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  • 1
    Online Resource
    Online Resource
    Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae
    American Society for Microbiology ; 2013
    In:  Applied and Environmental Microbiology Vol. 79, No. 5 ( 2013-03), p. 1500-1507
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 79, No. 5 ( 2013-03), p. 1500-1507
    Abstract: Saccharomyces cerevisiae cannot utilize cellobiose, but this yeast can be engineered to ferment cellobiose by introducing both cellodextrin transporter ( cdt-1 ) and intracellular β-glucosidase ( gh1-1 ) genes from Neurospora crassa . Here, we report that an engineered S. cerevisiae strain expressing the putative hexose transporter gene HXT2.4 from Scheffersomyces stipitis and gh1-1 can also ferment cellobiose. This result suggests that HXT2.4p may function as a cellobiose transporter when HXT2.4 is overexpressed in S. cerevisiae . However, cellobiose fermentation by the engineered strain expressing HXT2.4 and gh1-1 was much slower and less efficient than that by an engineered strain that initially expressed cdt-1 and gh1-1 . The rate of cellobiose fermentation by the HXT2.4 -expressing strain increased drastically after serial subcultures on cellobiose. Sequencing and retransformation of the isolated plasmids from a single colony of the fast cellobiose-fermenting culture led to the identification of a mutation (A291D) in HXT2.4 that is responsible for improved cellobiose fermentation by the evolved S. cerevisiae strain. Substitutions for alanine (A291) of negatively charged amino acids (A291E and A291D) or positively charged amino acids (A291K and A291R) significantly improved cellobiose fermentation. The mutant HXT2.4(A291D) exhibited 1.5-fold higher K m and 4-fold higher V max values than those from wild-type HXT2.4, whereas the expression levels were the same. These results suggest that the kinetic properties of wild-type HXT2.4 expressed in S. cerevisiae are suboptimal, and mutations of A291 into bulky charged amino acids might transform HXT2.4p into an efficient transporter, enabling rapid cellobiose fermentation by engineered S. cerevisiae strains.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.03253-12
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2013
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity
    Portland Press Ltd. ; 2011
    In:  Biochemical Journal Vol. 434, No. 1 ( 2011-02-15), p. 171-180
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 434, No. 1 ( 2011-02-15), p. 171-180
    Abstract: Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC–MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20101473
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2011
    detail.hit.zdb_id: 1473095-9
    SSG: 12
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  • 3
    Online Resource
    Online Resource
    Decision between mitophagy and apoptosis by Parkin via VDAC1 ubiquitination
    Proceedings of the National Academy of Sciences ; 2020
    In:  Proceedings of the National Academy of Sciences Vol. 117, No. 8 ( 2020-02-25), p. 4281-4291
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 117, No. 8 ( 2020-02-25), p. 4281-4291
    Abstract: VDAC1 is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis. Here, we demonstrate that VDAC1 can be either mono- or polyubiquitinated by Parkin in a PINK1-dependent manner. VDAC1 deficient with polyubiquitination (VDAC1 Poly-KR) hampers mitophagy, but VDAC1 deficient with monoubiquitination (VDAC1 K274R) promotes apoptosis by augmenting the mitochondrial calcium uptake through the mitochondrial calcium uniporter (MCU) channel. The transgenic flies expressing Drosophila Porin K273R, corresponding to human VDAC1 K274R, show Parkinson disease (PD)-related phenotypes including locomotive dysfunction and degenerated dopaminergic neurons, which are relieved by suppressing MCU and mitochondrial calcium uptake. To further confirm the relevance of our findings in PD, we identify a missense mutation of Parkin discovered in PD patients, T415N, which lacks the ability to induce VDAC1 monoubiquitination but still maintains polyubiquitination. Interestingly, Drosophila Parkin T433N, corresponding to human Parkin T415N, fails to rescue the PD-related phenotypes of Parkin -null flies. Taken together, our results suggest that VDAC1 monoubiquitination plays important roles in the pathologies of PD by controlling apoptosis.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1909814117
