Biology subjects

Keane, J.

Publications and source records attributed to Keane, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Systematic analysis of genetic vulnerabilities caused by chromosome loss events in cancer

Chromosome loss events are common across tumor types but rarely occur in normal tissue, raising the possibility that these alterations could be exploited as cancer-specific therapeutic vulnerabilities. Here, we integrate genome-scale CRISPR screening data with chromosome-level copy number profiles from 1,150 cancer cell lines and identify 389 dependencies that are selectively associated with individual chromosome loss events. The most significant vulnerabilities uncovered by our analysis are paralogous gene pairs, in which the loss of a paralog on an aneuploid chromosome establishes a unique dependency on its redundant paralog encoded elsewhere in the genome. We experimentally validate two paralog dependencies: loss of chromosome 8p, which encodes the phosphatase PPP2CB, creates a specific dependency on its paralog PPP2CA, while loss of the Y chromosome, which encodes the helicase DDX3Y, creates a specific dependency on its paralog DDX3X. Systematic benchmarking of CRISPR screening data indicates that the selective dependency on DDX3X in loss-of-Y cells exceeds other published aneuploidy-associated vulnerabilities and matches or exceeds most clinically-validated biomarker-target relationships. Furthermore, we found that the acute degradation of DDX3X was sufficient to trigger apoptosis in vitro and tumor regressions in mice. Collectively, these findings establish chromosome loss as a recurrent source of biomarker-defined therapeutic targets, identify paralog insufficiency as a general vulnerability in aneuploid cells, and provide in vivo proof-of-concept for targeting DDX3X in cancers with Y chromosome loss.

cancer biology↗

bioRxiv2026

Neutrophil subsets in SLE exhibit increased glycolysis that correlates with disease activity

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by sustained type I interferon signalling and widespread immune dysregulation. Low-density neutrophils (LDNs) are expanded in SLE and display pro-inflammatory and tissue-damaging properties. However, their metabolic phenotype remains poorly defined. Here, we performed a comprehensive metabolic characterisation of circulating LDNs and normal-density neutrophils (NDNs) from patients with SLE and matched healthy individuals (HC). Neutrophil subsets were isolated from peripheral blood of SLE patients and HC donors using a two-step protocol of negative selection and Percoll density centrifugation. Immunophenotyping phenotype was carried out by flow cytometry to assess phenotypic expression of common neutrophil markers CD15, CD16, CD10, CD66b, CD62L, MPO, and IL-1{beta}. Bioenergetic profiling of LDNs and NDNs was performed in situ using the Seahorse MitoStress test to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Metabolic flexibility and phenotypic alterations were assessed in LDNs and NDNs following inhibiting mitochondrial metabolism with oligomycin and glycolysis with 2DG. We found that SLE LDNs exhibit an immature phenotype compared with autologous and healthy NDNs, as determined transcriptionally by C/EBP{varepsilon} and by surface protein expression levels of CD10. Both LDNs and NDNs from SLEDAI[≥]4 patients demonstrated significantly elevated ECAR relative to HC neutrophils. Further, SLE LDNs displayed enhanced metabolic flexibility, with the capacity to switch towards a glycolytic phenotype under metabolic stress conditions. Inhibition of glycolysis altered the inflammatory and maturation-associated phenotype of both SLE neutrophil subsets, indicating a direct link between cellular metabolism and pathogenic neutrophil function. Collectively, these findings identify fundamental metabolic alterations in SLE neutrophil subsets and support neutrophil immunometabolism as a potential therapeutic target in SLE.

