Quantification Of Gene Expression Levels Research Articles

AKT1 and MAPK8: New Targets for Gestational Diabetes Mellitus?

Objective: Gestational diabetes mellitus (GDM) disrupts placental function and increases risks for pregnancy. This study investigates the potential involvement of AKT1 and MAPK8 genes, known for their roles in insulin resistance and cell signaling, in GDM pathophysiology. Methods: Placental tissues from GDM patients and healthy controls were analyzed using real-time PCR to quantify gene expression levels. In silico analysis further explored the functional implications of expression changes. Results: AKT1 and MAPK8 displayed significantly altered expression in GDM placentas compared to controls (p = 0.047 and p = 0.007, respectively). In silico analysis suggests potential functional consequences related to diabetes-associated pathways. Conclusion: This study identifies differential expression of AKT1 and MAPK8 in GDM placentas, suggesting their potential roles in the disease process. Further investigation into their functional contributions could provide valuable insights into GDM pathophysiology and potential therapeutic targets.

RNA-clique: a method for computing genetic distances from RNA-seq data

BackgroundAlthough RNA-seq data are traditionally used for quantifying gene expression levels, the same data could be useful in an integrated approach to compute genetic distances as well. Challenges to using mRNA sequences for computing genetic distances include the relatively high conservation of coding sequences and the presence of paralogous and, in some species, homeologous genes.ResultsWe developed a new computational method, RNA-clique, for calculating genetic distances using assembled RNA-seq data and assessed the efficacy of the method using biological and simulated data. The method employs reciprocal BLASTn followed by graph-based filtering to ensure that only orthologous genes are compared. Each vertex in the graph constructed for filtering represents a gene in a specific sample under comparison, and an edge connects a pair of vertices if the genes they represent are best matches for each other in their respective samples. The distance computation is a function of the BLAST alignment statistics and the constructed graph and incorporates only those genes that are present in some complete connected component of this graph. As a biological testbed we used RNA-seq data of tall fescue (Lolium arundinaceum), an allohexaploid plant (2n=14Gb\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$2n = 14\ ext { Gb}$$\\end{document}), and bluehead wrasse (Thalassoma bifasciatum), a teleost fish. RNA-clique reliably distinguished individual tall fescue plants by genotype and distinguished bluehead wrasse RNA-seq samples by individual. In tests with simulated RNA-seq data, the ground truth phylogeny was accurately recovered from the computed distances. Moreover, tests of the algorithm parameters indicated that, even with stringent filtering for orthologs, sufficient sequence data were retained for the distance computations. Although comparisons with an alternative method revealed that RNA-clique has relatively high time and memory requirements, the comparisons also showed that RNA-clique’s results were at least as reliable as the alternative’s for tall fescue data and were much more reliable for the bluehead wrasse data.ConclusionResults of this work indicate that RNA-clique works well as a way of deriving genetic distances from RNA-seq data, thus providing a methodological integration of functional and genetic diversity studies.

ScCDC: a computational method for gene-specific contamination detection and correction in single-cell and single-nucleus RNA-seq data

In droplet-based single-cell and single-nucleus RNA-seq assays, systematic contamination of ambient RNA molecules biases the quantification of gene expression levels. Existing methods correct the contamination for all genes globally. However, there lacks specific evaluation of correction efficacy for varying contamination levels. Here, we show that DecontX and CellBender under-correct highly contaminating genes, while SoupX and scAR over-correct lowly/non-contaminating genes. Here, we develop scCDC as the first method to detect the contamination-causing genes and only correct expression levels of these genes, some of which are cell-type markers. Compared with existing decontamination methods, scCDC excels in decontaminating highly contaminating genes while avoiding over-correction of other genes.

MiR-374a/b-5p Suppresses Cell Growth in Papillary Thyroid Carcinoma Through Blocking Exosomal ANXA1-Induced Macrophage M2 Polarization.

