Bioinformatics Data Research Articles

Klotho exerts protection in chronic kidney disease associated with regulating inflammatory response and lipid metabolism.

The anti-aging protein Klotho plays a protective role in kidney disease, but its potential as a biomarker for chronic kidney disease (CKD) is controversial. Additionally, the main pathways through which Klotho exerts its effects on CKD remain unclear. Therefore, we used bioinformatics and clinical data analysis to determine its role in CKD. We analyzed the transcriptomic and clinical data from the Nephroseq v5 database and found that the Klotho gene was mainly expressed in the tubulointerstitium, and its expression was significantly positively correlated with estimated glomerular filtration rate (eGFR) and negatively correlated with blood urea nitrogen (BUN) in CKD. We further found that Klotho gene expression was mainly negatively associated with inflammatory response and positively associated with lipid metabolism in CKD tubulointerstitium by analyzing two large sample-size CKD tubulointerstitial transcriptome datasets. By analyzing 10-year clinical data from the National Health and Nutrition Examination Survey (NHANES) 2007-2016, we also found that Klotho negatively correlated with inflammatory biomarkers and triglyceride and positively correlated with eGFR in the CKD population. Mediation analysis showed that Klotho could improve renal function in the general population by modulating the inflammatory response and lipid metabolism, while in the CKD population, it primarily manifested by mediating the inflammatory response. Restricted cubic spline (RCS) analysis showed that the optimal concentration range for Klotho to exert its biological function was around 1000pg/ml. Kaplan-Meier curves showed that lower cumulative hazards of all-cause mortality in participants with higher levels of Klotho. We also demonstrated that Klotho could reduce cellular inflammatory response and improve cellular lipid metabolism by establishing an in vitro model similar to CKD. Our results suggest that Klotho exerts protection in CKD, which may be mainly related to the regulation of inflammatory response and lipid metabolism, and it can serve as a potential biomarker for CKD.

Developmental and Housekeeping Genes: Two Types of Genetic Organization in the Drosophila Genome.

We developed a procedure for locating genes on Drosophila melanogaster polytene chromosomes and described three types of chromosome structures (gray bands, black bands, and interbands), which differed markedly in morphological and genetic properties. This was reached through the use of our original methods of molecular and genetic analysis, electron microscopy, and bioinformatics data processing. Analysis of the genome-wide distribution of these properties led us to a bioinformatics model of the Drosophila genome organization, in which the genome was divided into two groups of genes. One was constituted by 65, in which the genome was divided into two groups, 62 genes that are expressed in most cell types during life cycle and perform basic cellular functions (the so-called "housekeeping genes"). The other one was made up of 3162 genes that are expressed only at particular stages of development ("developmental genes"). These two groups of genes are so different that we may state that the genome has two types of genetic organization. Different are the timings of their expression, chromatin packaging levels, the composition of activating and deactivating proteins, the sizes of these genes, the lengths of their introns, the organization of the promoter regions of the genes, the locations of origin recognition complexes (ORCs), and DNA replication timings.

Scientific figures interpreted by ChatGPT: strengths in plot recognition and limits in color perception

Emerging studies underscore the promising capabilities of large language model-based chatbots in conducting basic bioinformatics data analyses. The recent feature of accepting image inputs by ChatGPT, also known as GPT-4V(ision), motivated us to explore its efficacy in deciphering bioinformatics scientific figures. Our evaluation with examples in cancer research, including sequencing data analysis, multimodal network-based drug repositioning, and tumor clonal evolution, revealed that ChatGPT can proficiently explain different plot types and apply biological knowledge to enrich interpretations. However, it struggled to provide accurate interpretations when color perception and quantitative analysis of visual elements were involved. Furthermore, while the chatbot can draft figure legends and summarize findings from the figures, stringent proofreading is imperative to ensure the accuracy and reliability of the content.

RAB32 Ser71Arg in autosomal dominant Parkinson's disease: linkage, association, and functional analyses

