Biology subjects

Skinner, D.

Publications and source records attributed to Skinner, D..

9 recordsLinked to original sources

bioRxiv2025

Gene-Corrected Basal Cells Restore CFTR In Vitro; Transplants Regenerate Epithelium in a Preclinical Sinus Model

BackgroundCystic fibrosis (CF) is caused by mutations in the CFTR gene, leading to epithelial dysfunction and progressive lung disease. Although CFTR modulators have transformed care, [~]10% of people with CF remain without effective therapy. Durable, mutation-agnostic approaches are urgently needed. MethodWe used a lentiviral (LV) vector to deliver wild-type CFTR to airway basal cells derived from 13 paediatric CF participants with a range of genotypes. Transduced cells were assessed for transgene expression, epithelial differentiation, and CFTR function using air-liquid interface (ALI) cultures. Separately, to evaluate regenerative capacity in vivo, LVGFP-transduced rabbit airway basal cells were transplanted into the denuded nasal septum of healthy New Zealand white rabbits using a biocompatible scaffold. ResultsTransduced basal cells retained multilineage differentiation capacity, forming well-organized, pseudostratified epithelium with intact barrier function and ciliary activity. CFTR channel activity was restored to levels comparable to or exceeding those achieved with elexacaftor/tezacaftor/ivacaftor (ETI), including in individuals with nonsense mutations. Combined CFTR transduction plus ETI treatment showed additive benefit. In vivo, transplanted rabbit basal cells engrafted and differentiated to regenerate a mucociliary epithelium, with improved nasal potential difference and mucociliary clearance compared to scaffold-only controls. ConclusionOur study demonstrates that LV-mediated CFTR gene addition restores CFTR function in vitro across genotypes and supports epithelial regeneration in a clinically relevant animal airway model. This two-part platform offers a scalable path toward cell therapies for all people with CF and may have broader applications in upper airway epithelial repair.

cell biology↗

bioRxiv2025

A revision of the Neotropical Costaceae: results from sixty years of taxonomic struggle

Here we present a taxonomic revision of all neotropical Costaceae represented by 90 species, distributed in four monophyletic genera, Chamaecostus (9 spp.), Costus (76 spp.), Dimerocostus (5 spp.), and Monocostus (1 sp.). The work resulted in three new combinations, several nomenclatural changes, and four insufficiently known species are mentioned to demonstrate the need for continued and ongoing work on this charismatic yet often cryptic lineage of plants. We highlight the seventeen species of Costus and one species of Chamaecostus that were recently described (Maas et al. 2023) based on knowledge that emerged during this complete revisionary work, 60 years in the making. Included in this revision are details concerning the morphology, distribution, ecology, and phylogenetic relationships among the species studied. All currently recognized species are fully described, including notes on taxonomic synonymy, lectotypification, information on distribution and ecology, conservation status (either as evaluated and published in the IUCN Red List of Threatened Species or as estimated for this publication), vernacular names (when given) and short taxonomic notes. For each species, a distribution map is prepared and at least one colour photograph is included to assist in species identification. Dichotomous keys that include all species are provided, as is a list of all (c. 11.000) exsiccatae studied and an index to scientific names.

evolutionary biology↗

bioRxiv2024

Physical modeling of embryonic transcriptomes identifies collective modes of gene expression

Starting from one totipotent cell, complex multicellular organisms form through a series of differentiation and morphogenetic events, culminating in a multitude of cell types arranged in a functional and intricate spatial pattern. To do so, cells coordinate with each other, resulting in dynamics which follow a precise developmental trajectory, constraining the space of possible embryo-to-embryo variation. Using recent single-cell sequencing data of early ascidian embryos, we leverage natural variation together with modeling and inference techniques from statistical physics to investigate development at the level of a complete interconnected embryo - an embryonic transcriptome. After developing a robust and biophysically motivated approach to identifying distinct transcriptomic states or cell types, a statistical analysis reveals correlations within embryos and across cell types demonstrating the presence of collective variation. From these intra-embryo correlations, we infer minimal networks of cell-cell interactions, which reveal the collective modes of gene expression. Our work demonstrates how the existence and nature of spatial interactions along with the collective modes of expression that they give rise to can be inferred from single-cell gene expression measurements, opening up a wider range of biological questions that can be addressed using sequencing-based modalities.

developmental biology↗

bioRxiv2024

Development of subunit selective proteasome substrates for Schistosoma species

Schistosomiasis, or bilharzia, is a neglected tropical disease caused by Schistosoma spp. blood flukes that infects over 200 million people worldwide. Just one partially effective drug is available, and new drugs and drug targets would be welcome. The 20S proteasome is a validated drug target for many parasitic infections, including those caused by Plasmodium and Leishmania. We previously showed that anticancer proteasome inhibitors that act through the Schistosoma mansoni 20S proteasome (Sm20S) kill the parasite in vitro. To advance these initial findings, we employed Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to define the substrate cleavage specificities of the three catalytic {beta} subunits of purified Sm20S. The profiles in turn were used to design and synthesize subunit-specific optimized substrates that performed two to eight fold better than the equivalent substrates used to measure the activity of the constitutive human proteasome (c20S). These specific substrates also eliminated the need to purify Sm20S from parasite extracts - a single step enrichment was sufficient to accurately measure substrate hydrolysis and its inhibition with proteasome inhibitors. Finally, we show that the substrate and inhibition profiles for the 20S proteasome from the three medically important schistosome species are similar, suggesting that data arising from an inhibitor development campaign that focuses on Sm20S can be extrapolated to the other two targets with consequent time and cost savings.