Papillary thyroid carcinoma (PTC), comprising 85% of all thyroid cancers, is an epithelial malignancy. The potential for malignant transformation in normal cells by thyroid cancer cells via exosomal Annexin A1 (ANXA1) delivery is investigated in this study. Our aim is to determine the impact of PTC cells on macrophage polarization through exosomal ANXA1 secretion and its implications for tumor progression.Exosomes in PTC cells were examined using transmission electron microscopy, exosome labeling, and nanoparticle tracking analysis. Real-time quantitative polymerase chain reaction was employed to quantify gene expression levels. Protein levels were determined through Western blot analysis. The interplay between genes was assessed using luciferase reporter and RNA pull-down assays. Functional experiments were conducted to investigate PTC cell proliferation and apoptosis.Our findings reveal that ANXA1 promotes PTC cell proliferation and inhibits apoptosis. Exosomes derived from PTC cells were found to promote macrophage M2 polarization. ANXA1 stimulates M2 polarization through the activation of the PI3K/AKT pathway. MicroRNA-374a-5p (miR-374a-5p) and microRNA-374b-5p (miR-374b-5p) were identified as inhibitors of ANXA1 expression and PI3K/AKT pathway activity, thereby inhibiting macrophage M2 polarization. Furthermore, miR-374a-5p and miR-374b-5p were observed to suppress PTC cell proliferation through their regulatory action on ANXA1.Our study suggests that miR-374a/b-5p inhibits PTC cell growth by blocking the macrophage M2 polarization induced by exosomal ANXA1.

Monocyte-derived transcriptomes explain the ineffectiveness of abatacept in rheumatoid arthritis

BackgroundThe biological mechanisms underlying the differential response to abatacept in patients with rheumatoid arthritis (RA) are unknown. Here, we aimed to identify cellular, transcriptomic, and proteomic features that predict resistance to abatacept in patients with RA.MethodsBlood samples were collected from 22 RA patients treated with abatacept at baseline and after 3 months of treatment. Response to treatment was defined by the European League Against Rheumatism (EULAR) response criteria at 3 months, and seven patients were classified as responders and the others as non-responders. We quantified gene expression levels by RNA sequencing, 67 plasma protein levels, and the expression of surface molecules (CD3, 19, and 56) by flow cytometry. In addition, three gene expression data sets, comprising a total of 27 responders and 50 non-responders, were used to replicate the results.ResultsAmong the clinical characteristics, the number of monocytes was significantly higher in the non-responders before treatment. Cell type enrichment analysis showed that differentially expressed genes (DEGs) between responders and non-responders were enriched in monocytes. Gene set enrichment analysis, together with single-cell analysis and deconvolution analysis, identified that Toll-like receptor 5 (TLR5) and interleukin-17 receptor A (IL17RA) pathway in monocytes was upregulated in non-responders. Hepatocyte growth factor (HGF) correlated with this signature showed higher concentrations in non-responders before treatment. The DEGs in the replication set were also enriched for the genes expressed in monocytes, not for the TLR5 and IL17RA pathway but for the oxidative phosphorylation (OXPHOS) pathway.ConclusionsMonocyte-derived transcriptomic features before treatment underlie the differences in abatacept efficacy in patients with RA. The pathway activated in monocytes was the TLR5 and IL17RA-HGF signature in the current study, while it was the OXPHOS pathway in the replication set. Elevated levels of HGF before treatment may serve as a potential biomarker for predicting poor responses to abatacept. These findings provide insights into the biological mechanisms of abatacept resistance, contributing valuable evidence for stratifying patients with RA.

Identification of Tumorigenic Markers in Head and Neck Cancer Patients, Exploring their Correlation with Tumor Aggressiveness

