RNA-based Approaches Research Articles

Patient selection using novel RNA-based approaches: Early experience from a phase I study with the pan-FGFR inhibitor BAY 1163877 in patients with urothelial bladder cancer.

386 Background: FGFRs play a role in a variety of malignancies, including urothelial bladder cancer (UBC). Whereas genetic alterations of FGFRs in UBC are known, non-genetic and epigenetic activation of FGFR gene expression have also been described. BAY 1163877 is an oral inhibitor of FGFRs 1-3 with antitumor activity in FGFR mRNA overexpressing xenograft models not limited to DNA alterations. We report the results from a phase I expansion cohort in UBC patients pre-screened for treatment with BAY 1163877 based on FGFR1-3 mRNA overexpression and/or activating mutations in the FGFR3 gene. Methods: FGFR mRNA levels were assessed in paraffin-embedded archival or fresh biopsy specimens by RNAscope and NanoString technology and activating mutations by RT-PCR-based mutation assay. Tumor response to treatment was assessed by RECIST, v1.1. Adverse events were assessed using CTCAE v4.03 criteria. Results: A total of 80 patients from multiple indications were enrolled in the phase I study; 800 mg BID was established as the recommended phase 2 dose. Sixty seven UBC patients were screened, with 31 tumor biopsies (46%) determined to overexpress FGFR mRNA: 2 patients each with FGFR1 and FGFR2, and 27 patients with FGFR3. Of the 27 patients with FGFR3 mRNA overexpression, 5 had in parallel an activating mutation in FGFR3 gene. Eight UBC patients met eligibility criteria and entered treatment; all had elevated FGFR3 mRNA expression and 4 had alterations in FGFR3 DNA (3 mutations and 1 translocation). Seven patients (87.5%) had tumor shrinkage in target lesions as best response, with 3/8 partial responses (PR; 37.5%). One patient with a PR presented with elevated FGFR3 mRNA without evidence of aberrant DNA. BAY 1163877 was generally well tolerated and AEs manageable with dose modification. The most common AEs in the overall population were increased phosphorus (66% all grade, 1 grade-3 event) and diarrhea (33% all grade, 2 grade-3 events). Conclusions: Selection of patients for treatment with BAY 1163877 based on FGFR mRNA expression levels in archival tissue was feasible. BAY 1163877 had a favorable safety profile and promising anti-tumor activity in UBC patients. Clinical trial information: NCT01976741.

Human γδ T cells are quickly reconstituted after stem-cell transplantation and show adaptive clonal expansion in response to viral infection.

To investigate how the human γδ T cell pool is shaped during ontogeny and how it is regenerated after transplantation of hematopoietic stem cells (HSCs), we applied an RNA-based next-generation sequencing approach to monitor the dynamics of the repertoires of γδ T cell antigen receptors (TCRs) before and after transplantation in a prospective cohort study. We found that repertoires of rearranged genes encoding γδ TCRs (TRG and TRD) in the peripheral blood of healthy adults were stable over time. Although a large fraction of human TRG repertoires consisted of public sequences, the TRD repertoires were private. In patients undergoing HSC transplantation, γδ T cells were quickly reconstituted; however, they had profoundly altered TCR repertoires. Notably, the clonal proliferation of individual virus-reactive γδ TCR sequences in patients with reactivation of cytomegalovirus revealed strong evidence for adaptive anti-viral γδ T cell immune responses.

Artificial MicroRNA-Based Specific Gene Silencing of Grain Hardness Genes in Polyploid Cereals Appeared to Be Not Stable Over Transgenic Plant Generations.

Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were designed to silence Puroindoline a (Pina) and Puroindoline b (Pinb) hardness genes in wheat and their orthologues Secaloindoline a (Sina) and Secaloindoline b (Sinb) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions of Pina/Sina or Pinb/Sinb genes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using the Pinb-derived amiR vector for silencing of Pinb or Sinb, respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using the Pina-derived amiR cassette, despite large numbers of embryos attempted. Silencing of Pinb in wheat and Sinb in triticale was highly efficient in the T1 generation. The transcript level of Pinb in wheat was reduced up to 92% and Sinb in triticale was reduced up to 98%. Moreover, intended silencing of Pinb/Sinb with Pinb-derived amiR cassette was highly correlated with simultaneous silencing of Pina/Sina in the same transgenic plants. High downregulation of Pinb/Pina genes in T1 plants of wheat and Sinb/Sina genes in T1 plants of triticale was associated with strong expression of Pinb-derived amiR. Silencing of the target genes correlated with increased grain hardness in both species. Total protein content in the grains of transgenic wheat was significantly lower. Although, the Pinb-derived amiR cassette was stably inherited in the T2 generation of wheat and triticale the silencing effect including strongly decreased expression of silenced genes as well as strong expression of Pinb-derived amiR was not transmitted. Advantages and disadvantages of posttranscriptional silencing of target genes by means of amiR and siRNA-based approaches in polyploid cereals are discussed.

Emerging roles for RNA-binding proteins as effectors and regulators of cardiovascular disease.

The cardiovascular system comprises multiple cell types that possess the capacity to modulate their phenotype in response to acute or chronic injury. Transcriptional and post-transcriptional mechanisms play a key role in the regulation of remodelling and regenerative responses to damaged cardiovascular tissues. Simultaneously, insufficient regulation of cellular phenotype is tightly coupled with the persistence and exacerbation of cardiovascular disease. Recently, RNA-binding proteins such as Quaking, HuR, Muscleblind, and SRSF1 have emerged as pivotal regulators of these functional adaptations in the cardiovascular system by guiding a wide-ranging number of post-transcriptional events that dramatically impact RNA fate, including alternative splicing, stability, localization and translation. Moreover, homozygous disruption of RNA-binding protein genes is commonly associated with cardiac- and/or vascular complications. Here, we summarize the current knowledge on the versatile role of RNA-binding proteins in regulating the transcriptome during phenotype switching in cardiovascular health and disease. We also detail existing and potential DNA- and RNA-based therapeutic approaches that could impact the treatment of cardiovascular disease in the future.

Duplex nested reverse transcriptase polymerase chain reaction for simultaneous detection of type 2 porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 from tissue samples.

There are high levels of co-incidence of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in porcine tissue. This study established a duplex nested reverse transcriptase polymerase chain reaction (RT-PCR) method that targets the genomic RNA of type 2 PRRSV and the mRNA of PCV2 in infected tissues. The method amplified discriminative bands of 347 bp and 265 bp specific for type 2 PRRSV and PCV2, respectively. The limits of detection of the duplex nested RT-PCR were 101.5 TCID50/mL for type 2 PRRSV and 102 infected cells/mL for PCV2. The kappa statistic, which measures agreement between methods, was 0.867, indicating a good level of agreement. This RNA-based duplex RT-PCR approach can be another way to detect type 2 PRRSV and PCV2 simultaneously and with improved convenience.

Chitosan Nanoparticles for miRNA Delivery.

RNA interference techniques represent a promising strategy for therapeutic applications. In addition to small interfering RNA-based approaches, which have been widely studied and translated into clinical investigations, microRNA-based approaches are attractive owing to their "one hit, multiple targets" concept. To overcome challenges with in vivo delivery of microRNAs related to stability, cellular uptake, and specific delivery, our group has developed and characterized chitosan nanoparticles for nucleotide delivery. This platform allows for robust target modulation and antitumor activity following intravenous administration.

A Versatile DNA/RNA NGS Targeted Capture Strategy for Detection of Fusion Genes in Pediatric ALL

