Publications by authors named "Satoru Watanabe"

175 Publications

Identification of Transcription Factors and the Regulatory Genes Involved in Triacylglycerol Accumulation in the Unicellular Red Alga .

Plants (Basel) 2021 May 13;10(5). Epub 2021 May 13.

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8503, Japan.

Microalgal triacylglycerols (TAGs) are a good feedstock for liquid biofuel production. Improving the expression and/or function of transcription factors (TFs) involved in TAG accumulation may increase TAG content; however, information on microalgae is still lacking. In this study, 14 TFs in the unicellular red alga were identified as candidate TFs regulating TAG accumulation using available transcriptome and phosphoproteome data under conditions driving TAG accumulation. To investigate the roles of these TFs, we constructed TF-overexpression strains and analyzed lipid droplet (LD) formation and TAG contents in the cells grown under standard conditions. Based on the results, we identified four TFs involved in LD and TAG accumulation. RNA-Seq analyses were performed to identify genes regulated by the four TFs using each overexpression strain. Among the TAG biosynthesis-related genes, only the gene encoding the endoplasmic reticulum-localized lysophosphatidic acid acyltransferase 1 (LPAT1) was notably increased among the overexpression strains. In the LPAT1 overexpression strain, TAG accumulation was significantly increased compared with the control strain under normal growth conditions. These results indicate that the four TFs positively regulate TAG accumulation by changing their target gene expression in .
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http://dx.doi.org/10.3390/plants10050971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8152781PMC
May 2021

Visualization of the seasonal shift of a variety of airborne pollens in western Tokyo.

Sci Total Environ 2021 May 8;788:147623. Epub 2021 May 8.

Colorado State University, Department of Atmospheric Sciences, 80523, USA.

Airborne pollens cause pollinosis and have the potential to affect microphysics in clouds; however, the number of monitored species has been very limited due to technical difficulties for the morphotype identification. In this study, we applied an eDNA approach to the airborne pollen communities in the suburbs of the Tokyo metropolitan area in Japan, within a mixed urban, rural, and mountain landscape, revealing pollen seasonality of various taxa (a total of 78 families across the period) in the spring season (February to May). Those taxa distinctly shifted in the season, especially in the beginning of February and the middle of April. Air temperature shift was an obvious key factor to affect the airborne pollen community, while the influence of other meteorological factors, such as wind speed, humidity, and precipitation, was not clear. Taxonomic classification of major Amplicon Sequence Variants (ASVs) indicates multiple pollen sources, including natural forest, planted forest, roadside, park lands, and horticultural activities. Most major ASV belongs to Japanese cedar (Cryptomeria japonica), which is the most notable allergen that causes pollinosis in Japan, peaking in mid-February to March. Backward trajectory analysis of air masses suggests that the Japanese cedar and other Cupressaceae plantation forests in the western mountains were a significant source of airborne pollen communities detected at our sampling site. Other major plant pollen sources, including Japanese zelkova (Zelkova serrata) and ginkgo (Ginkgo biloba), emanated from the nearby parks or roadside regions. This study's approach enables us to visualize the phenology of multiple pollen, including timing and duration. Long-term monitoring of this type would provide additional insight into understanding the role of climate change on pollen transmission and links to flowering events.
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http://dx.doi.org/10.1016/j.scitotenv.2021.147623DOI Listing
May 2021

DiRect: Site-directed mutagenesis method for protein engineering by rational design.

Biochem Biophys Res Commun 2021 04 13;551:107-113. Epub 2021 Mar 13.

Laboratory for Cellular Structural Biology, RIKEN Center for Biosystems Dynamics Research (BDR), 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan; Laboratory for Biomolecular Structure and Dynamics, RIKEN Quantitative Biology Center (QBiC), 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan. Electronic address:

Site-directed mutagenesis (SDM), an indispensable method in molecular biology and protein engineering, is rather time-consuming and laborious. Protein engineering, especially that of enzymes, nowadays increasingly relies on rational design approaches in which both SDM and protein expression are the bottlenecks because they are generally based on the recombinant DNA technology. Here, we developed a new PCR-based mutagenesis method, DiRect, that achieves high performance in product quality (≥99% substitution) without recombinant DNA technology. We applied DiRect in combination with a cell-free protein expression system to an industrially relevant enzyme, nicotinamide adenine dinucleotide phosphate-dependent 3-quinuclidinone reductase from Rhodotorula rubra. In a single round of screening, 90 newly designed mutant proteins were produced within two days, and an unreported mutant (Q135I) exhibiting much higher thermostability than the wild-type enzyme was successfully identified within one extra day. Thus, DiRect is a simple, efficient, and potentially scalable SDM method.
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http://dx.doi.org/10.1016/j.bbrc.2021.03.021DOI Listing
April 2021

Evolution of Ribosomal Protein S14 Demonstrated by the Reconstruction of Chimeric Ribosomes in Bacillus subtilis.

J Bacteriol 2021 Apr 21;203(10). Epub 2021 Apr 21.

Department of Life Science, College of Science, Rikkyo University, Tokyo, Japan.

Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn binding motif and which are ca. 90 residues and 100 residues in length, respectively. In the present study, the C+ type S14 from ribosomes (S14BsC+) were completely replaced by the heterologous C- long type of S14 from (S14Ec) or (S14Se). Surprisingly, S14Ec and S14Se were incorporated fully into 70S ribosomes in However, the growth rates as well as the sporulation efficiency of the mutants harboring heterologous S14 were significantly decreased. In these mutants, the polysome fraction was decreased and the 30S and 50S subunits accumulated unusually, indicating that cellular translational activity of these mutants was decreased. analysis showed a reduction in the translational activity of the 70S ribosome fraction purified from these mutants. The abundance of ribosomal proteins S2 and S3 in the 30S fraction in these mutants was reduced while that of S14 was not significantly decreased. It seems likely that binding of heterologous S14 changes the structure of the 30S subunit, which causes a decrease in the assembly efficiency of S2 and S3, which are located near the binding site of S14. Moreover, we found that S3 from cannot function in unless S14Se is present. S14, an essential ribosomal protein, may have evolved to adapt bacteria to zinc-limited environments by replacement of a zinc-binding motif with a zinc-independent sequence. It was expected that the bacterial ribosome would be tolerant to replacement of S14 because of the previous prediction that the spread of C- type S14 involved horizontal gene transfer. In this study, we completely replaced the C+ type of S14 in ribosome with the heterologous C- long type of S14 and characterized the resulting chimeric ribosomes. Our results suggest that the ribosome is permissive for the replacement of S14, but coevolution of S3 might be required to utilize the C- long type of S14 more effectively.
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http://dx.doi.org/10.1128/JB.00599-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088600PMC
April 2021

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers for In Situ Live-Cell Molecular Imaging of Dengue Virus Replication.

