No abstract

Herbaspirillum seropedicae is a diazotrophic and endophytic bacterium that associates with economically important grasses promoting plant growth and increasing productivity. To identify genes related to bacterial ability to colonize plants, wheat seedlings growing hydroponically in Hoagland's medium were inoculated with H. seropedicae and incubated for 3 days. Total mRNA from the bacteria present in the root surface and in the plant medium were purified, depleted from rRNA and used for RNA-seq profiling. RT-qPCR analyses were conducted to confirm regulation of selected genes. Comparison of RNA profile of root attached and planktonic bacteria revealed extensive metabolic adaptations to the epiphytic life style. These adaptations include expression of specific adhesins and cell wall re-modeling to attach to the root. Additionally, the metabolism was adapted to the microxic environment and nitrogen-fixation genes were expressed. Polyhydroxybutyrate (PHB) synthesis was activated, and PHB granules were stored as observed by microscopy. Genes related to plant growth promotion, such as auxin production were expressed. Many ABC transporter genes were regulated in the bacteria attached to the roots. The results provide new insights into the adaptation of H. seropedicae to the interaction with the plant.

Key Points Expression of Hippo component TAZ is downregulated in MM through hypermethylation. TAZ reexpression, exogenously or pharmacologically, causes apoptosis and enhances sensitivity to anti-MM therapies by downregulating MYC.

Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

Lung cancer is one of the most frequent types of cancer worldwide. Most patients are diagnosed at advanced stage and thus have poor prognosis. Smoking is a risk factor for lung cancer, however most smokers do not develop lung cancer while 20% of women with lung adenocarcinoma are non-smokers. Therefore, it is possible that these two groups present differences besides the smoking status, including differences in their gene expression signature. The altered expression patterns of non-coding RNAs in complex diseases make them potential biomarkers for diagnosis and treatment. We analyzed data from differentially and constitutively expressed PIWI-interacting RNAs and small nucleolar RNAs from publicly available small RNA high-throughput sequencing data in search of an expression pattern of non-coding RNA that could differentiate these two groups. Here, we report two sets of differentially expressed small non-coding RNAs identified in normal and tumoral tissues of women with lung adenocarcinoma, that discriminate between smokers and non-smokers. Our findings may offer new insights on metabolic alterations caused by tobacco and may be used for early diagnosis of lung cancer.

PAX5 and EBF1 work synergistically to regulate genes that are involved in B lymphocyte differentiation. We used the KIS-1 diffuse large B cell lymphoma cell line, which is reported to have elevated levels of PAX5 expression, to investigate the mechanism of EBF1- and PAX5-regulated gene expression. We demonstrate the lack of expression of hallmark B cell genes, including CD19, CD79b, and EBF1, in the KIS-1 cell line. Upon restoration of EBF1 expression we observed activation of CD19, CD79b and other genes with critical roles in B cell differentiation. Mass spectrometry analyses of proteins co-immunoprecipitated with PAX5 in KIS-1 identified components of the MLL H3K4 methylation complex, which drives histone modifications associated with transcription activation. Immunoblotting showed a stronger association of this complex with PAX5 in the presence of EBF1. Silencing of KMT2A, the catalytic component of MLL, repressed the ability of exogenous EBF1 to activate transcription of both CD19 and CD79b in KIS-1 cells. We also find association of PAX5 with the MLL complex and decreased CD19 expression following silencing of KMT2A in other human B cell lines. These data support an important role for the MLL complex in PAX5-mediated transcription regulation.

The Colorado potato beetle Leptinotarsa decemlineata is an insect pest that threatens potato crops globally. The primary method to control its damage on potato plants is the use of insecticides, including imidacloprid, chlorantraniliprole and spinosad. However, insecticide resistance has been frequently observed in Colorado potato beetles. The molecular targets and the basis of resistance to imidacloprid and chlorantraniliprole have both been previously quantified. This work was undertaken with the overarching goal of better characterizing the molecular changes associated with spinosad exposure in this insect pest. Next-generation sequencing was conducted to identify transcripts that were differentially expressed between Colorado potato beetles exposed to spinosad versus control insects. Results showed several transcripts that exhibit different expression levels between the two conditions, including ones coding for venom carboxylesterase-6, chitinase 10, juvenile hormone esterase and multidrug resistance-associated protein 4. In addition, several microRNAs, such as miR-12-3p and miR-750-3p, were also modulated in the investigated conditions. Overall, this work reveals a molecular footprint underlying spinosad response in Colorado potato beetles and provides novel leads that could be targeted as part of RNAi-based approaches to control this insect pest.

Cell-derived extracellular vesicles (EVs) participate in cell-cell communication via transfer of molecular cargo including genetic material like miRNAs. In mammals, it has previously been established that EV-mediated transfer of miRNAs can alter the development or function of immune cells, such as macrophages. Our previous research revealed that Atlantic salmon head kidney leukocytes (HKLs) change their morphology, phagocytic ability and miRNA profile from primarily “monocyte-like” at Day 1 to primarily “macrophage-like” at Day 5 of culture. Therefore, we aimed to characterize the miRNA cargo packaged in EVs released from these two cell populations. We successfully isolated EVs from Atlantic salmon HKL culture supernatants using the established Vn96 peptide-based pull-down. Isolation was validated using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. RNA-sequencing identified 19 differentially enriched (DE) miRNAs packaged in Day 1 versus Day 5 EVs. Several of the highly abundant miRNAs, including those that were DE (e.g. ssa-miR-146a, ssa-miR-155 and ssa-miR-731), were previously identified as DE in HKLs and are associated with macrophage differentiation and immune response in other species. Interestingly, the abundance relative of the miRNAs in EVs, including the most abundant miRNA (ssa-miR-125b), was different than the miRNA abundance in HKLs, indicating selective packaging of miRNAs in EVs. Further study of the miRNA cargo in EVs derived from fish immune cells will be an important next step in identifying EV biomarkers useful for evaluating immune cell function, fish health, or response to disease.