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Adzuki bean (Vigna angularis) is an important legume crop cultivated in over 30 countries worldwide. We developed a high-quality chromosome-level reference genome of adzuki bean cultivar Jingnong6 by combining PacBio Sequel long-read sequencing with short-read and Hi-C technologies. The assembled genome covers 97.8% of the adzuki bean genome with a contig N50 of approximately 16 Mb and a total of 32 738 protein-coding genes. We also generated a comprehensive genome variation map of adzuki bean by whole-genome resequencing (WGRS) of 322 diverse adzuki beans accessions including both wild and cultivated. Furthermore, we have conducted comparative genomics and a genome-wide association study (GWAS) on key agricultural traits to investigate the evolution and domestication. GWAS identified several candidate genes, including VaCycA3;1, VaHB15, VaANR1 and VaBm, that exhibited significant associations with domestication traits. Furthermore, we conducted functional analyses on the roles of VaANR1 and VaBm in regulating seed coat colour. We provided evidence for the highest genetic diversity of wild adzuki (Vigna angularis var. nipponensis) in China with the presence of the most original wild adzuki bean, and the occurrence of domestication process facilitating transition from wild to cultigen. The present study elucidates the genetic basis of adzuki bean domestication traits and provides crucial genomic resources to support future breeding efforts in adzuki bean., (© 2024 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Eukaryotic genes contain introns that are removed by the spliceosomal machinery during mRNA maturation. Introns impose a huge energetic burden on a cell; therefore, they must play an essential role in maintaining genome stability and/or regulating gene expression. Many genes (> 50%) in Plasmodium parasites contain predicted introns, including introns in 5' and 3' untranslated regions (UTR). However, the roles of UTR introns in the gene expression of malaria parasites remain unknown., Methods: In this study, an episomal dual-luciferase assay was developed to evaluate gene expression driven by promoters with or without a 5'UTR intron from four Plasmodium yoelii genes. To investigate the effect of the 5'UTR intron on endogenous gene expression, the pytctp gene was tagged with 3xHA at the N-terminal of the coding region, and parasites with or without the 5'UTR intron were generated using the CRISPR/Cas9 system., Results: We showed that promoters with 5'UTR introns had higher activities in driving gene expression than those without 5'UTR introns. The results were confirmed in recombinant parasites expressing an HA-tagged gene (pytctp) driven by promoter with or without 5'UTR intron. The enhancement of gene expression was intron size dependent, but not the DNA sequence, e.g. the longer the intron, the higher levels of expression. Similar results were observed when a promoter from one strain of P. yoelii was introduced into different parasite strains. Finally, the 5'UTR introns were alternatively spliced in different parasite development stages, suggesting an active mechanism employed by the parasites to regulate gene expression in various developmental stages., Conclusions: Plasmodium 5'UTR introns enhance gene expression in a size-dependent manner; the presence of alternatively spliced mRNAs in different parasite developmental stages suggests that alternative slicing of 5'UTR introns is one of the key mechanisms in regulating parasite gene expression and differentiation., (© 2024. The Author(s).)

Background: Cerebral ischemia leads to linguistic and motor dysfunction, as the death of neurons in ischemic core is permanent and non-renewable. An innovative avenue is to induce and/or facilitate reprogramming of adjacent astrocytes into neurons to replace the lost neurons and re-establish brain homeostasis., Purpose: This study aimed to investigate whether the p-hydroxy benzaldehyde (p-HBA), a phenolic compound isolated from Gastrodia elata Blume, could facilitate the reprogramming of oxygen-glucose deprivation/reperfusion (OGD/R)-damaged astrocytes into neurons., Study Design/methods: The primary parenchymal astrocytes of rat were exposure to OGD and reperfusion with define culture medium. Cells were then incubated with different concentration of p-HBA (1, 10, 100, 400 μM) and collected at desired time point for reprogramming process analysis., Results: OGD/R could elicit endogenous neurogenic program in primary parenchymal astrocytes of rat under define culture condition, and these so-called reactive astrocytes could be reprogrammed into neurons. However, the neonatal neurons produced by this endogenous procedure could not develop into mature neurons, and the conversion rate was only 1.9%. Treatment of these reactive astrocytes with p-HBA could successfully promote the conversion rate to 6.1%, and the neonatal neurons could develop into mature neurons within 14 days. Further analysis showed that p-HBA down-regulated the Notch signal component genes Dll1, Hes1 and SOX2, while the transcription factor NeuroD1 was up-regulated., Conclusion: The results of this study demonstrated that p-HBA facilitated the astrocyte-to-neuron conversion. This chemical reprogramming was mediated by inhibition of Notch1 signaling pathway and transcriptional activation of NeuroD1.

