Fold Enrichment Research Articles

Abstract P2-25-01: TRPS1 Regulates angiogenesis via JAG1 and TYMP in Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC), the most difficult subtype to treat, is defined as estrogen receptor (ER), HER2, and progesterone receptor (PR) negative and comprises roughly 15-20% of all breast cancers. Patients with a TNBC diagnosis have worse outcomes and poorer survival rates when compared to women with other breast cancers, despite adjuvant chemotherapy. One of the factors contributing to its metastatic ability is the angiogenic process it undergoes. Clinical trials with anti-angiogenic therapeutics showed to be inadequate, with moderate response rates and insignificant survival gains for patients. TRPS1, a GATA and GATA-like transcription factor, has been proven to play a role in the epithelial to mesenchymal transition (EMT) which ultimately promotes tumor growth and metastasis. It has previously been shown that TRPS1 regulates angiogenesis via the expression of VEGF proteins in breast cancer. Our purpose in this study was to further investigate the functional role of TRPS1-regulated genes involved in TNBC angiogenic pathways. Methods: Sleeping-Beauty (SB) transposons help detect cancer progression genes which participate in tumorigenesis and metastasis, not easily identified with current sequencing technology. Using the SB transposon methodology, several candidate trunk drivers were elucidated using Pten mutant mice. Validation studies further showed TRPS1 as one of the eight TNBC tumor suppressor genes discovered by this technology. Then, ChIP-seq array studies were performed which showed that TRPS1 regulates expression of genes involved in the angiogenesis pathway. To corroborate the functional role of TRPS1 in angiogenesis, tube formation and sprouting assays were performed using overexpression and inactivation of TRPS1 in MDA-MD-231 and HCC70 cells, respectively. Furthermore, we performed a ChIP qPCR human angiogenesis array to determine TRPS1-regulated angiogenic genes. A luciferase reporter assay was performed to determine whether TRPS1 is a direct transcriptional regulator of these genes. Results: Inactivation of TRPS1 expression allowed tube formation and cell branching in the sprouting assays. Tumor xenografts overexpressing TRPS1 were analyzed by immunohistochemistry, showed a significant reduction of angiogenic vasculature when stained with CD31; and a substantial increase in blood vessels when TRPS1 was shRNA inactivated. Furthermore, human ChIP qPCR angiogenesis array identified 10 top candidate genes potentially regulated by TRPS1 transcription factor. Of these candidates, JAG1 and TYMP showed to have a higher fold enrichment compared to other angiogenic related genes. Finally, we validated its direct functional binding by using luciferase reporter assay, such demonstrated that TRPS1 is a direct transcriptional regulator of JAG1 and TYMP. Discussion: It has been previously established that the NOTCH ligand, JAG1 and the thymidine phosphorylase enzyme, TYMP are powerful genes participating in angiogenesis. Furthermore, elevated expression of JAG1 mRNA and protein has been associated with poor patient outcomes in breast cancer. Similarly, TYMP activity has been shown to be higher in triple-negative breast tumors, leading to poor outcomes as well. Although several anti-angiogenic drugs have been approved, tumor-acquired resistance to therapy limit their effectiveness. An increased understanding of tumor vessel mechanisms involved in tumorigenesis and metastasis is necessary to overcome the obstacles that prevent successful control of the angiogenic response in tumors. This would require as many paths as can be unraveled regarding the angiogenic mechanisms driving TNBC. Understanding the relationship between the function of TRPS1 in regulating the expression of important genes such as JAG1 and TYMP lays a better foundation for new drugs and drug targets that control and inhibit tumor angiogenesis. Citation Format: Liliana Guzman, Camila Ayerbe, Roberto Rangel, Jenny Chang, Roberto Rosato. TRPS1 Regulates angiogenesis via JAG1 and TYMP in Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-25-01.

Identification of radiation responsive RBC membrane associated proteins (RMAPs) in whole-body γ-irradiated New Zealand white rabbits

This study is aimed to identify radiation-responsive RBC Membrane Associated Proteins (RMAPs) in Rabbits in vivo. Male New Zealand White rabbits were exposed to a single acute total body γ-radiation dose of 2 Gy at a dose rate of 0.746 Gy/min. Following this, at early time points of 6 h till the 7 d, RMAPs were collected and analyzed by MALDI-TOF-MS. Bioinformatics analysis was conducted to explore the biological functions of these proteins. Based on fold change, radiation responsiveness, GO, pathway enrichment, and hub position in the PPI network, we identified seven RMAPs as potential biomarker candidates viz., PVALB, PRKCB, GPD1, CP2G1, CSNK2B, ATP1B1, TPI1. As per KEGG enrichment, most of the proteins were implicated in cellular radiation response, oxidative damage, DNA repair, apoptosis, immune response, and cell signaling. This study forms the foundation for RMAPs-based Proteomic strategies for high throughput radiation bio-dosimetry for triage in the case of a radiological/nuclear incident.

