Biology subjects

Conway, J.

Publications and source records attributed to Conway, J..

11 recordsLinked to original sources

bioRxiv2026

Expanding the Promoter Toolbox for Metabolic Engineering in the Lignocellulolytic Thermophile Anaerocellum bescii

Anaerocellum (formerly Caldicellulosiruptor) bescii, an anaerobic, extremely thermophilic (Topt [~]78 {degrees}C) lignocellulolytic bacterium, is a promising chassis for metabolic engineering and next-generation bioprocessing. Yet, a lack of well-characterized genetic parts in A. bescii has hampered metabolic engineering efforts. Here, using a previously developed hyperthermophilic {beta}-galactosidase reporter system, we screened a diverse panel of putative A. bescii promoter sequences, identifying promoters that drove reporter output across a broad range. For a select subset, we mapped their transcriptional start sites (TSSs) and evaluated ribosome binding site (RBS) regions using chimeric promoter constructs. By constructing truncated promoter variants, we defined functional regions within the widely used, high-expression S-layer protein promoter (Pslp) and engineered a compact 99 bp variant that retained substantial reporter activity. Finally, we demonstrated that these new promoters can be used for metabolic engineering by using two newly characterized promoters to express an established thermostable alcohol dehydrogenase from Thermoclostridium stercorarium to drive ethanol production in A. bescii. Together, this work expands and diversifies the A. bescii genetic toolkit, opening doors to future metabolic engineering efforts in this species.

Synthetic Biology↗

bioRxiv2026

Combining xenium in situ spatial transcriptomics and imaging mass cytometry on a single tissue section

Spatial imaging technologies provide an expansive view of tissue microenvironments through high-plex profiling of protein and molecular targets in situ. Imaging mass cytometry (IMC; Standard BioTools) is a trusted method for defining immune phenotypes based on up to 40 protein targets, whilst Xenium in situ spatial transcriptomics (Xenium; 10x Genomics) is an emerging platform that can measure up to 5000 mRNA markers simultaneously. Although these platforms can reveal valuable insights on their own, there is an increasing need to analyse samples using a multi-omics approach to further our understanding of complex biological processes. To address this, we have assessed a novel dual-platform workflow that combines Xenium and IMC on a single formalin-fixed paraffin-embedded tissue section to enable the spatial profiling of both mRNA and protein targets at single-cell resolution. The feasibility of the workflow was determined by comparing the staining quality of IMC performed after Xenium to that of IMC performed alone on an adjacent tissue section, confirming that Xenium has little to no negative impact on subsequent IMC protein staining. Although the location of transcripts picked up by Xenium correlated with the corresponding proteins picked up by IMC at a global scale, discrepancies between the two technologies were apparent at the single-cell level. This is to be expected, as biologically transcript expression does not always correlate with protein, and both platforms have their own technical limitations. However, when we analyse T cells identified by both technologies, as opposed to T cells identified by Xenium or IMC alone, it produces the most biologically meaningful results at both the transcript and protein level for specific T cell markers. These results highlight how integration of the two platforms, identifying the presence of both RNA and protein, can foster a more comprehensive view of cellular landscapes and provide a greater depth of functional capabilities and cellular interactions.

immunology↗

bioRxiv2023

Cultivation, chemistry, and genome of Psilocybe zapotecorum

Psilocybe zapotecorum is a strongly blue-bruising psilocybin mushroom used by indigenous groups in southeastern Mexico and beyond. While this species has a rich history of ceremonial use, research into its chemistry and genetics have been limited. Herein, we detail mushroom morphology and report on cultivation parameters, chemical profile, and the full genome sequence of P. zapotecorum. First, growth and cloning methods are detailed that are simple, and reproducible. In combination with high resolution microscopic analysis, the strain was barcoded, confirming species-level identification. Full genome sequencing reveals the architecture of the psilocybin gene cluster in P. zapotecorum, and can serve as a reference genome for Psilocybe Clade I. Characterization of the tryptamine profile revealed a psilocybin concentration of 17.9{+/-}1.7 mg/g, with a range of 10.6-25.7 mg/g (n=7), and similar tryptamines (psilocin, baeocystin, norbaeocystin, norpsilocin, aeruginascin, 4-HO-tryptamine, and tryptamine) in lesser concentrations for a combined tryptamine concentration of 22.5{+/-}3.2 mg/g. These results show P. zapotecorum to be a potent - and variable - Psilocybe mushroom. Chemical profiling, genetic analysis, and cultivation assist in demystifying these mushrooms. As clinical studies with psilocybin gain traction, understanding the diversity of psilocybin mushrooms will assure that psilocybin therapy does not become synonymous with psilocybin mushrooms.

molecular biology↗

bioRxiv2023

Cell-type-specific nuclear morphology predicts genomic instability and prognosis in multiple cancer types

