Regulation of stomatal movement by cortical microtubule organization in response to darkness and ABA signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Microtubule dynamics are essential for plant cell development and in producing responses to external stimuli. However, little is known about the regulation of microtubule dynamics or crosstalk between microtubule and stomatal movement. Here we identified microtubule reorganization as a crucial factor determining guard cell responses to dark and abscisic acid (ABA) signaling. As stomata opened, guard cells exhibited radially arranged cortical microtubules, which depolymerized into the cytosol when exposed to darkness and ABA. Suppression of microtubule disassembly by paclitaxel, a microtubule-stabilizing drug, significantly enhanced stomatal aperture under light, and partially blocked ABA- or darkness-induced stomatal closure. However, treatment with only the anti-microtubule drug, oryzalin, did not affect stomatal movement with or without external stimuli. Phosphatidic acid (PA) bound to a clade A type 2C protein phosphatase (PP2C), PP2CA, and deletion of PP2CA partially inhibited PA-induced microtubule depolymerization and stomatal closure. Moreover, microtubule reorganization was altered in the ABA-insensitive mutant pldα1, but not in the ABA-hypersensitive mutant pp2ca. We propose that a faithfully balanced reorganization of microtubules fulfills fundamental functions to enable the fast change of stomata in plant adaptive responses to developmental and environmental cues.     

A cluster pattern algorithm for the analysis of multiparametric cell assays.

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.     

Heterochromatin restricts the mobility of nuclear bodies

Nuclear bodies are relatively immobile organelles. Here, we investigated the mechanisms underlying their movement using experimentally induced interphase prenucleolar bodies (iPNBs). Most iPNBs demonstrated constrained diffusion, exhibiting infrequent fusions with other iPNBs and nucleoli. Fusion events were actin-independent and appeared to be the consequence of stochastic collisions between iPNBs. Most iPNBs were surrounded by condensed chromatin, while fusing iPNBs were usually found in a single heterochromatin-delimited compartment (“cage”). The experimentally induced over-condensation of chromatin significantly decreased the frequency of iPNB fusion. Thus, the data obtained indicate that the mobility of nuclear bodies is restricted by heterochromatin.     

Decoding the effects of synonymous variants

Synonymous single nucleotide variants (sSNVs) are common in the human genome but are often overlooked. However, sSNVs can have significant biological impact and may lead to disease. Existing computational methods for evaluating the effect of sSNVs suffer from the lack of gold-standard training/evaluation data and exhibit over-reliance on sequence conservation signals. We developed synVep (synonymous Variant effect predictor), a machine learning-based method that overcomes both of these limitations. Our training data was a combination of variants reported by gnomAD (observed) and those unreported, but possible in the human genome (generated). We used positive-unlabeled learning to purify the generated variant set of any likely unobservable variants. We then trained two sequential extreme gradient boosting models to identify subsets of the remaining variants putatively enriched and depleted in effect. Our method attained 90% precision/recall on a previously unseen set of variants. Furthermore, although synVep does not explicitly use conservation, its scores correlated with evolutionary distances between orthologs in cross-species variation analysis. synVep was also able to differentiate pathogenic vs. benign variants, as well as splice-site disrupting variants (SDV) vs. non-SDVs. Thus, synVep provides an important improvement in annotation of sSNVs, allowing users to focus on variants that most likely harbor effects.     

Role of Microstructure in Oxygen Induced Photodegradation of Methylammonium Lead Triiodide Perovskite Films

This paper investigates the impact of microstructure on the degradation rate of methylammonium lead triiodide (MAPbI₃) perovskite films upon exposure to light and oxygen. By comparing the oxygen induced degradation of perovskite films of different microstructure–fabricated using either a lead acetate trihydrate precursor or a solvent engineering technique–it is demonstrated that films with larger and more uniform grains and better electronic quality show a significantly reduced degradation compared to films with smaller, more irregular grains. The effect of degradation on the optical, compositional, and microstructural properties of the perovskite layers is characterized and it is demonstrated that oxygen induced degradation is initiated at the layer surface and grain boundaries. It is found that under illumination, irreversible degradation can occur at oxygen levels as low as 1%, suggesting that degradation can commence already during the device fabrication stage. Finally, this work establishes that improved thin‐film microstructure, with large uniform grains and a low density of defects, is a prerequisite for enhanced stability necessary in order to make MAPbI₃ a promising long lived and low cost alternative for future photovoltaic applications.     