Background: Globally, head and neck cancer (HNC) considered the sixth most severe cancer associated with multiple risk factors. Due to lack of personalized therapies and undefined biomarkers, HNC shows low overall survival rate. Long non-coding RNA (LncRNA) helps to regulate multiple gene expressions in multiple biological responses. LncRNA tumor biomarkers stimulated tumor aggressiveness, cellular proliferation and inhibit apoptosis with poor prognosis. BLACAT-1, NEAT-1, HIF-1α, Caspase-3, Caspase-9 and Telomerase were associated with metastasis, tumor staging (TNM stage) activated proliferation, invasion and migration. Objective: The presented study aims to analyze tumorigenic biomarkers, (BLACAT-1, NEAT-1 and HIF-1α, Caspase-3, Caspase-9 and Telomerase) in development and progression of head and neck cancer. Meanwhile, to observe the correlation between these biomarkers in promoting head and neck cancer. Methods: In this comprehensive study, EDTA blood samples (5ml each) were collected from a cohort comprising 50 head and neck cancer (HNC) patients and an additional 30 healthy controls. The primary objective was to extract RNA from these samples for a detailed analysis. The study focused on evaluating the correlation between various tumor biomarkers across different HNC tumor subtypes. This correlation was meticulously analyzed using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR), a method known for its precision in quantifying gene expression levels. Results: BLACAT-1 shows a significant increase expression in squamous cell carcinoma. NEAT-1, Caspase-3 and Caspase-9 are non-significant in HNC tumor types whereas, inverse correlation was observed between NEAT-1 with Caspase-9 and direct proportion between NEAT-1 with Caspase-3. However, HIF-1α represented an insignificant data among all tumor subtypes under hypoxic microenvironment. Conclusion: Higher expression levels are suggesting potential biomarkers, therefore a gene signature can be developed for determining the severity of breast tumor; therefore, can be exploited as novel therapeutic approaches to block tumor metastasis and angiogenesis.

(Invited) COVID-19 As a Technology Accelerator for High-Speed Polymerase Chain Reaction (PCR)

Limit of detection (LOD), speed and cost for some of the most important diagnostics tools for COVID-19 i.e., lateral flow assays (LFA), enzyme-linked immunosorbent assay (ELISA) and Polymerase Chain Reaction (PCR) all benefitted from both the financial and regulatory support brought about by the pandemic. Before COVID-19, LFA was the fastest and least expensive but came with the poorest limit of detection (LOD). The slowest, most expensive but with the best (lowest) LOD was PCR. In between those two extremes, in terms of cost, LOD and speed, was an ELISA . Today, through the “COVID-19 accelerator effect” there are point of care (POC) reverse transcriptase quantitative PCR (ie., RT-qPCR) COVID-19 tests on the market that provide sample to answer in under 20 minutes, while retaining the same very low LOD but at a lower cost [1]. There are even POC PCR tests under development that come in at less than 10 minutes: beating both LFA and ELISAs in terms of time and LOD [2]. Although RT-qPCR has gained the most in overall performance, signal-amplified POC LFAs are possible now with an LOD that rivals that of an ELISA.However, implementing the next generation PCR equipment in point-of-care settings, remains very challenging because of difficulties in reaching a 10 minute or below sample to answer time , instrument complexity and cost. In this work, we discuss the engineering challenges involved in the development of high-speed microfluidic PCR equipment. To guide this review/comparison of PCR advances we introduce three parameters. The first one is the overall sample to answer time (t), the second is called lambda (λ) and is a measure of the degree to which stochastic effects interfere with obtaining analytical results and thereby setting the minimal number of copies required per volume. The third parameter gamma (γ), introduced first by Dong et al [3], is the ratio of thermal cycling time (in min) to the heat cycled sample volume (in mL). It describes the trade-off between the speed of the PCR system and the desired level of sensitivity. It can be interpreted as a system efficiency: the most efficient system heat cycles the largest amount of sample in the smallest amount of time.With these three criteria in mind we set out to suggest designs for an optimized POC reverse transcriptase (RT) quantitative qPCR or RT-qPCR system. Applications of RT-qPCR include quantifying gene expression levels, validating RNA interference (RNAi), and detecting pathogens such as viruses. The aim of this specific POC RT-qPCR system is to provide sample to answer in ten minutes or less for a respiratory pathogens panel, with an LOD of 500 copies/mL and the capability to multiplex (8 chambers) while remaining portable and low cost.Finally we introduce two of the most daunting, remaining, microfluidics challenges that both need to be tackled to implement an ideal microfluidic platform like the POC RT-qPCR system described above. One is liquid storage and the other is proportional valves on a disposable microfluidic platform. Liquid storage is essential when the assay requires repeated washes, if these washes can come from one and the same reservoir via a proportional valve, real estate is gained and system complexity is reduced. The storage issue is further complicated when the design also calls for storing lyophilized or dried reagents on the same disposable. We compare some of the most recent approaches to liquid storage and proportional valving that we believe will enable the fast POC diagnostic platforms of the future. 1. Tsang, Hin Fung, Wai Ming Stanley Leung, Lawrence Wing Chi Chan, William Chi Shing Cho, and Sze Chuen Cesar Wong. “Performance Comparison of the Cobas® Liat® and Cepheid® GeneXpert® Systems on SARS-CoV-2 Detection in Nasopharyngeal Swab and Posterior Oropharyngeal Saliva.” Expert Review of Molecular Diagnostics 21, no. 5 (May 4, 2021): 515–18. 2. BUCKLAND, Justin, Tom JELLICOE, Alex STOKOE, and Amaru ARAYA-WILLIAMS. “VARIABLE TEMPERATURE REACTOR, HEATER AND CONTROL CIRCUIT FOR THE SAME.” Patent Application, January 30, 2020. 3. Dong, Xiaobin, Luyao Liu, Yunping Tu, Jing Zhang, Guijun Miao, Lulu Zhang, Shengxiang Ge, Ningshao Xia, Duli Yu, and Xianbo Qiu. “Rapid PCR Powered by Microfluidics: A Quick Review under the Background of COVID-19 Pandemic.” Trends in Analytical Chemistry 143 (October 2021): 116377.