While alterations in nucleus size, shape, and color are ubiquitous in cancer, comprehensive quantification of nuclear morphology across a whole-slide histologic image remains a challenge. Here, we describe the development of a pan-tissue, deep learning-based digital pathology pipeline for exhaustive nucleus detection, segmentation, and classification and the utility of this pipeline for nuclear morphologic biomarker discovery. Manually-collected nucleus annotations were used to train an object detection and segmentation model for identifying nuclei, which was deployed to segment nuclei in H&E-stained slides from the BRCA, LUAD, and PRAD TCGA cohorts. Interpretable features describing the shape, size, color, and texture of each nucleus were extracted from segmented nuclei and compared to measurements of genomic instability, gene expression, and prognosis. The nuclear segmentation and classification model trained herein performed comparably to previously reported models. Features extracted from the model revealed differences sufficient to distinguish between BRCA, LUAD, and PRAD. Furthermore, cancer cell nuclear area was associated with increased aneuploidy score and homologous recombination deficiency. In BRCA, increased fibroblast nuclear area was indicative of poor progression-free and overall survival and was associated with gene expression signatures related to extracellular matrix remodeling and anti-tumor immunity. Thus, we developed a powerful pan-tissue approach for nucleus segmentation and featurization, enabling the construction of predictive models and the identification of features linking nuclear morphology with clinically-relevant prognostic biomarkers across multiple cancer types.

cancer biology↗

bioRxiv2023

Analysis of evolutionary dynamics and clonal architecture in prostate cancer

The extent to which clinical and genomic characteristics associate with prostate cancer clonal architecture, tumor evolution, and therapeutic response remains unclear. Here, we reconstructed the clonal architecture and evolutionary trajectories of 845 prostate cancer tumors with harmonized clinical and molecular data. We observed that tumors from patients who self-reported as Black had more linear and monoclonal architectures, despite these men having higher rates of biochemical recurrence. This finding contrasts with prior observations relating polyclonal architecture to adverse clinical outcomes. Additionally, we utilized a novel approach to mutational signature analysis that leverages clonal architecture to uncover additional cases of homologous recombination and mismatch repair deficiency in primary and metastatic tumors and link the origin of mutational signatures to specific subclones. Broadly, prostate cancer clonal architecture analysis reveals novel biological insights that may be immediately clinically actionable and provide multiple opportunities for subsequent investigation. Statement of significanceTumors from patients who self-reported as Black demonstrate linear and monoclonal evolutionary trajectories yet experience higher rates of biochemical recurrence. In addition, analysis of clonal and subclonal mutational signatures identifies additional tumors with potentially actionable alterations such as deficiencies in mismatch repair and homologous recombination.

cancer biology↗

bioRxiv2023

Insights into a viral motor: the structure of the HK97 packaging termination assembly

Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.

molecular biology↗

bioRxiv2022

Mutational footprint of platinum chemotherapy in a secondary thyroid cancer

Although papillary thyroid carcinoma (PTC) is the most frequent endocrine tumor with a generally excellent prognosis, a patient developed a clinically aggressive PTC eleven years after receiving platinum chemotherapy for ovarian endometrioid adenocarcinoma. Germline and somatic analyses of multi-temporal and multi-regional molecular profiles indicated that ovarian and thyroid tumors did not share common genetic alterations. PTC tumors had driver events associated with aggressive PTC behavior, an RBPMS-NTRK3 fusion and a TERT promoter mutation. Spatial and temporal genomic heterogeneity analysis indicated a close link between anatomical locations and molecular patterns of PTC. Mutational signature analyses demonstrated a molecular footprint of platinum exposure, and that aggressive molecular drivers of PTC were linked to prior platinum-associated mutagenesis. This case provides a direct association between platinum chemotherapy exposure and secondary solid tumor evolution, in specific aggressive thyroid carcinoma, and suggests that uniform clinical assessments for secondary PTC after platinum chemotherapy may warrant further evaluation.

genomics↗

bioRxiv2021

Dissecting tumor cell programs through group biology estimation in clinical single-cell transcriptomics

Given the growing number of clinically integrated cancer single-cell transcriptomic studies, robust differential enrichment methods for gene signatures to dissect tumor cellular states for discovery and translation are critical. Current analysis strategies neither adequately represent the hierarchical structure of clinical single-cell transcriptomic datasets nor account for the variability in the number of recovered cells per sample, leading to results potentially confounded by sample-driven biology with high false positives instead of accurately representing true differential enrichment of group-level biology (e.g., treatment responders vs. non-responders). This problem is especially prominent for single-cell analyses of the tumor compartment, because high intra-patient similarity (as opposed to inter-patient similarity) results in stricter hierarchical structured data that confounds enrichment analysis. Furthermore, to identify signatures which are truly representative of the entire group, there is a need to quantify the robustness of otherwise statistically significant signatures to sample exclusion. Here, we present a new nonparametric statistical method, BEANIE, to account for these issues, and demonstrate its utility in two cancer cohorts stratified by clinical groups to reduce biological hypotheses and guide translational investigations. Using BEANIE, we show how the consideration of sample-specific versus group biology greatly decreases the false positive rate and guides identification of robust signatures that can also be corroborated across different cell type compartments.