immunology↗

bioRxiv2025

Sex-Specific Metabolic Programming in Human Neutrophil Subsets

BackgroundSex differences in immune responses are well-documented, with females exhibiting more robust immunity against infections but higher susceptibility to autoimmune diseases, while males often demonstrate more severe inflammatory pathology. Neutrophils which are key players in the innate immune response, display sex-based functional differences, but whether these extend to metabolic programming, particularly in low-density neutrophils (LDNs), remains unknown. MethodsWe isolated LDNs and normal-density neutrophils (NDNs) from healthy human donors using density gradient centrifugation and negative selection. Cellular metabolism was assessed via Seahorse XF analysis (measuring oxygen consumption rate, OCR, and extracellular acidification rate, ECAR), alongside flow cytometry for maturity markers (CD16hi/lo). ResultsMale LDNs exhibited significantly higher basal OCR and ATP production than female LDNs, while no sex differences were observed in NDNs. Strikingly, male LDNs had higher OCR and glycolysis than their matched NDNs, whereas female NDNs were more oxidative than their LDNs. These metabolic differences were independent of neutrophil maturity, as CD16hi frequencies did not differ between subsets or sexes. ConclusionsOur study reveals, for the first time, subset-specific sexual dimorphism in neutrophil metabolism whereby male LDNs adopt a hypermetabolic (oxidative/glycolytic) phenotype, while female NDNs retain higher oxidative capacity. This reprogramming occurs independently of developmental stage, suggesting sex hormones or epigenetic regulation may drive these differences. These findings provide a metabolic basis for sex-biased immune responses and highlight the need for sex-stratified approaches in neutrophil-targeted therapies. Plain English SummaryMen and women fight infections differently, and our study could reveal why--their immune cells produce energy in distinct ways. We examined two types of neutrophils (infection-fighting cells), normal cells (NDNs) and low-density cells (LDNs) found in inflammation. Using advanced metabolic measurements, we discovered that mens LDNs use more oxygen and generate more energy than womens. Surprisingly, while mens LDNs were more active than their normal neutrophils, women showed the opposite--their normal neutrophils were more energetic than their LDNs. These differences werent due to cell maturity, suggesting biological distinctions between sexes. This may help explain why men often have worse outcomes in diseases like sepsis (where oxygen-driven inflammation dominates), while women are more prone to autoimmune diseases like lupus. Our findings could lead to better sex-specific treatments by dampening overactive immune responses in men or adjusting metabolism in women to prevent autoimmune flares. This research highlights why medical studies must consider sex differences, as one-size-fits-all treatments may miss key biological variations. HighlightsO_LICD16hi frequencies show no sex differences, however female (but not male) NDNs contain significantly more mature CD15+ CD16hi cells than their LDNs, revealing female-specific maturation differences between subsets. C_LIO_LIFirst report of sex-specific metabolic differences in LDNs where males exhibit significantly higher basal respiration and ATP production than female LDNs. C_LIO_LIMale LDNs show higher OCR than matched NDNs, while female NDNs are more oxidative than their LDNs. C_LI

immunology↗

bioRxiv2025

Human macrophages secrete both interferon α and interferon β protein during infection with Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) infection activates type I interferons (IFNs) which are crucial mediators of tuberculosis (TB) pathogenesis. Despite assumptions that IFN and IFN{beta} are secreted by macrophages, direct protein quantification in primary human monocyte-derived macrophages is surprisingly lacking. Here, we demonstrate measurable IFN and IFN{beta} secretion by MDMs infected with both virulent (H37Rv) and attenuated (H37Ra) Mtb strains as early as 48 h post-infection, with levels persisting at 120 h. These findings challenge existing assumptions about type I IFN kinetics and highlight the importance of timing in experimental designs and provides a foundation for exploring their role in host-pathogen interactions.

immunology↗

bioRxiv2025

Dexamethasone impairs glycolysis but improves mycobacterial killing in primary human macrophages

