RNA-based Approaches Research Articles

RNA Interference and CRISPR/Cas Gene Editing for Crop Improvement: Paradigm Shift towards Sustainable Agriculture.

With the rapid population growth, there is an urgent need for innovative crop improvement approaches to meet the increasing demand for food. Classical crop improvement approaches involve, however, a backbreaking process that cannot equipoise with increasing crop demand. RNA-based approaches i.e., RNAi-mediated gene regulation and the site-specific nuclease-based CRISPR/Cas9 system for gene editing has made advances in the efficient targeted modification in many crops for the higher yield and resistance to diseases and different stresses. In functional genomics, RNA interference (RNAi) is a propitious gene regulatory approach that plays a significant role in crop improvement by permitting the downregulation of gene expression by small molecules of interfering RNA without affecting the expression of other genes. Gene editing technologies viz. the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (CRISPR/Cas) have appeared prominently as a powerful tool for precise targeted modification of nearly all crops’ genome sequences to generate variation and accelerate breeding efforts. In this regard, the review highlights the diverse roles and applications of RNAi and CRISPR/Cas9 system as powerful technologies to improve agronomically important plants to enhance crop yields and increase tolerance to environmental stress (biotic or abiotic). Ultimately, these technologies can prove to be important in view of global food security and sustainable agriculture.

Leveraging histone modifications to improve genome annotations.

Accurate genome annotations are essential to modern biology; however, they remain challenging to produce. Variation in gene structure and expression across species, as well as within an organism, make correctly annotating genes arduous; an issue exacerbated by pitfalls in current in silico methods. These issues necessitate complementary approaches to add additional confidence and rectify potential misannotations. Integration of epigenomic data into genome annotation is one such approach. In this study, we utilized sets of histone modification data, which are precisely distributed at either gene bodies or promoters to evaluate the annotation of the Zea mays genome. We leveraged these data genome wide, allowing for identification of annotations discordant with empirical data. In total, 13,159 annotation discrepancies were found in Z. mays upon integrating data across three different tissues, which were corroborated using RNA-based approaches. Upon correction, genes were extended by an average of 2128 base pairs, and we identified 2529 novel genes. Application of this method to five additional plant genomes identified a series of misannotations, as well as identified novel genes, including 13,836 in Asparagus officinalis, 2724 in Setaria viridis, 2446 in Sorghum bicolor, 8631 in Glycine max, and 2585 in Phaseolous vulgaris. This study demonstrates that histone modification data can be leveraged to rapidly improve current genome annotations across diverse plant lineages.

RNA Is a Double-Edged Sword in ALS Pathogenesis.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that affects upper and lower motor neurons. Familial ALS accounts for a small subset of cases (<10–15%) and is caused by dominant mutations in one of more than 10 known genes. Multiple genes have been causally or pathologically linked to both ALS and frontotemporal dementia (FTD). Many of these genes encode RNA-binding proteins, so the role of dysregulated RNA metabolism in neurodegeneration is being actively investigated. In addition to defects in RNA metabolism, recent studies provide emerging evidence into how RNA itself can contribute to the degeneration of both motor and cortical neurons. In this review, we discuss the roles of altered RNA metabolism and RNA-mediated toxicity in the context of TARDBP, FUS, and C9ORF72 mutations. Specifically, we focus on recent studies that describe toxic RNA as the potential initiator of disease, disease-associated defects in specific RNA metabolism pathways, as well as how RNA-based approaches can be used as potential therapies. Altogether, we highlight the importance of RNA-based investigations into the molecular progression of ALS, as well as the need for RNA-dependent structural studies of disease-linked RNA-binding proteins to identify clear therapeutic targets.

Abstract 1031: Translatome and protein synthesis machinery remodeling by rocaglates drives antitumor activity