Introduction: It is widely acknowledged that acute lymphoblastic leukemia (ALL) is caused by acquisition of mutations, with a central role for chromosomal translocations. Therefore, genomic profiling of ALL has the potential to identify important new prognostic markers and druggable fusion genes.Aim: (i) setting up a Next Generation Sequencing-Targeted Capture (NGS-TC) panel focused on ALL, identifying both novel and conventional fusion genes; (ii) providing a user-friendly but powerful, knowledge-based bioinformatics platform for its analysis.Methods: NGS-TC approaches on MiSeq platform (Illumina) were evaluated: (i) on DNA, by Nextera Rapid Capture Custom panel (Illumina), 17 genes; (ii) on RNA, by comparing alternative panels: the Ovation Fusion Panel Target Enrichment System (Nugen, pre-designed 500 or custom 77 genes) and the TruSight Pan-Cancer (Illumina, pre-designed 1385 genes). Both panels include probes for genes involved in B-cell leukemogenesis (such as ABL1, JAK2, EBF1, PAX5).DNA datasets were analyzed by our purpose-built bioinformatics platform, 'BreakingPoint'. RNA results were analyzed by both 'BreakingPoint' and other similar tools available on 'Illumina BaseSpace' Cloud, such as 'TopHat'.Results: 'BreakingPoint' detects fusion genes from datasets derived from any sequencing technology and any biological material (DNA/RNA). Its distinctive feature is that it initially aligns reads on targets to efficiently and effectively find candidate breakpoints, then merges relevant reads into longer (thus more informative) consensus sequences, which are then studied across the genome to confirm the breakpoints and identify fusion partners. 'BreakingPoint' also uses an ever-expanding user database to filter out potential false positives and highlights known or novel true fusions.Firstly, we targeted 17 genes on N=65 DNA of ALL patients and we evaluated 'BreakingPoint' on a subset of 10 samples with 7 known rearrangements: 6 of them were detected with almost no false positives. P2RY8/CRLF2 known fusion was not detected mainly due to a non-homogenous target coverage which affected all the DNA NGS-TC experiments: coverage analyses showed high variability among different genes and in a sample-specific manner. Nevertheless, we detected N=6 novel fusions: 3 involving PAX5 and 3 involving ABL1.Secondly, the two candidate RNA protocols were transversally analyzed in a subset (N=5) of patients with known fusion genes. In this setting, we compared library preparation flowcharts in term of amount of RNA (Ovation kit 200ng vs. PanCancer kit 100ng), timeline and man-effort (2 up to 3 days), cost (similar), single run output (v2 vs. v3 MiSeq reagent kit with a mean of 15M reads/8samples and 25M reads/8samples, respectively) and capturing: both methods efficiently detected known fusion genes.Subsequently, the Ovation Fusion Panel was used to analyze N=59 ALL patients, enrolled consecutively to the protocol to mimic the diagnostic practice. From routinely RT-PCR screening, 14/59 patients carried a previously known fusion gene. By Ovation Nugen NGS-TC, they were all confirmed, such as BCR/ABL1 (N=1), ETV6/RUNX1 (N=8), TCF3/PBX1 (N=4), and P2RY8/CRLF2 (N=1). More interestingly, N=1 gene fusion involving TCF3 and N=2 cases carrying NUP214/ABL1 were detected for the first time.The TruSight Pan-Cancer panel was used to process N=42 newly enrolled cases. Previously known fusion genes were confirmed (N=2 ETV6/RUNX1, N=3 BCR/ABL1, N=3 TCF3/PBX, N=2 P2RY8/CLRF2). Moreover, we detected N=7 previously unknown fusions, mostly confirmed by RT-PCR (validation in progress): N=1 MLL/MLLT10, N=1 EBF1/PDGFRB, N=1 TCF3/OAZ1, N=2 JAK2-fusions, N=1 PAX5-fusion, and N=1 involving MLL with a new partner. Importantly, and in contrast to DNA capture, both RNA-based approaches were successful in detecting the P2RY8/CRLF2 cases.Conclusion: Our results show that RNA NGS-TC assays offer the most feasible solution towards a routine diagnostic protocol and that target coverage is the most important condition for success. Consequently, we have built our bioinformatics approach, 'BreakingPoint', to sensitively and reliably detect fusion genes even in cases of low coverage, irrespective of biological material or protocol. We consider our approach complete and clinically-relevant for identifying known and novel targetable fusion genes in pediatric ALL. DisclosuresBiondi:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees.

Low abundance of Archaeorhizomycetes among fungi in soil metatranscriptomes.