Glucocorticoids (GC) are useful adjunctive host directed therapies for sub-types of tuberculosis (TB). Macrophages play a central role in controlling Mycobacterium tuberculosis (Mtb) infection, relying on glycolytic reprogramming to support an effective host defense, yet the influence of GC on these important phagocytes is poorly understood. Here, we examined the impact of dexamethasone on metabolic and functional responses of primary human airway macrophages (AM) from bronchoalveolar lavage fluid and monocyte-derived macrophages (MDM). We found that dexamethasone significantly reduced basal and compensatory glycolysis in both AM and MDM, and decreased expression of the glycolytic enzyme PFKFB3. Oxidative metabolism was lower in dexamethasone AM but not MDM, indicating different specific metabolic sensitivity of macrophages. Dexamethasone also inhibited the glycolytic response to Mtb and reduced secretion of IL-1{beta}, TNF, IL-6, IL-8, and IL-10. Dexamethasone-treated macrophages showed enhanced survival following Mtb infection and these cells had a significant reduction in bacterial burden. This antimicrobial effect was impaired when macrophages were pre-treated with bafilomycin A1, implicating that phagosomal acidification may at least in part mediate dexamethasone-induced bacterial control. Collectively, these findings demonstrate that dexamethasone reprograms human macrophage metabolism toward a less glycolytic state while preserving their ability to limit Mtb growth. These results may offer a basis for the clinical benefit of GC in some TB presentations and support the development of targeting GC therapies to macrophages, thereby mitigating inflammation without compromising host antimicrobial defense.

immunology↗

bioRxiv2025

Succinate enhances mitochondrial metabolism and phagocytosis in human airspace monocytes

Airspace macrophages (AM) are crucial to host defence and to maintenance of lung homeostasis, with smoking drastically compromising these functions. Airspace monocytes, precursors of the differentiated AM, are found in increased numbers in the lungs of smokers yet little is known about their metabolic regulation and function. Here, we develop a click chemistry-based single cell analysis platform to characterise human airspace monocytes and AM ex vivo, identifying distinct metabolic profiles and a key role for oxidative phosphorylation in supporting phagocytic function. While blood and newly recruited CD93+ airspace monocytes show low mitochondrial dependency, AM rely heavily on oxidative phosphorylation. Acute succinate supplementation enhanced mitochondrial metabolism and phagocytosis in monocytes and promoted their differentiation into highly oxidative macrophages with enhanced function. Succinate emerges as a promising candidate to restore lung immune function, particularly in the smokers lung where airspace monocytes are enriched. Overall, we identify mitochondrial metabolism as a key modulator of lung immune function and a target for therapeutic intervention, with potential applications in systemic monocyte-targeted therapies and metabolic preconditioning for adoptive cell therapies. One Sentence Summary: Mitochondrial metabolism regulates phagocytic function in human lung monocytes and macrophages and can be targeted using succinate. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/659271v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@18c8716org.highwire.dtl.DTLVardef@f8f4b8org.highwire.dtl.DTLVardef@8a806forg.highwire.dtl.DTLVardef@f5f8b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Mitochondrial Bioenergetic Failure in SLE Immunocytes: Targeting Fitness for Therapy

BackgroundSystemic Lupus Erythematosus (SLE) is characterized by dysregulated immune responses linked to immunometabolic perturbations. While mitochondrial dysfunction has been implicated in SLE, its cell-type-specific impact on immune subsets remains underexplored. MethodsWe repurposed existing RNA-seq data from SLE patient peripheral blood mononuclear cells, with a focus on nuclear-encoded mitochondrial (NEmt) genes, as well as mitochondrial genes themselves, to identify differentially expressed genes compared to healthy controls. Mitochondrial stress tests were performed on freshly isolated CD4+ T cells, CD8+ T cells, B cells, and monocytes from SLE patients and healthy donors to assess bioenergetic function. ResultsRNA-seq revealed that both NEmt genes and mitochondrial genes were downregulated in the PBMC population of SLE patients. In situ mitochondrial stress tests revealed significant reductions in oxygen consumption rate (OCR), indicating impaired oxidative phosphorylation (OXPHOS) across all immune subsets, while extracellular acidification rate (ECAR), a marker of glycolysis, remained unchanged. These findings highlight immune-cell-specific mitochondrial bioenergetic failure in SLE, without compensatory glycolytic adaptation. ConclusionOur results position mitochondrial fitness as a novel therapeutic target in SLE. We propose leveraging high-throughput screening of mitochondria-targeted compounds, including FDA-approved agents, to enhance OXPHOS, regulate mitophagy, or mitigate oxidative stress. This precision-based approach offers a paradigm shift from conventional immunosuppression to metabolic recalibration, with the potential to restore immune homeostasis in SLE. Key messagesO_LIWhat is already known on this topic: Mitochondrial dysfunction and metabolic reprogramming have been linked to SLE pathogenesis, but the cell-type-specific extent of mitochondrial impairment and its therapeutic potential remained unclear. C_LIO_LIWhat this study adds: We demonstrate reduced OXPHOS in CD4+ T cells, CD8+ T cells, B cells, and monocytes from SLE patients for the first time, highlighting cell-type-specific mitochondrial dysfunction as a possible driver of immune dysregulation and target for therapy. C_LIO_LIHow this study might affect research, practice, or policy: This study advocates for drug repurposing to restore mitochondrial fitness, offering a novel therapeutic strategy to complement existing SLE treatments and improve patient outcomes. C_LI

