Reads Per Kilobase Per Million Research Articles

A multi-cell model for the C4 photosynthetic pathway in developing wheat grains based upon tissue-specific transcriptome data

A non-Kranz C4 photosynthesis of the NAD-ME subtype, specifically in developing wheat grains (14 dpa, days post-anthesis) was originally demonstrated using transcriptome-based RNA-seq. Here we present a re-examination of evidence for C4 photosynthesis in the developing grains of wheat and, more broadly, the Pooideae and an investigation of the evolutionary processes and implications. The expression profiles for the genes associated with C4 photosynthesis (C4- and C3-specific) were evaluated using published transcriptome data for the outer pericarp, inner pericarp, and endosperm tissues of the developing wheat grains. The expression of the C4-specific genes across these three tissues revealed the involvement of all three tissues in an orderly fashion to accomplish the non-Kranz NAD-ME-dependent C4 photosynthesis. Based on their expression levels in RPKM (reads per kilobase per million mapped reads) values, a model involving multiple cell- and tissue-types is proposed for C4 photosynthesis involved in the refixation of the respired CO2 from the endosperm tissues in the developing wheat grains. This multi-cell C4 model, proposed to involve more than two cell types, requires further biochemical validation.

Identification and Characterization of the CPP Gene Family in the Genome of Aedes aegypti L. (Yellow Fever Mosquito) (Diptera: Culicidae)

Aedes aegypti is an important vector organism responsible for carrying numerous arboviral pathogens and serious diseases, including yellow fever, Zika, Chikungunya, and Dengue fever. The CPP gene family, one of the crucial molecular defense systems, plays a significant role in the regulation of growth and development by controlling the production of proteins. In this study, a comprehensive genome analysis of the CPP gene family in Ae. aegypti was conducted. Each gene was thoroughly characterized, gene structures were examined, and conserved motifs were investigated. Additionally, the properties of these proteins were comprehensively analyzed. Expression analyses were performed to reveal the effects of CPP genes on development by calculating Reads Per Kilobase Million (RPKM) values. The findings emphasize the importance of CPP genes in controlling arboviral pathogens and understanding general stress responses in insects. The information derived from this research could contribute to the development of more effective intervention strategies for Ae. aegypti and other vector carriers to cope with stress. In conclusion, the systematic analysis of the CPP gene family in the Ae. aegypti genome is a crucial step in the management and development of effective disease prevention strategies for this species. Moreover, this study provides a significant foundation for future functional genomics research in understanding the structure and function of CPP genes.

Highly sensitive detection of antimicrobial resistance genes in hospital wastewater using the multiplex hybrid capture target enrichment.

Wastewater can be useful in monitoring the spread of antimicrobial resistance (AMR) within a hospital. The abundance of antibiotic resistance genes (ARGs) in hospital effluent was assessed using metagenomic sequencing (mDNA-seq) and hybrid capture (xHYB). mDNA-seq analysis and subsequent xHYB targeted enrichment were conducted on two effluent samples per month from November 2018 to May 2021. Reads per kilobase per million (RPKM) values were calculated for all 1,272 ARGs in the constructed database. The monthly numbers of patients with presumed extended-spectrum β-lactamase (ESBL)-producing and metallo-β-lactamase (MBL)-producing bacteria, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE) were compared with the monthly RPKM values of blaCTX-M, blaIMP, mecA, vanA, and vanB by xHYB. The average RPKM value for all ARGs detected by xHYB was significantly higher than that of mDNA-seq (665, 225, and 328, respectively, and P < 0.05). The average number of patients with ESBL producers and RPKM values of blaCTX-M-1 genes in 2020 were significantly higher than that in 2019 (17 and 13 patients per month and 921 vs 232 per month, respectively, both P < 0.05). The average numbers of patients with MBL-producers, MRSA, and VRE were 1, 28, and 0 per month, respectively, while the average RPKM values of blaIMP, mecA, vanA, and vanB were 6,163, 6, 0, and 126 per month, respectively. Monitoring ARGs in hospital effluent using xHYB was found to be more useful than conventional mDNA-seq in detecting ARGs including blaCTX-M, blaIMP, and vanB, which are important for infection control.IMPORTANCEEnvironmental ARGs play a crucial role in the emergence and spread of AMR that constitutes a significant global health threat. One major source of ARGs is effluent from healthcare facilities, where patients are frequently administered antimicrobials. Culture-independent methods, including metagenomics, can detect environmental ARGs carried by non-culturable bacteria and extracellular ARGs. mDNA-seq is one of the most comprehensive methods for environmental ARG surveillance; however, its sensitivity is insufficient for wastewater surveillance. This study demonstrates that xHYB appropriately monitors ARGs in hospital effluent for sensitive identification of nosocomial AMR dissemination. Correlations were observed between the numbers of inpatients with antibiotic-resistant bacteria and the ARG RPKM values in hospital effluent over time. ARG surveillance in hospital effluent using the highly sensitive and specific xHYB method could improve our understanding of the emergence and spread of AMR within a hospital.