The infection of Aeromonas hydrophila activated Multiple programmed cell death pathways in red blood cells of Clarias fuscus

In contrast to mammalian red blood cells (RBCs), Osteichthyes RBCs contain a nucleus and organelles, suggesting the involvement of more intricate mechanisms, particularly in the context of ferroptosis. In this study, we utilized RBCs from Clarias fuscus (referred to as Cf-RBCs) as a model system. We conducted RNA-seq analysis to quantify gene expression levels in Cf-RBCs after exposure to both Aeromonas hydrophila and lipopolysaccharides. Our analysis unveiled 1326 differentially expressed genes (DEGs) in Cf-RBCs following 4 h of incubation with A. hydrophila, comprising 715 and 611 genes with upregulated and downregulated expression, respectively. These DEGs were further categorized into functional clusters: 292 related to cellular processes, 241 involved in environmental information processing, 272 associated with genetic information processing, and 399 linked to organismal systems. Additionally, notable changes were observed in genes associated with the autophagy pathway at 4 h, and alterations in the ferroptosis pathway were observed at 8 h following A. hydrophila incubation. To validate these findings, we assessed the expression of cytokines (DMT1, TFR1, LC3, and GSS). All selected genes were significantly upregulated after exposure to A. hydrophila. Using flow cytometry, we evaluated the extent of ferroptosis, and the group incubated with A. hydrophila for 8 h exhibited higher levels of lipid peroxidation compared with the 4-h incubation group, even under baseline conditions. An evaluation of the glutathione redox system through GSSG/GSH ratios indicated an increased ratio in Cf-RBCs after exposure to A. hydrophila. In summary, our data suggest that A. hydrophila may induce ferroptosis in Cf-RBCs, potentially by triggering the cystine/glutamate antiporter system (system XC−), while Cf-RBCs counteract ferroptosis through the regulation of the glutathione redox system. These findings contribute to our understanding of the iron overload mechanism in Osteichthyes RBCs, provide insights into the management of bacterial diseases in Clarias fuscus, and offer potential strategies to mitigate economic losses in aquaculture.

