Sensing and inactivation of Bacillus anthracis Sterne by polymer–bromine complexes

We report on the performance of brominated poly(N-vinylpyrrolidone) (PVP-Br), brominated poly(ethylene glycol) (PEG-Br), and brominated poly(allylamine-co-4-aminopyridine) (PAAm-APy-Br) for their ability to decontaminate Bacillus anthracis Sterne spores in solution while also allowing for the sensing of the spores. The polymers were brominated by bromine using carbon tetrachloride or potassium tribromide as solvents, with bromine loadings ranging from 1.6 to 4.2 mEq/g of polymer. B. anthracis Sterne spores were exposed to increasing concentrations of brominated polymers for 5 min, while the kinetics of the sporicidal activity was assessed. All brominated polymers demonstrated spore log-kills of 8 within 5 min of exposure at 12 mg/mL aqueous polymer concentration. Sensing of spores was accomplished by measuring the release of dipicolinic acid (DPA) from the spore using time-resolved fluorescence. Parent, non-brominated polymers did not cause any release of DPA and the spores remained viable. In contrast, spores exposed to the brominated polymers were inactivated and the release of DPA was observed within minutes of exposure. Also, this release of DPA continued for a long time after spore inactivation as in a controlled release process. The DPA release was more pronounced for spores exposed to brominated PVP and brominated PEG-8000 compared to brominated PAAm-APy and brominated PEG-400. Using time-resolved fluorescence, we detected as low as 2500 B. anthracis spores, with PEG-8000 being more sensitive to low spore numbers. Our results suggest that the brominated polymers may be used effectively as decontamination agents against bacterial spores while also providing the sensing capability.Defense Threat Reduction Agency (DTRA

A triple helix stabilizes the 3' ends of long noncoding RNAs that lack poly(A) tails

The MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) locus is misregulated in many human cancers and produces an abundant long nuclear-retained noncoding RNA. Despite being transcribed by RNA polymerase II, the 3' end of MALAT1 is produced not by canonical cleavage/polyadenylation but instead by recognition and cleavage of a tRNA-like structure by RNase P. Mature MALAT1 thus lacks a poly(A) tail yet is expressed at a level higher than many protein-coding genes in vivo. Here we show that the 3' ends of MALAT1 and the MEN beta long noncoding RNAs are protected from 3'-5' exonucleases by highly conserved triple helical structures. Surprisingly, when these structures are placed downstream from an ORF, the transcript is efficiently translated in vivo despite the lack of a poly(A) tail. The triple helix therefore also functions as a translational enhancer, and mutations in this region separate this translation activity from simple effects on RNA stability or transport. We further found that a transcript ending in a triple helix is efficiently repressed by microRNAs in vivo, arguing against a major role for the poly(A) tail in microRNA-mediated silencing. These results provide new insights into how transcripts that lack poly(A) tails are stabilized and regulated and suggest that RNA triple-helical structures likely have key regulatory functions in vivo

An Unexpected Path for Malat1 in Neurons: Trafficking Out of the Nucleus for Translation

The Malat1 (metastasis-associated lung adenocarcinoma transcript 1) long noncoding RNA is highly and broadly expressed in mammalian tissues, accumulating in the nucleus where it modulates expression and pre-mRNA processing of many protein-coding genes. In this issue of Genes & Development, Xiao and colleagues (doi:10.1101/gad.351557.124) report that a significant fraction of Malat1 transcripts in cultured mouse neurons are surprisingly exported from the nucleus. These transcripts are packaged with Staufen proteins in RNA granules and traffic down the lengths of neurites. They then can be released in a stimulus-dependent manner to be locally translated into a microprotein that alters neuronal gene expression patterns

CRISPR/Cas13 Effectors Have Differing Extents of Off-Target Effects That Limit Their Utility in Eukaryotic Cells

CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as the level of on-target RNA knockdown. The extent of off-target effects is positively correlated with target RNA expression levels, and collateral damage can be observed even after reducing RxCas13d/guide RNA levels. The PspCas13b effector showed improved specificity and, unlike RxCas13d, can be used to deplete a Drosophila circular RNA without affecting the expression of the associated linear RNA. PspCas13b nonetheless still can have off-target effects and we notably found that the extent of off-target effects for Cas13 effectors differs depending on the cell type and target RNA examined. In total, these results highlight the need for caution when designing and interpreting Cas13-based knockdown experiments

Chimeric peptide nucleic acid compounds modulate splicing of the bcl-x gene in vitro and in vivo

Alternative splicing of the bcl-x gene generates two transcripts: the anti-apoptotic bcl-xL isoform and the pro-apoptotic bcl-xS isoform. The ratio between the two isoforms is a key factor in development and in cancer progression. Here, we show that a short antisense chimeric peptide nucleic acid (PNA) oligonucleotide conjugated to a polypeptide containing eight Ser-Arg repeats (SR)(8) can modulate splicing of bcl-x both in vitro and in vivo and induces apoptosis in HeLa cells. The PNA-SR oligo was targeted to a region of bcl-x that does not contain splicing regulatory sequences and was able to override the complex network of splicing enhancers and silencers that regulates the ratio between the two bcl-x isoforms. Thus, PNA-SR oligos are powerful tools that can potentially modulate splice site choice in endogenous genes independent of the presence of other splicing regulatory mechanisms on the target gene

Circular STAG2 RNA Modulates Bladder Cancer Progression via miR-145-5p/TAGLN2 and Is Considered as a Biomarker for Recurrence

Simple Summary Bladder cancer (BCa) is a major urologic malignancy with a high potential for death-threatening complications. Successful treatment relies on timely diagnostics and prediction of clinical outcomes. Molecular markers can be helpful for earlier diagnosis and for making treatment decisions. The biomarker landscape for BCa is not satisfactory yet. The discovery of novel diagnostic indicators for making treatment decisions is critical. Biological fluids are the most convenient and reliable source of these molecules. Circular RNAs (CircRNAs), special non-coding RNAs with a stable structure that are formed by a unique backsplicing process, recently became popular candidates for biomarkers in cancer biology. CircRNAs are widely distributed within the body including biological fluids. Their biological functions include gene regulation via sponging microRNAs, another type of small regulatory RNA. Our study characterized the circRNA hsa_circ_0139697 (circSTAG(16–25)) derived from exons 16 to 25 of STAG2, a gene known to play an important role in BCa biology that can serve as a biomarker predicting recurrence. We found that circSTAG2(16–25) could promote the proliferation, migration, and invasion of BCa cells by binding miR-145-5p, which causes the upregulation of the potential onco-gene TAGLN2. In addition, circSTAG(16–25) can serve as a biomarker for recurrence prediction. Abstract The current study aimed to elucidate the regulatory mechanisms of the circRNA hsa_circ_0139697 (circSTAG2(16–25)) in BCa and to consider the opportunity of using circSTAG2(16–25) isolated from BCa patient urine as a marker for disease development prediction. The selection of this circRNA was determined by the special role of its parental gene STAG2 in BCa biology. The circRNA hsa_circ_0139697 was chosen from 25 STAG2 circRNAs due to its differential expression in the urine of BCa patients and healthy volunteers. Higher levels of circSTAG2(16–25) were detected in urine samples obtained from patients with recurrent tumors. A higher expression of circSTAG2(16–25) was also detected in more tumorigenic BCa cell lines. The overexpression of circSTAG2(16–25) in BCa cells induced the elevation of proliferation, motility, and invasion. To study the mechanisms of circSTAG2(16–25) activity, we confirmed that circSTAG2(16–25) can bind miR-145-5p in vitro as was predicted by bioinformatic search. miR-145-5p was shown to suppress some genes that promoted BCa progression. One of these genes, TAGLN2, encodes the protein Transgelin 2, which plays a role in BCa cell motility and invasion. Therefore, the possible mechanism of action of circSTAG2(16–25) could be sponging the tumor suppressor miR-145-5p, which results in activation of TAGLN2. In addition, circSTAG2(16–25) might be considered as a potential biomarker for recurrence prediction

