Diffusion of water in palm leaf materials.

Diffusion of water into plant materials is known to decrease their mechanical strength and stiffness but improve formability. Here, we characterize water diffusion through areca palm leaf-sheath-a model plant material, with hierarchical structure, used in eco-friendly foodware. The diffusion process is studied using mass gain measurements and in situ imaging of water transport. By treating the areca sheath as homogeneous ensemble, and incorporating effects of material swelling due- to water absorption, a factor typically neglected in prior studies, the diffusion coefficient Dw for water is estimated as (6.5 ± 2.2) × 10-4 mm2 s-1. It is shown that neglecting the swelling results in gross underestimation of Dw. Microstructural effects (e.g. fibre, matrix) on the diffusion are characterized using in situ imaging of the water transport at high resolution. The observations show that the water diffuses an order of magnitude faster in the matrix (8.63 × 10-4 mm2 s-1) than in the fibres (7.19 × 10-5 mm2 s-1). This non-uniformity is also reflected in the swelling-induced strain in the leaf, mapped by image correlation. Lastly, we vary salt concentration by controlled additions of NaCl and note a non-monotonic dependence of the diffusion on concentration. Implications of the results for improving foodware manufacturing processes and product life are discussed.

The transition structure of chromatin fibers at the nanoscale probed by cryogenic electron tomography.

The naked DNA inside the nucleus interacts with proteins and RNAs forming a higher order chromatin structure to spatially and temporally control transcription in eukaryotic cells. The 30 nm chromatin fiber is one of the most important determinants of the regulation of eukaryotic transcription. However, the transition of chromatin from the 30 nm inactive higher order structure to the actively transcribed lower order nucleosomal arrays is unclear, which limits our understanding of eukaryotic transcription. Using a method to extract near-native eukaryotic chromatin, we revealed the chromatin structure at the transitional state from the 30 nm chromatin to multiple nucleosomal arrays by cryogenic electron tomography (cryo-ET). Reproducible electron microscopy images revealed that the transitional structure is a branching structure that the 30 nm chromatin hierarchically branches into lower order nucleosomal arrays, indicating chromatin compaction at different levels to control its accessibility during the interphase. We further observed that some of the chromatin fibers on the branching structure have a helix ribbon structure, while the others randomly twist together. Our finding of the chromatin helix ribbon structure on the extracted native chromatin revealed by cryo-ET indicates a complex higher order chromatin organization beyond the beads-on-a-string structure. The hierarchical branching and helix ribbon structure may provide mechanistic insights into how chromatin organization plays a central role in transcriptional regulation and other DNA-related biological processes during diseases such as cancer.

Skeletal muscle-derived exosomes regulate endothelial cell functions via reactive oxygen species-activated nuclear factor-κB signalling.

NEW FINDINGS: What is the central question of this study? Capillary rarefaction is found in diabetic and aged muscle, whereas exercise increases skeletal muscle angiogenesis. The association implies a crosstalk between muscle cells and endothelial cells. The underlying mechanisms mediating the crosstalk between these cells remains to be elucidated fully. What is the main finding and its importance? Endothelial cell functions are regulated by skeletal muscle cell-derived exosomes via a vascular endothelial growth factor-independent pathway. This study reveals a new mechanism mediating the crosstalk between skeletal muscle cells and endothelial cells. ABSTRACT: Loss of skeletal muscle capillarization, known as capillary rarefaction, is found in type 2 diabetes, chronic heart failure and healthy ageing and is associated with impaired delivery of substrates to the muscle. However, the interaction and communication of skeletal muscle with endothelial cells in the regulation of capillaries surrounding the muscle remains elusive. Exosomes are a type of secreted extracellular vesicle containing mRNAs, proteins and, especially, microRNAs that exert paracrine and endocrine effects. In this study, we investigated whether skeletal muscle-derived exosomes (SkM-Exo) regulate the endothelial cell functions of angiogenesis. We demonstrated that C2C12 myotube-derived exosomes improved endothelial cell functions, assessed by the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs), which were increased by 20, 23 and 40%, respectively, after SkM-Exo exposure. The SkM-Exo failed to activate HUVEC vascular endothelial growth factor (VEGF) signalling. The SkM-Exo increased HUVEC reactive oxygen species and activated the nuclear factor-κB pathway, suggesting that SkM-Exo-induced angiogenesis was mediated by a VEGF-independent pathway. In addition, several angiogenic microRNAs were packaged in SkM-Exo, with miR-130a being particularly enriched and successfully transferred from SkM-Exo to HUVECs. Delivery of miRNAs into endothelial cells might explain the enhancement of reactive oxygen species production and angiogenesis by SkM-Exo. The potential angiogenic effect of SkM-Exo could provide an effective therapy for promoting skeletal muscle angiogenesis in diseases characterized by capillary rarefaction or inadequate angiogenesis.

Protein particulate retention and microorganism recovery for rapid detection of Salmonella.