Int J Mol Sci 2020 Dec 4;21(23). Epub 2020 Dec 4.

Laboratory for Translational and Molecular Imaging, Cancer and Stem Cell Biology Programme, Duke-NUS Medical School, Singapore 169857, Singapore.

Current methods to detect and monitor pathogens in biological systems are largely limited by the tradeoffs between spatial context and temporal detail. A new generation of molecular tracking that provides both information simultaneously involves in situ detection coupled with non-invasive imaging. An example is antisense imaging that uses antisense oligonucleotide probes complementary to a target nucleotide sequence. In this study, we explored the potential of repurposing antisense oligonucleotides initially developed as antiviral therapeutics as molecular probes for imaging of viral infections in vitro and in vivo. We employed nuclease-resistant phosphorodiamidate synthetic oligonucleotides conjugated with cell-penetrating peptides (i.e., PPMOs) previously established as antivirals for dengue virus serotype-2 (DENV2). As proof of concept, and before further development for preclinical testing, we evaluated its validity as in situ molecular imaging probe for tracking cellular DENV2 infection using live-cell fluorescence imaging. Although the PPMO was designed to specifically target the DENV2 genome, it was unsuitable as in situ molecular imaging probe. This study details our evaluation of the PPMOs to assess specific and sensitive molecular imaging of DENV2 infection and tells a cautionary tale for those exploring antisense oligonucleotides as probes for non-invasive imaging and monitoring of pathogen infections in experimental animal models.
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http://dx.doi.org/10.3390/ijms21239260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730579PMC
December 2020

F-fluorodeoxyglucose positron emission tomography as a window into human dengue pathophysiology.

Antiviral Res 2021 01 3;185:104991. Epub 2020 Dec 3.

Department of Infectious Diseases, Singapore General Hospital, Singapore; Programme in Emerging Infectious Diseases, Duke-National University of Singapore (NUS), Medical School, Singapore. Electronic address:

In mouse models of dengue virus (DENV) infection, F-FDG PET is able to sensitively detect tissue-specific sites of inflammation and disease activity, as well as track therapeutic response to anti- DENV agents. However, the use of F-FDG PET to study the pathogenesis of inflammation and disease activity in DENV infection in humans, has not been clinically validated. Here we report the F-FDG PET imaging results of two patients during the febrile phase of acute DENV infection, paired with serial serum viral load, NS1 and proinflammatory cytokine measurements. Our findings demonstrate that F-FDG PET is able to sensitively detect and quantify organ-specific inflammation in the lymph nodes and spleen, in classic acute dengue fever. This raises the potential for F-FDG PET to be used as a research tool that may provide further insights into disease pathogenesis.
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http://dx.doi.org/10.1016/j.antiviral.2020.104991DOI Listing
January 2021

Development of a Weight-Drop Impact Testing Method for Dental Applications.

Polymers (Basel) 2020 Nov 26;12(12). Epub 2020 Nov 26.

Department of Dental Materials Science, School of Life Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan.

For evaluating the impact strength of dental materials, the Izod test or Charpy test has been used, but specimen preparation for these tests is difficult due to the adjustment of a notch on them. By contrast, a weight-drop impact test does not require notched specimens. Therefore, it might be possible to measure the impact strength more accurately than conventional methods. This study aimed to establish appropriate conditions for applying the weight-drop impact test on small specimens of acrylic resin. To determine the most reliable impact fracture energy of acrylic resins, different diameters and thicknesses of PMMA resin specimens, diameters and weights of the striker, and diameters of the supporting jig were compared. For all specimen thicknesses, when the striker diameter was 6-10 mm, the impact fracture energy was constant when the inner diameter of the specimen-supporting jig was 8-10 mm. In addition, the measured E value was mostly equal to the median value of the impact fracture energy. Thus, for the weight-drop impact test, this method was effective for material testing of small specimens, by clearly specifying the test conditions, such as the thickness of disc-shaped specimens, the diameter of the striker, and the inner diameter of the specimen-supporting jig.
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http://dx.doi.org/10.3390/polym12122803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760405PMC
November 2020

Mechanical Properties of Selective Laser Sintering Pure Titanium and Ti-6Al-4V, and Its Anisotropy.

Materials (Basel) 2020 Nov 11;13(22). Epub 2020 Nov 11.

Department of Dental Materials Science, School of Life Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan.

Selective laser sintering (SLS) is being developed for dental applications. This study aimed to investigate the properties of Ti-6Al-4V and pure titanium specimens fabricated using the SLS process and compare them with casting specimens. Besides, the effect of the building direction on the properties of the SLS specimens was also investigated. Specimens were prepared by SLS using Ti-6Al-4V powder or pure titanium powder. Casting specimens were also prepared using Ti-6Al-4V alloys and pure titanium. The mechanical properties (tensile strength and elongation), physical properties (surface roughness, contact angle, and Vickers hardness); corrosion resistors (color difference and corrosion), and surface properties (chemical composition and surface observation) were examined. Both Ti-6Al-4V and pure titanium specimens produced using the SLS process had comparable or superior properties compared with casting specimens. In comparing the building directions, specimens fabricated horizontally to the printing platform showed the greatest tensile strength, and the surface roughness scanned in the horizontal direction to the platform showed the smallest. However, there was no significant effect on other properties. Thus, the SLS process with Ti-6Al-4V powder and pure titanium powder has great performance for the fabrication of dental prosthesis, and there is a possibility for it to take the place of conventional methods.
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http://dx.doi.org/10.3390/ma13225081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696245PMC
November 2020

Live vaccine infection burden elicits adaptive humoral and cellular immunity required to prevent Zika virus infection.

