Quantification Of Transcripts Research Articles

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Expression of alternatively spliced PD-L1 RNA with skipping of transmembrane domain.

e14596 Background: Programmed death-ligand 1 (PD-L1) is a transmembrane ligand for the programmed cell death protein 1 (PD-1), a receptor that inhibits T-cell activity. The PD-L1/PD-1 immune checkpoint axis has been successfully targeted to enhance antitumor immune responses. This gene is composed of seven exons. Exons 1-4 are responsible for the extracellular domain, exon 5 for the transmembrane domain, and exons 6-7 for the intracellular domain. Emerging data indicates that alternative splicing in genes is an important mechanism for expression of different isoforms that may influence antibody-based therapies. We explored the potential of the presence of alternative splicing in PD-L1 transcripts via sequencing of PD-L1 RNA in patients with various type of solid tumor cancers. Methods: RNA was extracted from 3443 FFPE solid tumor samples including lung, colon, pancreas, breast, and other cancer types. In addition, cfRNA was extracted from 532 peripheral blood plasma samples from patients with solid tumor cancers. RNA was sequenced using a hybrid capture targeted RNA panel on FFPE samples and peripheral blood with analysis focused on PD-L1 gene transcript. Quantification of RNA transcript was done using Salmon algorithm. Results: Of the 3443 FFPE samples, 38 (1.2%) samples showed alternative splicing involving deletion of exon 5 (PD-L1∆Ex5). The alternative splicing was in 2.10% of lung cancer, 2% of colon cancer, 0.70% of pancreatic cancer, 1.40% of gastric cancer, 0.38% of breast cancer, 0.30% of ovarian cancer, 1.10% of sarcoma cases, 1.50% of glioblastomas, 3.30% of thyroid cancer and 2% of urothelial cancer. Of the 532 cfRNA samples, 20 (3.7%) showed PD-L1∆Ex5. The median percentage of PD-L1∆Ex5 transcripts was 2.2% of total PD-L1 transcripts in tissue samples as compared with 6.6% in cfDNA samples. In tissue samples, there was a correlation between levels of PD-L1 and PD-L1∆Ex5 (r = .567, p < .001). In contrast, cfRNA showed no significant correlation between levels of PD-L1 and levels of PD-L1∆Ex5 (r = .268). Conclusions: PD-L1 exon 5 skipping was detected in a number of patients with different types of solid tumor cancers. The percentage of skipping transcripts is low in cells and relatively higher in cfRNA. Since exon 5 skipping deletes the transmembrane domain, the PD-L1 protein may remain in the cytoplasm or perhaps is secreted as cell-free PD-L1 protein. The demonstration of relatively higher levels of PD-L1∆Ex5 in cfRNA is likely reflecting higher turnover of cells and raises the possibility that cells with this isoform of PD-L1 are more aggressive than cells without the expression of this isoform. Further studies are needed to explore the clinical relevance of the expression of such abnormal PD-L1 protein on treatment with immune checkpoint inhibitors.

Technical challenges for standardization of molecular monitoring in chronic myeloid leukaemia in developing countries

Quantification of BCR-ABL after treatment with tyrosine kinase inhibitors is an important therapeutic indicator for patients with chronic myeloid leukaemia (CML). An international scale (IS) for BCR-ABL was established in 2012 to improve the comparability of results between different laboratories. BCR-ABL measurement is technically challenging, especially in developing countries where the process of standardization isn’t easily available. The aim of this study was to assess the technical advantages of reporting results on the IS using the GeneXpert BCR-ABL assay, compared to the RQ-PCR standard method currently recommended by the European Leukaemia Network but not standardised in our laboratory and to compare the results obtained by both methods. BCR-ABL transcript quantification was performed in 51 samples from CML patients by both methods. The operator hands-on time was reduced using the automated method. A good linear correlation was observed in the results between the two methods. The concordance at logarithmic intervals reached 64%. When the major molecular response (MMR) and the early molecular response (EMR) were analysed, 90% and 80% agreement, respectively, were achieved. However, the discordances in the remaining cases could have misled the therapeutic approach if only a standard method was used. The complexity of the process to establish a laboratory-specific conversion factor in developing countries can be overcome by the use of the GeneXpert automated system, which represents a rapid, accurate and reliable clinical tool to correctly define molecular responses on the IS and to allow clinicians to achieve better patient management.

