COVID-19 serological survey using micro blood sampling.

During August 2020, we carried out a serological survey among students and employees at the Okinawa Institute of Science and Technology Graduate University (OIST), Japan, testing for the presence of antibodies against SARS-CoV-2, the causative agent of COVID-19. We used a FDA-authorized 2-step ELISA protocol in combination with at-home self-collection of blood samples using a custom low-cost finger prick-based capillary blood collection kit. Although our survey did not find any COVID-19 seropositive individuals among the OIST cohort, it reliably detected all positive control samples obtained from a local hospital and excluded all negatives controls. We found that high serum antibody titers can persist for more than 9 months post infection. Among our controls, we found strong cross-reactivity of antibodies in samples from a serum pool from two MERS patients in the anti-SARS-CoV-2-S ELISA. Here we show that a centralized ELISA in combination with patient-based capillary blood collection using as little as one drop of blood can reliably assess the seroprevalence among communities. Anonymous sample tracking and an integrated website created a stream-lined procedure. Major parts of the workflow were automated on a liquid handler, demonstrating scalability. We anticipate this concept to serve as a prototype for reliable serological testing among larger populations.

A new KSRP-binding compound suppresses distant metastasis of colorectal cancer by targeting the oncogenic KITENIN complex.

BACKGROUND: Distant metastasis is the major cause of death in patients with colorectal cancer (CRC). Previously, we identified KITENIN as a metastasis-enhancing gene and suggested that the oncogenic KITENIN complex is involved in metastatic dissemination of KITENIN-overexpressing CRC cells. Here, we attempted to find substances targeting the KITENIN complex and test their ability to suppress distant metastasis of CRC. METHODS: We screened a small-molecule compound library to find candidate substances suppressing the KITENIN complex in CRC cells. We selected a candidate compound and examined its effects on the KITENIN complex and distant metastasis through in vitro assays, a molecular docking model, and in vivo tumor models. RESULTS: Among several compounds, we identified DKC1125 (Disintegrator of KITENIN Complex #1125) as the best candidate. DKC1125 specifically suppressed KITENIN gain of function. After binding KH-type splicing regulatory protein (KSRP), DKC1125 degraded KITENIN and Dvl2 by recruiting RACK1 and miRNA-124, leading to the disintegration of the functional KITENIN-KSRP-RACK1-Dvl2 complex. A computer docking model suggested that DKC1125 specifically interacted with the binding pocket of the fourth KH-domain of KSRP. KITENIN-overexpressing CRC cells deregulated certain microRNAs and were resistant to 5-fluorouracil, oxaliplatin, and cetuximab. DKC1125 restored sensitivity to these drugs by normalizing expression of the deregulated microRNAs, including miRNA-124. DKC1125 effectively suppressed colorectal liver metastasis in a mouse model. Interestingly, the combination of DKC1125 with 5-fluorouracil suppressed metastasis more effectively than either drug alone. CONCLUSION: DKC1125 targets the KITENIN complex and could therefore be used as a novel therapeutic to suppress liver metastasis in CRC expressing high levels of KITENIN.

Biomimetic Crack Sensors Using Electrohydrodynamic Technology.

This paper proposes a new mechanism for detecting microscopic damage of structures based on imitating the sensory organs of spiders. Therefore, it is essential to manufacture sensors that can react sensitively to the micro deformations of structures. Numerous cracks were intentionally generated to improve the sensitivity of the proposed sensor, and an increase in the gap of the crack was observed by scanning electron microscopy (SEM) observation. Electrohydrodynamic technology is used to detect deformations in a structure of depositing Ag nano paste on a polyethylene terephtha-late (PET) substrate. Ag nano lines are also observed by SEM images. The sensor is constructed as a grid structure, by forming layers patterned horizontally and vertically. An impact tester is used to verify the mechanism for structural health monitoring using the developed sensor. The resistance changes of the sensors are applied to estimate the structure's damaged location. The intersections of the lines with varying resistance can be used to accurately detect crack initiation. The proposed mechanism is a powerful methodology for estimating and detecting microscopic deformations and damage to structures.

MYO1D binds with kinase domain of the EGFR family to anchor them to plasma membrane before their activation and contributes carcinogenesis.

