Biology subjects

Clarke, C.

Publications and source records attributed to Clarke, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Continued genomic surveillance of Vibrio cholerae O1 isolates from cholera cases in Europe, 2023-2024

Background: Following the global resurgence of cholera in 2022, 51 cholera cases were reported by European countries. We previously characterised 49 Vibrio cholerae O1 isolates from these cases using whole genome sequencing. In 2023 and 2024, 59 additional cholera cases were reported by 11 European countries. Aim: We aimed to confirm that V. cholerae O1 isolates associated with cholera cases reported in Europe in 2023-2024 belonged to the epidemic seventh pandemic El Tor (7PET) lineage rather than to non-epidemic V. cholerae O1 lineages, and to characterise their virulence, antimicrobial resistance (AMR) determinants and phylogenetic relationships. Methods: Fifty V. cholerae O1 isolates were available for whole genome sequencing, of which 49 yielded genomes of sufficient quality for analysis. Genomes were analysed together with more than 1,500 publicly available 7PET genomes to place the European isolates into a global phylogenetic context. Results: All 49 genomes belonged to the 7PET lineage and Wave 3. Four sub-lineages were identified: BD1.2 (2/49), AFR12 (6/49), AFR13 (8/49) and Pre-AFR15 (33/49). The predominance of Pre-AFR15 confirms its continued contribution to the ongoing global cholera resurgence. In contrast to our 2022 survey, eight highly drug-resistant AFR13 isolates were detected, documenting the international spread of this highly drug-resistant clone resistant to multiple first-line antimicrobial agents. Conclusion: Whole genome sequencing should be routinely used by reference laboratories to distinguish epidemic 7PET from non-epidemic V. cholerae O1 lineages and to monitor the emergence and international spread of AMR clones. Continued collaborative genomic surveillance of travel-associated cholera cases can provide early insights into the emergence, international spread and antimicrobial resistance of 7PET sub-lineages.

genomics↗

bioRxiv2026

Oncogene-driven metabolic regulation of Dihydroceramide Desaturase 1 (DES1) converges on GAPDH in matrix-detached conditions

Deregulation of sphingolipid (SL) metabolism is well-established across many cancers, yet the underlying mechanisms that drive changes in SLs are poorly understood. We previously identified dihydroceramide desaturase 1 (DES1) as a downstream target of HER2 and implicated DES1 as a driver of anchorage-independent survival in breast cancer. In this study, we expand on these results to establish the oncogenic PI3K pathway as a driver of post-translational DES1 activity following cell detachment from the extracellular matrix. PI3K activation of DES1 required glucose uptake and metabolism through both glycolysis and the pentose phosphate pathway. However, it did not require glucose flux into the TCA cycle and was independent of antioxidant capacity of the cell. Moreover, Instead, results identify GAPDH - a point of convergence between glycolysis and PPP - as important for oncogene-driven DES1 activity. Overall, this study defines a novel pathway of DES1 regulation and establishes DES1 as a point of crosstalk between glucose and SL metabolic pathways.

Cancer Biology↗

bioRxiv2026

Genomic history and selection in Roman and early medieval Britain

Leading biomedical resources rely on genome variation in Britain1-3, but the historical processes that shaped present-day fine-scale diversity remain debated4-13. Here we sequenced 1039 ancient shotgun genomes from Britain (median 1.4-fold coverage), primarily dating to the first millennium CE. We imputed [~]660 million variants in the UK Biobank14-16 and employed genealogy-based ancestry reconstruction. We found an association between Iron Age consanguinity and matrilineal burial practices17, later disrupted following the Roman Conquest. Despite this societal impact, only 20% of Roman-period individuals carried detectable ancestry from outside Britain. In contrast, from the 6th century CE we detect widespread influx of ancestry in over 70% of individuals in southernAnglo-Saxon Britain, with limited local admixture. We find previously underappreciated heterogeneity, with ancestries associated with Central and Southern Europe rising in prevalence from the 7th century CE. We demonstrate distinct Scandinavian-related ancestry in many Viking-associated contexts, but show that the population-level impact of the Viking Age in Britain was limited. Finally, we detect pre-medieval selection on variants linked with key immunity genes TLR10-TLR1 and IRF8. These results identify population-level and selective processes that shape variation and disease risk in Britain today.

genomics↗

bioRxiv2025

Human colonization with Phytobacter co-harbouring blaIMP-4 and mcr-9.1 highlights its potential as emerging human pathogen.

