Discovery of novel pyrrole derivatives as potent agonists for the niacin receptor GPR109A.

Novel pyrrole derivatives were discovered as potent agonists of the niacin receptor, GPR109A. During the derivatization, compound 16 was found to be effective both in vitro and in vivo. The compound 16 exhibited a significant reduction of the non-esterified fatty acid in human GPR109A transgenic rats, and the duration of its in vivo efficacy was much longer than niacin.

Generation and Characterization of a Novel Small Biologic Alternative to Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Antibodies, DS-9001a, Albumin Binding Domain-Fused Anticalin Protein.

Since it was recently reported that an antibody for proprotein convertase subtilisin/kexin type 9 (PCSK9) reduces the risk of cardiovascular events in a clinical context, PCSK9 inhibition is thought to be an attractive therapy for dyslipidemia. In the present study, we created a novel small biologic alternative to PCSK9 antibodies called DS-9001a, comprising an albumin binding domain fused to an artificial lipocalin mutein (ABD-fused Anticalin protein), which can be produced by a microbial production system. DS-9001a strongly interfered with PCSK9 binding to low-density-lipoprotein receptor (LDL-R) and PCSK9-mediated degradation of LDL-R.

Hyperactive FOXO1 results in lack of tip stalk identity and deficient microvascular regeneration during kidney injury.

Loss of the microvascular (MV) network results in tissue ischemia, loss of tissue function, and is a hallmark of chronic diseases. The incorporation of a functional vascular network with that of the host remains a challenge to utilizing engineered tissues in clinically relevant therapies. We showed that vascular-bed-specific endothelial cells (ECs) exhibit differing angiogenic capacities, with kidney microvascular endothelial cells (MVECs) being the most deficient, and sought to explore the underlying mechanism.

Maintenance of vascular integrity by pericytes is essential for normal kidney function.

Pericytes are tissue-resident mesenchymal progenitor cells anatomically associated with the vasculature that have been shown to participate in tissue regeneration. Here, we tested the hypothesis that kidney pericytes, derived from FoxD1 mesodermal progenitors during embryogenesis, are necessary for postnatal kidney homeostasis. Diphtheria toxin delivery to FoxD1Cre::RsDTR transgenic mice resulted in selective ablation of >90% of kidney pericytes but not other cell lineages.

A Novel Three-Dimensional Human Peritubular Microvascular System.

Human kidney peritubular capillaries are particularly susceptible to injury, resulting in dysregulated angiogenesis, capillary rarefaction and regression, and progressive loss of kidney function. However, little is known about the structure and function of human kidney microvasculature. Here, we isolated, purified, and characterized human kidney peritubular microvascular endothelial cells (HKMECs) and reconstituted a three-dimensional human kidney microvasculature in a flow-directed microphysiologic system.

Detection of protein tyrosine phosphorylation by open sandwich fluoroimmunoassay.

In the era of proteomics, high-throughput screening of posttranslational modification states of proteins, especially protein phosphorylation, is considered of utmost importance. However, current protein phosphorylation detection methods depend on either the combination of proteolysis and mass spectrometry, or time-consuming immunoassay that requires inevitable washing processes. As a way to rapidly assay protein phosphorylation events, here we propose the use of Open Sandwich immunoassay that detects antigen-dependent stabilization of antibody variable region (Fv).

The role of interface framework residues in determining antibody V(H)/V(L) interaction strength and antigen-binding affinity.

While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region fragment (V(H))/antibody light chain variable region fragment (V(L)) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak V(H)/V(L) interaction strength, in the absence of antigen. To investigate the possible relationship between antigen-binding affinity and V(H)/V(L) interaction strength, a novel phage display system that can switch two display modes was employed. We focused on the two framework region 2 regions of the HyHEL-10 V(H) and V(L), facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.

A general method to select antibody fragments suitable for noncompetitive detection of monovalent antigens.

Previously, immunological detection of a small hapten was only possible in competitive format, which needed a competitor antigen either labeled by a reporter or attached to a carrier protein. Recently, we proposed the open sandwich (OS) immunoassay, a simple immunoassay that can noncompetitively determine monovalent antigen concentration by measuring the antigen-dependent change in a heavy-chain variable region (VH)/light-chain variable region (VL) interaction of an antibody. However, there was a limitation in the assay that the antibody used should have a suitable property such that the VH/VL interaction would become fairly strong along with the addition of antigen.

Luminescent and substrate binding activities of firefly luciferase N-terminal domain.

Firefly luciferase catalyzes highly efficient emission of light from the substrates luciferin, Mg-ATP, and oxygen. A number of amino acid residues are identified to be important for the luminescent activity, and almost all the key residues are thought to be located in the N-terminal domain (1-437), except one in the C-terminal domain, Lys529, which is thought to be critical for efficient substrate orientation. Here we show that the purified N-terminal domain still binds to the substrates luciferin and ATP with reduced affinity, and retains luminescent activity of up to 0.

Importance of a CDR H3 basal residue in V(H)/V(L) interaction of human antibodies.

Although the cooperativity of the V(H) and V(L) domains of an antibody in antigen binding has been extensively studied, the interaction between the V(H) and V(L) domains had not received sufficient attention. To systematically investigate the relationship between the amino acid sequence and V(H)/V(L) interaction strength, we here used a set of anti-bovine serum albumin antibodies having a single human framework for V(H) (V3-23/DP-47 and JH4b) and Vk (O12/O2/DPK9 and Jk1), but with different V(H)/V(L) interaction strengths. By phage display of a V(H) mini-library and analysis of the interaction of amino acids with immobilized V(L) fragments, the residue at H95 (Kabat numbering) at the beginning of seven CDR H3 residues was found to play a key role in determining the V(H)/V(L) interaction.