contribution of cfDNA from distinct immune cell types. We anticipate our analysis to illuminate the utility of plasma cfDNA as a biomarker for cancer patient’s immune competency, enabling improved patient stratification for treatment, leveraging their distinct immune capabilities. Citation Format: Laura Andersen, Amanda Frydendahl, Tenna Vesterman Henriksen, Christina Demuth, Mads Heilskov Rasmussen, Iver Nordentoft, Karin Birkenkamp-Demtröder, Jakob Skou Pedersen, Søren Besenbacher, Lars Dyrskjøt, Claus L. Andersen, Nicolai J. Birkbak. Plasma cell-free DNA profiling for deciphering cellular origins and immune competence in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3668.

The impact of ovarian stimulation on the human endometrial microenvironment.

How does ovarian stimulation (OS), which is used to mature multiple oocytes for ART procedures, impact the principal cellular compartments and transcriptome of the human endometrium in the periovulatory and mid-secretory phases? During the mid-secretory window of implantation, OS alters the abundance of endometrial immune cells, whereas during the periovulatory period, OS substantially changes the endometrial transcriptome and impacts both endometrial glandular and immune cells. Pregnancies conceived in an OS cycle are at risk of complications reflective of abnormal placentation and placental function. OS can alter endometrial gene expression and immune cell populations. How OS impacts the glandular, stromal, immune, and vascular compartments of the endometrium, in the periovulatory period as compared to the window of implantation, is unknown. This prospective cohort study carried out between 2020 and 2022 included 25 subjects undergoing OS and 25 subjects in natural menstrual cycles. Endometrial biopsies were performed in the proliferative, periovulatory, and mid-secretory phases. Blood samples were processed to determine serum estradiol and progesterone levels. Both the endometrial transcriptome and the principal cellular compartments of the endometrium, including glands, stroma, immune, and vasculature, were evaluated by examining endometrial dating, differential gene expression, protein expression, cell populations, and the three-dimensional structure in endometrial tissue. Mann-Whitney U tests, unpaired t-tests or one-way ANOVA and pairwise multiple comparison tests were used to statistically evaluate differences. In the periovulatory period, OS induced high levels of differential gene expression, glandular-stromal dyssynchrony, and an increase in both glandular epithelial volume and the frequency of endometrial monocytes/macrophages. In the window of implantation during the mid-secretory phase, OS induced changes in endometrial immune cells, with a greater frequency of B cells and a lower frequency of CD4 effector T cells. The data underlying this article have been uploaded to the Genome Expression Omnibus/National Center for Biotechnology Information with accession number GSE220044. A limited number of subjects were included in this study, although the subjects within each group, natural cycle or OS, were homogenous in their clinical characteristics. The number of subjects utilized was sufficient to identify significant differences; however, with a larger number of subjects and additional power, we may detect additional differences. Another limitation of the study is that proliferative phase biopsies were collected in natural cycles, but not in OS cycles. Given that the OS cycle subjects did not have known endometrial factor infertility, and the comparisons involved subjects who had a similar and robust response to stimulation, the findings are generalizable to women with a normal response to OS. OS substantially altered the periovulatory phase endometrium, with fewer transcriptomic and cell type-specific changes in the mid-secretory phase. Our findings show that after OS, the endometrial microenvironment in the window of implantation possesses many more similarities to that of a natural cycle than does the periovulatory endometrium. Further investigation of the immune compartment and the functional significance of this cellular compartment under OS conditions is warranted. Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (R01AI148695 to A.M.B. and N.C.D.), Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD109152 to R.A.), and the March of Dimes (5-FY20-209 to R.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or March of Dimes. All authors declare no conflict of interest.

Evaluation of Biological Plant Protection Products for Their Ability to Induce Olive Innate Immune Mechanisms and Control Colletotrichum acutatum, the Causal Agent of Olive Anthracnose.

Olive anthracnose is the most important fungal disease of the olive fruit worldwide, with the fungus Colletotrichum acutatum as the main cause of the disease in Greece. A total of 11 commercial biological plant protection products (bioPPPs) (Amylo-X®, Botector®, FytoSave®, LBG 01F34®, Mevalone®, Polyversum®, Remedier®, Serenade® ASO, Sonata®, Trianum-P®, Vacciplant®), with various modes of action against the fungus C. acutatum, were evaluated by bioassays using detached fruits of two important olive Greek varieties, cv. Koroneiki and cv. Kalamon. Subsequently, the most effective bioPPPs were evaluated for their ability to induce plant defense mechanisms, by determining the expression levels of ten Olea europaea defense genes (Pal, CuaO, Aldh1, Bglu, Mpol, Lox, Phely, CHI-2, PR-10, PR-5). Remedier®, Trianum-P®, Serenade® ASO, Sonata®, and Mevalone® were the most effective in reducing disease severity, and/or inhibiting the conidia production by the fungus at high rates. Post bioPPPs application, high expression levels of several olive plant defense genes were observed. This study provides insights into commercial bioPPPs' effectiveness in controlling olive anthracnose, as well as biocontrol-agents-mediated modulation of olive defense mechanisms.

