Biology subjects

Gilbert, A.

Publications and source records attributed to Gilbert, A..

11 recordsLinked to original sources

bioRxiv2026

A massively parallel reporter screen defines the syntax of SECReTE, a secretion-enhancing cis regulatory targeting element, in mammalian cells

Messenger RNA (mRNA) localization to specific subcellular regions is crucial for proper protein translation and localization. Potential means to localize mRNAs include cis-acting or structural motifs embedded within the sequence. We previously identified a secretion-enhancing cis regulatory targeting element (SECReTE) enriched in mRNAs encoding secreted proteins from prokaryotes, eukaryotes, and viruses, found to enhance both protein synthesis and secretion by localizing mRNAs to the endoplasmic reticulum (ER) membrane. Defined as at least 10 consecutive NNY triplets (where N is any nucleotide and Y is a pyrimidine), this highly degenerate motif can be specified by approximately ~10^15 different sequence combinations. Yet, some combinations may prove more efficient than others in regulating mRNA localization and protein secretion. To better understand SECReTE syntax and to identify robust combinations, we designed and synthesized a library of ~5 x 10^4 variants and inserted them into the 3' untranslated region (3'UTR) of two reporter genes, green fluorescent protein (GFP) and immunoglobulin light chain. We expressed each variant in Chinese hamster ovary cells and assayed their activity using parallel assays to measure the effect of each variant upon mRNA partitioning between the ER and cytoplasm, and protein secretion. Our results reveal that ER-associated mRNAs are enriched with SECReTE elements of a defined syntax. Notably, RRY repeats (where R is purine) were superior to NNY repeats. Variants conferring a high mRNA ER localization score consistently enhanced protein secretion, confirming the mechanistic relationship between mRNA targeting to the ER and secretion efficiency. The correlation between variant performances across both reporters suggests a shared regulatory mechanism. Additional analysis identified a novel secretion-promoting cis element, called "split NNS" (where S is G or C). This work refines the syntactic definition of mRNA localization motifs that regulate protein secretion in both natural and heterologous expression systems.

cell biology↗

bioRxiv2026

Seeing Just Enough: The Contribution of Hands, Objects and Visual Features to Egocentric Action Recognition

Humans recognize everyday actions without conscious effort despite challenges such as poor viewing conditions and visual similarity between actions. Yet the visual features contributing to action recognition remain unclear. To address this, we combined semantic modelling and feature reduction methods to identify critical features for recognizing actions from challenging egocentric perspectives. We first identified egocentric action videos from home environments that a motion-focused action classification network could correctly classify (Easy videos) or not (Hard videos). In Experiment 1, participants (N=136) labelled the action and object in the videos. Using a language model framework, we derived human ground truth labels for each video and quantified its recognition consistency based on semantic similarity. Participants recognized actions and objects in Easy videos more consistently than in Hard videos. In Experiment 2, we recursively reduced the Easy and Hard videos with high recognition consistency to extract minimal recognizable configurations (MIRCs), in which any further spatial or temporal reductions disrupted recognition. The data was collected using a large-scale online study (N=4360). We extracted information related to the hand, objects, scene background and visual features (e.g., orientation or motion signals) from the 474 MIRCs. Binary classification showed that recognition was disrupted when regions containing the manipulated object and strong orientation signals were removed, while temporal reduction by frame-scrambling disrupted recognition in 73% of MIRCs. The active hand had some marginal contribution. Our results highlight the importance of both mid- and high-level information for egocentric action recognition and link hierarchical feature theories with naturalistic human perception.

neuroscience↗

bioRxiv2025

Sensitivity to ATR-CHK1 pathway inhibition in AML/MDS is enhanced by SRSF2 mutations and reduced by RUNX1 loss

