Functional rejuvenation of aged neural stem cells by Plagl2 and anti-Dyrk1a activity.

The regenerative potential of neural stem cells (NSCs) declines during aging, leading to cognitive dysfunctions. This decline involves up-regulation of senescence-associated genes, but inactivation of such genes failed to reverse aging of hippocampal NSCs. Because many genes are up-regulated or down-regulated during aging, manipulation of single genes would be insufficient to reverse aging. Here we searched for a gene combination that can rejuvenate NSCs in the aged mouse brain from nuclear factors differentially expressed between embryonic and adult NSCs and their modulators. We found that a combination of inducing the zinc finger transcription factor gene Plagl2 and inhibiting Dyrk1a, a gene associated with Down syndrome (a genetic disorder known to accelerate aging), rejuvenated aged hippocampal NSCs, which already lost proliferative and neurogenic potential. Such rejuvenated NSCs proliferated and produced new neurons continuously at the level observed in juvenile hippocampi, leading to improved cognition. Epigenome, transcriptome, and live-imaging analyses indicated that this gene combination induces up-regulation of embryo-associated genes and down-regulation of age-associated genes by changing their chromatin accessibility, thereby rejuvenating aged dormant NSCs to function like juvenile active NSCs. Thus, aging of NSCs can be reversed to induce functional neurogenesis continuously, offering a way to treat age-related neurological disorders.

Enhancement of Vivid-based photo-activatable Gal4 transcription factor in mammalian cells.

The Gal4/UAS system is a versatile tool to manipulate exogenous gene expression of cells spatially and temporally in many model organisms. Many variations of light-controllable Gal4/UAS system are now available, following the development of photo-activatable (PA) molecular switches and integration of these tools. However, many PA-Gal4 transcription factors have undesired background transcription activities even in dark conditions, and this severely attenuates reliable light-controlled gene expression. Therefore, it is important to develop reliable PA-Gal4 transcription factors with robust light-induced gene expression and limited background activity. By optimization of synthetic PA-Gal4 transcription factors, we have validated configurations of Gal4 DNA biding domain, transcription activation domain and blue light-dependent dimer formation molecule Vivid (VVD), and applied types of transcription activation domains to develop a new PA-Gal4 transcription factor we have named eGAV (enhanced Gal4-VVD transcription factor). Background activity of eGAV in dark conditions was significantly lower than that of hGAVPO, a commonly used PA-Gal4 transcription factor, and maximum light-induced gene expression levels were also improved. Light-controlled gene expression was verified in cultured HEK293T cells with plasmid-transient transfections, and in mouse EpH4 cells with lentivirus vector-mediated transduction. Furthermore, light-controlled eGAV-mediated transcription was confirmed in transfected neural stem cells and progenitors in developing and adult mouse brain and chick spinal cord, and in adult mouse hepatocytes, demonstrating that eGAV can be applied to a wide range of experimental systems and model organisms.Key words: optogenetics, Gal4/UAS system, transcription, gene expression, Vivid.

Separate optogenetic manipulation of Nerve/glial antigen 2 (NG2) glia and mural cells using the NG2 promoter.

Nerve/glial antigen 2 (NG2) is a protein marker of NG2 glia and mural cells, and NG2 promoter activity is utilized to target these cells. However, the NG2 promoter cannot target NG2 glia and mural cells separately. This has been an obstacle for NG2 glia-specific manipulation. Here, we developed transgenic mice in which either cell type can be targeted using the NG2 promoter. We selected a tetracycline-controllable gene induction system for cell type-specific transgene expression, and generated NG2-tetracycline transactivator (tTA) transgenic lines. We crossed tTA lines with the tetO-ChR2 (channelrhodopsin-2)-EYFP line to characterize tTA-dependent transgene induction. We isolated two unique NG2-tTA mouse lines: one that induced ChR2-EYFP only in mural cells, likely due to the chromosomal position effect of NG2-tTA insertion, and the other that induced it in both cell types. We then applied a Cre-mediated set-subtraction strategy to the latter case and eliminated ChR2-EYFP from mural cells, resulting in NG2 glia-specific transgene induction. We further demonstrated that tTA-dependent ChR2 expression could manipulate cell function. Optogenetic mural cell activation decreased cerebral blood flow, as previously reported, indicating that tTA-mediated ChR2 expression was sufficient to impact cellular function. ChR2-mediated depolarization was observed in NG2 glia in acute hippocampal slices. In addition, ChR2-mediated depolarization of NG2 glia inhibited their proliferation but promoted their differentiation in juvenile mice. Since the tTA-tetO combination is expandable, the mural cell-specific NG2-tTA line and the NG2 glia-specific NG2-tTA line will permit us to conduct observational and manipulation studies to examine in vivo function of these cells separately.