Identification and functional analysis of arabinogalactan protein expressed in pear pollen tubes

Arabinogalactan proteins (AGPs) are widely distributed in the plant kingdom and play a vital role during the process of plant sexual reproduction. In this study, we performed a comprehensive identification of the PbrAGPs expressed in pear pollen and further explored their influences on pollen tube growth. Among the 187 PbrAGPs that were found to be expressed in pear pollen tubes, 38 PbrAGPs were specifically expressed in pollen according to the RNA-seq data. The PbrAGPs were divided into two groups of highly expressed and specifically expressed in pear pollen. We further tested their expression patterns using RT-PCR and RT-qPCR. Most of the PbrAGPs were expressed in multiple tissues and their expression levels were consistent with Reads Per Kilobase per Million (RPKM) values during pollen tube growth, implying that PbrAGPs might be involved in the regulation of pear pollen tube growth. We also constructed phylogenetic trees to identify the functional genes in pear pollen tube growth. Therefore, 9 PbrAGPs (PbrAGP1 to PbrAGP19) were selected to test their influences on pollen tube growth. Recombinant proteins of the 19 PbrAGP-his were purified and used to treat pear pollen, and 11 of the PbrAGP-his recombinant proteins could promote pear pollen tube growth. Additionally, pollen tube growth was inhibited when the expression levels of PbrAGP1 and PbrAGP5 were knocked down using an antisense oligonucleotide assay. PbrAGP1 and PbrAGP5 were localized in the plasma membrane and might not alter the distribution of pectin in the pollen tube. In summary, this study identified the PbrAGPs expressed in pear pollen and lays the foundation for further exploring their functions in pollen tube growth.

Genome-wide identification and expression analysis of mitogen-activated protein kinase (MAPK) genes in response to salinity stress in channel catfish (Ictalurus punctatus).

The mitogen-activated protein kinase (MAPK) gene family has been systematically described in several fish species, but less so in channel catfish (Ictalurus punctatus), which is an important global aquaculture species. In this study, 16 MAPK genes were identified in the channel catfish genome and classified into three subfamilies based on phylogenetic analysis, including six extracellular signal regulated kinase (ERK) genes, six p38-MAPK genes and four C-Jun N-terminal kinase (JNK) genes. All MAPK genes were distributed unevenly across 10 chromosomes, of which three (IpMAPK8, IpMAPK12 and IpMAPK14) underwent teleost-specific whole genome duplication during evolution. Gene expression profiles in channel catfish during salinity stress were analysed using transcriptome sequencing and qRT-PCR (quantitative reverse transcription PCR). Results from reads per kilobase million (RPKM) analysis showed IpMAPK13, IpMAPK14a and IpMAPK14b genes were differentially expressed when compared with other genes between treatment and control groups. Furthermore, three of these genes were validated by qRT-PCR, of which IpMAPK14a expression levels were significantly upregulated in treatment groups (high and low salinity) when compared with the control group, with the highest expression levels in the low salinity group (P < 0.05). Therefore, IpMAPK14a may have important response roles to salinity stress in channel catfish.

Detection of multiple types of cancer driver mutations using targeted RNA sequencing in NSCLC.