Parkinson's disease is a progressive neurodegenerative disorder with multifactorial causes, among which genetic risk factors play a part. The RAB GTPases are regulators and substrates of LRRK2, and variants in the LRRK2 gene are important risk factors for Parkinson's disease. We aimed to explore genetic variability in RAB GTPases within cases of familial Parkinson's disease. We did whole-exome sequencing in probands from families in Canada and Tunisia with Parkinson's disease without a genetic cause, who were recruited from the Centre for Applied Neurogenetics (Vancouver, BC, Canada), an international consortium that includes people with Parkinson's disease from 36 sites in 24 countries. 61 RAB GTPases were genetically screened, and candidate variants were genotyped in relatives of the probands to assess disease segregation by linkage analysis. Genotyping was also done to assess variant frequencies in individuals with idiopathic Parkinson's disease and controls, matched for age and sex, who were also from the Centre for Applied Neurogenetics but unrelated to the probands or each other. All participants were aged 18 years or older. The sequencing and genotyping findings were validated by case-control association analyses using bioinformatic data obtained from publicly available clinicogenomic databases (AMP-PD, GP2, and 100 000 Genomes Project) and a private German clinical diagnostic database (University of Tübingen). Clinical and pathological findings were summarised and haplotypes were determined. In-vitro studies were done to investigate protein interactions and enzyme activities. Between June 1, 2010, and May 31, 2017, 130 probands from Canada and Tunisia (47 [36%] female and 83 [64%] male; mean age 72·7 years [SD 11·7; range 38-96]; 109 White European ancestry, 18 north African, two east Asian, and one Hispanic] underwent whole-exome sequencing. 15 variants in RAB GTPase genes were identified, of which the RAB32 variant c.213C>G (Ser71Arg) cosegregated with autosomal dominant Parkinson's disease in three families (nine affected individuals; non-parametric linkage Z score=1·95; p=0·03). 2604 unrelated individuals with Parkinson's disease and 344 matched controls were additionally genotyped, and five more people originating from five countries (Canada, Italy, Poland, Turkey, and Tunisia) were identified with the RAB32 variant. From the database searches, in which 6043 individuals with Parkinson's disease and 62 549 controls were included, another eight individuals were identified with the RAB32 variant from four countries (Canada, Germany, UK, and USA). Overall, the association of RAB32 c.213C>G (Ser71Arg) with Parkinson's disease was significant (odds ratio [OR] 13·17, 95% CI 2·15-87·23; p=0·0055; I2=99·96%). In the people who had the variant, Parkinson's disease presented at age 54·6 years (SD 12·75, range 31-81, n=16), and two-thirds had a family history of parkinsonism. RAB32 Ser71Arg heterozygotes shared a common haplotype, although penetrance was incomplete. Findings in one individual at autopsy showed sparse neurofibrillary tangle pathology in the midbrain and thalamus, without Lewy body pathology. In functional studies, RAB32 Arg71 activated LRRK2 kinase to a level greater than RAB32 Ser71. RAB32 Ser71Arg is a novel genetic risk factor for Parkinson's disease, with reduced penetrance. The variant was found in individuals with Parkinson's disease from multiple ethnic groups, with the same haplotype. In-vitro assays show that RAB32 Arg71 activates LRRK2 kinase, which indicates that genetically distinct causes of familial parkinsonism share the same mechanism. The discovery of RAB32 Ser71Arg also suggests several genetically inherited causes of Parkinson's disease originated to control intracellular immunity. This shared aetiology should be considered in future translational research, while the global epidemiology of RAB32 Ser71Arg needs to be assessed to inform genetic counselling. National Institutes of Health, the Canada Excellence Research Chairs program, Aligning Science Across Parkinson's, the Michael J Fox Foundation for Parkinson's Research, and the UK Medical Research Council.

CDCA5 promoted cell invasion and migration by activating TGF-β1 pathway in human ovarian cancer cells

BackgroundThe gene cell division cycle associated 5 (CDCA5), also called sororin, has oncogenic characteristics and is upregulated in various carcinomas. Nevertheless, the involvement of CDCA5 in ovarian cancer (OC), a highly aggressive form of cancer, and the underlying mechanism of metastasis remain inadequately investigated.ResultsThe bioinformatics data revealed a negative correlation between the patient’s survival and CDCA5 expression, which was overexpressed in OC. Functional assays also confirmed high expression levels of CDCA5 in OC tissues and cells. This suggests that CDCA5 may potentially enhance the motility, migration, and proliferation of OC cells invitro. It impedes DNA damage and apoptosis in OC cells, inhibiting xenograft development in nude mice. The RNA sequencing results suggest CDCA5 is majorly associated with biological functions related to the extracellular matrix (ECM) and influences the transforming growth factor (TGF) signaling pathway. Moreover, subsequent functional investigations elucidated that CDCA5 facilitated the migration and invasion of OC cells viathe TGF-β1/Smad2/3 signaling pathway activation.ConclusionsCDCA5 may be a strong potential therapeutic target for the treatment and management of OC.

The homozygous pathogenic variant of the POMGNT1 gene identified using whole-exome sequencing in Iranian family with congenital hydrocephalus

