Biology subjects

Hamada, A.

Publications and source records attributed to Hamada, A..

6 recordsLinked to original sources

bioRxiv2025

Functional impact of pathogenic Runt domain mutations in Runx2 in vivo: Insights into the skeletal and dental anomalies of cleidocranial dysplasia

Runt-related transcription factor 2 (RUNX2) is essential for skeletogenesis, and mutations in its gene cause cleidocranial dysplasia (CCD), an autosomal dominant skeletal disorder. The evolutionarily conserved 128-amino acid Runt homology domain (RHD) of human RUNX2 is essential for DNA binding and heterodimerization, and serves as a mutation hotspot associated with severe CCD phenotypes. To elucidate the functional impact of pathogenic RHD mutations in vivo, we generated two novel mouse lines: one carrying a missense mutation, c.695G>A (p.R232Q) (Runx2m/+), corresponding to the human RUNX2 c.674G>A (p.R225Q), and the other harboring a frameshift mutation, c.697_698delGA (p.E233TfsTer9) (Runx2112/+), causing a premature stop codon. Homozygous Runx2m/mand Runx2112/112 mice lacked membranous ossification, whereas heterozygous Runx2m/+ and Runx2112/+ mice displayed typical CCD-like skeletal features, including an open anterior fontanelle and clavicle hypoplasia. Unexpectedly, heterozygotes carrying pathogenic mutations in RHD developed an accessory root-like protrusion at the furcation of three-rooted maxillary first molars, representing a previously unrecognized dental phenotype during root development. Dual luciferase assays revealed impaired transactivation of the p.R232Q mutant Runx2 on the osteocalcin enhancer/promoter. Wild-type Runx2 was robustly expressed in osteoblasts and hypertrophic chondrocytes during bone formation, but the mutant Runx2 exhibited reduced expression in hypertrophic chondrocytes and partially impaired nuclear localization, resulting in arrested osteoblast and chondrocyte maturation. Our mutant mouse model provides a valuable in vivo platform to study CCD pathogenesis, mechanisms of tooth root furcation, and therapeutic interventions targeting dysfunctional RHD.

genetics↗

bioRxiv2025

Metabolism of L-threonate, an ascorbate degradation product, requires a protein with L-threonate metabolizing domains in Arabidopsis

L-Threonate is one of the major degradation products of ascorbate in plants. While bacteria can utilize L-threonate as a sole carbon source by converting it to dihydroxyacetone phosphate, a glycolysis intermediate, through a three- or four-step metabolic pathway, the corresponding processes in plants remain uncharacterized. Remarkably, an Arabidopsis gene encodes a unique protein containing domains homologous to all three enzymes involved in the bacterial three-step pathway. We designated this protein as L-threonate metabolizing domains (LTD) and investigated its functional role in plant L-threonate metabolism. Despite extensive efforts, recombinant expression of LTD was unsuccessful, likely due to its large protein size. Therefore, a reverse genetic approach was employed, using ltd knockout Arabidopsis lines to explore LTD function. Under continuous dark conditions, where ascorbate degradation is facilitated, LTD transcription was significantly upregulated, leading to increased L-threonate dehydrogenase (LtnD) activity. Knockout lines of LTD exhibited no detectable LtnD activity under both light and dark conditions, alongside elevated levels of L-threonate compared to wild-type plants. These results indicate that LTD is essential for L-threonate metabolism in Arabidopsis. The LTD gene is highly conserved among land plants but is absent in green algae, providing a hypothesis that the rise in ascorbate concentrations during plant evolution necessitated a more active metabolism of ascorbate degradation products.

plant biology↗

bioRxiv2025

Foliar ascorbate degradation in the dark occurs independently of ascorbate peroxidase/oxidase, recycling enzymes, and senescence signaling