SRSF2 mutations occur in up to 25% of acute myeloid leukemia (AML) and 17% of myelodysplastic syndrome (MDS) cases and are associated with poor prognosis, yet no mutation-directed therapy exists. Here, we aimed to identify therapeutically targetable vulnerabilities in MDS/AML with SRSF2 mutations. Ex vivo drug-sensitivity testing of bone marrow cells from AML patients and healthy donors showed that SRSF2-mutant cells are sensitive to inhibitors of CHK1, and WEE1 DNA damage response (DDR) kinases. To test causality, we engineered isogenic K562 cell line clones expressing SRSF2P95H/L/R mutations. RNA sequencing confirmed splicing aberrations characteristic of MDS/AML in these clones. We found that SRSF2P95H/L/R sensitize leukemia cells to ATR-CHK1-WEE1 inhibition. Bone marrow progenitors from Srsf2P95H and U2AF1S34F knock-in mice showed heightened sensitivity to CHK1 inhibition, corroborating the human data. In contrast, RUNX1 mutations were linked to resistance against CHK1 and WEE1 inhibition in SRSF2-mutant AML samples. Runx1 loss also caused resistance to CHK1 inhibitors in knock-in mouse progenitors harboring Srsf2P95H or U2AF1S34F, indicating that RUNX1 loss is a mechanism of resistance. In conclusion, SRSF2 and U2AF1 mutations are biomarkers of sensitivity to ATR-CHK1 pathway inhibitors, while RUNX1 mutations cause resistance. These biomarkers can support patient stratification in MDS/AML.

cancer biology↗

bioRxiv2025

Maize kernel composition and morphology influences pericarp retention during nixtamalization

Background and ObjectivesPericarp retention during nixtamalization directly influences masa quality, affecting texture, machinability, and nutritional content of staple foods such as tortillas and chips. Despite its industrial relevance, the underlying kernel traits that govern pericarp retention remain poorly characterized. This study aimed to assess an existing staining method to quantify pericarp retention based on visual evaluation, and identify the compositional and morphological characteristics of maize kernels that most strongly predict pericarp retention during nixtamalization. FindingsStain-based scoring of pericarp retention showed moderate correlation with directly measured pericarp mass, achieving a Pearsons correlation coefficient of 0.77 in the rapid cook test and 0.56 in the benchtop cook test. Pearson correlation coefficients of 18 compositional and morphological traits range from -0.294 to 0.538. Ground kernel ash content and initial pericarp quantity were the most correlated and predictive variables associated with pericarp retention. ConclusionsStain-based methods lack the resolution necessary for quantitative pericarp assessment. Initial pericarp quantity and ground kernel ash content are likely key determinants of nixtamalization pericarp retention. Significance and NoveltyThis study provides a first assessment on the impacts of morphological and compositional variation for pericarp retention, which can be built upon to develop improved varieties and cooking methods for product optimization.

plant biology↗

bioRxiv2024

Interferon signaling is enhanced by ATR inhibition in glioblastoma cells irradiated with X-rays, protons or carbon ions

Background and purposeInterferon signaling plays an important role in antitumor immune responses. Inhibitors of the DNA damage response, such as ATR inhibitors, can increase interferon signaling upon conventional radiotherapy with X-rays. However, whether such inhibitors also increase interferon (IFN) signaling after high linear energy transfer (LET) particle irradiation is not known. Materials and methodsHuman glioblastoma U-251 and T98G cells were treated with X-rays, protons (linear energy transfer (LET): 7 and 38 keV/m) and carbon ions (LET: 28 and 73 keV/m), with and without ATR inhibitor (VE822) or ATM inhibitor (AZD1390). DNA damage signaling and cell cycle distribution were assayed by immunoblotting and flow cytometry, and radiosensitivity by clonogenic survival. IFN-{beta} secretion was measured by ELISA and STAT1 activation by immunoblotting. ResultsHigh-LET protons and carbon ions caused stronger activation of the DNA damage response compared to low-LET protons andX-rays at similar radiation dose. G2 checkpoint arrest was abrogated by the ATR inhibitor and prolonged by the ATM inhibitor after all radiation types. The inhibitors increased radiosensitivity, as measured after X- and carbon-ion-irradiation. ATR inhibition increased IFN signaling after both low-LET and high-LET irradiation in both cell lines. In T98G, IFN signaling was also enhanced by ATM inhibition. Notably, T98G cells secreted markedly more IFN-{beta} when the inhibitors were combined with high-LET compared to low-LET irradiation. ConclusionOur results show that ATR inhibition can increase IFN signaling after both X-, proton- and carbon-ion-irradiation. Additionally, IFN induction is strongly dependent on LET in one of the tested cell lines.