Phytobacter is a recently delineated, frequently misidentified genus within the order Enterobacterales. Following two rare cases of patient colonization with multidrug resistant Phytobacter in Ireland, this study presents a genus-wide genomic analysis that aims to define the pathogenic potential of Phytobacter species, with emphasis on their role as emerging human pathogens and reservoirs of carbapenemases. Two carbapenemase-encoding isolates were recovered from rectal swabs in Ireland in 2024 and were initially identified as Phytobacter by MALDI-ToF. Whole-genome sequencing with in silico species typing (dDDH, ANI) provided definitive taxonomic resolution. A genus-wide maximum-likelihood core-genome phylogeny was reconstructed, and the plasmidome and resistome were bioinformatically profiled across all available Phytobacter genomes. Phenotypic susceptibility of the Irish isolates was determined through minimum inhibitory concentration (MIC) testing. The Irish isolates (P. diazotrophicus E787336 and P. ursingii E980862) are the first reported Phytobacter strains carrying both plasmid-borne blaIMP-4 and mcr-9.1 in the genus. MIC testing confirmed resistance to aztreonam, aminoglycosides, cephalosporins, fluoroquinolones, and the {beta}-lactam/{beta}-lactamase inhibitor combination piperacillin-tazobactam. Across 34 Phytobacter genomes examined, 22 distinct plasmid replicon types were identified in 22 isolates, often shared across species. The genus-wide resistome encompassed 71 genes, more than half predicted to be acquired, with carbapenemases detected in 26.5% (9/34) of the genomes. In summary, Phytobacter harbors a diverse, plasmid-borne resistome including carbapenemases, with documented cases of human colonization and infection. These findings support its recognition as an emerging pathogen and reservoir of antimicrobial resistance, underscoring the need for improved clinical identification, genomic surveillance, and preparedness for limited treatment options. Author summarySince Phytobacter was first characterized in 2007, this bacterial genus has mainly been associated with plant growth promotion. More recently, however, increasing reports of human infections have raised concerns about its potential as emerging bacterial pathogen. These are further underscored by the description of multidrug resistant isolates capable to withstand different classes of antimicrobials, including carbapenems which are often used as a last line of treatment. Following the rare finding of multidrug resistant Phytobacter in two patients in Ireland, we combined bioinformatics and laboratory testing to characterize the antimicrobial resistance profile of this overlooked bacterial genus. Our results uncover the variety of resistance determinants, including to carbapenems, which is encoded in the genomes of Phytobacter. This shows its potential as hidden reservoir of drug resistance and emerging bacterial pathogen. We encourage improved clinical recognition and monitoring of Phytobacter to better anticipate infections with limited therapeutic options.

genomics↗

bioRxiv2025

Plasmidome, resistome, and virulence-associated genes characterization of Acinetobacter johnsonii in NASA cleanrooms and a clinical setting.

