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Walker, T.

Publications and source records attributed to Walker, T..

At least 19 recordsLinked to original sources

Development and translational validation of a novel mouse model for allergic rhinitis

Background: Approximately 30% of the North American population are affected by seasonal or perennial rhinitis. Allergens from cats are common triggers of allergic rhinitis. Importantly, cat allergic rhinitis (cat-AR) is a major risk factor for more severe allergic diseases, including asthma. Current therapies rarely achieve full symptom control and development of novel therapeutics is constrained by the lack of physiologically relevant preclinical models. Objective: Establishment of a clinically-informed mouse model of cat allergen exposure in humans. Methods: Controlled human exposure was performed using airborne natural cat dander (CD) in a validated environmental exposure facility. Nasal immune cells and IgE concentrations in serum and nasal samples were assessed. In mice, we established a 14-day intermittent intranasal sensitization and challenge protocol using the same CD allergens. Clinical signs in mice were objectively quantified using AI-based video detection. Cellular changes in the nose were assessed with level-specific, spatial resolution. Lastly, systemic sensitization was confirmed through serum IgE and intradermal ear challenge. Measurements and Main Results: Following CD exposure, cat-allergic participants displayed increased nasal symptoms and eosinophil influx. CD-sensitized mice recapitulated key clinical features, including increased nasal rubbing, elevated nasal immune cell infiltration, and systemic sensitization. Importantly, spatial investigation of the cellular changes revealed major changes in the turbinate and olfactory regions of the nose. Conclusions: This novel mouse model provides a translationally valuable platform for investigating local and systemic mechanisms of cat-AR. It will serve for mechanistic and therapeutic evaluations with the goal of improving clinical translation and therapeutic precision.

immunology↗

Multi-site phenotypic characterisation of Kisumu colonies for comparison of insecticide susceptibility between testing centres

Testing of insecticides and insecticide-based vector control tools relies on the use of bioassays whereby insects are exposed and mortality, or another relevant entomological endpoint, is measured. To be able to compare results between products and across time and space standard laboratory strains are used. Insecticide-susceptible strains are often used as comparators in studies involving insecticide-resistant populations. Kisumu is a long-established, widely-used insecticide-susceptible strain of Anopheles gambiae. In this study, 16 facilities across different countries exposed their Kisumu colonies aged 3-5 days to four insecticides (permethrin, alpha-cypermethrin, DDT and pirimiphos-methyl) at a range of concentrations in WHO tube assays to assess susceptibility. Insecticide-treated filter papers were made at the Liverpool School of Tropical Medicine and sent to testing centres, along with a standard protocol and testing conditions requirements, to standardise testing as much as possible. A first observation was that DDT results were affected by the use of Risella oil, the WHO-recommended carrier oil but immiscible in acetone, leading to uneven coating of papers and increased variability in bioassay results (40x variance between colonies). Bioassay data generated by the testing facilities suggest a wide range of susceptibility to different insecticides between colonies of Kisumu: LC50 values between colonies differed, though not significantly, by up to 4x for permethrin, 6x for pirimiphos-methyl, and 15x for alpha-cypermethrin. Some colonies are classified as insecticide resistant based on the WHO definition of <98% mortality on exposure to a discriminating concentration. The assumption that Kisumu is a standard susceptible strain, consistent between facilities is challenged, which has implications when comparing results between studies. We propose genotypic analysis of the colonies to identify signs of resistance markers indicative of contamination from resistant strains or wild mosquitoes, and generation of guidance to support the maintenance and monitoring of susceptible mosquito strains.

zoology↗

Rapid Determination of Drug-to-Antibody Ratios in Antibody Drug Conjugates Using Ultrafast Microdroplet Digestion Technology

