Housekeeping Genes Research Articles

Comprehensive analysis of housekeeping genes, tissue-specific genes, and dynamic regulation across developmental stages in pearl millet

BackgroundPearl millet (Pennisetum glaucum (L.) R. Br.) is a vital cereal crop, predominantly cultivated in arid and semi-arid regions of Asia and Africa. It serves as a staple food for millions, while also being utilized as forage and an energy crop. The crop’s resistance to heat and drought, coupled with its high biomass, positions it as a promising candidate for climate-resilient agriculture. A detailed understanding of its gene expression patterns across various tissues and developmental stages is essential for enhancing its yield and quality. This study aims to fill this knowledge gap by employing RNA-seq to identify housekeeping genes (HKGs) and tissue-specific genes (TSGs) in pearl millet.ResultsOur analysis of RNA-seq data from nine tissues (seed, germ, radicle, leaf, root, tillering tissue, stem, spike, and grain) across eight developmental stages in pearl millet accession Tifleaf3 revealed a comprehensive gene expression profile. We identified 461 HKGs that exhibited stable expression across all tissues and stages, providing robust internal references for RT-qPCR. Additionally, 8091 TSGs were discovered, many of which showed distinctive expression patterns in tissues such as spike, stem, and leaf. Functional enrichment analysis of these genes using GO and KEGG pathways highlighted their roles in key biological processes and pathways, indicating their potential in crop trait enhancement. Protein-protein interaction networks constructed for stem and leaf tissues further illuminated the regulatory mechanisms underlying the transition from vegetative to reproductive growth stages.ConclusionThis study presents a detailed transcriptomic landscape of pearl millet, identifying a set of HKGs and TSGs that are crucial for understanding the molecular basis of its growth and development. We provided valuable options for transcript normalization and crucial targets for exploring gene function for the plant growth and development in pearl millet. The insights gained from this work are instrumental for breeding programs aimed at enhancing the productivity of pearl millet, thereby contributing to food and energy security.

A maternal sweet diet is associated with the gut dysbiosis in the first trimester of pregnancy

BackgroundThe composition of maternal gut phylum in each trimester of pregnancy has been associated with fetal development, separately. Diet is a main effective factor on the gut composition of phylum. However, associations between dietary glycemic index (GI), load (GL) and total antioxidant capacity (TAC) not studied with the gut population of phylum in mothers at the first trimester of pregnancy.Materials and methodsNinety healthy pregnant women aged 18–40 yrs, in the first trimester, were participated. Stool samples were gathered in a fasting state. Population of dominant phylum was determined after DNA extraction based on the 16SrRNA expression, as a housekeeping gene. Dietary intake was collected by a validated food frequency questionnaire and dietary indices were computed.ResultsThe Proteobacteria population was significantly higher in the gut of pregnant mothers than the other phylum (p < 0.001). Participants in the highest level of dietary GI had lower Bacteroidetes (p < 0.001) and Actinobacteria (p = 0.04) in their gut compared to the lowest level. Participants in the lowest level of dietary GL had higher Bacteroidetes (p < 0.001) and lower proteobacteria (p = 0.04) in their gut than the highest level. Dietary selenium showed a significant negative effect on the Firmicutes (p = 0.04) and Proteobacteria (p = 0.04), however positively affected the Actinobacteria (p = 0.01) population. Dietary zinc and manganese showed a negative effect on the Firmicutes population (p = 0.01 and p = 0.003). Zinc and vitamin E showed a negative effect on the Proteobacteria population (p = 0.04 and p = 0.03).ConclusionsA maternal diet with high GI and GL have been associated with the gut dysbiosis, however dietary intake of selenium, zinc, manganese and vitamin E act in favor of the intestinal eubiosis in the first trimester of pregnancy.

Modulation of signature cancer-related genes in oral cancer cells (Ca9-22) by anethole treatment: Insights into therapeutic potential.

