MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS): a global mechanism for the regulation of alternative splicing.

While RNA secondary structures are critical to regulate alternative splicing of long-range pre-mRNA, the factors that modulate RNA structure and interfere with the recognition of the splice sites are largely unknown. Previously, we identified a small, non-coding microRNA that sufficiently affects stable stem structure formation of Nmnat pre-mRNA to regulate the outcomes of alternative splicing. However, the fundamental question remains whether such microRNA-mediated interference with RNA secondary structures is a global molecular mechanism for regulating mRNA splicing. We designed and refined a bioinformatic pipeline to predict candidate microRNAs that potentially interfere with pre-mRNA stem-loop structures, and experimentally verified splicing predictions of three different long-range pre-mRNAs in the Drosophila model system. Specifically, we observed that microRNAs can either disrupt or stabilize stem-loop structures to influence splicing outcomes. Our study suggests that MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing, increases the repertoire of microRNA function and further indicates cellular complexity of post-transcriptional regulation.

Cysteine conjugates of acetaminophen and p-aminophenol are potent inducers of cellular impairment in human proximal tubular kidney HK-2 cells.

Acetaminophen (APAP) belong among the most used analgesics and antipyretics. It is structurally derived from p-aminophenol (PAP), a potent inducer of kidney toxicity. Both compounds can be metabolized to oxidation products and conjugated with glutathione. The glutathione-conjugates can be cleaved to provide cysteine conjugates considered as generally nontoxic. The aim of the present report was to synthesize and to purify both APAP- and PAP-cysteine conjugates and, as the first study at all, to evaluate their biological effects in human kidney HK-2 cells in comparison to parent compounds. HK-2 cells were treated with tested compounds (0-1000 µM) for up to 24 h. Cell viability, glutathione levels, ROS production and mitochondrial function were determined. After 24 h, we found that both APAP- and PAP-cysteine conjugates (1 mM) were capable to induce harmful cellular damage observed as a decrease of glutathione levels to 10% and 0%, respectively, compared to control cells. In addition, we detected the disappearance of mitochondrial membrane potential in these cells. In the case of PAP-cysteine, the extent of cellular impairment was comparable to that induced by PAP at similar doses. On the other hand, 1 mM APAP-cysteine induced even larger damage of HK-2 cells compared to 1 mM APAP after 6 or 24 h. We conclude that cysteine conjugates with aminophenol are potent inducers of oxidative stress causing significant injury in kidney cells. Thus, the harmful effects cysteine-aminophenolic conjugates ought to be considered in the description of APAP or PAP toxicity.

MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS): a global mechanism for the regulation of alternative splicing.

While RNA secondary structures are critical to regulate alternative splicing of long-range pre-mRNA, the factors that modulate RNA structure and interfere with the recognition of the splice sites are largely unknown. Previously, we identified a small, non-coding microRNA that sufficiently affects stable stem structure formation of Nmnat pre-mRNA to regulate the outcomes of alternative splicing. However, the fundamental question remains whether such microRNA-mediated interference with RNA secondary structures is a global molecular mechanism for regulating mRNA splicing. We designed and refined a bioinformatic pipeline to predict candidate microRNAs that potentially interfere with pre-mRNA stem-loop structures, and experimentally verified splicing predictions of three different long-range pre-mRNAs in the Drosophila model system. Specifically, we observed that microRNAs can either disrupt or stabilize stem-loop structures to influence splicing outcomes. Our study suggests that MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing, increases the repertoire of microRNA function and further indicates cellular complexity of post-transcriptional regulation. One-Sentence Summary: MicroRNA-Mediated Obstruction of Stem-loop Alternative Splicing (MIMOSAS) is a novel regulatory mechanism for the transcriptome-wide regulation of alternative splicing.

Optimization of gradient reversed phase high performance liquid chromatography analysis of acetaminophen oxidation metabolites using linear and non-linear retention model.

Acetaminophen (paracetamol, APAP) is one of the most widely used drugs worldwide. Unfortunately, its overdose, which is caused by predominant oxidation of APAP, can lead to acute liver injury. In liver, oxidized APAP is conjugated with glutathione, leading to APAP-glutathione conjugate, which is metabolized to APAP-cysteine and APAP-N-acetylcysteine conjugates. Thus, all of those compounds could be used to monitor APAP metabolism in the overdosed patients. To date, only a limited number of rapid and accurate methods have been reported for the assessment of APAP oxidation metabolites using simple instrumentation, and thus this work was aimed at developing a fast and convenient gradient HPLC-UV/MS method. For this purpose, APAP conjugates with glutathione, cysteine, and N-acetylcysteine were synthesized, purified by preparative liquid chromatography, and characterized by NMR and high-resolution mass spectrometry. The gradient elution conditions were optimized using the window diagram approach and the effects of mobile phase composition and additives on separation and detection sensitivity were evaluated using two, i.e., linear and non-linear isocratic retention models. Quantitative parameters of the developed method were evaluated and the effectiveness, sensitivity, and specificity of the method were demonstrated on the analysis of human kidney HK-2 cell lysates, confirming the suitability of the method for routine use in studies on APAP toxicity.

Not that kind of tree: Assessing the potential for decision tree-based plant identification using trait databases.

PREMISE: Advancements in machine learning and the rise of accessible "big data" provide an important opportunity to improve trait-based plant identification. Here, we applied decision-tree induction to a subset of data from the TRY plant trait database to (1) assess the potential of decision trees for plant identification and (2) determine informative traits for distinguishing taxa. METHODS: Decision trees were induced using 16 vegetative and floral traits (689 species, 20 genera). We assessed how well the algorithm classified species from test data and pinpointed those traits that were important for identification across diverse taxa. RESULTS: The unpruned tree correctly placed 98% of the species in our data set into genera, indicating its promise for distinguishing among the species used to construct them. Furthermore, in the pruned tree, an average of 89% of the species from the test data sets were properly classified into their genera, demonstrating the flexibility of decision trees to also classify new species into genera within the tree. Closer inspection revealed that seven of the 16 traits were sufficient for the classification, and these traits yielded approximately two times more initial information gain than those not included. DISCUSSION: Our findings demonstrate the potential for tree-based machine learning and big data in distinguishing among taxa and determining which traits are important for plant identification.

Epigenomic signatures underpin the axonal regenerative ability of dorsal root ganglia sensory neurons.

Axonal injury results in regenerative success or failure, depending on whether the axon lies in the peripheral or the CNS, respectively. The present study addresses whether epigenetic signatures in dorsal root ganglia discriminate between regenerative and non-regenerative axonal injury. Chromatin immunoprecipitation for the histone 3 (H3) post-translational modifications H3K9ac, H3K27ac and H3K27me3; an assay for transposase-accessible chromatin; and RNA sequencing were performed in dorsal root ganglia after sciatic nerve or dorsal column axotomy. Distinct histone acetylation and chromatin accessibility signatures correlated with gene expression after peripheral, but not central, axonal injury. DNA-footprinting analyses revealed new transcriptional regulators associated with regenerative ability. Machine-learning algorithms inferred the direction of most of the gene expression changes. Neuronal conditional deletion of the chromatin remodeler CCCTC-binding factor impaired nerve regeneration, implicating chromatin organization in the regenerative competence. Altogether, the present study offers the first epigenomic map providing insight into the transcriptional response to injury and the differential regenerative ability of sensory neurons.