ZFP36L1 negatively regulates plasmacytoid differentiation of BCL1 cells by targeting BLIMP1 mRNA

The ZFP36/Tis11 family of zinc-finger proteins regulate cellular processes by binding to adenine uridine rich elements in the 3′ untranslated regions of various mRNAs and promoting their degradation. We show here that ZFP36L1 expression is largely extinguished during the transition from B cells to plasma cells, in a reciprocal pattern to that of ZFP36 and the plasma cell transcription factor, BLIMP1. Enforced expression of ZFP36L1 in the mouse BCL1 cell line blocked cytokine-induced differentiation while shRNA-mediated knock-down enhanced differentiation. Reconstruction of regulatory networks from microarray gene expression data using the ARACNe algorithm identified candidate mRNA targets for ZFP36L1 including BLIMP1. Genes that displayed down-regulation in plasma cells were significantly over-represented (P = <0.0001) in a set of previously validated ZFP36 targets suggesting that ZFP36L1 and ZFP36 target distinct sets of mRNAs during plasmacytoid differentiation. ShRNA-mediated knock-down of ZFP36L1 in BCL1 cells led to an increase in levels of BLIMP1 mRNA and protein, but not for mRNAs of other transcription factors that regulate plasmacytoid differentiation (xbp1, irf4, bcl6). Finally, ZFP36L1 significantly reduced the activity of a BLIMP1 3′ untranslated region-driven luciferase reporter. Taken together, these findings suggest that ZFP36L1 negatively regulates plasmacytoid differentiation, at least in part, by targeting the expression of BLIMP1

Aedes aegypti uses RNA interference in defense against Sindbis virus infection

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is an important anti-viral defense mechanism. The <it>Aedes aegypti </it>genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use <it>Ae. aegypti </it>as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family <it>Togaviridae</it>, genus <it>Alphavirus</it>).</p> <p>Results</p> <p>SINV (TR339-eGFP) (+) strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate <it>Ago2</it>, <it>Dcr2</it>, or <it>TSN </it>mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP) with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut <it>Ago2</it>, <it>Dcr2</it>, and <it>TSN </it>transcript levels during infection revealed that only <it>TSN </it>transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner.</p> <p>Conclusion</p> <p>We show that silencing RNAi components in <it>Ae. aegypti </it>results in transient increases in SINV replication. Furthermore, <it>Ae. aegypti </it>RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in <it>Ae. aegypti</it>.</p

A transcriptomic analysis of Echinococcus granulosus larval stages:implications for parasite biology and host adaptation

The cestode Echinococcus granulosus--the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide--is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval stages. We report the salient features of this study with a focus on genes reflecting physiological adaptations of different parasite stages.We generated ~10,000 ESTs from two sets of full-length enriched libraries (derived from oligo-capped and trans-spliced cDNAs) prepared with three parasite materials: hydatid cyst wall, larval worms (protoscoleces), and pepsin/H(+)-activated protoscoleces. The ESTs were clustered into 2700 distinct gene products. In the context of the biology of E. granulosus, our analyses reveal: (i) a diverse group of abundant long non-protein coding transcripts showing homology to a middle repetitive element (EgBRep) that could either be active molecular species or represent precursors of small RNAs (like piRNAs); (ii) an up-regulation of fermentative pathways in the tissue of the cyst wall; (iii) highly expressed thiol- and selenol-dependent antioxidant enzyme targets of thioredoxin glutathione reductase, the functional hub of redox metabolism in parasitic flatworms; (iv) candidate apomucins for the external layer of the tissue-dwelling hydatid cyst, a mucin-rich structure that is critical for survival in the intermediate host; (v) a set of tetraspanins, a protein family that appears to have expanded in the cestode lineage; and (vi) a set of platyhelminth-specific gene products that may offer targets for novel pan-platyhelminth drug development.This survey has greatly increased the quality and the quantity of the molecular information on E. granulosus and constitutes a valuable resource for gene prediction on the parasite genome and for further genomic and proteomic analyses focused on cestodes and platyhelminths