The Archaeorhizomycetes are recently discovered fungi with poorly resolved ecology. Even their abundance in soil fungal communities is currently disputed. Here we applied a PCR-independent, RNA-based metatranscriptomic approach to determine their abundance among fungi in eleven different soils across Europe. Using small subunit (SSU) ribosomal RNA transcripts as marker, we detected Archaeorhizomycetes in 17 out of 28 soil metatranscriptomes. They had average relative SSU rRNA abundance of 2.0% with a maximum of 9.4% among fungal SSU rRNAs. Network analysis revealed that they co-occur with arbuscular mycorrhizal Glomerales, which is in line with their previously suggested association with plant roots. Moreover, Archaeorhizomycetes ranked among the potential keystone taxa. This metatranscriptomic survey exemplifies the usage of non-targeted molecular approaches for the study of soil fungi. It provides PCR- and DNA-independent evidence for the low abundance of Archaeorhizomycetes in soil fungal communities, although they might be non-negligible players despite their low abundance.

Active community structure of microeukaryotes in a rice (Oryza sativa L.) rhizosphere revealed by RNA-based PCR-DGGE

ABSTRACTThe rhizosphere is one of the hot spots in soil ecosystems for a variety of microorganisms. In this study, we explored the seasonal change of the microeukaryotic community of a rice rhizosphere focusing on the active members through an RNA-based molecular approach. Rice plants (Oryza sativa L.) were grown in a pot where the rhizosphere was compartmented from bulk soil with a nylon gauze. The Eh in the rhizosphere compartment indicated that the rhizosphere was under oxic conditions in the initial stage of plant growth and then suddenly became anoxic or suboxic. Denaturing gradient gel electrophoresis targeting 18S rRNA-transcribed cDNA demonstrated that the active community of microeukaryotes in the rice rhizosphere was different from that in the bulk soil. The rhizosphere community showed a temporal shift in accordance with the shift of the redox conditions having three stages: the oxic before maximum tillering stage, anoxic/suboxic stage before maximum tillering stage, and anoxic/suboxic stage in the panicle initiation stage and thereafter. Active members specific to the rhizosphere at either the oxic or anoxic/suboxic stage were found: Heterolobosea amoeba, ciliates, and Chytridiomycota fungi for the oxic stage and oomycetes, ciliates and Ascomycota fungi in the anoxic/suboxic stage. The present results demonstrate that a specific group of microeukaryotes inhabit the rice rhizosphere even under anoxic/suboxic conditions and play various ecological roles as plant parasites, microbial grazers and organic decomposers.

Synthetic long non-coding RNAs [SINEUPs] rescue defective gene expression in vivo.

Non-coding RNAs provide additional regulatory layers to gene expression as well as the potential to being exploited as therapeutic tools. Non-coding RNA-based therapeutic approaches have been attempted in dominant diseases, however their use for treatment of genetic diseases caused by insufficient gene dosage is currently more challenging. SINEUPs are long antisense non-coding RNAs that up-regulate translation in mammalian cells in a gene-specific manner, although, so far evidence of SINEUP efficacy has only been demonstrated in in vitro systems. We now show that synthetic SINEUPs effectively and specifically increase protein levels of a gene of interest in vivo. We demonstrated that SINEUPs rescue haploinsufficient gene dosage in a medakafish model of a human disorder leading to amelioration of the disease phenotype. Our results demonstrate that SINEUPs act through mechanisms conserved among vertebrates and that SINEUP technology can be successfully applied in vivo as a new research and therapeutic tool for gene-specific up-regulation of endogenous functional proteins.

The host-pathogen interaction between wheat and yellow rust induces temporally coordinated waves of gene expression.

