Harb, J. | bioRxiv Science

bioRxiv2026

Profiling of Secondary Metabolites of Leaves of Indigenous and Introduced Grapes Collected from Hebron and Bethlehem Regions in the West Bank-Palestine.

Grape (Vitis vinifera L.) is one of the most cultivated plant species and has a long history in the Levant. Grape products, including leaves, are highly appreciated as healthy products, mainly because of their high levels of nutraceuticals. The significance of these products lies in the fact that poor diet is the primary cause of malnutrition, which is associated with severe noncommunicable diseases. Accordingly, this study aimed to profile secondary metabolites in a selection of grape genotypes from Hebron and Bethlehem regions in the West Bank-Palestine that include both indigenous and introduced genotypes. Fresh, delicate leaves from each genotype-region combination were analyzed for their content of secondary metabolites via LC-MS. The results revealed that the collection regions had a negligible impact, whereas the genotype impact was high and significant. More importantly, the secondary metabolites profiles of leaves allow for the clustering of the assessed genotypes into a few clusters, each with a specific set of metabolites that can serve as fingerprint profile. In conclusion, the results of the present study revealed the diversity of grape genotypes at the metabolomics level, which will help preserve indigenous grape genotypes and aid in the development of grape varieties that can cope with the adverse impacts of climate change.

plant biology↗

bioRxiv2022

CRISPR-Mediated Generation and Characterization of a Gaa Homozygous c.1935C>A (p.D645E) Pompe Disease Knock-in Mouse Model Recapitulates Human Infantile Onset-Pompe Disease

Pompe disease (PD) is an autosomal recessive disorder caused by deficient lysosomal acid -glucosidase (GAA), leading to reduced degradation and subsequent accumulation of intra-lysosomal glycogen in tissues, especially skeletal and oftentimes cardiac muscle. The c.1935C>A (p.Asp645Glu) variant is the most frequent GAA pathogenic mutation in people of Taiwanese and Southern Chinese ethnicity, causing infantile-onset PD (IOPD), which presents neonatally with severe hypertrophic cardiomyopathy, profound muscle hypotonia, and respiratory failure leading to premature death if untreated. To further investigate the pathogenic mechanism and facilitate development of therapies pertaining to this variant, we applied CRISPR-Cas9 homology-directed repair (HDR) using a novel dual sgRNA approach flanking the target site to generate a GaaEm1935C>A knock-in mouse model as well as a myoblast cell line carrying the Gaa c.1935C>A mutation. Herein we describe the molecular, biochemical, physiological, histological, and behavioral characterization of 3-month-old homozygous GaaEm1935C>A mice. Homozygous GaaEm1935C>A knock-in mice exhibited normal Gaa mRNA expression levels relative to wild-type mice, but GAA enzymatic activity was almost completely abolished, leading to a substantial increase in tissue glycogen storage, and significant concomitant impairment of autophagy. Echocardiography of 3-month-old knock-in mice revealed significant cardiac hypertrophy. The mice also demonstrated skeletal muscle weakness but, paradoxically, not early mortality. Longitudinal studies of this model, including assessment of its immune response to exogenously supplied GAA enzyme, are currently underway. In summary, the GaaEm1935C>A knock-in mouse model recapitulates the molecular, biochemical, histopathologic, and phenotypic aspects of human IOPD caused by the GAA c.1935C>A pathogenic variant. It is an ideal model to assess innovative therapies to treat IOPD, including personalized therapeutic strategies that correct pathogenic variants, restore GAA activity and produce functional phenotypes.

genetics↗