Splenic abscess diagnosed following relapsing sterile peritonitis in a peritoneal dialysis patient: A case report with literature review.

Peritoneal dialysis (PD)-related peritonitis is sometimes complicated with other infections; however, few cases of splenic abscess have been reported. We present the case of a 64-year-old PD patient with complicated splenic abscesses diagnosed following relapsing sterile peritonitis. After PD induction, he presented with turbid peritoneal fluid and was diagnosed with PD-related peritonitis. A plain abdominal computed tomography (CT) did not reveal any intra-abdominal focus of infection. After empiric intravenous antibiotics, the peritoneal dialysate was initially cleared, with a decrease in dialysate white blood cells (WBC) to 20/µL. However, WBC and C-reactive protein (CRP) levels remained elevated. A contrast-enhanced abdominal CT showed two areas of low-density fluid with no enhancement in a mildly enlarged spleen, making it difficult to distinguish abscesses from cysts. Due to relapsing sterile peritonitis, we performed an abdominal ultrasonography, and suspected splenic abscesses due to rapid increase in size. Repeated imaging tests were useful in establishing a diagnosis of splenic abscesses. Considering the persistent elevation of WBC and CRP levels, imaging findings, and episodes of relapsing peritonitis, we comprehensively formed the diagnosis, and performed a splenectomy as a rescue therapy. We should consider the possibility of other infectious foci with persistent inflammation after resolving PD-related peritonitis.

Vitamin D receptor is overexpressed in the duodenum of patients with irritable bowel syndrome.

BACKGROUND AND AIM: Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders, and bile acids are thought to be associated with the pathogenesis of IBS. Bile acid receptors are expressed on intestinal epithelial cells. However, no study has assessed bile acid receptor proteins in IBS. Therefore, we examined the intestinal mucosal expression of bile acid receptors in patients with IBS. METHODS: Intestinal biopsies were performed in patients with IBS and controls. Mast cells, vitamin D receptor (VDR), and somatostatin were stained with specific antibodies. Levels of VDR, farnesoid X receptor (FXR), takeda-G-protein-receptor-5 (TGR5), claudins, and transient-receptor-potential-cation-channel-subfamily-V-member 6 (TRPV6) were assessed by western blotting. RESULTS: 3Mast cell counts in the second part of the duodenum were significantly higher in patients with IBS than in controls. VDR protein levels were significantly elevated in the duodenum and terminal ileum of patients with IBS compared with controls, although this difference was not seen in the cecum or rectum. FXR and TGR5 protein levels did not differ in any part of the intestine. VDR-positive cryptal epithelia in IBS were distributed not only at basal crypt but also along the upper part of the basal crypt epithelial cells. In contrast, the pattern of gut somatostatin-positive cells, claudins, and TRPV6 levels did not differ. CONCLUSIONS: The number of mast cells in the duodenum was significantly increased, and the protein expression levels of VDR, but not those of FXR or TGR5, were elevated in the duodenal epithelial crypt in patients with IBS.

Glutamine Blocks Interleukin-13-Induced Intestinal Epithelial Barrier Dysfunction.

INTRODUCTION: Impaired intestinal epithelial barrier function is a hallmark of a variety of pathological conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). IBD patients with IBS-like symptoms show higher interleukin-13 (IL-13) serum levels and poor psychological well-being. Supplementary glutamine reduced the daily bowel movement frequency, improved the stool form, and normalized intestinal hyperpermeability. This study was aimed at assessing the effects of IL-13 and supplementary glutamine on human intestinal epithelial function in vitro. METHODS: Caco-2 cells were grown on TranswellTM inserts. -IL-13 was added to the basolateral compartment, and transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) labeled-dextran permeability measured. Effects of glutamine or the phosphatidylinositol-3-kinase inhibitor LY294002 were assessed. Involvement of tight junction proteins was assessed using Western blotting and immunofluorescence staining. RESULTS: IL-13 significantly decreased TEER and increased FITC labeled-dextran epithelial permeability. IL-13 stimulation decreased the claudin-1 expression and increased the claudin-2 expression. Glutamine alleviated IL-13-induced decrease of TEER and increase of FITC labeled-dextran permeability. Further, the phosphatidylinositol-3-kinase inhibitor showed this alleviating effect while the signal transducer and activator of transcription 6 inhibitor did not. CONCLUSIONS: IL-13 induced barrier integrity impairment by decreasing claudin-1 and increasing claudin-2. Glutamine alleviated IL-13-induced barrier dysfunction by increasing claudin-1 expression, via disruption of the phosphatidylinositol-3-kinase/Akt signaling pathway.