BackgroundUnderstanding how plants and pathogens modulate gene expression during the host-pathogen interaction is key to uncovering the molecular mechanisms that regulate disease progression. Recent advances in sequencing technologies have provided new opportunities to decode the complexity of such interactions. In this study, we used an RNA-based sequencing approach (RNA-seq) to assess the global expression profiles of the wheat yellow rust pathogen Puccinia striiformis f. sp. tritici (PST) and its host during infection.ResultsWe performed a detailed RNA-seq time-course for a susceptible and a resistant wheat host infected with PST. This study (i) defined the global gene expression profiles for PST and its wheat host, (ii) substantially improved the gene models for PST, (iii) evaluated the utility of several programmes for quantification of global gene expression for PST and wheat, and (iv) identified clusters of differentially expressed genes in the host and pathogen. By focusing on components of the defence response in susceptible and resistant hosts, we were able to visualise the effect of PST infection on the expression of various defence components and host immune receptors.ConclusionsOur data showed sequential, temporally coordinated activation and suppression of expression of a suite of immune-response regulators that varied between compatible and incompatible interactions. These findings provide the framework for a better understanding of how PST causes disease and support the idea that PST can suppress the expression of defence components in wheat to successfully colonize a susceptible host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2684-4) contains supplementary material, which is available to authorized users.

Microbiota of an Italian Grana-Like Cheese during Manufacture and Ripening, Unraveled by 16S rRNA-Based Approaches.

The microbial ecology of cheese involves a rich and complex interaction between starter lactic acid bacteria and nonstarter lactic acid bacteria (NSLAB), mainly originating from raw milk and/or from the environment, that can contribute to the final characteristics of cheese. The aim of the present research was the exploration of the active microbiota by RNA-based approaches during the manufacturing and ripening of a Grana-like cheese. Reverse transcriptase PCR (RT-PCR)-denaturing gradient gel electrophoresis (DGGE) and RNA-based high-throughput sequencing were applied to profile microbial populations, while the enumeration of active bacteria was carried out by using quantitative PCR (qPCR). Three different cheese productions (named D, E, and F) collected in the same month from the same dairy plant were analyzed. The application of the qPCR protocol revealed the presence of 7 log CFU/ml of bacterial load in raw milk, while, during ripening, active bacterial populations ranged from <4 to 8 log CFU/ml. The natural whey starters used in the three productions showed the same microbiota composition, characterized by the presence of Lactobacillus helveticus and Lactobacillus delbrueckii Nevertheless, beta-diversity analysis of the 16S rRNA sequencing data and RT-PCR-DGGE showed a clear clustering of the samples according to the three productions, probably driven by the different milks used. Milk samples were found to be characterized by the presence of several contaminants, such as Propionibacterium acnes, Acidovorax, Acinetobacter, Pseudomonas, and NSLAB. The core genera of the starter tended to limit the development of the spoilage bacteria only in two of the three batches. This study underlines the influence of different factors that can affect the final microbiota composition of the artisanal cheese. This study highlights the importance of the quality of the raw milk in the production of a hard cheese. Independent from the use of a starter culture, raw milk with low microbiological quality can negatively affect the populations of lactic acid bacteria and, as a consequence, impact the quality of the final product due to metabolic processes associated with spoilage bacteria.

A direct quantification method for measuring plasma MicroRNAs identified potential biomarkers for detecting metastatic breast cancer.

Circulating miRNAs are protected from ribonuclease degradation by assembly into microvesicles and exosomes. Releasing miRNAs completely from these particles is the key step to quantify the circulating miRNAs. Currently purified RNA-based quantitative analysis is widely used while it is time and cost consuming with high risk for those circulating miRNAs with low abundance due to partial loss of RNA during the steps of total RNA extraction and small RNA enrichment. Herein, we optimized a simple, effective and time-saving method to directly measure plasma miRNAs without RNA isolation. It is based on complete miRNA release from the protein complexes, followed by miRNA-specific reverse transcription and quantitative real-time PCR amplification. By comparison to the RNA-based approach, the direct quantification method showed more efficiency for circulating miRNA analysis, higher accuracy and specificity. By application of the direct quantification method to clinical samples combined with the RNA-based miRNA screening analysis, upregulation of miR-106a in blood was validated in metastatic breast cancer patients, indicating miR-106a are a potential biomarker for metastatic breast cancer.