The cell surface receptor tyrosine kinase (RTK) exists in a dynamic state, however, it remains unknown how single membrane-spanning RTK proteins are retained in the plasma membrane before their activation. This study was undertaken to investigate how RTK proteins are anchored in the plasma membrane before they bind with their respective extracellular ligands for activation through protein-protein interaction, co-localization, and functional phenotype studies. Here we show that unconventional myosin-I MYO1D functions to hold members of the EGFR family (except ErbB3) at the plasma membrane. MYO1D binds only with unphosphorylated EGFRs and anchors them to underlying actin cytoskeleton at the plasma membrane. The C-terminal end region of the MYO1D tail domain containing a ß-meander motif is critical for direct binding with kinase domain of the EGFR family, and expression of the tail domain alone suppresses the oncogenic action of full-length MYO1D. Overexpressed MYO1D increases colorectal and breast cancer cell motility and viability through upregulating EGFR level, and thereby promotes colorectal tumor progression in a syngeneic mouse model. MYO1D is upregulated in human colorectal cancer tissues from advanced stages. Collectively, molecular motor MYO1D plays a distinct role in the dynamic regulation of EGFR family levels by holding them at the plasma membrane before their activation. Overexpressed MYO1D contributes to colorectal carcinogenesis possibly as a novel oncogene and thus may serve as an additional target for suppression of RTK signaling in cancer treatment.

Structures of three ependymin-related proteins suggest their function as a hydrophobic molecule binder.

Ependymin was first discovered as a predominant protein in brain extracellular fluid in fish and was suggested to be involved in functions mostly related to learning and memory. Orthologous proteins to ependymin called ependymin-related proteins (EPDRs) have been found to exist in various tissues from sea urchins to humans, yet their functional role remains to be revealed. In this study, the structures of EPDR1 from frog, mouse and human were determined and analyzed. All of the EPDR1s fold into a dimer using a monomeric subunit that is mostly made up of two stacking antiparallel ß-sheets with a curvature on one side, resulting in the formation of a deep hydrophobic pocket. All six of the cysteine residues in the monomeric subunit participate in the formation of three intramolecular disulfide bonds. Other interesting features of EPDR1 include two asparagine residues with glycosylation and a Ca2+-binding site. The EPDR1 fold is very similar to the folds of bacterial VioE and LolA/LolB, which also use a similar hydrophobic pocket for their respective functions as a hydrophobic substrate-binding enzyme and a lipoprotein carrier, respectively. A further fatty-acid binding assay using EPDR1 suggests that it indeed binds to fatty acids, presumably via this pocket. Additional interactome analysis of EPDR1 showed that EPDR1 interacts with insulin-like growth factor 2 receptor and flotillin proteins, which are known to be involved in protein and vesicle translocation.

The carboxy-terminal region of the TBC1D4 (AS160) RabGAP mediates protein homodimerization.

TBC1D4 (also known as AS160) is a Rab·GTPase-activating protein (RabGAP) which functions in insulin signaling. TBC1D4 is critical for translocation of glucose transporter 4 (GLUT4), from an inactive, intracellular, vesicle-bound site to the plasma membrane, where it promotes glucose entry into cells. The TBC1D4 protein is structurally subdivided into two N-terminal phosphotyrosine-binding (PTB) domains, a C-terminal catalytic RabGAP domain, and a disordered segment in between containing potential Akt phosphorylation sites. Structural predictions further suggest that a region C-terminal to the RabGAP domain adopts a coiled-coil motif. We show that C-terminal region (CTR) region is largely α-helical and mediates TBC1D4 RabGAP dimerization. RabGAP catalytic activity and thermal stability appear to be independent of CTR-mediated dimerization.

In Streptomyces coelicolor SigR, methionine at the -35 element interacting region 4 confers the -31'-adenine base selectivity.

In Gram-positive Streptomyces coelicolor A3(2), SigR (Sc σ(R)) of the group IV ECF sigma factor singly activates expression of more than 30 oxidation responsive genes. Of the two promoter-binding domains--individually called region 2 and region 4 - within Sc σ(R), we hereby report a 2.6 Å resolution structure of the -35 element interacting carboxyl-terminal region 4 (Sc σ(R)4). Structural comparison of Sc σ(R)4 with the Escherichia coli SigE (Ec σ(E)) in complex with Ec σ(E) -35 element suggested that a single residue (Sc σ(R) Met188 and Ec σ(E) Arg171) may be responsible for distinguishing the one-base pair difference of the -35 elements--Sc σ(R)(-31')ATTCC(-35') ((-31')A) vs. Ec σ(E)(-31')GTTCC(-35') ((-31')G)--by interacting with the -31'-base. Further studies using expressed Sc σ(R) indicate that the wild-type Sc σ(R) with Met188 selectively interacted with the (-31')A sequence over the (-31')G sequence, whereas a mutation of Met188 to arginine resulted in interaction with both (-31')A and (-31')G sequences. Hence, we conclude that Met188 of Sc σ(R) confers the (-31')A-selectivity in -35 element interaction by disfavoured interaction with the (-31')G base.