e15043 Background: DNA NGS sequencing has been widely used as a companion diagnostic method for matching NSCLC patients with appropriate personalized target therapy. RNA-based NGS has been proved to be valuable in detecting fusion and exon skipping mutations and therefore recommended by NCCN guidelines for these mutation types. It would be helpful to have a diagnostic method that suits different types of clinically actionable mutations in a single tube. Methods: We developed an RNA-based hybridization panel targeting actionable driver oncogenes in solid tumors. Experimental and bioinformatics pipelines were optimized for detection of fusion, SNV and indel mutations, as well as gene expression quantification. Reference materials were used for analytical validation of the panel, and 1253 FFPE samples from NSCLC patients were analyzed by DNA panel sequencing and RNA panel sequencing in parallel to assess the performance of the RNA panel in detection of multiple types of clinically actionable mutations. Results: Analytical validation showed a detection specificity of 100.0% for SNVs and fusions. The targeted RNA panel achieved a 1.45-3.15 copies/ng limit of detection (LOD) for SNVs and a 0.21-6.48 copies/ng LOD for fusions. Gene expression analysis revealed a correlation greater than 0.9 between the RNA-seq and targeted RNA panel results. Among 1253 NSCLC FFPE samples, multiple mutation types were called at both DNA and RNA levels and compared between the two assays. Twenty-one fusion and six METex14 skipping events were only detected by the RNA panel. The positive percent agreement (PPA) and positive prediction value (PPV) were 98.08% and 98.62% for targetable SNVs and 98.15% and 99.38% for targetable Indels, respectively. We also explored the relationship of RNA expression with gene copy number and protein expression. The RPKM (reads per kilobase per million mapped reads) of EGFR transcripts exhibited a linear relationship with copy number, whereas MET gene expression seemed to be regulated in a more complexed manner. In IHC analysis, all MET IHC 3+ samples had significantly higher RPKM levels than samples with MET IHC level of 2+/1+. Conclusions: Parallel DNA and RNA systematic analysis demonstrated the accuracy and robustness of the RNA sequencing panel for detection of multiple types of clinically actionable mutations. The simplified experimental workflow, lowered cost and sample consumption, along with the superior performance in simultaneous detection of multiple types of mutations will make RNA panel sequencing an effective method in clinical testing for NSCLC.

Identification and Tissue Expression Profiles of Odorant Receptor Genes in the Green Peach Aphid Myzus persicae.

Simple SummaryThe green peach aphid is a pest that mainly endangers vegetables. It spreads a variety of plant viruses through pricking and sucking plant juice. At the same time, it also secretes honeydew, which causes mildew and pollution and leads to the loss of many crops. Olfaction plays a very important role in the aphids’ search for hosts, avoidance of natural enemies, mating and oviposition. However, the molecular mechanism of olfactory recognition in the green peach aphid is not clear. Odorant receptors (ORs) are a type of olfactory neuron membrane receptor expressed on insect antennae and they play a role in identifying odorants from the environment. We identified 33 OR genes from the genome and transcriptomes of the green peach aphid and analyzed their phylogenetic relationship and genomic structure. We further examined the expression patterns in different tissues by real-time quantitative PCR (RT-qPCR). This study enriches our understanding of the olfactory system of the green peach aphid and will help to better apply it to the integrated control of a variety of pests.The green peach aphid Myzus persicae (Hemiptera: Aphididae) relies heavily on its olfactory system to locate plant hosts, find mates, and avoid parasitoids or predators. The insect odorant receptors (ORs) have been proven to play a critical role in the perception of odorants from the environment. In the present study, 33 odorant receptor candidate genes including the Orco gene were identified from the antennal, head, legs and body transcriptomes of M. persicae. Phylogenetic analysis of ORs from seven different orders of insect species suggests that ORs from different insect species are highly divergent and most ORs from the same species formed monophyletic groups. In addition, the aphid ORs were clustered into six different sub-clades in the same clade. Furthermore, the genomic structure of the OR genes also tends to be consistent, suggesting that ORs from the family Aphididae have a relatively close evolutionary relationship. Reads per kilobase per million (RPKM) and tissue expression profiles analyses revealed that 27 out of the 33 MperORs were uniquely or primarily expressed in the antennae, indicating their putative roles in chemoreception. This work provides a foundation to further investigate the molecular and ecological functions of MperORs in the aphid–aphid, aphid–plant and aphid–natural enemy interactions.

Overexpression of an ethylene-forming ACC oxidase (ACO) gene precedes the Minute Hilum seed coat phenotype in Glycine max

BackgroundTo elucidate features of seed development, we investigated the transcriptome of a soybean isoline from the germplasm collection that contained an introgressed allele known as minute hilum (mi) which confers a smaller hilum region where the seed attaches to the pod and also results in seed coat cracking surrounding the hilum region.ResultsRNAs were extracted from immature seed from an extended hilum region (i.e., the hilum and a small ring of tissue surrounding the hilum in which the cracks form) at three different developmental stages:10–25, 25–50 and 50–100 mg seed fresh weight in two independent replicates for each stage. The transcriptomes of these samples from both the Clark isoline containing the mi allele (PI 547628, UC413, iiR t mi G), and its recurrent Clark 63 parent isoline (PI 548532, UC7, iiR T Mi g), which was used for six generations of backcrossing, were compared for differential expression of 88,648 Glyma models of the soybean genome Wm82.a2. The RNA sequence data obtained from the 12 cDNA libraries were subjected to padj value < 0.05 and at least two-fold expression differences to select with confidence genes differentially expressed in the hilum-containing tissue of the seed coat between the two lines. Glyma.09G008400 annotated as encoding an ethylene forming enzyme, ACC oxidase (ACO), was found to be highly overexpressed in the mi hilum region at 165 RPKMs (reads per kilobase per million mapped reads) compared to the standard line at just 0.03 RPKMs. Evidence of changes in expression of genes downstream of the ethylene pathway included those involved in auxin and gibberellin hormone action and extensive differences in expression of cell wall protein genes. These changes are postulated to determine the restricted hilum size and cracking phenotypes.ConclusionsWe present transcriptome and phenotypic evidence that substantially higher expression of an ethylene-forming ACO gene likely shifts hormone balance and sets in motion downstream changes resulting in a smaller hilum phenotype and the cracks observed in the minute hilum (mi) isoline as compared to its recurrent parent.