Background: The formation of glial scar around the ischemic core following cerebral blood interruption exerts a protective effect in the subacute phase but impedes neurorepair in the chronic phase. Therefore, the present study aimed to explore whether p-hydroxy benzaldehyde (p-HBA), a phenolic compound isolated from Gastrodia elata Blume, can cut the Gordian knot of glial scar and promote brain repair after cerebral ischemia., Methods: The effects of p-HBA on neurorepair were evaluated using a rat model of transient middle cerebral artery occlusion (tMCAO). The motor functions were evaluated by neurobehavioral tests, the pathophysiological processes in the peri-infarct cortex (PIC) were detected by viral-based lineage tracking or immunofluorescence staining, and the putative signaling pathway was analyzed by western blot., Results: Administration of p-HBA in the acute stage after stroke onset alleviated the motor impairment in tMCAO rats in a time-dependent manner. The corresponding cellular events were inhibition of astrogliosis, facilitating the conversion of reactive astrocytes (RAs) into neurons, and prompting angiogenesis in PIC, thereby protecting the structure of the neurovascular unit (NVU). One of the underlying molecular mechanisms is the activation of the neurogenic switch of the Wnt/β-catenin signaling pathway. Notably, p-HBA only promotes astrocyte-to-neuron conversion in the PIC, and only partial RAs were converted to neurons. This pattern of conversion ensures that the brain structure remains unaltered, and the beneficial role of glial scarring is preserved during the subacute phase after ischemia., Conclusions: These results provided a potential approach to address the dilemma of glial scarring after brain injury, i.e., the pharmacological promotion of astrocyte-to-neuron conversion in the PIC without interfering with normal brain tissue, which mitigates but does not eliminate the glial scar. Subsequently, the neuron rescue-unfriendly environment is switched to a beneficial reconstruction milieu in PIC, which is conducive to neurorepair. Moreover, p-HBA could be a candidate for pharmacological intervention., (Copyright © 2022. Published by Elsevier GmbH.)

CRISPR/Cas9 is a powerful genome editing method that has greatly facilitated functional studies in many eukaryotic organisms including malaria parasites. Due to the lack of genes encoding enzymes necessary for the non-homologous end joining DNA repair pathway, genetic manipulation of malaria parasite genomes is generally accomplished through homologous recombination requiring the presence of DNA templates. Recently, an alternative double-strand break repair pathway, microhomology-mediated end joining, was found in the Plasmodium falciparum parasite. Taking advantage of the MMEJ pathway, we developed a MMEJ-based CRISPR/Cas9 (mCRISPR) strategy to efficiently generate multiple mutant parasites simultaneously in genes with repetitive sequences. As a proof of principle, we successfully produced various size mutants in the central repeat region of the Plasmodium yoelii circumsporozoite surface protein without the use of template DNA. Monitoring mixed parasite populations and individual parasites with different sizes of CSP-CRR showed that the CSP-CRR plays a role in the development of mosquito stages, with severe developmental defects in parasites with large deletions in the repeat region. However, the majority of the csp mutant parasite clones grew similarly to the wild type P. yoelii 17XL parasite in mice. This study develops a useful technique to efficiently generate mutant parasites with deletions or insertions, and shows that the CSP-CRR plays a role in parasite development in mosquito., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: East Asian dog breeds are one of the most ancient groups of dogs that radiated after the domestication of the dog and represent the most basal lineages of dog evolution. Among these, the Chow Chow is an ancient breed that embodies very distinct morphological and physiological features, such as sturdy build, dense coat, and blue/purple tongue., Results: Using a Restricted site Associated DNA (RAD) sequencing approach, we sequenced the genomes of nine Chow Chows from China. Combined with a dataset of 37 canid whole genome sequencing (WGS) from several published works, we found that the Chow Chow is one of the most basal lineages, which originated together with other East Asian breeds, such as the Shar-Pei and Akita. Demographic analysis found that Chow Chows originated from the Chinese indigenous dog about 8300 years ago. The bottleneck leading to Chow Chows was not strong and genetic migration between Chow Chows and other populations is low. Two classes of genes show strong evidence of positive selection along the Chow Chow lineage, namely genes related to metabolism and digestion as well as muscle/heart development and differentiation., Conclusions: Dog breeds from East Asia, including the Chow Chow, originated from Chinese indigenous dogs very early in time. The genetic bottleneck leading to Chow Chows and migrations with other populations are found to be quite mild. Our current study represents an early endeavor to characterize the origin of East Asian dog breeds and establishes an important reference point for understanding the origin of ancient breeds in Asia.

A rapid, sensitive and selective high-performance liquid chromatography mass spectrometric method has been developed and validated for the simultaneous determination of oxymatrine and its active metabolite matrine in human plasma after administration of oxymatrine oral solution. Analytes were extracted from the plasma by liquid-liquid extraction with chloroform. The chromatographic separation was accomplished on a Venusil C18 column (150 mm × 4.6 mm, 5 μm) protected by a C18 guard column (4.0 mm × 2.0 nm; Phenomenex, Torrance, CA, USA). Analytes were detected on a single quadruple mass spectrometer by selected ion monitoring mode via electrospray ionization source. The assay had a lower limit of quantification of 1.5 ng·mL(-1) for oxymatrine and 3 ng·mL(-1) for matrine in plasma. The calibration curves were linear in the measured range. The overall precision and accuracy for all concentrations of quality controls and standards were within ±15%. The proposed method enabled unambiguous identification and quantification of oxymatrine and its active metabolite matrine in vivo. The results provided a meaningful basis for evaluating the clinical applications of the oxymatrine oral solution., (Copyright © 2013. Published by Elsevier B.V.)