Andrographis Reverses Gemcitabine Resistance through Regulation of ERBB3 and Calcium Signaling Pathway in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, primarily due to intrinsic or acquired resistance to chemotherapy, such as Gemcitabine (Gem). Naturally occurring botanicals, including Andrographis (Andro), can help enhance the anti-tumorigenic therapeutic efficacy of conventional chemotherapy through time-tested safety and cost-effectiveness. Accordingly, we hypothesized that Andro might reverse Gem resistance in PDAC. The critical regulatory pathways associated with Gem resistance in PDAC were identified by analyzing publicly available transcriptomic profiling and PDAC tissue specimens. A series of systematic in vitro experiments were performed using Gem-resistant (Gem-R) PDAC cells and patient-derived 3D-organoids to evaluate the Andro-mediated reversal of Gem resistance in PDAC. Transcriptomic profiling identified the calcium signaling pathway as a critical regulator of Gem-resistance (Fold enrichment: 2.8, p = 0.002). Within this pathway, high ERBB3 expression was significantly associated with poor prognosis in PDAC patients. The combination of Andro and Gem exhibited superior anti-cancer potential in Gem-R PDAC cells through potentiating cellular apoptosis. The combined treatment down-regulated ERBB3 and decreased intracellular calcium concentration in Gem-R PDAC cells. Finally, these findings were successfully interrogated in patient-derived 3D-organoids. In conclusion, we demonstrate novel evidence for Andro-mediated reversal of chemoresistance to Gem in PDAC cells through the regulation of ERBB3 and calcium signaling.

Maize cytolines as models to study the impact of different cytoplasms on gene expression under heat stress conditions

BackgroundCrops are under constant pressure due to global warming, which unfolds at a much faster pace than their ability to adapt through evolution. Agronomic traits are linked to cytoplasmic-nuclear genome interactions. It thus becomes important to understand the influence exerted by the organelles on gene expression under heat stress conditions and profit from the available genetic diversity. Maize (Zea mays) cytolines allow us to investigate how the gene expression changes under heat stress conditions in three different cytoplasmic environments, but each having the same nucleus. Analyzing retrograde signaling in such an experimental set-up has never been done before. Here, we quantified the response of three cytolines to heat stress as differentially expressed genes (DEGs), and studied gene expression patterns in the context of existing polymorphism in their organellar genomes.ResultsOur study unveils a plethora of new genes and GO terms that are differentially expressed or enriched, respectively, in response to heat stress. We report 19,600 DEGs as responding to heat stress (out of 30,331 analyzed), which significantly enrich 164 GO biological processes, 30 GO molecular functions, and 83 GO cell components. Our approach allowed for the discovery of a significant number of DEGs and GO terms that are not common in the three cytolines and could therefore be linked to retrograde signaling. Filtering for DEGs with a fold regulation > 2 (absolute values) that are exclusive to just one of the cytolines, we find a total of 391 up- and down-DEGs. Similarly, there are 19 GO terms with a fold enrichment > 2 that are cytoline-specific. Using GBS data we report contrasting differences in the number of DEGs and GO terms in each cytoline, which correlate with the genetic distances between the mitochondrial genomes (but not chloroplast) and the original nuclei of the cytolines, respectively.ConclusionsThe experimental design used here adds a new facet to the paradigm used to explain how gene expression changes in response to heat stress, capturing the influence exerted by different organelles upon one nucleus rather than investigating the response of several nuclei in their innate cytoplasmic environments.

Genome-wide search reveals the uniqueness of DNA regions associated with coat color and innate immunity in Hokkaido Native Horse.

Hokkaido Native Horse (HKD) is a horse breed native to Hokkaido in Japan known for the traits such as coat color with no white spots and adaptability to the local cold climate. To examine whether those traits of HKD are conferred at the DNA level, we attempted to identify fixed DNA regions in HKD individuals, that is, the selection signatures of HKD. A comparison of genome-wide single nucleotide polymorphism genotypes in 58 HKD individuals by principal component analysis, and cluster analysis between breeds, including HKD, and within the HKD individuals indicated the genetic independence of HKD as a breed. Tajima's D analysis and runs of homozygosity analysis identified 23 selection signatures unique to HKD (P < 0.05), and following database search found 20 traits that were associated with those selection signatures; among these traits, coat color traits, face and body markings, showed the highest important value (0.50 and 0.46). Enrichment analysis of genes in the selection signatures identified six gene ontology terms (P < 0.05), and a term related to innate immunity (regulation of defense response; GO:0031347) showed the highest positive fold enrichment value (7.13). These results provide the first scientific evidence of a genetic basis for the traits of HKD.

