Biology subjects

Sapir, T.

Publications and source records attributed to Sapir, T..

8 recordsLinked to original sources

bioRxiv2026

Intelliwaste: NMR of 13C-labeled Spent Media Enables Non-Invasive Metabolic Fingerprinting of Pluripotent Stem Cells and LIS1-Associated Neuropathology

Metabolic dysregulation is increasingly recognized as a key contributor to neurodevelopmental disorders. Here, we present Intelliwaste, a non-invasive, cost-effective method for profiling carbon metabolism in pluripotent stem cells and brain organoids using 13C-labeled metabolites and 1H and 13C NMR spectroscopy. This approach enables longitudinal analysis of extracellular fluxes without disrupting cell viability. We apply Intelliwaste to human embryonic stem cells (hESCs) cultured in a defined media enriched with >95% 13C1-Glucose. Under these conditions, 13C3-lactate emerged as the most abundant labeled product, with 20-50-fold lower fluxes to 13C3-alanine, 13C2-acetate, 13C3-serine, and 13C3-pyruvate, and 100-300-fold lower fluxes to 13C1-formate and multiple 13C-labeled glutamate species. These profiles allow for precise quantification of fractional metabolic isotopic labeling and glucose-derived carbon flow. To demonstrate biological utility, we first examine the effect of L-glutamine omission, which selectively reduces 13C3-alanine/13C3-lactate and 13C4-glutamate/13C3-lactate flux ratios, while the 13C3-Glutamate/13C3-Lactate and 13C2-Glutamate/13C3-Lactate flux ratios remained unchanged. These findings suggest a specific role for extracellular glutamine in modulating the activity of alanine aminotransferase and pyruvate carboxylase. We then characterized LIS1 mutant hESCs--a model of lissencephaly--and observed significantly increased flux ratios involving 13C4-, 13C3-, and 13C2-glutamate relative to 13C3-lactate, indicating enhanced glutamate production via the TCA cycle. These findings establish Intelliwaste as a powerful tool for metabolic profiling in the study of human neurodevelopment and disease. Its non-destructive nature makes it particularly well-suited for tracking metabolic changes during differentiation and in patient-derived organoid models of neurological disorders.

cell biology↗

bioRxiv2025

Neural Progenitors as a Novel Pathogenic Mechanism in Microcephaly

Despite their significance, the genetic and molecular bases of neurodevelopmental disorders remain poorly understood. In this study, using human brain organoids and mouse models, we show that loss of NDE1, a gene closely associated with microcephaly, disrupts progenitor identity, prolongs mitosis, and alters regional patterning in the forebrain. NDE1 knockout leads to a caudal identity shift of neural progenitor cells in the organoids and mouse brains, coinciding with aberrant ERK signaling. Notably, downstream activation of the ERK pathway restored rostral PAX6 expression in human brain organoids. Parallel analyses of Nde1 knockout mice confirmed disrupted regional patterning of the forebrain. Together, our data establish NDE1 as a critical regulator of early human brain regionalization and elucidate molecular mechanisms underlying the structural abnormalities observed in NDE1-associated microcephaly.

developmental biology↗

bioRxiv2024

Parallel Evolution of Salinity Tolerance in Arabidopsis thaliana Accessions from Cape Verde Islands

Soil salinization poses a significant threat to crop production impacting one fifth of all cultivated land. The Cape Verde Islands are located 600 km from the coast of Africa and are characterized by high salinity soils and inland water sources. In this study we find that Arabidopsis thaliana plants native to these islands accumulate a metabolite that protects them from salt stress. We partially characterized this metabolite as glucuronyl-mannose. We find that the ability to produce glucuronyl-mannose evolved independently in two different islands from the same archipelago through mutations in the same gene, an alpha glycosidase protein that we named GH38cv. These cases of parallel evolution suggest positive selection towards the increase of salt tolerance with low fitness costs. Indeed, plants carrying derived alleles of GH38cv do not present growth defects or low defenses under normal conditions, but show better germination rates, longer roots and better hydric status than non-mutated plants when exposed to salt stress. These findings provide a knowledge-based method to develop salt resilient crops using natural mechanisms, which could be attractive both to conventional and organic agriculture.

plant biology↗

bioRxiv2024

Altered Extracellular Matrix Structure and Elevated Stiffness in a Brain Organoid Model for Disease

The viscoelasticity of tissues impacts their shape, as well as the growth and differentiation of their cells. Nevertheless, little is known about changes in viscoelastic properties during brain malformations. Lissencephaly is a severe malformation of cortical development caused by LIS1 mutations, which results in a lack of cortical convolutions. Here, we show that human-derived brain organoids with LIS1 mutation are stiffer than control ones at multiple developmental times. This stiffening is accompanied by abnormal ECM expression and organization, as well as elevated water content, as measured by diffusion-weighted MRI. Proteolytic cleavage of ECM components by short-term treatment with the catalytic subunit of MMP9 reduced the stiffening and water diffusion levels of mutated organoids to control levels. Finally, based on the molecular and rheological properties obtained, we generated a computational microstructure mechanical model that can successfully predict mechanical changes that follow differential ECM localization and integrity in the developing brain. Overall, our study reveals that LIS1 is essential for the expression and organization of ECM proteins during brain development, and its mutation leads to a substantial viscoelastic change. To our knowledge, this is the first study to elucidate how tissue mechanics change in disease states using human brain organoids.