Accurate determination of drug-to-antibody ratios (DARs) is essential for the development, quality control, and performance evaluation of antibody-drug conjugates (ADCs); yet conventional analytical approaches often require extensive sample preparation, long analysis time, and substantial sample consumption. The peak distribution of intact ADCs is highly complex due to inherent glycosylation heterogeneity and variable drug conjugation. By applying enzymatic digestion, ADC can be converted into smaller subunits or deglycosylated species, thereby significantly simplifying the mass spectral profile. This reduction in structural heterogeneity facilitates clearer peak assignment and enables more accurate and reliable DAR quantification. Herein, we report an ultrafast microdroplet digestion-mass spectrometry strategy for rapid DAR characterization of ADCs. Microdroplet enzymatic digestion of antibodies and ADCs occurs within microsecond-time scales during spray ionization, enabling direct online subunit analysis with minimal sample preparation. The method was validated using NIST monoclonal antibody (mAb) conjugated to ADC mimics spanning low to high DAR (0-14) ranges, Cetuximab-derived ADC mimics (DAR[~]5) with complex glycosylation, and the commercial ADC Kadcyla (DAR[~]3.5). Consistent DAR values were obtained across multiple enzymatic workflows (IdeS, EndoS2, and EndoF3) with good reproducibility (%CV typically <5%). This approach substantially reduces analysis time while maintaining analytical accuracy and structural specificity, providing a rapid, sensitive platform for high-throughput ADC characterization and process monitoring. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/729562v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@23145corg.highwire.dtl.DTLVardef@10ddd88org.highwire.dtl.DTLVardef@14b1370org.highwire.dtl.DTLVardef@1e94ad6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Dichotomous SMAD2/3 regulation and selective anti-hypertrophic activity of heparin during in vitro chondrogenesis of mesenchymal stromal cells

BackgroundEndochondral instead of chondral differentiation hinders mesenchymal stromal cell (MSC) application for clinical cartilage regeneration. We previously showed that heparin-polyethylene glycol (PEG) hydrogels loaded with transforming growth factor beta (TGF-{beta}) instructed stable chondral MSC development in vivo. We here assessed this approach in vitro, utilizing heparin-PEG hydrogels or soluble heparin supplementation of chondrogenic medium. MethodsHuman MSCs were cultured in heparin-PEG hydrogels (22.4 mg/mL crosslinked heparin, 120ng TGF-{beta}1) or as hydrogel-free pellet cultures treated with soluble heparin (0, 10, 100, 700 g/mL) in TGF-{beta}1-containing (10 ng/mL) chondrogenic medium. Chondral and endochondral signaling (1-3 h, 4 weeks) and cartilage matrix formation (4 weeks) were analyzed using Western blot, histology, qPCR, ELISA, and enzyme activity. ResultsUnlike in vivo, human MSCs differentiated in heparin-PEG hydrogels into type X collagen and alkaline phosphatase-positive hypertrophic chondrocytes in vitro. Interestingly, treatment with soluble heparin (10-700 {micro}g/mL) revealed reduced TGF-{beta}-small mother against decapentaplegic (SMAD)3 but not SMAD2 activation at unaffected type II collagen and proteoglycan/DNA levels. We propose that the stimulation of the insulin-AKT pathway by heparin aided in maintaining SMAD2 activation which apparently plays a more prominent role than SMAD3 for MSC chondrogenesis. Heparin treatment inhibited the pro-hypertrophic WNT/{beta}-catenin pathway in vitro but insufficiently silenced TGF-{beta}-SMAD1/5/9 activation and unfortunately reduced anti-hypertrophic prostaglandin E2 (PGE2) levels. Ultimately, treatment with 10 {micro}g/mL heparin reduced the upregulation of several hypertrophy markers (MEF2C, IHH, IBSP mRNAs, alkaline phosphatase activity) below control levels, but type X collagen remained unresponsive. Thus, soluble heparin treatment was similarly selective and effective as previous anti-hypertrophic interventions (parathyroid-hormone related protein (PTHrP)-pulses, wingless-int (WNT)-inhibition), while offering technical simplicity, reduced cost, and solvent-free formulation. ConclusionsTaken together, heparin-TGF-{beta} showed a novel dichotomous SMAD2/3 inhibition at maintained chondrogenic power and context-dependent lineage-instructive properties: permitting endochondral differentiation in vitro but chondral development in vivo. Thus, environmental contributions are mandatory to allow heparin-PEG-guided chondral versus endochondral lineage commitment of MSCs in vivo, potentially involving SMAD1/5/9 suppressors and PGE2 sources. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/673657v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@140313aorg.highwire.dtl.DTLVardef@160049dorg.highwire.dtl.DTLVardef@cee22corg.highwire.dtl.DTLVardef@62e961_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

Optimising the use of Oxford Nanopore Sequencing technology for detection of Wolbachia bacterial endosymbionts in Anopheles mosquitoes.