Specification of spatial identities of cerebellar neuron progenitors by ptf1a and atoh1 for proper production of GABAergic and glutamatergic neurons.

In the cerebellum, the bHLH transcription factors Ptf1a and Atoh1 are expressed in distinct neuroepithelial regions, the ventricular zone (VZ) and the rhombic lip (RL), and are required for producing GABAergic and glutamatergic neurons, respectively. However, it is unclear whether Ptf1a or Atoh1 is sufficient for specifying GABAergic or glutamatergic neuronal fates. To test this, we generated two novel knock-in mouse lines, Ptf1a(Atoh1) and Atoh1(Ptf1a), that are designed to express Atoh1 and Ptf1a ectopically in the VZ and RL, respectively. In Ptf1a(Atoh1) embryos, ectopically Atoh1-expressing VZ cells produced glutamatergic neurons, including granule cells and deep cerebellar nuclei neurons. Correspondingly, in Atoh1(Ptf1a) animals, ectopically Ptf1a-expressing RL cells produced GABAergic populations, such as Purkinje cells and GABAergic interneurons. Consistent results were also obtained from in utero electroporation of Ptf1a or Atoh1 into embryonic cerebella, suggesting that Ptf1a and Atoh1 are essential and sufficient for GABAergic versus glutamatergic specification in the neuroepithelium. Furthermore, birthdating analyses with BrdU in the knock-in mice or with electroporation studies showed that ectopically produced fate-changed neuronal types were generated at temporal schedules closely simulating those of the wild-type RL and VZ, suggesting that the VZ and RL share common temporal information. Observations of knock-in brains as well as electroporated brains revealed that Ptf1a and Atoh1 mutually negatively regulate their expression, probably contributing to formation of non-overlapping neuroepithelial domains. These findings suggest that Ptf1a and Atoh1 specify spatial identities of cerebellar neuron progenitors in the neuroepithelium, leading to appropriate production of GABAergic and glutamatergic neurons, respectively.

Two Cases of Children With Black Hairy Tongue and Tooth Discoloration Caused by Antibacterial Agents.

Black hairy tongue (BHT) is a lesion in which the filiform papillae of the tongue are significantly extended by hyperkeratosis, thereby giving the tongue a hairy appearance. Here, we report two rare cases of children with BHT and tooth discoloration caused by antimicrobial agents. Case 1: A four-year-old female patient received intravenous linezolid after spinal surgery, and BHT developed on day eight of treatment. Subsequently, the patient developed teeth discoloration. Linezolid was continually administered for 50 days, and BHT and teeth discoloration improved 10 days after the end of linezolid treatment. Case 2: A two-year-old male patient with a brain abscess received intravenous meropenem and vancomycin. On the fourth day of treatment, BHT developed, and teeth discoloration was subsequently observed. Antibiotic therapy was continued for 82 days, and BHT and tooth discoloration improved 20 days after the treatment was discontinued.

Health state utility estimation of Mycobacterium avium complex pulmonary disease using a time trade-off approach.

AIMS: To obtain appropriate health state utility values for cost-effectiveness analyses of new Mycobacterium avium complex pulmonary disease (MAC-PD) treatments. The impact of MAC-PD severity and symptoms on quality of life (QoL) also were quantified. METHODS: A questionnaire describing four health states, MAC-positive severe, MAC-positive moderate, MAC-positive mild, and MAC-negative, was developed based on St. George's Respiratory Questionnaire (SGRQ) Symptom and Activity scores from the CONVERT trial. The time trade-off (TTO) method with ping-pong titration procedure was used to estimate health state utilities. Regression analyses assessed the impacts of covariates. RESULTS: Of 319 Japanese adults (49.8% female, mean age 44.8 years), mean (95% CI) health state utility scores (MAC-positive severe, MAC-positive moderate, MAC-positive mild, and MAC-negative) were 0.252 (0.194-0.310), 0.535 (0.488-0.582), 0.816 (0.793-0.839), and 0.881 (0.866-0.896), respectively. MAC-negative state utility scores were significantly higher than MAC-positive severe (mean difference [95% CI], 0.629 [0.574-0.684]), MAC-positive moderate (0.346 [0.304-0.389]), and MAC-positive mild (0.065 [0.048-0.082]) scores (p < .001 each). Most participants would trade survival duration to avoid MAC-positive states (97.5% to avoid MAC-positive severe; 88.7% MAC-positive moderate; 61.4% MAC-positive mild). Regression analyses to investigate the impact of background characteristics showed similar utility differences between health states when not adjusted for covariates. LIMITATIONS: Some participant demographics differed from the general population; however, this did not impact utility differences among health states as regression analyses adjusting for demographics did not affect these differences. Similar investigations are needed among patients with MAC-PD and in other countries. CONCLUSIONS: This study evaluating the impact of MAC-PD on utilities using the TTO method demonstrates that differences in utilities are dependent on the severity of respiratory symptoms and their impacts on daily activities and QoL. These results could contribute to a better quantification of the value of MAC-PD treatments and improve assessments of cost-effectiveness.

Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses.

Nonfibrillar assemblies of amyloid ß-protein (Aß) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aß labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aß assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aß(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aß assembly steps at which inhibition may be beneficial.

A molecular MRI probe to detect treatment of cardiac apoptosis in vivo.

Cell death by apoptosis is critical in myocardial diseases, and noninvasive detection of early, reversible apoptosis might be useful clinically. Exogenous Annexin-V (ANX) protein binds membrane phosphatidylserine, which is externalized in early apoptosis. A molecular MRI probe was constructed with superparamagnetic iron oxide (SPIO) conjugated to recombinant human ANX (ANX-SPIO). Apoptosis was induced with doxorubicin, a cardiotoxic cancer drug, in culture in neonatal rat ventricular myocytes, cardiac fibroblasts, and mesenchymal stem cells, and in vivo in the mouse heart. ANX-SPIO was validated using T2*-weighted 3T MRI. ANX-SPIO produced T2* signal loss, reflecting iron content, that correlated highly with independent apoptosis markers; bound with high affinity to apoptotic myocytes by competition assay (Ki 69 nM); detected apoptosis in culture much earlier than did TUNEL stain; and revealed fibroblast resistance to apoptosis. With apoptosis in vivo, ANX-SPIO produced diffuse myocardial T2* signal loss that correlated with increased iron stain and caspase activity. Treatment with an alpha-1-adrenergic agonist in vivo reversed apoptosis and eliminated the ANX-SPIO MRI signal. It is concluded that cardiac MRI of ANX-SPIO detects early, nonischemic cardiac apoptosis in culture and in vivo, and can identify reversibly injured cardiac cells in diseased hearts, when treatment is still possible.

Adherence and Acceptability of an Oral Antibiotic Used for the Prevention of Pediatric Urinary Tract Infection in Japan.

Urinary tract infection (UTI) is a common health care-associated adverse event and the leading nosocomial complication following pediatric urological surgery. While continuous antimicrobial prophylaxis effectively reduces the risk of UTI following such a surgery, non-adherence is common and represents a distinct clinical entity that is associated with renal scarring. Acceptability is likely to have a significant impact on patient adherence. Herein we used a validated data-driven approach-the ClinSearch acceptability score test (CAST)-to investigate the acceptability of cefaclor, an oral antibiotic widely used for the prevention of pediatric UTI in Japan. Standardized observer reports were collected for 58 intakes of cefaclor 10% fine granules in patients aged from 0 to 17 years. The medicine was classified as positively accepted on the acceptability reference framework. According to the percentage of the prescribed dose taken reported at the end of the treatment, patients exhibited good adherence to this well-accepted medicine. Nonetheless, requirements for greater dosing frequency or poor acceptability in certain patients could affect adherence. Acceptability should be established to ensure patient adherence to medicines used for long-term prophylaxis and consequently guarantee the safety and efficacy of the treatment.

Cell cycle arrest determines adult neural stem cell ontogeny by an embryonic Notch-nonoscillatory Hey1 module.

Quiescent neural stem cells (NSCs) in the adult mouse brain are the source of neurogenesis that regulates innate and adaptive behaviors. Adult NSCs in the subventricular zone are derived from a subpopulation of embryonic neural stem-progenitor cells (NPCs) that is characterized by a slower cell cycle relative to the more abundant rapid cycling NPCs that build the brain. Yet, how slow cell cycle can cause the establishment of adult NSCs remains largely unknown. Here, we demonstrate that Notch and an effector Hey1 form a module that is upregulated by cell cycle arrest in slowly dividing NPCs. In contrast to the oscillatory expression of the Notch effectors Hes1 and Hes5 in fast cycling progenitors, Hey1 displays a non-oscillatory stationary expression pattern and contributes to the long-term maintenance of NSCs. These findings reveal a novel division of labor in Notch effectors where cell cycle rate biases effector selection and cell fate.

