Biology subjects

Joseph, P.

Publications and source records attributed to Joseph, P..

10 recordsLinked to original sources

bioRxiv2026

Selective Laser Etching technology for reconfigurable microfluidic and electrochemistry-on-chip

Polydimethylsiloxane (PDMS) is widely used in academic microfluidics due to its favorable biocompatible properties and compatibility with soft lithography. Moreover, the recent developments of reconfigurable microfluidics rely on the microfabrication of sliding elements, which are 3D objects insertable inside a microfluidic chip to provide a given function. However, the complexity of microfluidic device geometries or sliding elements remains largely limited by the traditional microfabrication methods such as, among others, on SU-8 photolithography or dry epoxy films. Such methods are suited for simple, planar "2.5D" structures with uniform depths, yet struggle to produce more advanced architectures due to material and alignment constraints. To address these limitations, we propose to investigate advanced laser-based approaches, such as Direct Laser Writing (DLW) and Selective Laser Etching (SLE), which enable the construction of high-resolution, 3D designs. We further explore the use of SLE to fabricate sliding elements that enhance chip functionality, including chamber reconfiguration and biological sample manipulation. As a proof of concept, we show that these elements can be functionalized into pH microsensors, paving the way to reusable and reconfigurable electrochemistry-on-chip.

biophysics↗

bioRxiv2024

Single Cell Integration Characterises Metaplasia in Inflammatory Intestinal Diseases

The gastrointestinal (GI) tract consists of connected organs, from the oral cavity to rectum, which function to ensure efficient nutrient uptake and barrier immunity. Diseases of the GI tract affect millions worldwide and as such there are now over 25 published single cell RNA-sequencing (scRNAseq) datasets surveying the GI tract, profiling specific anatomical regions, cell lineages, ages and diseases. To consolidate these efforts, we harmonised and integrated scRNAseq datasets across the whole GI tract from developing and adult human tissues, as well as newly generated data from preterm gut. We uniformly processed 385 samples from 189 healthy controls using a newly developed automated QC approach (scAutoQC). In total, our healthy reference contains [~]1.1 million cells which we annotated to a total of 137 fine-grained cell states. We anchor 13 published and 1 unpublished GI disease datasets covering gastric and colorectal (CRC) cancers, celiac disease, ulcerative colitis (UC) and Crohns disease (CD) to this reference, taking our atlas to a total of 1.6 million cells. We provide our atlas as a valuable resource to the community (available at gutcellatlas.org). Using this resource, we discover epithelial cell metaplasia arising from stem cells across intestinal inflammatory diseases (celiac, UC and CD) and CRC with transcriptional similarity to cells of the gastric and Brunners glands. Whilst previously linked to mucosal healing, we now implicate these cells in inflammation through recruitment of immune cells including T cells and neutrophils, and through direct interactions with T cells. Overall, we discover a shift in paradigm whereby changes in stem cells during inflammation lead to altered mucosal tissue architecture, which in turn contributes to ongoing inflammation. These findings highlight that in addition to barrier function, epithelial cells actively contribute to progression of inflammation which may be a function applicable to other tissues and diseases.

genomics↗

bioRxiv2023

Parallel on-chip micropipettes enabling quantitative multiplexed characterization of vesicle mechanics and cell aggregates rheology

Micropipette aspiration (MPA) is one of the gold standards to quantify biological samples mechanical properties, which are crucial from the cell membrane scale to the multicellular tissue. However, relying on the manipulation of individual home-made glass pipettes, MPA suffers from low throughput and difficult automation. Here, we introduce the sliding insert micropipette aspiration (SIMPA) method, that permits parallelization and automation, thanks to the insertion of tubular pipettes, obtained by photolithography, within microfluidic channels. We show its application both at the lipid bilayer level, by probing vesicles to measure membrane bending and stretching moduli, and at the tissue level by quantifying the viscoelasticity of 3D cell aggregates. This approach opens the way to high-throughput, quantitative mechanical testing of many types of biological samples, from vesicles and individual cells to cell aggregates and explants, under dynamic physico-chemical stimuli.

