Biology subjects

Shimamura, M.

Publications and source records attributed to Shimamura, M..

8 recordsLinked to original sources

bioRxiv2026

Improved adenine-HPLC method for quantifying yeast based on cellular DNA content

Accurate quantification of fungi is important for a myriad of applications but remains challenging. Previously, we demonstrated that an approach called the adenine-HPLC method can quantify bacteria, including those with aggregating properties that are difficult to quantify using conventional methods, by measuring cellular adenine derived from DNA and converting the adenine amount to genome copy number, without being influenced by cell morphology. However, in this study, when this adenine-HPLC method was applied to the quantification of budding yeast as a model fungus, accurate measurement proved impossible. This limitation was attributed to adenine release from other adenine-containing biomolecules, such as RNA and ATP, and we therefore developed a method that suppresses adenine release from these molecules. This method involves reducing the temperature of the acid treatment and prewashing the cells before acid treatment. In addition, we incorporated a process that corrects for the naturally occurring free adenine level as background during total adenine measurement. The improved adenine-HPLC method based on these modifications enables accurate quantification of budding yeast using genomic DNA content in whole cells as the quantification unit.

microbiology↗

bioRxiv2026

Initiation of asexual reproduction by the AP2/ERF gene GEMMIFER in Marchantia polymorpha

Plants can propagate their own clones through asexual reproduction. Genetic and hormonal factors regulating asexual reproduction have begun to be elucidated in the liverwort Marchantia polymorpha, which produce asexual propagules called gemmae within the gemma cups. Here, we report an AP2/ERF family gene, GEMMIFER (MpGMFR), as a key regulator of asexual reproduction in M. polymorpha. Suppression of MpGMFR function using genome editing and amiRNA results in the loss of gemma and gemma cup formation. In contrast, activation of MpGMFR function using a dexamethasone inducible system promotes gemma and/or gemma cup formation depending on the induction conditions. Notably, transient strong activation of MpGMFR induced gemma initial cells at the meristem, which develop into mature gemmae capable of reproducing as new individuals after detachment. These results show that MpGMFR is necessary and sufficient for the initiation of asexual reproduction at the meristem. MpGMFR expression was detected from the meristem through to the early stages of gemma development including the gemma cup floor cells. MpGMFR expression precedes the expression of GEMMA CUP-ASSOCIATED MYB1 (MpGCAM1) in early gemma development, supporting the notion that MpGMFR initiates asexual reproduction. We anticipate this finding to be a foundation to study the evolution of extra meristem formation in plant bodies that may have made a significant contribution to the prosperity of plants on land.

plant biology↗

bioRxiv2024

Population genomics of Marchantia polymorpha subspecies ruderalis reveals evidence of climate adaptation

Sexual reproduction results in the development of haploid and diploid cell states during the life cycle. In bryophytes the dominant multicellular haploid phase produces motile sperm that swim through water to the egg to effect fertilization from which a relatively small diploid phase develops. In angiosperms, the reduced multicellular haploid phase produces non-motile sperm that is delivered to the egg through a pollen tube to effect fertilization from which the dominant diploid phase develops. These different life cycle characteristics are likely to impact the distribution of genetic variation among populations. However, little is known about the distribution of genetic variation among populations of bryophytes. To understand how genetic variation is distributed among populations of a bryophyte and to establish the foundation for population genetics research in bryophytes, we described the genetic diversity of collections of Marchantia polymorpha subspecies ruderalis, a cosmopolitan ruderal liverwort. We explored genetic diversity of this species using 78 genetically unique (non-clonal) accessions from a total of 209 collected from 37 sites in Europe and Japan. There was no detectable population structure among European populations but significant genetic differentiation between Japanese and European populations. By associating genetic variation across the genome with global climate data, we identified summer temperature and precipitation as climate factors influencing the frequency of adaptative alleles. We speculate that the requirement for water through which motile sperm swim imposes a constraint on the life cycle to which the plant genetically adapts.

plant biology↗

bioRxiv2021

An ancestral function of strigolactones as symbiotic rhizosphere signals

In flowering plants, carotenoid-derived strigolactones (SLs) have dual functions as hormones that regulate growth and development, and as rhizosphere signaling molecules that induce symbiosis with arbuscular mycorrhizal (AM) fungi. Here, we report the identification of bryosymbiol (BSB), a previously unidentified SL from the bryophyte Marchantia paleacea. BSB is also found in vascular plants, indicating that it is ancestral in land plants. BSB synthesis is enhanced at AM symbiosis permissive conditions and BSB deficient mutants are impaired in AM symbiosis. In contrast, the absence of BSB synthesis has little effect on the growth and gene expression. We show that the introduction of the SL receptor of Arabidopsis renders M. paleacea cells BSB-responsive. These results suggest that BSB is not perceived by M. paleacea cells due to the lack of cognate SL receptors. We propose that SLs originated as AM symbiosis-inducing rhizosphere signaling molecules and were later recruited as plant hormone.

plant biology↗

bioRxiv2021

Th1/17 Cells Infiltrate Murine Cytomegalovirus-Infected Renal Allografts via Virus-Induced CCL20 and Promote Th1 Cells through IL-17A.