Expression and clinical role of PRDX6 in lung adenocarcinoma.

To explore the levels of expression and clinical role of peroxiredoxin 6 (PRDX6) in lung adenocarcinoma. This retrospective study used a series of bioinformatics methods to detect the levels of expression of and mutations in the PRDX6 gene in a range of cancers and lung adenocarcinoma. Immunohistochemistry was used to verify the levels of expression of PRDX6 protein in samples of lung adenocarcinoma compared with normal adjacent tissue. The effect of PRDX6 gene knockdown on the in vitro proliferation of a lung adenocarcinoma cell line was measured. Bioinformatics methods were used to determine the diagnostic value and impact on survival of the PRDX6 gene in patients with lung adenocarcinoma. The results showed that the PRDX6 gene was highly expressed in lung adenocarcinoma and there were five mutations at different sites on the gene. PRDX6 promoted the proliferation of the lung adenocarcinoma cell line. The survival duration of lung adenocarcinoma patients with high levels of PRDX6 gene expression was significantly shorter than that of patients with low PRDX6 gene expression. PRDX6 is highly expressed in lung adenocarcinoma and higher levels of expression of the PRDX6 gene were associated with a poorer prognosis.

Identification of Highly Repetitive Enhancers with Long-range Regulation Potential in Barley via STARR-seq.

Enhancers are DNA sequences that can strengthen transcription initiation. However, the global identification of plant enhancers is complicated due to uncertainty in the distance and orientation of enhancers, especially in species with large genomes. In this study, we performed self-transcribing active regulatory region sequencing (STARR-seq) for the first time to identify enhancers across the barley genome. A total of 7323 enhancers were successfully identified, and among 45 randomly selected enhancers, over 75% were effective as validated by a dual-luciferase reporter assay system in the lower epidermis of tobacco leaves. Interestingly, up to 53.5% of the barley enhancers were repetitive sequences, especially transposable elements (TEs), thus reinforcing the vital role of repetitive enhancers in gene expression. Both the common active mark H3K4me3 and repressive mark H3K27me3 were abundant among the barley STARR-seq enhancers. In addition, the functional range of barley STARR-seq enhancers seemed much broader than that of rice or maize and extended to ±100 kb of the gene body, and this finding was consistent with the high expression levels of genes in the genome. This study specifically depicts the unique features of barley enhancers and provides available barley enhancers for further utilization.

Functional genetic characterization of stress tolerance and biofilm formation in Nakaseomyces (Candida) glabrata via a novel CRISPR activation system.

The overexpression of genes frequently arises in Nakaseomyces (formerly Candida) glabrata via gain-of-function mutations, gene duplication, or aneuploidies, with important consequences on pathogenesis traits and antifungal drug resistance. This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system in N. glabrata for targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in N. glabrata, and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly used N. glabrata genetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications.IMPORTANCENakaseomyces (formerly Candida) glabrata is an important fungal pathogen that is now the second leading cause of candidiasis infections. A common strategy that this pathogen employs to resist antifungal treatment is through the upregulation of gene expression, but we have limited tools available to study this phenomenon. Here, we develop, optimize, and apply the use of CRISPRa as a means to overexpress genes in N. glabrata. We demonstrate the utility of this system to overexpress key genes involved in antifungal susceptibility, stress tolerance, and biofilm growth. This tool will be an important contribution to our ability to study the biology of this important fungal pathogen.

In vitro culture and tissue-derived specific expression of melanocytes from ovary of adult Silky Fowl

BackgroundThe presence of a significant number of melanocytes in the ovary and follicular membrane of Silky Fowl suggests their potential involvement in follicle development. Currently, there is a lack of available data regarding to the isolation of primary melanocytes from adult chickens. To date, primary melanocytes and their in vitro culture system have been successfully conducted in the peritoneum of chicken embryos. Herein, melanocytes from silky fowl ovaries were isolated and identified. MethodsSilky Fowl ovaries were obtained by mixed digestion of 0.1% collagenase II and 0.25% trypsin-EDTA. Melanocytes could be further purified and cultured up to five generations in vitro. RNA-seq analysis was used to investigate whether there were differences in the functional status of melanocytes in different tissues and developmental stages. ResultsConsequently, differential gene expressions between peritoneal and ovarian melanocytes were compared. These findings demonstrated that the Silky Fowl ovary had higher expression levels of genes involved in the production of sexual hormones and melanogenesis, while those of melanocytes derived from the peritoneum were involved in amino acid metabolism, lipid synthesis, and overall metabolic rates. This suggests that the role of melanocytes is dependent on the origin tissue and developmental stage, and is tightly connected to the function of the specific source tissue from which the cells were derived. ConclusionsThis study provides a method for isolating adult melanocytes and serve as a basis for further investigate the effect of SFOM on germ cells.