Biomethane production and microbial community response according to influent concentration of molasses wastewater in a UASB reactor

This study aimed to investigate the interaction between methane production performance and active microbial community dynamics at different loading rates by increasing influent substrate concentration. The model system was an upflow anaerobic sludge blanket (UASB) reactor using molasses wastewater. The active microbial community was analyzed using a ribosomal RNA-based approach in order to reflect active members in the UASB system. The methane production rate (MPR) increased with an increase in organic loading rate (OLR) from 3.6 to 5.5g COD·L(-1)·day(-1) and then it decreased with further OLR addition until 9.7g COD·L(-1)·day(-1). The UASB reactor achieved a maximum methane production rate of 0.48L·L(-1)·day(-1) with a chemical oxygen demand (COD) removal efficiency of 91.2% at an influent molasses concentration of 16g COD·L(-1) (OLR of 5.5g COD·L(-1)·day(-1)). In the archaeal community, Methanosarcina was predominant irrespective of loading rate, and the relative abundance of Methanosaeta increased with loading rate. In the bacterial community, Firmicutes and Eubacteriaceae were relatively abundant in the loading conditions tested. The network analysis between operation parameters and microbial community indicated that MPR was positively associated with most methanogenic archaea, including the relatively abundant Methanosarcina and Methanosaeta, except Methanofollis. The most abundant Methanosarcina was negatively associated with Bifidobacterium and Methanosaeta, whereas Methanosaeta was positively associated with Bifidobacterium.

Charged MVB protein 5 is involved in T-cell receptor signaling.

Charged multivesicular body protein 5 (CHMP5) has a key role in multivesicular body biogenesis and a critical role in the downregulation of signaling pathways through receptor degradation. However, the role of CHMP5 in T-cell receptor (TCR)–mediated signaling has not been previously investigated. In this study, we utilized a short hairpin RNA-based RNA interference approach to investigate the functional role of CHMP5. Upon TCR stimulation, CHMP5-knockdown (CHMP5KD) Jurkat T cells exhibited activation of TCR downstream signaling molecules, such as PKCθ and IKKαβ, and resulted in the activation of nuclear factor-κB and the marked upregulation of TCR-induced gene expression. Moreover, we found that activator protein-1 and nuclear factor of activated T-cells transcriptional factors were markedly activated in CHMP5KD Jurkat cells in response to TCR stimulation, which led to a significant increase in interleukin-2 secretion. Biochemical studies revealed that CHMP5 endogenously forms high-molecular-weight complexes, including TCR molecules, and specifically interacts with TCRβ. Interestingly, flow cytometry analysis also revealed that CHMP5KD Jurkat T cells exhibit upregulation of TCR expression on the cell surface compared with control Jurkat T cells. Taken together, these findings demonstrated that CHMP5 might be involved in the homeostatic regulation of TCR on the cell surface, presumably through TCR recycling or degradation. Thus CHMP5 is implicated in TCR-mediated signaling.

Full-length HLA-DRB1 coding sequences generated by a hemizygous RNA-SBT approach.

Currently 1582 HLA-DRB1 alleles have been identified in the IMGT/HLA database (v3.18). Among those alleles, more than 90% have incomplete allele sequences, which complicates the analysis of the functional relevance of polymorphism beyond exon 2. The polymorphic index of each individual exon of the currently known allele sequences, shows that polymorphism is present in all exons, albeit not equally abundant. Full-length HLA-DRB1 RNA sequencing identifies polymorphism of the complete coding region. Here we describe a hemizygous full-length RNA sequence-based typing (SBT) approach based on group-specific HLA-DRB1 amplification and subsequent sequencing. RNA full-length sequences can easily be accessed because of the short amplicon length (801 bp). The RNA-SBT approach was successfully validated on a panel of DRB1 alleles having fully known coding sequences according to the IMGT/HLA database, and cover all serological equivalents. Subsequently, the approach was applied on a panel of 54 alleles with incomplete allele sequences, resulting in full-length coding sequences and the identification of one new and one corrected allele. This study shows the universal applicability of the RNA-based sequencing approach to identify full-length coding sequences and to define the polymorphic content of HLA-DRB1 alleles.