Abstract Rocaglates are under active development for cancer therapy, with the first-in-class member zotatifin recently entering Phase I/II clinical trial for advanced solid tumor malignancies (NCT04092673). Initiation of this trial was based purely on rocaglates' inhibitory activity against translation initiation factor and RNA helicase eIF4A. Rapid development of these compounds towards clinical use urgently requires a complete understanding of their mechanisms. Critically, however, the full effects of rocaglates on protein synthesis are unknown, especially with regards to up-regulated proteins and rocaglate-activated translation factors that mediate their potent anticancer activity. Past rocaglate studies focused exclusively on mechanisms and targets of translational inhibition, relying exclusively on indirect RNA-based approaches that do not reliably capture proteome-level changes, a caveat proven in cancer cells responding to drugs. Formal proteomic investigations of rocaglate effects are non-existent in the literature. We addressed these vital knowledge gaps in this study. An original proteome-level analysis using metabolic pulse-labeling and TMT-mass spectrometry revealed extensive rocaglate-induced changes in protein output (translatome), including myriad rocaglate-up-regulated proteins that drive cytotoxicity through currently uncharacterized mechanisms. Specifically, we find in vitro and in vivo that rocaglates, including zotatifin, induce GEF-H1, activating anti-survival RHOA/JNK signaling. Intriguingly, these responses occur regardless of eIF4A expression. Beyond translatome remodeling, mass spectrometry-based MATRIX interrogation of the protein synthesis machinery revealed widespread rocaglate-dependent changes. As a prime example, rocaglate-augmented eEF1ϵ1 activity mediates initiator methionine-tRNAiMet delivery to drive synthesis of rocaglate-inducible proteins and phenotypes. Overall, this study transforms the current definition of rocaglates from one-dimensional eIF4A inhibitors to translation remodelers that globally reprogram protein output and the translation machinery. As human patients begin treatment with these compounds, our discovery of rocaglate-inducible proteins and rocaglate-activated translation factors that drive drug-specific responses highlights an entirely uncharacterized yet critical aspect of rocaglate potency. These timely findings emphasize systemic translational modeling as a newer concept for cancer therapy, and advocate for a systematic overhaul in our understanding of drugs traditionally defined as translation inhibitors. Citation Format: Jyun David Ho, Jr, Tyler A. Cunningham, Caroline A. Coughlin, Artavazd Arumov, Evan R. Roberts, Daniel Bilbao, Jonathan R. Krieger, Stephen Lee, Jonathan H. Schatz. Translatome and protein synthesis machinery remodeling by rocaglates drives antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1031.

Elevated Atmospheric CO2 Modifies Mostly the Metabolic Active Rhizosphere Soil Microbiome in the Giessen FACE Experiment

Elevated levels of atmospheric CO2 lead to the increase of plant photosynthetic rates, carbon inputs into soil and root exudation. In this work, the effects of rising atmospheric CO2 levels on the metabolic active soil microbiome have been investigated at the Giessen free-air CO2 enrichment (Gi-FACE) experiment on a permanent grassland site near Giessen, Germany. The aim was to assess the effects of increased C supply into the soil, due to elevated CO2, on the active soil microbiome composition. RNA extraction and 16S rRNA (cDNA) metabarcoding sequencing were performed from bulk and rhizosphere soils, and the obtained data were processed for a compositional data analysis calculating diversity indices and differential abundance analyses. The structure of the metabolic active microbiome in the rhizospheric soil showed a clear separation between elevated and ambient CO2 (p = 0.002); increased atmospheric CO2 concentration exerted a significant influence on the microbiomes differentiation (p = 0.01). In contrast, elevated CO2 had no major influence on the structure of the bulk soil microbiome (p = 0.097). Differential abundance results demonstrated that 42 bacterial genera were stimulated under elevated CO2. The RNA-based metabarcoding approach used in this research showed that the ongoing atmospheric CO2 increase of climate change will significantly shift the microbiome structure in the rhizosphere.

A novel RNA-based approach to counteract EMT.

A novel RNA-based approach to counteract EMT.

Role of MicroRNAs, Aptamers in Neuroinflammation and Neurodegenerative Disorders.

Exploring the microRNAs and aptamers for their therapeutic role as biological drugs has expanded the horizon of its applicability against various human diseases, explicitly targeting the genetic materials. RNA-based therapeutics are widely being explored for the treatment and diagnosis of multiple diseases, including neurodegenerative disorders (NDD). Latter includes microRNA, aptamers, ribozymes, and small interfering RNAs (siRNAs), which control the gene expression mainly at the transcriptional strata. One RNA transcript translates into different protein types; hence, therapies targeted at the transcriptional sphere may have prominent and more extensive effects than alternative therapeutics. Unlike conventional gene therapy, RNAs, upon delivery, can either altogether abolish or alter the synthesis of the protein of interest, therefore, regulating their activities in a controlled and diverse manner. NDDs like Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, Prion disease, and others are characterized by deposition of misfolded protein such as amyloid-ß, tau, α-synuclein, huntingtin and prion proteins. Neuroinflammation, one of the perquisites for neurodegeneration, is induced during neurodegenerative pathogenesis. In this review, we discuss microRNAs and aptamers' role as two different RNA-based approaches for their unique ability to regulate protein production at the transcription level, hence offering many advantages over other biologicals. The microRNA acts either by alleviating the malfunctioning RNA expression or by working as a replacement to lost microRNA. On the contrary, aptamer act as a chemical antibody and forms an aptamer-target complex.