CSIG-07. GAIN-OF-FUNCTION MUTANT P53 REGULATES LONG-NONCODING RNAS IN GLIOBLASTOMA

Abstract P53 is frequently mutated in most human cancers, including glioblastoma (GBM). Many p53 mutants acquire gain-of-function oncogenic effects through only partially understood mechanisms. To investigate the role of gain-of-function mutant p53 (MUT-p53) in GBM, we performed ChIP-seq of wildtype p53 (WT-p53) and MUT-p53 GBM cell lines. Among 2834 unique peaks reads in MUT-p53 cells, we found 242 long non-coding RNAs (lncRNAs) with up to 145 fold enrichment relative to WT-p53. LncRNAs regulate many molecular and cellular functions, including gene expression, cell proliferation, death, cancer stem cell renewal and differentiation. We selected lncRNAs SOX21-AS1 and LINC00643 with highly enriched binding by MUT-p53 and investigated their expressions and functions in the p53 pathway. We performed ChIP confirmation of MUT-p53 binding to the promoters of these lncRNAs. We found that these lncRNAs are deregulated in GBM and correlated with GBM patient survival in the TCGA database. To investigate the functions of these LncRNAs, we knocked down their expressions by siRNA, and found significant cell death induced by si-SOX21-AS1, but not by si-LINC00643. Overexpression of LINC00643 in GBM cells led to inhibition of GBM cell proliferation, migration, invasion and in vivo xenograft growth. LINC00643 mediated the effects of MUT-p53. Co-expression of human LINC00643 and its mouse homologous in a RCAS transgenic mouse model of GBM reduced tumor growth and improved animal survival. To elucidate the mechanisms of action of the lncRNA, we performed Chromatin Isolation by RNA purification high-throughput sequencing (CHIRP-seq) to identify its binding targets. We found that LINC00643 binds to HIF1a 5’ promoter/enhancer region. Overexpression of LINC00643 in GBM cells at hypoxia growth condition reduced HIF1a mRNA and protein expression. Our study shows for the first time that gain-of-function mutant p53 regulates a subset of lncRNAs and that the lncRNAs mediate the oncogenic effects of the MUT-p53 in GBM.

Downregulation of ACE, AGTR1, and ACE2 genes mediating SARS-CoV-2 pathogenesis by gut microbiota members and their postbiotics on Caco-2 cells

IntroductionCoronavirus disease-2019 (COVID-19) is a complex infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can cause also gastrointestinal symptoms. There are various factors that determine the host susceptibility and severity of infection, including the renin-angiotensin system, the immune response, and the gut microbiota. In this regard, we aimed to investigate the gene expression of ACE, AGTR1, ACE2, and TMPRSS2, which mediate SARS-CoV-2 pathogenesis by Akkermansia muciniphila, Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, and Bacteroides fragilis on Caco-2 cells. Also, the enrichment analysis considering the studied genes was analyzed on raw data from the microarray analysis of COVID-19 patients. Materials and methodsCaco-2 cells were treated with live, heat-inactivated form and cell free supernatants of A. muciniphila, F. prausnitzii, B. thetaiotaomicron and B. fragilis for overnight. After RNA extraction and cDNA synthesis, the expression of studied genes was assessed by RT-qPCR. DNA methylation of studied genes was analyzed by Partek® Genomics Suite® software on the GSE174818 dataset. We used GSE164805 and GSE166552 datasets from COVID-19 patients to perform enrichment analysis by considering the mentioned genes via GEO2R, DAVID. Finally, the related microRNAs to GO terms concerned on the studied genes were identified by miRPath. ResultsThe downregulation of ACE, AGTR1, and ACE2 genes by A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis in live, heat-inactivated, and cell-free supernatants was reported for the first time. These genes had hypomethylated DNA status in COVID-19 patients' raw data. The highest fold enrichment in upregulated RAS pathways and immune responses belonged to ACE, AGTR1, and ACE2 by considering the protein-protein interaction network. The common miRNAs targeting the studied genes were reported as miR-124-3p and miR-26b-5p. ConclusionIn combination with our experimental data and bioinformatic analysis, we showed the potential of A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis and their postbiotics to reduce ACE, ATR1, and ACE2 expression, which are essential genes that drive upregulated biological processes in COVID-19 patients.Accordingly, due to the potential of studied bacteria on the alteration of ACE, AGTR1, ACE2 genes expression, understanding their correlation with demonstrated miRNAs expression could be valuable. These findings suggest the importance of considering targeted gut microbiota intervention when designing the possible therapeutic strategy for controlling the COVID-19.