BackgroundHydrocephalus is one of the most common pathophysiological disabilities with a high mortality rate, which occurs both congenitally and acquired. It is estimated that genetic components are the etiology for up to 40% of hydrocephalus cases; however, causal mutations identified until now could only explain approximately 20% of congenital hydrocephalus (CH) patients, and most potential hydrocephalus-associated genes have yet to be determined. This study sought to find causal variations in a consanguineous family with four affected children diagnosed with hydrocephalus.Material and methodsIn this study, we evaluated twenty-five members of an extended family consisting of a nuclear family with four affected children resulting from a consanguineous couple and eighteen of their relatives, including one hydrocephalus case. The mother of this family was experiencing her 15th week of pregnancy, and cytogenetic evaluation was performed using amniocentesis to identify fetal chromosomal abnormalities. We conducted whole-exome sequencing (WES) on the genomic DNA of the proband to detect the CH-causing variants, followed by confirmation and segregation analysis of the detected variant in the proband, fetus, and family members through Sanger sequencing.ResultsFollowing the bioinformatic analysis and data filtering, we found a homozygous variant [NM_001243766.2:c.74G>A:p.W25X] within the protein O-mannose beta-1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) gene confirmed by Sanger sequencing in the proband and segregated with the hydrocephalus in the family. The variant was described as pathogenic and regarded as a nonsense-mediated mRNA decay (NMD) due to the premature stop codon, which results in a truncated protein.ConclusionThe results of the current study broadened the mutational gene spectrum of CH and our knowledge of the hydrocephalus etiology by introducing a novel homozygous variant within the POMGNT1 gene, which had never been previously reported solitary in these patients.

BnAP2-12 overexpression delays ramie flowering: evidence from AP2/ERF gene expression.

The APETALA2/ethylene response factor (AP2/ERF) superfamily plays a significant role in regulating plant gene expression in response to growth and development. To date, there have been no studies into whether the ramie AP2/ERF genes are involved in the regulation of flower development. Here, 84 BnAP2/ERF members were identified from the ramie genome database, and various bioinformatics data on the AP2/ERF gene family, structure, replication, promoters and regulatory networks were analysed. BnAP2-12 was transferred into Arabidopsis through the flower-dipping method. Phylogenetic analysis classified the 84 BnAP2/ERF members into four subfamilies: AP2 (18), RAV (3), ERF (42), and DREB (21). The functional domain analysis of genes revealed 10 conserved motifs. Genetic mapping localised the 84 members on 14 chromosomes, among which chromosomes 1, 3, 5, and 8 had more members. Collinearity analysis revealed that 43.37% possibly resulted from replication events during the evolution of the ramie genome. Promoter sequence analysis identified classified cis-acting elements associated with plant growth and development, and responses to stress, hormones, and light. Transcriptomic comparison identified 3,635 differentially expressed genes (DEGs) between male and female flowers (1,803 and 1,832 upregulated and downregulated genes, respectively). Kyoto Encyclopaedia of Genes and Genomes pathway analysis categorised DEGs involved in metabolic pathways and biosynthesis of secondary metabolites. Gene Ontology enrichment analysis further identified enriched genes associated with pollen and female gamete formations. Of the 84 BnAP2/ERFs genes, 22 and 8 upregulated and downregulated genes, respectively, were present in female flowers. Co-expression network analysis identified AP2/ERF members associated with flower development, including BnAP2-12. Subcellular localisation analysis showed that the BnAP2-12 protein is localised in the nucleus and cell membrane. Overexpression BnAP2-12 delayed the flowering time of Arabidopsis thaliana. These findings provide insights into the mechanism of ramie flower development.

Abstract 6220: TRIP13 augments pancreatic cancer progression- An integrated systems biology study

Abstract Background- Pancreatic cancer (PanCa) is one of the most aggressive forms of cancer and its incidence rate is continuously increasing every year. It is expected that by 2030, PanCa will become the 2nd leading cause of cancer related deaths in the United States due to the lack of understanding of the complexity of disease, early diagnostic methods, and extremely poor survival. Despite great advancements in biomedical research, there are very limited modalities available for the early detection of PanCa and its treatment. Thus, understanding of disease biology and identification of newer diagnostic and therapeutic modalities are high priority research in the field. Our group has identified Thyroid Receptor Interacting Protein -13 (TRIP13) as an oncogenic protein which contributes to early events of PanCa. Methodology- High dimensional omics and structural elucidation of TRIP13 was conducted using publicly available databases like Consurf, Phosphosite, GTEx and GEPIA2. Molecular biology techniques were applied to cross validate all our bioinformatics data. qPCR, immunoblotting and IHC analysis was performed in progressive PDAC cell lines and human tissues to decipher the expression level of TRIP13 in various pathological staging including functional enrichment analysis using Linkdomics. Results- The structural elucidation analysis revealed that most of the functionally exposed residues, phosphorylation and ubiquitylation sites situated at AAA domain of TRIP13. As compared to normal, TRIP13 is significantly over expressed in pancreatic tumor samples. Pathological staging 2 and 1 (early stages) showed relatively higher expression of TRIP13 as compared advanced stage. Of the total 7 isoforms of TRIP13, only one isoform (TRIP13-001) has shown the over expression in pancreatic tumor samples and shown association with poor survival. In correlation studies, TRIP13 has shown positive association with other pancreatic cancer biomarkers (CEACAM5, S100A4, MUC1, MSLN, CA125). The functional enrichment analyses suggest that TRIP13 is involved in important patho-physiological pathways like DNA repair, viral carcinogenesis, cellular senescence, and cell cycle. Our wet lab experiments also support our computational biology observation. Conclusion- This integrated computational biology study suggests a potential role of TRIP13 in pancreatic cancer progression via modulation of important oncological pathways. Wet lab experimentations also validated the involvement of TRIP13 in pancreatic cancer progression. TRIP13 expression may be associated with patient prognosis. Thus, its inhibitory small molecules can be useful for boosting pancreatic cancer therapies in clinical settings. Key words- TRIP13, Pancreatic cancer, Early events of cancer, Integrative Biology, Transcriptomics, Computational biology Citation Format: Swati Dhasmana, Anupam Dhasmana, Stella Rios, Iris A. Perez, Sheema Khan, Farrukh Afaq, Upender Manne, Murali M. Yallapu, Subhash C. Chauhan. TRIP13 augments pancreatic cancer progression- An integrated systems biology study [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 6220.

