Microfluidics of binary liquid mixtures with temperature-dependent miscibility

Liquid–liquid microfluidic systems rely on the intricate control over the fluid properties of either miscible or immiscible mixtures. Herein, we report on the use of partially miscible binary liquid mixtures that lend their microfluidic properties from a highly temperature-sensitive mixing and phase separation behaviour. For a blend composed of the thermotropic liquid crystal 4-cyano-4′-pentylbiphenyl (5CB) and methanol, mixing at temperatures above the upper critical solution temperature (UCST; 24.4 °C) leads to a uniform single phase while partial mixing can be achieved at temperatures below the UCST. Thermally-driven phase separation inside the microfluidic channels results in the spontaneous formation of very regular phase arrangements, namely in droplets, plug, slug and annular flow. We map different flow regimes and relate findings to the role of interfacial tension and viscosity and their temperature dependence. Importantly, different flow regimes can be achieved at constant channel architecture and flow rate by varying the temperature of the blend. A consistent behaviour is observed for a binary liquid mixture with lower critical solution temperature, namely 2,6-lutidine and water. This temperature-responsive approach to microfluidics is an interesting candidate for multi-stage processes, selective extraction and sensing applications

Structural Characterization of Mesoporous Thin Film Architectures: A Tutorial Overview

Mesoporous thin film architectures are an important class of materials that exhibit unique properties, which include high surface area, versatile surface functionalization, and bicontinuous percolation paths through a broad library of pore arrangements on the 10 nm length scale. Although porosimetry of bulk materials via sorption techniques is common practice, the characterization of thin mesoporous films with small sample volumes remains a challenge. A range of techniques are geared toward providing information over pore morphology, pore size distribution, surface area and overall porosity, but none of them offers a holistic evaluation and results are at times inconsistent. In this work, we present a tutorial overview for the reliable structural characterization of mesoporous films. Three model samples with variable pore size and porosity prepared by block copolymer (BCP) coassembly serve for a rational comparison. Various techniques are assessed side-by-side, including scanning electron microscopy (SEM), atomic force microscopy (AFM), grazing incidence small-angle X-ray scattering (GISAXS), and ellipsometric porosimetry (EP). We critically discuss advantages and limitations of each technique and provide guidelines for reliable implementation

TMX2 Is a Crucial Regulator of Cellular Redox State, and Its Dysfunction Causes Severe Brain Developmental Abnormalities.

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation and dendritic and axonal growth. The relevance of endoplasmic reticulum (ER)-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by bi-allelic loss-of-function variants in thioredoxin (TRX)-related transmembrane-2 (TMX2); these variants were detected by exome sequencing in 14 affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria, and other migration disorders. TMX2 encodes one of the five TMX proteins of the protein disulfide isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER mitochondria-associated membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM-associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2-deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as a signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with an in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking an increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration, and organization in the developing brain

Insights into the Function of the CRM1 Cofactor RanBP3 from the Structure of Its Ran-Binding Domain

Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins

Matrin 3 is a co-factor for HIV-1 Rev in regulating post-transcriptional viral gene expression

Post-transcriptional regulation of HIV-1 gene expression is mediated by interactions between viral transcripts and viral/cellular proteins. For HIV-1, post-transcriptional nuclear control allows for the export of intron-containing RNAs which are normally retained in the nucleus. Specific signals on the viral RNAs, such as instability sequences (INS) and Rev responsive element (RRE), are binding sites for viral and cellular factors that serve to regulate RNA-export. The HIV-1 encoded viral Rev protein binds to the RRE found on unspliced and incompletely spliced viral RNAs. Binding by Rev directs the export of these RNAs from the nucleus to the cytoplasm. Previously, Rev co-factors have been found to include cellular factors such as CRM1, DDX3, PIMT and others. In this work, the nuclear matrix protein Matrin 3 is shown to bind Rev/RRE-containing viral RNA. This binding interaction stabilizes unspliced and partially spliced HIV-1 transcripts leading to increased cytoplasmic expression of these viral RNAs

Nucleo-cytoplasmic transport of proteins and RNA in plants

Merkle T. Nucleo-cytoplasmic transport of proteins and RNA in plants. Plant Cell Reports. 2011;30(2):153-176.Transport of macromolecules between the nucleus and the cytoplasm is an essential necessity in eukaryotic cells, since the nuclear envelope separates transcription from translation. In the past few years, an increasing number of components of the plant nuclear transport machinery have been characterised. This progress, although far from being completed, confirmed that the general characteristics of nuclear transport are conserved between plants and other organisms. However, plant-specific components were also identified. Interestingly, several mutants in genes encoding components of the plant nuclear transport machinery were investigated, revealing differential sensitivity of plant-specific pathways to impaired nuclear transport. These findings attracted attention towards plant-specific cargoes that are transported over the nuclear envelope, unravelling connections between nuclear transport and components of signalling and developmental pathways. The current state of research in plants is summarised in comparison to yeast and vertebrate systems, and special emphasis is given to plant nuclear transport mutants

In vitro nuclear interactome of the HIV-1 Tat protein

<p>Abstract</p> <p>Background</p> <p>One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86–101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry.</p> <p>Results</p> <p>Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied <it>in silico </it>analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture.</p> <p>Conclusion</p> <p>We have completed the <it>in vitro </it>Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will provide a framework to further advance our understanding of the mechanisms of HIV-1 proviral gene silencing and activation.</p

Phase behaviour and applications of a binary liquid mixture of methanol and a thermotropic liquid crystal

Herein, we report on the phase behaviour of a binary liquid mixture composed of methanol (MeOH) and the thermotropic liquid crystal 4-cyano-4′-pentylbiphenyl (5CB). The corresponding phase diagram combines features of a conventional liquid–liquid mixture with characteristics that are particular to the nematic liquid crystal. We observe four arrangements as a function of composition and temperature, namely monophasic isotropic, monophasic nematic, biphasic isotropic–isotropic and biphasic isotropic–nematic, with an upper critical solution temperature of 24.4 ± 0.5 °C. The interplay of nematogenic and non-nematogenic species offers tunability of phase mixing and phase composition in an accessible temperature window and provides novel routes for the extraction of target compounds, here exemplarily shown for Crystal Violet, Doxorubicin, Eosin Y, Rhodamine 6G and Sudan IV