Malaria, a mosquito-borne disease predominantly affecting sub-Saharan Africa, remains a significant global health challenge with 263 million cases in 2023. Although progress in vaccine development has recently occurred, insecticide resistance has reduced the effectiveness of the main stay method of traditional vector control and novel innovative vector control strategies against Anopheles mosquitoes is crucial to combat the spread of malaria. Wolbachia is an endosymbiotic bacterium that can invade mosquito populations and inhibit human pathogens. Historically Wolbachia was thought to not occur naturally within wild Anopheles An.) populations, but recent evidence has confirmed the presence of at least two strains in genuine symbiosis. Detection of Wolbachia strains has pre-dominantly relied on PCR amplification of Wolbachia genes and/or sanger and illumina sequencing. In this study, we assess the use of Oxford Nanopore Technology (ONT) for detecting bacteria within wild Anopheles mosquitoes and determine the ability to detect the endosymbiotc bacterium Wolbachia. We assessed Wolbachia detection from library preparations using the ONT Field sequencing kit for MinION sequencing on the Mk1C and found a pool size of 10 mosquitoes is required preventing individual mosquito analysis. There were roughly 13 times more Wolbachia reads in pooled Anopheles demeilloni compared to An. gambiae. Utilizing the more economical Flongle flow cells and Mk1B device, we further evaluated the effectiveness of two library preparation methods that are compatible: Rapid PCR Barcoding and 16S Barcoding kits. Our findings indicate that the 16S Barcoding Kit in conjunction with the Flongle flowcell effectively identified Wolbachia in both Anopheles demeilloni and Anopheles moucheti but we found no evidence of Wolbachia in Anopheles gambiae. Our study demonstrates the feasibility of developing ONT for field-based sequencing in malaria-endemic regions, providing a potentially lower cost and field friendly method for monitoring mosquito populations and associated endosymbionts. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/659891v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@18ee91dorg.highwire.dtl.DTLVardef@16a71c4org.highwire.dtl.DTLVardef@15330edorg.highwire.dtl.DTLVardef@b13e3d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

Platelet concentrate-derived extracellular vesicles promote adult hippocampal neurogenesis

Platelet-derived materials are emerging as promising, cell-free biotherapies for regenerative medicine. While platelet lysates have shown neuroprotective activity in preclinical models, the neurogenic potential of platelet concentrate-derived extracellular vesicles (pEVs) remains underexplored. Here, we evaluated the effects of human pEVs and a neuroprotective heat-treated human platelet lysate (HPPL) on adult hippocampal neurogenesis using both an ex vivo neurosphere assay and an in vivo intranasal administration model. pEVs selectively enhanced dentate gyrus (DG)-derived neurosphere growth, even in the absence of exogenous growth factors, and were internalized by neural precursors. In vivo, short-term pEV delivery increased EdU proliferating cells in the DG, while long-term administration (28 days) elevated the proportion of newborn mature neurons. By contrast, HPPL primarily promoted early neurogenesis by expanding immature DCX neurons. Quantitative proteomics of DG tissue after pEV treatment revealed 111 differentially expressed proteins, with enrichment in pathways related to oxidative phosphorylation, Notch4 signaling, myelination, and MHC class I-mediated antigen presentation. Downregulated proteins included cytoskeletal and translation-related regulators, suggesting a shift toward neuronal differentiation and circuit integration. Biophysical characterization confirmed the purity and vesicular nature of pEVs, with a defined protein cargo including immune modulators and ECM-interacting molecules such as CD44, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), and complement proteins. These findings identify allogeneic pEVs as multifunctional agents that modulate neural precursor cell fate and brain tissue remodeling through coordinated metabolic and immunoregulatory mechanisms. This work supports the translational potential of pEV-based therapeutics for promoting hippocampal neurogenesis and cognitive repair in neurodegenerative and age-related brain disorders.