The Effector Repertoire of the Hop Downy Mildew Pathogen Pseudoperonospora humuli.

Pseudoperonospora humuli is an obligate biotrophic oomycete that causes downy mildew (DM), one of the most destructive diseases of cultivated hop that can lead to 100% crop loss in susceptible cultivars. We used the published genome of P. humuli to predict the secretome and effectorome and analyze the transcriptome variation among diverse isolates and during infection of hop leaves. Mining the predicted coding genes of the sequenced isolate OR502AA of P. humuli revealed a secretome of 1,250 genes. We identified 296 RXLR and RXLR-like effector-encoding genes in the secretome. Among the predicted RXLRs, there were several WY-motif-containing effectors that lacked canonical RXLR domains. Transcriptome analysis of sporangia from 12 different isolates collected from various hop cultivars revealed 754 secreted proteins and 201 RXLR effectors that showed transcript evidence across all isolates with reads per kilobase million (RPKM) values > 0. RNA-seq analysis of OR502AA-infected hop leaf samples at different time points after infection revealed highly expressed effectors that may play a relevant role in pathogenicity. Quantitative RT-PCR analysis confirmed the differential expression of selected effectors. We identified a set of P. humuli core effectors that showed transcript evidence in all tested isolates and elevated expression during infection. These effectors are ideal candidates for functional analysis and effector-assisted breeding to develop DM resistant hop cultivars.

Meta-Transcriptomic Analysis of RNAseq Data Reveals Pacu and Loach Fish with Unusually High Levels of Myoglobin Expression in Skeletal Muscles.

Simple SummaryOxygen-binding proteins that mediate oxygen-binding for storage and consumption, to reduce energy, are very diverse in fish, depending on their habitats. In the present study, oxygen-binding protein gene expression in the skeletal muscle of 25 diverse fish species was examined by a meta-transcriptomic approach. By using RNAseq data, this is the first study to examine the high level of myoglobin, one of the oxygen-binding proteins, transcripts in pacu and loach fish that might be related to their high tolerance for the oxygen-deficient environment. In addition, this study presents the power of the current method to compare the fish oxygen-binding protein expression and its putative gene expansion event.Oxygen-binding proteins, such as myoglobin, hemoglobin, neuroglobin, and cytoglobin, play a role in oxygen binding and delivery to tissues. In icefish, the loss of myoglobin and hemoglobin genes has been reported to be an adaptive evolution event. This interesting finding prompted us to exam oxygen-binding protein expression in diverse fish species. Taking advantage of substantial RNAseq data deposited in the NCBI (National Center for Biotechnology Information) database, we adopted a meta-transcriptomic approach to explore and compare four oxygen-binding protein gene expression levels in the skeletal muscle of 25 diverse fish species for the first time. RNAseq data were downloaded from the NCBI Sequence Read Archive (SRA) database, and de novo assembly was performed to generate transcript contigs. The genes encoding oxygen-binding proteins were then identified by the BLAST search, and the relative expression level of oxygen-binding protein genes was normalized by the RPKM (Reads per Kilobase Million) method. By performing expression profiling, hierarchy clustering, and principal component analysis, pacu and loach fish were noticed by their high myoglobin expression levels in skeletal muscle tissues among 25 diverse fish species. In conclusion, we demonstrated that meta-transcriptomic analysis of RNAseq data is an informative approach to compare the oxygen-binding protein expression and putative gene expansion event in fish.

