Biology subjects

Fujii, S.

Publications and source records attributed to Fujii, S..

41 records · Page 3Linked to original sources

bioRxiv2020

Crosstalk between repair pathways elicits Double-Strand Breaks in alkylated DNA: implications for the action of temozolomide

Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG). Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-Methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg extracts. We show that in this system, mismatch repair (MMR) proteins are enriched on MNU-treated DNA and we observe robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increased linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving BER acting at a nearby N-alkylation adducts. We propose a new, replication-independent mechanism of action of TMZ, that operates in addition to the well-studied cell cycle dependent mode of action.

biochemistry↗

bioRxiv2020

Biofilm Formation and Virulence of Shigella flexneri is Modulated by pH of Gastrointestinal Tract

Shigella infections remain a major public health issue in developing countries. One model of Shigella pathogenesis suggests that the microfold epithelial cells in the small intestine are the preferred initial site of infection. However, a growing body of evidence supports an alternative model whereby Shigella primarily infects a much wider range of epithelial cells that reside primarily within the colon. Here, we investigated whether the luminal pH difference between the small intestine and colon could provide evidence in support of either model of Shigella flexneri pathogenesis. As virulence factors leading to cellular invasion are linked to biofilms in S. flexneri, we examined the effect of pH on S. flexneris ability to form and maintain adherent biofilms when induced by deoxycholate. We showed that a basic pH inhibited formation and dispersed pre-assembled mature biofilms while an acidic pH (similar to the colonic environment) did not have either of these effects. To further elucidate this phenomenon at the molecular level, we probed the transcriptomes of biofilms and S. flexneri grown in different pH conditions. We identified specific amino acid metabolic pathways (cysteine and arginine) that were enriched in the bacteria that formed the biofilms, but decreased upon pH increase. We then utilized a type III secretion system reporter strain to show that increasing pH reduced deoxycholate-induced virulence of S. flexneri in a dose dependent manner. Taken together, these experiments support a model whereby Shigella infection is favored in the colon because of the local pH differences in these organs.

microbiology↗

bioRxiv2020

Reassembly of soil fungal communities under reforestation and herbivore exclusion

Soil fungi can help improve ecosystem restoration, yet our understanding of how they reassemble in degraded land is limited. Here, we studied fungal community structure using DNA metabarcoding in reforested sites following agricultural abandonment and overgrazing. Two treatments, namely reforestation using different numbers of tree species and deer exclusion, have been applied for multiple decades in the study sites. We found that local fungal richness (alpha diversity) and total fungal richness (gamma diversity) were 1.9-2.9 and 1.3-1.9 times greater, respectively, in reforested stands than in natural forests. These results were regardless of the number of tree species planted in the reforested stands. Conversely, reforested stands had a spatially homogenized community structure with relatively lower degrees of compositional dissimilarity among sites within each stand (beta diversity). These findings were attributable to lower environmental heterogeneity, stronger dispersal limitation, and a comparatively shorter time since the onset of community assembly in reforested stands. Deer exclosures had no detectable effect on fungal community structure. Overall, the agricultural legacy in fungal community structure appears to have persisted for decades, even under proactive restoration of aboveground vegetation. Direct human intervention belowground may therefore be necessary for the recovery of soil biota once altered.

ecology↗

bioRxiv2019

Targeting dormant ovarian cancer cells in vitro and in an in vivo model of platinum resistance

ObjectiveOvarian cancer cells often exist in vivo as multicellular spheroids. Spheroid formation in vitro has been used to enrich for cancer stem cell populations from primary tumors. Such spheroids exhibit drug resistance and slow proliferation, suggesting involvement in disease recurrence. Our objectives were to characterize cancer spheroid phenotypes, determine gene expression profiles associated with spheroid forming capacity and to evaluate the responsiveness of spheroids to commonly used and novel therapeutic agents.\n\nMethodsTumorigenic potential was assessed using anchorage independent growth assays in 24 cell lines. Spheroids from cell lines (N=12) and from primary cancers (N=8) were grown on non-adherent tissue culture plates in serum-free media. Cell proliferation was measured using MTT assays and Ki67 immunostaining. Affymetrix HT U133A gene expression data was used to identify differentially expressed genes based on spheroid forming capacity. Matched monolayers and spheroids (N=7 pairs) were tested for response to cisplatin, paclitaxel and 7-hydroxystaurosporine (UCN-01) while mitochondrial inhibition was performed using oligomycin. Xenograft tumors from intraperitoneal injection of CAOV2-GFP/LUC ovarian cancer cells into nude mice were treated with carboplatin to reduce tumor burden followed by secondary treatment with carboplatin, UCN-01, or Oltipraz. Tumor formation and response was monitored using live imaging.\n\nResultsOf 12 cell lines with increased anchorage-independent growth, 8 also formed spheroids under serum-free spheroid culture conditions. Spheroids showed reduced proliferation (p<0.0001) and Ki67 immunostaining (8% versus 87%) relative to monolayer cells. Spheroid forming capacity was associated with increased mitochondrial pathway activity (p [≤] 0.001). The mitochondrial inhibitors, UCN-01 and Oligomycin, demonstrated effectiveness against spheroids, while spheroids were refractory to cisplatin and paclitaxel. By live in vivo imaging, ovarian cancer xenograft tumors were reduced after primary treatment with carboplatin. Continued treatment with carboplatin was accompanied by an increase in tumor signal while there was little or no increase in tumor signal observed with subsequent treatment with UCN-01 or Oltipraz.\n\nConclusionsOur findings suggest that the mitochondrial pathway in spheroids may be an important therapeutic target in preventing disease recurrence.

cancer biology↗

bioRxiv2017

Blue Light Negatively Regulates Tolerance to Phosphate Deficiency in Arabidopsis

Plants have evolved mechanisms to improve utilization efficiency or acquisition of inorganic phosphate (Pi) in response to Pi deficiency, such as altering root architecture, secreting acid phosphatases, and activating the expression of genes related to Pi uptake and recycling. Although many genes responsive to Pi starvation have been identified, transcription factors that affect tolerance to Pi deficiency have not been well characterized. We show here that defect in the ELONGATED HYPOCOTYL 5 (HY5) transcription factor gene results in tolerance to Pi deficiency in Arabidopsis. The primary root length of hy5 was only slightly inhibited under Pi deficient condition and its fresh weight was significantly higher than that of wild type. The Pi deficiency-tolerant phenotype of hy5 was similarly observed when grown on the medium without Pi. In addition, a double mutant, hy5slr1, without lateral roots also showed tolerance to phosphate deficiency, indicating that the tolerance of hy5 does not result from increase of external Pi uptake and may be related to internal Pi utilization or recycling. Moreover, we found that blue light negatively regulates tolerance to Pi-deficiency and that hy5 exhibits tolerance to Pi deficiency due to blockage of blue-light responses. Collectively, this study points out light quality may play an important role in the regulation of internal Pi recycling and utilization efficiency. Also, it may contribute to reducing Pi fertilizer requirements in plants through a proper illumination.

plant biology↗