To explore an alternative strategy to chemotherapy to combat oral cancer, natural products and their derivates constitute one promising approach. In the last previous study, we have demonstrated the potential anti-tumor properties of anethole; an aromatic compound abundantly present in nature that serves as a major active ingredient found in plants like anise and fennel. In the current study, we aimed to investigate how this molecule inhibits oral cancer cell proliferation and induces apoptosis. This will be carried out by a transcriptomic study of its effects on the expression profile of cell cycle and apoptosis regulation genes in gingival cancer cells. cell cycle. Ca9-22 cells were treated with 10 μM of anethole (IC50) and cell proliferation was evaluated by MTT assay. The percentage of cells in different stages of the cell cycle was measured by flow cytometry. Cytotoxicity was evaluated by LDH assay and apoptosis was investigated by Pi/Annexin V assay following 24-hour treatment. Furthermore, we employed PCR array analysis to investigate alterations in the expression levels of oncogenes and tumor suppressor genes associated with cell cycle regulation and apoptosis. Finally, Gene-gene interactions were examined using the Gene MANIA database. Our findings demonstrate that anethole significantly attenuated the proliferation of Ca9-22 cells, leading to disturbances in cell cycle progression and eliciting cellular toxicity and apoptosis. By a double normalizing with two housekeeping genes (Actin and GAPDH), we show that, treatment with 10 μM of anethole alters (more than two-fold) the expression of 13 genes involved in the control of the cell cycle (8 were up regulated and 5 were down regulated) and 7 genes involved in the regulation of apoptosis (4 were up regulated and 3 downregulated by anethole). Finally, each group of genes modulated by anethole forms a network of connections between them or with other genes. Our study suggests that anethole holds promise as a potential alternative treatment for oral cancer by its ability to modify numerous oncogenes and tumor suppressor genes implicated in the cell cycle regulation and induction of apoptosis in oral cancer cells. These findings underscore the significance of further research into the potential therapeutic application of anethole as an alternative drug for managing oral cancer.

Developmental gene expression in skull-base chordomas and chondrosarcomas.

Chordomas are malignant tumors of the axial spine and skull base, and they are notorious for their poor treatment response. Differentiating these tumors from comparatively less malignant chondrosarcomas is crucial for treatment and prognostication. Both tumor types differ in their developmental origin. Chordomas are considered to be derived from notochordal remnants and chondrosarcomas from mesenchymal cells. Here, we evaluated the differential expression of developmental transcription factors in these skull base tumors. Histopathologically-confirmed tumor biopsies were obtained from 12 chordoma and 7 chondrosarcoma patients. Following RNA extraction, samples were submitted to real-time quantitative PCR (RT-qPCR) for the evaluation of 32 evolutionary conserved genes that are known to associate with notochord, mesoderm, and axial spine development. Gene expression levels were normalized to housekeeping genes ACTB and RS27a. Fifteen genes were either exclusively expressed (n = 12) or overexpressed (n = 3; 2.21-4.43 fold increase) in chordoma, compared to chondrosarcoma. Brachyury and CD24 were highly and exclusively expressed in chordoma. Other novel genes exclusive to chordomas included chordin, HOXA5 and ACAN. Vice versa, ten genes were either exclusively expressed (n = 2) or overexpressed (n = 8; 0.01-0.66 fold increase) in chondrosarcoma, compared to chordoma. As chordoma patients demonstrate a worse prognosis compared to chondrosarcoma patients, the differential expression of chordin, HOXA5 and ACAN and CD24 could be relevant for the pathophysiology of chordomas and may have diagnostic and treatment value. Further study on role of these genes in tumorigenesis is therefore warranted.

Characterisation of Itersonilia spp. from Parsnip and Other Hosts

Parsnips (Pastinaca sativa) are a speciality UK crop with an economic value of at least 31M GBP annually. Currently, the major constraints to production are losses associated with root canker disease due to a range of fungal pathogens, among which Itersonilia pastinacae is of most concern to growers. With limited research conducted on this species, this work aimed to provide a much-needed characterisation of isolates from across the UK, continental Europe, and New Zealand. Previously, up to four separate Itersonilia species have been proposed based on the formation of chlamydospores and host specificity: I. pastinacae, I. perplexans, I. pyriformans, and I. pannonica. However, Itersonilia spp. isolates principally from parsnip, but also from a range of other hosts, which were found to infect both parsnip roots and leaves in pathogenicity tests. In growth rate assays, isolates were found to grow at temperatures of 0–25 °C and produce both chlamydospores and ballistospores across the same range of temperatures, although chlamydospore production was found to decrease as temperature increased. Following whole genome sequencing, specific primers were designed for the molecular characterisation of the isolates using six housekeeping genes and three highly variable functional genes. Phylogenetic analysis separated isolates into two and six clades, respectively, but the grouping was not associated with hosts or locations. Based on the results of this research, there was no evidence to support more than a single species of Itersonilia among the isolates studied.

Lasia spinosa Stem Aqueous Extract Potentiates Antidiabetic Effects Exhibiting Antioxidant Genes Upregulation and DNA-damage Protection.