developmental biology↗

bioRxiv2023

Maize (Zea mays L.) interaction with the arbuscular mycorrhizal fungus Rhizophagus irregularis allows mitigation of nitrogen deficiency stress: physiological and molecular characterization

Maize is currently the most productive cereal crop in the world (www.faostat.org). Maize can form a symbiotic relationship with the Arbuscular Mycorrhizal Fungus (AMF), Rhizophagus irregularis. In this relationship, the fungus provides the plant with additional water and mineral nutrients, while the plant supplies carbon compounds to the fungus. Little is known about the N metabolism disruption during symbiosis in both partners. To address this issue, two genetically distant maize lines were studied in terms of physiological and molecular responses to AMF inoculation by dual RNA-seq, metabolomics and phenotyping. Interestingly, the beneficial effects of the AMF were observed mainly under conditions of limited N fertilization. Under such conditions, the AMF helped maintain plant biomass production. The availability of nitrogen was found to be a crucial factor influencing all the traits studied showing that the level of N supply plays a pivotal role in determining how maize plants interact with the AMF. Despite the two maize lines showing different transcriptomic and metabolomic responses to R. irregularis, their agro-physiological traits remained similar. Both the plant and fungal transcriptomes were more significantly influenced by the level of N nutrition rather than the specific maize genotype. This suggests that N availability has a more profound impact on gene expression in both organisms than the genetic makeup of the maize plant. To understand the metabolic implications of this symbiotic relationship, we integrated transcriptomic data into our recently built multi-organ Genome-scale metabolic model (GSM) called iZMA6517. Remarkably, this modelling approach was supported by metabolomics profiling, in particular increased leaf pyrimidine levels in response to AMF inoculation under limiting N supply. Consistently, fungal genes involved in pyrimidine de novo synthesis and salvage were found to be expressed in symbiotic roots. Our work highlights nucleotide and ureides metabolism as previously unrecognized factors contributing to the symbiotic N nutrition facilitated by R. irregularis, thereby enhancing maize growth. This study demonstrates the effectiveness of integrating multi-omics approaches with mathematical modelling to uncover novel metabolic mechanisms associated with AM symbiosis, without a priori.

plant biology↗

bioRxiv2022

LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the activity of the molecular motor cytoplasmic dynein. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it regulates the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the expression of extracellular matrix (ECM) and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post- transcriptional regulation underlying development and mechanosensitive processes.

cell biology↗

bioRxiv2022

Interaction between photoperiod and variation in circadian rhythms in tomato

Many biological processes follow circadian rhythmicity and are controlled by the circadian clock. Predictable environmental changes such as seasonal variation in photoperiod can modulate circadian rhythms, allowing organisms to adjust to the time of the year. Modification of circadian clocks is especially relevant in crops to enhance their cultivability in specific regions by changing their sensibility to photoperiod. In tomato, the appearance of mutations in EMPFINDLICHER IM DUNKELROTEN LICHT 1 (EID1, Solyc09g075080) and NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED GENE 2 (LNK2, Solyc01g068560) during domestication delayed its circadian rhythms, and allowed its expansion outside its equatorial origin. Here we study how variation in circadian rhythms in tomato affects its perception of photoperiod. To do this, we create near isogenic lines carrying combinations of wild alleles of EID1 and LNK2 and perform transcriptomic profiling under two different photoperiods. We observe that EID1, but not LNK2, has a large effect on the tomato transcriptome and its response to photoperiod. This large effect of EID1 is likely a consequence of the global phase shift elicited by this gene in tomatos circadian rhythms.

plant biology↗

bioRxiv2017

Converting a broad matrix metalloproteinase family inhibitor into a specific inhibitor of MMP-9 and MMP-14

MMP-14 and MMP-9 are two well established cancer targets for which no specific clinically relevant inhibitor is available. Using a powerful combination of computational design and yeast surface display technology, we engineered such an inhibitor starting from a non-specific MMP inhibitor, N-TIMP2. The engineered purified N-TIMP2 variants showed enhanced specificity towards MMP-14 and MMP-9 relative to a panel of off-target MMPs. MMP-specific N-TIMP2 sequence signatures were obtained that could be understood from the structural perspective of MMP/N-TIMP2 interactions. Our MMP-9 inhibitor exhibited 1000-fold preference for MMP-9 vs. MMP-14, which is likely to translate into significant differences under physiological conditions. Our results provide new insights regarding evolution of promiscuous proteins and optimization strategies for design of inhibitors with single-target specificities.

biophysics↗