Stress granule related-genes during blastogenetic cycle of two colonial ascidians: Botryllus schlosseri compared to Botryllus primigenus.

AbstractBackgroundColonial ascidians are chordates known to reproduce both sexually and asexually. In the present study, we used the Mediterranean species Botryllus schlosseri and the Japanese Botryllus primigenus to investigate the possible role of tiar, ttp, and g3bp in the periodical renewal of the colonies, defined by generation changes or takeovers. In this scenario, the above genes, which codify key components for the formation of stress granules, storing specific mRNAs, can play a pivotal role, allowing the regulation of processes such as stress responses, cell proliferation, and stem cell development.ResultsWe characterized tiarx1, ttp, and g3bp2 sequences in B. schlosseri and B. primigenus. Then, we analyzed gene transcription by in situ hybridization in hemolymph cells and colony tissues, and we proceeded with quantification of the gene transcription by quantitative real‐time PCR, during the phases of the colonial blastogenetic cycle.ConclusionsOur results allowed us to assign a role in the development of the new colonial generations to the studied genes.

The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

Strigolactones promote flowering by inducing the miR319-LA-SFT module in tomato

Strigolactones are a class of phytohormones with various functions in plant development, stress responses, and in the interaction with (micro)organisms in the rhizosphere. While their effects on vegetative development are well studied, little is known about their role in reproduction. We investigated the effects of genetic and chemical modification of strigolactone levels on the timing and intensity of flowering in tomato (Solanum lycopersicum L.) and the molecular mechanisms underlying such effects. Results showed that strigolactone levels in the shoot, whether endogenous or exogenous, correlate inversely with the time of anthesis and directly with the number of flowers and the transcript levels of the florigen-encoding gene SINGLE FLOWER TRUSS (SFT) in the leaves. Transcript quantifications coupled with metabolite analyses demonstrated that strigolactones promote flowering in tomato by inducing the activation of the microRNA319-LANCEOLATE module in leaves. This, in turn, decreases gibberellin content and increases the transcription of SFT. Several other floral markers and morpho-anatomical features of developmental progression are induced in the apical meristems upon treatment with strigolactones, affecting floral transition and, more markedly, flower development. Thus, strigolactones promote meristem maturation and flower development via the induction of SFT both before and after floral transition, and their effects are blocked in plants expressing a miR319-resistant version of LANCEOLATE. Our study positions strigolactones in the context of the flowering regulation network in a model crop species.

MIR4435-2HG as a possible novel predictive biomarker of chemotherapy response and death in pediatric B-cell ALL.

Introduction: Although B-cell acute lymphoblastic leukemia (B-cell ALL) survival rates have improved in recent years, Hispanic children continue to have poorer survival rates. There are few tools available to identify at the time of diagnosis whether the patient will respond to induction therapy. Our goal was to identify predictive biomarkers of treatment response, which could also serve as prognostic biomarkers of death, by identifying methylated and differentially expressed genes between patients with positive minimal residual disease (MRD+) and negative minimal residual disease (MRD-). Methods: DNA and RNA were extracted from tumor blasts separated by immunomagnetic columns. Illumina MethlationEPIC and mRNA sequencing assays were performed on 13 bone marrows from Hispanic children with B-cell ALL. Partek Flow was used for transcript mapping and quantification, followed by differential expression analysis using DEseq2. DNA methylation analyses were performed with Partek Genomic Suite and Genome Studio. Gene expression and differential methylation were compared between patients with MRD-/- and MRD+/+ at the end of induction chemotherapy. Overexpressed and hypomethylated genes were selected and validated by RT-qPCR in samples of an independent validation cohort. The predictive ability of the genes was assessed by logistic regression. Survival and Cox regression analyses were performed to determine the association of genes with death. Results: DAPK1, BOC, CNKSR3, MIR4435-2HG, CTHRC1, NPDC1, SLC45A3, ITGA6, and ASCL2 were overexpressed and hypomethylated in MRD+/+ patients. Overexpression was also validated by RT-qPCR. DAPK1, BOC, ASCL2, and CNKSR3 can predict refractoriness, but MIR4435-2HG is the best predictor. Additionally, higher expression of MIR4435-2HG increases the probability of non-response, death, and the risk of death. Finally, MIR4435-2HG overexpression, together with MRD+, are associated with poorer survival, and together with overexpression of DAPK1 and ASCL2, it could improve the risk classification of patients with normal karyotype. Conclusion: MIR4435-2HG is a potential predictive biomarker of treatment response and death in children with B-cell ALL.