Diverse and active archaea communities occur in non-disinfected drinking water systems–Less activity revealed in disinfected and hot water systems

The knowledge about the members of active archaea communities in DWDS is limited. The current understanding is based on high-throughput 16S ribosomal RNA gene (DNA-based) amplicon sequencing that reveals the diversity of active, dormant, and dead members of the prokaryote (bacteria, archaea) communities. The sequencing primers optimized for bacteria community analysis may underestimate the share of the archaea community. This study characterized archaea communities at five full-scale drinking water distribution systems (DWDS), representing a variety of drinking water production units (A-E); A&B use artificially recharged non-disinfected groundwater (ARG), the other DWDS's supplied water disinfected by using ultraviolet (UV) light and chlorine compounds, C&D were surface waterworks and E was a ground waterworks. For the first time for archaea community analyses, this study employed the archaea-specific high-throughput sequencing primers for 16S ribosomal RNA (rRNA) as a target (reverse-transcribed cDNA; an RNA-based approach) in addition to the previously used 16S rRNA gene target (rDNA; a DNA-based approach) to reveal the active fraction of the archaea present in DWDS. The archaea community structure in varying environmental conditions in the water and biofilm of the five DWDSs were investigated by taking into consideration the system properties (cold or hot water system) and water age (distance from the treatment plants) in samples from each season of one year.The RNA-based archaea amplicon reads were obtained mostly from cold water samples from DWDSs (A–B) distributing water without disinfection where the DNA-based and RNA-based analysis created separate clusters in a weighted beta-diversity analysis. The season and location in DWDS A further affected the diversity of these archaea communities as was seen by different clusters in beta-diversity plots. The recovery of archaea reads was not adequate for analysis in any of the disinfected samples in DWDSs C–E or non-disinfected hot water in DWDSs A–B when utilizing RNA-based template. The metabolically active archaea community of DWDSs thus seemed to be effectively controlled by disinfection of water and in the hot water systems by the temperature. All biofilms regardless of DWDS showed lower species richness values (mainly Nitrososphaeria class) than non-disinfected water from DWDSs A–B where several archaea classes occurred (e.g. Woesearchaeia, Nitrososphaeria, Micrarchaeia, Methanomicrobia, Iairchaeia, Bathyarchaeia) indicating only part of the archaea members were able to survive in biofilms. Thus, Archaea has been shown as a significant part of normal DWDS biota, and their role especially in non-disinfected DWDS may be more important than previously considered.

Detection of gene fusions using targeted next-generation sequencing: a comparative evaluation

BackgroundGene fusions represent promising targets for cancer therapy in lung cancer. Reliable detection of multiple gene fusions is therefore essential.MethodsFive commercially available parallel sequencing assays were evaluated for their ability to detect gene fusions in eight cell lines and 18 FFPE tissue samples carrying a variety of known gene fusions. Four RNA-based assays and one DNA-based assay were compared; two were hybrid capture-based, TruSight Tumor 170 Assay (Illumina) and SureSelect XT HS Custom Panel (Agilent), and three were amplicon-based, Archer FusionPlex Lung Panel (ArcherDX), QIAseq RNAscan Custom Panel (Qiagen) and Oncomine Focus Assay (Thermo Fisher Scientific).ResultsThe Illumina assay detected all tested fusions and showed the smallest number of false positive results. Both, the ArcherDX and Qiagen panels missed only one fusion event. Among the RNA-based assays, the Qiagen panel had the highest number of false positive events. The Oncomine Focus Assay (Thermo Fisher Scientific) was the least adequate assay for our purposes, seven fusions were not covered by the assay and two fusions were classified as uncertain. The DNA-based SureSelect XT HS Custom Panel (Agilent) missed three fusions and nine fusions were only called by one software version. Additionally, many false positive fusions were observed.ConclusionsIn summary, especially RNA-based parallel sequencing approaches are potent tools for reliable detection of targetable gene fusions in clinical diagnostics.