Evidence shows persistence of non-spore-forming Acinetobacter johnsonii in high-stakes controlled and nutrient-limited environments. This study aims to explore the mechanisms underpinning such adaptability through a comprehensive genomic analysis of 22 isolates of A. johnsonii from NASAs Payload Hazardous Servicing Facility (PHSF) and one carbapenem-resistant strain (E154408A) from patient colonization in Ireland. Core-genome phylogeny revealed clustering of PHSF-originating isolates in a monophyletic clade divergent from the main species lineage. Species-wide virulence-associated genes and metabolic profiling indicated the unique presence in PHSF-originating isolates of two complete efflux pumps and of a conserved allantoin racemase, suggesting adaptability for multiple environmental stresses. Observed ubiquity of blaOXA in investigated genomes (n=112) and phenotypically-validated multidrug-resistant profile of E154408A strain highlight A. johnsoniis potential as antimicrobial resistance (AMR) reservoir. Plasmidome analysis suggested gain/loss events across the monophyletic population and potential AMR acquisition pathways. Genome-to-metagenome mapping identified genomic signatures of A. johnsonii in PHSF >10 years post initial isolation. ImportanceAcinetobacter johnsonii is increasingly recognized as an emerging human pathogen, with growing evidence of its ability to persist in controlled, high-stakes environments, posing risks as both persisting environmental contaminant and antimicrobial resistance (AMR) reservoir. Yet, gaps remain in our understanding of its AMR profile and the mechanisms that enable its enhanced environmental adaptability. This knowledge is necessary in contexts where biological cleanliness is a priority such as clinical settings and spacecraft assembly facilities cleanrooms, where contamination of hardware with terrestrial microorganisms is concerning. In this study, we aim to address some of key knowledge gaps by providing genomic insights into a rare multi-drug resistant clinical isolate and 22 NASA cleanroom isolates that persisted for over a decade in extremely clean conditions. Our findings will help evaluate the contamination risk of A. johnsonii in high-stakes environments and ultimately strengthen our ability to manage this microbial contaminant across terrestrial and extraterrestrial settings. HighlightsO_LICleanrooms-derived A. johnsonii genomes show favorable traits for increased adaptability C_LIO_LIGenomic signatures of A. johnsonii persisted in the cleanrooms for >10 years C_LIO_LIblaOXA is ubiquitously found in the genome of all A. johnsonii C_LIO_LIE154408A is the first patient colonization by carbapenem-resistant A. johnsonii in Europe C_LI

genomics↗

bioRxiv2025

Evolutionarily divergent DUF4465 domains have a common vitamin B12-binding function

Domains of Unknown Function (DUFs) comprise a large portion of the bacterial proteome, yet their biological roles remain poorly understood. We recently identified two DUF4465 proteins (IPR027828 family proteins), BtuJ1 and BtuJ2, in the vitamin B12-auxotrophic gut commensal Bacteroides thetaiotaomicron, which act as high-affinity B12-binding proteins that scavenge the cofactor to ensure survival. Such B12 capture is essential for bacteria that have lost the ability to synthesize B12 de novo. The DUF4465 family contains more than 1,000 members distributed across eight bacterial clades in gut microbiome and marine environments, raising the question of whether B12-binding is ubiquitous across this family. Here, we show that B12-binding is conserved across five additional sequence-diverse DUF4465 proteins bringing the total we have characterized to seven. Structural and biochemical analyses, including the crystal structure of D5EK51 from Coraliomargarita akajimensis bound to B12, reveal a conserved augmented {beta}-jellyroll fold and a shared B12-binding motif. Together, these findings establish DUF4465 as a structurally conserved family of B12-binding proteins and point to their widespread role in microbial competition for this essential cofactor.

biochemistry↗

bioRxiv2025

Extracellular Vesicle-Linked Vitamin B12 Acquisition via Novel Binding Proteins in Bacteroides thetaiotaomicron

The acquisition and utilization of vitamin B12 (cobalamin) are essential for the metabolic functions of many gut bacteria, including Bacteroides thetaiotaomicron, which relies on external sources of cobalamin due to its inability to synthesize it de novo. To scavenge cobalamin efficiently, B. thetaiotaomicron employs a sophisticated cobamide uptake system comprising multiple operons encoding outer membrane binding proteins and transporters. This study identifies and characterizes several novel cobalamin-binding proteins and elucidates their roles in cobamide uptake and delivery via bacterial extracellular vesicles (BEVs), highlighting their siderophore-like function for competitive nutrient acquisition. We demonstrate that BtuJ1 and BtuJ2, members of the IPR027828 protein family, bind cobalamin with distinct structural and kinetic properties and are key components of BEVs responsible for cobamide capture and transfer to cells. Structural analyses reveal a conserved augmented {beta}-jellyroll architecture in these proteins, with tyrosine residues playing a central role in stabilizing cobalamin binding. Comparative proteomics of BEVs and cells under cobamide starvation underscore the selective enrichment of BtuJ proteins in BEVs, suggesting a specialized mechanism for nutrient acquisition. Additionally, we identify another novel B12-binding protein, BtuK1. We further establish BtuL as a critical player in early BEV release and propose a theoretical model in which BEVs function similarly to siderophores, scavenging cobalamin in the environment and delivering it to cells via specific receptors. This study provides new insights into the interplay between BEV-mediated transport, cobamide uptake, and the metabolic strategies employed by gut bacteria to thrive in nutrient-limited environments.

