Gata6 Promotes GLI3 Repressor Activities in the Limb

Gli3 is a major regulator of Hedgehog signaling during limb development. In the anterior mesenchyme, GLI3 is proteolytically processed into GLI3R, a truncated repressor form that inhibits Hedgehog signaling. Although numerous studies have identified mechanisms that regulate Gli3 function in vitro, it is not completely understood how Gli3 function is regulated in vivo. In this study, we show a novel mechanism of regulation of GLI3R activities in limb buds by Gata6, a member of the GATA transcription factor family. We show that conditional inactivation of Gata6 prior to limb outgrowth by the Tcre deleter causes preaxial polydactyly, the formation of an anterior extra digit, in hindlimbs. A recent study suggested that Gata6 represses Shh transcription in hindlimb buds. However, we found that ectopic Hedgehog signaling precedes ectopic Shh expression. In conjunction, we observed Gata6 and Gli3 genetically interact, and compound heterozygous mutants develop preaxial polydactyly without ectopic Shh expression, indicating an additional prior mechanism to prevent polydactyly. These results support the idea that Gata6 possesses dual roles during limb development: enhancement of Gli3 repressor function to repress Hedgehog signaling in the anterior limb bud, and negative regulation of Shh expression. Our in vitro and in vivo studies identified that GATA6 physically interacts with GLI3R to facilitate nuclear localization of GLI3R and repressor activities of GLI3R. Both the genetic and biochemical data elucidates a novel mechanism by Gata6 to regulate GLI3R activities in the anterior limb progenitor cells to prevent polydactyly and attain proper development of the mammalian autopod

A connecter-like factor, CacA, links RssB/RpoS and the CpxR/CpxA two-component system in Salmonella

BACKGROUND: Bacteria integrate numerous environmental stimuli when generating cellular responses. Increasing numbers of examples describe how one two-component system (TCS) responds to signals detected by the sensor of another TCS. However, the molecular mechanisms underlying this phenomenon remain poorly defined. RESULTS: Here, we report a connector-like factor that affects the activity of the CpxR/CpxA two-component system in Salmonella enterica serovar Typhimurium. We isolated a clone that induced the expression of a cpxP-lac gene fusion from a high-copy-number plasmid pool of random Salmonella genomic fragments. A 63-amino acid protein, CacA, was responsible for the CpxA/CpxR-dependent activation of the cpxP gene. The CpxR-activated genes cpxP and spy exhibited approximately 30% and 50% reductions in transcription, respectively, in a clean cacA deletion mutant strain in comparison to wild-type. From 33 response regulator (RR) deletion mutants, we identified that the RssB regulator represses cacA transcription. Substitution mutations in a conserved -10 region harboring the RNA polymerase recognition sequence, which is well conserved with a known RpoS -10 region consensus sequence, rendered the cacA promoter RpoS-independent. The CacA-mediated induction of cpxP transcription was affected in a trxA deletion mutant, which encodes thioredoxin 1, suggesting a role for cysteine thiol-disulfide exchange(s) in CacA-dependent Cpx activation. CONCLUSIONS: We identified CacA as an activator of the CpxR/CpxA system in the plasmid clone. We propose that CacA may integrate the regulatory status of RssB/RpoS into the CpxR/CpxA system. Future investigations are necessary to thoroughly elucidate how CacA activates the CpxR/CpxA system

Bioluminescence Microscopy: Design and Applications

Bioluminescence imaging by microscopy is performed using an ultra-low-light imaging camera. Although imaging devices such as sensor and camera have been greatly improved over time, such improvements have not been attained commercially which are available for microscopes now. We previously optimized the optical system of a microscope for bioluminescence imaging using a short-focal-length imaging lens and evaluated this system with a conventional color charge-coupled device camera. Here, we describe the concept of bioluminescence microscope design using a short-focal-length imaging lens and some representative applications, including intracellular calcium imaging, imaging of clock gene promoter assays, and three-dimensional reconstruction of Drosophila larva. This system facilitates the acquisition of bioluminescence images of single live cells using luciferase, which is similar to fluorescence microscopy using a fluorescent protein

Design of engineered active zymogen of microbial transglutaminase

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Negative-chirality order in S=1/2S=1/2 kagome antiferromagnet CdCu3_{3}(OH)6_{6}(NO3_{3})2⋅_{2}\cdot H2_{2}O