Biosecurity risks associated with vaccine platform technologies

Vaccine platforms have been critical for accelerating the timeline of COVID-19 vaccine development. Faster vaccine timelines demand further development of these technologies. Currently investigated platform approaches include virally vectored and RNA-based vaccines, as well as DNA vaccines and recombinant protein expression system platforms, each featuring different advantages and challenges. Viral vector-based and DNA vaccines in particular have received a large share of research funding to date. Platform vaccine technologies may feature dual-use potential through informing or enabling pathogen engineering, which may raise the risk for the occurrence of deliberate, anthropogenic biological events. Research on virally vectored vaccines exhibits relatively high dual-use potential for two reasons. First, development of virally vectored vaccines may generate insights of particular dual-use concern such as techniques for circumventing pre-existing anti-vector immunity. Second, while the amount of work on viral vectors for gene therapy exceeds that for vaccine research, work on virally vectored vaccines may increase the number of individuals capable of engineering viruses of particular concern, such as ones closely related to smallpox. Other platform vaccine approaches, such as RNA vaccines, feature relatively little dual-use potential. The biosecurity risk associated with platform advancement may be minimised by focusing preferentially on circumventing anti-vector immunity with non-genetic rather than genetic modifications, using vectors that are not based on viruses pathogenic to humans, or preferential investment into promising RNA-based vaccine approaches. To reduce the risk of anthropogenic pandemics, structures for the governance of biotechnology and life science research with dual-use potential need to be reworked. Scientists outside of the pathogen research community, for instance those who work on viral vectors or oncolytic viruses, need to become more aware of the dual-use risks associated with their research. Both public and private research-funding bodies need to prioritise the evaluation and reduction of biosecurity risks.

Efficient Ocular Delivery of VCP siRNA via Reverse Magnetofection in RHO P23H Rodent Retina Explants.

The use of synthetic RNA for research purposes as well as RNA-based therapy and vaccination has gained increasing importance. Given the anatomical seclusion of the eye, small interfering RNA (siRNA)-induced gene silencing bears great potential for targeted reduction of pathological gene expression that may allow rational treatment of chronic eye diseases in the future. However, there is yet an unmet need for techniques providing safe and efficient siRNA delivery to the retina. We used magnetic nanoparticles (MNPs) and magnetic force (Reverse Magnetofection) to deliver siRNA/MNP complexes into retinal explant tissue, targeting valosin-containing protein (VCP) previously established as a potential therapeutic target for autosomal dominant retinitis pigmentosa (adRP). Safe and efficient delivery of VCP siRNA was achieved into all retinal cell layers of retinal explants from the RHO P23H rat, a rodent model for adRP. No toxicity or microglial activation was observed. VCP silencing led to a significant decrease of retinal degeneration. Reverse Magnetofection thus offers an effective method to deliver siRNA into retinal tissue. Used in combination with retinal organotypic explants, it can provide an efficient and reliable preclinical test platform of RNA-based therapy approaches for ocular diseases.

LncRNA LINC00518 Acts as an Oncogene in Uveal Melanoma by Regulating an RNA-Based Network.