biophysics↗

bioRxiv2023

Mechanical characterization of regenerating Hydra tissue spheres

Hydra vulgaris, long known for its remarkable regenerative capabilities, is also a longstanding source of inspiration for models of spontaneous patterning. Recently, it became clear that early patterning during Hydra regeneration is an integrated mechano-chemical process where morphogen dynamics is influenced by tissue mechanics. One roadblock to understand Hydra self-organization is our lack of knowledge about the mechanical properties of these organisms. In this paper, we combined microfluidic developments to perform parallelized microaspiration rheological experiments and numerical simulations to characterize these mechanical properties. We found three different behaviors depending on the applied stresses: an elastic response, a visco-elastic one and tissue rupture. Using models of deformable shells, we quantify their Youngs modulus, shear viscosity as well as the critical stresses required to switch between behaviors. Based on these experimental results, we propose a description of the tissue mechanics during normal regeneration. Our results provide a first step towards the development of original mechano-chemical models of patterning grounded in quantitative, experimental data. Statement of significanceHydra vulgaris is a remarkable organism thanks to its regenerative abilities. One can cut this animal into several pieces which will reform a full Hydra in a few days. In this process, the pieces have to define a new organizing axis. Recently, researchers have shown that this axis definition is under mechanical control. One roadblock to understand the relationship between tissue mechanics and Hydra biology is our lack of knowledge about the mechanical state of this organism. Here, we perform a mechanical characterization using a combination of microaspiration setups and numerical simulations. We finally propose a description of what happens at the mechanical level during Hydra regeneration, allowing quantitative approaches questioning the role of mechanical cues in axis definition.

biophysics↗

bioRxiv2022

Small molecule mediated stabilization of PP2A modulates the Homologous Recombination pathway and potentiates DNA damage-induced cell death

High-Grade Serous Carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP-inhibitors (PARPi) have become the mainstay of HGSC targeted therapy, given that these tumors are driven by a high degree of genomic instability and Homologous Recombination (HR) defects. Nonetheless, only [~]30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a Small Molecule Activator of Protein Phosphatase 2A (PP2A) (SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in Patient-Derived HGSC cells and Xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for transformation (B56,B56{gamma} and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061 stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and -deficient cells and in patient-derived xenograft models. Our studies identify PP2A as novel regulator of HR and introduces PP2A activators as a potential treatment for HGSC tumors. Our studies further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 has presented benefits in overcoming PARPi-resistance driven by BRCA1/2 mutation reversions.

cancer biology↗

bioRxiv2021

SYS-Mut: Decoding the Functional Significance of Rare Somatic Mutations in Cancer

Current tailored-therapy efforts in cancer are largely focused on a small number of highly recurrently-mutated driver genes but therapeutic targeting of these oncogenes remains challenging. On the other hand, the vast number of genes mutated infrequently across cancers have received less attention, in part, due to a lack of understanding of their biologic significance. Here we present SYS-Mut, a systems biology platform that can robustly infer the biologic consequences of somatic mutations by integrating routine multi-omic profiles in primary tumors. We established the accuracy of SYS-Mut by recapitulating the functional impact of known driver genes in PanCancer datasets. Subsequent application of SYS-Mut on low-frequency gene mutations in Head and Neck Cancers (HNSC), followed by molecular and pharmacogenetic validation, revealed the lipidogenic network as a novel therapeutic vulnerability in aggressive HNSC. SYS-Mut is thus a robust scalable framework that enables discovery of new targetable avenues in cancer.

cancer biology↗

bioRxiv2021

The EphB2-MYC Axis is a Major Determinant of Barrett's Pathobiology and a Therapeutic Vulnerability in Esophageal Cancer

Esophageal adenocarcinoma (EAC), a highly aggressive cancer with limited therapeutic options, often arises in the backdrop of a molecularly-complex esophageal metaplasia disorder, Barretts Esophagus (BE). Using transcriptomics and systems biology analyses of treatment-naive malignant/pre-malignant biopsy tissues, we found Eph receptor B2 (EphB2) tyrosine kinase signaling to be frequently hyperactivated during early stages of EAC development, and across the BE-EAC continuum. Functional studies revealed EphB2 to be an upstream post-translational regulator of c-MYC activity and as a key molecular dependency in BE/EAC. Single-cell transcriptomics in a porcine esophageal 3D spheroid model showed enhanced EphB2 and MYC activity to be significantly associated with BE-like cell fate. shRNA-based knockdown of EphB2 or small molecule inhibitors of MEK, that modulate MYC protein stability, proved effective in suppressing EAC tumor growth in vivo. These findings point to EphB2-MYC axis as an early promoter of EAC and a novel therapeutic vulnerability in this increasingly-prevalent esophageal malignancy. STATEMENT OF SIGNIFICANCEWe identify EphB2 signaling as a potential master regulator and early promoter of esophageal adenocarcinoma, and the proto-oncogene MYC as a key downstream effector of EphB2 function. Targeting the EphB2-MYC axis could be a promising therapeutic strategy for these often refractory and lethal EAC tumors.