biochemistry↗

bioRxiv2025

Emergence of Dual β-lactam and Colistin Resistance via blaFRI-8 and mcr10.2 co-carriage on an IncFII Family Plasmid in Enterobacter vonholyi

ObjectivesEnterobacter vonholyi isolate E323169 represents a rare case of co-carriage of the antimicrobial resistance genes (ARGs) blaFRI-8, mcr-10.2. isolated from a clinical rectal swab. E323169 is one of only 12 available E. vonholyi genomes. To date, and across over two million pathogenic genomes scanned, only three harbour blaFRI-8 and two encode mcr-10.2, yet none exhibit both genes together. This study analyzes the genomic, phenotypic and epidemiological importance of this rare co-occurrence. MethodsSpecies-ID for E323169 was assigned by MALDI-TOF and then re-assigned and confirmed using a multifactorial genomic workflow. Antimicrobial susceptibilities were determined by MIC assay. The genome of E323169 was sequenced on an Illumina-NextSeq-1000, assembled, and annotated for ARGs, virulence factors, and plasmid replicons detection. Comparative phylogenomics used 12 E. vonholyi RefSeq assemblies, and NCBI metadata were analysed for plasmid distributions of blaFRI-8 and mcr-10.2. ResultsE323169 carried six ARGs: four chromosomally encoded (blaACT-91, fosA, oqxA10, oqxB9) and two plasmid-borne (blaFRI-8 and mcr-10.2) co-located on IncFII(p14)_1_p14 replicon. Additional plasmid replicons: Col(MG828)_1 and ColRNAI_1 were also identified. By mining the NCBI Pathogen Detection pipeline, we identified blaFRI-8 on IncFII replicon in E. asburiae JBIWA002, and mcr-10.2 on a multi-replicon (IncFIB/IncFII) plasmid in E. kobei 11778-yvys. ConclusionThis report documents the first E. vonholyi isolate co-harboring blaFRI-8 and mcr-10.2 on a single IncFII family plasmid, showing the widening host range of plasmid-mediated resistance to carbapenems/colistin. No prior similar co-occurrences reported, underscoring its rarity. These findings highlight the urgent need for enhanced clinical screening and genomic surveillance to curb the spread.

microbiology↗

bioRxiv2024

Metabolic adaptations of micrometastases alter EV production to generate invasive microenvironments

Altered cellular metabolism has been associated with acquisition of invasive phenotypes during metastasis. To study this, we combined a genetically engineered mouse model of mammary carcinoma with syngeneic transplantation and primary tumour resection to generate isogenic cells from primary tumours and their corresponding lung micrometastases. Metabolic analyses indicated that micrometastatic cells increase proline production at the expense of glutathione synthesis leading to a reduction in total glutathione levels. Micrometastatic cells also have altered sphingomyelin metabolism leading to increased intracellular levels of specific ceramides. The combination of these two metabolic adaptations alters small extracellular vesicle (sEV) production to drive generation of an invasive microenvironment. Indeed, micrometastatic cells shut-down Rab27-dependent production of sEVs and, instead, switch-on neutral sphingomyelinase-2 (nSM2)-dependent sEV release. sEVs released in a nSM2-dependent manner from micrometastatic cells, in turn, influence the ability of fibroblasts to deposit extracellular matrix which promotes cancer cell invasiveness. These data provide evidence that metabolic rewiring drives invasive processes in metastasis by influencing sEV release. SummaryBreast cancer cells isolated from lung micrometastases have altered metabolism which influences extracellular vesicle production to generate invasive microenvironments.