Biochemical profile and oocyte quality of primiparous Bos indicus cows submitted to a timed artificial insemination protocol

The objective of this study was to verify the causes of the lower response of primiparous Bos indicus cows to the ovulation synchronization protocol. Two experiments were performed to evaluate the biochemical profile, oocyte and follicular cell quality (Experiment 1) and pregnancy (Experiment 2). In Experiment 1, suckled primiparous (n = 24) and multiparous cows (n = 24) were submitted to ovum pick up (OPU). On Day 0 (D0), all cows received 2 mg of estradiol benzoate (EB) and an intravaginal progesterone insert (P4). Five days (D5) after the first hormonal administration (EB + P4), all follicles larger than 3 mm were counted on each ovary, and ovum pick-up (OPU) was performed. On day 12 (D12), the intravaginal progesterone insert was removed, and measurement and aspiration of the largest follicle were performed. Blood samples were collected on D5 and D12 to evaluate the concentrations of glucose, cholesterol, NEFA, IGF-1 and insulin. In Experiment 2, suckled primiparous (n = 50) and multiparous (n = 50) cows were subjected to an ovulation synchronization protocol based on E2/P4 (D0: 2 mg EB plus P4 intravaginal insert; D8: 500 μg of cloprostenol, 1 mg cypionate estradiol and 300UI of eCG; D10: artificial insemination). In addition, blood samples were collected on D10 for evaluation of the same hormones and metabolites described in Experiment 1. In all studies, calves remained with the cows during the experimental period. In experiment 1, the number of oocytes grade 1 (P = 0.83), grade 2 (P = 0.23) and grade 3 (P = 0.51), total number of retrieved oocytes (P = 0.14), oocyte quality index (P = 0.93) and total viable oocytes (P = 0.38) did not differ between primiparous and multiparous cows. The number of follicles at the time of follicular aspiration (20.7 ± 1.5 vs. 18.0 ± 1.9; P = 0.05) and the diameter of the largest follicle on D12 (13.5 ± 0.6 vs. 11.4 ± 0.6; P = 0.04) were greater in multiparous cows, and the number of degenerated oocytes was greater in primiparous cows (1.9 ± 0.7 vs. 1.2 ± 0.3; P = 0.05). On D5, the concentrations of IGF-1 (P = 0.47), insulin (P = 0.08), total cholesterol (P = 0.47), NEFA (P = 0.77) and glucose (P = 0.55) in the blood and IGF-1 (P = 0.97) and insulin (P = 0.11) in the follicular fluid did not differ between parity groups. On D12, there was no difference in the concentrations of IGF-1 (P = 0.26), total cholesterol (P = 0.32), NEFAs (P = 0.31) and glucose (P = 0.93) in the blood between primiparous and multiparous cows, however, the serum insulin concentration (P = 0.04) was higher in primiparous cows. There was no correlation between serum and follicular fluid insulin concentrations (r = 0.17; P = 0.31), however, there was a low correlation between serum and follicular fluid IGF-1 concentrations (r = 0.47; P = 0.002). Quantification of transcripts did not differ between parity groups. In experiment 2, concentrations of NEFA (P = 0.12) and insulin (P = 0.16) in the blood and P/AI (P = 0.93) did not differ between parity [60 % (30/50) primiparous vs. 60 % (30/50) multiparous]. In contrast, blood concentrations of IGF-1 (P = 0.0001), total cholesterol (P = 0.005) and glucose (P = 0.01) were greater in primiparous cows. It was concluded that the oocyte quality and expression of the genes evaluated in the granulosa cells were not different between primiparous and multiparous cows. Unexpectedly, the pregnancy rate did not differ between parity. Nevertheless, the blood concentrations of IGF-1, total cholesterol and glucose were greater in primiparous cows.