EBioMedicine 2020 Nov 9;61:103028. Epub 2020 Oct 9.

Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore 169857, Singapore; Viral Research and Experimental Medicine Centre, SingHealth Duke-NUS Academic Medical Centre, Singapore 169857, Singapore; Saw Swee Hock School of Public health, National University of Singapore, Singapore 117549, Singapore; Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore. Electronic address:

Background: The emergence of Zika virus (ZIKV) as an important cause of congenital and childhood developmental disorders presents another challenge to global health. Efforts to develop a Zika vaccine have begun although vaccine development against flaviviruses, of which ZIKV belongs to, has proven to be time-consuming and challenging. Defining the vaccine attributes that elicit adaptive immune response necessary for preventing ZIKV infection could provide an evidence-based guide to Zika vaccine development.

Methods: We used a previously described attenuated ZIKV DN-2 strain in a type-I interferon receptor deficient mouse model and tested the hypothesis that duration of vaccine burden rather than peak level of infection, is a determinant of immunogenicity. We quantified both humoral and cellular responses against ZIKV using plaque reduction neutralisation test and flow cytometry with ELISPOT assays, respectively. Vaccinated mice were challenged with wild-type ZIKV (H/PF/2013 strain) to determine the level of protection against infection.

Findings: We found that the overall vaccine burden is directly correlated with neutralising antibody titres. Reduced duration of vaccine burden lowered neutralising antibody titres that resulted in subclinical infection, despite unchanged peak vaccine viraemia levels. We also found that sterilising immunity is dependant on both neutralising antibody and CD8T cell responses; depletion of CD8T cells in vaccinated animals led to wild-type ZIKV infection, especially in the male reproductive tract.

Interpretation: Our findings indicate that duration of attenuated virus vaccine burden is a determinant of humoral and cellular immunity and also suggest that vaccines that elicit both arms of the adaptive immune response are needed to fully prevent ZIKV transmission.

Funding: This study was supported by the National Medical Research Council through the Clinician-Scientist Award (Senior Investigator) to E.E.O. Salary support for S.W. was from a Competitive Research Programme grant awarded by the National Research Foundation of Singapore.
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http://dx.doi.org/10.1016/j.ebiom.2020.103028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553235PMC
November 2020

H, C and N resonance assignment of the YTH domain of YTHDC2.

Biomol NMR Assign 2021 Apr 15;15(1):1-7. Epub 2020 Sep 15.

RIKEN Center for Life Science and Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.

In humans, YTH (YT521-B homology) domain containing protein 2 (YTHDC2) plays a crucial role in the phase-shift from mitosis to meiosis. YTH domains bind to methylated adenosine nucleotides such as mA. In a phylogenic tree, the YTH domain of YTHDC2 (YTH2) and that of the YTH containing protein YTHDC1 (YTH1) belong to the same sub-group. However, the binding affinity of mA differs between these proteins. Here, we report H, C and N resonance assignment of YTH2 and its solution structure to examine the difference of the structural architecture and the dynamic properties of YTH1 and YTH2. YTH2 adopts a β1-α1-β2-α2-β3-β4-β5-α3-β6-α4 topology, which was also observed in YTH1. However, the β4-β5 loops of YTH1 and YTH2 are distinct in length and amino acid composition. Our data revealed that, unlike in YTH1, the structure of mA-binding pocket of YTH2 formed by the β4-β5 loop is stabilized by electrostatic interaction. This assignment and the structural information for YTH2 will provide the insight on the further functional research of YTHDC2.
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http://dx.doi.org/10.1007/s12104-020-09974-3DOI Listing
April 2021

mRNA localization, reaction centre biogenesis and thylakoid membrane targeting in cyanobacteria.

Nat Plants 2020 09 7;6(9):1179-1191. Epub 2020 Sep 7.

School of Biological and Chemical Sciences, Queen Mary University of London, London, UK.

The thylakoid membranes of cyanobacteria form a complex intracellular membrane system with a distinctive proteome. The sites of biogenesis of thylakoid proteins remain uncertain, as do the signals that direct thylakoid membrane-integral proteins to the thylakoids rather than to the plasma membrane. Here, we address these questions by using fluorescence in situ hybridization to probe the subcellular location of messenger RNA molecules encoding core subunits of the photosystems in two cyanobacterial species. These mRNAs cluster at thylakoid surfaces mainly adjacent to the central cytoplasm and the nucleoid, in contrast to mRNAs encoding proteins with other locations. Ribosome association influences the distribution of the photosynthetic mRNAs on the thylakoid surface, but thylakoid affinity is retained in the absence of ribosome association. However, thylakoid association is disrupted in a mutant lacking two mRNA-binding proteins, which probably play roles in targeting photosynthetic proteins to the thylakoid membrane.
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http://dx.doi.org/10.1038/s41477-020-00764-2DOI Listing
September 2020

Diagnostic Use of Post-therapy I-Meta-Iodobenzylguanidine Scintigraphy in Consolidation Therapy for Children with High-Risk Neuroblastoma.

Diagnostics (Basel) 2020 Sep 2;10(9). Epub 2020 Sep 2.

Department of Nuclear Medicine, Kanazawa University Hospital, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8641, Japan.