Aagab is required for zebrafish larval development by regulating neural activity

Clathrin-mediated endocytosis has been implicated in various physiological processes, including nutrient uptake, signal transduction, synaptic vesicle recycling, maintenance of cell polarity, and antigen presentation. Despite prior knowledge of its importance as a key regulator in promoting clathrin-mediated endocytosis, the physiological function of α- and γ-adaptin binding protein (aagab) remains elusive. In this study, we investigate the biological function of aagab during zebrafish development. We establish a loss-of-function mutant of aagab in zebrafish, revealing impaired swimming and early larval mortality. Given the high expression level of aagab in the brain, we probe into its physiological role in the nervous system. aagab mutants display subdued calcium responses and local field potential in the optic tectal neurons, aligning with reduced neurotransmitter release (e.g., norepinephrine) in the tectal neuropil of aagab mutants. Overexpressing aagab mRNA or nervous stimulant treatment in mutants restores neurotransmitter release, calcium responses, swimming ability, and survival. Furthermore, our observations show delayed release of FM 1–43 in AAGAB knockdown differentiated neuroblastoma cells, pointing towards a probable link to defective clathrin-mediated synaptic vesicle recycling. In conclusion, our study underscores the significance of Aagab in neurobiology and suggests its potential impacts on neurological disorders.

Kaposi's sarcoma herpesvirus latency-associated nuclear antigen broadly regulates viral gene expression and is essential for lytic infection.

Kaposi's sarcoma herpesvirus (KSHV) is a leading cause of malignancy in AIDS and current therapies are limited. Like all herpesviruses, KSHV infection can be latent or lytic. KSHV latency-associated nuclear antigen (LANA) is essential for viral genome persistence during latent infection. LANA also maintains latency by antagonizing expression and function of the KSHV lytic switch protein, RTA. Here, we find LANA null KSHV is not capable of lytic replication, indicating a requirement for LANA. While LANA promoted both lytic and latent gene expression in cells partially permissive for lytic infection, it repressed expression in non-permissive cells. Importantly, forced RTA expression in non-permissive cells led to induction of lytic infection and LANA switched to promote, rather than repress, most lytic viral gene expression. When basal viral gene expression levels were high, LANA promoted expression, but repressed expression at low basal levels unless RTA expression was forcibly induced. LANA's effects were broad, but virus gene specific, extending to an engineered, recombinant viral GFP under control of host EF1α promoter, but not to host EF1α. Together, these results demonstrate that, in addition to its essential role in genome maintenance, LANA broadly regulates viral gene expression, and is required for high levels of lytic gene expression during lytic infection. Strategies that target LANA are expected to abolish KSHV infection.

Abstract A028: Multiomic modelling of pancreatic IPMN stroma reveals distinct tertiary lymphoid structure distribution: Mapping the transcriptomic landscape via regional bulk, single-cell and subcellular approaches

