Biology subjects

Carpenter, R.

Publications and source records attributed to Carpenter, R..

6 recordsLinked to original sources

bioRxiv2026

Efficient and Robust Genomic DNA Isolation and Next-Generation Sequencing Library Preparation from Recalcitrant Wild Grape Species

This protocol details the extraction of high-molecular-weight genomic DNA from grapevine tissues (wild and cultivated Vitis spp., including pathogen-infected samples) and the subsequent preparation of Illumina(R) whole-genome sequencing libraries using bead-bound Tn5 transposase. It is designed to overcome challenges from polyphenolic compounds and secondary metabolites in wild plants, providing a cost-effective workflow for large-scale population genomics. It includes recipes for buffers, incubation times, critical notes, and troubleshooting tips to maximize yield and library quality. Although designed for the grapevine DNA, this protocol is potentially applicable to other similar wild plant species HighlightsO_LIOptimized CTAB-PTB DNA extraction protocol for field-collected wild plant tissues. C_LIO_LIEffective removal of polyphenols and secondary metabolites associated with DNA using PTB. C_LIO_LICost-effective Illumina DNA Prep library preparation using bead-bound Tn5 transposase (Tagmentation). C_LIO_LIScalable workflow suitable for large-scale population genomics in Vitis species. C_LIO_LIValidated method for high-molecular-weight DNA and high-quality sequencing data. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/713680v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@b637d4org.highwire.dtl.DTLVardef@10b563aorg.highwire.dtl.DTLVardef@14a32caorg.highwire.dtl.DTLVardef@4c9577_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

Brain Mecp2 Gene Dosage and Gene Therapy Shape Multi-Omic Signatures and Biomarkers in Rett Syndrome

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Like other genetic neurodevelopmental disorders, it lacks protein biomarkers to evaluate disease and therapeutic outcomes. We present a strategy to define putative biomarkers of MeCP2 dysfunction in brain with potential to delineate mechanisms and monitor therapeutic interventions. This strategy relies on a library of proteins responsive to Mecp2 gene dosage and correlated with molecular and clinical outcomes after AAV9-mediated MECP2 gene therapy in Mecp2-KO mice. Gene rescue restored MeCP2 in brain, improved clinical phenotypes, and reverted transcriptome and proteome abnormalities. We identified 327 shared proteins among 1852 cortical and hippocampal proteins responsive to Mecp2/MECP2. Of these, 119 also displayed Mecp2/MECP2-dependent transcript changes. Both the Mecp2-responsive proteome and transcript-protein pairs were enriched in synaptic and metabolic pathways, including central carbon and NAD+ metabolism. We used this therapy-responsive protein library to guide selection of candidate cerebrospinal fluid (CSF) biomarkers in RTT. CSF composition from neurotypical and RTT groups was analyzed using ultrasensitive nucleic acid-based multiplexed ELISA. Twenty-eight proteins were altered in RTT, nine overlapping with Mecp2 dosage- and therapy-sensitive proteins. Multivariate regression linked several candidates to Mecp2/MeCP2 abundance and phenotypic improvement in mice. This paradigm provides a rigorous molecular systems-level framework integrating genetics, preclinical gene therapy, and clinical metrics to define robust cross-species putative biomarkers and possible mechanisms in RTT, with potential applicability to other neurodevelopmental disorders. One Sentence SummaryGenetic Identification of cross-species biomarkers and mechanisms in Rett Syndrome

neuroscience↗

bioRxiv2025

Deep-learning-enabled multi-omics analyses for prediction of future metastasis in cancer