Comparative Analysis of Single-Cell RNA Sequencing Methods with and without Sample Multiplexing.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful technique for investigating biological heterogeneity at the single-cell level in human systems and model organisms. Recent advances in scRNA-seq have enabled the pooling of cells from multiple samples into single libraries, thereby increasing sample throughput while reducing technical batch effects, library preparation time, and the overall cost. However, a comparative analysis of scRNA-seq methods with and without sample multiplexing is lacking. In this study, we benchmarked methods from two representative platforms: Parse Biosciences (Parse; with sample multiplexing) and 10x Genomics (10x; without sample multiplexing). By using peripheral blood mononuclear cells (PBMCs) obtained from two healthy individuals, we demonstrate that demultiplexed scRNA-seq data obtained from Parse showed similar cell type frequencies compared to 10x data where samples were not multiplexed. Despite relatively lower cell capture affecting library preparation, Parse can detect rare cell types (e.g., plasmablasts and dendritic cells) which is likely due to its relatively higher sensitivity in gene detection. Moreover, a comparative analysis of transcript quantification between the two platforms revealed platform-specific distributions of gene length and GC content. These results offer guidance for researchers in designing high-throughput scRNA-seq studies.

Abstract 6251: Validation and implementation of whole-transcriptome sequencing assay into cancer care: An experience from a cancer center

Abstract Introduction Transcriptome sequencing is a well-established diagnostic tool to characterize and quantify gene expression profiles and to detect fusion transcripts. The utilization of whole-transcriptome sequencing (WTS), in clinical workflows can tremendously help improve diagnosis, risk stratification, and therapy selection. However, there are multiple challenges to widespread clinical implementation of WTS including nucleic acid input, defining appropriate quality control (QC) criteria, tumor specific baseline controls and depth of sequencing. Here, we present a validation study to deploy routine whole transcriptome sequencing with automated analysis in a mid-size academic center. Methods Nucleic acid material was obtained through clinical residual samples in the Center for Genomics and Advanced Technology. RNA samples were extracted using the Purigen Ionic® FFPE to Pure RNA Kit. RNA input sample quantities ranged from 34-100ng. WTS libraries were prepared using the Illumina® RNA Prep with Enrichment (L) Tagmentation kit with the Illumina Exome Panel for FFPE tumor samples. The validation was performed on a set of patient samples with known clinically reported variants from the Illumina TruSight® Tumor 170 assay. A total of 84 true positives with reportable fusions, 5 splicing events, 16 negative samples, and 15 limit of detection (LOD) samples were used. Libraries were prepared in a three-plex format. Sequencing was performed on the Novaseq-6000 system using S2 and S4 flow cells with a target read count of 100 million. All data was processed through the AUGMET informatics platform for demultiplexing, quality control, fusion detection, transcript quantification and determination of alternatively spliced transcript. Results The WTS validation assay demonstrated 98% (n=49/50) sensitivity, with only one false negative, and 100% (n=68/68) specificity. A final review of our data was determined to develop stable QC standards for RNA sequenced libraries to be > 60M reads and > 130bp read length. When analyzing (LOD) for WTS, interestingly, read length was inversely proportional to input and as a result detection of the clinically relevant fusion. Based on our LOD experiments, a minimum input threshold of 20ng of RNA was determined to maintain the sensitivity and specificity. An LOD study for tumor purity was not performed due to the known lack of correlation between tumor purity and fusion transcript quantity. Discussion WTS can detect all clinically reported fusions and splicing events with comparable sensitivity to panel assays. Moreover, simultaneously available transcript quantification and variant calling provide the ability to implement several disease classifiers. This work lays the foundation for integration with our clinically available tumor exome sequencing assay to allow joint exome transcriptome analysis in a single informatics platform. Citation Format: Jillian A. Moran, Edward G. Hughes, Donald C. Green, Shrey Sukhadia, Gregory J. Tsongalis, Samantha F. Allen, Laura Tafe, Brianna Houde, Nevena B. Ognjenovic, Jennifer N. Barbuto, Ivy Riano, Parth Shah. Validation and implementation of whole-transcriptome sequencing assay into cancer care: An experience from a cancer center [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 6251.