Exploring the mutational landscape of genes associated with inherited retinal disease using large genomic datasets: identifying loss of function intolerance and outlying propensities for missense changes

BackgroundLarge databases permit quantitative description of genes in terms of intolerance to loss of function (‘haploinsufficiency’) and prevalence of missense variants. We explored these parameters in inherited retinal disease (IRD)...

Abstract 1104: Loss of SUV420H2 drives EGFR inhibitor resistance in EGFR mutant NSCLC cell lines

Abstract Although important advancements have been achieved in the treatment of cancer, lung cancer is still the leading cause of cancer-related deaths in both men and women, with an estimated 1.6 million deaths each year. Non-small cell lung cancer (NSCLC), which makes up ~85% of lung cancer cases, is mainly treated with radiotherapy, chemotherapies, and targeted agents. Epidermal growth factor receptor (EGFR) is commonly mutated in NSCLC patients and leads to increased proliferation and cell survival. The standard-of-care treatment for patients harboring activating mutations in EGFR is treatment with tyrosine kinase inhibitors (TKI), such as erlotinib. While tumors initially respond to TKIs, after 1-2 years most of the patients develop resistance. In ~60% of patients, TKI resistance is the results of a secondary mutation in EGFR, leading to sustained receptor activation. Whereas, in the remaining 20%, tumors turn on bypass tracks-signals to overcome inhibition of the EGFR pathway. However, in 15-20% of the cases the mechanisms underlying resistance are unknown. Hence, there is a need to identify additional mechanisms involved in TKIs resistance. Our laboratory performed a genome-wide CRISPR Cas9 knock-out screen to identify genes that when knocked-out would drive erlotinib resistance. Fold enrichment analysis of sgRNAs, identified the protein coding gene SUV420H2 as a top candidate. SUV420H2 is a histone methyltransferase that trimethylates H4K20 (H4K20me3), enabling the establishment of constitutive and facultative heterochromatin. The process by which SUV420H2 is reduced in tumors is unknown, yet data from human samples suggests that SUV420H2 is globally downregulated in NSCLC. Additionally, loss of H4K20me3, the modification made by SUV420H2, in preneoplasia influences prognosis of NSCLC, indicating that loss of SUV420H2 function is a crucial mechanism in carcinogenesis. SUV420H2 expression has historically been associated with generating constitutive heterochromatin. However, more recently SUV420H2 was found to silence euchromatin regions. Our recent data suggests that SUV420H2 leads to reduced transcription of the oncogene MET by silencing the oncogenic long non-coding RNA LINC01510 upstream of MET. These findings led us to hypothesize that loss of SUV420H2 leads to erlotinib resistance through to upregulation of oncogenes including MET. Moreover, we aim to identify genes that are dynamically regulated by SUV420H2 using chromatin immunoprecipitation in NSCLC cell lines to further understand SUV420H2-mediated gene regulation and its role in TKIs resistance. Citation Format: Alejandra Agredo, Arpita Pal, Nadia Lanman, Jihye Son, Andrea L. Kasinski. Loss of SUV420H2 drives EGFR inhibitor resistance in EGFR mutant NSCLC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1104.

Transcriptome-Wide Analysis of RNA N6-Methyladenosine Modification in Adriamycin-Resistant Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is one of the most aggressive hematopoietic malignancies. Patients still suffer from refractory/relapsed disease after anthracycline-based therapy, which leads to a poor prognosis. N6-Methyladenosine (m6A) is the most abundant post-transcriptional modification in eukaryotes, the imbalance of which is reported to be associated with various pathological processes, including drug resistance. However, the relationship between m6A modification and drug resistance has not been well defined in AML. In this study, we analyzed the sequencing data of HL60 and its Adriamycin-resistant cell line HL60/ADR. We found a total of 40,550 m6A-methylated peaks, representing 15,640 genes in HL60, and 38,834 m6A-methylated peaks, representing 15,285 genes in HL60/ADR. KEGG pathway analysis showed that pathways were enriched in the FoxO signaling pathway, p53 signaling pathway, and Notch signaling pathway. MeRIP-seq results showed that the fold enrichment of the global m6A level in HL60/ADR was higher than that in HL60, and dot blot assay results indicated that the global m6A level was elevated in HL60/ADR cells compared with that in HL60 cells. Further analysis revealed that the expression level of METTL3 was elevated in HL60/ADR cells compared with that in HL60 cells. After a combined treatment of STM2457 (an inhibitor of METTL3) and Adriamycin, the proliferation of HL60/ADR was inhibited. Thus, we hypothesized that the abnormality of m6A modification played an important role in Adriamycin-resistant AML.