This research elucidated the hypoglycemic effect correlated with DNA protective and antioxidative activity of Lasia spinosa stem aqueous extract (LSSAE) using streptozotocin-induced type 2 diabetic rat models. LSSAE, characterized by phytochemical screening, GC-MS, and FTIR analyses, was investigated for its DNA protective activity by exposing PBR322 plasmid DNA to Fenton's reagents. Long-Evans rats, treated by LSSAE were found to be improved for body weight, fasting blood glucose level, and oral glucose load. A 30-day supplement of LSSAE significantly recovered serum biochemical markers, including hepatic (i.e., ALP, AST, ALT, and TB); renal (i.e., creatinine and uric acid); and lipid profiles (i.e., TC, TG, HDLc, LDLc, and VLDLc). An increased level of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) and a decreased level of malondialdehyde (MDA) were observed in LSSAE-treated rat liver. Antioxidant enzyme activities were evaluated by mRNA expression level of antioxidant genes (CAT, SOD2, GPX1, PON1, PFK1, GAPDH, using the 2-ΔΔCT method, normalized with housekeeping gene, ß-ACTIN) using qRT-PCR. Additionally, histological examination confirmed the restoration of morphology, and function of pancreatic β-cell, kidney, liver, and spleen. Results reveal that LSSAE exerts an antidiabetic effect through upregulation of antioxidant genes, DNA protection and modulation of biochemical and histological parameters.

Pseudomonas boreofloridensis sp. nov., and Pseudomonas citrulli sp. nov., isolated from watermelon in Florida.

Three fluorescent bacterial strains, K1, K13 and K18, were obtained from watermelon (Citrullus lanatus) foliage symptomatic of bacterial leaf spot of cucurbits in Florida. The strains underwent phenotypic characterization, including LOPAT (levan production, oxidase activity, pectolytic activity on potato, arginine dihydrolase production and hypersensitive response (HR) on both tobacco and tomato) and pathogenicity testing on watermelon and squash seedlings. Whole-genome sequencing of the isolates was performed, and multi-locus sequence analysis (MLSA) utilizing housekeeping genes gltA, rpoD, gapA and gyrB placed the isolates into two distinct clades within the Pseudomonas genus. Average nucleotide identity based on blast (ANIb) was used to compare the isolates to Pseudomonas reference genomes. Using ANIb, the closest relatives to the novel strains were identified as Pseudomonas wayambapalatensis (K1 : 82.58%; K13 : 83.77%) and Pseudomonas kilonensis (K18 : 87.16%), although ANIb values were below the 95% threshold for species delineation. DNA-DNA hybridization (genome-genome distance calculation method), comparison to the online Type Genome Server, Biolog biochemical profiling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were also unable to identify the isolates as any known species of Pseudomonas. Based on the combination of genetic and phenotypic data, we conclude that these isolates represent two novel Pseudomonas species, for which we propose the names Pseudomonas boreofloridensis sp. nov. (K1, K13T, NCPPB 4759=LMG 33364) and Pseudomonas citrulli sp. nov. (K18T, NCPPB 4761=LMG 33365). The specific epithet boreofloridensis was chosen for the geographic location of isolation (northern Florida), while citrulli designates the host of origin (C. lanatus).

Cut-offs for relapse detection in men with stage I testicular germ cell tumors during active surveillance within a prospective multicentre cohort study using either raw or housekeeper normalized miR-371a-3p serum levels

PurposeMiR-371a-3p represents a novel liquid biomarker that detects all histologies of germ-cell tumors (GCT) except teratoma. However, it is currently unclear whether miR-371a-3p results obtained directly from RT-PCR (raw Cq) or normalized for housekeeper genes and transformed into the relative quantity (RQ) value should be used and at what cut-off level. The purpose of this research was to evaluate, which values should be used, and a potential cut-off level for relapse-detection to inform subsequent studies. Materials and MethodsWe applied a CE-certified qRT-PCR test to measure miR-371a-3p at each follow-up visit during active surveillance in 34 men with stage I testicular GCT. MiR-371a-3p levels were calculated by the ΔΔ method. ResultsAbout 18 Patients had pure seminoma and 16 had mixed or nonseminomatous testicular GCT. Recurrences were detected in 10 patients and were correctly identified by both raw and housekeeper-normalized miR-371a-3p serum levels. The raw Cq, with a cut-off value of <28, resulted in only 1 false positive (3%), whereas RQ, with a cut-off value of >15, produced 6 false positive results (17%). Most of these false positive results normalized in subsequent measurements. The RQ approach detected recurrence in 1 patient 6 months earlier than the raw Cq approach. ConclusionOur preliminary data suggest that this CE-certified assay, using previously suggested cut-off values, is a promising method for detecting disease recurrence, provided a confirmatory second test is conducted to identify false positive results. To avoid unnecessary scans or overtreatment, we are currently validating this assay and cut-offs in a prospective cohort study.

