Characterizing Microbubble-Mediated Permeabilization in a Vessel-on-a-Chip Model.

Drug transport from blood to extravascular tissue can locally be achieved by increasing the vascular permeability through ultrasound-activated microbubbles. However, the mechanism remains unknown, including whether short and long cycles of ultrasound induce the same onset rate, spatial distribution, and amount of vascular permeability increase. Accurate models are necessary for insights into the mechanism so a microvessel-on-a-chip is developed with a membrane-free extravascular space.

Issues with lipid probes in flip-flop measurements: A comparative study using sum-frequency vibrational spectroscopy and second-harmonic generation.

Fluorescent lipid probes such as 1-palmitoyl-2-(6-[7-nitro-2-1,3-benzoxadiazol-4-yl]amino-hexanoyl)-sn-glycero-3-phosphocholine (C6 NBD-PC) have been used extensively to study the kinetics of lipid flip-flop. However, the efficacy of these probes as reliable reporters of native lipid translocation has never been tested. In this study, sum-frequency vibrational spectroscopy (SFVS) was used to measure the kinetics of C6 NBD-PC lipid flip-flop and the flip-flop of native lipids in planar supported lipid bilayers.

How cytoskeletal crosstalk makes cells move: Bridging cell-free and cell studies.

Cell migration is a fundamental process for life and is highly dependent on the dynamical and mechanical properties of the cytoskeleton. Intensive physical and biochemical crosstalk among actin, microtubules, and intermediate filaments ensures their coordination to facilitate and enable migration. In this review, we discuss the different mechanical aspects that govern cell migration and provide, for each mechanical aspect, a novel perspective by juxtaposing two complementary approaches to the biophysical study of cytoskeletal crosstalk: live-cell studies (often referred to as top-down studies) and cell-free studies (often referred to as bottom-up studies).

Sum-frequency vibrational spectroscopy, a tutorial: Applications for the study of lipid membrane structure and dynamics.

Planar supported lipid bilayers (PSLBs) are an ideal model for the study of lipid membrane structures and dynamics when using sum-frequency vibrational spectroscopy (SFVS). In this paper, we describe the construction of asymmetric PSLBs and the basic SFVS theory needed to understand and make measurements on these membranes. Several examples are presented, including the determination of phospholipid orientation and measuring phospholipid transmembrane translocation (flip-flop).

Modelling metastatic colonization of cholangiocarcinoma organoids in decellularized lung and lymph nodes.

Cholangiocarcinoma (CCA) is a type of liver cancer with an aggressive phenotype and dismal outcome in patients. The metastasis of CCA cancer cells to distant organs, commonly lung and lymph nodes, drastically reduces overall survival. However, mechanistic insight how CCA invades these metastatic sites is still lacking.

Inhibitory Effect of Lanthanides on Native Lipid Flip-Flop.

The influence of ytterbium ions (Yb), a commonly used paramagnetic NMR chemical shift reagent, on the physical properties and flip-flop kinetics of dipalmitoylphosphatidylcholine (DPPC) planar supported lipid bilayers (PSLBs) was investigated. Langmuir isotherm studies revealed that Yb interacts strongly with the phosphate headgroup of DPPC, evidenced by the increases in shear and compression moduli. Using sum-frequency vibrational spectroscopy, changes in the acyl chain ordering and phase transition temperature were also observed, consistent with Yb interacting with the phosphate headgroup of DPPC.

Influence of very-long-chain polyunsaturated fatty acids on membrane structure and dynamics.

The unique attributes of very-long-chain polyunsaturated fatty acids (VLC-PUFAs), their long carbon chains (n > 24) and high degree of unsaturation, impart unique chemical and physical properties to this class of fatty acids. The changes imparted by VLC-PUFA 32:6 n-3 on lipid packing and the compression moduli of model membranes were evaluated from π-A isotherms of VLC-PUFA in 1,2-distearoyl-sn-3-glycero-phosphocholine (DSPC) lipid monolayers. To compare the attractive or repulsive forces between VLC-PUFA and DSPC lipid monolayers, the measured mean molecular areas (MMAs) were compared with the calculated MMAs of an ideal mixture of VLC-PUFA and DSPC.

A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient.

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR.

Unannotated splicing regulatory elements in deep intron space.

Deep intron space harbors a diverse array of splicing regulatory elements that cooperate with better-known exon-proximal elements to enforce proper tissue-specific and development-specific pre-mRNA processing. Many deep intron elements have been highly conserved through vertebrate evolution, yet remain poorly annotated in the human genome. Recursive splicing exons (RS-exons) and intraexons promote noncanonical, multistep resplicing pathways in long introns, involving transient intermediate structures that are greatly underrepresented in RNA-seq datasets.

Retinal bioavailability and functional effects of a synthetic very-long-chain polyunsaturated fatty acid in mice.

Rare, nondietary very-long-chain polyunsaturated fatty acids (VLC-PUFAs) are uniquely found in the retina and a few other vertebrate tissues. These special fatty acids play a clinically significant role in retinal degeneration and development, but their physiological and interventional research has been hampered because pure VLC-PUFAs are scarce. We hypothesize that if Stargardt-3 or age-related macular degeneration patients were to consume an adequate amount of VLC-PUFAs that could be directly used in the retina, it may be possible to bypass the steps of lipid elongation mediated by the retina's ELOVL4 enzyme and to delay or prevent degeneration.

Selective effects of protein 4.1N deficiency on neuroendocrine and reproductive systems.

Protein 4.1N, a member of the protein 4.1 family, is highly expressed in the brain.

Revealing the Kinetic Advantage of a Competitive Small-Molecule Immunoassay by Direct Detection.