Abstract 2917: Purifying cancer-associated fibroblasts (CAFs) from glioblastoma and defining their role in therapeutic resistance

Abstract Glioblastoma (GBM), a challenging brain tumor, necessitates the development of new therapies due to poor patient prognoses, with a median survival of 14-16 months. Standard of care, as established almost 20 years ago, remains to be surgical resection followed by chemotherapy and radiation and till date there is no defined standard of care for recurrent GBM, highlighting the urgent need for the development of novel therapies. Recent studies have implicated the tumor microenvironment in these poor prognoses. While cancer associated fibroblasts (CAFs) are a key cellular component of the microenvironment of many systemic tumors, their presence in GBM had been doubted until we recently developed intriguing data identifying cells in GBM that express fibroblast markers, have fibroblast-like morphology, and, most importantly, transcriptomically resemble CAFs from systemic cancers rather than other stromal cells that express similar surface markers such as pericytes (Jain et al. 2023). Similar to other solid cancers, GBM CAFs increase tumor growth and drive immunosuppression. The role of CAFs in regulating these processes could have significant implications in response to standard of care therapy as well as novel immunotherapies. While our initial CAF isolation had just 50% purity, we have developed a novel flow-sorting algorithm for CAF enrichment driven by our bioinformatic data. Through this protocol, we have successfully established, the first of its kind, a biobank of patient derived GBM CAFs with greater than 90% purity. These CAFs are now being utilized to investigate their contribution in conferring resistance to chemotherapy (Temozolomide) and radiotherapy. The CAFs are also being assessed for their role in suppressing T cell function and resistance to immunotherapies. In parallel, the origin of GBM CAFs and their development trajectory is being analyzed to find molecular markers to specifically target CAFs in the GBM microenvironment. The work put together will provide insight on mechanisms employed by CAFs in altering the GBM microenvironment and a basis for developing novel combination approaches, alleviating therapeutic resistance caused due to the presence of CAFs in the GBM microenvironment. The results of this study have the potential to significantly attenuate therapeutic resistance in GBM and provide much needed treatment options and improvement in survival, for what remains one of the most dismal diagnoses in the clinic. Citation Format: Ankita Sati, Aakriti Mathur, Meeki Lad, Atul Saha, Manish K. Aghi. Purifying cancer-associated fibroblasts (CAFs) from glioblastoma and defining their role in therapeutic resistance [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 2917.

Abstract 2022: E7386, a Wnt/β-catenin signaling modulator, suppresses the differentiation of regulatory T cells in combination with lenvatinib plus anti-PD-1 antibody