cancer biology↗

bioRxiv2021

Predicting Moisture Content During Maize Nixtamalization Using Machine Learning with NIR Spectroscopy

Lack of high throughput phenotyping systems for determining moisture content during the maize nixtamalization cooking process has led to difficulty in breeding for this trait. This study provides a high throughput, quantitative measure of kernel moisture content during nixtamalization based on NIR scanning of uncooked maize kernels. Machine learning was utilized to develop models based on the combination of NIR spectra and moisture content determined from a scaled-down benchtop cook method. A linear support vector machine (SVM) model with a Spearmans rank correlation coefficient of 0.852 between wet lab and predicted values was developed from 100 diverse temperate genotypes grown in replicate across two environments. This model was applied to NIR data from 501 diverse temperate genotypes grown in replicate in five environments. Analysis of variance revealed environment explained the highest percent of the variation (51.5%), followed by genotype (15.6%) and genotype-by-environment interaction (11.2%). A genome-wide association study identified 26 significant loci across five environments that explained between 5.04% and 16.01% (average = 10.41%). However, genome-wide markers explained 10.54% to 45.99% (average = 31.68%) of the variation, indicating the genetic architecture of this trait is likely complex and controlled by many loci of small effect. This study provides a high-throughput method to evaluate moisture content during nixtamalization that is feasible at the scale of a breeding program and provides important information about the factors contributing to variation of this trait for breeders and food companies to make future strategies to improve this important processing trait. Key MessageMoisture content during nixtamalization can be accurately predicted from NIR spectroscopy when coupled with a support vector machine (SVM) model, is strongly modulated by the environment, and has a complex genetic architecture.

plant biology↗

bioRxiv2021

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC); this rare type of leukodystrophy is characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes (PvAPs, which along with blood vessels form the gliovascular unit (GVU)). We analyzed the GVU in the Mlc1 knock-out mouse model of MLC. The absence of MLC1 led to an accumulation of fluid in the brain but did not modify the endothelial organization or the integrity of the blood-brain barrier. From P10 onward, the postnatal acquisition of vascular smooth muscle cell contractility was altered, resulting in a marked reduction in arterial perfusion and neurovascular coupling. These anomalies were correlated with alterations in astrocyte morphology, astrocyte polarity and the structural organization of the PvAPs perivascular coverage, and poor intraparenchymal circulation of the cerebrospinal fluid (CSF). Hence, MLC1 is required for the postnatal development and organization of PvAPs and controls vessel contractility and intraparenchymal interstitial fluid clearance. Our data suggest that (i) MLC is a developmental disorder of the GVU, and (ii) PvAP and VSMC maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

neuroscience↗

bioRxiv2021

In mice and humans, the brain's blood vessels mature postnatally to acquire barrier and contractile properties

The brain dense vascular network is essential for distributing oxygen and nutrients to neural cells. The network develops during embryogenesis and leads to the formation of the endothelial blood-brain barrier (BBB). This barrier is surrounded by mural cells (pericytes and vascular smooth muscle cells (VSMCs)) and fibroblasts. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (ECs), VSMCs and fibroblasts follow specific molecular maturation programs over this time period. In particular, ECs acquire P-glycoprotein (P-gP)-mediated efflux capacities. The arterial VSMC network expands, acquires contractile proteins (such as smooth muscle actin (SMA) and myosin heavy chain 11 (Myh11)) and becomes contractile. We also analyzed samples of human brain cortex from the early prenatal stage through to adulthood: the expression of endothelial P-gP increased at birth and Myh11 in VSMCs acts as a developmental switch (as in the mouse) at birth and up to the age of 2 of 5 years. Thus, in both mice and humans, the early postnatal phase is a critical period during which the essential properties of cerebral blood vessels (i.e. the endothelial efflux of xenobiotics and other molecules, and the VSMC contractility required for vessel tone and brain perfusion) are acquired and mature.

neuroscience↗

bioRxiv2021

Genetic Architecture of Kernel Compositional Variation in a Maize Diversity Panel

