Real-time observation of a metal complex-driven reaction intermediate using a porous protein crystal and serial femtosecond crystallography.

Determining short-lived intermediate structures in chemical reactions is challenging. Although ultrafast spectroscopic methods can detect the formation of transient intermediates, real-space structures cannot be determined directly from such studies. Time-resolved serial femtosecond crystallography (TR-SFX) has recently proven to be a powerful method for capturing molecular changes in proteins on femtosecond timescales. However, the methodology has been mostly applied to natural proteins/enzymes and limited to reactions promoted by synthetic molecules due to structure determination challenges. This work demonstrates the applicability of TR-SFX for investigations of chemical reaction mechanisms of synthetic metal complexes. We fix a light-induced CO-releasing Mn(CO)3 reaction center in porous hen egg white lysozyme (HEWL) microcrystals. By controlling light exposure and time, we capture the real-time formation of Mn-carbonyl intermediates during the CO release reaction. The asymmetric protein environment is found to influence the order of CO release. The experimentally-observed reaction path agrees with quantum mechanical calculations. Therefore, our demonstration offers a new approach to visualize atomic-level reactions of small molecules using TR-SFX with real-space structure determination. This advance holds the potential to facilitate design of artificial metalloenzymes with precise mechanisms, empowering design, control and development of innovative reactions.

Displaying a Protein Cage on a Protein Crystal by In-Cell Crystal Engineering.

The development of solid biomaterials has rapidly progressed in recent years in applications in bionanotechnology. The immobilization of proteins, such as enzymes, within protein crystals is being used to develop solid catalysts and functionalized materials. However, an efficient method for encapsulating protein assemblies has not yet been established. This work presents a novel approach to displaying protein cages onto a crystalline protein scaffold using in-cell protein crystal engineering. The polyhedra crystal (PhC) scaffold, which displays a ferritin cage, was produced by coexpression of polyhedrin monomer (PhM) and H1-ferritin (H1-Fr) monomer in Escherichia coli. The H1-tag is derived from the H1-helix of PhM. Our technique represents a unique strategy for immobilizing protein assemblies onto in-cell protein crystals and is expected to contribute to various applications in bionanotechnology.

Percutaneous trans-bladder drainage of a pelvic abscess caused by sigmoid diverticulitis: Two case reports.

Sigmoid diverticulitis can potentially lead to the development of pelvic abscesses. Mortality rate from intra-abdominal abscesses is as high as 35%, and patients with diverticulitis complicated by an abscess are at a high risk of recurrent diverticulitis. Recently, image-guided drainage has been performed using computed tomography or ultrasonography for the treatment of pelvic abscesses. Standard radiological pelvic abscess image-guided drainage methods consist of transabdominal, transgluteal, or cranio-caudal puncture approaches. However, these standard approaches have technical limitations due to intrapelvic organ structures. Therefore, we performed image-guided drainage using a percutaneous trans-bladder approach with a simpler 1-step puncture method and a less invasive 7-Fr drainage catheter in 2 cases of a 72-year-old female and a 53-year-old female with relapsed pelvic abscesses complicated by sigmoid diverticulitis due to difficulties involving the standard approach. The abscesses in both cases disappeared on follow-up computed tomography scans, demonstrating no pelvic abscess recurrence. Our results showed that trans-bladder image-guided drainage is an effective alternative method for treating pelvic abscesses.

Direct measurement of the evolution of magnetism and superconductivity toward the quantum critical point.

Nontrivial quantum states can be realized in the vicinity of the quantum critical point (QCP) in many strongly correlated electron systems. In particular, an emergence of unconventional superconductivity around the QCP strongly suggests that the quantum critical fluctuations play a central role in the superconducting pairing mechanism. However, a clear signature of the direct coupling between the superconducting pairing states and the quantum criticality has not yet been elucidated by the microscopic probes. Herein, we present muon spin rotation/relaxation and neutron diffraction measurements in the superconducting dome of CeCo(In1 - xZnx)5. It was found that a magnetically ordered state develops at x≥ 0.03, coexisting with the superconductivity. The magnitude of the ordered magnetic moment is continuously reduced with decreasing x, and it disappears below x∼ 0.03, indicating a second-order phase transition and the presence of the QCP at this critical Zn concentration. Furthermore, the magnetic penetration depth diverges toward the QCP. These facts provide evidence for the intimate coupling between quantum criticality and Cooper pairing.

Percutaneous transhepatic obliteration of a large portosystemic shunt associated with hepatic encephalopathy using a technique of n-butyl-2-cyanoacrylate injection inside hydrogel-coated coils: A case report.