Development of RT-qPCR assay for assessing the expression of ACTB and SDHA housekeeping genes in the cell cultures of mammalian hosts of zoonotic infections

Background. The molecular mechanisms behind the maintenance of zoonotic pathogens in nature can be better understood by examining the gene expression in host cells in response to the infection. Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) is a powerful method to study gene expression, especially with the use of endogenous reference genes (RG) to normalize the data. Therefore, it is critical to develop the reliable qRT-PCR assay and validate that selected RGs are stably expressed in the studied organism or cell culture.The aim. In this work, we aimed to develop the real-time qRT-PCR method for detecting mRNA of two candidate RGs, succinate dehydrogenase subunit A (SDHA) and beta-actin (ACTB), in the cell lines of mammalian hosts of zoonotic infections.Materials and methods. The SPEV (porcine embryonic kidney cell line) and ApnK (Korean field mouse kidney cell line) cells were grown in 24-well culture plates. Total RNA/DNA was isolated from trypsin-detached cell monolayers. Genomic DNA in the samples was removed with RNase-free DNase I, and one-step RT-qPCR was performed using primers for SDHA and ACTB gene fragments and the corresponding TaqMan hydrolysis probes. The experiment was performed in 4 independent replicates.Results. In the Korean field mouse cells, the linearity, efficiency, repeatability, and reproducibility of the RT-qPCR for ACTB gene mRNA corresponded to the modern requirements. However, RT-qPCR for SDHA exhibited good linearity and efficiency of the reaction, but CV values for repeatability and reproducibility slightly exceeded the recommended standards. In porcine cells, both assays had acceptable parameters. Thus, to use the SDHA as RG for ApnK cells, a detailed study of the stability of its expression in this particular model is required.Conclusions. New qRT-PCR assay was developed to assess the expression of housekeeping genes ACTB and SDHA in the cells of the Korean field mouse and domestic pig. Further research is necessary to validate these genes as references for quantitative assessment of gene expression in the cells of mammalian hosts of zoonotic infections.

Reference gene selection for real-time qPCR in European flounder (Platichthys flesus) using organ-specific RNA-seq data

BackgroundThe European flounder is readily chosen as an experimental subject and model in physiological and ecotoxicological studies mostly because of its adaptability to laboratory conditions. Many studies utilise a quantitative PCR (qPCR) approach to ascertain the expression of target genes under experimental conditions. Such an approach relies heavily on the selection of reference genes with stable expression. Yet certain housekeeping genes are commonly used in this role, often without due consideration of their overall expression patterns. Therefore, new approaches should be developed to identify stable reference genes for a given species and to expand the general pool of genes suitable for the reference in qPCR analysis.Methods and resultsHere RNA-seq data of nine flounder organs led to identify four candidate genes of the most stable expression. It was achieved by differential expression analysis and tritoconstrictor script. Specific primers were designed for the complete ORF as well as for qPCR analysis. RT-qPCR efficiencies were tested on ORF amplicon templates. Most of the genes tested showed good amplification in a wide range of template dilutions (107-101), with a correlation coefficient (R2) ranging from 0.991 to 0.998 and a consistent efficiency (E) (Sybr Green I staining and TaqMan molecular probe).ConclusionsThe proposed approach based on differential expression analysis and a new bioinformatic tool is an appropriate selection method of candidates for reference genes in qPCR. The proposed approach, combining differential expression analysis with a new bioinformatics tool, provides an effective method for selecting reference gene candidates for qPCR. As a result, we can propose four genes (polr2f, yif1a, sf3b6, uba52), each with a set of validated primers, as suitable for consideration as reference genes in qPCR analysis in European flounder, an emerging model species.

Antisense transcription is associated with expression of metal resistance determinants in Cupriavidus metallidurans CH34.

Cupriavidus metallidurans is able to thrive in metal-rich environments but also survives metal starvation. Expression of metal resistance determinants in C. metallidurans was investigated on a global scale. C. metallidurans was challenged with a MultiTox metal mix specifically designed for the wildtype strain CH34 and its plasmid-free derivative AE104, including treatment with ethylenediamintetraacetate (EDTA), or without challenge. The sense and antisense transcripts were analyzed in both strains and under all three conditions by RNASeq. A total of 10 757 antisense transcripts (ASTs) were assigned to sense signals from genes and untranslated regions, and 1 319 of these ASTs were expressed and were longer than 50 bases. Most of these (82%) were dual-use transcripts that contained antisense and sense regions, but ASTs (16%) were also observed that had no sense regions. Especially in metal-treated cells of strains CH34 and AE104, up- or down-regulated sense transcripts were accompanied by antisense transcription activities that were also regulated. The presence of selected asRNAs was verified by RT-PCR. Following metal stress, expression of genes encoding components of the respiratory chain, motility, transcription, translation and protein export were down-regulated. This should also affect the integration of the metal efflux pumps into the membrane and the supply of the energy required to operate them. To solve this dilemma, transcripts for the metal efflux pumps may be stabilized by interactions with ASTs to allow their translation and import into the membrane. Alternatively, metal stress possibly causes recruitment of RNA polymerase from housekeeping genes for preferential expression of metal resistance determinants.