cancer biology↗

bioRxiv2023

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanosine triphosphate (GTP) synthesis and is controlled by feedback inhibition and allosteric regulation. IMPDH assembles into micron-scale filaments in cells, which desensitizes the enzyme to feedback inhibition by GTP and boosts nucleotide production. The vertebrate retina expresses two tissue-specific splice variants IMPDH1(546) and IMPDH1(595). IMPDH1(546) filaments adopt high and low activity conformations, while IMPDH1(595) filaments maintain high activity. In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of phosphorylation in IMPDH1 variants. The S477D mutation re-sensitized both variants to GTP inhibition, but only blocked assembly of IMPDH1(595) filaments and not IMPDH1(546) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of the high activity assembly interface, still allowing assembly of low activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, phosphorylation at S477 acts as a mechanism for downregulating retinal GTP synthesis in the dark, when nucleotide turnover is decreased. Like IMPDH1, many other metabolic enzymes dynamically assemble filamentous polymers that allosterically regulate activity. Our work suggests that posttranslational modifications may be yet another layer of regulatory control to finely tune activity by modulating filament assembly in response to changing metabolic demands. SIGNIFICANCE STATEMENTOver 20 different metabolic enzymes form micron-scale filaments in cells, suggesting that filament assembly is a conserved mechanism for regulating diverse metabolic pathways. Filament assembly regulates catalytic activity of many of these enzymes, including inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP biosynthesis. The vertebrate retina expresses two IMPDH1 splice variants that are critical for maintaining nucleotide levels required for phototransduction. Here, we show that filament assembly by these variants is itself controlled by phosphorylation at a single residue, adding further complexity to the tight regulation of nucleotide metabolism in the retina. Phosphorylation and other posttranslational modifications are likely to be a general regulatory mechanism controlling filament assembly by enzymes in many different metabolic pathways.

biochemistry↗

bioRxiv2023

Regulation of Human PINK1 ubiquitin kinase by Serine167, Serine228 and Cysteine412 phosphorylation

Loss-of-function mutations in the human PINK1 kinase (hPINK1) are causative of early-onset Parkinsons disease (PD). Activation of hPINK1 induces phosphorylated ubiquitin to initiate removal of damaged mitochondria by autophagy. Previously we solved the structure of the insect PINK1 orthologue, Tribolium castaneum PINK1, and showed that autophosphorylation of Ser205 was critical for ubiquitin interaction and phosphorylation (Kumar, Tamjar, Waddell et al., 2017). In this advance, we report new findings on the regulation of hPINK1 by phosphorylation. We reconstitute E. coli expressed hPINK1 activity in vitro by direct incorporation of phosphoserine at the equivalent site Serine 228 (pSer228), providing direct evidence for a role for Ser228 phosphorylation in hPINK1 activation. Furthermore, using mass spectrometry, we identify six novel Ser/Thr autophosphorylation sites including regulatory Serine167 phosphorylation (pSer167), which in addition to pSer228 is required for ubiquitin recognition and phosphorylation. Strikingly, we also detect phosphorylation of a conserved Cysteine412 (pCys412) residue in the hPINK1 activation segment. Structural modelling suggests that pCys412 inhibits ubiquitin recognition and we demonstrate that mutation of Cys412 to Ala renders hPINK1 more active towards ubiquitin when expressed in human cells. These results outline new insights into hPINK1 activation by pSer167 and pSer228 and a novel inhibitory mechanism mediated by pCys412. These findings will aid in the development of small molecule activators of hPINK1.

biochemistry↗

bioRxiv2022

Understanding the transcriptional response to ER stress in Chinese hamster ovary cells using multiplexed single cell RNA-seq