Portosystemic shunts with cirrhosis may lead to hepatic encephalopathy (HE), which is often pharmacotherapy-resistant. We report a case of a 66-year-old female patient diagnosed with alcoholic cirrhosis and uncontrolled HE. She underwent percutaneous transhepatic obliteration (PTO) for treatment of a large portosystemic shunt from the left and right gastric veins to the azygos vein. We embolized the target veins using hydro-coated coils and filled them with n-butyl-2-cyanoacrylate (NBCA), leading to firmed obstruction of the large portosystemic shunt without NBCA migration, thus reducing the number of coils required. The HE symptoms improved after PTO and could thereafter be controlled with conservative therapy. Our results showed that PTO using an NBCA injection inside hydrogel-coated coils for a large portosystemic shunt associated with HE is effective and safe.

Amphotericin B assembles into seven-molecule ion channels: An NMR and molecular dynamics study.

Amphotericin B, an antifungal drug with a long history of use, forms fungicidal ion-permeable channels across cell membranes. Using solid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we experimentally elucidated the three-dimensional structure of the molecular assemblies formed by this drug in membranes in the presence of the fungal sterol ergosterol. A stable assembly consisting of seven drug molecules was observed to form an ion conductive channel. The structure is somewhat similar to the upper half of the barrel-stave model proposed in the 1970s but substantially different in the number of molecules and in their arrangement. The present structure explains many previous findings, including structure-activity relationships of the drug, which will be useful for improving drug efficacy and reducing adverse effects.

Simultaneous generation of the second- and third-order OAM modes by using a high-order helical long-period fiber grating.

An all-fiber orbital angular momentum (OAM) mode generator enabling simultaneous generation of the second- and the third-order OAM modes with conversion efficiencies larger than 95% has been proposed and experimentally demonstrated, which is realized by using a high-order helical long-period fiber grating (HLPG) written in a thinned four-mode fiber. This is the first time, to the best of our knowledge, that two such OAM modes have been simultaneously obtained at wavelengths ranging from 1450 to 1620 nm by using only one fiber component, i.e., the HLPG. The proposed method provides a new way to simultaneously generate different orders of the OAM modes, which would further expand the OAM's applications to the fields of the optical tweezers, microscopy, and fiber communication, etc.

The Perpendicular Orientation of Amphotericin B Methyl Ester in Hydrated Lipid Bilayers Supports the Barrel-Stave Model.

The clinically important antibiotic amphotericin B (AmB) is a membrane-active natural product that targets membrane sterol. The antimicrobial activity of AmB is generally attributed to its membrane permeabilization, which occurs when a pore is formed across a lipid bilayer. In this study, the molecular orientation of AmB was investigated using solid-state nuclear magnetic resonance (NMR) to better understand the mechanism of antifungal activity. The methyl ester of AmB (AME) labeled with NMR isotopes, d3-AME, and its fluorinated and/or 13C-labeled derivatives were prepared. All of the AmB derivatives showed similar membrane-disrupting activities and ultraviolet spectra in phospholipid liposomes, suggesting that their molecular assemblies in membranes closely mimic those of AmB. Solid-state 2H NMR measurements of d3-AME in a hydrated membrane showed that the mobility of AME molecules depends on concentration and temperature. At a 1:5:45 AME:Erg:dimyristoylphosphatidylcholine ratio, AME became sufficiently mobilized to observe the motional averaging of quadrupole coupling. On the basis of the rotational averaging effect of 19F chemical shift anisotropy, 2H quadrupolar splitting, and 13C-19F dipolar coupling of 14ß-F-AMEs, we deduced that the molecular axis of AME is predominantly parallel to the normal of a lipid bilayer. This result supports the barrel-stave model as a molecular assembly of AmB in membranes.

Hypoxia-responsive transgene expression system using RTP801 promoter and synthetic transactivator fused with oxygen-dependent degradation domain.

Precise control of gene expression using an artificial gene circuit is a major challenge in the application of synthetic biology. Here, we designed a hypoxia-responsive transgene expression system by combining a hypoxia-inducible RTP801 promoter and a tetracycline-responsive transactivator fused with an oxygen-dependent degradation domain (TA-ODD). The reporter gene expression was highly induced by hypoxia when a transactivator-expression plasmid, pRTP801/TA-ODD, harboring a TA-ODD gene driven by the RTP801 promoter, was cotransfected with a reporter plasmid, pTRE/EGFP, harboring an EGFP gene controlled under the transactivator-responsive promoter. A stable cell line into which the expression units RTP801/TA-ODD and TRE/EGFP had been introduced responded to hypoxia and expressed the reporter gene in an oxygen-concentration-dependent manner. Moreover, the cells demonstrated potential as sensors to detect hypoxic conditions in a three-dimensional tissue culture in vitro. These results indicate that the hypoxia-responsive transgene expression system is useful for constructing cell-based hypoxia detection systems.