Fragment Size-Based Enrichment of Viral Sequences in Plasma Cell-Free DNA

Sequencing of plasma cell-free DNA (cfDNA) is a promising milieu for broad-based cancer and infectious disease diagnostics. The performance of cfDNA sequencing for infectious disease diagnostics is chiefly limited by inadequate analytical sensitivity. The current study investigated whether the analytical sensitivity of cfDNA sequencing for viral diagnostics could be improved by selective sequencing of short cfDNA fragments, given prior observations of shorter fragment size distribution in microbial and cytomegalovirus-derived cfDNA compared with human-derived cfDNA. It shows that the shorter plasma cfDNA fragment size distribution is a general feature of multiple DNA viruses, including adenovirus [interquartile range (IQR), 87 to 165 bp], herpes simplex virus 2 (IQR, 114 to 195 bp), human herpesvirus 6 (IQR, 145 to 176 bp), and varicella zoster virus (IQR, 98 to 182 bp), compared with human (IQR, 148 to 178 bp). It was used to further optimize a size selection-based cfDNA sequencing method, demonstrating an enrichment of viral sequences up to 16.6-fold, with a median fold enrichment of 6.7×, 4.6×, 2.2×, and 10.3× for adenovirus, herpes simplex virus 2, human herpesvirus 6, and varicella zoster virus, respectively. These findings demonstrate a simple yet scalable method for enhanced detection of DNA viremia that maintains the unbiased nature of cfDNA sequencing.

Age-related differences in hamstring tendon used as autograft in reconstructive anterior cruciate ligament surgery

The hamstring tendon is the most commonly used autograft material in reconstructive surgeries of anterior cruciate ligament (ACL) tears. Younger patients have worse surgical outcomes, with a higher risk of re-rupture. We hypothesized that age-related changes in hamstring tendon properties affect the tendon's propensity to rupture when used as an autograft in ACL reconstructions. The purpose of this study was to compare hamstring tendon samples obtained from people aged 20years or younger to samples obtained from older people. Superfluous hamstring tendon material was collected from 13 young donors (aged 16-20years) and 17 older donors undergoing ACL reconstructive surgery. Sections of the tendon samples were used for biomechanical testing, structural analysis of collagen fibrils by electron microscopy, and global analysis of gene expression by microarrays. We found that tendon samples from the older group had lower Young's modulus than the younger group (P = 0.015), whereas the stress to failure was similar in the two groups. We found no difference in the average diameter of collagen fibrils between the two groups. Microarray analysis identified 162 differentially expressed genes (fold change ≥ 1.5, P < 0.05), with overrepresentation of several biological processes, including regulation of adhesion, migration, inflammation, and differentiation (fold enrichment > 2.0, false discovery rate P < 0.05). The hamstring tendon from younger people has higher stiffness than tendon from older people, and the profile of gene expression in tendon varies with age. These differences may negatively affect the performance of the hamstring tendon in ACL reconstructions in younger people.

993. A CRISPR-Powered Universal Infectious Disease Assay

BackgroundThe COVID-19 pandemic has brought awareness to the dangers of emerging pathogens to global human health and welfare. Sensitivity and flexibility are important features for methods used to detect emerging pathogens. While PCR testing is rapid and sensitive, a significant advantage next generation sequencing (NGS) approaches have over PCR-based analyses is the ability to detect previously undiscovered pathogens while also providing genomic information that can detect SARS-CoV-2 genome sequence, identify source of co-infection, and the host transcriptional response in a single workflow. The critical component enabling this approach is Jumpcode CRISPRclean technology which removes abundant human and bacterial ribosomal RNA sequences from NGS libraries.CRISPRclean workflow easily integrates into next generation sequencing projects Schematic of the Jumpcode CRISPRclean protocolMethodsCRISPRclean was applied to contrived infected tissue samples including human lung RNA spiked with serially diluted amounts of SARS-CoV-2 RNA and bacterial RNA. NEB RNA libraries were prepared and treated with CRISPRclean protocol, then sequenced on Illumina instruments. Data analysis was performed using Jumpcode proprietary software to measure alignment and depletion rates, the Silva database for rRNA read alignment, and Kraken2 and CosmosID pipelines for k-mer based metagenomic investigation. Fold enrichment of SARS-CoV-2 reads after CRISPRclean depletion of libraries prepared from contrived samples. CRISPRclean treatment of the fully contrived samples increases the fraction of reads that map to the SARS-CoV-2 genome by an average of ~10-foldResultsCRISPRclean treatment of the contrived samples increases ~10 fold of reads that map to the SARS-CoV-2 genome. For the 60 viral copies of SARS-CoV-2 sample, the number of reads mapping to the SARSCoV-2 genome increases from ~10,000 reads to ~70,000 reads. A similar increase in reads occurs for S. aureus. The percentage of SARS-CoV-2 genome covered at 1X and 10X also increases. Similar results were achieved even after downsampling the datasets to 5M reads. There is a ~4-fold increase in bacterial species detection in these stool samples after CRISPRclean treatment. Percentage of SARS-CoV-2 genome covered at 1X and 10X increases as a result of rRNA depletion. Coverage of the SARS-CoV-2 genome at 50 million reads.Number of reads aligning to the S. aureus and SARS-CoV-2 genomes increases after CRISPRclean depletion. For the sample containing 0.0001% SARS-CoV-2, (60 viral copies), the number of reads mapping to the SARS-CoV-2 genome increases from ~10,000 reads to ~70,000 reads.CosmosID Shotgun Metagenomics Analysis heat map showing read alignments to viral genomes. The yellow color indicates high read counts. The CosmosID shotgun metagenomic analysis software was used to analyze the sequencing data, classify the sequences and generate the heat map.ConclusionMetatranscriptomics powered by CRISPR-mediated rRNA depletion offers a robust methodology to acquire viral genomic data, microbiome composition, co-infection information, and the transcriptional status of the host immune response in a single workflow. This sequencing-based approach can be available on the first day of the next viral outbreak and should be considered as a first-line test for novel zoonotic virus detection. Bacterial species composition of patient stool samples before and after CRISPRclean depletion. ~4-fold increase in bacterial species detection in these stool samples after CRISPRclean treatment. Sequencing data downsampled to 20 million reads.Disclosures Keith Brown, n/a, Jumpcode Genomics (Board Member, Employee, Shareholder)