immunology↗

bioRxiv2025

HIV inhibits Warburg metabolism in human macrophages infected with Mycobacterium tuberculosis.

Tuberculosis (TB)-associated mortality remains disproportionately high among people living with HIV (PLWH), with macrophage dysfunction representing a key mechanism of impaired host defence against Mycobacterium tuberculosis (Mtb) infection. Using the U1 chronically HIV-infected macrophage cell line model coupled with primary human monocyte-derived macrophages (MDMs) exposed to HIV-1 gp120, we systematically characterized immunometabolic perturbations during Mtb infection. Nanostring RNA analysis revealed that Mtb monoinfection upregulated glycolytic genes while suppressing oxidative phosphorylation (OXPHOS) transcripts, consistent with a Warburg-type metabolic shift. Conversely, HIV infection downregulated glycolytic enzymes and enhanced mitochondrial respiratory chain components. Coinfection studies demonstrated HIV-mediated suppression of Mtb-induced glycolytic reprogramming. Extracellular flux analysis demonstrated that gp120 exposure increased basal oxygen consumption rate while impairing spare respiratory capacity in Mtb-infected MDMs, effectively blocking the Warburg metabolic transition. Notably, gp120 concentrations equivalent to those observed in antiretroviral therapy (ART)-treated PLWH significantly disrupted metabolic plasticity and high-dose gp120 attenuated Mtb-induced TNF- secretion. ImportanceThis study provides mechanistic insight into HIV-associated susceptibility to TB by demonstrating that HIV-1 infection fundamentally alters macrophage immunometabolic responses to Mtb. We establish that HIV-1, through gp120-mediated signaling, subverts the critical glycolytic induction required for effective antimicrobial responses against Mtb. The persistence of this metabolic dysregulation at clinically relevant gp120 concentrations, comparable to those observed in virologically suppressed PLWH, suggests ongoing immunological vulnerability despite ART. These findings identify HIV-induced metabolic reprogramming as a potential contributor to the persistently elevated TB risk in ART-treated individuals and highlight macrophage immunometabolism as a promising therapeutic target for host-directed therapies in HIV/TB coinfection. The dissociation between metabolic and cytokine responses suggests complex, multifactorial mechanisms underlying HIV-associated impairment of anti-mycobacterial immunity, warranting further investigation into the molecular pathways connecting cellular metabolism and immune effector functions.