Stair climbing/descending exercise for a short time decreases blood glucose levels after a meal in people with type 2 diabetes.

OBJECTIVE: We examined whether stair climbing-descending exercise (ST-EX), a convenient method to increase physical activity in daily life, for a short period would acutely improve the postprandial blood glucose (BG) response in people with type 2 diabetes (T2D). METHODS: 16 people with T2D (age 65.4±1.1 years) participated in 2 separate sessions. After an overnight fast, each participant consumed a test meal and then kept resting for 180 min, except when performing each 3 min bout of ST-EX at 60 and 120 min after the meal (ST-EX session), or kept resting for 180 min (REST session). ST-EX comprised 6 continuous repetitions of climbing to the second floor (21 steps) at a rate of 80-110 steps/min followed by walking down slowly to the first floor at a free step rate. RESULTS: The BG at 60 min after the meal during the ST-EX session (immediately before the first ST-EX) did not differ from that during the REST session, but analysis of variance revealed a significant interaction between time and treatment (p<0.01). The BG at 150 min after the meal (30 min after the second ST-EX) was significantly lower than that during the REST session (p<0.01). The area under the curve was also 18% lower during the ST-EX session than during the REST session (p<0.05). The heart rate and blood lactate levels indicated that the actual intensity of ST-EX was 'hard'. In contrast, the rating of perceived exertion (RPE) indicated that the overall intensity of ST-EX was 'moderate' because of decreased RPE scores during descent. CONCLUSIONS: The present findings suggest that performing 3 min ST-EX 60 and 120 min after a meal may be a useful strategy to accelerate the decrease in postprandial BG levels in people with T2D.

DNA damage-responsive transgene expression mediated by the p53 promoter with transcriptional amplification.

We constructed a DNA damage-responsive transgene expression system mediated by the p53 promoter. We incorporated a transactivation system to generate transcriptional amplification via a positive feedback loop. Higher levels of DNA damage-responsive transgene expression were observed when transactivation was active.

Exportin 4 interacts with Sox9 through the HMG Box and inhibits the DNA binding of Sox9.

Sox9 is a transcription factor that is required for tissue development in mammals. In general, such transcription factors require co-regulators for precise temporal and spatial control of the activation and inactivation of the numerous genes necessary for precise development during embryogenesis. Here we identify a new Sox9 co-regulator: Using affinity chromatography with immobilized Sox9 protein, we identified exportin 4 (Exp4) as an interacting protein of Sox9 in human cultured cells. Interaction between endogenous Exp4 and Sox9 proteins was confirmed in the human osteosarcoma U2OS cells by immunoprecipitation experiments using anti-Sox9 antibody. siRNA depletion of Exp4 enhanced transcription of Sox9 target genes in U2OS cells, but did not affect nuclear localization of Sox9. These results suggest that Exp4 regulates Sox9 activity in the nucleus. Furthermore we found that the HMG box of Sox9 was responsible for binding to Exp4, and the HMG box was required for suppression of Sox9-mediated transcription. This contrasts with the known Sox9 co-regulators which bind to its transcriptional activation domain. Chromatin immunoprecipitation analyses revealed that Exp4 prevents Sox9 binding to the enhancers of its target genes. These results demonstrate that Exp4 acts as a Sox9 co-regulator that directly regulates binding of Sox9 to its target genes.

Liver receptor homologue-1 (LRH-1) activates the promoter of brain aromatase (cyp19a2) in a teleost fish, the medaka, Oryzias latipes.

The medaka, Oryzias latipes, like other fish, have two distinct aromatase genes, the ovarian (cyp19a1) and brain (cyp19a2) forms. We previously reported that Ad4BP/SF-1, a member of the NR5A subfamily, plays an important role in the regulation of cyp19a1 expression in medaka ovarian follicles during vitellogenesis. In the present study, we investigated whether liver receptor homologue-1 (LRH-1), another NR5A subfamily member, is involved in the regulation of cyp19a2 expression in the medaka brain. In situ hybridization analysis revealed that LRH-1 was expressed in the hypothalamus, where it colocalized with aromatase (cyp19a2). We then showed by transient transfection assays that LRH-1 was able to increase expression of a cyp19a2 reporter gene in various mammalian cell lines, and that mutation of a putative LRH-1 binding site within the cyp19a2 promoter abolished this effect. Taken together, these findings suggest that LRH-1 plays a role in regulating cyp19a2 expression in the medaka brain. This is the first to demonstrate in vitro the activation of brain aromatase by LRH-1 in the vertebrate brain.

Role of the LXXLL-motif and activation function 2 domain in subcellular localization of Dax-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1).

Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (Dax-1, NR0B1) is an orphan nuclear receptor that represses transcription by Ad4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1, NR5A1). Observations on human diseases and the phenotypes of mice, in which the corresponding genes have been disrupted, have elucidated essential roles of these two nuclear receptors in differentiation of steroidogenic tissues. However, little is known about how the functions of these factors are regulated. Here we have examined their subcellular localization and have clarified the molecular mechanisms regulating subcellular localization of Dax-1. Prompted by the finding that nuclear localization of Dax-1 correlates with the presence of Ad4BP/SF-1 in the early stages of pituitary development, we have tested the possibility that interaction between the two factors is essential for the nuclear localization of Dax-1. In vitro studies with cultured cells demonstrated that an interaction involving the LXXLL motifs in the N-terminal repeat region of Dax-1 plays a key role in its subcellular localization. In addition, we found that a mutant form of DAX-1 (L466R), from a patient with adrenal hypoplasia congenita, was defective in nuclear localization in spite of having an intact N terminus. Taken together, the results reveal that the subcellular localization of Dax-1 is influenced by the presence of Ad4BP/SF-1, and that two regions of Dax-1 have important roles for this process.

LXXLL-related motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1.

The orphan receptor Ad4BP/SF-1 (NR5A1) is a constitutive activator, and its activity is repressed by another orphan receptor, Dax-1 (NR0B1). In the present study, we investigated the molecular mechanisms underlying this repression by Dax-1. Yeast two-hybrid and transient-transfection assays confirmed the necessity of three LXXLL-related motifs in Dax-1 for interaction with and repression of Ad4BP/SF-1. In vitro pull-down experiments confirmed that Dax-1 interacts with Ad4BP/SF-1 and also with LRH-1 (NR5A2). The target specificity of the LXXLL-related motifs was indicated by the observations that Ad4BP/SF-1, ERalpha (NR3A1), LRH-1, ERR2 (NR3B2), and fly FTZ-F1 (NR5A3) interacted through their ligand binding domains with all the LXXLL-related motifs in Dax-1 whereas HNF4 (NR2A1) and RORalpha (NR1F1) did not. Transcriptional activities of the receptors whose DNA binding domains (DBDs) were replaced by the GAL4 DBD were repressed by Dax-1 to various levels, which correlated with the strength of interaction. Amino acid substitutions revealed that Ad4BP/SF-1 and LRH-1 preferentially interact with L(+1)XXLL-related motifs containing serine, tyrosine, serine, and threonine at positions -2, +2, +3, and +6, respectively. Taken together, our results indicate that the specificities of LXXLL-related motifs in Dax-1 based on their amino acid sequences play an important role in regulation of orphan receptors.

Molecular cloning of DAX1 and SHP cDNAs and their expression patterns in the Nile tilapia, Oreochromis niloticus.

Piscine DAX1 and SHP cDNAs with an open reading frame encoding 296 and 258 amino acid residues, respectively, as well as SHP partial gene fragment, were cloned from Nile tilapia. Phylogenetic analyses of DAX1s, SHPs, and homologous EST fragments indicate that DAX1 and SHP are conserved in gene structure and are present throughout vertebrates. A single band of approximately 1.4kb for DAX1 and of approximately 1.2kb for SHP was detected in the Northern blot analysis. Tissue distribution analysis by RT-PCR showed that fish DAX1 and SHP mRNAs are widely expressed in adult tissues, with the most abundant expression in gonads and liver, respectively. DAX1 and SHP were also detected in gonads of both sexes at 5-90 days after hatching (dah). However, the expression of DAX1 is weak at 5 and 10dah and then significantly up-regulated between 10 and 15dah, whereas the expression of SHP is moderate and consistent during the ontogeny.

Concerted regulation of gonad differentiation by transcription factors and growth factors.

It is well known that signals from growth factors regulate gene transcription thus initiating certain steps of cellular and tissue differentiation during development. In gonad differentiation several transcription factors have been identified as the genes underlying human diseases displaying gonadal defects and as the genes necessary for gonad differentiation as demonstrated by gene disruption studies. In addition, one of the growth factors, WNT4, is known to be involved in gonadal differentiation. However, it remains unclear which gene is directly downstream of the WNT4 signal. We have recently demonstrated that Dax1 (NR0B1) gene transcription is significantly up-regulated by the presence of SF1 (NR5A1). Functional analysis showed that DAX1 acts as a repressor against SF1 through direct interaction between the repeated sequences at the N-terminus of DAX1 and a ligand-binding domain of SF1. Considering that the expressions of these factors during gonad differentiation show a sexually dimorphic pattern, it is likely that the Dax1 gene transcription is up-regulated by WNT4 signal and thereafter DAX1 suppresses the genes downstream of SF1 such as Amh and steroidogenic genes in female gonads.