Extracellular vesicles-derived microRNAs expression as biomarkers for neurological radiation injury: Risk assessment for space exploration

Space missions pose threats to the health of the astronauts due to long-term exposure to galactic cosmic rays and solar particle events comprised predominantly of medium to high energy protons, energetic helium ions, and energetic high atomic number particles (HZEs). While the tissue-specific effects of radiation have been studied extensively, the changes in exosomal miRNA expression levels in response to acute radiation exposure have not been assessed. Extracellular vesicles (EVs) originate from the host cells and contain nucleic acid and proteins that can modify the physiology of the receiving cells via the transfer of genomic, proteomic, and lipids cargo. Detection and analysis of miRNA cargo of circulating EVs is an emerging method for non-invasive diagnosis and monitoring of neurological disorders. This study characterizes the EV-derived miRNA expression profiles of human astrocytes to identify those that are altered after treatment with 3 Gy proton radiation as biomarkers of neurological radiation injury. The relationship between radiation and miRNA extracellular vesicles expression levels was investigated in human astrocytes after treatment with 3 Gy proton radiation at Willis-Knighton Cancer Center. Microarray analysis was performed using miRNA from the EVs enriched fraction in the cell culture medium collected from sham-control and radiation-treated cells. The exosomal levels of 13 miRNAs were significantly (FDR p < 0.05) down-regulated after exposure to high-energy radiation. The computational analysis identified hsa-miR-762, hsa-let-7c-5p, and has-let-7b-5p regulate the highest number of genes being associated with cognitive, mental, and motor delay. These miRNAs target the same subset of genes (Amd1, CCNF, COX6B, PLXND1) that are associated with epileptic encephalopathy; frontotemporal dementia; mitochondrial complex iv deficiency, and a rare neurological condition (Moebius syndrome) respectively. GO enrichment analysis of the biological processes identified overrepresentation in mRNA polyadenylation and regulation of glutamine and long fatty acids transport. Gene expression analysis confirmed the upregulation of the glutamine synthetase after irradiation. Significant fold enrichment of GO l-glutamine transmembrane transporter activity was identified in the molecular function category as well indicating exosome-mediated regulation of this important pathway after proton radiation exposure.

Enhanced propagation of intracellular and extracellular antibiotic resistance genes in municipal wastewater by microplastics