Abstract 7488: Detection of PD-L1 mRNA expression in circulating tumor cells (CTCs) from patients with metastatic NSCLC, HNSCC and melanoma using a novel highly sensitive commercially available CE-IVD kit

Abstract Background: We have previously reported the development of an RT-qPCR assay for PD-L1 expression in CTCs (Strati et al, Ann Oncol, 2017). This assay is now commercially available as Oncolipsy PD-L1 kit (CE-IVD, Pharmassist, Greece) and we report here it’s application in size-based enriched CTCs from patients with metastatic NSCLC, HNSCC and melanoma, before and during PD-1/PD-L1 blockade therapies. Patients and Methods: The Oncolipsy PD-L1 kit was analytically validated using Mini-Genes as positive controls (IDT, USA). The analytical sensitivity was tested by spiking known numbers of H1975 cells in peripheral blood (PB) samples from healthy donors (HD). In total 95 patients were enrolled in the study: 69 patients with metastatic NSCLC, 19 patients with metastatic HNSCC and 7 patients with metastatic melanoma. 40 PB samples from HD were used as a control group. 10 ml of PB was collected at baseline (V0), two months after immunotherapy (V1) and at the time of disease progression (PD) or 18 months after initiation of immunotherapy (V2). PD-L1 and B2M transcripts were quantified in cDNAs obtained from CTCs isolated with the size-based PARSORTIX (ANGLE, UK) device. A sample was considered as CTC-positive when cDNAs were positive for CK-8, CK-18 or CK-19 expression. Results: Spiking experiments have shown that 10 H1975cells/10ml PB can be detected using the kit. In CTC-positive samples, overexpression of PD-L1 was detected in 15/58(25.8%) patients at V0, in 6/34(17.6%) patients at V1 and in 3/11(27.3%) at V2. Patients with elevated expression of PD-L1 at V1 in respect to V0 and detectable CTCs had longer PFS (18.5mo vs. 13.2mo, p=0.033) and longer OS (24.8mo vs. 12.8mo, p = 0.071) after initiation of immunotherapy. Patients with elevated PD-L1 at V1 compared to V0 benefited from immunotherapy, with a longer DFS (17.8mo vs. 13.4mo) and OS (29.4mo vs. 26.9mo). Conclusions: The commercially available Oncolipsy PD-L1 kit can be used for the quantification of PD-L1 transcripts in CTCs. Expression of PD-L1 in size-based enriched CTCs can predict response to PD-1/PD-L1 blockade therapy. Acknowledgements: This study has been financially supported by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T1RCI-02935). Trial registration: ClinicalTrials.gov Identifier: NCT04490564 Citation Format: Areti Strati, Martha Zavridou, Stavroula Smilkou, Victoria Tserpeli, Eleni Tzanikou, Dimitra Stergiopoulou, Eleni Efthimiadou, Emily Tsaroucha, Amanda Psyrri, Aggeliki Sfika, Evangelos Bournakis, Ioanna Balgkouranidou, Stylianos Kakolyris, Ioannis Boukovinas, Christos Papadimitriou, Loukas Kaklamanis, Ioanna Koukli, Evi Lianidou. Detection of PD-L1 mRNA expression in circulating tumor cells (CTCs) from patients with metastatic NSCLC, HNSCC and melanoma using a novel highly sensitive commercially available CE-IVD kit [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 7488.

Targeted DNA sequencing of high-grade serous ovarian carcinoma reveals association of TP53 mutations with platinum resistance when combined with gene expression.

