JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates

Recent genetic studies in Drosophila identified a novel non-canonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements

Identification and Characterization of Vancomycin-resistant Enterococcus species Frequently Isolated from Laboratory Mice

To determine the prevalence of drug resistant bacteria colonizing laboratory mice, we isolated and characterized vancomycin-resistant Enterococcus species (VRE) from commercially available mice. A total of 24 VRE isolates were obtained from 19 of 21 mouse strains supplied by 4 commercial breeding companies. Of these, 19 isolates of E. gallinarum and 5 isolates of E. casseliflavus possessing the vanC1 and vanC2/3 genes intrinsically, exhibited intermediate resistance to vancomycin respectively. In addition, these isolates also exhibited diverse resistant patterns to erythromycin, tetracycline, and ciprofloxacin, whereas the use of antibiotics had not been undertaken in mouse strains tested in this study. Although 6 virulence-associated genes (ace, asa, cylA, efaA, esp, and gelE) and secretion of gelatinase and hemolysin were not detected in all isolates, 23 of 24 isolates including the isolates of E. casselifalvus secreted ATP into culture supernatants. Since secretion of ATP by bacteria resident in the intestinal tract modulates the local immune responses, the prevalence of ATP-secreting VRE in mice therefore needs to be considered in animal experiments that alter the gut microflora by use of antibiotics

Phosphate-dependent luminal ATP metabolism regulates transcellular calcium transport in intestinal epithelial cells

Extracellular low phosphate strongly enhances intestinal calcium absorption independently of active vitamin D [1,25(OH)2D3] signaling, but the underlying mechanisms remain poorly characterized. To elucidate the phosphate-dependent regulation of calcium transport, we investigated part of the enteral environment that is involved in 1,25(OH)2D3-independent calcium absorption, which responds to dietary phosphate levels in mice that lack intestinal vitamin D receptor ( Vdr) activity. Impaired calcium absorption in intestinal Vdr-null mice was improved by dietary phosphate restriction. Accordingly, calcium transport in cultured intestinal epithelial cells was increased when the apical side was exposed to low phosphate levels (0.5 mM) compared with normal or high phosphate levels (1.0 or 5.0 mM, respectively). Mechanistically, low phosphate increased ATP in the apical side medium and allowed calcium entry into epithelial cells via the P2X7 purinoreceptor, which results in increased calcium transport. We found that luminal ATP was regulated by the release and degradation of ATP at the epithelium, and phosphate restriction increased ATP release from epithelial cells via connexin-43 hemichannels. Furthermore, ATP degradation by ectonucleotide pyrophosphatase-1 was reduced, which was caused by the reduction of the MAPK cascade. These findings indicate that luminal ATP metabolism regulates transcellular calcium transport in the intestine by an 1,25(OH)2D3-independent mechanism in response to dietary phosphate levels.-Uekawa, A., Yamanaka, H., Lieben, L., Kimira, Y., Uehara, M., Yamamoto, Y., Kato, S., Ito, K., Carmeliet, G., Masuyama, R. Phosphate-dependent luminal ATP metabolism regulates transcellular calcium transport in intestinal epithelial cells.status: publishe

Genetic regions affecting the replication and pathogenicity of dengue virus type 2.

Dengue is a mosquito-borne disease that has spread to over 100 countries. Its symptoms vary from the relatively mild acute febrile illness called dengue fever to the much more severe dengue shock syndrome. Dengue is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. There are four serotypes of DENV, i.e., DENV1 to DENV4, and each serotype is divided into distinct genotypes. Thailand is an endemic area where all four serotypes of DENV co-circulate. Genome sequencing of the DENV2 that was isolated in Thailand in 2016 and 2017 revealed the emergence of the Cosmopolitan genotype and its co-circulation with the Asian-I genotype. However, it was unclear whether different genotypes have different levels of viral replication and pathogenicity. Focus-forming assay (FFA) results showed that clinical isolates of these genotypes differed in focus size and proliferative capacity. Using circular polymerase extension reaction, we generated parental and chimeric viruses with swapped genes between these two DENV2 genotypes, and compared their focus sizes and infectivity titers using FFA. The results showed that the focus size was larger when the structural proteins and/or non-structural NS1-NS2B proteins were derived from the Cosmopolitan virus. The infectious titers were consistent with the focus sizes. Single-round infectious particle assay results confirmed that chimeric viruses with Cosmopolitan type structural proteins, particularly prM/E, had significantly increased luciferase activity. Replicon assay results showed that Cosmopolitan NS1-NS2B proteins had increased reporter gene expression levels. Furthermore, in interferon-receptor knock-out mice, viruses with Cosmopolitan structural and NS1-NS2B proteins had higher titers in the blood, and caused critical disease courses. These results suggested that differences in the sequences within the structural and NS1-NS2B proteins may be responsible for the differences in replication, pathogenicity, and infectivity between the Asian-I and Cosmopolitan viruses