Bacterial Communities Nodulating Lupinus cosentinii Gus. and Their Inputs in the Worldwide Phylogeography of Lupine Endosymbionts

Genetic variability in bacterial populations that nodulate Lupinus cosentinii in Tunisia was investigated. Phylogenetic studies of 40 isolates using recA partial sequences categorized them into three clusters within the Bradyrhizobium genus. Twenty-three strains selected from the three clusters were thoroughly examined through housekeeping genes (recA, glnII and rpoB) multilocus sequence analysis (MLSA). Our results showed that 23 representative strains were distributed in five distinct clusters, with 13 strains belonging to Bradyrhizobium canariense BTA-1T/Bradyrhizobium lupini USDA3051T (10 strains) and Bradyrhizobium hipponense aSej3T (three strains) lineages. Interestingly, eight strains occupied a separate position and could belong to two putative novel Bradyrhizobium species. The nodC phylogeny placed the 23 strains within three symbiovars: genistearum (19 strains), lupini (two strains) and, for the first time, the symbiovar cyanophyllae (two strains). Based on the worldwide phylogeography of rhizobial symbionts nodulating lupine (14 species), our results reported that eight species occurred in more than one continent, and six species were specific for one continent, e.g., Bradyrhizobium rifense, Bradyrhizobium diazoefficiens, Phyllobacterium sp. and Devosia sp. were specific to the African continent, the Bradyrhizobium iriomotense/Bradyrhizobium stylosanthis group to America, and Bradyrhizobium valentinum to the European continent.

Putative evolution of Myxococcus fulvus 124B02 plasmid pMF1 from a chromosomal segment in another Myxococcus species.

Myxobacteria or order Myxococcales (old nomenclature) or phylum Myxococcota (new terminology) are fascinating organisms well known for their diverse peculiar physiological, taxonomic, and genomic properties. Researchers have long sought to identify plasmids within these organisms, yet thus far, only two organisms from different families have been found to harbor a plasmid. This study delves into the putative evolution of one of these plasmids, i.e., pMF1 present in Myxococcus fulvus 124B02 in the suborder Cystobacterineae and family Myxococcaceae. Here, we first reannotated the pMF1 plasmid genome sequence and identified two additional open reading frames or putative genes which were not annotated until now. We further reported that all pMF1 plasmid genes depict homology with Myxococcus stipitatus CYD1 draft genome (contig 28) and a chromosomal segment of M. stipitatus DSM14675 in a syntenic manner, implying the presence of plasmid-like structure in M. stipitatus CYD1, integrated into its chromosome. To comprehend the relationship among these three species, we conducted phylogenetic analyses using 16S and concatenated housekeeping genes and genome-to-genome distance calculator (GGDC) analysis, which confirmed that M. stipitatus CYD1 is a distinct and novel species within the genus Myxococcus. Overall, this comparative genomic study sheds light on the putative emergence of the pMF1 plasmid from a common ancestor of closely related yet distinct species, M. stipitatus CYD1, possibly through the partition from its chromosome as a segment.IMPORTANCEMyxobacteria are not well known to have plasmids. Until now, only two organisms have been shown to have plasmids, raising a pertinent question about how these plasmids evolved randomly within the phylum Myxococcota. The study presented in this manuscript delves into the emergence of the pMF1 plasmid found in Myxococcus fulvus 124B02, a member of the suborder Cystobacterineae and family Myxococcaceae. Our research addresses this intriguing topic of plasmid identification and evolution within myxobacteria, which are a group of fascinating organisms that have garnered significant interest due to their diverse physiological, taxonomic, and genomic properties.