1662-P: Lower Expression of Bile Acid Transporters and Fibroblast Growth Factor 19 in Mucosa Biopsies from the Ileocecal Region in Persons with Type 2 Diabetes Compared with Healthy Controls

Background and Aims: Bile acids stimulate the secretion of gluco-regulatory and appetite-regulating gut hormones (including glucagon-like peptide 1 and fibroblast factor 19 (FGF19)) and increase energy expenditure. Changes in postprandial concentrations of circulating bile acids (primarily determined by the apical sodium-dependent bile acid transporter (ASBT) and organic solute transporter α/β (OSTα/β) in the terminal ileum) have been linked to type 2 diabetic pathophysiology. Methods: In a cross-sectional study, 12 persons with type 2 diabetes (T2D) (mean[range] age 51[34-63] years, BMI 26.8[23.7-31.5] kg/m2, HbA1c 48[36-85] mmol/mol) and 12 gender, age and BMI-matched nondiabetic controls (CTRLs) (age 50[41-67] years, BMI 27.1[20.3-30.8] kg/m2, HbA1c 34[29-43] mmol/mol) underwent upper and lower double-balloon enteroscopy with retrieval of mucosal biopsies from every 30 cm along the entire intestinal tract. The biopsies were subjected to full mRNA sequencing and here, the expression of ASBT, OSTα/β and FGF19 is reported. Results: For both groups, robust expression (reads per kilobase million (RPKM) ≥1) of ASBT and OSTα/β was evident in the duodenum and this increased along the small intestine, with highest expression of ASBT in the ileocecal region and highest expression of OSTα/β in terminal ileum. Compared to healthy controls, persons with T2D exhibited significantly lower ASBT and OSTα/β expression in the ileocecal region (P&amp;lt;0.05). Compared to persons with T2D in whom expression of FGF19 was below 1 RPKM in all locations, the control group had robust and significantly greater expression of FGF19 in the terminal ileum (P&amp;lt;0.01). Conclusions: T2D is associated with reduced expression of ASBT, OSTα/β and FGF19 in the terminal ileum and the proximal colon. This may point to a link between dysfunctional enterohepatic circulation of bile acids and type 2 diabetic pathophysiology. Disclosure H.H. Nerild: Employee; Spouse/Partner; Novo Nordisk A/S. A. Brønden: None. A. El Haddouchi: None. T. Jorsal: None. D.P. Sonne: None. J. Jelsing: Board Member; Self; Gubra. Stock/Shareholder; Self; Gubra. K. Rigbolt: Employee; Self; Gubra. T. Vilsbøll: Advisory Panel; Self; AstraZeneca, Mundipharma International, Novo Nordisk A/S, Sun Pharmaceutical Industries Ltd. Consultant; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Medscape, Merck Sharp &amp; Dohme Corp., Sanofi. F.K. Knop: Advisory Panel; Self; AstraZeneca, Merck Sharp &amp; Dohme Corp., Mundipharma International, Novo Nordisk A/S, Sanofi. Consultant; Self; Carmot Therapeutics, Inc., Eli Lilly and Company, Novo Nordisk A/S. Research Support; Self; AstraZeneca, Gubra, Novo Nordisk A/S, Sanofi, Zealand Pharma A/S. Speaker’s Bureau; Self; AstraZeneca, Lupin Pharmaceuticals, Inc., Merck Sharp &amp; Dohme Corp., Norgine B.V., Novo Nordisk A/S.

Drought‐tolerant transcription factors identified in Arachis dardani and Arachis ipaënsis

AbstractIn this study, a greenhouse transcriptome study with 7‐d‐imposed drought was conducted on Arachis dardani Krapov. &amp; W.C. Greg. (GK 12946) and the reference species A. ipaënsis (Krapov. and W.C. Greg.) (K 30076) (B genome donor of the cultivated peanut) to identify candidate genes associated with drought. Differential gene expression analysis (EdgeR Test) of the normalized reads per kilobase million (RPKM) values was conducted with a fold value ≥ |2| at the P ≤ .05 level using CLC Genomics Workbench v8.1. Statistically significant transcript levels associated with drought tolerance were identified in relation to the putative drought species A. dardani GK 12946 and the reference species A. ipaënsis K 30076 which have not been reported previously in peanut and will serve as candidate genes for marker development and gene introgression in future research.

Transcriptome & viral growth analysis of SARS-CoV-2-infected Vero CCL-81 cells.