Abstract Background Intraductal Papillary Mucinous Neoplasms (IPMN) remain the largest subtype of cystic pancreatic cancer precursors. Cancer risk has been observed to vary between histological grades and histological subtypes. Spatial characterisation has revealed variation in immune cell distribution disease subtypes. Methods A cohort of 14 surgically resected IPMN tumors across a range of histological grades (Low-grade LG, high-grade HG and invasive IPMN cancers IPMN-PDAC) and subtypes (gastric, intestinal, pancreaticobiliary) underwent multimodal spatial interrogation. After expert histopathological annotation; tissue sections underwent regional whole transcriptome analysis with TempO-seq and 10x Visium. Extensive region analysis with NanoString GeoMx employing segmentation using staining for epithelium (PanCK) fibroblast stroma (aSMA) was performed. A tissue microarray was then constructed, and representative cores underwent single-cell and subcellular spatial transcriptomic analysis with NanoString CosMx. Raw count data and digital images were exported and analysed using Seurat, Giotto, SPATA2 and custom R pipelines. Region deconvolution, Harmony integration, Leiden Clustering, Gene Set Enrichment Analysis, Trajectory Mapping, and Moran’s I Analysis were performed. Results TempO-seq revealed up-regulation of canonical oncogene expression in all IPMN-PDAC cases as compared to LG and HG IPMN. There were no statistically significant differences in expression comparing LG and HG IPMN lesions. Visium differential gene expression and clustering models identified cancer epithelium, stroma and lymphatic components that correlated with histopathological annotations. Immune cell rich spots were identified via gene ontology of top 50 marker genes. The spatial trajectory of B-cell expression signatures in IPMN-PDAC showed greater concentration and less variability than HG tumors, Moran’s I -0.34 vs 0.12 (P = 0.021). B-cells in HG IPMN were showed greater concentration and less variability than LG IPMN, Moran’s I -0.15 vs 0.02 (P=0.05). Much of this concentration was in identified lymphoid aggregates or tertiary lymphoid structures. The spatial trajectory of T-cell expression. The spatial trajectory of macrophage expression showed greater concentration and less variability than in LG and HG tumors. GeoMx analysis of stromal regions identified up regulation of B cell signatures in IPMN PDAC and HG IPMN when compared to LG IPMN. Markers associated with TLS formation were found to be significantly upregulated including CXCL13, CXCR5 and LAMP3. Pancreaticobiliary subtypes were found to have the least concentrated B-cell distribution across subtypes. Conclusions Immune cell composition was found to vary across histological grades and subtypes of IPMN. A paradoxical relationship was observed between T-cell infiltrates and B-cell and macrophage populations. The greater proportion of tertiary lymphoid structures, and high levels of TLS specific gene expression, may indicate a propensity for b-cell activation in IPMN tumors which progress to malignancy. Citation Format: Andrew J. Cameron, Assya Legrini, Colin S. Wood, Craig Nourse, Yoana Doncheva, Claire Kennedy Dietrich, Colin Nixon, Jennifer Hay, Fraser Duthie, Pawel Herzyk, Jennifer Morton, Nigel B. Jamieson. Multiomic modelling of pancreatic IPMN stroma reveals distinct tertiary lymphoid structure distribution: Mapping the transcriptomic landscape via regional bulk, single-cell and subcellular approaches [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 A028.

Quantifying Dynamic Phenotypic Heterogeneity in Resistant Escherichia coli under Translation-Inhibiting Antibiotics.

Understanding the phenotypic heterogeneity of antibiotic-resistant bacteria following treatment and the transitions between different phenotypes is crucial for developing effective infection control strategies. The study expands upon previous work by explicating chloramphenicol-induced phenotypic heterogeneities in growth rate, gene expression, and morphology of resistant Escherichia coli using time-lapse microscopy. Correlating the bacterial growth rate and cspC expression, four interchangeable phenotypic subpopulations across varying antibiotic concentrations are identified, surpassing the previously described growth rate bistability. Notably, bacterial cells exhibiting either fast or slow growth rates can concurrently harbor subpopulations characterized by high and low gene expression levels, respectively. To elucidate the mechanisms behind this enhanced heterogeneity, a concise gene expression network model is proposed and the biological significance of the four phenotypes is further explored. Additionally, by employing Hidden Markov Model fitting and integrating the non-equilibrium landscape and flux theory, the real-time data encompassing diverse bacterial traits are analyzed. This approach reveals dynamic changes and switching kinetics in different cell fates, facilitating the quantification of observable behaviors and the non-equilibrium dynamics and thermodynamics at play. The results highlight the multi-dimensional heterogeneous behaviors of antibiotic-resistant bacteria under antibiotic stress, providing new insights into the compromised antibiotic efficacy, microbial response, and associated evolution processes.

BRCA1 mutation promotes sprouting angiogenesis in inflammatory cancer-associated fibroblast of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with inferior outcomes owing to its low treatment response and high invasiveness. Based on abundant cancer-associated fibroblasts (CAFs) and frequent mutation of breast cancer-associated 1 (BRCA1) in TNBC, the characteristics of CAFs in TNBC patients with BRCA1 mutation compared to wild-type were investigated using single-cell analysis. Intriguingly, we observed that characteristics of inflammatory CAFs (iCAFs) were enriched in patients with BRCA1 mutation compared to the wild-type. iCAFs in patients with BRCA1 mutation exhibited outgoing signals to endothelial cells (ECs) clusters, including chemokine (C-X-C motif) ligand (CXCL) and vascular endothelial growth factor (VEGF). During CXCL signaling, the atypical chemokine receptor 1 (ACKR1) mainly interacts with CXCL family members in tumor endothelial cells (TECs). ACKR1-high TECs also showed high expression levels of angiogenesis-related genes, such as ANGPT2, MMP1, and SELE, which might lead to EC migration. Furthermore, iCAFs showed VEGF signals for FLT1 and KDR in TECs, which showed high co-expression with tip cell marker genes, including ZEB1 and MAFF, involved in sprouting angiogenesis. Moreover, BRCA1 mutation patients with relatively abundant iCAFs and tip cell gene expression exhibited a limited response to neoadjuvant chemotherapy, including cisplatin and bevacizumab. Importantly, our study observed the intricate link between iCAFs-mediated angiogenesis and chemoresistance in TNBC with BRCA1 mutation.