Abstract A003: Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing

Abstract Small nucleolar RNAs (snoRNAs) a class of ncRNAs that canonically guide post-transcriptional modifications, including 2'-O-methylation, on ribosomal RNA (rRNA) and small nuclear RNA (snRNA). While traditionally considered housekeeping genes, snoRNAs have increasingly been found to function in diverse physiologic and pathologic processes, including cancer. In an immune-competent murine model of triple negative breast cancer (TNBC) lymphatic metastasis, we identified the snoRNA Snord67 as one of the most upregulated noncoding RNAs in axillary LN (AxLN) tumors relative to mammary fat pad (MFP) tumors and lung metastases. Loss of Snord67 resulted in decreased colony formation and spheroid size in murine and human TNBC cell lines, and led to decreased lymph node tumor growth and decreased distant metastases in two immune-competent murine models of TNBC lymphatic dissemination. To determine the mechanism by which Snord67 promotes tumor growth and metastasis, we examined the impact of Snord67 on 2'-O-methylation, gene expression, and alternative splicing. Loss of Snord67 in TNBC cell lines led to decreased 2'-O-methylation at the C60 nucleotide (Cm60) in the core spliceosome component U6 snRNA. Re-introduction of wild-type Snord67 rescued Cm60 in U6 snRNA and rescued the colony formation and spheroid phenotypes of the Snord67 knockout cell lines. However, a mutant Snord67 incapable of guiding U6 Cm60 only partially rescued these in vitro phenotypes, suggesting that Snord67 promotes in vitro tumor cell growth at least in part by guiding Cm60 in U6 snRNA. We then performed RNA sequencing of Snord67 knockout and wild-type murine and human TNBC cell lines. We found that loss of Snord67 led to widespread changes in alternative splicing, consistent with its role in guiding 2'-O-methylation of the core spliceosome component U6. We further demonstrated that the inclusion of alternatively spliced cassette exons in MYO18A and NFYA was not only downregulated upon Snord67 knockout in a human TNBC cell line, but also positively correlated with Snord67 expression levels in primary breast tumors and lymph node metastases from breast cancer patients. Based on these results, we propose a model in which Snord67 guides U6 Cm60, which leads to a pro-metastatic alternative splicing program and thereby promotes lymph node tumor growth and distant metastasis. Citation Format: Katherine I Zhou, Yvonne Chao, Kwame K Forbes, Alessandro Porrello, Gabrielle M Gentile, Aaron C Chack, Dixcy J.S. John Mary, Haizhou Liu, Yinzhou Zhu, Eric Cockman, Lincy Edatt, Grant A Goda, Justin Zhao, Hala Abou Assi, Hannah J Wiedner, Yi-Hsuan Tsai, Lily Wilkinson, Amanda E Van Swearingen, Lisa A Carey, Jimena Giudice, Daniel Dominguez, Christopher L Holley, Chad V Pecot. Snord67 promotes breast cancer metastasis through U6-mediated alternative splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A003.

Abstract B061: Assessing saliva for cancer biomarker discovery: A liquid biopsy approach

Abstract Introduction: Liquid biopsy has been gaining traction as a replacement for invasive tumor biopsies in oncology. Several blood-based in vitro diagnostics have been approved by the FDA and other regulatory agencies, and efforts are being made to validate urine, saliva, and buccal swabs as alternative matrices. The primary aim of this study was to investigate the relative mRNA expression in paired blood and saliva samples from cancer patients and healthy individuals. Methods: We collected matched saliva and blood samples from 35 subjects, comprising 26 patients with prostate, breast, colon, lung, melanoma, and pancreatic cancers, and 9 healthy individuals. Samples were collected using Wren's proprietary stabilization buffer kit, and total RNA extracted by TRIzol-based Qiagen reagents. RNA sequencing (RNA-Seq) was performed on a Novaseq 6000 platform, and data was analyzed using high-performance computing (HPC) with PartekFlow software, employing the BWA aligner (hg38). We focused on comparing gene expression in cancers (as a group) compared to controls. Results: The total number of genes detected in saliva was 3,985, compared to 14,459 genes detected in blood samples. A substantial overlap was observed, with more than 92.8% of saliva-detected genes also found in blood. We also examined housekeeping gene detection in both matrices. In this set of 1,003 housekeeping genes, 30% (n=305) were expressed in both saliva and blood. When we narrowed the scope to only include the 1,154 cancer-related genes as listed by the OncoKB database, 88.3% were detected in saliva and/or blood, with 37.1% of these genes (n=429) were expressed in both saliva and blood. Lastly, we examined a set of 241 cancer-associated biomarkers utilized in RT-PCR-based diagnostic blood assays clinically validated by our laboratory. This subset analysis revealed that 23.2% of these biomarkers were expressed in both saliva and blood. Conclusion: Our data indicate that the majority of mRNA detected in saliva was also detected in blood, with differential expression observed in cancer patients compared to normal controls. Given the relatively high limit of detection (low sensitivity) using RNA-Seq technology compared to PCR and the relatively small number of subjects, the data may suggest that saliva could function as a surrogate biomarker matrix for blood. Citation Format: Srinivas V Koduru, Mark Kidd, Abdel B Halim. Assessing saliva for cancer biomarker discovery: A liquid biopsy approach [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B061.

Functional characteristics and computational model of abundant hyperactive loci in the human genome.