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2020
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
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  • 4
    Online Resource
    Online Resource
    Role of intermediate phase for stable cycling of Na 7 V 4 (P 2 O 7 ) 4 PO 4 in sodium ion battery
    Proceedings of the National Academy of Sciences ; 2014
    In:  Proceedings of the National Academy of Sciences Vol. 111, No. 2 ( 2014-01-14), p. 599-604
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 111, No. 2 ( 2014-01-14), p. 599-604
    Abstract: Sodium ion batteries offer promising opportunities in emerging utility grid applications because of the low cost of raw materials, yet low energy density and limited cycle life remain critical drawbacks in their electrochemical operations. Herein, we report a vanadium-based ortho-diphosphate, Na 7 V 4 (P 2 O 7 ) 4 PO 4 , or VODP, that significantly reduces all these drawbacks. Indeed, VODP exhibits single-valued voltage plateaus at 3.88 V vs. Na/Na + while retaining substantial capacity ( 〉 78%) over 1,000 cycles. Electronic structure calculations reveal that the remarkable single plateau and cycle life originate from an intermediate phase (a very shallow voltage step) that is similar both in the energy level and lattice parameters to those of fully intercalated and deintercalated states. We propose a theoretical scheme in which the reaction barrier that arises from lattice mismatches can be evaluated by using a simple energetic consideration, suggesting that the presence of intermediate phases is beneficial for cell kinetics by buffering the differences in lattice parameters between initial and final phases. We expect these insights into the role of intermediate phases found for VODP hold in general and thus provide a helpful guideline in the further understanding and design of battery materials.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1316557110
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2014
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
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  • 5
    Online Resource
    Online Resource
    Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase
    American Society for Microbiology ; 2008
    In:  Applied and Environmental Microbiology Vol. 74, No. 21 ( 2008-11), p. 6563-6569
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 74, No. 21 ( 2008-11), p. 6563-6569
    Abstract: The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase ( Neq DNA polymerase) produced λ DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 × 10 −6 ) than Taq DNA polymerase (11.98 × 10 −6 ). Uniquely, Neq DNA polymerase also amplified λ DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00624-08
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2008
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 6
    Online Resource
    Online Resource
    Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease
    American Society for Microbiology ; 2014
    In:  Applied and Environmental Microbiology Vol. 80, No. 24 ( 2014-12-15), p. 7694-7701
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 80, No. 24 ( 2014-12-15), p. 7694-7701
    Abstract: Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3 , trp1 , leu2 , and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02310-14
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 7
    Online Resource
    Online Resource
    Submillisecond organic synthesis: Outpacing Fries rearrangement through microfluidic rapid mixing
    American Association for the Advancement of Science (AAAS) ; 2016
    In:  Science Vol. 352, No. 6286 ( 2016-05-06), p. 691-694
    Details
    In: Science, American Association for the Advancement of Science (AAAS), Vol. 352, No. 6286 ( 2016-05-06), p. 691-694
    Abstract: In chemical synthesis, rapid intramolecular rearrangements often foil attempts at site-selective bimolecular functionalization. We developed a microfluidic technique that outpaces the very rapid anionic Fries rearrangement to chemoselectively functionalize iodophenyl carbamates at the ortho position. Central to the technique is a chip microreactor of our design, which can deliver a reaction time in the submillisecond range even at cryogenic temperatures. The microreactor was applied to the synthesis of afesal, a bioactive molecule exhibiting anthelmintic activity, to demonstrate its potential for practical synthesis and production.
