Biology subjects

Nakamura, S.

Publications and source records attributed to Nakamura, S..

4 recordsLinked to original sources

bioRxiv2018

Cas9/CRISPR genome editing to demonstrate the contribution of Cyp51A Gly138Ser to azole resistance in Aspergillus fumigatus

Azole resistance in Aspergillus fumigatus is predominantly associated with increased expression of Cyp51A (lanosterol 14-demethylase), the target enzyme of azole antifungal agents, or with single-nucleotide polymorphisms (SNPs) in cyp51A. Although several SNPs that may be linked to low susceptibility in azole-resistant isolates have previously been reported, few studies have been conducted to conclusively demonstrate the contribution of SNPs to decreased azole susceptibility. An A. fumigatus strain was isolated from the sputum of a 74-year-old male receiving long-term voriconazole treatment for chronic progressive pulmonary aspergillosis. Etest antifungal susceptibility testing showed low susceptibility to voriconazole, itraconazole, and posaconazole. Nucleotide sequencing of cyp51A from this isolate revealed the mutations Gly138Ser (GGC[->]AGC) and Asn248Lys (AAT[->]AAA) compared with the cyp51A of azole-susceptible isolates. PCR-amplified DNA fragments containing cyp51A with or without the mutations of interest and a hygromycin marker were simultaneously introduced along with the Cas9 protein and in vitro-synthesized single-guide RNA into protoplasts of the azole-resistant/susceptible strains. Etest azole susceptibility testing of recombinant strains showed an increased susceptibility via the replacement of Ser138 by glycine. In contrast, azole susceptibility was slightly decreased when a Ser138 mutation was introduced into the azole-susceptible strain AfS35, indicating that the serine at position 138 of Cyp51A contributes to low susceptibility in the azole-resistant isolate. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using Cas9/CRISPR genome editing. This technique could be useful to investigate the contribution of other SNPs of cyp51A to azole resistance.

microbiology↗

bioRxiv2018

The expansion in lymphoid organs of IL-4+ BATF+ T follicular helper cells is linked to IgG4 class switching in vivo

Distinct TFH subsets that influence specific class-switching events are assumed to exist, but the accumulation of isotype-specific TFH subsets in secondary and tertiary lymphoid organs has not been hitherto demonstrated. IL-4 expressing TFH cells are surprisingly sparse in human secondary lymphoid organs. In sharp contrast, in IgG4-related disease (IgG4-RD), a disorder characterized by polarized Ig class switching, most TFH cells in tertiary and secondary lymphoid organs make IL-4. Human IL-4+ TFH cells do not express GATA-3 but express nuclear BATF, and the transcriptomes of IL-4 secreting TFH cells differ both from PD1hi TFH cells that do not secrete IL-4 and IL4-secreting non-TFH cells. Unlike IgG4-RD, IL-4+ TFH cells are rarely found in tertiary lymphoid organs in Sjogrens syndrome, a disorder in which IgG4 is not elevated. The proportion of CD4+IL-4+BATF+ T cells as well as of CD4+IL-4+CXCR5+ T cells in IgG4-RD tissues correlates tightly with tissue IgG4 plasma cell numbers and plasma IgG4 levels in patients but not with the total plasma levels of other isotypes. These data describe a disease-related TFH sub-population in human tertiary and secondary lymphoid organs that is linked to IgG4 class switching.

immunology↗

bioRxiv2017

In situ genome editing method suitable for routine generation of germline modified animal models

Animal genome engineering experimental procedures involve three major steps: isolation of zygotes from pregnant females; microinjection of zygotes, and; transfer of injected zygotes into recipient females, that have been practiced for over three decades. The laboratory set ups intending to performing these procedures require to have sophisticated equipment as well as highly skilled technical personnel. Because of these reasons, animal transgenesis experiments are typically performed at centralized core facilities in most research organizations. We recently showed that all three steps, of animal transgensis, can be bypassed using a method termed GONAD (Genome-editing via Oviductal Nucleic Acids Delivery), by directly electroporating genome editing components into zygotes in situ. Although our first report demonstrated the genome-editing capability, its efficiency was lower than the standard methods using microinjection. Here we investigated critical parameters of GONAD to make it suitable for creating animal models of large genomic deletions, single nucleotide corrections and long sequence insertions. The efficiency of genome editing in the improved GONAD (i-GONAD) method reached to the levels comparable to traditional microinjection methods. The streamlined parameters, and the simplified experimental steps, in the i-GONAD method makes it suitable for routine genome editing applications performed both at centralized facilities as well as at the laboratories that lack highly skilled personnel and the sophisticated equipment.

genomics↗

bioRxiv2016

Autophagy mitigates high-temperature injury during microsporogenesis in Arabidopsis thaliana

Autophagy is one of the cellular processes that break down cellular components during senescence, starvation, and stress. The susceptibility of plant pollen development to high-temperature (HT) stress is well known, but the involvement of autophagy in HT injury is yet to be clarified. Here, we found that following transfer to 30 {degrees}C, all autophagy-deficient (atg) mutants (atg2-1, 5-1, 7-2, and 10-1) of Arabidopsis thaliana tested displayed visibly impaired pollen development and anther dehiscence. HT-induced male sterility significantly increased in the atg mutants, but the degree of HT-induced obstacles did not change between the wild type (WT) and mutants from the seedling stage to the bolting stage. Cytological analyses showed that 30 {degrees}C promoted autophagy and autolysosome formation in both anther wall cells and microspores in developing anthers of WT, but the atg5-1 mutant did not show completion of tapetum degeneration and microspore maturation. HT upregulated hydrogen peroxide and dehydroascorbate reductase 1 production in both WT and atg5-1 anthers, but the basal levels were already higher in the mutant. HT repressed expression of UNDEAD and its regulator MYB80, which are required for tapetal programmed cell death (PCD) for proper pollen development. Taken together, our results suggest that autophagy functions in tapetum degeneration and pollen development during HT-caused tapetal PCD abortion.\n\nHighlightsO_LIIn Arabidopsis, autophagy is not essential for completion of the life cycle under normal temperatures.\nC_LIO_LIHigh temperature (HT) stress induces autophagy in developing anther wall cells and microspores.\nC_LIO_LIAutophagy deficient atg mutants become almost completely male-sterile at moderate HT.\nC_LIO_LIAutophagy plays a role in tapetum degeneration and pollen development during HT-caused abortion of tapetal program cell death.\nC_LI

plant biology↗