A model for integrative study of human gastric acid secretion.

We have developed a unique virtual human model of gastric acid secretion and its regulation in which food provides a driving force. Food stimulus triggers neural activity in central and enteric nervous systems and G cells to release gastrin, a critical stimulatory hormone. Gastrin stimulates enterochromaffin-like cells to release histamine, which, together with acetylcholine, stimulates acid secretion from parietal cells. Secretion of somatostatin from antral and corpus D cells comprises a negative-feedback loop. We demonstrate that although acid levels are most sensitive to food and nervous system inputs, somatostatin-associated interactions are also important in governing acidity. The importance of gastrin in acid secretion is greatest at the level of transport between the antral and corpus regions. Our model can be applied to study conditions that are not yet experimentally reproducible. For example, we are able to preferentially deplete antral or corpus somatostatin. Depletion of antral somatostatin exhibits a more significant elevation of acid release than depletion of corpus somatostatin. This increase in acid release is likely due to elevated gastrin levels. Prolonged hypergastrinemia has significant effects in the long term (5 days) by promoting enterochromaffin-like cell overgrowth. Our results may be useful in the design of therapeutic strategies for acid secretory dysfunctions such as hyper- and hypochlorhydria.     

miR‐516a‐3p inhibits breast cancer cell growth and EMT by blocking the Pygo2/Wnt signalling pathway

miR‐516a‐3p has been reported to play a suppressive role in several types of human tumours. However, the expression level, biological function and fundamental mechanisms of miR‐516a‐3p in breast cancer remain unclear. In the present study, we found that miR‐516a‐3p expression was down‐regulated and Pygopus2 (Pygo2) expression was up‐regulated in human breast cancer tissues and cells. Through analysing the clinicopathological characteristics, we demonstrated that low miR‐516a‐3p expression or positive Pygo2 expression was a predictor of poor prognosis for patients with breast cancer. The results of a dual luciferase reporter assay and Western blot analysis indicated that Pygo2 was a target gene of miR‐516a‐3p. Moreover, overexpression of miR‐516a‐3p inhibited cell growth, migration and invasion as well as epithelial‐mesenchymal transition (EMT) of breast cancer cells, whereas reduced miR‐516a‐3p expression promoted breast cancer cell growth, migration, invasion and EMT. Furthermore, we showed that miR‐516a‐3p suppressed cell proliferation, metastasis and EMT of breast cancer cells by inhibiting Pygo2 expression. We confirmed that miR‐516a‐3p exerted an anti‐tumour effect by inhibiting the activation of the Wnt/β‐catenin pathway. Finally, xenograft tumour models were used to show that miR‐516a‐3p inhibited breast cancer cell growth and EMT via suppressing the Pygo2/Wnt signalling pathway. Taken together, these results show that miR‐516a‐3p inhibits breast cancer cell growth, metastasis and EMT by blocking the Pygo2/ Wnt/β‐catenin pathway.     

Function‐based assessment of structural similarity measurements using metal co‐factor orientation

Structure comparison is widely used to quantify protein relationships. Although there are several approaches to calculate structural similarity, specifying significance thresholds for similarity metrics is difficult due to the inherent likeness of common secondary structure elements. In this study, metal co‐factor location is used to assess the biological relevance of structural alignments. The distance between the centroids of bound co‐factors adds a chemical and function‐relevant constraint to the structural superimposition of two proteins. This additional dimension can be used to define cut‐off values for discriminating valid and spurious alignments in large alignment sets. The hypothesis underlying our approach is that metal coordination sites constrain structural evolution, thus revealing functional relationships between distantly related proteins. A comparison of three related nitrogenases shows the sequence and fold constraints imposed on the protein structures up to 18 Å away from the centers of their bound metal clusters. Proteins 2014; 82:648–656. © 2013 Wiley Periodicals, Inc.