Maize (Zea mays L.) is a multi-purpose row crop grown worldwide, which overtime has often been bred for increased yield at the detriment of lower composition grain quality. Some knowledge of the genetic factors that affect quality traits has been discovered through the study of classical maize mutants. However, much of the underlying genetic architecture controlling these traits and the interaction between these traits remains unknown. To better understand variation that exists for grain compositional traits in maize, we evaluated 501 diverse temperate maize inbred lines in five unique environments and predicted 16 compositional traits (e.g. carbohydrates, protein, starch) based on the output of near-infrared (NIR) spectroscopy. Phenotypic analysis found substantial variation for compositional traits and the majority of variation was explained by genetic and environmental factors. Correlations and trade-offs among traits in different maize types (e.g. dent, sweetcorn, popcorn) were explored and significant differences and correlations were detected. In total, 22.9-71.1% of the phenotypic variation across these traits could be explained using 2,386,666 single nucleotide polymorphism (SNP) markers generated from whole genome resequencing data. A genome-wide association study (GWAS) was conducted using these same markers and found 70 statistically significant loci for 12 compositional traits. This study provides valuable insights in the phenotypic variation and genetic architecture underlying compositional traits that can be used in breeding programs for improving maize grain quality. Core IdeasO_LIUnderstanding kernel compositional variation is important for food grade corn improvement. C_LIO_LIGenetic and environmental factors account for most of the variation in compositional traits. C_LIO_LIA broad range in trait heritabilities was observed across compositional traits. C_LIO_LICompositional trade-offs will be important to consider when conducting multitrait breeding. C_LIO_LICompositional traits are mostly controlled by a large number of small effect loci. C_LI

plant biology↗

bioRxiv2020

Population Level Variation of Transposable Elements in a Maize Diversity Panel

Intact transposable elements (TEs) account for 65% of the maize genome and can impact gene function and regulation. Although TEs comprise the majority of the maize genome and affect important phenotypes, genome wide patterns of TE polymorphisms in maize have only been studied in a handful of maize genotypes, due to the challenging nature of assessing highly repetitive sequences. We implemented a method to use short read sequencing data from 509 diverse inbred lines to classify the presence/absence of 445,418 non-redundant TEs that were previously annotated in four genome assemblies including B73, Mo17, PH207, and W22. Different orders of TEs (i.e. LTRs, Helitrons, TIRs) had different frequency distributions within the population. LTRs with lower LTR similarity were generally more frequent in the population than LTRs with higher LTR similarity, though high frequency insertions with very high LTR similarity were observed. LTR similarity and frequency estimates of nested elements and the outer elements in which they insert revealed that most nesting events occurred very near the timing of the outer element insertion. TEs within genes were at higher frequency than those that were outside of genes and this is particularly true for those not inserted into introns. Many TE insertional polymorphisms observed in this population were tagged by SNP markers. However, there were also 19.9% of the TE polymorphisms that were not well tagged by SNPs (R2 < 0.5) that potentially represent information that has not been well captured in previous SNP based marker-trait association studies. This study provides a population scale genome-wide assessment of TE variation in maize, and provides valuable insight on variation in TEs in maize and factors that contribute to this variation.

plant biology↗

bioRxiv2020

Role of astrogial Connexin 43 in pneumococcal meningitis and pneumolysin cytotoxicity

Introductory paragraphStreptococcus pneumoniae or pneumococcus (PN) is a major causative agent of bacterial meningitis with high mortality in young infants and elderly people. The mechanism underlying PN crossing of the blood brain barrier (BBB) remains poorly understood. Here, we show that the gap junctional component connexin 43 expressed in astrocytes (aCx43) plays a major role in PN meningitis. Following intravenous PN challenge, mice deficient for aCx43 developed milder symptoms and showed severely reduced bacterial counts in the brain. We show a role for aCx43 in the PN-induced fragmentation of astrocytic GFAP filaments associated with bacterial translocation across endothelial vessels and replication in the brain cortex. aCx43 triggers the PN- and Ply-dependent GFAP fragmentation and nuclear shrinkage in in vitro cultured astrocytes. We showed that purified pneumolysin (Ply) co-opted Cx43 to promote the permeabilization and cytosolic calcium (Ca2+) increase of host cells, a process sensitive to extracellular ATP depletion. These results point to aCx43 as a major player during bacterial meningitis and extend cytolytic mechanisms implicating other host cell plasma membrane channels proposed for small pore-forming toxins, to Ply, a cholesterol-dependent cytolysin, at concentrations relevant to bacterial infection.

microbiology↗