Biology subjects

D'Souza, R. N.

Publications and source records attributed to D'Souza, R. N..

8 recordsLinked to original sources

bioRxiv2024

Tendon-associated gene expression precedes osteogenesis in mid-palatal suture establishment

Orthodontic maxillary expansion relies on intrinsic mid-palatal suture mechanobiology to induce guided osteogenesis, yet establishment of the mid-palatal suture within the continuous secondary palate and causes of maxillary insufficiency remain poorly understood. In contrast, advances in cranial suture research hold promise to improve surgical repair of prematurely fused cranial sutures in craniosynostosis to potentially restore the obliterated signaling environment and ensure continual success of the intervention. We hypothesized that mid-palatal suture establishment is governed by shared principles with calvarial sutures and involves functional linkage between expanding primary ossification centres with the midline mesenchyme. We characterized establishment of the mid-palatal suture from late embryonic to early postnatal timepoints. Suture establishment was visualized using histological techniques and multimodal transcriptomics. We identified that mid-palatal suture formation depends on a spatiotemporally controlled signalling milieu in which tendon-associated genes play a significant role. We mapped relationships between extracellular matrix-encoding gene expression, tenocyte markers, and novel suture patency candidate genes. We identified similar expression patterns in FaceBase-deposited scRNA-seq datasets from cranial sutures. These findings demonstrate shared biological principles for suture establishment, providing further avenues for future development and understanding of maxillofacial interventions.

developmental biology↗

bioRxiv2024

Filamin A mediates embryonical palatal fusion by linking mechanotransduction with β-Catenin/Smad2

To decipher potential mechanisms underlying cleft palate (CP), we used advanced bioinformatic integrated with literature mining and genome-wide association study (GWAS). Re-analysis of RNA-seq data (GSE45568, GSE185279) combined with literature mining highlighted the roles of Filamin A (Flna) and Epithelial-Mesenchymal Transition (EMT) in palatal development. Immunofluorescence of in vivo palatal shelves showed increased Flna in medial edge epithelial (MEE) cells and EMT cells located in an epithelial triangle. Inhibition of TGF-{beta} or RhoA and mechanical stimuli impacted Flna expression in ex vivo cultured palatal shelves. Re-analysis of scRNA-seq data (GSE155928) highlighted a correlation between Flna and Ctnnb1 in EMT cells. Flna knockdown affected {beta}-catenin/Smad2 expression in cultured palatal shelves and HaCaT cells. Epithelium-specific knockout of Flna delayed palatal fusion in female mice but not males. Mendelian randomization analysis suggested that parental habitual physical activity (HPA) was causally associated with lower risk of CP in their offspring. Together, these findings suggested that parental HPA could benefit their offsprings palatal development through Flna by linking mechanotransduction with the Wnt/TGF-{beta}/Smad signaling pathways during palatal fusion.

developmental biology↗

bioRxiv2023

Dkk2 interacts with Pax9 in palate mesenchyme to pattern and tune osteogenesis

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which constitutes the most common among the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study utilized the Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. While prior research had identified upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 in the mesenchyme. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

developmental biology↗

bioRxiv2023

Integrated spatiotemporal transcriptomic resolution of embryonic palate osteogenesis

The differentiation of osteoblasts and the subsequent formation of bone marks an important terminal phase in palate formation that leads to the separation of the oral and nasal cavities. While the developmental events that precede palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms that lead to the bony union of fusing palatal shelves. Herein, the timeline of osteogenic transcriptional programming is unveiled in the embryonic palate by way of integrated bulk, single-cell, and spatially resolved RNA-seq analyses. We define spatially restricted expression patterns of key marker genes, both regulatory and structural, that are differentially expressed during palatal fusion, including the identification of several novel genes (Deup1, Dynlrb2, Lrrc23) spatially restricted in expression to the palate, providing a relevant framework for future studies that identify new candidate genes for cleft palate anomalies in humans as well as the timing of mammalian embryonic palatal osteogenesis.

developmental biology↗

bioRxiv2020

Supervised and Unsupervised Classification of Cocoa Bean Origin and Processing using Liquid Chromatography-Mass Spectrometry

Liquid Chromatography-Mass Spectrometry (LC-MS) provides an unprecedented wealth of metabolomics information for food products, including insights into compositional changes during food processing. Here, we employed the largest available LC-MS dataset of around 300 cocoa bean samples to assess the capability of two popular multivariate classification methods, principal component analysis (PCA) and linear decomposition analysis (LDA), for studying bean geographic origin and responsible characteristic compounds. The unsupervised method, PCA, only provides a limited separation in bean origin. Expectedly, the supervised method, LDA, provides a better origin clustering. However, it suffers from a strong, nonlinear dependence on the set of compounds used in the analysis. We show that for LDA a compound filtering criterion based on Gaussian intensity distributions dramatically enhances origin clustering of samples, thus increasing its predictive efficiency. In this form, the supervised method of LDA holds the possibility to identify potential markers of a specific origin.