Enhancers and promoters are classically considered to be bound by a small set of transcription factors (TFs) in a sequence-specific manner. This assumption has come under increasing skepticism as the datasets of ChIP-seq assays of TFs have expanded. In particular, high-occupancy target (HOT) loci attract hundreds of TFs with often no detectable correlation between ChIP-seq peaks and DNA-binding motif presence. Here, we used a set of 1003 TF ChIP-seq datasets (HepG2, K562, H1) to analyze the patterns of ChIP-seq peak co-occurrence in combination with functional genomics datasets. We identified 43,891 HOT loci forming at the promoter (53%) and enhancer (47%) regions. HOT promoters regulate housekeeping genes, whereas HOT enhancers are involved in tissue-specific process regulation. HOT loci form the foundation of human super-enhancers and evolve under strong negative selection, with some of these loci being located in ultraconserved regions. Sequence-based classification analysis of HOT loci suggested that their formation is driven by the sequence features, and the density of mapped ChIP-seq peaks across TF-bound loci correlates with sequence features and the expression level of flanking genes. Based on the affinities to bind to promoters and enhancers we detected five distinct clusters of TFs that form the core of the HOT loci. We report an abundance of HOT loci in the human genome and a commitment of 51% of all TF ChIP-seq binding events to HOT locus formation thus challenging the classical model of enhancer activity and propose a model of HOT locus formation based on the existence of large transcriptional condensates.

Functional characteristics and computational model of abundant hyperactive loci in the human genome

Enhancers and promoters are classically considered to be bound by a small set of transcription factors (TFs) in a sequence-specific manner. This assumption has come under increasing skepticism as the datasets of ChIP-seq assays of TFs have expanded. In particular, high-occupancy target (HOT) loci attract hundreds of TFs with often no detectable correlation between ChIP-seq peaks and DNA-binding motif presence. Here, we used a set of 1003 TF ChIP-seq datasets (HepG2, K562, H1) to analyze the patterns of ChIP-seq peak co-occurrence in combination with functional genomics datasets. We identified 43,891 HOT loci forming at the promoter (53%) and enhancer (47%) regions. HOT promoters regulate housekeeping genes, whereas HOT enhancers are involved in tissue-specific process regulation. HOT loci form the foundation of human super-enhancers and evolve under strong negative selection, with some of these loci being located in ultraconserved regions. Sequence-based classification analysis of HOT loci suggested that their formation is driven by the sequence features, and the density of mapped ChIP-seq peaks across TF-bound loci correlates with sequence features and the expression level of flanking genes. Based on the affinities to bind to promoters and enhancers we detected five distinct clusters of TFs that form the core of the HOT loci. We report an abundance of HOT loci in the human genome and a commitment of 51% of all TF ChIP-seq binding events to HOT locus formation thus challenging the classical model of enhancer activity and propose a model of HOT locus formation based on the existence of large transcriptional condensates.

Abstract 4133040: Cardiac Favored Transcription of the Exogenous Gene in Mice after A Single Systemic Delivery of AAV9-cBIN1

Introduction: Heart failure (HF) is associated with high mortality and morbidity, and there are limited therapeutic options available to improve cardiac muscle function. Recent studies suggest that the impairment of cardiomyocyte T-tubule microdomains is a key factor in HF pathophysiology. AAV9-cBIN1 gene therapy effectively addresses this impairment. However, the kinetics and biodistribution of AAV9-cBIN1 have not been explored, limiting the knowledge of the transcription efficiency and organ specificity of cBIN1 gene therapy when delivered systemically. Aim: This study aims to elucidate the kinetics and biodistribution of AAV9-cBIN1 in a murine model. Method: Adult male C57BL/6J mice (n=20) were administered a single retro-orbital injection of AAV9-CMV-cBIN1-V5 at a dose (2x10^12 vg/kg) known to effectively transduce the majority of cardiomyocytes. Mice were sacrificed at pre-determined time points: prior to injection (Day 0, n=2) and post-injection at Days 3, 7, 14, 28, 56, and 84 (n=3/group). Tissues were harvested from the heart, lung, liver, kidney, skeletal muscle, and spleen. Genomic DNA was extracted and analyzed using qPCR to determine vector genome (vg) copies. For transcriptional analysis of the AAV9-transduced exogenous gene, mRNA was extracted and converted to cDNA for qRT-PCR quantification of cBin1-V5 , normalized to the housekeeping gene Hprt1 . Results: All mice survived to their designated sacrifice times. Kinetic analysis demonstrated a time-dependent expression of the exogenous cBin1-V5 gene, with peak expression observed at 4 weeks in both the heart and liver, with expression declining in the liver after this peak (Figure 1A). Genomic DNA analysis showed a gradual increase and plateau of viral genome distribution in the heart between 2-4 weeks, while in the liver, distribution peaked sharply between Days 3-7 and then decreased. When correcting the gene transcription of cBin1-V5 (ΔCq of V5/Hprt1 ) for vector DNA vg copies in the tissue, the transcription efficiency in the heart was higher than in the liver (Figure 1B). Outside of the liver and heart, cBin1-V5 mRNA expression was undetectable in all other tissues by Day 56. Conclusion: In adult male mice, a single systemic administration of AAV9-cBIN1 results in robust transduction and higher transcription efficiency in the heart compared to the liver. AAV9-cBIN1 holds promise for targeted gene therapy in cardiac tissues, with minimal off-target expression in other organs.