    Type of Medium: Online Resource
    ISSN: 0036-8075 , 1095-9203
    URL: Article
    DOI: 10.1126/science.aaf1389
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: American Association for the Advancement of Science (AAAS)
    Publication Date: 2016
    detail.hit.zdb_id: 128410-1
    detail.hit.zdb_id: 2066996-3
    detail.hit.zdb_id: 2060783-0
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  • 8
    Online Resource
    Online Resource
    Efficient Statistical Method for Association Analysis of X-Linked Variants
    S. Karger AG ; 2016
    In:  Human Heredity Vol. 82, No. 1-2 ( 2016), p. 50-63
    Details
    In: Human Heredity, S. Karger AG, Vol. 82, No. 1-2 ( 2016), p. 50-63
    Abstract: Background/Aims: Unlike the gene-poor Y chromosome, the X chromosome contains over 1,000 genes that are essential for viability of cells. Females have 2 X chromosomes, and thus female X-linked gene expression would be expected to be twice that of males. To adjust this imbalance, one of the 2 X-linked genes is often inactivated, and this is known as X-chromosome inactivation (XCI). However, recent studies described that a gene can be nonrandomly selected for inactivation from 2 X-linked genes and that XCI is not observed in some X-linked genes. Since this complex biological process has prevented efficient statistical association analyses, we propose a new statistical method against this uncertain biological process. Methods: The proposed method consists of 2 steps. First, p values for various biological processes are calculated and then combined into a single p value with the modified Fisher method and a minimum p value. Results: Our simulation results show that the proposed method is generally the most statistically efficient and is not sensitive to the unknown biological model. Conclusion: Therefore, we can conclude that the proposed approaches are robust against the various XCI processes for testing the association of X-linked single nucleotide polymorphisms with the disease of interest and the proposed method is a practical solution.
    Type of Medium: Online Resource
    ISSN: 0001-5652 , 1423-0062
    URL: Article
    DOI: 10.1159/000478048
    RVK:
    WA 15000
    Language: English
    Publisher: S. Karger AG
    Publication Date: 2016
    detail.hit.zdb_id: 1482710-4
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  • 9
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    Online Resource
    Metabolic Engineering of Probiotic Saccharomyces boulardii
    American Society for Microbiology ; 2016
    In:  Applied and Environmental Microbiology Vol. 82, No. 8 ( 2016-04-15), p. 2280-2287
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 82, No. 8 ( 2016-04-15), p. 2280-2287
    Abstract: Saccharomyces boulardii is a probiotic yeast that has been used for promoting gut health as well as preventing diarrheal diseases. This yeast not only exhibits beneficial phenotypes for gut health but also can stay longer in the gut than Saccharomyces cerevisiae . Therefore, S. boulardii is an attractive host for metabolic engineering to produce biomolecules of interest in the gut. However, the lack of auxotrophic strains with defined genetic backgrounds has hampered the use of this strain for metabolic engineering. Here, we report the development of well-defined auxotrophic mutants ( leu2 , ura3 , his3 , and trp1 ) through clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-based genome editing. The resulting auxotrophic mutants can be used as a host for introducing various genetic perturbations, such as overexpression or deletion of a target gene, using existing genetic tools for S. cerevisiae . We demonstrated the overexpression of a heterologous gene ( lacZ ), the correct localization of a target protein (red fluorescent protein) into mitochondria by using a protein localization signal, and the introduction of a heterologous metabolic pathway (xylose-assimilating pathway) in the genome of S. boulardii . We further demonstrated that human lysozyme, which is beneficial for human gut health, could be secreted by S. boulardii . Our results suggest that more sophisticated genetic perturbations to improve S. boulardii can be performed without using a drug resistance marker, which is a prerequisite for in vivo applications using engineered S. boulardii .
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00057-16
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2016
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 10
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    Analysis of cellodextrin transporters from Neurospora crassa in Saccharomyces cerevisiae for cellobiose fermentation
    Springer Science and Business Media LLC ; 2014
    In:  Applied Microbiology and Biotechnology Vol. 98, No. 3 ( 2014-2), p. 1087-1094
    Details
    In: Applied Microbiology and Biotechnology, Springer Science and Business Media LLC, Vol. 98, No. 3 ( 2014-2), p. 1087-1094
    Type of Medium: Online Resource
    ISSN: 0175-7598 , 1432-0614
    URL: Article
    DOI: 10.1007/s00253-013-5339-2
    RVK:
    WA 15000
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2014
    detail.hit.zdb_id: 1464336-4
    SSG: 12
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