cancer biology↗

bioRxiv2020

ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

Diabetes is associated with increased mortality from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Given literature suggesting a potential association between SARS-CoV-2 infection and diabetes induction, we examined pancreatic expression of the key molecule for SARS-CoV-2 infection of cells, angiotensin-converting enzyme-2 (ACE2). Specifically, we analyzed five public scRNAseq pancreas datasets and performed fluorescence in situ hybridization, Western blotting, and immunolocalization for ACE2 with extensive reagent validation on normal human pancreatic tissues across the lifespan, as well as those from coronavirus disease 2019 (COVID-19) patients. These in silico and ex vivo analyses demonstrated pancreatic expression of ACE2 is prominent in pancreatic ductal epithelium and the microvasculature, with rare endocrine cell expression of this molecule. Pancreata from COVID-19 patients demonstrated multiple thrombotic lesions with SARS-CoV-2 nucleocapsid protein expression primarily limited to ducts. SARS-CoV-2 infection of pancreatic endocrine cells, via ACE2, appears an unlikely central pathogenic feature of COVID-19 as it relates to diabetes.

pathology↗

bioRxiv2020

Studies of human twins reveal genetic variation that affects dietary fat perception

To learn more about the mechanisms of human dietary fat perception, 398 human twins rated fattiness and liking for six types of potato chips that differed in triglyceride content (2.5, 5, 10, and 15% corn oil); reliability estimates were obtained from a subset (n = 50) who did the task twice. Some chips also had a saturated long-chain fatty acid (hexadecanoic acid, 16:0) added (0.2%) to evaluate its effect on fattiness and liking. We computed the heritability of these measures and conducted a genome-wide association study (GWAS) to identify regions of the genome that co-segregate with fattiness and liking. Perceived fattiness and liking for the potato chips were reliable (r = 0.31-0.62, p < 0.05) and heritable (up to h2 = 0.29, p < 0.001, for liking). Adding hexadecanoic acid to the potato chips significantly increased ratings of fattiness but decreased liking. Twins with the G allele of rs263429 near GATA3-AS1 or the G allele of rs8103990 within ZNF729 reported more liking for potato chips than did twins with the other allele (multivariate GWAS, p < 1x10-5), with results reaching genome-wide suggestive but not significance criteria. Person-to-person variation in the perception and liking of dietary fat was (a) negatively affected by the addition of a saturated fatty acid and (b) related to inborn genetic variants. These data suggest liking for dietary fat is not due solely to fatty acid content and highlight new candidate genes and proteins within this sensory pathway.

genetics↗

bioRxiv2019

Complete blood count with differential: An effective diagnostic for IBS subtype in the context of BMI?

The Complete Blood Count with Differential hematological assay is a mainstay diagnostic for point-of-care clinical diagnoses for a spectrum of diseases including infection, inflammation, anemia, and leukemia, and CBC-D profiles are under investigation as early prognostic biomarkers for leukemias and other diseases. Chronic abdominal pain (CAP) and irritable bowel syndrome (IBS) are prevalent gastrointestinal disorders in the United States, with obesity among the most common comorbidities. Often, IBS-like gastrointestinal (GI) symptoms persist after resolution of known inflammation and/or enteropathogenic infection, and current literature contains significant discussion of the extent to which IBS is within the biological spectrum of inflammatory disease. Obesity is also associated with generalized signatures of inflammation and may confound accurate diagnoses. We performed ANOVA, multiple means comparisons, statistical analyses of CBC data from our \"Brain-Gut Natural History\" (BGNH) clinical cohort, with additional ELISA assays for lipopolysaccharide binding protein (LBP), IL-10, cortisol, and ACTH, signatures of immune-inflammatory response and Hypothalamic-Pituitary-Adrenal (HPA) axis activity, respectively. BGNH cohort includes healthy and overweight individuals diagnosed with IBS diarrhea-(IBS-D) and constipation-predominant (IBS-C) subtypes. We identified several potentially significant markers for IBS-D and IBS-C, notably IL-10, mean platelet volume (MPV), with LBP and monocyte percent also showing some statistical significance. Weight also showed significant results for overweight vs. normal weight, regardless of IBS subtype, particularly for Cortisol. CBC-D predictive profiles for IBS subtype and weight were identified using discriminant functions analysis and show that predictivity of marker profiles have poor performance relative to their normal weight subsets. Further refinement of this analysis will be performed utilizing increased sample size, additional molecular profiles, and enhanced statistical analysis.

immunology↗