Abstract Background: E7386, a selective inhibitor of the interaction between β-catenin and CREB binding protein (CBP), showed antitumor activity in preclinical tumor models with aberrant activation of the Wnt/β-catenin signaling pathway. Previously, we reported the enhanced antitumor activity of a triple combination (E7386 in combination with Lenvatinib [LEN, a multiple receptor tyrosine kinase inhibitor mainly targeting VEGFRs and FGFRs] plus anti-PD-1 antibody) versus a 2-drug combination (LEN plus anti-PD-1 antibody). In this study, we further investigated the immunomodulatory functions of E7386 as a triple combination by single-cell RNA-sequencing (scRNA-Seq) analysis and subsequent functional assays. Methods: Mice bearing subcutaneous RAG (mouse renal cell carcinoma) tumors were randomized into 4 groups (n=6 in each group), including ⑴ Nontreatment, ⑵ E7386 at 25 mg/kg (PO, QD), ⑶ LEN at 10 mg/kg (PO, QD) plus anti-PD-1 antibody at 10 mg/kg (IP, BIW), and ⑷ triple combination. Tumor tissues were collected after a 2-week treatment period. CD45+ immune cells were isolated from the tumors by magnetic cell separation (MACS) and subjected to scRNA-Seq using 10x Genomics pipelines. Bioinformatics data analysis was conducted with Clarivate Analytics. To evaluate the effects of E7386 on Treg differentiation, an in vitro-induced regulatory T cells (Tregs) differentiation assay was conducted. Naïve CD4+ T cells were isolated by MACS and plated under iTreg differentiation conditions (stimulation of TGF-β and IL-2) with or without treatment of E7386. Induced Tregs and conditional culture medium were collected for evaluation of Treg differentiation, including gene expression and protein analysis. Results: CD45+ immune cells were clustered into 14 cell-type groups. Increased proportion of monocytes, NK cells, Tregs, CD4+ T cells, CD8+ T cells, eosinophils, and neutrophils were observed after LEN plus anti-PD-1 treatment. Among these subpopulations, Treg were most significantly suppressed by E7386 plus LEN plus anti-PD-1. Gene expression analysis revealed that the triple combination suppressed the activated gene expression of the Stat5/Foxp3 axis in CD4+ T cells, particularly Tregs, induced by LEN plus anti-PD-1 treatment. Consistently, the iTreg differentiation assay confirmed the suppressive function of E7386 on the Treg differentiation rate. Multiple subsequent in vitro assays also demonstrated the inhibitory effects of E7386 on TGF-β secretion and the expression levels of TGF-β downstream target genes. Conclusion: In summary, these data suggest a suppressive role of E7386 on Treg differentiation, which leads to antitumor activity of E7386 in combination with lenvatinib and anti-PD-1 antibody in a preclinical tumor model. Citation Format: Jialing Shen, Ryoga Ishida, Masahiko Kume, Takayuki Kimura, Yusuke Adachi, Yu Kato. E7386, a Wnt/β-catenin signaling modulator, suppresses the differentiation of regulatory T cells in combination with lenvatinib plus anti-PD-1 antibody [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 2022.

Abstract 6257: Genome wide, high-throughput, high-resolution structural variation detection at low variant allele fraction for oncology samples

Abstract Structural variation detection remains an elusive undertaking. For decades, G-banded karyotyping has remained the standard practice for nearly all hematological malignancy samples. Cancers are diseases of the genome and structural variants (SVs) are a hallmark of nearly all cancers, are often drivers of oncogenicity, and can sometimes be targeted for treatment, as highlighted by the discovery of the Philadelphia chromosome and later the development of targeted kinase inhibitors. One of the most important limitations of karyotyping, its requirement for cells to divide in culture, has prevented its application for most solid tumors and therefore, there is considerably less understanding of the impact of SVs in solid tumors. Optical genome mapping is a technique that is revolutionizing cytogenomics and unlocking the understanding of SVs in the genome at a level never seen before. It not only can detect nearly all variants that a karyotype can, but it can provide 10,000x higher resolution and does not require cell culture. Because of this, it is being adopted for oncology studies across hematological malignancies and solid tumors. This study demonstrates the performance of a new high throughput Stratys instrument for generation of optical genome map data at extraordinarily high coverage depth. With this instrument, 12 samples can be loaded at once, each loaded onto its own cartridge and the instrument can collect 400x genome coverage for each of these samples in 24 hours. The instrument has random access to the cartridges so that completed samples can be swapped out to facilitate optimal instrument run efficiency. To date, this system has been demonstrated to run 60 samples in a week, each at 400x coverage. Data from the instrument can be processed by Bionano Access™ and VIA™ software to call SVs, copy number variants (CNVs), regions of loss of heterozygosity (LOH), on a genome wide basis, and to classify those variants automatically according to guidelines such as the National Comprehensive Cancer Network (NCCN), World Health Organization (WHO), and National Health Services (NHS, United Kingdom). This new high throughput system with advanced bioinformatic data analysis has the potential to further transform structural variation analysis in cancer research. Citation Format: Alex R. Hastie, Pat Lynch. Genome wide, high-throughput, high-resolution structural variation detection at low variant allele fraction for oncology samples [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 6257.

Abstract 2026: Single-cell RNA-sequencing analysis of tumor endothelial cells reveals suppression of specific endothelial subtypes by the combination treatment of Wnt/β-catenin signaling modulator E7386 plus lenvatinib