Background & objectives:The genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), belonging to the family Coronaviridae, encodes for structural, non-structural, and accessory proteins, which are required for replication of the virus. These proteins are encoded by different genes present on the SARS-CoV-2 genome. The expression pattern of these genes in the host cells needs to be assessed. This study was undertaken to understand the transcription pattern of the SARS-CoV-2 genes in the Vero CCL-81 cells during the course of infection.Methods:Vero CCL-81 cells were infected with the SARS-CoV-2 virus inoculum having a 0.1 multiplicity of infection. The supernatants and cell pellets were harvested after centrifugation at different time points, post-infection. The 50% tissue culture infective dose (TCID50) and cycle threshold (Ct) values of the E and the RdRp-2 genes were calculated. Next-generation sequencing of the harvested sample was carried out to observe the expression pattern of the virus by mapping to the SARS-CoV-2 Wuhan HU-1 reference sequence. The expressions were in terms of the reads per kilobase million (RPKM) values.Results:In the inital six hours post-infection, the copy numbers of E and RdRp-2 genes were approximately constant, which raised 10 log-fold and continued to increase till the 12 h post-infection (hpi). The TCID50 was observed in the supernatant after 7 hpi, indicating the release of the viral progeny. ORF8 and ORF7a, along with the nucleocapsid transcript, were found to express at higher levels.Interpretation & conclusions:This study was a step towards understanding the growth kinetics of the SARS-CoV-2 replication cycle. The findings indicated that ORF8 and ORF7b gene transcripts were expressed in higher amounts indicating their essential role in viral replication. Future studies need to be conducted to explore their role in the SARS-CoV-2 replication.

Transcriptome analysis and targeted metabolic profiling for pathway elucidation and identification of a geraniol synthase involved in iridoid biosynthesis from Gardenia jasminoides

Iridoid compounds have been reported to be accumulated in the dried fruits of Gardenia jasminoides (Rubiaceae), which are used in traditional Chinese medicine. These compounds exhibit obvious pharmacological activities, including effective protective effects on the liver and therapeutic efficacy for cardiovascular diseases. However, the biosynthetic pathway of iridoid remains uninvestigated. In this study, 12 transcriptomes from four tissues (lower leaves, top leaves, flowers, and fruits) were analyzed to characterize the accumulation of active constituents and to identify the corresponding genes involved in iridoid biosynthesis. Almost all genes in the mevalonate (MVA) pathway and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway were found in our transcriptome database; these pathways comprise upstream pathways of iridoid compounds (monoterpene derivatives), most of which are abundant in flowers and fruits. Geraniol synthase (GES), which is involved in the first branch step in the iridoid biosynthetic pathway, was cloned, and quantitative real-time PCR (qRT-PCR) revealed that GjGES showed a similar expression pattern to that in the transcriptome in terms of reads per kilobase per million (RPKM) mapped reads. The recombinant GjGES protein efficiently converted geranyl diphosphate (GPP) to geraniol. Subsequently, the metabolic profiling of seven iridoids and iridoid glycosides, namely geniposide, gardenoside, genipin, geniposidic acid, genipin-1-β-gentiobioside, shanzhiside, and shanzhiside methyl ester, revealed that these compounds were highly accumulated in fruits. Consequently, the metabolites and speculated unigenes involved in iridoid metabolic pathways enabled the identification of glucosyltransferase, hydroxylase, and O-methyltransferase candidate genes responsible for the biosynthesis of iridoid and iridoid glycosides. The results of the present study can serve as a reference for the functional characterization of enzyme-coding genes and are beneficial for the engineering of biosynthetic pathways of iridoids and iridoid glycosides in the future.

Progerin expression induces a significant downregulation of transcription from human repetitive sequences in iPSC-derived dopaminergic neurons.

Repetitive DNA sequences represent about half of the human genome. They have a central role in human biology, especially neurobiology, but are notoriously difficult to study. The purpose of this study was to quantify the transcription from repetitive sequences in a progerin-expressing cellular model of neuronal aging. Progerin is a nuclear protein causative of the Hutchinson-Gilford progeria syndrome that is also incrementally expressed during the normal aging process. A dedicated pipeline of analysis allowed to quantify transcripts containing repetitive sequences from RNAseq datasets oblivious of their genomic localization, tolerating a sufficient degree of mutational noise, all with low computational requirements. The pipeline has been applied to a published panel of RNAseq datasets derived from a well-established and well-described cellular model of aging of dopaminergic neurons. Progerin expression strongly downregulated the transcription from all the classes of repetitive sequences: satellites, long and short interspersed nuclear elements, human endogenous retroviruses, and DNA transposon. The Alu element represented by far the principal source of transcript originating either from repetitive sequences or from canonical coding genes; it was expressed on average at 192,493.5 reads per kilobase million (RPKM) (SE = 21,081.3) in the control neurons and dropped to 43,760.1 RPKM (SE = 5315.0) in the progerin-expressing neurons, being significant downregulated (p = 0.0005). The results highlighted a global perturbation of transcripts derived from repetitive sequences in a cellular model of aging and provided a direct link between progerin expression and alteration of transcription from human repetitive elements.

Whole gene transcriptomic analysis of PCB/biphenyl degrading Rhodococcus jostii RHA1.