bioinformatics↗

bioRxiv2020

Correlation network analysis based on untargeted LC-MS profiles of cocoa reveals processing stage and origin country

In order to implement quality control measures and create fine flavor products, an important objective in cocoa processing industry is to realize standards for characterization of cocoa raw materials, intermediate and finished products with respect to their processing stages and countries of origin. Towards this end, various works have studied separability or distinguishability of cocoa samples belonging to various processing stages in a typical cocoa processing pipeline or to different origins. Limited amount of success has been possible in this direction in that unfermented and fermented cocoa samples have been shown to group into separate clusters in PCA. However, a clear clustering with respect to the country of origin has remained elusive. In this work we suggest an alternative approach to this problem through the framework of correlation networks. For 140 cocoa samples belonging to eight countries and three progressive stages in a typical cocoa processing pipeline we compute pairwise Spearman and Pearson correlation coefficients based on the LC-MS profiles and derive correlation networks by retaining only correlations higher than a threshold. Progressively increasing this threshold reveals, first, processing stage (or sample type) modules (or network clusters) at low and intermediate values of correlation threshold and then country specific modules at high correlation thresholds. We present both qualitative and quantitative evidence through network visualization and node connectivity statistics. Besides demonstrating separability of the two data properties via this network-based method, our work suggests a new approach for studying classification of cocoa samples with nested attributes of processing stage sample types and country of origin along with possibility of including additional factors, e.g., hybrid variety, etc. in the analysis.

systems biology↗

bioRxiv2018

Feasibility of clinician-facilitated 3D printing of synthetic cranioplasty flaps

Objectives3D scanning and stereolithographic printing technology becoming increasingly common, however its implementation into clinical practice is in its primacy. These technologies may be esoteric to the practicing neurosurgeon. We explored a range of 3D scanning and stereolithographic techniques to create patient-specific synthetic implants.\n\nMethodsWe simulated bilateral craniectomies from a single cadaveric specimen to create 3 methods of creating stereolithographically-viable virtual models. Firstly, we used pre-and-post operative CT derived bony windows to create a virtual skull model, from which the flap was extracted. Secondly, we used an entry-level 3D light-scanner to scan and render models of the individual bone pieces. Thirdly, we used an arm-mounted, 3D laser-scanner to create virtual models using a real-time approach.\n\nResultsFlaps were printed from the CT scanner and laser scanner models only, in a UV-cured polymer. The light scanner did not produce suitable virtual models for printing. The CT scanner derived models required extensive post-fabrication modification to fit the existing defects. The laser-scanner models assumed good fit within the defects without any modification.\n\nConclusionsThe methods presented varying levels of complexity in acquisition and model rendering. Each technique required hardware at varying in price points from $0 to [~]$100,000. The laser-scanner models produced the best quality parts which bore near-perfect fit with the original defects. We discuss potential neurosurgical applications of this technology.

synthetic biology↗

bioRxiv2017

The Pax9/Wnt pathway regulates secondary palate formation in mice

Clefts of the palate and/or lip arise in about 1/700 human live births and are caused by multiple genetic and environmental factors. Studies of mouse knockout models of cleft palate have improved our understanding of the molecular control of palatogenesis. While it is known that Pax9 regulates palatogenesis through Bmp, Fgf and Shh signaling, there is still much to learn about its precise relationship with other pathways. Here we show that alterations of Wnt expression and decreased Wnt activity in Pax9-/- palatal shelves are a result of Pax9s ability to directly bind and repress the promoters of Dkk1 and Dkk2, proteins that antagonize Wnt signaling. The delivery of small-molecule Dkk inhibitors (Wnt agonists) into the tail-veins of pregnant Pax9+/- mice from E10.5 to E14.5 restored Wnt signaling, promoted cell proliferation, bone formation and restored the fusion of palatal shelves in Pax9-/- embryos. In contrast, other organ defects in Pax9 mutants were not corrected. These data uncover a unique molecular relationship between Pax9 and Wnt genes in palatogenesis and offer a new approach for treating cleft palates in humans.\n\nSummary StatementThese studies demonstrate that the Pax9/Wnt genes regulate murine palatogenesis. This unique molecular relationship is proven by the correction of cleft defects in Pax9-deficient mice through Wnt agonist therapies.

developmental biology↗