The rapid detection of Salmonella in ground meat requires that living microorganisms be brought to levels detectable by PCR, immunoassays, or similar techniques within 8 h. Previously, we employed microfiltration using hollow fiber membranes to rapidly process and concentrate viable bacteria in food extracts through a combination of enzyme treatment and prefiltration in order to prevent blockage or fouling of the hollow fiber membranes. However, scanning electron microscopy and particle size analysis of enzyme hydrolysates showed that enzyme treatment followed by filtration caused submicron particles to form and be trapped within the prefiltration media, which in turn, retained about 80% of the bacteria. Filtering prior to enzyme treatment resulted in formation of a filter cake consisting of protein particles retained on the surface of the filter, while facilitating passage of the much smaller microorganisms through the filter, separating them from particulates. Subsequent enzyme treatment of the filtrate resulted in an extract that was microfiltered in less than an hour, while concentrating viable microorganisms in the extract by 500×. An inoculum of Salmonella enterica cells into turkey burger containing of 1-20 CFU/mL, consisting of spiked cells plus cells already present in the turkey burger sample, was rapidly brought to levels detectable by conventional PCR and BAX® PCR assays. The entire procedure from sample processing to detection of Salmonella enterica was achieved in less than 8 h. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:687-695, 2017.

Understanding the Impact of Water on the Miscibility and Microstructure of Amorphous Solid Dispersions: An AFM-LCR and TEM-EDX Study.

Miscibility is critical for amorphous solid dispersions (ASDs). Phase-separated ASDs are more prone to crystallization, and thus can lose their solubility advantage leading to product failure. Additionally, dissolution performance can be diminished as a result of phase separation in the ASD matrix. Water is known to induce phase separation during storage for some ASDs. However, the impact of water introduced during preparation has not been as thoroughly investigated to date. The purpose of this study was to develop a mechanistic understanding of the effect of water on the phase behavior and microstructure of ASDs. Evacetrapib and two polymers were selected as the model system. Atomic force microscopy coupled with Lorentz contact resonance, and transmission electron microscopy with energy dispersive X-ray spectroscopy were employed to evaluate the microstructure and composition of phase-separated ASDs. It was found that phase separation could be induced via two routes: solution-state phase separation during ASD formation caused by water absorption during film formation by a hydrophilic solvent, or solid-phase separation following exposure to high RH during storage. Water contents of as low as 2% in the organic solvent system used to dissolve the drug and polymer were found to result in phase separation in the resultant ASD film. These findings have profound implications on lab-scale ASD preparation and potentially also for industrial production. Additionally, these high-resolution imaging techniques combined with orthogonal analyses are powerful tools to visualize structural changes in ASDs, which in turn will enable better links to be made between ASD structure and performance.

Parasite-induced TH1 cells and intestinal dysbiosis cooperate in IFN-γ-dependent elimination of Paneth cells.

Activation of Toll-like receptors (TLRs) by pathogens triggers cytokine production and T cell activation, immune defense mechanisms that are linked to immunopathology. Here we show that IFN-γ production by CD4(+) T(H)1 cells during mucosal responses to the protozoan parasite Toxoplasma gondii resulted in dysbiosis and the elimination of Paneth cells. Paneth cell death led to loss of antimicrobial peptides and occurred in conjunction with uncontrolled expansion of the Enterobacteriaceae family of Gram-negative bacteria. The expanded intestinal bacteria were required for the parasite-induced intestinal pathology. The investigation of cell type-specific factors regulating T(H)1 polarization during T. gondii infection identified the T cell-intrinsic TLR pathway as a major regulator of IFN-γ production in CD4(+) T cells responsible for Paneth cell death, dysbiosis and intestinal immunopathology.

Type III effector VopC mediates invasion for Vibrio species.

Vibrio spp. are associated with infections caused by contaminated food and water. A type III secretion system (T3SS2) is a shared feature of all clinical isolates of V. parahaemolyticus and some V. cholerae strains. Despite its being responsible for enterotoxicity, no molecular mechanism has been determined for the T3SS2-dependent pathogenicity. Here, we show that although Vibrio spp. are typically thought of as extracellular pathogens, the T3SS2 of Vibrio mediates host cell invasion, vacuole formation, and replication of intracellular bacteria. The catalytically active effector VopC is critical for Vibrio T3SS2-mediated invasion. There are other marine bacteria encoding VopC homologs associated with a T3SS; therefore, we predict that these bacteria are also likely to use T3SS-mediated invasion as part of their pathogenesis mechanisms. These findings suggest a new molecular paradigm for Vibrio pathogenicity and modify our view of the roles of T3SS effectors that are translocated during infection.