High-grade serous ovarian carcinoma (HGSC) is the most common subtype of ovarian cancer and is among the most fatal gynecological malignancies worldwide, due to late diagnosis at advanced stages and frequent therapy resistance. In 47 HGSC patients, we assessed somatic and germline genetic variability of a custom panel of 144 known or suspected HGSC-related genes by high-coverage targeted DNA sequencing to identify the genetic determinants associated with resistance to platinum-based therapy. In the germline, the most mutated genes were DNAH14 (17%), RAD51B (17%), CFTR (13%), BRCA1 (11%), and RAD51 (11%). Somatically, the most mutated gene was TP53 (98%), followed by CSMD1/2/3 (19/19/36%), and CFTR (23%). Results were compared with those from whole exome sequencing of a similar set of 35 HGSC patients. Somatic variants in TP53 were also validated using GENIE data of 1287 HGSC samples. Our approach showed increased prevalence of high impact somatic and germline mutations, especially those affecting splice sites of TP53, compared to validation datasets. Furthermore, nonsense TP53 somatic mutations were negatively associated with patient survival. Elevated TP53 transcript levels were associated with platinum resistance and presence of TP53 missense mutations, while decreased TP53 levels were found in tumors carrying mutations with predicted high impact, which was confirmed in The Cancer Genome Atlas data (n = 260). Targeted DNA sequencing of TP53 combined with transcript quantification may contribute to the concept of precision oncology of HGSC. Future studies should explore targeting the p53 pathway based on specific mutation types and co-analyze the expression and mutational profiles of other key cancer genes.

Unveiling unique alternative splicing responses to low temperature in Zoysia japonica through ZjRTD1.0, a high-quality reference transcript dataset.

Inadequate reference databases in RNA-seq analysis can hinder data utilization and interpretation. In this study, we have successfully constructed a high-quality reference transcript dataset, ZjRTD1.0, for Zoysia japonica, a widely-used turfgrass with exceptional tolerance to various abiotic stress, including low temperatures and salinity.This dataset comprises 113,089 transcripts from 57,143 genes. BUSCO analysis demonstrates exceptional completeness (92.4%) in ZjRTD1.0, with reduced proportions of fragmented (3.3%) and missing (4.3%) orthologs compared to prior datasets. ZjRTD1.0 enables more precise analyses, including transcript quantification and alternative splicing assessments using public datasets, which identified a substantial number of differentially expressed transcripts (DETs) and differential alternative splicing (DAS) events, leading to several novel findings on Z. japonica's responses to abiotic stresses. First, spliceosome gene expression influenced alternative splicing significantly under abiotic stress, with a greater impact observed during low-temperature stress. Then, a significant positive correlation was found between the number of differentially expressed genes (DEGs) encoding protein kinases and the frequency of DAS events, suggesting the role of protein phosphorylation in regulating alternative splicing. Additionally, our results suggest possible involvement of serine/arginine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) in generating inclusion/exclusion isoforms under low-temperature stress. Furthermore, our investigation revealed a significantly enhanced overlap between DEGs and differentially alternatively spliced genes (DASGs) in response to low-temperature stress, suggesting a unique co-regulatory mechanism governing transcription and splicing in the context of low-temperature response. In conclusion, we have proven that ZjRTD1.0 will serve as a reliable and useful resource for future transcriptomic analyses in Z. japonica.

Immunological and Molecular Investigation of BCR-ABL1 in Samples of Iraqi Chronic Myeloid Leukemia Patients

Background: Chronic myelogenous leukemia (CML) is a blood malignancy that affects hematopoietic stem cells. Also, it is commonly identified by the Philadelphia chromosome. This chromosome produces a protein with strong tyrosine kinase activity and functions as a tumorigenic factor. Objective: This study aimed to investigate the Immunological and Molecular of BCR-ABL1 gene expression and ABL1 protein in patients admitted to the Oncology and Hematology Center of the Kirkuk Governorate, Iraq. Materials and Methods: A study was conducted in Kirkuk Governorate, Iraq, from the beginning of November 2022 to May 2023 on Chronic Myeloid Leukemia patients in the Oncology and Hematology Center, which was hospital-based and cross-sectional. The study comprised fifty-three patients with Chronic Myeloid Leukemia. They were diagnosed using a complete blood count (CBC), a molecular test BCR-ABL1, and an immunological test ABL1 protein. The research did not include patients with missing data and those without consent. Fifty-three peripheral blood samples from 53 CML patients at chronic stages of CML were taken, and using automated Gene Xpert BCR-ABL1 Ultra assay, BCR-ABL1 transcript quantification was conducted on them. Results: The research included fifty-three patients in total (27 males, 28 females), with a mean age of 47.3 12.8 years. The average of BCR\_ABL1 by RT\_ qPCR 3.22 0.09 and the mean of ABL1 by Enzyme-linked Immune sorbent assay (ELISA) reigned is 981 891. Conclusion: Values of the quantitative real-time reverse transcription polymerase chain reaction (RT\_ qPCR) by Gene Xpert Ultra level range between 38.62 and 0.00 with a mean of 3.22 0.09. The RT\_ qPCR level showed that 22% of the patients were in good condition, 11% of them had a strong response to treatment, and other 11% showed a complete cytogenetic response (CCyR), which is also a good outcome; however, 78% of the patients either not taking their medication, or there is true resistance. These two methods used different techniques; the former is a nuclear-derived method that detects the presence of specific genetic materials of any pathogen; in the current research, BCR-ABL1 is the target. The latter is a test that detects and measures antibodies, antigens, proteins, and glycoproteins in biological samples, so ABL1 is the target.