Direct detection of the mercury-nitrogen bond in the thymine-Hg-II-thymine base-pair with Hg-199 NMR spectroscopy

One-bond 199Hg–15N J-coupling.</p

Evaluation of infectivity by a single-round infectious particle (SRIP) assay.

(A) Schematic representation of the reporter vector showing the position of the cytomegalovirus promoter (CMV), NanoLuc gene, 2A protein sequence of foot-and-mouth disease virus (FMDV2A) for self-excision, hepatitis delta virus ribozyme (HDV-RZ), and polyadenylation signal (pA). The structural protein-expressing plasmids (prME) used to generate SRIPs are also shown. Blue and red indicate the sequences derived from the Asian-I and Cosmopolitan viruses, respectively. (B, C) Luciferase activity of the SRIPs produced by the transfection of Lenti-X 293T cells with the reporter plasmid and structural protein-expressing plasmids as indicated. The multiple t test results with a statistically significant difference are indicated by asterisks (*:P < 0.05, **:P < 0.01). The means and standard deviation of triplicate samples are shown. Representative results of three independent experiments are shown.</p

Generation of DENV2 by the circular polymerase extension reaction (CPER) method.

(A) Schematic representation of the fragmentation of the whole dengue virus genome for the CPER method. In a previous publication by Setoh et al. (mSphere.2017; 2(3)) seven fragments were designed with an overlapping region of about 22 nt at the end. In the present study, five fragments were used, and each fragment was designed to have a 25-nt-overlapping region at the end. These fragments were mixed with a linker fragment containing the cytomegalovirus (CMV) promoter, hepatitis D virus ribozyme (HDVr), and a poly(A) tail (pA). (B) Comparison of the amounts of viral RNA in the culture supernatants at 6 days after the transfection of CPER products into BHK-21 cells. (C) Amounts of RNA in viruses propagated in C6/36 cells with the culture supernatants of transduced BHK-21 cells. Asian-I and Cosmopolitan viruses were infected at a multiplicity of infection of 0.01 copies/cell. We used NS5-C709A mutants as non-replicating negative controls.</p

Characteristics of the chimeric DENV2 between the Cosmopolitan and Asian-I genotypes.

(A) Schematic representation of 30 chimeric viruses with two different parental strains. The fragments derived from the Asian-I [A] genotype and Cosmopolitan [C] genotype are shown in blue and orange, respectively. AAAAA and CCCCC indicate the parental Asian-I and Cosmopolitan viruses, respectively. (B) The FFU of the generated recombinant viruses 3 days after the infection of Vero cells at a multiplicity of infection of 0.5 copies/cell. Viruses with titers equal to or lower than that of the parental Asian-I strain are shown in blue. Viruses with titers equal to or higher than that of the parental Cosmopolitan strain are shown in orange. Viruses with titers in between those of the two parental viruses are shown in green. The means and standard deviation of triplicate samples are shown. (C) Images of the foci of all recombinant viruses. The chimeric viruses are grouped according to the number of Cosmopolitan virus-derived fragments and whether the structural and NS1, NS2A, and NS2B proteins were derived from the Cosmopolitan or Asian-I viruses. Red CC and blue AA indicate the viruses with structural and NS1, NS2A, and NS2B proteins derived from the Cosmopolitan and Asian-I viruses, respectively.</p