Abstract Background: Aberrant activation of Wnt/β-catenin signaling plays an important role in both carcinogenesis and modulation of the tumor microenvironment. E7386, a novel orally active antitumor agent, inhibits the interaction between β-catenin and CREB-binding protein (CBP) and thereby modulates Wnt/β-catenin signaling. We previously reported that E7386 in combination with lenvatinib (LEN), a multiple receptor tyrosine kinase inhibitor mainly targeting VEGFRs and FGFRs, showed a greater antitumor activity and reduction of tumor microvessels than each treatment alone in preclinical tumor models. Multiple clinical studies are in progress including the combination of E7386 plus LEN (NCT04008797). Here, we further tried to elucidate the mechanism underlying the antitumor and antiangiogenic effect of the combination of E7386 plus LEN by using single-cell RNA sequencing (scRNA-seq) of tumor endothelial cells (ECs). Methods: Mice bearing subcutaneous RAG (mouse renal cell carcinoma cell line) tumors were treated with E7386 at 25 mg/kg and/or with LEN at 10 mg/kg by daily oral gavage for 8 days. Tumor ECs (CD31+/CD45−) were isolated by magnetic cell separation and flow cytometry and were subjected to scRNA-seq using 10x Genomics pipeline. Bioinformatics data analysis was conducted by Clarivate Analytics. After quality control and filtering, ECs were gated by their expression of pan-EC markers and were further classified into 9 subtypes (tip, capillary, arterial, venous, proliferating, immature, interferon, activated artery, and lymphatic) according to previously reported markers for each subtype. Results: Among classified subtypes, proliferating and immature subtypes showed clear reduction in the percentage of total ECs with combination of E7386 plus LEN versus LEN monotherapy. In both proliferating and immature subtypes, a subset of Wnt/β-catenin downstream genes showed increased expression with LEN monotreatment. Conversely, these Wnt/β-catenin downstream genes were downregulated with the combination of E7386 plus LEN, suggesting that Wnt/β-catenin signaling may be associated with a drug resistance mechanism against LEN in these subtypes. Pathway analysis indicated that genes involved in cell proliferation and survival were downregulated by the E7386 plus LEN combination versus LEN monotreatment in both proliferating and immature subtypes. Conclusion: Taken together, these results suggest a role for E7386 in suppressing the activation of Wnt/β-catenin signaling induced by LEN monotreatment in specific EC subtypes; this activity leads to enhanced antiangiogenic and antitumor activity with E7386 plus LEN combination therapy in a preclinical tumor model. Citation Format: Masahiko Kume, Ryoga Ishida, Yoichi Ozawa, Jialing Shen, Hiroto Konishi, Megumi Kuronishi, Takayuki Kimura, Yusuke Adachi, Yu Kato. Single-cell RNA-sequencing analysis of tumor endothelial cells reveals suppression of specific endothelial subtypes by the combination treatment of Wnt/β-catenin signaling modulator E7386 plus lenvatinib [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 2026.

Aberrantly Glycosylated GLUT1 as a Poor Prognosis Marker in Aggressive Bladder Cancer.

Muscle-invasive bladder cancer (MIBC) remains a pressing health concern due to conventional treatment failure and significant molecular heterogeneity, hampering the development of novel targeted therapeutics. In our quest for novel targetable markers, recent glycoproteomics and bioinformatics data have pinpointed (glucose transporter 1) GLUT1 as a potential biomarker due to its increased expression in tumours compared to healthy tissues. This study explores this hypothesis in more detail, with emphasis on GLUT1 glycosylation patterns and cancer specificity. Immunohistochemistry analysis across a diverse set of human bladder tumours representing all disease stages revealed increasing GLUT1 expression with lesion severity, extending to metastasis, while remaining undetectable in healthy urothelium. In line with this, GLUT1 emerged as a marker of reduced overall survival. Revisiting nanoLC-EThcD-MS/MS data targeting immature O-glycosylation on muscle-invasive tumours identified GLUT1 as a carrier of short glycosylation associated with invasive disease. Precise glycosite mapping uncovered significant heterogeneity between patient samples, but also common glycopatterns that could provide the molecular basis for targeted solutions. Immature O-glycosylation conferred cancer specificity to GLUT1, laying the molecular groundwork for enhanced targeted therapeutics in bladder cancer. Future studies should focus on a comprehensive mapping of GLUT1 glycosites for highly specific cancer-targeted therapy development for bladder cancer.

MicroRNA-155 mediates multiple gene regulations pertinent to the role of human adipose-derived mesenchymal stem cells in skin regeneration.

Introduction: The role of Adipose-derived mesenchymal stem cells (AD-MSCs) in skin wound healing remains to be fully characterized. This study aims to evaluate the regenerative potential of autologous AD-MSCs in a non-healing porcine wound model, in addition to elucidate key miRNA-mediated epigenetic regulations that underlie the regenerative potential of AD-MSCs in wounds. Methods: The regenerative potential of autologous AD-MSCs was evaluated in porcine model using histopathology and spatial frequency domain imaging. Then, the correlations between miRNAs and proteins of AD-MSCs were evaluated using an integration analysis in primary human AD-MSCs in comparison to primary human keratinocytes. Transfection study of AD-MSCs was conducted to validate the bioinformatics data. Results: Autologous porcine AD-MSCs improved wound epithelialization and skin properties in comparison to control wounds. We identified 26 proteins upregulated in human AD-MSCs, including growth and angiogenic factors, chemokines and inflammatory cytokines. Pathway enrichment analysis highlighted cell signalling-associated pathways and immunomodulatory pathways. miRNA-target modelling revealed regulations related to genes encoding for 16 upregulated proteins. miR-155-5p was predicted to regulate Fibroblast growth factor 2 and 7, C-C motif chemokine ligand 2 and Vascular cell adhesion molecule 1. Transfecting human AD-MSCs cell line with anti-miR-155 showed transient gene silencing of the four proteins at 24h post-transfection. Discussion: This study proposes a positive miR-155-mediated gene regulation of key factors involved in wound healing. The study represents a promising approach for miRNA-based and cell-free regenerative treatment for difficult-to-heal wounds. The therapeutic potential of miR-155 and its identified targets should be further explored in-vivo.