Microplastics (MPs) are an emerging global concern as they are abundant in the environment and can act as vectors of various contaminants. However, whether and how MPs can be vectors of antibiotic resistance genes (ARGs), especially extracellular ARGs (eARGs), remains far from explicit. This study addresses the adsorption of both intracellular ARGs (iARGs) and eARGs by four types of MPs in municipal wastewater, and then explores the potential horizontal gene transfer of iARGs and eARGs exposed to MPs. Results indicate that though MPs significantly adsorbed both iARGs and eARGs, eARGs were adsorbed with a significantly higher fold enrichment (2.0–5.0 log versus 2.0–3.3 log) and rate (0.0056 min−1 versus 0.0037 min−1) than iARGs. While all four types of MPs adsorbed ARGs, polypropylene MPs showed the highest adsorption capacity for ARGs. Background constituents such as humic acid and antibiotics significantly inhibited adsorption of iARGs, but not eARGs on MPs. The presence of sodium chloride didn't significantly affect adsorption of iARGs or eARGs. The adsorption of ARGs was well explained by the extended Derjaguin–Landau–Verwey–Overbeek (XDLVO) interaction energy profile. Higher eARG adsorption was attributed to a lower energy barrier between MPs and eARGs than that between MPs and iARGs. Exposure to MPs enhanced horizontal gene transfer of both iARGs and eARGs by 1.5 and 2.0 times, respectively. The improved contact potential between donors and recipients, as well as the increased cell permeability of recipients induced the improved horizontal gene transfer by MPs. This study underscores the need to address ARG propagation through adsorption to MPs.

SNAI1-mediated transcriptional regulation of epithelial-to-mesenchymal transition genes in breast cancer stem cells

BackgroundTriple-negative breast cancer (TNBC) tumors are composed of a heterogeneous population containing both cancer cells and cancer stem cells (CSCs). These CSCs are generated through an epithelial-to-mesenchymal transition (EMT), thus making it pertinent to identify the unique EMT-molecular targets that regulate this phenomenon. Methods and resultsIn the present study, we performed in silico analysis of microarray data from luminal, Her2+, and TNBC cell lines and identified 15 relatively unexplored EMT-related differentially expressed genes (DEGs) along with the markedly high expression of EMT-transcription factor (EMT-TF), SNAI1. Interestingly, stable overexpression of SNAI1 in MCF-7 induced the expression of DEGs along with increased migration, invasion, and in vitro tumorigenesis that was comparable to TNBCs. Next, stable SNAI1 overexpression led to increased expression of DEGs that was reverted with SNAI1 silencing in both breast cancer cells and CSCs sorted from various TNBC cell lines. Higher fold enrichment of SNAI1 on E-boxes in the promoter regions suggested a positive regulation of ALCAM, MMP2, MMP13, MMP14, VCAN, ANKRD1, KRT16, CTGF, TGFRIIβ, PROCR negative regulation of CDH1, DSP and DSC3B by SNAI1 leading to EMT. Furthermore, SNAI1-mediated increased migration, invasion, and tumorigenesis in these sorted cells led to the activation of signaling mediators, ERK1/2, STAT3, Src, and FAK. Finally, the SNAI1-mediated activation of breast CSC phenotypes was perturbed by inhibition of downstream target, MMPs using Ilomastat. ConclusionThus, the molecular investigation for the gene regulatory framework in the present study identified MMPs, a downstream effector in the SNAI1-mediated EMT regulation.

Abstract 556: Highly complex DNA sequencing library preparation for cfDNA enrichment panels using a single-stranded approach

Abstract DNA sequencing library preparation is a crucial step for next-generation sequencing of cell free DNA of plasma for cancer diagnostics. We present SRSLY, a robust single-stranded DNA library preparation method, that generates libraries with unique molecule identifiers (UMIs), with advantages over traditional double-stranded DNA preparations. Using 5 ng of plasma-derived cfDNA from acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and gastrointestinal cancer cases, we prepared both SRSLY and double-stranded DNA (dsDNA) sequencing libraries with UMIs. The libraries were enriched using a Twist Biosciences custom hybridization probe set for an 800 kilobase cancer mutation panel. We sequenced to a panel depth of 1000x to 2000x, after correcting UMI sequences and removing duplicate reads. With comparable fold enrichment and on-target percentages, we observe complexities of 11-21 million unique molecules with SRSLY. This is approximately four times higher than dsDNA complexity of 2.8-4.9 million unique molecules. Using duplicate reads from the same unique molecule, we perform error correction on template read bases and are able to call low-variant allele frequency mutations. In particular, we show an increase in KRAS p.G12D variant allele fraction during progression from MDS to AML. SRSLY shows increased recovery of small fragments over dsDNA preparations and retains the native lengths and termini of all fragments. Apart from improved library complexity, we show that MDS and AML samples can be distinguished from healthy samples by their disrupted genomic profile of short to long fragments, which has been previously demonstrated in solid tumors but not for MDS or AML. Citation Format: Charles J. Vaske, Chris Troll, Camille Schwartz, Colin Naughton, Abdullah Mahmood Ali, Azra Raza, Varsha Rao, Kelly Harkins-Kincaid, Richard Edward Green. Highly complex DNA sequencing library preparation for cfDNA enrichment panels using a single-stranded approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 556.

Influenza Virus-Induced Novel miRNAs Regulate the STAT Pathway.