Simple SummaryUveal melanoma (UM) is the most frequent primary tumor of the eye in adults. Although molecular alterations on protein-coding genes have been associated with the development of UM, the role of non-coding RNAs and their competitive endogenous networks remain poorly investigated. Starting from a computational analysis on UM expression dataset deposited in The Cancer Genome Atlas, we identified the long non-coding RNA LINC00518 as a potential oncogene. We then experimentally evaluated LINC00518 and its supposed RNA signaling in human biopsies and in vitro functional assays. The results obtained suggest that LINC00518, under potential transcriptional control by MITF, regulates an RNA–RNA network promoting cancer-related processes (i.e., cell proliferation and migration). These findings open the way to the characterization of the unknown RNA signaling associated with UM and pave the way to the exploitation of a potential target for RNA-based therapeutics.Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults; little is known about the contribution of non-coding RNAs (ncRNAs) to UM pathogenesis. Competitive endogenous RNA (ceRNA) networks based on RNA–RNA interactions regulate physiological and pathological processes. Through a combined approach of in silico and experimental biology, we investigated the expression of a set of long non-coding RNAs (lncRNAs) in patient biopsies, identifying LINC00518 as a potential oncogene in UM. The detection of LINC00518 dysregulation associated with several in vitro functional assays allowed us to investigate its ceRNA regulatory network and shed light on its potential involvement in cancer-related processes, such as epithelial to mesenchymal transition (EMT) and CoCl2-induced hypoxia-like response. In vitro transient silencing of LINC00518 impaired cell proliferation and migration, and affected mRNA expression of LINGO2, NFIA, OTUD7B, SEC22C, and VAMP3. A “miRNA sponge” and “miRNA protector” model have been hypothesized for LINC00518-induced regulation of mRNAs. In vitro inhibition of MITF suggested its role as a potential activator of LINC00518 expression. Comprehensively, LINC00518 may be considered a new oncogene in UM and a potential target for RNA-based therapeutic approaches.

Significantly Improved Recovery of Recombinant Sonchus Yellow Net Rhabdovirus by Expressing the Negative-Strand Genomic RNA

Generation of recombinant negative-stranded RNA viruses (NSVs) from plasmids involves in vivo reconstitution of biologically active nucleocapsids and faces a unique antisense problem where the negative-sense viral genomic RNAs can hybridize to viral messenger RNAs. To overcome this problem, a positive-sense RNA approach has been devised through expression of viral antigenomic (ag)RNA and core proteins for assembly of antigenomic nucleocapsids. Although this detour strategy works for many NSVs, the process is still inefficient. Using Sonchus yellow net rhabdovirus (SYNV) as a model; here, we develop a negative-sense genomic RNA-based approach that increased rescue efficiency by two orders of magnitude compared to the conventional agRNA approach. The system relied on suppression of double-stranded RNA induced antiviral responses by co-expression of plant viruses-encoded RNA silencing suppressors or animal viruses-encoded double-stranded RNA antagonists. With the improved approach, we were able to recover a highly attenuated SYNV mutant with a deletion in the matrix protein gene which otherwise could not be rescued via the agRNA approach. Reverse genetics analyses of the generated mutant virus provided insights into SYNV virion assembly and morphogenesis. This approach may potentially be applicable to other NSVs of plants or animals.

α- and β-Santalols Delay Aging in Caenorhabditis elegans via Preventing Oxidative Stress and Protein Aggregation.

α- and β-Santalol (santalol isomers) are the most abundant sesquiterpenoids found in sandalwood, contributing to its pleasant fragrance and wide-spectrum bioactivity. This study aimed at identifying the antiaging and antiaggregation mechanism of α- and β-santalol using the genetic tractability of an in vivo model Caenorhabditis elegans. The results showed that santalol isomers retard aging, improved health span, and inhibited the aggregation of toxic amyloid-β (Aβ1–42) and polyglutamine repeats (Q35, Q40, and HtnQ150) in C. elegans models for Alzheimer’s and Huntington’s disease, respectively. The genetic study, reporter gene expression, RNA-based reverse genetic approach (RNA interferences/RNAi), and gene expression analysis revealed that santalol isomers selectively regulate SKN-1/Nrf2 and EOR-1/PLZF transcription factors through the RTK/Ras/MAPK-dependent signaling axis that could trigger the expression of several antioxidants and protein aggregation inhibitory genes, viz., gst-4, gcs-1, gst-10, gsr-1, hsp-4, and skr-5, which extend longevity and help minimize age-induced protein oxidation and aggregation. We believe that these findings will further promote α- and β-santalol to become next-generation prolongevity and antiaggregation molecules for longer and healthier life.