Cooperatively inhibition effect of miR-143-5p and miR-145-5p in tumorigenesis of glioblastoma cells through modulating AKT signaling pathway

Introduction: As the most common aggressive primary brain tumor, glioblastoma is inevitably a recurrent malignancy whose patients’ prognosis is poor. miR-143 and miR-145, as tumor suppressor miRNAs, are downregulated through tumorigenesis of multiple human cancers, including glioblastoma. These two miRNAs regulate numerous cellular processes, such as proliferation and migration. This research was intended to explore the simultaneous replacement effect of miR-143, and miR-145 on in vitro tumorgenicity of U87 glioblastoma cells. Methods: U87 cells were cultured, and transfected with miR-143-5p and miR-145-5p. Afterward, the changes in cell viability, and apoptosis induction were determined by MTT assay and Annexin V/PI staining. The accumulation of cells at the cell cycle phases was assessed using the flow cytometry. Wound healing and colony formation assays were performed to study cell migration. qRT-PCR and western blot techniques were utilized to quantify gene expression levels. Results: Our results showed that miR-143-5p and 145-5p exogenous upregulation cooperatively diminished cell viability, and enhanced U-87 cell apoptosis by modulating Caspase-3/8/9, Bax, and Bcl-2 protein expression. The combination therapy increased accumulation of cells at the sub-G1 phase by modulating CDK1, Cyclin D1, and P53 protein expression. miR-143/145-5p significantly decreased cell migration, and reduced colony formation ability by the downregulation of c-Myc and CD44 gene expression. Furthermore, the results showed the combination therapy of these miRNAs could remarkably downregulate phosphorylated-AKT expression levels. Conclusion: In conclusion, miR-143 and miR-145 were indicated to show cooperative anti- cancer effects on glioblastoma cells via modulating AKT signaling as a new therapeutic approach.

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species.

Comparisons of parametric and non-parametric methods for analyzing RT-PCR experiment data

The real-time reverse-transcript polymerase chain reaction (RT-PCR) test is a widely used laboratory technique that is highly sensitive and reliable for measuring the quantification of gene expression levels and diagnosing various of diseases, including COVID-19. The RT-PCR experiments often have correlated technical replicates of a small number of samples. However, current statistical analysis of RT-PCR assumes a large sample size and does not account for correlated structure across the replicates. In this paper, we review popular statistical methods for analyzing RT-PCR data and propose a permutation method that accounts for the small sample size and the correlated structure of RT-PCR data. Our proposed method provides a more accurate and efficient analysis of RT-PCR data. We provide an R program to implement our method for practitioners.

Evaluation of Gene Expression Level of miRNA-29c, miRNA-125, miRNA-141, miRNA-145 and miRNA-205 as Predisposing Factors for Transitional Cell Carcinoma-Bladder Cancer in Iraqi Patients

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. Transitional cell carcinoma(TCC) of the bladder Cancer (C) account 95 percent of bladder malignancies, with males having a greater prevalence than females. The current study sought to determine whether there is a link between miRNA-29c, miRNA-125, miRNA-141, miRNA-145 and miRNA205 expression levels and TCC/BC risk in Iraqi bladder cancer patients. In the current prospective cross-sectional investigation, 149 samples were collected (95 urine and 54 tissue biopsies). From November 2018 to August 2019, 37/95 urine samples were randomly taken from healthy persons. Total RNA was extracted from tissue and urine samples, and then converted to cDNA via reverse transcription. Quantitate Real-Time-PCR was done using specific primers for quantification of gene expression level of the studied miRNAs. The results showed that 32/49 (65%) patients had non-muscles invasive bladder cancer (stage T1), while 17/49 (35%) patients had muscles invasive bladder cancer (stage T2-T4). Fold change in miRNA-125 expression level showed highly significant differences between non-muscles invasive bladder cancer (T1) and muscles invasion bladder cancer (T2-T4) in both urine and tissue samples biopsies using Chi-square test at p ≤ 0.01. Whereas miR-29c, miR-141, miR-145 and miR-205 showed no significant differences at p > 0.05 between muscles invasive bladder cancer (T1) and muscles invasive bladder cancer (T2-T4) in both urine and tissue biopsies samples. Thus, miR-125 can be associated with the development of invasive stages of TCC-BC as there is an increase in miR-125 expression level in the urine of patients during the final stage of cancer.Hence, this gene could be considered a good predictor in advanced stages of bladder cancer.