biochemistry↗

bioRxiv2022

Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 {micro}M and 66 {micro}M. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 {micro}M to 46 {micro}M. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

biochemistry↗

bioRxiv2021

Discovery of Potent Triple Inhibitors of Both SARS-CoV-2 Proteases and Human Cathepsin L

There are currently no FDA approved inhibitors of SARS-CoV-2 viral proteases with specific treatment for post-exposure of SARS-CoV-2. Here, we discovered inhibitors containing thiuram disulfide or dithiobis-(thioformate) tested against three key proteases in SARS CoV-2 replication including SARS CoV-2 Main Protease (Mpro), SARS CoV-2 Papain Like Protease (PLpro), and human cathepsin L. The use of thiuram disulfide and dithiobis-(thioformate) covalent inhibitor warheads was inspired by disulfiram, a currently prescribed drug commonly used to treat chronic alcoholism that at the present time is in Phase 2 clinical trials against SARS-CoV-2. At the maximal allowed dose, disulfiram is associated with adverse effects. Our goal was to find more potent inhibitors that target both viral proteases and one essential human protease to reduce the dosage and minimize the adverse effects associated with these agents. We found that compounds coded as RI175, JX 06, and RI172 are the most potent inhibitors from an enzymatic assay against SARS-CoV-2 Mpro, SARS-CoV-2 PLpro, and human cathepsin L with IC50s of 330, 250 nM, and 190 nM about 4.5, 17, and 11.5-fold more potent than disulfiram, respectively. The identified protease inhibitors in this series were also tested against SARS CoV-2 in a cell-based and toxicity assay and were shown to have similar or greater antiviral effect than disulfiram. The identified triple protease inhibitors and their derivatives are promising candidates for treatment of the Covid-19 virus and related variants.

pharmacology and toxicology↗

bioRxiv2020

Potent in vitro anti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L

The emergence of SARS-CoV-2 in late 2019, and the subsequent COVID-19 pandemic, has led to substantial mortality, together with mass global disruption. There is an urgent need for novel antiviral drugs for therapeutic or prophylactic application. Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is recognized as a promising drug target. The marine natural product, gallinamide A and several synthetic analogues, were identified as potent inhibitors of cathepsin L activity with IC50 values in the picomolar range. Lead molecules possessed selectivity over cathepsin B and other related human cathepsin proteases and did not exhibit inhibitory activity against viral proteases Mpro and PLpro. We demonstrate that gallinamide A and two lead analogues potently inhibit SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range, thus further highlighting the potential of cathepsin L as a COVID-19 antiviral drug target.

biochemistry↗

bioRxiv2020

A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 M inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of [≤] 100 M. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing. SIGNIFICANCEThe virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.

biochemistry↗

bioRxiv2019

Quantifying the mechanics of locomotion of the schistosome pathogen with respect to changes in its physical environment

Schistosomiasis is a chronic and morbid disease of poverty affecting approximately 200 million people worldwide. Mature schistosome flatworms wander in the hosts hepatic portal and mesenteric venous system where they encounter a range of blood flow conditions and geometrical confinement. However, the mechanisms that support schistosome locomotion and underlie the pathogens adaptation to its physical environment are largely unknown. By combining microfabrication and traction force microscopy, we developed various in vitro assays to quantify the mechanics of locomotion of adult male S. mansoni in different physiologically relevant conditions. We show that in unconfined settings, the parasite undergoes two-anchor marching mediated by the coordinated action of its oral and ventral suckers. This mode of locomotion is maintained when the worm faces an external flow, to which it responds by adjusting the strength of its suckers. In geometrically confined conditions, S. mansoni switches to a different crawling modality by generating retrograde peristaltic waves along its body, a mechanism shared with terrestrial and marine worms. But while the surface of most worms has backward-pointing bristles that rectify peristaltic waves and facilitate forward locomotion, S mansoni has isotropically oriented tubercles. This requires tight coordination between muscle contraction and substrate friction but confers S. mansoni the ability to reverse its direction of locomotion without turning its body, which is likely advantageous to maneuver in narrow bore vessels. We show that the parasite can also coordinate the action of its suckers with its peristaltic body contractions to increase crawling speed. Throughout this study, we report on a number of biomechanical parameters to quantify the motility of adult schistosomes (e.g. sucker grabbing strength, rate of detachment under flow, peristaltic wave properties and traction stresses). The new series of in vitro assays make it possible to quantify key phenotypical aspects of S. mansoni motility that could guide the discovery of new drugs to treat schistosomiasis.

biophysics↗