TMIC-57. EFFECT OF HYPOXIA ON SPHINGOSINE-1-PHOSPHATE (S1P) METABOLIZING ENZYMES IN DIFFUSE INTRINSIC PONTINE GLIOMA CELL LINES

Abstract Diffuse Midline Glioma H3K27 altered (DMG) is a devastating and incurable paediatric brain tumour. Previously a bioinformatic approach was taken to evaluate genes involved in the sphingosine-1-phosphate (S1P) biosynthesis pathway in diffuse intrinsic pontine gliomas (DIPG). S1P is known to interact with epigenetic partners (HDAC1) as well as play a role in proliferation, immune cell trafficking, angiogenesis and apoptosis. As a part of a broad bioinformatic approach evaluating the expression of genes associated with the sphingomyelin (SM) biosynthesis pathway, S1P metabolism, S1P receptors and downstream targets, 14 of 25 genes were found to be dysregulated in DIPG relative to normal brain (p&amp;lt;0.0002). Herein we focus on the imbalance of the S1P metabolic enzymes sphingosine kinase 2 (SPHK2) and sphingosine-1-phosphate lyase (SGPL1). Bioinformatic data revealed higher levels of SGPL1 and lower levels of SPHK2 compared with normal brain. DIPG cell lines (SU-DIPG-IV, VUMC-DIPG-08, VUMC-DIPG-A) were used to validate gene and protein expression and to evaluate changes following exposure to hypoxic conditions. Confocal images reveal an increase in nuclear SPHK2 staining in cells cultured for 24- and 48-hours under hypoxic conditions. HDAC1 immunostaining was localised in the nucleus. SGPL1 immunostaining, while mostly cytoplasmic, nuclear staining was observed. qRT-PCR assays were used to evaluate changes in mRNA expression in SPHK2, SGPL1 and HDAC1 in DIPG cell lines incubated under hypoxic conditions. The Pfaffl method was used to analyse gene expression relative to the housekeeping gene TBP. Under hypoxic conditions, the levels of SPHK2 and HDAC1 were higher in DIPG cells cultured in hypoxic conditions for 48 hours compared to DIPG cells cultured in normoxic conditions, whereas SGPL1 expression was lower in hypoxic treated cells. Current experiments targeting the modulation of these S1P-metabolizing enzymes are underway to determine the role of the S1P signalling pathway and determine the potential advantage of targeting this pathway in DIPG.

Bifidobacterium favimelis sp. nov., isolated from black comb honey.

A strain of the bacterial genus Bifidobacterium, IMAU50987T, that was isolated from black comb honey from Lianghe County, Dehong Autonomous Prefecture, Yunnan Province, China, could not be accurately identified as a known species using 16S rRNA gene sequencing. In this study, the taxonomic status of strain IMAU50987T was evaluated using whole-genome sequencing combined with phenotypic analyses. Strain IMAU50987T was Gram-positive, non-motile and non-spore-forming. Comparative of 16S rRNA gene sequence similarity analysis indicated that strain IMAU50987T could be attributed to the genus Bifidobacterium and most closely related to the B. mellis Bin7NT (98.5% identity). Comparative sequence analysis of eight housekeeping genes (pyrG, thrS, glnA1, recA, tuf, dnaB, rpoC and xfp) showed that strain IMAU50987T was different from closely related species. Phylogenomic analysis based on the 88 single-copy genes in the phylogenetic tree showed that strain IMAU50987T belonged to the B. asteroides group. The highest average nucleotide identity value for strain IMAU50987T was 77.0% in comparison with B. choladohabitans B14384H11T and B. apis F753-1T. However, the highest digital DNA-DNA hybridization value was 23.1% compared with B. apis F753-1T and B. xylocopae XV2T; the highest average amino acid identity value was 73.2% compared with B. polysaccharolyticum W8117T. These values are below the accepted threshold for novel species boundaries. The draft genome size of strain IMAU50987T was 1.9 Mb, and the DNA G+C content was 63.5%. The major cellular fatty acids of strain IMAU50987T were C16 : 0, C18 : 1 ω9c and Sum in Feature 7 (C19 : 1 ω7c/C19 : 1 ω6c). The cell-wall peptidoglycan was of the A3α l-Lys-Gly type. Based on data from this study, a novel species, Bifidobacterium favimelis sp. nov., is proposed with the type strain IMAU50987T (= GDMCC 1.4189T = JCM 36315T).