Abstract 3307: A novel targeted RNA sequencing approach for identification of aggressive prostate cancer

Abstract Introduction: Prostate cancer (PC) is a highly heterogeneous disease and can be either aggressive, requiring immediate treatment, or remain slow growing and even without treatment present no significant symptoms throughout the man’s life. Currently, there is an unmet need for new tools that can clearly distinguish indolent from aggressive PC in order to guide treatment decisions.PC is a highly heritable disease and so far, approximately 450 genomic regions associated with PC risk have been defined in genome-wide association studies. Most of these regions are however devoid of annotated genes and consequently the underlying mechanisms behind the associations are not easily elucidated. Accordingly, yet uncharacterized genes in PC-associated regions could be involved in PC development/progression. Methods: We quantified gene expression levels by Kallisto on total RNAseq data from fresh frozen prostate tissue samples from 134 localized PCs, 33 matched adjacent normal (AN) areas as well as 15 PC cell lines. Furthermore, we performed targeted RNAseq covering approximately seven million base pairs divided among 101 PC-risk associated regions (GWAS hits) for in-depth transcriptomic analyses in the same set of samples. HISAT and StringTie were used to identify novel transcripts (genes) with the criteria that a given gene had to have at least 10 reads in more than 50% of all samples to be included. Patient genotyping was performed on Illumina’s global screening array. Results: In totalRNA seq data we observed significant dysregulation of 10,256 genes (5,685 up- and 4,571 downregulated) between AN and localized PC, including upregulation of know PC-associated genes such as SIM2, PCAT7, TDRD1, and TFF3. Interestingly, we found a correlation between high transcription of TFF3 and high risk of biochemical recurrence (BCR) after radical prostatectomy and furthermore a trans-acting eQTL, the PC-risk associated SNP rs1938781. Here, heterozygote patients with the risk-allele had higher risk of BCR than patients homozygous for the reference allele, indicating a potential functional relationship between the PC risk-SNP and TFF3. Additionally, we detected 207 previously unannotated non-coding genes in the capture RNAseq data, where 148 (71.5%) were undetectable in the matched total RNAseq data indicating the existence of stably albeit lowly expressed genes in the genomic areas containing PC-risk SNPs. Citation Format: Jacob Fredsøe, Christa Haldrup, Iver Nordentoft, Benedicte Ulhøi, Michael Borre, Karina D. Sørensen. A novel targeted RNA sequencing approach for identification of aggressive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3307.

Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells.

Nitrogen gas (N2) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH4+) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (-0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N2 fixation genes. At -0.15 V, the expression of nitrogenase genes, such as nifH, nifD, and nifK, significantly increased relative to that at +0.15 V, as well as genes associated with NH4+ uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at -0.15 V. N2 fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at -0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at -0.15 V, suggesting that these mechanisms may be involved in N2 fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N2 fixation rates. We hypothesize that at -0.15 V, they increase N2 fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use. IMPORTANCE Biological nitrogen fixation coupled with ammonium recovery provides a sustainable alternative to the carbon-, water-, and energy-intensive Haber-Bosch process. Aerobic biological nitrogen fixation technologies are hindered by oxygen gas inhibition of the nitrogenase enzyme. Electrically driving biological nitrogen fixation in anaerobic microbial electrochemical technologies overcomes this challenge. Using Geobacter sulfurreducens as a model exoelectrogenic diazotroph, we show that the anode potential in microbial electrochemical technologies has a significant impact on nitrogen gas fixation rates, ammonium assimilation pathways, and expression of genes associated with nitrogen gas fixation. These findings have important implications for understanding regulatory pathways of nitrogen gas fixation and will help identify target genes and operational strategies to enhance ammonium production in microbial electrochemical technologies.

Stepwise Optimization of Real-Time RT-PCR Analysis.