FcγRIIB Gene Polymorphisms Are Associated with Disease Risk and Clinical Manifestations of Systemic Lupus Erythematosus in Koreans.

Systemic lupus erythematosus (SLE) is chronic autoimmune disease with various autoantibodies, which are involved in tissue damage. Fc gamma receptors (FcγRs) bind the constant region of the immunoglobulin G and transmit stimulatory or inhibitory signal to immune cells. The FcγR genes map to 1q23, a susceptible locus for SLE. We have screened single nucleotide polymorphisms (SNPs) in one of FcγR gene, FcγRIIB, which is the only inhibitory receptor, after considering gene map and reported SNPs. There were 3 SNPs in FcγRIIB: 10849 T>C (rs1050501) in exon 5 and 10950 T>G (rs6666965) and 11045 G>T (rs12117530) in intron 5 in Koreans. The frequency of the minor allele (T) of rs12117530 was significantly higher in SLE patients (50 patients, 20.4%) than healthy controls (17 patients, 12%, p = 0.041). Leukopenia occurred more frequently in SLE patients carrying the minor allele (T) of rs12117530 (p = 0.032). Among 5 haplotypes, the frequency of decreased complement was significantly lower in SLE patients with haplotype 1 [TTG] (p = 0.045). Nephritis, lymphopenia and anti-dsDNA antibody were significantly less frequent in SLE patients with haplotype 2 [TGG] (p = 0.046, p = 0.018, p = 0.002, respectively). The frequency of thrombocytopenia and anti-dsDNA antibody was significantly higher in SLE patients with haplotype 3 [CTG] (p < 0.001, p = 0.04, respectively). These data reveal that genetic polymorphisms within FcγRIIB are associated with disease susceptibility and phenotypes of SLE in Koreans. Furthermore, FcγRIIB rs12117530 polymorphism (T allele) may be an important risk factor in SLE.

Overproduction, crystallization and preliminary X-ray crystallographic analysis of Escherichia coli tRNA N6-threonylcarbamoyladenosine dehydratase.

Escherichia coli tRNA N6-threonylcarbamoyladenosine dehydratase (TcdA), previously called CsdL or YgdL, was overproduced and purified from E. coli and crystallized using polyethylene glycol 3350 as a crystallizing agent. X-ray diffraction data were collected to 2.70 Šresolution under cryoconditions using synchrotron X-rays. The crystals belonged to space group P21, with unit-cell parameters a=65.4, b=96.8, c=83.3 Å, ß=111.7°. According to the Matthews coefficient, the asymmetric unit may contain up to four subunits of the monomeric protein, with a crystal volume per protein mass (VM) of 2.12 Å3 Da(-1) and 42.1% solvent content.

Crystallization and preliminary X-ray crystallographic analysis of carboxyl-terminal region 4 of SigR from Streptomyces coelicolor A3(2).

Full-length SigR from Streptomyces coelicolor A3(2) was overexpressed in Escherichia coli, purified and submitted to crystallization trials using either polyethylene glycol 3350 or 4000 as a precipitant. X-ray diffraction data were collected to 2.60 Šresolution under cryoconditions using synchrotron X-rays. The crystal packs in space group P43212, with unit-cell parameters a=b=42.14, c=102.02 Å. According to the Matthews coefficient, the crystal asymmetric unit cannot contain the full-length protein. Molecular replacement with the known structures of region 2 and region 4 as independent search models indicates that the crystal contains only the -35 element-binding carboxyl-terminal region 4 of full-length SigR. Mass-spectrometric analysis of the harvested crystal confirms this, suggesting a crystal volume per protein weight (VM) of 2.24 Å3 Da(-1) and 45.1% solvent content.

Discovery and the structural basis of a novel p21-activated kinase 4 inhibitor.