I-meta-iodobenzylguanidine (I-mIBG) scintigraphy is used for evaluating disease extent in children with neuroblastoma. I-mIBG therapy has been used for evaluation in children with high-risk neuroblastoma, and post-therapy I-mIBG scintigraphy may detect more lesions compared with diagnostic I-mIBG scintigraphy. However, no studies have yet revealed the detection rate of hidden mIBG-avid lesions on post-therapy I-mIBG whole-body scan (WBS) and SPECT images in neuroblastoma children without mIBG-avid lesions as demonstrated by diagnostic I-mIBG scintigraphy. We retrospectively examined the diagnostic utility of post-therapy I-mIBG scintigraphy in children who received I-mIBG as consolidation therapy. Nineteen children with complete response to primary therapy were examined. Post-therapy I-mIBG scintigraphy was performed four days after injection. The post-therapy I-mIBG scintigraphy, 4 children exhibited abnormal uptake on the WBS. Post-therapy I-mIBG SPECT/CT provided additional information in 2 cases. In total, 6 children exhibited abnormal uptake. The site of abnormal accumulation was on the recurrence site in one case, operation sites in five cases, and bone metastasis in one case. Post-therapy I-mIBG scintigraphy could detect residual disease that was not recognized using diagnostic I-mIBG scintigraphy in 32% of children with high-risk neuroblastoma and ganglioneuroblastoma. The diagnostic use of post-therapy I-mIBG scintigraphy can provide valuable information for detecting residual disease.
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http://dx.doi.org/10.3390/diagnostics10090663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555271PMC
September 2020

High-dose I-mIBG as consolidation therapy in pediatric patients with relapsed neuroblastoma and ganglioneuroblastoma: the Japanese experience.

Ann Nucl Med 2020 Nov 29;34(11):840-846. Epub 2020 Aug 29.

Department of Nuclear Medicine, Kanazawa University Hospital, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8641, Japan.

Objective: Children with relapsed neuroblastoma have a poor prognosis despite modern multimodality therapy. Novel and more effective therapeutic strategies are required for relapsed neuroblastoma. We retrospectively examined the utility of consolidation therapy with high-dose I-meta-iodo-benzyl-guanidine (I-mIBG) in relapsed neuroblastoma or ganglioneuroblastoma patients with complete response (CR) to induction therapy as demonstrated by diagnostic I-mIBG scintigraphy.

Methods: Between December 2009 and 2014, five patients with relapsed neuroblastoma and one with relapsed ganglioneuroblastoma received high-dose I-mIBG therapy. Overall and progression-free survival rates at five years after I-mIBG therapy were analyzed by the Kaplan-Meier method.

Results: During follow-up, three children showed no signs of disease relapse, whereas three died. One child without a relapse died from post-transplant side effects, and two children with a relapse died owing to tumor progression. The 5-year progression-free and overall survival rates after I-mIBG therapy were 44% and 67%, respectively.

Conclusions: Consolidation therapy with high-dose I-mIBG for patients with 2 CR showed good overall and progression-free survival. While the risks of radiation exposure must be considered, high-dose I-mIBG therapy as consolidation therapy needs to be further investigated.
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http://dx.doi.org/10.1007/s12149-020-01514-2DOI Listing
November 2020

Protective Immune Responses Elicited by Deglycosylated Live-Attenuated Simian Immunodeficiency Virus Vaccine Are Associated with IL-15 Effector Functions.

J Immunol 2020 09 3;205(5):1331-1344. Epub 2020 Aug 3.

AIDS Research Center, National Institute of Infectious Diseases, Tokyo 162-8640, Japan;

Deglycosylated, live-attenuated SIV vaccines elicited protective immune responses against heterologous SIVsmE543-3, which differs from the vaccine strain SIVmac239 to levels similar to those across HIV-1 clades. Two thirds of the vaccinees contained the chronic SIVsmE543-3 infection (controllers), whereas one third did not (noncontrollers). In this study, we investigated immune correlates of heterologous challenge control in rhesus macaques of Burmese origin. Because depletion of CD8 cells in the controllers by administration of anti-CD8α Ab abrogated the control of viral replication, CD8 cells were required for the protective immune response. However, classical SIV-specific CD8 T cells did not account for the protective immune response in all controllers. Instead, IL-15-responding CD8α cells, including CD8 T and NK cells, were significantly higher in the controllers than those in the noncontrollers, before and after vaccination with deglycosylated SIV. It is well established that IL-15 signal transduction occurs through "-presentation" in which IL-15 complexed with IL-15Rα on monocytes, macrophages, and dendritic cells binds to IL-15 Rβ/γ expressed on CD8 T and NK cells. Accordingly, levels of IL-15 stimulation were strongly affected by the depletion of monocytes from PBMCs, implying key roles of innate immune cells. These results suggest that intrinsic IL-15 responsiveness may dictate the outcome of protective responses and may lead to optimized formulations of future broadly protective HIV vaccines.
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http://dx.doi.org/10.4049/jimmunol.1901431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484436PMC
September 2020

Ion Migration-Induced Degradation and Efficiency Roll-off in Quasi-2D Perovskite Light-Emitting Diodes.

ACS Appl Mater Interfaces 2020 Jul 7;12(29):33004-33013. Epub 2020 Jul 7.

Center for Organic Photonics and Electronics Research (OPERA), Kyushu University, 744 Motooka, Nishi, Fukuoka 819-0395, Japan.

Quasi-2D perovskites have attracted wide attention as the emitter of light-emitting diodes (LEDs) in recent years because of the ease of obtaining high external quantum efficiencies (EQEs). However, the quick degradation under continuous operation and significant EQE roll-off at high current densities are issues that need to be overcome for future practical applications using quasi-2D perovskite LEDs (PeLEDs). In this context, we discuss the mechanism of the degradation and EQE roll-off on the basis of ion migration. The migration of ligand cations though domain boundaries of quasi-2D perovskite films induces the gradual loss of defect passivation at the boundaries, which results in the reversible PeLED degradation and severe EQE roll-off. When the device operation time is long, the mobile cations enter and interact with the electron transport layer, leading to the stage of irreversible PeLED degradation. The device degradation mechanisms we discovered here are constructive for developing quasi-2D PeLEDs with better operational durability.
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http://dx.doi.org/10.1021/acsami.0c06737DOI Listing
July 2020

The CRP-family transcriptional regulator DevH regulates expression of heterocyst-specific genes at the later stage of differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

Mol Microbiol 2020 10 21;114(4):553-562. Epub 2020 Jun 21.

Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Tokyo, Japan.

Heterocysts are terminally differentiated cells of filamentous cyanobacteria, which are specialized for nitrogen fixation. Because nitrogenase is easily inactivated by oxygen, the intracellular environment of heterocysts is kept microoxic. In heterocysts, the oxygen-evolving photosystem II is inactivated, a heterocyst-specific envelope with an outer polysaccharide layer and an inner glycolipid layer is formed to limit oxygen entry, and oxygen consumption is activated. Heterocyst differentiation, which is accompanied by drastic morphological and physiological changes, requires strictly controlled gene expression systems. Here, we investigated the functions of a CRP-family transcriptional regulator, DevH, in the process of heterocyst differentiation. A devH-knockdown strain, devH-kd, was created by replacing the original promoter with the gifA promoter, which is repressed during heterocyst differentiation. Although devH-kd formed morphologically distinct cells with the heterocyst envelope polysaccharide layer, it was unable to grow diazotrophically. Genes involved in construction of the microoxic environment, such as cox operons and the hgl island, were not upregulated in devH-kd. Moreover, expression of the nif gene cluster was completely abolished. Although CnfR was expressed in devH-kd, the nif gene cluster was not induced even under microoxic conditions. Thus, DevH is necessary for the establishment of a microoxic environment and induction of nitrogenase in heterocysts.
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http://dx.doi.org/10.1111/mmi.14558DOI Listing
October 2020

Proteomic analysis of haem-binding protein from and .

Philos Trans R Soc Lond B Biol Sci 2020 06 4;375(1801):20190488. Epub 2020 May 4.

Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan.

Chloroplast biogenesis involves the coordinated expression of the plastid and nuclear genomes, requiring information to be sent from the nucleus to the developing chloroplasts and vice versa. Although it is well known how the nucleus controls chloroplast development, it is still poorly understood how the plastid communicates with the nucleus. Currently, haem is proposed as a plastid-to-nucleus (retrograde) signal that is involved in various physiological regulations, such as photosynthesis-associated nuclear genes expression and cell cycle in plants and algae. However, components that transduce haem-dependent signalling are still unidentified. In this study, by using haem-immobilized high-performance affinity beads, we performed proteomic analysis of haem-binding proteins from and . Most of the identified proteins were non-canonical haemoproteins localized in various organelles. Interestingly, half of the identified proteins were nucleus proteins, some of them have a similar function or localization in either or both organisms. Following biochemical analysis of selective proteins demonstrated haem binding. This study firstly demonstrates that nucleus proteins in plant and algae show haem-binding properties. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.
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http://dx.doi.org/10.1098/rstb.2019.0488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7209954PMC
June 2020

Regulation of the groESL1 transcription by the HrcA repressor and a novel transcription factor Orf7.5 in the cyanobacterium Synechococcus elongatus PCC7942.

J Gen Appl Microbiol 2020 Jun 10;66(2):85-92. Epub 2020 Apr 10.

Molecular Biology Course, Graduate School of Science and Engineering, Saitama University.

The CIRCE/HrcA system is highly conserved in cyanobacterial genomes. We have shown that heat-shock induction of the groESL1 operon in the cyanobacterium Synechocystis sp. PCC6803 is negatively regulated by the CIRCE/HrcA system. In Synechococcus elongatus PCC7942, a novel heat shock protein, Orf7.5, is involved in positive regulation of the groESL1 transcription. However, Orf7.5 is not conserved in some cyanobacteria, including Synechocystis sp. PCC6803. The purpose of this study is to evaluate the functional conservation of the CIRCE/HrcA system in S. elongatus PCC7942 and to understand the interplay between the CIRCE/HrcA system and the Orf7.5 regulatory system. We constructed single and double mutants of S. elongatus orf7.5, hrcA and orf7.5/hrcA and heat induction of the groESL1 transcription in these mutants was analyzed. Unexpectedly, derepression of the groESL1 transcription in an hrcA mutant was not observed. In all these mutants, the transcription was greatly suppressed under both normal and heat stress conditions, indicating that both HrcA and Orf7.5 are involved in regulation of the groESL1 transcription in a positive way. Consistent with the decrease in the groESL1 mRNA level, all the single and double mutants showed a great loss of acquired thermotolerance. Heat induction of the orf7.5 promoter activity was totally diminished in the orf7.5 mutant, indicating that Orf7.5 activates its own transcription. Yeast two hybrid analysis showed that the principle sigma factor RpoD1 interacts with Orf7.5. These results indicate that Orf7.5 enhances the transcription of groESL1 and orf7.5 by interacting with RpoD1.
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http://dx.doi.org/10.2323/jgam.2020.02.001DOI Listing
June 2020

High-dose I-metaiodobenzylguanidine therapy in patients with high-risk neuroblastoma in Japan.

Ann Nucl Med 2020 Jun 26;34(6):397-406. Epub 2020 Mar 26.

Department of Nuclear Medicine, Kanazawa University Hospital and Department of Nuclear Medicine, Institute of Medical, Pharmaceutical and Health Science, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920-8641, Japan.

Objective: The aim of the study was to investigate the outcomes and prognostic factors of high-dose I-metaiodobenzylguanidine (I-MIBG) therapy in patients with refractory or relapsed neuroblastoma (NBL) in Japan.

Methods: We retrospectively analyzed 20 patients with refractory or relapsed high-risk NBL who underwent I-MIBG therapy with an administration dose ranging from 444 to 666 MBq/kg at Kanazawa University Hospital, Japan, between September 2008 and September 2013. We focused on measurements regarding their initial responses, prognostic factors, survivals, and toxicities following I-MIBG therapy using our hospital data and questionnaires from the hospitals that these patients were initially referred from. Furthermore, we performed Kaplan-Meier survival analysis to evaluate event-free survival (EFS) and overall survival (OS).