Quantitative real-time reverse transcription PCR (qRT-PCR) analysis has been used routinely to quantify gene expression levels. Primer design and the optimization of qRT-PCR parameters are critical for the accuracy and reproducibility of qRT-PCR analysis. Computational tool-assisted primer design often overlooks the presence of homologous sequences of the gene of interest and the sequence similarities between homologous genes in a plant genome. This sometimes results in skipping the optimization of qRT-PCR parameters due to the false confidence in the quality of the designed primers. Here we present a stepwise optimization protocol for single nucleotide polymorphisms (SNPs)-based sequence-specific primer design and sequential optimization of primer sequences, annealing temperatures, primer concentrations, and cDNA concentration range for each reference and target gene. The goal of this optimization protocol is to achieve a standard cDNA concentration curve with an R2≥0.9999 and efficiency (E)=100±5% for the best primer pair of each gene, which serves as the prerequisite for using the 2-ΔΔCT method for data analysis.

Comparison of Metagenomics and Metatranscriptomics Tools: A Guide to Making the Right Choice.

The study of microorganisms is a field of great interest due to their environmental (e.g., soil contamination) and biomedical (e.g., parasitic diseases, autism) importance. The advent of revolutionary next-generation sequencing techniques, and their application to the hypervariable regions of the 16S, 18S or 23S ribosomal subunits, have allowed the research of a large variety of organisms more in-depth, including bacteria, archaea, eukaryotes and fungi. Additionally, together with the development of analysis software, the creation of specific databases (e.g., SILVA or RDP) has boosted the enormous growth of these studies. As the cost of sequencing per sample has continuously decreased, new protocols have also emerged, such as shotgun sequencing, which allows the profiling of all taxonomic domains in a sample. The sequencing of hypervariable regions and shotgun sequencing are technologies that enable the taxonomic classification of microorganisms from the DNA present in microbial communities. However, they are not capable of measuring what is actively expressed. Conversely, we advocate that metatranscriptomics is a "new" technology that makes the identification of the mRNAs of a microbial community possible, quantifying gene expression levels and active biological pathways. Furthermore, it can be also used to characterise symbiotic interactions between the host and its microbiome. In this manuscript, we examine the three technologies above, and discuss the implementation of different software and databases, which greatly impact the obtaining of reliable results. Finally, we have developed two easy-to-use pipelines leveraging Nextflow technology. These aim to provide everything required for an average user to perform a metagenomic analysis of marker genes with QIMME2 and a metatranscriptomic study using Kraken2/Bracken.

A Nile rat transcriptomic landscape across 22 organs by ultra-deep sequencing and comparative RNA-seq pipeline (CRSP)

RNA sequencing (RNA-seq) has been a widely used high-throughput method to characterize transcriptomic dynamics spatiotemporally. However, RNA-seq data analysis pipelines typically depend on either a sequenced genome and/or corresponding reference transcripts. This limitation is a challenge for species lacking sequenced genomes and corresponding reference transcripts. The Nile rat (Arvicanthis niloticus) has two key features – it is daytime active, and it is prone to diet-induced diabetes, which makes it more similar to humans than regular laboratory rodents. However, at the time of this study, neither a Nile rat genome nor a reference transcript set were available, making it technically challenging to perform large-scale RNA-seq based transcriptomic studies. This genome-independent work progressed concurrently with our generation of a Nile rat genome. A well-annotated genome requires several iterations of manually reviewing curated transcripts and takes years to achieve. Here, we developed a Comparative RNA-Seq Pipeline (CRSP), integrating a comparative species strategy independent of a specific sequenced genome or species-matched reference transcripts. We performed benchmarking to validate that our CRSP tool can accurately quantify gene expression levels. In this study, we generated the first ultra-deep (2.3 billion × 2 paired-end) Nile rat RNA-seq data from 59 biopsy samples representing 22 major organs, providing a unique resource and spatial gene expression reference for Nile rat researchers. Importantly, CRSP is not limited to the Nile rat species and can be applied to any species without prior genomic knowledge. To facilitate a general use of CRSP, we also characterized the number of RNA-seq reads required for accurate estimation via simulation studies. CRSP and documents are available at: https://github.com/pjiang1105/CRSP.