RNA-Based next generation sequencing complements but does not replace fluorescence in situ hybridization studies for the classification of aggressive B-Cell lymphomas

Aggressive B-cell lymphomas are currently classified based in part upon the presence or absence of translocations involving BCL2, BCL6, and MYC. Most clinical laboratories employ fluorescence in situ hybridization (FISH) analysis for the detection of these rearrangements. The potential role of RNA-based sequencing approaches in the evaluation of malignant lymphoma is currently unclear. In this study, we performed RNA sequencing (RNAseq) in 37 cases of aggressive B-cell lymphomas using a commercially available next generation sequencing assay and compared results to previously performed FISH studies. RNAseq detected 1/7 MYC (14%), 3/8 BCL2 (38%) and 4/8 BCL6 (50%) translocations identified by FISH. RNAseq also detected 1 MYC/IGH fusion in a case not initially tested by FISH due to low MYC protein expression and 2 BCL6 translocations that were not detected by FISH. RNAseq identified the partner gene in each detected rearrangement, including a novel EIF4G1-BCL6 rearrangement. In summary, RNAseq complements FISH for the detection of rearrangements of BCL2, BCL6 and MYC in the evaluation and classification of aggressive B-cell lymphomas by detecting rearrangements that may be cryptic by FISH methods and by identifying the rearrangement partner genes. Detection of these clinically important translocations may be optimized by combined use of FISH and RNAseq.

Dominant and Active Methanogens in the Production Waters From a High-Temperature Petroleum Reservoir by DNA- and RNA-Based Analysis

Methane metabolism in deep subsurface petroleum reservoirs is of paramount interest in the global biogeochemical cycle of carbon. Methanogenesis in such habitats is driven mostly by methanogens in syntrophic association with bacteria. In the present study, methanogenic communities in production water samples from six wells of a high-temperature petroleum reservoir were analyzed based on both genomic 16S rDNA and metabolically active 16S rRNA. The PCR-amplified mcrA gene analysis showed that Methanosaeta (26%) and Methanomassiliicoccus (56%) were separately the dominant members in sample W2_71 and W9_18 at the DNA level. In comparison, the RNA-based analysis showed that Methanosaeta (63 ∼ 83%) followed by Methanolinea (16 ∼ 23%) were the most active methanogens, which were different from the communities of genomic DNA. While several lines of studies indicated that CO2-reducing methanogens of the genus Methanothermobacter was the most frequently detected phylotype in deep-subsurface petroleum reservoirs. Redundancy analysis (RDA) showed the possible correlation between active methanogens (Methanosaeta) and environmental factors (CO3 2-). The datasets indicated the importance of investigating methanogenic community by integrating DNA- and RNA-based approaches. These results provide new insight into active methanogens in high-temperature petroleum reservoirs, and promote a comprehensive understanding of methanogens as well as their potential methanogenic pathways in such environments.

Effect of chicory inulin-type fructan–containing snack bars on the human gut microbiota in low dietary fiber consumers in a randomized crossover trial

The low intake of dietary fiber compared to recommended amounts has been referred to as the dietary fiber gap. The addition of fiber to snack foods could favorably alter gut microbiota and help individuals meet intake recommendations. Our objective was to examine the effect of low- and moderate-dose fiber-containing snack bars, comprising mainly chicory root inulin-type fructans (ITF), on gut microbiota in healthy adults with habitual low dietary fiber intake using 16S ribosomal RNA-based approaches. In 2 separate 4-wk, placebo-controlled, double-blind, crossover trials, 50 healthy adults with low dietary fiber intake were randomly assigned to receive isocaloric snack bars of either moderate-dose fiber (7 g/d) or control in Trial 1 (n=25) or low-dose fiber (3 g/d) or control in Trial 2 (n=25), with 4-wk washout periods. Fecal microbiota composition and inferred function, fecal SCFA concentration, gastrointestinal (GI) symptoms, dietary intake, and quality of life were measured. Compared with the control group, the moderate-dose group showed significant differences across multiple microbial taxa, most notably an increased relative abundance of the Bifidobacterium genus from (mean±SEM) 5.3%±5.9% to 18.7%±15.0%. With low-dose ITF, significant increases in Bifidobacterium were no longer present after correction for multiple comparisons but targeted analysis with qPCR showed a significant increase in Bifidobacterium. Predictive functional profiling identified changes in predicted function after intake of the moderate- but not the low-dose bar. Fecal SCFAs were affected by time but not treatment. There were no between-group differences in GI symptoms. Importantly, fiber intake increased significantly with the moderate- and low-dose bars. In healthy adults, adding 3 or 7 g ITF to snack bars increased Bifidobacterium, a beneficial member of the gut microbial community. The addition of ITF to food products could help reduce the dietary fiber gap prevalent in modern life.This trial was registered at clinicaltrials.gov as NCT03042494.