MicroRNAs (miRNAs) are essential regulators of gene expression in humans and can control pathogenesis and host–virus interactions. Notably, the role of specific host miRNAs during influenza virus infections are still ill-defined. The central goal of this study was to identify novel miRNAs and their target genes in response to influenza virus infections in airway epithelium. Human airway epithelial cells exposed to influenza A virus (IAV) induced several novel miRNAs that were identified using next-generation sequencing (NGS) and their target genes by biochemical methods. NGS analysis predicted forty-two RNA sequences as possible miRNAs based on computational algorithms. The expression patterns of these putative miRNAs were further confirmed using RT-PCR in human bronchial epithelial cells exposed to H1N1, H9N1(1P10), and H9N1 (1WF10) strains of influenza virus. A time-course study showed significant downregulation of put-miR-34 in H1N1 and put-miR-35 in H9N1(1P10)-infected cells, which is consistent with the NGS data. Additionally, put-miR-34 and put-miR-35 showed a high fold enrichment in an argonaute-immunoprecipitation assay compared to the controls, indicating their ability to form a complex with argonaute protein and RNA-induced silencing complex (RISC), which is a typical mode of action found with miRNAs. Our earlier studies have shown that the replication and survival of influenza virus is modulated by certain transcription factors such as NF-ĸB. To identify the target(s) of these putative miRNAs, we screened 84 transcription factors that have a role in viral pathogenesis. Cells transfected with mimic of the put-miR-34 showed a significant decrease in the expression of Signal Transducers and Activators of Transcription 3 (STAT3), whereas the inhibitor of put-miR-34 showed a significant increase in STAT3 expression and its phosphorylation. In addition, put-miR-34 had 76% homology to the untranslated region of STAT3. NGS and PCR array data submitted to the Gene Ontology project also predicted the role of transcription factors modulated by put-miR-34. Our data suggest that put-miR-34 may be a good target for antiviral therapy.

Sex hormones regulate lipid metabolism in adult Sertoli cells: A genome-wide study of estrogen and androgen receptor binding sites

Optimal functioning of Sertoli cells is crucial for spermatogenesis which is under tight regulation of sex hormones, estrogen and androgen. Adult rat Sertoli cells expresses estrogen receptor beta (ERβ) and androgen receptor (AR), both of which regulate gene transcription by binding to the DNA. The present study is aimed to acquire a genome-wide map of estrogen- and androgen-regulated genes in adult Sertoli cells. ChIP-Seq was performed for ERβ and AR in Sertoli cells under physiological conditions. 30,859 peaks in ERβ and 9,594 peaks in AR were identified with a fold enrichment >2 fold. Pathway analysis for the genes revealed metabolic pathways to be significantly enriched. Since Sertoli cells have supportive functions and provide energy substrates to germ cells during spermatogenesis, significantly enriched metabolic pathways were explored further. Peaks of the genes involved in lipid metabolism, like fatty acid, glyceride, leucine, and sphingosine metabolism were validated. Motif analysis confirmed the presence of estrogen- and androgen-response elements (EREs and AREs). Moreover, transcript levels of enzymes involved in the lipid metabolic pathways were significantly altered in cultured Sertoli cells treated with estrogen and androgen receptor agonists, demonstrating functional significance of these binding sites. This study elucidates a mechanism by which sex hormones regulate lipid metabolism in Sertoli cells by transcriptionally controlling the expression of these genes, thereby shedding light on the roles of these hormones in male fertility.

Interactions of dextransucrase purified from Streptococcus mutans 890 with plant polyphenols

Plant polyphenols have been extensively studied for their chemopreventive properties for human health. Dextransucrase plays an essential role in synthesizing exopolysaccharides from its exclusive substrate sucrose in Streptococcus mutans. In the present study, the effect of polyphenols gallic acid and tannic acid was investigated on the dextransucrase activity. The enzyme was purified by ethanol precipitation followed by column chromatography by Sephadex G-200 gel chromatography, followed by PEG-400 treatment. The purified enzyme exhibited 52 fold enrichment with 17.5% yield and specific activity of 3.54 Units/mg protein. On SDS-PAGE enzyme protein gave a single band with a molecular weight of 160 kDa. Dextransucrase activity was inhibited 80–90% by 0.04 mM tannic acid (TA) or 0.4 mM gallic acid (GA) suggesting that tannic acid has 10- fold more inhibitory potential than gallic acid on the activity of dextransucrase. CD/ORD studies revealed modifications in the tertiary structure of enzyme protein in presence of tannic acid and gallic acid, which were further confirmed by fluorescence spectra of the protein in presence of tannic acid. These results suggest that inhibition of dextransucrase activity in S. mutans by polyphenols may have potential applications in the prevention and control of dental caries.