Evaluation of candidate reference genes for RT‐qPCR analysis during developmental stages in striped murrel ( Channa striata )

Real-time quantitative PCR (RT-qPCR) is the most advanced and commonly used method for quantification of gene expression levels because of its high sensitivity, specificity and accuracy. The analytical capability of RT-qPCR, on the other hand, is dependent on precise normalization by using the most stable reference genes. The present study was undertaken to find the best-suited reference gene for normalization of qPCR data for ontogenetic studies of striped murrel. Four commonly used and constitutively expressed genes, namely beta-actin (β-actin), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (EF1α) and 18 S ribosomal protein (18 S RNA) were screened and evaluated for their expression stability in developing larvae from 1 to 35 days post hatch (dph). The stability and suitability of these genes were determined by using four different algorithms; namely, delta Ct, BestKeeper, geNorm and NormFinder. The four algorithms together generated a comprehensive ranking EF1α being the most stable, followed by GAPDH and β-actin as the least stable gene. This is the first report evaluating the most suitable reference gene for mRNA expression studies in C. striata, in the context of ontogenetic development.

Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.)

Quantitative real-time polymerase chain reaction (qRT-PCR) is a sensitive and widely used technique for quantifying gene expression levels, and its accuracy depends on the reference genes used for data normalization. To date, no reference gene has been reported in the nutritious and functional vegetable okra (Abelmoschus esculentus L.). Herein, 11 candidates of reference genes were selected and evaluated for their expression stability in okra in different tissues at different developmental stages by using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Among them, eukaryotic initiation factor 4 alpha (eIF4A) and protein phosphatase 2A (PP2A) showed the highest stability, while TUA5 had the lowest stability. The combined usage of these two most stable reference genes was sufficient to normalize gene expression in okra. Then, the above results were further validated by normalizing the expression of the cellulose synthase gene CesA4. This work provides appropriate reference genes for transcript normalization in okra, which will facilitate subsequent functional gene research on this vegetable crop.

Biomarker discovery for practice of precision medicine in hypopharyngeal cancer: a theranostic study on response prediction of the key therapeutic agents

BackgroundHypopharyngeal cancer is a relatively rare malignancy with poor prognosis. Current chemotherapeutic algorithm is still far from personalized medicine, and the identification of the truly active therapeutic biomarkers and/or targets is eagerly awaited.MethodsVenturing to focus on the conventional key chemotherapeutic drugs, we identified the most correlative genes (and/or proteins) with cellular sensitivity to docetaxel (TXT), cisplatin (CDDP) and 5-fluorouracil (5-FU) in the expression levels, through 3 steps approach: genome-wide screening, confirmation study on the quantified expression levels, and knock-down and transfection analyses of the candidates. The probable action pathways of selected genes were examined by Ingenuity Pathway Analysis using a large-scale database, The Cancer Genome Atlas.ResultsThe first genome-wide screening study derived 16 highly correlative genes with cellular drug sensitivity in 15 cell lines (|R| > 0.8, P < 0.01 for CDDP and 5-FU; |R| > 0.5, P < 0.05 for TXT). Among 10 genes the observed correlations were confirmed in the quantified gene expression levels, and finally knock-down and transfection analyses provided 4 molecules as the most potent predictive markers-AGR2 (anterior gradient 2 homolog gene), and PDE4D (phosphodiesterase 4D, cAMP-specific gene) for TXT; NINJ2 (nerve Injury-induced protein 2); CDC25B (cell division cycle 25 homolog B gene) for 5-FU- in both gene and protein expression levels. Overexpression of AGR2, PDE4D signified worse response to TXT, and the repressed expression sensitized TXT activity. Contrary to the findings, in the other 2 molecules, NINJ2 and CDC25, there observed opposite relationship to cellular drug response to the relevant drugs. IPA raised the potential that each selected molecule functionally interacts with main action pathway (and/or targets) of the relevant drug such as tubulin β chain genes for TXT, DNA replication pathway for CDDP, and DNA synthesis pathway and thymidylate synthetase gene for 5-FU.ConclusionWe newly propose 4 molecules -AGR2, PDE4D,NINJ2 and CDC25B) as the powerful exploratory markers for prediction of cellular response to 3 key chemotherapeutic drugs in hypopharyngeal cancers and also suggest their potentials to be the therapeutic targets, which could contribute to the development of precision medicine of the essential chemotherapy in hypopharyngeal patients. (339 words).