Serum proteins differentially expressed in gestational diabetes mellitus assessed using isobaric tag for relative and absolute quantitation proteomics.

As a well-known fact to the public, gestational diabetes mellitus (GDM) could bring serious risks for both pregnant women and infants. During this important investigation into the linkage between GDM patients and their altered expression in the serum, proteomics techniques were deployed to detect the differentially expressed proteins (DEPs) of in the serum of GDM patients to further explore its pathogenesis, and find out possible biomarkers to forecast GDM occurrence. To investigation serum proteins differentially expressed in GDM were assessed using isobaric tag for relative and absolute quantitation (iTRAQ) proteomics and bioinformatics analyses. Subjects were divided into GDM and normal control groups according to the IADPSG diagnostic criteria. Serum samples were randomly selected from four cases in each group at 24-28 wk of gestation, and the blood samples were identified by applying iTRAQ technology combined with liquid chromatography-tandem mass spectrometry. Key proteins and signaling pathways associated with GDM were identified by bioinformatics analysis, and the expression of key proteins in serum from 12 wk to 16 wk of gestation was further verified using enzyme-linked immunosorbent assay (ELISA). Forty-seven proteins were significantly differentially expressed by analyzing the serum samples between the GDM gravidas as well as the healthy ones. Among them, 31 proteins were found to be upregulated notably and the rest 16 proteins were downregulated remarkably. Bioinformatic data report revealed abnormal expression of proteins associated with lipid metabolism, coagulation cascade activation, complement system and inflammatory response in the GDM group. ELISA results showed that the contents of RBP4, as well as ANGPTL8, increased in the serum of GDM gravidas compared with the healthy ones, and this change was found to initiate from 12 wk to 16 wk of gestation. GDM symptoms may involve abnormalities in lipid metabolism, coagulation cascade activation, complement system and inflammatory response. RBP4 and ANGPTL8 are expected to be early predictors of GDM.

Strengths and limitations of reduced representation bisulfite sequencing (RRBS) in the perspective of DNA methylation analysis in fish: a case-study on rainbow trout spermatozoa.

DNA methylation in CpG dinucleotides is an important epigenetic mark in fish spermatozoa since it has been shown that some sperm methylome features are transmitted to the offspring. Reduced representation bisulfite sequencing (RRBS) is one genome-scale methods developed to assess DNA methylation at CpG sites. It allows the sequencing of a reduced fraction of the genome expected to be enriched in CpGs. The aim of this study is to characterize the extent of the CpG sites that can be identified in the RRBS-reduced sequenced fraction of rainbow trout spermatozoa, in order to evaluate the potential of RRBS for sperm DNA methylation studies. We observed that RRBS did provide a reduced amount of genomic data, the sum of the CpGs analyzed on 12 males spanning 9% of the total genomic CpGs. CpGs were only slightly enriched in the RRBS data (×1.7 times the sequenced nucleotides), the possible causes being linked to trout genome structure and sequenced fragments size. All genomic functional features were represented in our CpG dataset, with a noticeable enrichment in exons but, strikingly, not in promoters. The number of CpGs shared between biological replicates was low, but this proportion reached workable values from six biological replicates (46% of the analyzed cytosines) on. The choices that are to be made regarding fragment size selection and the options during bioinformatic data processing are discussed. In all, RRBS is a relevant first-approach method to scan the CpG DNA methylation status of spermatozoa along rainbow trout genome, although in a very reduced pattern among biological replicates.

Designing Potential Antisense Oligonucleotides (ASO) against Lung Cancer Associated microRNA and Studying their Interaction

Abstract: MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as gene regulators. They regulate diverse biological processes and bioinformatics data indicates that each miRNA can control hundreds of gene targets, underscoring the potential influence of miRNAs on almost every genetic pathway. miRNA mutations or mis-expression correlate with various human cancers and miRNAs can function as tumor suppressors and oncogenes. miRNAs have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of cancer. In this context, we identified various miRNAs associated with the prevalent cancer phenotype NSCLC/SCLC and used the respective target miRNA sequences as templates for designing antisense oligonucleotides (ASOs). We investigated the interaction of this cognate oligonucleotides and target miRNAs to hypotheses the inhibitory potential of such oligonucleotides through molecular docking studies.

Novel prognostic marker TGFBI affects the migration and invasion function of ovarian cancer cells and activates the integrin αvβ3-PI3K-Akt signaling pathway.