Implication of polymerase recycling for nascent transcript quantification by live cell imaging.

Transcription enables the production of RNA from a DNA template. Due to the highly dynamic nature of transcription, live-cell imaging methods play a crucial role in measuring the kinetics of this process. For instance, transcriptional bursts have been visualized using fluorescent phage-coat proteins that associate tightly with messenger RNA (mRNA) stem loops formed on nascent transcripts. To convert the signal emanating from a transcription site into meaningful estimates of transcription dynamics, the influence of various parameters on the measured signal must be evaluated. Here, the effect of gene length on the intensity of the transcription site focus was analyzed. Intuitively, a longer gene can support a larger number of transcribing polymerases, thus leading to an increase in the measured signal. However, measurements of transcription induced by hyper-osmotic stress responsive promoters display independence from gene length. A mathematical model of the stress-induced transcription process suggests that the formation of gene loops that favor the recycling of polymerase from the terminator to the promoter can explain the observed behavior. One experimentally validated prediction from this model is that the amount of mRNA produced from a short gene should be higher than for a long one as the density of active polymerase on the short gene will be increased by polymerase recycling. Our data suggest that this recycling contributes significantly to the expression output from a gene and that polymerase recycling is modulated by the promoter identity and the cellular state.

Importance of pre-mRNA splicing and its study tools in plants

AbstractAlternative splicing (AS) significantly enriches the diversity of transcriptomes and proteomes, playing a pivotal role in the physiology and development of eukaryotic organisms. With the continuous advancement of high-throughput sequencing technologies, an increasing number of novel transcript isoforms, along with factors related to splicing and their associated functions, are being unveiled. In this review, we succinctly summarize and compare the different splicing mechanisms across prokaryotes and eukaryotes. Furthermore, we provide an extensive overview of the recent progress in various studies on AS covering different developmental stages in diverse plant species and in response to various abiotic stresses. Additionally, we discuss modern techniques for studying the functions and quantification of AS transcripts, as well as their protein products. By integrating genetic studies, quantitative methods, and high-throughput omics techniques, we can discover novel transcript isoforms and functional splicing factors, thereby enhancing our understanding of the roles of various splicing modes in different plant species.

Gene copy number, gene configuration and LC/HC mRNA ratio impact on antibody productivity and product quality in targeted integration CHO cell lines.

The augmentation of transgene copy numbers is a prevalent approach presumed to enhance transcriptional activity and product yield. CHO cell lines engineered via targeted integration (TI) offer an advantageous platform for investigating the interplay between gene copy number, mRNA abundance, product yield, and product quality. Our investigation revealed that incrementally elevating the gene copy numbers of both IgG heavy chain (HC) and light chain (LC) concurrently resulted in the attainment of plateaus in mRNA levels and product titers, notably occurring beyond four to five gene copies integrated at the same TI site. Furthermore, maintaining a fixed gene copy number while varying the position of genes within the vector influenced the LC/HC mRNA ratio, which subsequently exerted a substantial impact on product titer. Moreover, manipulation of the LC/HC gene ratio through the introduction of surplus LC gene copies led to heightened LC mRNA expression and a reduction in the levels of high molecular weight species. It is noteworthy that the effects of excess LC on product titer were dependent on the specific molecule under consideration. The strategic utilization of PCR tags enabled precise quantification of transcription from each expression slot within the vector, facilitating the identification of highly expressive and less expressive slots. Collectively, these findings significantly enhance our understanding of stable antibody production in TI CHO cell lines.