An RNA-based approach to sequence the mitogenome of Hypoptopoma incognitum (Siluriformes: Loricariidae)

Hypoptopoma incognitum is a fish of the fifth most species-rich family of vertebrates and abundant in rivers from the Brazilian Amazon. Only two species of Loricariidae fish have their complete mitogenomes sequence deposited in the Genbank. An innovative RNA-based approach was used to assemble the complete mitogenome of H. incognitum with an average coverage depth of 5292×. The typical vertebrate mitochondrial features were found; 22 tRNA genes, two rRNA genes, 13 protein-coding genes, and a non-coding control region. Moreover, the use of this approach allowed the measurement of mtRNA expression levels, the punctuation pattern of editing, and the detection of heteroplasmies.

Deep-sequencing of the T-cell receptor repertoire in patients with haplo-cord and matched-donor transplants.

Haplo-cord transplant has emerged as a feasible and reliable approach for haematopoietic stem cell transplant in patients who are unable to find matched-donor. This approach provides fast myeloid recovery, low incidence of graft vs host disease (GVHD) and favorable graft versus leukemia (GVL) effects. T cell recovery plays an important role in preventing infectious complications; it also mediates the GVHD and the GVL effects. Here, we utilized a novel RNA-based sequencing approach to quantitatively characterize the T cell receptor (TCRs) repertoire in patients underwent haplo-cord transplant in comparison with those underwent matched-donor transplant. Our study shows that higher percentage of cord cells early post transplant were associated with significantly higher TCR diversity. TCR diversity was significantly lower in patients with GVHD and in relapsed patients. A larger cohort study is needed to validate these data and to provide useful information on the specific TCR clones correlated with clinical outcome.

The close relation between Lactococcus and Methanosaeta is a keystone for stable methane production from molasses wastewater in a UASB reactor.

The up-flow anaerobic sludge blanket (UASB) reactor is a promising method for the treatment of high-strength industrial wastewaters due to advantage of its high treatment capacity and settleable suspended biomass retention. Molasses wastewater as a sugar-rich waste is one of the most valuable raw material for bioenergy production due to its high organic strength and bioavailability. Interpretation for complex interactions of microbial community structures and operational parameters can help to establish stable biogas production. RNA-based approach for biogas production systems is recommended for analysis of functionally active community members which are significantly underestimated. In this study, methane production and active microbial community were characterized in an UASB reactor using molasses wastewater as feedstock. The UASB reactor achieved a stable process performance at an organic loading rate of 1.7~13.8-g chemical oxygen demand (COD,·L(-1) day(-1); 87-95 % COD removal efficiencies), and the maximum methane production rate was 4.01 L-CH4·at 13.8 g-COD L(-1) day(-1). Lactococcus and Methanosaeta were comprised up to 84 and 80 % of the active bacterial and archaeal communities, respectively. Network analysis of reactor performance and microbial community revealed that Lactococcus and Methanosaeta were network hub nodes and positively correlated each other. In addition, they were positively correlated with methane production and organic loading rate, and they shared the other microbial hub nodes as neighbors. The results indicate that the close association between Lactococcus and Methanosaeta is responsible for the stable production of methane in the UASB reactor using molasses wastewater.

Suppression of transient receptor potential melastatin 4 expression promotes conversion of endothelial cells into fibroblasts via transforming growth factor/activin receptor-like kinase 5 pathway.

To study whether transient receptor potential melastatin 4 (TRPM4) participates in endothelial fibrosis and to investigate the underlying mechanism. Primary human endothelial cells were used and pharmacological and short interfering RNA-based approaches were used to test the transforming growth factor beta (TGF-β)/activin receptor-like kinase 5 (ALK5) pathway participation and contribution of TRPM7 ion channel. Suppression of TRPM4 expression leads to decreased endothelial protein expression and increased expression of fibrotic and extracellular matrix markers. Furthermore, TRPM4 downregulation increases intracellular Ca levels as a potential condition for fibrosis. The underlying mechanism of endothelial fibrosis shows that inhibition of TRPM4 expression induces TGF-β1 and TGF-β2 expression, which act through their receptor, ALK5, and the nuclear translocation of the profibrotic transcription factor smad4. TRPM4 acts to maintain endothelial features and its loss promotes fibrotic conversion via TGF-β production. The regulation of TRPM4 levels could be a target for preserving endothelial function during inflammatory diseases.