Single cell RNA-seq (scRNA-seq) has recently been shown to provide a powerful method for the analysis of transcriptional heterogeneity in Chinese hamster ovary (CHO) cells. A potential drawback of current scRNA-seq platforms is that the cost can limit the complexity of experimental design and therefore the utility of the approach. In this manuscript, we report the use of oligonucleotide barcoding to perform multiplexed CHO cell scRNA-seq to study the impact of tunicamycin (TM), an inducer of the unfolded protein response (UPR). For this experiment, we treated a CHO-K1 GS cell line with 10g/ml tunicamycin and acquired samples at 1, 2, 4 and 8 hr post-treatment as well as a non-treated TM-control. We transfected cells with sample-specific polyadenylated ssDNA oligonucleotide barcodes enabling us to pool all cells for scRNA-seq. The sample from which each cell originated was subsequently determined by the oligonucleotide barcode sequence. Visualisation of the transcriptome data in a reduced dimensional space confirmed that cells were not only separable by sample but were also distributed according to time post-treatment. These data were subsequently utilised to perform weighted gene co-expression analysis (WGCNA) and uncovered groups of genes associated with TM treatment. For example, the expression of one group of coexpressed genes was found to increase over the time course and were enriched for biological processes associated with ER stress. The use of multiplexed single cell RNA-seq has the potential to reduce the cost associated with higher sample numbers and avoid batch effects for future studies of CHO cell biology. HighlightsO_LIPolyadenylated ssDNA oligonucleotide labelling is a viable strategy for multiplexed CHO cell scRNA-seq analysis. C_LIO_LITo demonstrate the effectiveness of the method we conducted an experiment to study the CHO cell response to tunicamycin treatment. C_LIO_LIscRNA-seq was carried out on an untreated control and at 4 time points post tunicamycin treatment. Cells from each sample were transfected with a unique oligonucleotide barcode and pooled for single cell transcriptomics. C_LIO_LIEach sample was demultiplexed post-sequencing and gene expression profiles of > 5,300 cells were obtained across the experiment. Following dimensionality reduction and visualisation, the cells were distributed according to sample identity. C_LIO_LIAnalysis of the resulting data enabled improved understanding of the transcriptional response to tunicamycin treatment. Three gene coexpression modules were found to be correlated with the tunicamycin time course. Gene set enrichment analysis revealed the over representation of genes related to biological processes associated with ER stress, and protein misfolding in one of these groups of coexpressed genes. C_LIO_LIFurther use of this approach will enable the CHO cell biology community to perform increasingly complex single cell experiments in a cost-effective manner. C_LI

cell biology↗

bioRxiv2022

Found in translation: Microproteins are a new class of potential host cell impurity in mAb drug products

Chinese hamster ovary (CHO) cells are used to produce almost 90% of therapeutic monoclonal antibodies (mAbs). The annotation of non-canonical translation events in these cellular factories remains incomplete, limiting not only our ability to study CHO cell biology but also detect host cell protein (HCP) contaminants in the final mAb drug product. We utilised ribosome footprint profiling (Ribo-seq) to identify novel open reading frames (ORFs) including N-terminal extensions and thousands of short ORFs (sORFs) predicted to encode microproteins. Mass spectrometry-based HCP analysis of four commercial mAb drug products using the extended protein sequence database revealed the presence of microprotein impurities for the first time. We also show that microprotein abundance varies with growth phase and can be affected by the cell culture environment. In addition, our work provides a vital resource to facilitate future studies of non-canonical translation as well as the regulation of protein synthesis in CHO cell lines.

genomics↗

bioRxiv2020

Tracing production instability in a clonally-derived CHO cell line using single cell transcriptomics

A variety of mechanisms including transcriptional silencing, gene copy loss and increased susceptibility to cellular stress have been associated with a sudden or gradual loss of monoclonal antibody (mAb) production in Chinese hamster ovary (CHO) cell lines. In this study, we utilised single cell RNA-seq (scRNA-seq) to study a clonally-derived CHO cell line that underwent production instability leading to a dramatic reduction of the levels of mAb produced. From the scRNA-seq data we identified sub clusters associated with variations in the mAb transgenes and observed that heavy chain gene expression was significantly lower than that of the light chain across the population. Using trajectory inference, the evolution of the cell line was reconstructed and was found to correlate with a reduction in heavy and light chain gene expression. Genes encoding for proteins involved in the response to oxidative stress and apoptosis were found to increase in expression as cells progressed along the trajectory. Future studies of CHO cell lines using this technology have the potential to dramatically enhance our understanding of the characteristics underpinning efficient manufacturing performance as well as product quality. HighlightsO_LIA clonally-derived CHO cell line in our laboratory had undergone production instability - in that the amount of intact monoclonal antibody had reduced dramatically to levels at which reliable quantitation was no longer possible. We were, however, able to detect mAb heavy and light chain protein, as well as dimerised light chain species in the cell culture media. C_LIO_LISingle cell RNA-seq was utilised to capture > 3,800 gene expression profiles from the cell line at 72hrs post seeding. C_LIO_LIAnalyses of the scRNA-seq data uncovered transcriptional heterogeneity and revealed the presence of multiple intra cell line clusters. The heavy chain transcript was detected at a significantly lower level in comparison to light chain transcripts. Light chain gene expression was not only more abundant, but also expressed more uniformly across the cell population. C_LIO_LIUsing unsupervised trajectory analysis, the emergence of heterogeneity in the cell population was traced from those cells most similar to the original isolated clone to those where transcription of the mAb heavy and light chain was undetectable. C_LIO_LISubsequent analysis of CHO cell gene expression patterns revealed a correlation between the progression of cells along the trajectory and the upregulation of genes involved in the cellular response to oxidative stress. C_LI

cell biology↗

bioRxiv2020

HybPhaser: a workflow for the detection and phasing of hybrids in target capture datasets