immunology↗

bioRxiv2025

Immunometabolic Reprogramming of Monocytes in Tuberculosis Infection and Disease

RationaleMonocytes are central to host defence against Mycobacterium tuberculosis (Mtb), yet their functional and metabolic profiles during latent TB infection (TBI) and active TB disease (TBD) remain poorly defined. Immunometabolic dysfunction may underlie ineffective responses in TB, but cell-specific mechanisms are unclear. ObjectivesTo compare the phenotypic, functional, and metabolic profiles of circulating monocytes from individuals with TBI, TBD, and healthy controls (HC), and assess the impact of treatment. MeasurementsPeripheral blood monocytes were profiled using high-dimensional flow cytometry, Luminex cytokine/chemokine assays, and SCENITH, a flow-based metabolic assay. Unstimulated and Mtb-stimulated monocytes from treatment-naive and treated individuals were analysed. Main ResultsMonocytes from TBI and TBD showed distinct phenotypes from HC, marked by elevated CD14 and CD45RA. HLA-DR was reduced in TBI versus HC and further decreased in TBD. TNF receptors were downregulated in TBI but unchanged in TBD. Baseline cytokine and chemokine profiles in TBI and TBD were similar (yet distinct from HC), but Mtb stimulation elicited a stronger cytokine response in TBI. Metabolically, TBI and TBD monocytes exhibited increased glycolysis and reduced mitochondrial dependence versus HC. Treatment partially restored mitochondrial function. Upon Mtb challenge, TBI monocytes had higher glycolytic capacity than TBD. ConclusionsMonocyte metabolic plasticity and cytokine production distinguish latent from active TB and are partially reversible with treatment. Circulating monocyte metabolism reflects TB immune status and may serve as a biomarker or therapeutic target. Reprogrammed glycolytic profiles in TBI contrast with impaired adaptability in TBD, suggesting dysfunctional myeloid activation during disease.

immunology↗

bioRxiv2024

Typhi Mykrobe: fast and accurate lineage identification and antimicrobial resistance genotyping directly from sequence reads for the typhoid fever agent Salmonella Typhi

BackgroundTyphoid fever results from systemic infection with Salmonella enterica serovar Typhi (Typhi) and causes 10 million illnesses annually. Disease control relies on prevention (water, sanitation, and hygiene interventions or vaccination) and effective antimicrobial treatment. Antimicrobial resistant (AMR) Typhi lineages have emerged and become established in many parts of the world. Knowledge of local pathogen populations informed by genomic surveillance, including of lineages (defined by the GenoTyphi scheme) and AMR determinants, is increasingly used to inform local treatment guidelines and to inform vaccination strategy. Current tools for genotyping Typhi require multiple read alignment or assembly steps and have not been validated for analysis of data generated with Oxford Nanopore Technologies (ONT) long-read sequencing devices. Here, we introduce Typhi Mykrobe, a command line software tool for rapid genotyping of Typhi lineages, AMR determinants, and plasmid replicons direct from sequencing reads. ResultsWe validated Typhi Mykrobe lineage genotyping by comparison with the current standard read mapping-based approach and demonstrated 99.8% concordance across nearly 13,000 genomes sequenced with Illumina platforms. For the few isolates with discordant calls, we show that Typhi Mykrobe results are better supported by the evidence from raw sequence read data than the results generated using the mapping-based approach. We also demonstrate 99.9% concordance for detection of AMR determinants compared with the current standard assembly-based approach, with similar results for plasmid marker detection. Typhi Mykrobe predicts clinical resistance categorisation (S/I/R) for eight drug classes, and we show strong agreement with phenotypic categorisations generated from reference laboratory minimum inhibitory concentration (MIC) data for n=1,572 Illumina-sequenced isolates (>99% agreement within one doubling dilution). We show strong concordance (>96% for genotype and >98% for AMR and plasmid) between calls made from ONT reads and those made from Illumina reads for isolates sequenced on both platforms (n =93 genomes). Typhi Mykrobe takes less than a minute per sample and is available at https://github.com/typhoidgenomics/genotyphi. ConclusionsTyphi Mykrobe provides rapid and sensitive genotyping of Typhi genomes direct from Illumina and ONT reads, although lower accuracy was observed for R9 ONT data. It demonstrated accurate assignment of GenoTyphi lineage, detection of AMR determinants and prediction of corresponding AMR phenotypes, and identification of plasmid replicons.

microbiology↗

bioRxiv2024

Human airway macrophages are metabolically reprogrammed by IFN-γ resulting in glycolysis dependent functional plasticity.