Individual patients with ovarian cancer show remarkably different prognosis. Present prognostic models for ovarian cancer mainly focus on clinico-pathological parameters, so quantifiable prognostic markers at molecular level are urgently needed. Platelets contribute to ovarian cancer progression, but have not been considered as biomarkers likely due to their instability. Here, we aimed to search for a stable prognostic marker from platelet-treated ovarian cancer cells, and explore its functions and mechanisms. Microarrays analysis was done with platelet-treated SKOV-3 ovarian cancer cells. Relevant studies were searched in the Gene Expression Omnibus (GEO) database. The candidate genes were determined by differentially expressed genes (DEGs), Venn diagram drawing, protein-protein interaction (PPI) network, Cox proportional hazards model and Kaplan-Meier analysis. The expression of TGFBI in clinical samples was assessed by immunehistochemical staining (IHC), and the association of TGFBI levels with the clinic-pathological characteristics and prognosis in ovarian cancer patients was evaluated by univariate and multivariate analysis. The functions of TGFBI were predicted using data from TCGA, and validated by in vitro and in vivo experiments. The mechanism exploration was performed based on proteomic analysis, molecular docking and intervention study. TGFBI was significantly higher expressed in the platelet-treated ovarian cancer cells. An analysis of bioinformatics data revealed that increased expression of TGFBI led to significant decrease of overall survival (OS), progression-free survival (PFS) and post-progression survival (PPS) in ovarian cancer patients. Tissue microarray results showed that TGFBI was an independent factor for ovarian cancer, and TGFBI expression predict poor prognosis. Functionally, TGFBI affected the migration and invasion of ovarian cancer cells by regulation of epithelial mesenchymal transition (EMT) markers (CDH1 and CDH2) and extracellular matrix (ECM) degradation proteins (MMP-2). Mechanistically, TGFBI phosphorylated PI3K and Akt by combining integrin αvβ3. We found out TGFBI as a novel prognostic indicator for ovarian cancer patients. TGFBI could promote metastasis in ovarian cancer by EMT induction and ECM remodeling, which might be associated with the activation of integrin αvβ3-PI3K-Akt signaling pathway.

Fitness benefits of a synonymous substitution in an ancient EF-Tu gene depend on the genetic background.

Synonymous mutations are changes to DNA sequence, which occur within translated genes but which do not affect the protein sequence. Although often referred to as silent mutations, evidence suggests that synonymous mutations can affect gene expression, mRNA stability, and even translation efficiency. A collection of both experimental and bioinformatic data has shown that synonymous mutations can impact cell phenotype, yet less is known about the molecular mechanisms and potential of beneficial or adaptive effects of such changes within evolved populations. Here, we report a beneficial synonymous mutation acquired via experimental evolution in an essential gene variant encoding the translation elongation factor protein EF-Tu. We demonstrate that this particular synonymous mutation increases EF-Tu mRNA and protein levels as well as global polysome abundance on RNA transcripts. Although presence of the synonymous mutation is clearly causative of such changes, we also demonstrate that fitness benefits are highly contingent on other potentiating mutations present within the genetic background in which the mutation arose. Our results underscore the importance of beneficial synonymous mutations, especially those that affect levels of proteins that are key for cellular processes.IMPORTANCEThis study explores the degree to which synonymous mutations in essential genes can influence adaptation in bacteria. An experimental system whereby an Escherichia coli strain harboring an engineered translation protein elongation factor-Tu (EF-Tu) was subjected to laboratory evolution. We find that a synonymous mutation acquired on the gene encoding for EF-Tu is conditionally beneficial for bacterial fitness. Our findings provide insight into the importance of the genetic background when a synonymous substitution is favored by natural selection and how such changes have the potential to impact evolution when critical cellular processes are involved.

Increasing of Compression Efficiency for Genomic Data by Manipulating Empirical Entropy

Sharing bio-informatics data is the key point to constructing a mobile and effective telemedicine network that brings with it various difficulties. A crucial challenge with this tremendous amount of information is storing it reversibly and analysing terabytes of data. Robust compression algorithms come up with a high rate of text and image compression ratios. However, the achievement of these advanced techniques has remained in a limited range since, intrinsically, the entropy contained by the raw data primarily determines the efficiency of compression. To enhance the performance of a compression algorithm, entropy of raw data needs to be reduced before any basic compression which reveals more effective redundancy. In this study, we use reversible sorting techniques to reduce the entropy thus providing higher efficiency in the case of integrating into compression technique for raw genomic data. To that end, permutation-based reversible sorting algorithms, such as Burrow-wheeler, are designed as a transform for entropy reduction. The algorithm achieves a low-entropy sequence by reordering raw data reversibly with low complexity and a fast approach. The empirical entropy, a quantitative analysis, shows a significant reduction of uncertainty has been achieved.