RhoA-inhibiting NSAIDs promote axonal myelination after spinal cord injury.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are extensively used to relieve pain and inflammation in humans via cyclooxygenase inhibition. Our recent research suggests that certain NSAIDs including ibuprofen suppress intracellular RhoA signal and improve significant axonal growth and functional recovery following axonal injury in the CNS. Several NSAIDs have been shown to reduce generation of amyloid-beta42 peptide via inactivation of RhoA signal, supporting potent RhoA-repressing function of selected NSAIDs. In this report, we demonstrate that RhoA-inhibiting NSAIDs ibuprofen and indomethacin dramatically reduce cell death of oligodendrocytes in cultures or along the white matter tracts in rats with a spinal cord injury. More importantly, we demonstrate that treatments with the RhoA-inhibiting NSAIDs significantly increase axonal myelination along the white matter tracts following a traumatic contusion spinal cord injury. In contrast, non-RhoA-inhibiting NSAID naproxen does not have such an effect. Thus, our results suggest that RhoA inactivation with certain NSAIDs benefits recovery of injured CNS axons not only by promoting axonal elongation, but by enhancing glial survival and axonal myelination along the disrupted axonal tracts. This study, together with previous reports, supports that RhoA signal is an important therapeutic target for promoting recovery of injured CNS and that RhoA-inhibiting NSAIDs provide great therapeutic potential for CNS axonal injuries in adult mammals.

Vibrio parahaemolyticus orchestrates a multifaceted host cell infection by induction of autophagy, cell rounding, and then cell lysis.

The bacterial pathogen Vibrio parahaemolyticus utilizes a type III secretion system to cause death of host cells within hours of infection. We report that cell death is completely independent of apoptosis and occurs by a mechanism in which injection of multiple type III effectors causes induction of autophagy, cell rounding, and the subsequent release of cellular contents. Autophagy is detected by the appearance of lipidated light chain 3 (LC3) and by increases in punctae and vacuole formation. Electron microscopy reveals the production of early autophagic vesicles during infection. Consistent with phosphoinositide 3 (PI3) kinase playing a role in autophagy, treatment of infected cells with a PI3 kinase inhibitor attenuates autophagy in infected cells. Because many effectors are injected during a V. parahaemolyticus infection, it is not surprising that the presence of a sole PI3 kinase inhibitor does not prevent inevitable host-cell death. Our studies reveal an infection paradigm whereby an extracellular pathogen uses its type III secretion system to cause at least three parallel events that eventually result in the proinflammatory death of an infected host cell.

Stroboscopic image capture: reducing the dose per frame by a factor of 30 does not prevent beam-induced specimen movement in paraffin.

Beam-induced specimen movement may be the major factor that limits the quality of high-resolution images of organic specimens. One of the possible measures to improve the situation that was proposed by Henderson and Glaeser [Ultramicroscopy 16 (1985) 139-150], which we refer to here as "stroboscopic image capture", is to divide the normal exposure into many successive frames, thus reducing the amount of electron exposure--and possibly the amount of beam-induced movement--per frame. The frames would then be aligned and summed. We have performed preliminary experiments on stroboscopic imaging using a 200-kV electron microscope that was equipped with a high dynamic range Charge-coupled device (CCD) camera for image recording and a liquid N2-cooled cryoholder. Single-layer paraffin crystals on carbon film were used as a test specimen. The ratio F(g)/F(0) of paraffin reflections, calculated from the images, serves as our criterion for the image quality. In the series that were evaluated, no significant improvement of the F(image)(g)/F(image)(0) ratio was found, even though the electron exposure per frame was reduced by a factor of 30. A frame-to-frame analysis of image distortions showed that considerable beam-induced movement had still occurred during each frame. In addition, the paraffin crystal lattice was observed to move relative to the supporting carbon film, a fact that cannot be explained as being an electron-optical effect caused by specimen charging. We conclude that a significant further reduction of the dose per frame (than was possible with this CCD detector) will be needed in order to test whether the frame-to-frame changes ultimately become small enough for stroboscopic image capture to show its potential.

SNARE-mediated lipid mixing depends on the physical state of the vesicles.

Reconstitution experiments have suggested that N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins constitute a minimal membrane fusion machinery but have yielded contradictory results, and it is unclear whether the mechanism of membrane merger is related to the stalk mechanism that underlies physiological membrane fusion. Here we show that reconstitution of solubilized neuronal SNAREs into preformed 100 nm liposomes (direct method) yields proteoliposomes with more homogeneous sizes and protein densities than the standard reconstitution method involving detergent cosolubilization of proteins and lipids. Standard reconstitutions yield slow but efficient lipid mixing at high protein densities and variable amounts of lipid mixing at moderate protein densities. However, the larger, more homogenous proteoliposomes prepared by the direct method yield almost no lipid mixing at moderate protein densities. These results suggest that the lipid mixing observed for standard reconstitutions is dominated by the physical state of the membrane, perhaps due to populations of small vesicles (or micelles) with high protein densities and curvature stress created upon reconstitution. Accordingly, changing membrane spontaneous curvature by adding lysophospholipids inhibits the lipid mixing observed for standard reconstitutions. Our data indicate that the lipid mixing caused by high SNARE densities and/or curvature stress occurs by a stalk mechanism resembling the mechanism of fusion between biological membranes, but the neuronal SNAREs are largely unable to induce lipid mixing at physiological protein densities and limited curvature stress.