Results: In 19 patients with complete follow-up data, the median age at first I-MIBG treatment was 7.9 years (range 2.5-17.7 years). Following I-MIBG therapy, 17 of the 19 patients underwent stem-cell transplantations, and their treatment response was either complete (CR) or partial (PR) in three and two cases, respectively. The EFS and OS rates at 1 year following I-MIBG therapy were 42% and 58%, respectively, and those at 5 years following I-MIBG therapy were 16% and 42%, respectively. Using the two-sample log-rank test, the OS time following I-MIBG therapy was significantly longer for < 3-year time interval between the initial diagnosis and I-MIBG therapy (p = 0.017), Curie score < 16 just before I-MIBG therapy (p = 0.002), without pain (p = 0.002), without both vanillylmandelic acid (VMA) and homovanillic acid (HVA) elevation (p = 0.037) at I-MIBG therapy, and with CR or PR following I-MIBG therapy (p = 0.015). Although severe hematological toxicities were identified in all 19 patients, severe nonhematological toxicity was not recorded in any patient, except for one patient with grade 3 anorexia and nausea.

Conclusions: High-dose I-MIBG therapy in patients with refractory or relapsed high-risk NBL can provide a favorable prognosis without severe nonhematological toxicities. Better prognosis may be anticipated in patients with the initial good response, no pain at I-MIBG therapy, no VMA and HVA elevation at I-MIBG therapy, low Curie score (< 16) just before I-MIBG therapy, and short time interval (< 3 years) between the initial diagnosis and I-MIBG therapy.
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http://dx.doi.org/10.1007/s12149-020-01460-zDOI Listing
June 2020

Cyanobacterial multi-copy chromosomes and their replication.

Authors:
Satoru Watanabe

Biosci Biotechnol Biochem 2020 Jul 11;84(7):1309-1321. Epub 2020 Mar 11.

Department of Bioscience, Tokyo University of Agriculture , Tokyo, Japan.

While the model bacteria and harbor single chromosomes, which is known as monoploidy, some freshwater cyanobacteria contain multiple chromosome copies per cell throughout their cell cycle, which is known as polyploidy. In the model cyanobacteria PCC 7942 and sp. PCC 6803, chromosome copy number (ploidy) is regulated in response to growth phase and environmental factors. In 7942, chromosome replication is asynchronous both among cells and chromosomes. Comparative analysis of 7942 and . sp. 6803 revealed a variety of DNA replication mechanisms. In this review, the current knowledge of ploidy and DNA replication mechanisms in cyanobacteria is summarized together with information on the features common with plant chloroplasts. It is worth noting that the occurrence of polyploidy and its regulation are correlated with certain cyanobacterial lifestyles and are shared between some cyanobacteria and chloroplasts.

Abbreviations: NGS: next-generation sequencing; Repli-seq: replication sequencing; BrdU: 5-bromo-2'-deoxyuridine; TK: thymidine kinase; GCSI: GC skew index; PET: photosynthetic electron transport; RET: respiration electron transport; Cyt complex: cytochrome complex; PQ: plastoquinone; PC: plastocyanin.
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http://dx.doi.org/10.1080/09168451.2020.1736983DOI Listing
July 2020

Acclimation process of the chlorophyll d-bearing cyanobacterium Acaryochloris marina to an orange light environment revealed by transcriptomic analysis and electron microscopic observation.

J Gen Appl Microbiol 2020 Jun 7;66(2):106-115. Epub 2020 Mar 7.

Department of Biological Science, Faculty of Science, Shizuoka University.

The cyanobacterium Acaryochloris marina MBIC 11017 (A. marina 11017) possesses chlorophyll d (Chl. d) peaking at 698 nm as photosystem reaction center pigments, instead of chlorophyll a (Chl. a) peaking at 665 nm. About 95% of the total chlorophylls is Chl. d in A. marina 11017. In addition, A. marina 11017 possesses phycobilisome (PBS) supercomplex to harvest orange light and to transfer the absorbing energy to the photosystems. In this context, A. marina 11017 utilizes both far-red and orange light as the photosynthetic energy source. In the present study, we incubated A. marina 11017 cells under monochromatic orange and far-red light conditions and performed transcriptional and morphological studies by RNA-seq analysis and electron microscopy. Cellular absorption spectra, transcriptomic profiles, and microscopic observations demonstrated that PBS was highly accumulated under an orange light condition relative to a far-red light condition. Notably, transcription of one cpcBA operon encoding the phycobiliprotein of the phycocyanin was up-regulated under the orange light condition, but another operon was constitutively expressed under both conditions, indicating functional diversification of these two operons for light harvesting. Taking the other observations into consideration, we could illustrate the photoacclimation processes of A. marina 11017 in response to orange and far-red light conditions in detail.
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http://dx.doi.org/10.2323/jgam.2019.11.008DOI Listing
June 2020

Specific binding of DnaA to the DnaA box motif in the cyanobacterium Synechococcus elongatus PCC 7942.

J Gen Appl Microbiol 2020 Jun 25;66(2):80-84. Epub 2020 Feb 25.

Department of Gene Function and Phenomics, National Institute of Genetics.

In bacterial DNA replication, the initiator protein DnaA binds to the multiple DnaA box sequences located at oriC to facilitate the unwinding of duplex DNA strands. The cyanobacterium Synechococcus elongatus PCC 7942, which contains multiple chromosomal copies per cell, has DnaA box (like sequences around the oriC region, which is located upstream of dnaN. We previously observed the binding of DnaA around the oriC region; however, the DNA-binding specificity of DnaA to DnaA box sequences has not been examined. Here, we analyzed the binding specificity of DnaA protein to the DnaA box in S. elongatus by using bio-layer interferometry (BLI), a method for monitoring intermolecular interactions. We observed that recombinant DnaA protein recognized specifically the DnaA box sequence TTTTCCACA in vitro. In addition, DNA binding activity was significantly increased by R328H mutation of DnaA. This is the first report to characterize DnaA binding to the DnaA box sequence in cyanobacteria.
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http://dx.doi.org/10.2323/jgam.2019.11.005DOI Listing
June 2020

Novel (p)ppGpp suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis.

Mol Microbiol 2020 06 25;113(6):1155-1169. Epub 2020 Feb 25.

Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan.

