Decreased Podocyte Vesicle Transcytosis and Albuminuria in APC C-Terminal Deficiency Mice with Puromycin-Induced Nephrotic Syndrome

In minimal change nephrotic syndrome, podocyte vesicle transport is enhanced. Adenomatous polyposis coli (APC) anchors microtubules to cell membranes and plays an important role in vesicle transport. To clarify the role of APC in vesicle transport in podocytes, nephrotic syndrome was induced by puromycin amino nucleoside (PAN) injection in mice expressing APC1638T lacking the C-terminal of microtubule-binding site (APC1638T mouse); this was examined in renal tissue changes. The kidney size and glomerular area of APC1638T mice were reduced (p = 0.014); however, the number of podocytes was same between wild-type (WT) mice and APC1638T mice. The ultrastructure of podocyte foot process was normal by electron microscopy. When nephrotic syndrome was induced, the kidneys of WT+PAN mice became swollen with many hyaline casts, whereas these changes were inhibited in the kidneys of APC1638T+PAN mice. Electron microscopy showed foot process effacement in both groups; however, APC1638T+PAN mice had fewer vesicles in the basal area of podocytes than WT+PAN mice. Cytoplasmic dynein-1, a motor protein for vesicle transport, and α-tubulin were significantly reduced in APC1638T+PAN mice associated with suppressed urinary albumin excretion compared to WT+PAN mice. In conclusion, APC1638T mice showed reduced albuminuria associated with suppressed podocyte vesicle transport when minimal change nephrotic syndrome was induced.     

Recombinant Long-Acting Thioredoxin Ameliorates AKI to CKD Transition via Modulating Renal Oxidative Stress and Inflammation

An effective strategy is highly desirable for preventing acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Thioredoxin-1 (Trx), a redox-active protein that has anti-oxidative and anti-inflammatory properties, would be a candidate for this but its short half-life limits its clinical application. In this study, we examined the renoprotective effect of long-acting Trx that is comprised of human albumin and Trx (HSA-Trx) against AKI to CKD transition. AKI to CKD mice were created by renal ischemia-reperfusion (IR). From day 1 to day 14 after renal IR, the recovery of renal function was accelerated by HSA-Trx administration. On day 14, HSA-Trx reduced renal fibrosis compared with PBS treatment. At the early phase of fibrogenesis (day 7), HSA-Trx treatment suppressed renal oxidative stress, pro-inflammatory cytokine production and macrophage infiltration, thus ameliorating tubular injury and fibrosis. In addition, HSA-Trx treatment inhibited G2/M cell cycle arrest and apoptosis in renal tubular cells. While renal Trx protein levels were decreased after renal IR, the levels were recovered by HSA-Trx treatment. Together, HSA-Trx has potential for use in the treatment of AKI to CKD transition via its effects of modulating oxidative stress and inflammation.     

Determination of atomistic deformation of tricalcium silicate paste with high-volume fly ash

We examined the effect of incorporating high-volume fly ash on the atomic arrangement and interatomic deformation behavior of calcium silicate hydrates in tricalcium silicate paste upon exposure to external forces. The interatomic structural changes and strains under compressive load were assessed using synchrotron in situ high-energy X-ray scattering-based atomic pair distribution function analysis. Three different types of strains, which were (a) macroscopic strains from gauges on the surfaces of specimen, (b) strains in a reciprocal space (Bragg peak shift), and (c) strains in real space (PDF peak shift), were compared to each other. All monitored and calculated strains for tricalcium silicate-fly ash (50 wt% fly ash) paste were compared with the counterparts of the pure tricalcium silicate paste. Pair distribution function analysis in the range of r < 10 Å indicated that the atomic arrangement of tricalcium silicate-fly ash was similar to that of synthetic calcium silicate hydrates followed by that of pure tricalcium silicate paste. Moreover, the pair distribution function refinement results revealed that the calcium silicate hydrate structure in tricalcium silicate-fly ash paste was similar to tobermorite 11 Å, unlike that in pure tricalcium silicate paste. The interatomic strain of tricalcium silicate-fly ash in the real space (r < 20 Å) was smaller than that of tricalcium silicate under compression, which suggested that the incompressibility of calcium silicate hydrates at atomistic scale was enhanced by the incorporation of fly ash into it. This was likely to be caused by the increased silicate polymerization of calcium silicate hydrates, which was attributed to the increase in the amount of silicate in their structure via the addition of fly ash.     

Effective Characterization of DNA Ligation Kinetics by High-Resolution Melting Analysis

High-resolution melting (HRM) analysis has been improved and applied for the first time to quantitative analysis of enzymatic reactions. By using the relative ratios of peak intensities of substrates and products, the quantitativity of conventional HRM analysis has been improved to allow detailed kinetic analysis. As an example, the ligation of sticky ends through the action of T4 DNA ligase has been kinetically analyzed, with comprehensive data on substrate specificity and other properties having been obtained. For the first time, the kinetic parameters (k_obs and apparent K_m) of sticky-end ligation were obtained for both fully matched and mismatched sticky ends. The effect of ATP concentration on sticky-end ligation was also investigated. The improved HRM method should also be applicable to versatile DNA-transforming enzymes, because the only requirement is that the products have T_m values different enough from the substrates.     