Microbial community composition and diversity in production water of a high-temperature offshore oil reservoir assessed by DNA- and RNA-based analyses

Microbial activity is ubiquitous and important in petroleum reservoir systems; however, current knowledge regarding the diversity and functions of microbial communities that are active in offshore petroleum reservoirs remains very limited. In the present work, high-throughput sequencing analysis of the 16S rRNA gene (DNA) and transcripts (RNA) was applied to respectively characterize the genomic and active microbial communities from two production wells in a high-temperature offshore oilfield in China. The results showed that the microbial community compositions in the two wells (X1 and X2) were very different. At the genus level, the dominant members in both genomic and active communities of sample X1 were Bosea, Acinetobacter, Sphingomonas, Candidatus Methanofastidiosum and Methanocalculus. Both genomic and active communities in sample X2 were mainly represented by Pseudomonas, Acinetobacter, Methanothermobacter, Methanosaeta, Candidatus Nitrosopumilus, Candidatus Methanomethylicus, Thermococcus, Candidatus Methanofastidiosum and Methanocalculus. Moreover, using an RNA-based approach, genera with a very low abundance and even undetected genera (based on a DNA approach) were shown to have increased activity. Besides the abundant active members, the comparison between the DNA- and RNA-based datasets also permitted the identification of genera that were relatively more active in the two production water samples. The existence of these active microorganisms suggested they might have potential functions associated with reservoir issues in the offshore oilfield. This study highlights the importance of assessing microbial diversity through a combination of DNA- and RNA-based analyses, and promotes a comprehensive and deep understanding of microbial compositions and their roles in offshore petroleum reservoirs.

Next-Generation Sequencing Approaches for the Identification of Pathognomonic Fusion Transcripts in Sarcomas: The Experience of the Italian ACC Sarcoma Working Group.

This work describes the set-up of a shared platform among the laboratories of the Alleanza Contro il Cancro (ACC) Italian Research Network for the identification of fusion transcripts in sarcomas by using Next Generation Sequencing (NGS). Different NGS approaches, including anchored multiplex PCR and hybrid capture-based panels, were employed to profile a large set of sarcomas of different histotypes. The analysis confirmed the reliability of NGS RNA-based approaches in detecting sarcoma-specific rearrangements. Overall, the anchored multiplex PCR assay proved to be a fast and easy-to-analyze approach for routine diagnostics laboratories.

CD4+ Memory Stem T Cells Recognizing Citrullinated Epitopes Are Expanded in Patients With Rheumatoid Arthritis and Sensitive to Tumor Necrosis Factor Blockade.

Memory stem T (Tscm) cells are long-lived, self-renewing T cells that play a relevant role in immunologic memory. This study was undertaken to investigate whether Tscm cells accumulate in rheumatoid arthritis (RA). The polarization and differentiation profiles of circulating T cells were assessed by flow cytometry. Antigen-specific T cells were characterized by staining with major histocompatibility complex class II tetramers. The T cell receptor (TCR) repertoire was analyzed by high-throughput sequencing using an unbiased RNA-based approach in CD4+ T cell subpopulations sorted by fluorescence-activated cell sorting. We analyzed the dynamics of circulating Tscm cells (identified as CD45RA+CD62L+CD95+ T cells) by flow cytometry in 27 RA patients, 16 of whom were also studied during treatment with the anti-tumor necrosis factor (anti-TNF) agent etanercept. Age-matched healthy donors were used as controls. CD4+ Tscm cells were selectively and significantly expanded in RA patients in terms of frequency and absolute numbers, and significantly contracted upon anti-TNF treatment. Expanded CD4+ Tscm cells displayed a prevalent Th17 phenotype and a skewed TCR repertoire in RA patients, with the 10 most abundant clones representing up to 53.7% of the detected sequences. CD4+ lymphocytes specific for a citrullinated vimentin (Cit-vimentin) epitope were expanded in RA patients with active disease. Tscm cells accounted for a large fraction of Cit-vimentin-specific CD4+ cells. Our results indicate that Tscm cells, including expanded clones specific for relevant autoantigens, accumulate in RA patients not exposed to biologic agents, and might be involved in the natural history of the disease. Further analysis of Tscm cell dynamics in autoimmune disorders may have implications for the design and efficacy assessment of innovative therapies.