neuroscience↗

Enhancement of spatial learning by 40 Hz visual stimulation requires parvalbumin interneuron-dependent hippocampal neurogenesis

Acute and short-term rhythmic 40 Hz light flicker stimulation has shown promising results in alleviating cognitive impairments in mouse models of Alzheimers disease (AD), stroke, and autism spectrum disorders (ASD). Understanding the long-term impacts and underlying mechanisms is crucial to progress this approach for potential human therapeutic applications. Here, we show that prolonged exposure to 40 Hz light flicker (1 hour per day for 30 days) significantly improved spatial learning and neurogenesis in the dentate gyrus (DG) without harmful behavioral side effects. Mice with transgenic deletion of doublecortin-positive cells (DCXDTR) in the adult hippocampus failed to exhibit enhanced neurogenesis and spatial learning with 40 Hz stimulation. Inactivation or knockout of GABAergic parvalbumin (PV) interneurons reduced the effects of 40 Hz entrainment and neurogenesis enhancement. Mechanistically, the stimulation did not alter the regional microvessel blood flow but significantly raised PV excitability and GABA levels and enhanced inhibitory transmission in the DG. Blocking GABAA receptors reversed the improvements in spatial learning and neurogenesis. These data showed that long-term exposure to 40 Hz light flicker enhances spatial learning through PV-dependent adult neurogenesis, which requires elevated GABA as a critical neurochemical mechanism for sustaining adult neurogenesis. In briefRhythmic 40 Hz light flicker alleviates cognitive impairments in diverse animal models of neurological diseases. Understanding its long-term effects and mechanisms is vital for advancing its therapeutic potential for humans. Here, we show that prolonged exposure to this flicker improves spatial learning and boosts adult neurogenesis in the dentate gyrus. Activation of PV interneurons and GABAergic support for the newborn immature neurons underlie this effect, demonstrating lasting benefits for the treatment of neurological diseases. HighlightsO_LILong-term exposure to 40 Hz light flicker significantly improved spatial learning and neurogenesis in the hippocampus, devoid of adverse effects. C_LIO_LIThe 40 Hz light flicker evoked adult neurogenesis requires the activity of GABAergic inhibitory PV interneurons. C_LIO_LIThe 40 Hz light flicker raised GABA levels and enhanced inhibitory transmission in the DG. C_LIO_LIIncreased GABA serves as a vital neurochemical mechanism to support adult neurogenesis. C_LI

neuroscience↗

Pathogenic IgE-fated B cell memory retains functional plasticity

Long-lived immunoglobulin (Ig) E responses against innocuous environmental and dietary antigens (Ags) are maintained by an IgG1-dominant memory B cell (MBC) compartment primed for IL-4 responsiveness. The plasticity of the MBC compartment destined for IgE class switch recombination (CSR), however, remains poorly understood. In this work, we report a critical IL-4/IL-13 dependency for the pathogenic IgE fate of type 2-polarized MBCs. Initiating a recall response in the absence of IL-4/IL-13 signaling diminished the type 2 MBC phenotype in mice and humans and, in mice, permitted the emergence of long-lived Ag-specific IgG2c+ MBCs. The divergence to a type 1-like response was dependent on IFN-{gamma} signaling and arose from both unswitched and class-switched Ag-specific B cells in vivo. This reprogrammed fate was sustained even beyond therapeutic intervention, revealing fundamental insight into the plasticity of the allergen-specific MBC response. One Sentence SummaryB cell responses to allergens can be reprogrammed away from a pathogenic fate through IL-4/IL-13 signaling blockade.

immunology↗

Sequential Switching Through IgG1 is Redundant for Allergic Reactivity and Memory to Allergens