Premise of the studyHybrids contain divergent alleles that can confound phylogenetic analyses but can provide insights into reticulated evolution when identified and phased. We developed a workflow to detect hybrids in target capture datasets and phase reads into parental lineages using a similarity and phylogenetic framework. MethodsWe used Angiosperms353 target capture data for Nepenthes including known hybrids to test the novel workflow. Reference mapping was used to assess heterozygous sites across the dataset, detect hybrid accessions and paralogous genes. Hybrid samples were phased by mapping reads to multiple references and sorting reads according to similarity. Phased accessions were included in the phylogenetic framework. ResultsAll known Nepenthes hybrids and nine more samples had high levels of heterozygous sites, reads associated with multiple divergent clades, and were phased into accessions resembling divergent haplotypes. Phylogenetic analysis including phased accessions increased clade support and confirmed parental lineages of hybrids. DiscussionHybPhaser provides a novel approach to detect and phase hybrids in target capture datasets, which can provide insights into reticulations by revealing origins of hybrids and reduce conflicting signal leading to more robust phylogenetic analyses.

evolutionary biology↗

bioRxiv2019

Sub physiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells

RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes, however alternative mRNA splicing, has thus far, received little attention. In this study, we utilised RNASeq for transcriptomic analysis of a monoclonal antibody producing CHOK1 cell line subjected to a temperature shift. More than 2,365 instances of differential splicing were observed 24hrs after the reduction of cell culture temperature. 1,163 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using qPCR in the monoclonal antibody producing CHOK1 line used for RNASeq and a further 2 CHOK1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilised to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.

cell biology↗

bioRxiv2019

Expanding the Chinese hamster ovary cell long non-coding RNA transcriptome using RNASeq

Our ability to study Chinese hamster ovary (CHO) cell biology has been revolutionised over the last decade with the development of next generation sequencing and the publication of reference DNA sequences for CHO cells and the Chinese hamster. RNA sequencing has not only enabled the association of transcript expression with bioreactor conditions and desirable bioprocess phenotypes but played a key role in the characterisation of protein coding and small non-coding RNAs. The annotation of long non-coding RNAs, and therefore our understanding of their role in CHO cell biology, has been limited to date. In this manuscript, we use high resolution RNASeq data to more than double the number of annotated lncRNA transcripts for the CHOK1 genome. In addition, the utilisation of strand specific sequencing enabled the identification of more than 1,000 new lncRNAs located antisense to protein coding genes. The utility of monitoring lncRNA expression is demonstrated through an analysis of the transcriptomic response to a reduction of cell culture temperature and identification of simultaneous sense/antisense differential expression for the first time in CHO cells. To enable further studies of lncRNAs, the transcripts annotated in this study have been made available for the CHO cell biology community.

cell biology↗

bioRxiv2019

Porous upconversion nanostructures as bimodal biomedical imaging contrast agents

Lanthanide ions doped upconversion nanoparticles (UCNPs) hold great promise as the imaging contrast agent for multimodal medical imaging techniques for diagnosis, including fluorescent bioimaging, magnetic resonance imaging, and computed tomography. However, the maximized signal values of fluorescence and MRI cannot be achieved simultaneously from the same upconversion nanoparticles structures because high specific surface areas can benefit the signal gaining of MRI while big size can induce brighter fluorescent imaging. In this work, we designed and fabricated novel core-porous shell structures for UCNPs with much-enhanced signal values for both fluorescent imaging and MRI. The core-porous shell UCNPs were synthesized via a post-treatment process after an inert shell was coated onto the core UCNPs. The formation mechanism was carefully investigated. The fluorescent and magnetic resonance properties have been detailed characterized and compared from core, core-shell and core-porous UCNPs. Large and bright UCNPs in fluorescence and MRI have been achieved and great potential as the dual-modal contrast agent.

biophysics↗