Airway macrophages (AM) are the predominant immune cell in the lung and play a crucial role in preventing infection, making them a target for host directed therapy. Macrophage effector functions are associated with cellular metabolism. A knowledge gap remains in understanding metabolic reprogramming and functional plasticity of distinct human macrophage subpopulations, especially in lung resident AM. We examined tissue-resident AM and monocyte derived macrophages (MDM; as a model of blood derived macrophages) in their resting state and after priming with IFN-{gamma} or IL-4 to model the Th1/Th2 axis in the lung. Human macrophages, regardless of origin, had a strong induction of glycolysis in response to IFN-{gamma} or upon stimulation. IFN-{gamma} significantly enhanced cellular energetics in both AM and MDM by upregulating both glycolysis and oxidative phosphorylation. Upon stimulation, AM do not decrease oxidative phosphorylation unlike MDM which shift to "Warburg"-like metabolism. IFN-{gamma} priming promoted cytokine secretion in AM. Blocking glycolysis with 2-deoxyglucose significantly reduced IFN-{gamma} driven cytokine production in AM, indicating that IFN-{gamma} induces functional plasticity in human AM, which is mechanistically mediated by glycolysis. Directly comparing responses between macrophages, AM were more responsive to IFN-{gamma} priming and dependent on glycolysis for cytokine secretion than MDM. Interestingly, TNF production was under the control of glycolysis in AM and not in MDM. MDM exhibited glycolysis-dependent upregulation of HLA-DR and CD40, whereas IFN-{gamma} upregulated HLA-DR and CD40 on AM independently of glycolysis. These data indicate that human AM are functionally plastic and respond to IFN-{gamma} in a manner distinct from MDM. These data provide evidence that human AM are a tractable target for inhalable immunomodulatory therapies for respiratory diseases.

immunology↗

bioRxiv2023

Interferon α and β induce differential transcriptional and functional metabolic phenotypes in human monocyte-derived macrophages and blunt glycolysis in response to antigenic stimuli.

In the context of acute settings, the roles of type I interferons (IFNs), notably subtypes IFN2a, 2b, and {beta}, in modulating macrophage metabolism and contributing to host defense against viral and bacterial pathogens are well-established. However, the impact of chronic exposure to type I IFNs on macrophage metabolism, intimately linked to macrophage function, remains less understood. This study aimed to unravel the nuanced host responses induced by type I IFN cytokines, offering insights for potential therapeutic approaches in diseases associated with these cytokines.Employing a combination of transcriptional profiling and real-time functional analysis, we delineated the temporal evolution of metabolic reprogramming in response to chronic interferon exposure. Our results reveal distinct transcriptional metabolic profiles between macrophages chronically exposed to IFN and IFN{beta}. Agilent Seahorse assays demonstrated that IFN{beta} significantly diminishes the oxygen consumption rate and glycolytic proton extrusion rate in macrophages. Conversely, IFN2b decreased parameters of mitochondrial fitness and induced a shift towards glutamine oxidation.Assessing the ability of macrophages to induce glycolysis in response to antigenic stimuli (LPS and iH37Rv), we found that chronic exposure to all IFN subtypes limited glycolytic induction. This study addresses a critical oversight in the literature, where individual roles of IFN subtypes are frequently amalgamated and lack distinction. These findings not only provide novel insights into the divergent effects of interferon 2a, 2b, and {beta} on macrophage metabolism but also highlight their potential implications for developing targeted therapeutic strategies. This is particularly relevant in autoimmune disorders where type I IFNs, particularly IFN, play a central role. The observed metabolic quiescence induced by chronic IFN exposure underscores its significance in macrophage functionality and its potential contribution to the pathophysiology of autoimmune disorders and susceptibility to infection.

immunology↗

bioRxiv2023

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor

Cellular senescence is now acknowledged as a key contributor to organismal ageing and late-life disease. Although popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and its paracrine effects. To address these issues, we repurposed the small molecule inhibitor inflachromene (ICM) to induce senescence to human primary cells. Within six days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered significant similarity of the two states. Notably though, ICM suppresses the proinflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM induces a senescence-like phenotype rapidly and synchronously thereby allowing the study of its core regulatory program without any confounding heterogeneity.