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.
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http://dx.doi.org/10.1111/mmi.14484DOI Listing
June 2020

Expression of Th1/Th2 cell-related chemokine receptors on CD4 lymphocytes under physiological conditions.

Int J Lab Hematol 2020 Feb 11;42(1):68-76. Epub 2019 Dec 11.

Department of Laboratory Sciences, Gunma University Graduate School of Health Sciences, Maebashi, Japan.

Introduction: Chemokine receptors (CRs) and the prostaglandin D receptor, CRTH2, have been used as surrogate markers for cytoplasmic Th1 and Th2 cytokines. The presence of regulatory T (Treg) and Th17 cells may affect the analysis of such surrogate markers, as they share several CRs with Th1 and Th2 cells. This study aimed to determine the optimal surrogate markers of Th1 and Th2 cells under physiological conditions.

Methods: Surface and cytoplasmic markers of CD4 peripheral lymphocytes were analyzed in healthy volunteers by flow cytometry. Th1, Th2, Th17, and Treg cells were identified as IFN-γ , IL-4 IL-13 , IL-17 , and CD25 FoxP3 CD4 lymphocytes, respectively.

Results: The percentages of CXCR3 and CCR5 CD4 lymphocytes clearly correlated with those of Th1 cells. The percentage of CRTH2 CD4 lymphocytes showed the closest correlation with that of Th2 cells. The percentages of Th2 cells correlated with those of CCR3 or CCR8 CD4 lymphocytes, with the majority of CCR3 and CCR8 cells unlikely to be Th2 cells, themselves. The proportions of CCR4 or CCR7 CD4 lymphocytes did not correlate with those of Th2 cells, possibly due to their expression on the surface of Treg and Th17 cells. Th2-related receptors were classified into three different groups for better understanding.

Conclusion: CXCR3 and CCR5 are useful markers of Th1 cells. With the exception of CCR4 and CCR7 expressed at measurable levels on Treg and Th17 cells, CRTH2 and CRs, CCR3, and CCR8 may be employed as surrogate markers of Th2 cells. The proposed surrogate markers may help physicians in interpreting the disease state.
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http://dx.doi.org/10.1111/ijlh.13141DOI Listing
February 2020

The Potential Role of the ZIKV NS5 Nuclear Spherical-Shell Structures in Cell Type-Specific Host Immune Modulation during ZIKV Infection.

Cells 2019 11 26;8(12). Epub 2019 Nov 26.

Program in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore 169857, Singapore.

The Zika virus (ZIKV) non-structural protein 5 (NS5) plays multiple viral and cellular roles during infection, with its primary role in virus RNA replication taking place in the cytoplasm. However, immunofluorescence assay studies have detected the presence of ZIKV NS5 in unique spherical shell-like structures in the nuclei of infected cells, suggesting potentially important cellular roles of ZIKV NS5 in the nucleus. Hence ZIKV NS5's subcellular distribution and localization must be tightly regulated during ZIKV infection. Both ZIKV NS5 expression or ZIKV infection antagonizes type I interferon signaling, and induces a pro-inflammatory transcriptional response in a cell type-specific manner, but the mechanisms involved and the role of nuclear ZIKV NS5 in these cellular functions has not been elucidated. Intriguingly, these cells originate from the brain and placenta, which are also organs that exhibit a pro-inflammatory signature and are known sites of pathogenesis during ZIKV infection in animal models and humans. Here, we discuss the regulation of the subcellular localization of the ZIKV NS5 protein, and its putative role in the induction of an inflammatory response and the occurrence of pathology in specific organs during ZIKV infection.
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http://dx.doi.org/10.3390/cells8121519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953166PMC
November 2019

The retrograde signaling protein GUN1 regulates tetrapyrrole biosynthesis.

Proc Natl Acad Sci U S A 2019 12 15;116(49):24900-24906. Epub 2019 Nov 15.

Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, 153-8902 Tokyo, Japan;

The biogenesis of the photosynthetic apparatus in developing seedlings requires the assembly of proteins encoded on both nuclear and chloroplast genomes. To coordinate this process there needs to be communication between these organelles, but the retrograde signals by which the chloroplast communicates with the nucleus at this time are still essentially unknown. The () mutants, that show elevated nuclear gene expression after chloroplast damage, have formed the basis of our understanding of retrograde signaling. Of the 6 reported mutations, 5 are in tetrapyrrole biosynthesis proteins and this has led to the development of a model for chloroplast-to-nucleus retrograde signaling in which ferrochelatase 1 (FC1)-dependent heme synthesis generates a positive signal promoting expression of photosynthesis-related genes. However, the molecular consequences of the strongest of the mutants, , are poorly understood, preventing the development of a unifying hypothesis for chloroplast-to-nucleus signaling. Here, we show that GUN1 directly binds to heme and other porphyrins, reduces flux through the tetrapyrrole biosynthesis pathway to limit heme and protochlorophyllide synthesis, and can increase the chelatase activity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabolism, supporting a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway.
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http://dx.doi.org/10.1073/pnas.1911251116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900522PMC
December 2019

Constitutive expression of phosphoketolase, a key enzyme for metabolic shift from homo- to heterolactic fermentation in QU 25.

Biosci Microbiota Food Health 2019 8;38(3):111-114. Epub 2019 May 8.

Department of Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.

Phosphoketolase (PK) is responsible for heterolactic fermentation; however, the PK gene of QU 25, , is transcribed constitutively, even under homolactic fermentation conditions. In order to deduce the regulatory mechanisms of PK activity in QU 25, XfpA levels in QU 25 cells under hetero- and homolactic fermentation conditions were tested using western blotting. The results showed that the XfpA protein expression was similar under both conditions and that the expression products formed complexes, most likely homodimers, indicating that the regulation of PK activity is downstream of translation.
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http://dx.doi.org/10.12938/bmfh.18-030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663511PMC
May 2019

Utility of I-MIBG Standardized Uptake Value in Patients with Refractory Pheochromocytoma and Paraganglioma.

