Biology subjects

Kagawa, H.

Publications and source records attributed to Kagawa, H..

3 recordsLinked to original sources

bioRxiv2024

Challenging the Astral mass analyzer - going beyond 5200 proteins per single-cell at unseen quantitative accuracy to study cellular heterogeneity.

A detailed proteome map is crucial for understanding molecular pathways and protein functions. Despite significant advancements in sample preparation, instrumentation, and data analysis, single-cell proteomics is currently limited by proteomic depth and quantitative performance. We combine a zero dead-end volume chromatographic column running at high throughput with the Thermo Scientific Orbitrap Astral mass spectrometer running in DIA mode. We demonstrate unprecedented depth of proteome coverage as well as accuracy and precision for quantification of ultra-low input amounts. Using a tailored library, we identify up to 7400 protein groups from as little as 250 pg HeLa at a throughput of 50 samples per day (SPD). We benchmark multiple data analysis strategies, estimate their influence on FDR and show that FDR on protein level can easily be maintained at 1 %. Using a two-proteome mix, we check for optimal parameters of quantification and show that fold change differences of 2 can still be successfully determined at single-cell level inputs. Eventually, we apply our workflow to A549 cells yielding a proteome coverage of up to 5300 protein groups from a single cell, which allows the observation of heterogeneity in a cellular population and studying dependencies between cell size and cell-cycle phase. Additionally, our work-flow enables us to distinguish between in vitro analogs of two human blastocyst lineages: naive human pluripotent stem cells (epiblast) and trophectoderm (TE)-like cells. Gene Ontology analysis of enriched proteins in TE-like cells harmoniously aligns with transcriptomic data, indicating that single-cell proteomics possesses the capability to identify biologically relevant differences between these two lineages within the blastocyst.

biochemistry↗

bioRxiv2021

Effects of domestication on neophobia: A comparison between the domesticated Bengalese finch (Lonchura striata var. domestica) and its wild ancestor, the white-rumped munia (Lonchura striata)

Bengalese finches (Lonchura striata var. domestica) have more complex song traits than their wild ancestors, white-rumped munias (Lonchura striata). Domesticated finches are likely able to allocate more resources to song development rather than allocating resources to mechanisms aimed at coping with predation, which are no longer needed under domesticated conditions. Here, we aimed to examine the effects of changes in selection pressure due to domestication on the behaviour of Bengalese finches and to contemplate the possible evolutionary mechanisms underlying these changes. To do so, we compared neophobic responses to novel-object conditions as an assessment of reactions to potential predators. We studied groups of Bengalese finches and white-rumped munias and found that Bengalese finches were more likely to eat the food provided to them under novel-object conditions. Bengalese finches had a shorter latency time to eat, and this latency time was less affected by the novel object in the case of Bengalese finches compared to white-rumped munias. Therefore, Bengalese finches have reduced neophobic responses due to domestication. The behavioural strategies of white-rumped munias appear to be more suitable for natural environments, which include unpredictable risks, whereas Bengalese finches have likely adapted their behaviour to the conditions of artificial selection.

animal behavior and cognition↗

bioRxiv2018

Effect of continuous compressive force on the expression of RANKL, OPG, and VEGF in MC3T3-E1 and MLO-Y4 cells

Osteocytes, known to have mechano-sensory functions, influence the regulation of bone remodeling. However, the mechanism by which osteocytes regulate bone metabolism when mechanical forces are being applied is still unclear. Osteoclastogenesis is mainly regulated by receptor activator of nuclear factor kappa-B ligand (RANKL); the protein osteoprotegerin (OPG) and angiogenesis also play important roles in osteogenesis. RANKL, OPG, and vascular endothelial growth factor (VEGF) are thought to be key factors for bone metabolism. In this study, we examined the effect of a continuous compressive force (CF) on the expression of RANKL, OPG, and VEGF in osteoblastic murine osteocytes (MLO-Y4) and osteoblastic (MC3T3-E1) cells. Gene and protein expression levels of RANKL, OPG, and VEGF in MLO-Y4 and MC3T3-E1 cells were quantitatively determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). Both cell types were also subjected to a CF of 1.0 g/cm2 for 1, 3, 6, and 12 hours. Furthermore, the effect of a stretch-activated (S-A) channel was examined by gadolinium (Gd3+) administration. The ratio of gene and protein expressions of RANKL, VEGF, and RANKL/OPG in MLO-Y4 cells were significantly higher than in MC3T3-E1 cells, while the expression of OPG was significantly lower. After CF application, both cell types showed significant increases in RANKL and VEGF expression as well as the RANKL/OPG ratio. Additionally, the upregulated gene and protein levels of these factors were reduced by Gd3+ administration.\n\nThese findings suggest that osteocytes play more important roles in bone metabolism and angiogenesis than osteoblasts. Osteocytes regulate the expression of RANKL, OPG, and VEGF via the S-A channel through the response to mechanical stress.

cell biology↗