Metastasis remains the leading cause of cancer-related mortality, yet predicting future metastasis is a major clinical challenge due to the lack of validated biomarkers and effective assessment methods. Here, we present EmitGCL, a deep-learning framework that accurately predicts future metastasis and its corresponding biomarkers. Based on a comprehensive benchmarking comparison, EmitGCL outperformed other computational tools across six cancer types from seven cohorts of patients with superior sensitivity and specificity. It captured occult metastatic cells in a patient with a lymph node-negative breast cancer, who was declared to have no evidence of disease by conventional imaging methods but was later confirmed to have a metastatic disease. Notably, EmitGCL identified HSP90AA1 and HSP90AB1 as predictable biomarkers for future breast cancer metastasis, which was validated across five independent cohorts of patients (n=420). Furthermore, we demonstrated YY1 transcription factor as a key driver of breast cancer metastasis which was validated through in-silico and CRISPR-based migration assays, suggesting that YY1 is a potential therapeutic target for deterring metastasis.

bioinformatics↗

bioRxiv2024

An In-Vitro Standardized Protocol for Preparing Smoke Extract Media from Cigarette, Electronic Cigarette and Waterpipe.

Cigarette smoke exposure is a major risk factor for various diseases, and in vitro studies using smoke medium (SM) are crucial for elucidating underlying mechanisms. However, the lack of standardized protocols for SM preparation has led to inconsistencies in experimental results. This paper presents an optimized protocol for reproducible generation of SM from conventional cigarette smoke extract (CSE), electronic cigarette smoke (ECS), and waterpipe vapour (WPV). The protocol involves: O_LIA custom-built apparatus for smoke capture in a cell culture medium C_LIO_LISpecific parameters mimicking human smoking topography C_LIO_LIRigorous aseptic techniques and safety measures C_LI Extensive details are provided for material preparation, apparatus setup, smoke generation procedures, and post-processing steps. This standardized method enables reliable in vitro exposure models, easing comparative studies across different tobacco products. To prove protocol consistency, we present optical. Additionally, we highlight the protocols utility by figuring out the SM quality through analysing O.D. data and morphological changes, cell viability assays, ELISA and gene expression analyses i.e., RNA sequencing through several in-vitro models, i.e., Human Umbilical Vein Endothelial Cells, (HUVECs, Merck), human leukaemia monocytic cell line (THP-1, Sigma) and induced pluripotent stem cells differentiated to cardiomyocytes (iPSC-CMs, Axol) respectively. This optimized protocol addresses variability in SM preparation, enabling consistent investigations into smoke-induced pathologies and potential therapeutic interventions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/606957v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@142ca65org.highwire.dtl.DTLVardef@1f5d797org.highwire.dtl.DTLVardef@11d1e63org.highwire.dtl.DTLVardef@b5fd55_HPS_FORMAT_FIGEXP M_FIG C_FIG Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@af8a3org.highwire.dtl.DTLVardef@49ccbforg.highwire.dtl.DTLVardef@121329forg.highwire.dtl.DTLVardef@828a3borg.highwire.dtl.DTLVardef@413930_HPS_FORMAT_FIGEXP M_TBL C_TBL

cell biology↗

bioRxiv2023

Systemic Metabolic and Mitochondrial Defects in Rett Syndrome Models

Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2. Transcriptomes and proteomes of organs and brain regions from Mecp2-null mice as well as diverse MECP2-null male and female human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2-null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2- and MECP2-sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2/MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.

neuroscience↗

bioRxiv2021

AusTraits: a curated plant trait database for the Australian flora

We introduce the AusTraits database - a compilation of measurements of plant traits for taxa in the Australian flora (hereafter AusTraits). AusTraits synthesises data on 375 traits across 29230 taxa from field campaigns, published literature, taxonomic monographs, and individual taxa descriptions. Traits vary in scope from physiological measures of performance (e.g. photosynthetic gas exchange, water-use efficiency) to morphological parameters (e.g. leaf area, seed mass, plant height) which link to aspects of ecological variation. AusTraits contains curated and harmonised individual-, species- and genus-level observations coupled to, where available, contextual information on site properties. This data descriptor provides information on version 2.1.0 of AusTraits which contains data for 937243 trait-by-taxa combinations. We envision AusTraits as an ongoing collaborative initiative for easily archiving and sharing trait data to increase our collective understanding of the Australian flora.

ecology↗