This study gives the first picture of whole RNA-Sequencing analysis of a PCB-degrading microbe, Rhodococcus jostii RHA1. Genes that were highly expressed in biphenyl-grown cells, compared with pyruvate-grown cells, were chosen based on the Reads Per Kilobase Million (RPKM) value and were summarized based on the criteria of RPKM ≥100 and fold change ≥2.0. Consequently, 266 total genes were identified as genes expressed particularly for the degradation of biphenyl. After comparison with previous microarray data that identified highly-expressed genes, based on a fold change ≥2.0 and p-value ≤0.05, 62 highly-expressed genes from biphenyl-grown cells were determined from both analytical platforms. As these 62 genes involve known PCB degradation genes, such as bph, etb, and ebd, the genes identified in this study can be considered as essential genes for PCB/biphenyl degradation. In the 62 genes, eleven genes encoding hypothetical proteins were highly expressed in the biphenyl-grown cells. Meanwhile, we identified several highly-expressed unannotated DNA regions on the opposite strand. In order to verify the encoded proteins, two regions were cloned into an expression vector. A protein was successfully obtained from one region at approximately 25 kDa from the unannotated strand. Thus, the genome sequence with transcriptomic analysis gives new insight, considering re-annotation of the genome of R. jostii RHA1, and provides a clearer picture of PCB/biphenyl degradation in this strain.

Bone Marrow T Cells Are Driven into Exhaustion By Acute Leukemia in Pediatric Patients Based on Protein and Transcriptome Analysis

Introduction: Acute leukemia is the most common malignancy in children and develops within the bone marrow. Consequently, bone marrow derived T cells of leukemia patients can be defined as tumor infiltrating lymphocytes (TILs). Dysfunctional TILs have been described in several other malignancies. However, in pediatric patients the interaction between leukemic blasts and TILs remains largely unknown. In order to understand the impact of leukemic blasts on bone marrow T cells we profiled T cells in the bone marrow of pediatric leukemia patients by surface marker and transcriptome wide analysis.Methods: First, artificial changes in marker expression due to cryopreservation and thawing were ruled out (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (HD, n=23) were identified in a local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T cells in primary (n=49) and relapse leukemia samples (n=28). As we could not detect surface CTLA4 expression on T cells, CTLA4 was stained intracellularly. Additionally, RNA-Seq on sorted bone marrow derived CD8 T cells (n=48; TCP-ALL: 12, AML: 20, HD: 16) was performed. Analysis of RNA-Seq data was based on Reads Per Kilobase Million (RPKM) normalization and False Discovery Rate (FDR, Benjamini-Hochberg) statistics. 172 differentially expressed genes were found when comparing bone marrow derived CD8 T cells from healthy donors (n=16) and leukemia patients (n=32) using the following criteria: RPKM>2 in both groups, fold change>2 and FDR<0.05).Results: The frequency of bone marrow T cells was reduced in patients with acute leukemia in comparison with healthy controls (5.9% vs. 24.4%, mean values, p<0.001). This reduction was more pronounced in BCP-ALL than in AML (0.9% vs. 8.4%, p<0.001). LAG3 and CTLA4 protein expression of T cells was increased in leukemia patients vs. healthy controls (LAG3: CD4: 2.6% vs. 0.7%, p<0.001; CD8: 8.6% vs. 2.2%, p<0.001; CTLA4: CD4: 7.3% vs. 3.8%, p=0.001; CD8: 1.2 vs. 0.3%, p<0.001). For CD8 T cells, those findings could be confirmed by RNA-Seq of sorted CD8 T cells (LAG3: 60.4 vs. 23.3 (RPKM), FDR=0.0044; CTLA4: 28.7 vs 4.7 (RPKM), FDR=0.046). Equally, TIM3 on T cells showed higher expression in leukemia patients vs. healthy controls (CD4: 3.7% vs. 1.3%, p=0.002; CD8: 8.5% vs. 3.3%, p<0.001). However, the same analysis of RNA-Seq data on sorted CD8 T cells did not yield a significant difference (18.1 vs. 5.6 (RPKM), FDR=0.29). PD1 was the only surface marker found to be more highly expressed in relapse samples than in primary diagnosis samples than in healthy controls (CD4: 42.3% vs. 28.9% vs. 19.8%, p<0.001; CD8: 45.2% vs. 33.3% vs. 26.5%, p=0.002). For CD8 T cells, RNA-Seq did not recapitulate this finding as no significant difference of PD1 transcript abundancy could be observed between leukemia patients and healthy donors by RNA-Seq (21.4 vs. 16.9 (RPKM), FDR=0.92). Finally, RNA-Seq on sorted CD8 T cells showed a pronounced overexpression of genes that are involved in the cytotoxic granule machinery in leukemia patients indicating an increase of effector phenotype in those cells. Contrarily, genes crucial for T cell function and memory formation were significantly downregulated in CD8 T cells from leukemia patients.Conclusion: By analyzing bone marrow samples from pediatric leukemia patients and healthy controls we confirm that bone marrow T cells of leukemia patients show signs of exhaustion compared to healthy individuals. Importantly, PD1 surface expression on T cells was identified as a marker that correlates with disease status (relapse > primary > healthy). A significant increase of exhaustion markers could be demonstrated both on protein and transcriptome level (LAG3, CTLA4) or on protein level only (TIM3, PD1). Moreover, we observed an increase of many elements of the cytotoxic granule machinery which is compatible with a loss of naïve/memory CD8 T cells. Additionally, genes essential for T cell memory formation were found to be downregulated in CD8 T cells from leukemia patients. These findings reflect an insufficient immune surveillance of pediatric leukemia by bone marrow T cells and may provide a rationale for future therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2289: Precision-weighted RNA-seq analyses of molecular abundance (RoMA) for detecting differential gene expression