Dynamic Stabilization of DNA Assembly by Using Pyrrole-Imidazole Polyamide

We used N-methylpyrrole (Py)-N-methylimidazole-(Im) polyamide as an exogenous agent to modulate the formation of DNA assemblies at specific double-stranded sequences. The concept was demonstrated on the hybridization chain reaction that forms linear DNA. Through a series of melting curve analyses, we demonstrated that the binding of Py−Im polyamide positively influenced both the HCR initiation and elongation steps. In particular, Py−Im polyamide was found to drastically stabilize the DNA duplex such that its thermal stability approached that of an equivalent hairpin structure. Also, the polyamide served as an anchor between hairpin pairs in the HCR assembly, thus improving the originally weak interstrand stability. We hope that these proof-of-concept results can inspire future use of Py−Im polyamide as a molecular tool to modulate the formation of DNA assemblies.     

Synthesis of polyamide-hydroxyapatite nanocomposites

Simultaneous one-pot syntheses of PA66 and HAp were carried out by extracting H₂O and CO₂ from PA66 monomers and HAp raw materials, respectively, resulting in the formation of a polyamide (PA) 66-hydroxyapatite (HAp) nanocomposite. During the process, a spherical nano-sized HAp particle was precipitated following dissolution of micro-sized CaHPO₄・2H₂O. The PA66 monomers were subsequently adsorbed onto the generated HAp product. Some of the adsorbed PA66 monomers formed a bound polymer on HAp, and an increase in the adhesiveness of the PA66-HAp interface was observed as the polymerization progressed. During this process, the synthesis of a nanocomposite from a micro-sized raw material and creation of an autonomous strong interface between the matrix and filler was achieved. In addition, the shape of the resultant HAp was controllable and could be modified to needle shape by the addition of F⁻ and Mg²⁺ ions to the raw material. HAp could also be changed to plate shape via octa-calcium phosphate (OCP). Notably, during the synthesis, the filler shape of the nanocomposite could be controlled to 0D (particle), 1D (needle), and 2D (plate).     

MiR-10a in Pancreatic Juice as a Biomarker for Invasive Intraductal Papillary Mucinous Neoplasm by miRNA Sequencing

New biomarkers are needed to further stratify the risk of malignancy in intraductal papillary mucinous neoplasm (IPMN). Although microRNAs (miRNAs) are expected to be stable biomarkers, they can vary owing to a lack of definite internal controls. To identify universal biomarkers for invasive IPMN, we performed miRNA sequencing using tumor-normal paired samples. A total of 19 resected tissues and 13 pancreatic juice samples from 32 IPMN patients were analyzed for miRNA expression by next-generation sequencing with a two-step normalization of miRNA sequence data. The miRNAs involved in IPMN associated with invasive carcinoma were identified from this tissue analysis and further verified with the pancreatic juice samples. From the tumor-normal paired tissue analysis of the expression levels of 2792 miRNAs, 20 upregulated and 17 downregulated miRNAs were identified. In IPMN associated with invasive carcinoma (INV), miR-10a-5p and miR-221-3p were upregulated and miR-148a-3p was downregulated when compared with noninvasive IPMN. When these findings were further validated with pancreatic juice samples, miR-10a-5p was found to be elevated in INV (p = 0.002). Therefore, three differentially expressed miRNAs were identified in tissues with INV, and the expression of miR-10a-5p was also elevated in pancreatic juice samples with INV. MiR-10a-5p is a promising additional biomarker for invasive IPMN.     

Possibility of Japanese Cedar Pollen Causing False Positives in the Deep Mycosis Test

Because Japanese cedar pollen (JCP) contains beta-1,3-d-glucan (BG), there is concern that its lingering presence in the atmosphere, especially during its scattering period, may cause false positives in the factor-G-based Limulus amebocyte lysate (LAL) assay used to test for deep mycosis (i.e., G-test). Hence, we examined whether the LAL assay would react positively with substances contained in JCP by using the G-test to measure JCP particles and extracts. BG was purified from the JCP extract on a BG-specific affinity column, and the percentage extractability was measured using three different BG-specific quantitative methods. The G-test detected 0.4 pg BG in a single JCP particle and 10 fg from a single particle in the extract. The percentage extractability of JCP-derived BG was not significantly different among the three quantitative methods. As the JCP particles should technically have been removed during serum separation, they should be less likely to be a direct false-positive factor. However, given that the LAL-assay-positive substances in the JCP extract were not distinguishable by the three BG-specific quantitative methods, we conclude that they may cause the background to rise. Therefore, in Japan false positives arising from JCP contamination should be considered when testing patients for deep mycosis.     

Fluorescence Correlation Spectroscopy Analysis of Effect of Molecular Crowding on Self-Assembly of β-Annulus Peptide into Artificial Viral Capsid

Recent progress in the de novo design of self-assembling peptides has enabled the construction of peptide-based viral capsids. Previously, we demonstrated that 24-mer β-annulus peptides from tomato bushy stunt virus spontaneously self-assemble into an artificial viral capsid. Here we propose to use the artificial viral capsid through the self-assembly of β-annulus peptide as a simple model to analyze the effect of molecular crowding environment on the formation process of viral capsid. Artificial viral capsids formed by co-assembly of fluorescent-labelled and unmodified β-annulus peptides in dilute aqueous solutions and under molecular crowding conditions were analyzed using fluorescence correlation spectroscopy (FCS). The apparent particle size and the dissociation constant (K_d) of the assemblies decreased with increasing concentration of the molecular crowding agent, i.e., polyethylene glycol (PEG). This is the first successful in situ analysis of self-assembling process of artificial viral capsid under molecular crowding conditions.