Allergic reactions to foods are driven by allergen-binding immunoglobulin (Ig)E antibodies. IgE- expressing cells can be generated through a sequential class switching pathway where activated B cells first switch to an intermediary isotype, most frequently IgG1, and then to IgE. It has been proposed that sequential class switch recombination is important in generating high affinity IgE, augmenting anaphylactic reactions, and in holding the memory of IgE responses. Here, we observed surprising redundancy of sequential switching through IgG1 for the functional affinity of the IgE repertoire against multiple food allergens as well as for the ability of IgE to elicit anaphylaxis. We further found that sequential switching via IgG1 was irrelevant for allergic memory. These results indicate that allergen-specific IgG1 B cells are redundant in sensitization, anaphylaxis, and food allergy persistence, thereby implicating other switching pathways as important considerations in the development of therapeutics for allergic diseases.

immunology↗

Global patterns of plant form and function are strongly determined by evolutionary relationships

Plants display an incredible variety of forms and functions, which sustains a vast diversity of ecosystems on Earth. This diversity is the result of two forces: environmental filtering, the selection of phenotypes fit to a specific habitat; and evolutionary history, genetic constraints that determine the potential adaptations that species may evolve. To date, most studies have focused on describing macroecological patterns of trait variation, while the role of evolutionary history in determining these patterns has remained much less explored. Here, we combine a traits dataset including 4,213 vascular plant species with a phylogeny of the plant kingdom, to determine the influence of evolutionary history on the patterns of trait variation. Our results show that commonly used plant traits, such as plant height or leaf nitrogen, are strongly constrained by evolutionary history. Accounting for phylogenetic relationships changes the variance explained by the main axes of trait variation in the Plant Kindgom --the leaf economic spectrum and the plant size and organ axis continuum-- with the former explaining more variance than the latter. Moreover, we demonstrate that the plant size continuum is more influenced by evolutionary history than the leaf economics spectrum. These results highlight the influence of evolutionary history in shaping plant life history strategies and the importance of accounting for phylogeny in trait-based studies.

ecology↗

Towards environmental detection, quantification, and molecular characterization of Anopheles stephensi and Aedes aegypti from larval breeding sites

The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 32 second instar larvae in 50ml of water. An. stephensi eDNA, derived from emergent pupae for 24 hours, was remarkably stable, and still detectable ~2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.

molecular biology↗

Binding of ATP to GroEL: a Case for Variable-Temperature Native Mass Spectrometry Thermodynamic Studies

Understanding how large, oligomeric protein complexes respond to the binding of small ligands and other proteins is essential for describing the molecular basis of life. This in turn requires a complete characterization of the binding energetics and correlation of thermodynamic data with interacting structures, including effects of small molecules and solvent. However, the size of many protein oligomers, the myriad intermediate ligation states they can populate, and their often complex allosteric regulation typically restrict analysis by traditional methods to low resolution, ensemble averages. Here, we employ variabletemperature electrospray ionization native mass spectrometry to determine the thermodynamics for stepwise binding of up to ATP molecules to the 801 kDa GroEL complex, a tetradecamer chaperonin complex. Binding thermodynamics reveal strong enthalpy-entropy compensation (EEC) and high degrees of cooperativity are observed for formation of GroEL-ATP7 and GroEL-ATP14. These are evidenced by entropically favored ATP binding to the cis ring (formation of GroEL-ATP1-7), with variations in EEC for subsequent binding of ATP to the trans ring (GroEL-ATP8-14), as expected for negative inter-ring cooperativity. Entropy driven ATP binding to the GroEL tetradecamer is consistent with ligand induced conformational changes of the GroEL tetradecamer, though the magnitude of the entropy change suggests that reorganization of GroEL-hydrating water molecules and/or expulsion of water from the GroEL cavity may also play a key role. By determining the thermodynamic signatures for individual ligand binding reactions to the large, nearly MDa GroEL complex, we expand our fundamental view of chaperonin functional chemistry. Moreover, this work and related studies of protein-ligand interactions illustrate unparalleled capabilities of vT-ESI-nMS for thermodynamics studies of protein interactions with ligands, and other molecules, such as proteins and drugs.