Deciphering Vanadium Speciation in Smelting Ash and Adaptive Responses of Soil Microorganisms.

Abundant smelting ash is discharged during pyrometallurgical vanadium (V) production. However, its associated V speciation and resultant ecological impact have remained elusive. In this study, V speciation in smelting ash and its influence on the metabolism of soil microorganisms were investigated. Smelting ashes from V smelters contained abundant V (19.6-115.9 mg/g). V(V) was the dominant species for soluble V, while solid V primarily existed in bioavailable forms. Previously unrevealed V nanoparticles (V-NPs) were prevalently detected, with a peak concentration of 1.3 × 1013 particles/g, a minimal size of 136.0 ± 0.6 nm, and primary constituents comprising FeVO4, VO2, and V2O5. Incubation experiments implied that smelting ash reshaped the soil microbial community. Metagenomic binning, gene transcription, and component quantification revealed that Microbacterium sp. and Tabrizicola sp. secreted extracellular polymeric substances through epsB and yhxB gene regulation for V-NPs aggregation to alleviate toxicity under aerobic operations. The V K-edge X-ray absorption near-edge structure (XANES) spectra suggested that VO2 NPs were oxidized to V2O5 NPs. In the anaerobic case, Comamonas sp. and Achromobacter sp. reduced V(V) to V(IV) for detoxification regulated by the napA gene. This study provides a deep understanding of the V speciation in smelting ash and microbial responses, inspiring promising bioremediation strategies to reduce its negative environmental impacts.

RNA-FISH for HIV Transcription/Localization Analysis.

RNA fluorescence in situ hybridization (FISH) serves as a method for visualizing specific RNA molecules within cells. Its primary utility lies in the observation of messenger RNA (mRNA) molecules associated with particular genes of significance. This technique can also be applied to examine viral transcription and the localization of said transcripts within infected cells. In this context, we provide a comprehensive protocol for the detection, localization, and quantification of HIV-1 transcripts in mammalian cell lines. This encompasses the preparation of required reagents, cellular treatments, visualization, and the subsequent analysis of the data acquired. These parameters play a pivotal role in enhancing our comprehension of the molecular processes during infection, particularly at the crucial transcription phase of the viral life cycle.

NOVEL GENE VARIANTS ASSOCIATED WITH HBA1C CHANGES OVER TIME AMONG NONDIABETIC SUBJECTS IN THE LONG LIFE FAMILY STUDY

Abstract Longitudinal changes in HbA1c (ΔHbA1c) are associated with insulin resistance, aging, cognition, and mortality. To identify novel gene variants underlying ΔHbA1c, we conducted linkage-guided sequence analysis on 17p12 (LODs=3.59) in the Long Life Family Study, a study with familial clustering of exceptional longevity in the US and Denmark. Subjects with clinical diagnosis of diabetes or diabetes treatment and undiagnosed diabetes cases whose fasting glucose ≥ 126 mg/dl or HbA1c ≥ 6.5% were excluded. ΔHbA1c, collected from two exams 7 years apart, was derived using growth curve modeling, adjusting for age, sex, BMI, smoking, field centers and PCs, and was blom-transformed to approximate normality. We identified a significant variant under the linkage peak (ARHGAP44 rs56340929, p=1.77E-06, MAF=6%, accounting for linkage=26.5%). Taking advantage of our currently available RNAseq data, we found a significant association between quantification of ARHGAP44 RNA transcript and adjusted ΔHbA1c using 16 linkage enriched families (n=176, β = -0.24, SE=0.09, p=0.01). We also assessed currently available lipidomics data (188 metabolites, 13 compound classes) and found phosphatidylcholine (p=0.025) and lysophosphatidylcholine (p=0.02) were marginally associated with adjusted ΔHbA1c. ARHGAP44 is reportedly associated with glycemic traits and is mainly expressed in the brain. Further complete omics-data analyses in the LLFS and a replication study using the Framingham Offspring Study are underway.