genomics↗

bioRxiv2023

Self-dyeing textiles grown from cellulose-producing bacteria with engineered tyrosinase expression

Environmental concerns are driving interests in post-petroleum synthetic textiles produced from microbial and fungal sources. Bacterial cellulose is a promising sustainable leather alternative, on account of its material properties, low infrastructure needs and biodegradability. However, for alternative textiles like bacterial cellulose to be fully sustainable, alternative ways to dye textiles need to be developed alongside alternative production methods. To address this, we here use genetic engineering of Komagataeibacter rhaeticus to create a bacterial strain that grows self-dyeing bacterial cellulose. Dark black pigmentation robust to material use is achieved through melanin biosynthesis in the bacteria from recombinant tyrosinase expression. Melanated bacterial cellulose production can be scaled up for the construction of prototype fashion products, and we illustrate the potential of combining engineered self-dyeing with tools from synthetic biology, via the optogenetic patterning of gene expression in cellulose-producing bacteria. With this work, we demonstrate that combining genetic engineering with current and future methods of textile biofabrication has the potential to create a new class of textiles.

synthetic biology↗

bioRxiv2022

Identifying the best PCR enzyme for library amplification in NGS

BackgroundPCR amplification is a necessary step in many next generation sequencing (NGS) library preparation methods[1] [2]. Whilst many PCR enzymes are developed to amplify single targets efficiently, accurately and with specificity, few are developed to meet the challenges imposed by NGS PCR, namely unbiased amplification of a wide range of different sizes and GC content. As a result PCR amplification during NGS library prep often results in bias toward GC neutral and smaller fragments. As NGS has matured, optimised NGS library prep kits and polymerase formulations have emerged and in this study we have tested a wide selection of available enzymes for both short read Illumina library preparation and long fragment amplification ahead of long-read sequencing. ResultsWe tested over 20 different Hi-fidelity PCR enzymes/NGS amplification mixes on a range of Illumina library templates of varying GC content and composition, and find that both yield and genome coverage uniformity characteristics of the commercially available enzymes varied dramatically. Three enzymes Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) "Equinox" and Takara Ex Premier were found to give a consistent performance, over all genomes, that mirrored closely that observed for PCR free datasets. We also test a range of enzymes for long read sequencing by amplifying size fractionated S. cerevisiae DNA of average size 21.6 and 13.4kb respectively. ConclusionThe enzymes of choice for short read (Illumina) library fragment amplification are Quantabio RepliQa Hifi Toughmix, Watchmaker Library Amplification Hot Start Master Mix (2X) "Equinox" and Takara Ex Premier, with RepliQa also being the best performing enzyme from the enzymes tested for long fragment amplification prior to long read sequencing.

genomics↗

bioRxiv2022

Chronic IFNα treatment induces leukopoiesis, increased plasma succinate and immune cell metabolic rewiring

Although clinically effective, the actions of IFN, either produced endogenously or by therapeutic delivery, remain poorly understood. Emblematic of this research gap is the disparate array of notable side effects that occur in susceptible individuals, such as neuropsychiatric consequences, autoimmune phenomena, and infectious complications. We hypothesised that these complications are driven at least in part by dysregulated cellular metabolism. Male Wistar rats were treated with either 170,000 IU/kg human recombinant IFN-2a or BSA/saline (0.9% NaCl) three times per week for three weeks. Bone marrow (BM) immune cells were isolated from the excised femurs for glycolytic rate and mitochondrial function assessment using Agilent Seahorse Technology. Frequencies of immune cell populations were assessed by flow cytometry to determine whether leukopoietic changes had occurred in both blood and BM. Plasma levels of lactate and succinate were also determined. BMDMs were metabolically assessed as above, as well as their metabolic response to an antigenic stimulus (iH37Rv). We observed that BM immune cells from IFN-treated rats exhibit a hypermetabolic state (increased basal OCR/GlycoPER) with decreased mitochondrial metabolic respiration and increased non-mitochondrial OCR. Flow cytometry results indicated an increase in immature granulocytes (RP1-SSChi CD45lo) and classical monocytes (CD43lo RP1hi) in the blood, together with increased succinate levels in the plasma. BMDMs from IFN-treated rats retained the hypermetabolic phenotype after differentiation and failed to induce a step-up in glycolysis and mitochondrial respiration after bacterial stimulation. This work provides the first evidence of the effects of IFN treatment in inducing hypermetabolic immune features that are associated with markers of inflammation, leukopoiesis, and defective responses to bacterial stimulation.