Functional versatility and elevated expression in cancers have endowed p21-activated kinase 4 (PAK4) as one of the first-in-class anti-cancer drug target. In this study, a novel PAK4 inhibitor, KY-04031 (N(2)-(2-(1H-indol-3-yl)ethyl)-N(4)-(1H-indazol-5-yl)-6-methoxy-1,3,5-triazine-2,4-diamine), was discovered using a high-throughput screening. Analysis of the complex crystal structure illustrated that both indole and indazole of KY-04031 are responsible for PAK4 hinge interaction. Moreover, the molecule's triazine core was found to mimic the ribose of the natural ATP substrate. The cell-based anti-cancer potency of KY-04031 was less effective than the pyrroloaminopyrazoles; however, the unique molecular feature of KY-04031 can be exploited in designing new PAK4 inhibitors.

RNA editing in RHOQ promotes invasion potential in colorectal cancer.

RNA editing can increase RNA sequence variation without altering the DNA sequence. By comparing whole-genome and transcriptome sequence data of a rectal cancer, we found novel tumor-associated increase of RNA editing in ras homologue family member Q (RHOQ) transcripts. The adenosine-to-inosine (A-to-I) editing results in substitution of asparagine with serine at residue 136. We observed a higher level of the RHOQ RNA editing in tumor compared with normal tissue in colorectal cancer (CRC). The degree of RNA editing was associated with RhoQ protein activity in CRC cancer cell lines. RhoQ N136S amino acid substitution increased RhoQ activity, actin cytoskeletal reorganization, and invasion potential. KRAS mutation further increased the invasion potential of RhoQ N136S in vitro. Among CRC patients, recurrence was more frequently observed in patients with tumors having edited RHOQ transcripts and mutations in the KRAS gene. In summary, we show that RNA editing is another mechanism of sequence alteration that contributes to CRC progression.

Affinity between TBC1D4 (AS160) phosphotyrosine-binding domain and insulin-regulated aminopeptidase cytoplasmic domain measured by isothermal titration calorimetry.

Uptake of circulating glucose into the cells happens via the insulin- mediated signalling pathway, which translocates the glucose transporter 4 (GLUT4) vesicles from the intracellular compartment to the plasma membrane. RabㆍGTPases are involved in this vesicle trafficking, where RabㆍGTPase-activating proteins (RabGAP) enhance the GTP to GDP hydrolysis. TBC1D4 (AS160) and TBC1D1 are functional RabGAPs in the adipocytes and the skeletonal myocytes, respectively. These proteins contain two phosphotyrosine-binding domains (PTBs) at the amino-terminus of the catalytic RabGAP domain. The second PTB has been shown to interact with the cytoplasmic region of the insulin-regulated aminopeptidase (IRAP) of the GLUT4 vesicle. In this study, we quantitatively measured the ∼µM affinity (KD) between TBC1D4 PTB and IRAP using isothermal titration calorimetry, and further showed that IRAP residues 1-49 are the major region mediating this interaction. We also demonstrated that the IRAP residues 1-15 are necessary but not sufficient for the PTB interaction.

Crystallization and preliminary X-ray crystallographic analysis of Thermotoga maritima CheA P3-P4-P5 domains in complex with CheW.

The CheA-CheW complex plays a key role in bacterial chemotaxis signal transduction by initiating phosphotransfer to response regulators via coupling to the chemoreceptors. CheA (P3-P4-P5 domains) and CheW from Thermotoga maritima were overexpressed in Escherichia coli and crystallized as a complex at 298 K using ammonium dihydrogen phosphate as a precipitant. X-ray diffraction data were collected to ~8 Å resolution at 100 K using synchrotron radiation. The crystal belonged to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 184.2, b = 286.4, c = 327.7 Å. The asymmetric unit may contain six to ten CheA-CheW molecules.

Crystallization and preliminary X-ray crystallographic analysis of Escherichia coli CheA P3 dimerization domain.

The chemotaxis histidine kinase CheA assembles into a dimer in which the P3 dimerization domain forms a four-helix bundle by the parallel association of two α-helical hairpins from each subunit. Ligand occupancy of the chemoreceptor regulates signal transduction by controlling the autophosphorylation activity of CheA. Autophosphorylation of CheA occurs in trans, i.e. one subunit phosphorylates the other. The P3 domain of CheA from Escherichia coli has been overexpressed in E. coli and crystallized at 298 K using PEG as a precipitant. X-ray diffraction data to 2.80 Šresolution have been collected at 100 K using synchrotron radiation. The crystal belonged to space group P1, with unit-cell parameters a = 59.271, b = 67.674, c = 82.815 Å, α = 77.568, ß = 86.073, γ = 64.436°. The asymmetric unit may contain up to ten dimeric units of P3 four-helix bundles.