An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation.

Fluorescent reporter mouse lines and Cre/Flp recombinase driver lines play essential roles in investigating various molecular functions in vivo. Now that applications of the CRISPR/Cas9 genome-editing system to mouse fertilized eggs have drastically accelerated these knock-in mouse generations, the next need is to establish easier, quicker, and cheaper methods for knock-in donor preparation. Here, we reverify and optimize the phospho-PCR method to obtain highly pure long single-stranded DNAs (ssDNAs) suitable for knock-in mouse generation via genome editing. The sophisticated sequential use of two exonucleases, in which double-stranded DNAs (dsDNAs) amplified by a pair of 5'-phosphorylated primer and normal primer are digested by Lambda exonuclease to yield ssDNA and the following Exonuclease III treatment degrades the remaining dsDNAs, enables much easier long ssDNA productions without laborious gel extraction steps. By microinjecting these donor DNAs along with CRISPR/Cas9 components into mouse zygotes, we have effectively generated fluorescent reporter lines and recombinase drivers. To further broaden the applicability, we have prepared long ssDNA donors in higher concentrations and electroporated them into mouse eggs to successfully obtain knock-in embryos. This classical yet improved method, which is regaining attention on the progress of CRISPR/Cas9 development, shall be the first choice for long donor DNA preparation, and the resulting knock-in lines could accelerate life science research.

Positive contrast with alternating repetition time SSFP (PARTS): a fast imaging technique for SPIO-labeled cells.

There has been recent interest in positive-contrast MRI methods for noninvasive tracking of cells labeled with superparamagnetic iron-oxide nanoparticles. Low-tip-angle balanced steady-state free precession sequences have been used for fast, high-resolution, and flow-insensitive positive-contrast imaging; however, the contrast can be compromised by the limited suppression of the on-resonant and fat signals. In this work, a new technique that produces positive contrast with alternating repetition time steady-state free precession is proposed to achieve robust background suppression for a broad range of tissue parameters. In vitro and in vivo experiments demonstrate the reliability of the generated positive contrast. The results indicate that the proposed method can enhance the suppression level by up to 18 dB compared with conventional balanced steady-state free precession.

Impact of mammalian target of rapamycin inhibition on lymphoid homing and tolerogenic function of nanoparticle-labeled dendritic cells following allogeneic hematopoietic cell transplantation.

Dendritic cells (DC) play a major role in the pathogenesis of graft-vs-host disease (GvHD). Directed modification of surface molecules on DC that provide instructive signals for T cells may create a tolerogenic DC phenotype that affects GvHD severity. To investigate the impact of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) on in vivo migratory capacities, tolerogenic function, and B7 superfamily surface expression on DC following allogeneic hematopoietic cell transplantation (aHCT), we generated a platform for magnetic resonance imaging and bioluminescence imaging based cell trafficking studies. Luciferase transgenic DC were labeled with superparamagnetic iron oxide nanoparticles bound to a murine IgG Ab that allowed for Fc-gammaR-mediated endocytosis. Locally injected luc(+) DC could be tracked within their anatomical context by bioluminescence imaging and magnetic resonance imaging after aHCT, based on stable intracellular localization of superparamagnetic iron oxide-IgG complexes. RAPA preconditioned DC (DC-R) displayed reduced expression of MHC class II, B7-1 (CD80), and B7-2 (CD86) but not B7-H4 whose ligation of T cells has a profound inhibitory effect on their proliferation and cytokine secretion. DC-R of recipient genotype reduced GvHD severity that is compatible with their tolerogenic phenotype. CCR5, CCR7, and CD62L expression was not affected by mTOR inhibition, which allowed for DC-R in vivo trafficking to secondary lymphoid compartments where immunregulation is required. This study is the first to delineate the impact of RAPA on DC migration and tolerogenic function after aHCT. Modification of the DC phenotype by mTOR inhibition may have therapeutic potential in an attempt to reduce GvHD following aHCT.

Light-mediated control of Gene expression in mammalian cells.