Ascorbate is a key antioxidant that protects plant cells from oxidative damage. While plants actively synthesize ascorbate during the day, its degradation becomes prominent under prolonged dark conditions. Since ascorbate degradation begins with its oxidized form, dehydroascorbate (DHA), this process inherently requires ascorbate oxidation. However, the molecular mechanisms underlying dark-induced ascorbate oxidation and subsequent degradation remain unclear. In this study, we investigated the role of intracellular and extracellular ascorbate redox regulation in controlling this process. Using Arabidopsis knockout mutants for key enzymes involved in ascorbate oxidation and recycling, including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and ascorbate oxidase (AO), as well as NADPH oxidases (rbohD and rbohF), we found that none of these enzymes significantly influenced the dark-induced decrease in ascorbate levels. Notably, ascorbate levels decreased similarly in newly generated multiple mutants, including a quintuple mutant ({Delta}dhar pad2 mdar5), which has severely impaired ascorbate recycling capacity, and the ao2 rbohD double mutant, which is strongly expected to exhibit a highly altered apoplastic redox state. Furthermore, we examined the potential involvement of senescence signaling, including ORESARA1 and ethylene signaling components, but found no evidence for their contribution. These findings indicate that the dark-induced decrease in ascorbate levels is not governed by conventional pathways for ascorbate oxidation and recycling or senescence signaling processes, suggesting an unidentified regulatory mechanism.

plant biology↗

bioRxiv2022

A rapid and universal liquid chromatograph-mass spectrometry-based platform, refmAb-Q nSMOL, for monitoring monoclonal antibody therapeutics

Accurate quantitation of antibody is critical for development of monoclonal antibody therapeutics (mAbs). Therapeutic drug monitoring has been applied to measure levels of mAbs in clinics for dose adjustment for autoimmune disease. Trough levels of mAbs can be a biomarker for cancer immunotherapy. Thus, the deployment of a rapid and universal platform for mAb monitoring may benefit processes ranging from drug development to clinical practice for a wide spectrum of diseases. However, mAb monitoring often requires development and conduct of an individual ligand binding assay such as ELISA, which is impractical to scale. We streamlined quantitation of antibody therapeutics by a nano-surface and molecular-orientation limited (nSMOL) proteolysis assay using LC-MS with a universal reference antibody (refmAb-Q), for accurate multiplexed quantitation of unique signature peptides derived from mAbs. This innovative refmAb-Q nSMOL platform may provide a practical solution for quantitating an ever-increasing number of mAbs from developmental to clinical use settings.

immunology↗

bioRxiv2021

Genomic style: yet another deep-learning approach to characterize bacterial genome sequences

MotivationBiological sequence classification is the most fundamental task in bioinformatics analysis. For example, in metagenome analysis, binning is a typical type of DNA sequence classification. In order to classify sequences, it is necessary to define sequence features. The k-mer frequency, base composition, and alignment-based metrics are commonly used. In contrast, in the field of image recognition using machine learning, image classification is broadly divided into those based on shape and those based on style. A style matrix was introduced as a method of expressing the style of an image (e.g., color usage and texture). ResultsWe propose a novel sequence feature, called genomic style, inspired by image classification approaches, for classifying and clustering DNA sequences. As with the style of images, the DNA sequence is considered to have a genomic style unique to the bacterial species, and the style matrix concept is applied to the DNA sequence. Our main aim is to introduce the genomics style as yet another basic sequence feature for metagenome binning problem in replace of the most commonly used sequence feature k-mer frequency. Performance evaluations show that our method using style matrix achieves the superior accuracy than state-of-the-art binning tools based on k-mer frequency.

bioinformatics↗

bioRxiv2020

Human dental pulp stem cells: a sanctuary for relapsing Bartonella quintana.

Bartonella quintana is a facultative intracellular bacterium responsible for relapsing fever, an example of non-sterilizing immunity. The cellular sanctuary of B. quintana in-between febrile relapses remains unknown but repeated detection of B. quintana in dental pulp specimens suggested long-term half-life dental pulp stem cells (DPSCs) as candidates. As the capacity of DPSCs to internalize microscopic particles was unknown, we confirmed that DPSCs internalized B. quintana bacteria: Gimenez staining and fluorescence microscopy localized B. quintana bacteria inside DPSCs and this internalization did not affect the cellular multiplication of DPSCs during a one-month follow-up despite the increase in the bacterial load. B. quintana-infected DPSCs did not produce Tumor Necrosis Factor- whereas an important production of Monocytes Chemoattractant Protein-1 was observed. These unprecedented observations suggested the possibility that DPSCs were shelters for the long-term persistence of B. quintana in the host, warranting further experimental and clinical investigations.

microbiology↗