biophysics↗

Quantum biological insights into CRISPR-Cas9 sgRNA efficiency from explainable-AI driven feature engineering

CRISPR-Cas9 tools have transformed genetic manipulation capabilities in the laboratory. Empirical rules-of-thumb have been established for only a narrow range of model organisms, and mechanistic underpinnings for sgRNA efficiency remain poorly understood. This work establishes a novel feature set and new public resource, produced with quantum chemical tensors, for interpreting and predicting sgRNA efficiency. Feature engineering for sgRNA efficiency is performed using an explainable-artificial intelligence model; iterative Random Forest (iRF). By encoding quantitative attributes of position-specific sequences for E. coli sgRNAs, we identify important traits for sgRNA design in bacterial species. Additionally, we show that expanding positional encoding to quantum descriptors of base-pair, dimer, trimer and tetramer sequences captures intricate interactions in local and neighboring nucleotides of the target DNA. These features highlight variation in CRISPR-Cas9 sgRNA dynamics between E. coli and H. sapien genomes. These novel encodings of sgRNAs greatly enhance our understanding of the elaborate quantum biological processes involved in CRISPR-Cas9 machinery.

systems biology↗

Genetic and microbial diversity of the invasive mosquito vector species Culex tritaeniorhynchus across its extensive inter-continental geographic range

Culex (Cx.) tritaeniorhynchus is a mosquito species with an extensive and expanding inter-continental geographic distribution, currently reported in over 50 countries, across Asia, Africa, the Middle East, Europe and now Australia. It is an important vector of medical and veterinary concern, capable of transmitting multiple arboviruses which cause significant morbidity and mortality in human and animal populations. In regions endemic for Japanese encephalitis virus (JEV) in Asia, Cx. tritaeniorhynchus is considered the major vector and this species has also been shown to contribute to the transmission of several other significant zoonotic arboviruses, including Rift Valley fever virus and West Nile virus. Significant variation in vectorial capacity can occur between different vector populations. Obtaining knowledge of a species from across its geographic range is crucial to understanding its significance for pathogen transmission across diverse environments and localities. Vectorial capacity can be influenced by factors including the mosquito genetic background, composition of the microbiota associated with the mosquito and the co-infection of human or animal pathogens. In addition to enhancing information on vector surveillance and potential risks for pathogen transmission, determining the genetic and microbial diversity of distinct populations of a vector species is also critical for the development and application of effective control strategies. In this study, multiple geographically dispersed populations of Cx. tritaeniorhynchus from countries within Europe, Africa, Eurasia and Asia were sampled. Molecular analysis demonstrated a high level of genetic and microbial diversity within and between populations, including genetic divergence in the mosquito CO1 gene, as well as diverse microbiomes identified by 16S rRNA gene amplicon sequencing. Evidence for the detection of the endosymbiotic bacteria Wolbachia in some populations was confirmed using Wolbachia-specific PCR detection and sequencing of Wolbachia MLST genes; in addition to PCR-based detection of insect-specific viruses. Laboratory vector competence showed Cx. tritaeniorhynchus from a Greek population are likely to be competent vectors of JEV. This study expands understanding of the diversity of Cx. tritaeniorhynchus across its inter-continental range, highlights the need for a greater focus on this invasive vector species and helps to inform potential future directions for development of vector control strategies.

genetics↗

Wolbachia endosymbionts in two Anopheles species indicates independent acquisitions and lack of prophage elements