Abstract In this study, we propose a novel method, RoMA, to accurately detect differential expression and unlock the integration with numerous upstream and downstream analyses on mRNA abundance in RNA-seq studies. Various methods have been proposed, each with its own limitations. Some naive normal-based tests have low testing power with the invalid normal distribution assumptions for RNA-seq read counts, whereas count-based methods lack a biologically meaningful interpretation and have limited capability for integration with other analysis packages for mRNA abundance. RoMA incorporates information from both mRNA abundance and raw counts by modeling RPKM (reads per kilobase per million), which represents the relative abundance of mRNA transcripts, and borrowing mean-variance dependency from CPM (counts per million) as a precision weight accounting for the variability in sequencing depth. Studies on simulated data and two real datasets showed that RoMA provides an accurate quantification of mRNA abundance and a value adjustment-tolerant DE analysis with high AUC, low FDR and a desirable type I error rate. This study provides a valid strategy for mRNA abundance modeling and data analysis integration for RNA-seq studies, which will greatly facilitate the identification and interpretation of DE genes. The methods is implemented in a user-friendly R package (RoMA). Citation Format: Guoshuai Cai, Jennifer M. Franks, Michael L. Whitfield. Precision-weighted RNA-seq analyses of molecular abundance (RoMA) for detecting differential gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2289.

GTSF-1 is required for formation of a functional RNA-dependent RNA Polymerase complex in Caenorhabditis elegans.

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C-terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf-1 and characterized it in the context of the sRNA pathways of C.elegans We report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, gtsf-1 mutants show a striking depletion of 26G-RNAs, a class of endogenous sRNAs, fully phenocopying rrf-3 mutants. We show, both invivo and invitro, that GTSF-1 interacts with RRF-3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex (ERIC), thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA-mediated silencing activities.

Functional characterization of naturally occurring wild soybean mutant (sg-5) lacking astringent saponins using whole genome sequencing approach

Triterpenoid saponins are one of the most highly accumulated groups of functional components in soybean (Glycine max) and the oxidative reactions during their biosynthesis are required for their aglycone diversity. Natural mutants of soyasaponins in wild soybean (Glycine soja) are valuable resources for establishing the soyasaponin biosynthesis pathway and breeding new soybean varieties. In this study, we investigated the genetic mechanism behind the absence of group A saponins in a Korean wild soybean mutant, CWS5095. Whole genome sequencing (WGS) of CWS5095 identified four point mutations [Val6 → Asp, Ile231 → Thr, His294 → Gln, and Arg376 → Lys] in CYP72A69 (Glyma15g39090), which oxygenate the C-21 position of soyasapogenol B or other intermediates to produce soyasapogenol A, leading to group A saponin production. An in vitro enzyme activity assay of single-sited mutated clones indicated that the Arg376 > Lys mutation (a highly conserved mutation based on a nucleotide change from G → A at the 1,127th position) may lead to loss of gene function in the sg-5 mutant. A very high normalized expression value of 377 reads per kilo base per million (RPKM) of Glyma15g39090 in the hypocotyl axis at the early maturation seed-development stage confirmed their abundant presence in seed hypocotyls. A molecular dynamics analysis of the Arg376 > Lys mutation based on the CYP3A4 (a human CYP450) protein structure found that it was responsible for the increase in axis length toward the heme (active site), which is critically important for biological activity and ligand binding. Our results provide important information on how to eradicate bitter and astringent saponins in soybean by utilizing the reported mutation in Glyma15g39090, and its importance for seed hypocotyl development based on transcript abundance.