The neutron diffraction and nuclear magnetic resonance (NMR) measurements have been used to microscopically analyze the magnetic structure in the $S = 1/2$ kagome antiferromagnet CdCu$_{3}$(OH)$_{6}$(NO$_{3}$)$_{2}\cdot$H$_{2}$O. Below the magnetic ordering temperature $T_N\simeq 4$ K, magnetic Bragg reflections at (110) and (100) were found in the neutron diffraction pattern, which suggests a $q=0$ magnetic structure. Furthermore, the vector spin chirality for the $q=0$ structure was successfully identified from the internal field direction obtained by the $^{14}$N-NMR measurement. Our findings point to a chirality-ordered magnetic structure with negative vector chirality and $\langle 100 \rangle$ anisotropy.Comment: 6 pages, 5 figure

Metformin directly binds the alarmin HMGB1 and inhibits its proinflammatory activity

Metformin is the first-line drug in the treatment of type 2 diabetes. In addition to its hypoglycemic effect, metformin has an anti-inflammatory function, but the precise mechanism promoting this activity remains unclear. High mobility group box 1 (HMGB1) is an alarmin that is released from necrotic cells and induces inflammatory responses by its cytokine-like activity and is, therefore, a target of anti-inflammatory therapies. Here we identified HMGB1 as a novel metformin-binding protein by affinity purification using a biotinylated metformin analogue. Metformin directly bound to the C-terminal acidic tail of HMGB1. Both in vitro and in vivo, metformin inhibited inflammatory responses induced by full-length HMGB1 but not by HMGB1 lacking the acidic tail. In an acetaminophen-induced acute liver injury model in which HMGB1 released from injured cells exacerbates the initial injury, metformin effectively reduced liver injury and had no additional inhibitory effects when the extracellular HMGB1 was blocked by anti-HMGB1-neutralizing antibody. In summary, we report for the first time that metformin suppresses inflammation by inhibiting the extracellular activity of HMGB1. Because HMGB1 plays a major role in inflammation, our results suggest possible new ways to manage HMGB1-induced inflammation

Cleavage of Toll-Like Receptor 9 Ectodomain Is Required for In Vivo Responses to Single Strand DNA

Mouse toll-like receptor 9 (TLR9) is an endosomal sensor for single-stranded DNA. TLR9 is transported from the endoplasmic reticulum to endolysosomes by a multiple transmembrane protein Unc93 homolog B1, and proteolytically cleaved at its ectodomain. The structure of TLR9 and its biochemical analyses have shown that the proteolytic cleavage of TLR9 ectodomain enables TLR9-dimerization and TLR9 activation. However, the requirement of TLR9 cleavage in vivo has not been studied. We here show that the 13 amino acids deletion at the cleavage site made TLR9 resistant to proteolytic cleavage. The deletion mutation in the Tlr9 gene impaired TLR9-dependent cytokine production in conventional dendritic cells from the mutant mice. Not only in vitro, in vivo production of inflammatory cytokines (TNF-α and IL-12p40), chemokine (CCR5/RANTES), and type I interferon (IFN-α) induced by administration of TLR9 ligand was also impaired. These results demonstrate that the TLR9 cleavage is required for TLR9 responses in vivo

Skin Tube Reconstruction for Esophageal Defects due to Postoperative Complications: Applying a skin flap in esophageal resection and reconstruction

Numerous improvements and advances in operational methods and techniques have occurred in the area of reconstruction for esophageal cancer. Patients with thoracic esophageal cancer who have previously had a gastrectomy usually undergo reconstruction using the colon and small intestine. The incidence of organ necrosis is not necessarily low after reconstruction with those organs. Generally, the main types of skin flaps and musculocutaneous flaps used for cervical and other esophageal reconstructions are deltopectoral (DP) flaps, pedicled musculocutaneous latissimus dorsi flaps and free anteriolateral thigh flaps. This kind of reconstruction is low invasive, relatively simple, and also causes very few fatal post-operative complications. Therefore, it is considered to be an effective reconstruction choice for the following types of patients: poor risk patients, patients whose gastrointestinal (GI) tract cannot be used for their reconstruction for some reason, and patients having a second reconstruction due to complications caused by organ necrosis after their first GI tract reconstruction