Taking advantage of the recent development of genetically-defined photo-activatable actuator molecules, cellular functions, including gene expression, can be controlled by exposure to light. Such optogenetic strategies enable precise temporal and spatial manipulation of targeted single cells or groups of cells at a level hitherto impossible. In this review, we introduce light-controllable gene expression systems exploiting blue or red/far-red wavelengths and discuss their inherent properties potentially affecting induced downstream gene expression patterns. We also discuss recent advances in optical devices that will extend the application of optical gene expression control technologies into many different areas of biology and medicine.

Recognition of glycan and protein substrates by N-acetylglucosaminyltransferase-V.

BACKGROUND: N-Glycosylation is crucial for protein folding, trafficking, and functions. N-Glycans have a different number of N-acetylglucosamine (GlcNAc) branches in a protein-selective manner, and the ß1,6-linked GlcNAc branch on specific proteins produced by N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) promotes cancer malignancy. However, little is known about how GnT-V acts on specific target proteins. METHODS: Based on our structural model, we hypothesized that GnT-V interacts with the N-glycan core or polypeptide moiety as well as the accepter site of N-glycan. To explore this possibility, we selected four candidate residues involved in the interaction with the glycan core or surrounding amino acids, created point mutants of these residues, and examined the in vitro and in vivo activities of the mutants. RESULTS: Our in vitro enzyme assays using various types of substrates including oligosaccharides and glycoproteins revealed that the V354N mutant had dramatically reduced activity for all tested substrates with an altered substrate preference and that K361A had reduced activity for an oligosaccharide with asparagine (Asn), but not a shorter oligosaccharide without the reducing end of GlcNAc and Asn. These results suggest that V354 and K361 are involved in the recognition of N-glycan core and surrounding amino acids. We further performed rescue experiments using GnT-V knockout HeLa cells and confirmed the importance of these residues for modifications of glycoproteins in cells. CONCLUSIONS: We identified several residues involved in the action of GnT-V toward N-glycan cores and surrounding amino acids. GENERAL SIGNIFICANCE: Our data provide new insights into how GnT-V recognizes glycoproteins.

Optimization of Light-Inducible Gal4/UAS Gene Expression System in Mammalian Cells.

Light-inducible gene expression systems represent powerful methods for studying the functional roles of dynamic gene expression. Here, we developed an optimized light-inducible Gal4/UAS gene expression system for mammalian cells. We designed photoactivatable (PA)-Gal4 transcriptional activators based on the concept of split transcription factors, in which light-dependent interactions between Cry2-CIB1 PA-protein interaction modules can reconstitute a split Gal4 DNA-binding domain and p65 transcription activation domain. We developed a set of PA-Gal4 transcriptional activators (PA-Gal4cc), which differ in terms of induced gene expression levels following pulsed or prolonged light exposure, and which have different activation/deactivation kinetics. These systems offer optogenetic tools for the precise manipulation of gene expression at fine spatiotemporal resolution in mammalian cells.

Dietary Intake of Carotenoid-Rich Vegetables Reduces Visceral Adiposity in Obese Japanese men-A Randomized, Double-Blind Trial.

Metabolic syndrome, whose main diagnostic component is obesity, is a risk factor for lifestyle-related diseases, type 2 diabetes, and cardiovascular disease. Diet is known to affect the prevalence of metabolic syndrome. However, the effect of diet on metabolic syndrome in Japanese subjects has not been thoroughly explored. In the present study, we investigated the effect of carotenoid-rich vegetables, particularly lycopene- and lutein-rich vegetables, on the metabolic syndrome in obese Japanese men. We conducted an 8-week long randomized, double-blinded, controlled clinical trial in which, 28 middle-aged (40 ≤ age < 65) Japanese men with high body mass index (BMI ≥ 25) were randomized into four dietary groups: high lycopene + high lutein (HLyHLu), high lycopene + low lutein (HLyLLu), low lycopene + high lutein (LLyHLu), and low lycopene + low lutein (LLyLLu). Our results showed that daily beverage-intake increased the plasma levels of carotenoids without adverse effects, and the visceral fat level was significantly decreased in all the groups. The waist circumference was significantly decreased only in the HLyLLu group, whereas the CoQ10 oxidation rate was decreased in all the groups. The gene expression profiles of whole blood samples before and after ingestion differed only in the LLyLLu group, indicating the effect of carotenoids on gene expression profile. In conclusion, our results suggest that dietary uptake of carotenoid-rich vegetables increases their concentration in blood and reduces the intra-abdominal visceral fat.