Cytomegalovirus (CMV) infection is associated with renal allograft failure by unknown mechanisms. In a murine renal transplant model, murine CMV (MCMV) induces intragraft infiltration of Th17 cells co-expressing Th1 cytokines, IFN-{gamma} and TNF-, but only a minority of intragraft Th17 cells are specific for MCMV antigens. Instead, MCMV promotes viral antigen-independent Th17 cell recruitment via CCL20-CCR6 and CXCL10-CXCR3 interactions. Th17 cells correlate directly with Th1 cell frequencies and inversely with Tregs in MCMV infected grafts. Pharmacologic inhibition of IL-17A reduces intragraft Th17 cells and neutrophils, increases Tregs, and reduces total but not MCMV-specific Th1 cells. Among a clinical renal transplant cohort with acute rejection, patients with CMV DNAemia had significantly higher serum IL-17A quantities compared to those without CMV DNAemia. Together, these findings indicate that CMV infection upregulates Th17 cell activity during acute rejection and suggests that inhibition of IL-17A may ameliorate CMV-associated allograft injury without impairing antiviral Th1 cells.

immunology↗

bioRxiv2020

Preclinical study of DNA vaccines targeting SARS-CoV-2

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFN{gamma} expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies via two different methods, a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and pseudovirus assay. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits, but not the S1 subunit. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

immunology↗

bioRxiv2020

Recovery of cryo-injured rabbit urethras by biofabricated C-shaped adipose-derived mesenchymal cell structures

Urethral tissue damage can cause stress urinary incontinence or urinary retention, and there are few long-term, effective treatments. For structural and functional recovery of urethral tissues, we designed and constructed C-shaped structures composed of adipose-derived mesenchymal cells (AMCs) by using a three-dimensional (3D)-bioprinter system, which could be transplanted in damaged urethras without obstructing the lumen. We determined if transplantation of the biofabricated structures could reconstruct the urethral tissues. AMCs were harvested from rabbits, cultured and labeled with PKH26 to form spheroids. The spheroids were assembled on a custom-designed C-shaped support by a 3D bioprinter. Urethras of rabbits were cryo-injured by spraying with liquid nitrogen for 20 seconds, incised and biofabricated structure was autologously transplanted. Control rabbits were treated similarly but without transplantation structure. Two and four weeks after surgery, the control urethras were partially constricted; however, the structure-transplanted urethras were patent. The AMCs within the structures differentiated into skeletal muscle, smooth muscle, nerve, or endothelial cells. Some cells contained growth factors and cytokines. Therefore, biofabricated C-shaped AMC structures have potential to be an effective treatment for urethral recovery. Summary StatementThree-dimensional bioprinter was used to biofabricate novel C-shaped structures composed of adipose-derived mesenchymal cells (AMCs) and have the potential to be an effective treatment for urethral recovery.

bioengineering↗

bioRxiv2019

Lateral organ diversification in plants mediated by the ALOG protein family of transcription factors

Land plant shoot structures evolved a diversity of lateral organs as morphological adaptations to the terrestrial environment, in which lateral organs independently evolved in each lineage in the sporophyte or gametophyte generation. The gametophyte meristem of the basally-diverging plant Marchantia polymorpha produces axes with non-photosynthetic scale-like lateral organs instead of leaves. Here we report that an ALOG (Arabidopsis LSH1 and Oryza G1) family protein in Marchantia, MpTAWAWA1 (MpTAW1), regulates meristem maintenance and lateral organ development. A mutation in MpTAW1, preferentially expressed in lateral organs, induces lateral organs with mis-specified identity and increased cell number, and furthermore, causes defects in apical meristem maintenance. Remarkably, MpTAW1 expression rescued the elongated-spikelet phenotype of a rice mutant of MpTAW1 homologue. This suggests that ALOG genes are co-opted to specify lateral organ identities in both gametophyte and sporophyte shoots by repressing lateral organ growth. We propose that the recruitment of ALOG-mediated lateral organ modification was in part responsible for the convergent evolution of independently-evolved lateral organs among highly divergent plant lineages and contributed to the morphological diversification of land plants.

plant biology↗