bioinformatics↗

bioRxiv2020

Improved bioavailability of montelukast through a novel oral mucoadhesive film in humans and mice

The leukotriene receptor antagonist Montelukast (MTK) is an approved medication for the treatment of asthma and allergic rhinitis. The existing marketed tablet forms of MTK exhibit inconsistent uptake and bioavailability, which partially explains the presence of a significant proportion of MTK low- and non-responders in the population. Besides that, tablets are suboptimal formulations for patients suffering of dysphagia, for example seen in COVID-19 patients or in patients with neurodegenerative diseases such as Alzheimers Disease. The increasing interest in repurposing of MTK for the treatment of such patients and the need for an improved bioavailability triggered us to reformulate MTK. The aim was to develop a mucoadhesive MTK film with a good safety and improved pharmacological, i.e. improved bioavailability, profile in humans as well as in a mouse model of Alzheimers Disease. We tested dissolution of the mucoadhesive film containing MTK in saliva buffer and assessed pharmacoexposure and -kinetics after acute and chronic oral application in mice. Furthermore, we performed a Phase I safety / bioavailability / pharmacokinetic analysis in healthy volunteers. The latter included a comparison with the marketed tablet form as well as a quantitative analysis of the MTK levels in the cerebrospinal fluid. The novel MTK film demonstrated significantly improved bioavailability compared to the marketed tablet in the clinical phase 1a study in healthy humans. Furthermore, there were measurable amounts of MTK present in the cerebrospinal fluid (CSF) at the 3.0 and 7.0 hour time points post drug administration in humans. In mice, MTK was detected in serum and CSF after acute and chronic exposure in a dose-dependent manner. The developed mucoadhesive film of MTK represents a promising alternative for the tablet delivery. The oral film might lower the non-responder rate in patients with asthma and might be an interesting product for repurposing of MTK in other diseases. As we demonstrate BBB penetrance in a preclinical model as well as in a clinical study, the oral film of MTK might find its use as a therapeutic for acute and chronic neurodegenerative diseases such as dementias and stroke.

pharmacology and toxicology↗

bioRxiv2020

Clinical interpretation of integrative molecular profiles to guide precision cancer medicine

Individual tumor molecular profiling is routinely used to detect single gene-variant ("first-order") genomic alterations that may inform therapeutic actions -- for instance, a tumor with a BRAF p.V600E variant might be considered for RAF/MEK inhibitor therapy. Interactions between such first-order events (e.g., somatic-germline) and global molecular features (e.g. mutational signatures) are increasingly associated with clinical outcomes, but these "second order" alterations are not yet generally accounted for in clinical interpretation algorithms and knowledge bases. Here, we introduce the Molecular Oncology Almanac (MOAlmanac), a clinical interpretation algorithm paired with a novel underlying knowledge base to enable integrative interpretation of genomic and transcriptional cancer data for point-of-care treatment decision-making and translational hypothesis generation. We compared MOAlmanac to first-order interpretation methodology in multiple retrospective patient cohorts and observed that the inclusion of preclinical and inferential evidence as well as second-order molecular features increased the number of nominated clinical hypotheses. MOAlmanac also performed matchmaking between patient molecular profiles and cancer cell lines to further expand individualized clinical actionability. When applied to a prospective precision oncology trial cohort, MOAlmanac nominated a median of two therapies per patient and identified therapeutic strategies administered in 46% of patient profiles. Overall, we present a novel computational method to perform integrative clinical interpretation of individualized molecular profiles. MOAlmanc increases clinical actionability over conventional approaches by considering second-order molecular features and additional evidence sources, and is available as an open-source framework.

cancer biology↗

bioRxiv2019

Should virus capsids assemble perfectly? A simple equilibrium model for defects

Although published structural models of viral capsids generally exhibit a high degree of regularity or symmetry, structural defects might be expected because of the fluctuating environment in which capsids assemble and the requirement of some capsids for disassembly prior to genome delivery. Defective structures are observed in computer simulations, and are evident in single-particle cryoEM studies. Here, we quantify the conditions under which defects might be expected, using a statistical mechanics model allowing for ideal, defective, and vacant sites. The model displays a threshold in affinity parameters below which there is an appreciable population of defective capsids. Even when defective sites are not allowed, there is generally some population of vacancies. Analysis of single particles in cryoEM micrographs yields a confirmatory {gtrsim}15% of defective particles. Our findings suggest structural heterogeneity in virus capsids may be under-appreciated, and also points to a non-traditional strategy for assembly inhibition.

biophysics↗