Small-molecule detection in an immunoassay format generally employs competition or labeling. A novel direct-detection label-free primary immunoassay utilizing second harmonic generation (SHG) has been developed and the utility of the method has been demonstrated for several small-molecule narcotics. Specifically, the binding of morphine, methadone, and cocaine to antimorphine, antimethadone, and anticocaine antibodies was measured by SHG, allowing binding affinities and rates of dissociation to be obtained.

Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts.

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression.

pre-mRNA splicing, revisited.

In this issue of , Ji et al have identified a novel regulatory mechanism that ensures proper splicing of human pre-messenger RNA (pre-mRNA).

Mechanistic Investigation of HIV-1 Gag Association with Lipid Membranes.

An extensive investigation into the initial association of HIV-1 Gag with lipid membranes was conducted with second harmonic generation. The roles of the lipid phase, phospholipid 1,2-dioleoyl- sn-glycero-3-phospho-(1-myo-inositol-4,5-bisphosphate) [PI(4,5)P], the presence of the myristoyl group on Gag, the C-terminus of Gag, and the presence of transfer ribonucleic acid (tRNA) in Gag-membrane association were examined using the physiologically most relevant full-length Gag protein studied thus far. The tighter packing of a bilayer composed of gel-phase lipids was found to have a lower relative amount of membrane-bound Gag in comparison to its fluid-phase counterpart.

An important class of intron retention events in human erythroblasts is regulated by cryptic exons proposed to function as splicing decoys.

During terminal erythropoiesis, the splicing machinery in differentiating erythroblasts executes a robust intron retention (IR) program that impacts expression of hundreds of genes. We studied IR mechanisms in the splicing factor gene, which expresses ∼50% of its transcripts in late erythroblasts as a nuclear isoform that retains intron 4. RNA-seq analysis of nonsense-mediated decay (NMD)-inhibited cells revealed previously undescribed splice junctions, rare or not detected in normal cells, that connect constitutive exons 4 and 5 to highly conserved cryptic cassette exons within the intron.

Circulating primitive erythroblasts establish a functional, protein 4.1R-dependent cytoskeletal network prior to enucleating.

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation.

Second Harmonic Correlation Spectroscopy: Theory and Principles for Determining Surface Binding Kinetics.

A novel application of second harmonic correlation spectroscopy (SHCS) for the direct determination of molecular adsorption and desorption kinetics to a surface is discussed in detail. The surface-specific nature of second harmonic generation (SHG) provides an efficient means to determine the kinetic rates of adsorption and desorption of molecular species to an interface without interference from bulk diffusion, which is a significant limitation of fluorescence correlation spectroscopy (FCS). The underlying principles of SHCS for the determination of surface binding kinetics are presented, including the role of optical coherence and optical heterodyne mixing.

Applications of Surface Second Harmonic Generation in Biological Sensing.

Surface second harmonic generation (SHG) is a coherent, nonlinear optical technique that is well suited for investigations of biomolecular interactions at interfaces. SHG is surface specific due to the intrinsic symmetry constraints on the nonlinear process, providing a distinct analytical advantage over linear spectroscopic methods, such as fluorescence and UV-Visible absorbance spectroscopies. SHG has the ability to detect low concentrations of analytes, such as proteins, peptides, and small molecules, due to its high sensitivity, and the second harmonic response can be enhanced through the use of target molecules that are resonant with the incident (ω) and/or second harmonic (2ω) frequencies.

RNA splicing during terminal erythropoiesis.

Purpose Of Review: Erythroid progenitors must accurately and efficiently splice thousands of pre-mRNAs as the cells undergo extensive changes in gene expression and cellular remodeling during terminal erythropoiesis. Alternative splicing choices are governed by interactions between RNA binding proteins and cis-regulatory binding motifs in the RNA. This review will focus on recent studies that define the genome-wide scope of splicing in erythroblasts and discuss what is known about its regulation.

The Ins and Outs of Lipid Flip-Flop.

Our current view of cellular membranes centers on the fluid-mosaic model, which envisions the cellular membrane as a "liquidlike" bilayer of lipids, cholesterol, and proteins that freely diffuse in two dimensions. In stark contrast, the exchange of materials between the leaflets of a bilayer was presumed to be prohibited by the large enthalpic barrier associated with translocating hydrophilic materials, such as a charged lipid headgroup, through the hydrophobic membrane core. This static picture with regard to lipid translocation (or "flip-flop" as it is affectionately known) has been a long-held belief in the study of membrane dynamics.

Developmental regulation of RNA processing by Rbfox proteins.

The Rbfox genes encode an ancient family of sequence-specific RNA binding proteins (RBPs) that are critical developmental regulators in multiple tissues including skeletal muscle, cardiac muscle, and brain. The hallmark of Rbfox proteins is a single high-affinity RRM domain, highly conserved from insects to humans, that binds preferentially to UGCAUG motifs at diverse regulatory sites in pre-mRNA introns, mRNA 3'UTRs, and pre-miRNAs hairpin structures. Versatile regulatory circuits operate on Rbfox pre-mRNA and mRNA to ensure proper expression of Rbfox1 protein isoforms, which then act on the broader transcriptome to regulate alternative splicing networks, mRNA stability and translation, and microRNA processing.

Structural Origins of Cholesterol Accelerated Lipid Flip-Flop Studied by Sum-Frequency Vibrational Spectroscopy.

The unique structure of cholesterol and its role in modulating lipid translocation (flip-flop) were examined using sum-frequency vibrational spectroscopy (SFVS). Two structural analogues of cholesterol--cholestanol and cholestene--were examined to explore the influence of ring rigidity and amphiphilicity on controlling distearoylphosphocholine (DSPC) flip-flop. Kinetic rates for DSPC flip-flop were determined as a function of sterol concentration and temperature.