Asia Ocean J Nucl Med Biol 2019 ;7(2):115-120

Department of Nuclear Medicine, Kanazawa University Hospital, Kanazawa, Japan.

Objectives: Single-photon emission computed tomography (SPECT) using metaiodobenzylguanidine (MIBG) is an important diagnostic tool for the treatment of refractory pheochromocytoma and paraganglioma (PPGL). Owing to the difficulty of SPECT quantification, the tumour-to-background ratio (TBR) is used to assess disease activity. However, the utility of TBR is limited owing to the background setting. A quantification technique of SPECT/computed tomography (CT) would facilitate image interpretation. This study aimed to assess the relationship between I-MIBG maximum standardized uptake value (SUV) and TBR and levels of urinary catecholamines and metabolites in patients with refractory PPGL.

Methods: This study included 15 patients with refractory PPGL who underwent I-MIBG therapy. Overall, 27 I-MIBG SPECT/CT images were acquired before and after the therapy. Lesions observed on whole-body images were analysed; the maximum number of lesions per scan was 10. I-MIBG SUV was semi-automatically calculated using Q. Metrix package (GE Healthcare). TBR was manually calculated according to the following formula: (max count in lesion - max count in background)/max count in background. Background was set in the contralateral area. When a background region of interest could not be set in the area, it was set in the thigh area. Urine was sampled for 24 h to measure catecholamine and metabolite levels. Increases of ≥3-fold were considered abnormal. TBR, I-MIBG SUV and urinary catecholamine and metabolite levels were compared using linear regression analysis.

Results: All patients had MIBG-avid lesions, as seen on I-MIBG SPECT/CT. A significant relationship between I-MIBG SUV and TBR was observed (correlation coefficient [r] =0.84, P < 0.0001). In 27 SPECT/CT examinations, normetanephrine (NMN) level was abnormally increased in 51% (14/27), but other catecholamine and other metabolites were abnormally increased in < 26% (7/27). I-MIBG SUV strongly correlated with NMN (r=0.76, P < 0.01) and log NMN (r=0.74, P < 0.01).

Conclusion: I-MIBG SUV demonstrated similar trends as TBR and reflected urinary NMN in patients with refractory PPGL. Semi-automatic quantification of SPECT/CT could be a useful tool for the evaluation of disease activity.
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http://dx.doi.org/10.22038/AOJNMB.2019.35953.1245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6661310PMC
January 2019

Integration of a plasma membrane sugar transporter enables heterotrophic growth of the obligate photoautotrophic red alga .

Plant Direct 2019 Apr 8;3(4):e00134. Epub 2019 Apr 8.

Department of Gene Function and Phenomics National Institute of Genetics Mishima Shizuoka Japan.

The unicellular thermoacidophilic red alga is an emerging model organism of photosynthetic eukaryotes. Its relatively simple genome (16.5 Mbp) with very low-genetic redundancy and its cellular structure possessing one chloroplast, mitochondrion, peroxisome, and other organelles have facilitated studies. In addition, this alga is genetically tractable, and the nuclear and chloroplast genomes can be modified by integration of transgenes via homologous recombination. Recent studies have attempted to clarify the structure and function of the photosystems of this alga. However, it is difficult to obtain photosynthesis-defective mutants for molecular genetic studies because this organism is an obligate autotroph. To overcome this issue in , we expressed a plasma membrane sugar transporter, GsSPT1, from , which is an evolutionary relative of and capable of heterotrophic growth. The heterologously expressed GsSPT1 localized at the plasma membrane. GsSPT1 enabled to grow mixotrophically and heterotrophically, in which cells grew in the dark with glucose or in the light with a photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and glucose. When the transgene multiplied on the chromosome via the selection marker, which can multiply itself and its flanking transgene, GsSPT1 protein level increased and the heterotrophic and mixotrophic growth of the transformant accelerated. We also found that GsSPT1 overexpressing efficiently formed colonies on solidified medium under light with glucose and DCMU. Thus, GsSPT1 overexpresser will facilitate single colony isolation and analyses of photosynthesis-deficient mutants produced either by random or site-directed mutagenesis. In addition, our results yielded evidence supporting that the presence or absence of plasma membrane sugar transporters is a major cause of difference in trophic properties between and .
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http://dx.doi.org/10.1002/pld3.134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589524PMC
April 2019

A T164S mutation in the dengue virus NS1 protein is associated with greater disease severity in mice.

Sci Transl Med 2019 06;11(498)

Program in Emerging Infectious Diseases, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore.

Dengue viruses cause severe and sudden human epidemics worldwide. The secreted form of the nonstructural protein 1 (sNS1) of dengue virus causes vascular leakage, a hallmark of severe dengue disease. Here, we reverse engineered the T164S mutation of NS1, associated with the severity of dengue epidemics in the Americas, into a dengue virus serotype 2 mildly infectious strain. The T164S mutant virus decreased infectious virus production and increased sNS1 production in mammalian cell lines and human peripheral blood mononuclear cells (PBMCs) without affecting viral RNA replication. Gene expression profiling of 268 inflammation-associated human genes revealed up-regulation of genes induced in response to vascular leakage. Infection of the mosquito vector with the T164S mutant virus resulted in increased viral load in the mosquito midgut and higher sNS1 production compared to wild-type virus infection. Infection of type 1 and 2 interferon receptor-deficient AG129 mice with the T164S mutant virus resulted in severe disease coupled with increased complement activation, tissue inflammation, and more rapid mortality compared to AG129 mice infected with wild-type virus. Molecular dynamics simulations predicted that mutant sNS1 formed stable dimers similar to the wild-type protein, whereas the hexameric mutant sNS1 was predicted to be unstable. Immunoaffinity-purified sNS1 from T164S mutant virus-infected mammalian cells was associated with different lipid classes compared to wild-type sNS1. Treatment of human PBMCs with sNS1 purified from T164S mutant virus resulted in a twofold higher production of proinflammatory cytokines, suggesting a mechanism for how mutant sNS1 may cause more severe dengue disease.
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http://dx.doi.org/10.1126/scitranslmed.aat7726DOI Listing
June 2019