immunology↗

bioRxiv2019

Transcriptome of the parasitic flatworm Schistosoma mansoni during intra-mammalian development

Schistosomes are parasitic blood flukes that survive for many years within the mammalian host vasculature. How the parasites establish a chronic infection in the hostile bloodstream environment, whilst evading the host immune response is poorly understood. The parasite develops morphologically and grows as it migrates to its preferred vascular niche, avoiding or repairing damage from the host immune system. In this study, we investigated temporal changes in gene expression during the intra-mammalian development of Schistosoma mansoni. RNA-seq data were analysed from parasites developing in the lung through to egg-laying mature adult worms, providing a comprehensive picture of in vivo intra-mammalian development. Remarkably, genes involved in signalling pathways, developmental control, and adaptation to oxidative stress were up-regulated in the lung stage. The data also suggested a potential role in immune evasion for a previously uncharacterised gene. This study not only provides a large and comprehensive data resource for the research community, but also reveals new directions for further characterising host-parasite interactions that could ultimately lead to new control strategies for this neglected tropical disease pathogen.\n\nAuthor SummaryThe life cycle of the parasitic flatworm Schistosoma mansoni is split between snail and mammalian (often human) hosts. An infection can last for more than 10 years, during which time the parasite physically interacts with its mammalian host as it moves through the bloodstream, travelling through the lungs and liver, to eventually establish a chronic infection in the blood vessels around the host gut. Throughout this complex journey, the parasite develops from a relatively simple larval form into a more complex, sexually reproducing adult. To understand the molecular basis of parasite interactions with the host during this complex journey we have produced genome-wide expression data from developing parasites. The parasites were collected from experimentally-infected mice over its developmental time-course from the poorly studied lung stage, to the fully mature egg-laying adult worm. The data highlight many genes involved in processes known to be associated with key stages of the infection. In addition, the gene expression data provide a unique view of interactions between the parasite and the immune system in the lung, including novel players in host-parasite interactions. A detailed understanding of these processes may provide new opportunities to design intervention strategies, particularly those focussed on the early stages of the infection that are not targeted by current chemotherapy.

microbiology↗

bioRxiv2018

Rapid multi-locus sequence typing direct from uncorrected long reads using Krocus

Genome sequencing is rapidly being adopted in reference labs and hospitals for bacterial outbreak investigation and diagnostics where time is critical. Seven gene multi-locus sequence typing is a standard tool for broadly classifying samples into sequence types, allowing, in many cases, to rule a sample in or out of an outbreak, or allowing for general characteristics about a bacterial strain to be inferred. Long read sequencing technologies, such as from PacBio or Oxford Nanopore, can produce read data within minutes of an experiment starting, unlike short read sequencing technologies which require many hours/days. However, the error rates of raw uncorrected long read data are very high. We present Krocus which can predict a sequence type directly from uncorrected long reads, and which was designed to consume read data as it is produced, providing results in minutes. It is the only tool which can do this from uncorrected long reads. We tested Krocus on over 600 samples sequenced with using long read sequencing technologies from PacBio and Oxford Nanopore. It provides sequence types on average within 90 seconds, with a sensitivity of 94% and specificity of 97%, directly from uncorrected raw sequence reads. The software is written in Python and is available under the open source license GNU GPL version 3.

bioinformatics↗