Wolbachia is a genus of obligate bacterial endosymbionts that infect a diverse range of arthropod species as well as filarial nematodes, with its single described species, Wolbachia pipientis, divided into several supergroups based on multilocus sequence typing. Wolbachia strains in mosquitoes have been shown to inhibit the transmission of human pathogens including Plasmodium malaria parasites and arboviruses. Despite their large host range, Wolbachia strains within the major malaria vectors of the Anopheles (A.) gambiae and A. funestus complexes appear at low density based solely on PCR-based methods. Questions have been raised as to whether this represents a true endosymbiotic relationship. However, recent definitive evidence for two distinct, high-density strains of supergroup B Wolbachia within A. demeilloni and A. moucheti has opened exciting possibilities to explore naturally occurring Wolbachia endosymbionts in Anopheles for biocontrol strategies to block Plasmodium transmission. Here we utilise genomic analyses to demonstrate that both Wolbachia strains have retained all key metabolic and transport pathways despite their smaller genome size. We further confirm the presence of cytoplasmic incompatibility factor genes, despite noticeably few prophage regions. Additionally, phylogenetic analysis indicates that these Wolbachia strains may have been introduced into these two Anopheles species via horizontal transmission events, and unlikely to be by ancestral acquisition and subsequent loss events in the Anopheles gambiae species complex. These are the first Wolbachia genomes that enable us to study the relationship between natural strains Plasmodium malaria parasites and their Anopheline hosts. Impact statementWolbachia naturally infects a wide range of arthropod species, including insect vectors of human pathogens, where they may play a role in inhibiting their replication. These bacteria have been commonly found within Aedes (Ae.) albopictus and Culex pipiens mosquitoes but have been noticeably absent in the Anopheles mosquito genera, which includes all species responsible for malaria transmission. Recent PCR-based methods have suggested the potential for natural Wolbachia strains within the A. gambiae species complex, which includes major malaria vector species including A. gambiae s.s., A. coluzzii and A. arabiensis. We recently reported the presence of stable Wolbachia strains naturally occurring within two different Anopheles species (A. demeilloni and A. moucheti). In this study, we perform comparative genomic analysis of these two Wolbachia genomes against each other and published Wolbachia strains. The current assemblies are some of the smallest sequenced Wolbachia strains of insects, although their metabolic pathway repertoire is comparable to other strains. Interestingly, prophage fragments were identified within only one of the two strains. The findings of this study will be of significant interest to researchers investigating Wolbachia as a potential malaria biocontrol strategy, giving greater insight into the evolution and diversity of this obligate intracellular endosymbiont. Data summarySequence data generated and used for this analysis are available in the National Centre for Biotechnology Information Sequence Read Archive (NCBI SRA bioproject number PRJNA642000). The two assembled Wolbachia genomes are available with genome accession numbers GCA_018491735.2 and GCA_018491625.2. Additional Wolbachia genomes used for comparative analysis are described in the supplementary material. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Additional supplementary data files used to generate several figures can be found at: https://figshare.com/projects/Wolbachia_endosymbionts_in_two_Anopheles_species_indicates_independent_acquisitions_and_lack_of_prophage_elements/126533

cell biology↗

The heterogeneity of the DNA damage response landscape determines patient outcomes in ovarian cancer

Defective DNA damage response (DDR) pathways allow cancer cells to accrue genomic aberrations and evade normal cellular growth checkpoints. Defective DDR also determines response to chemotherapy. However, the interaction and overlap between the two double strand repair pathways and the three single strand repair pathways is complex, and has remained poorly understood. Here we show that, in ovarian cancer, a disease hallmarked by chromosomal instability, explant cultures show a range of DDR abrogation patterns. Defective homologous recombination (HR) and non-homologous end joining (NHEJ) are near mutually exclusive with HR deficient (HRD) cells showing increased abrogation of the single strand repair pathways compared to NHEJ defective cells. When combined with global markers of DNA damage, including mitochondrial membrane functionality and reactive oxygen species burden, the pattern of DDR abrogation allows the construction of DDR signatures which are predictive of both ex vivo cytotoxicity, and more importantly, patient outcome. SignificanceHolistic assessment of the DDR is possible, shows improved ability to predict response to chemotherapy over single pathway assessment, and is applicable to a variety of ovarian cancer types. Such an assessment has clinical utility in settings of therapeutic dilemma such as retreatment for relapse.

cell biology↗

Overabundance of Asaia and Serratia bacteria is associated with deltamethrin insecticide susceptibiltiy in Anopheles coluzzii from Agboville, Cote d'Ivoire

BackgroundInsecticide resistance among mosquito species is now a pervasive phenomenon, which threatens to jeopardise global malaria vector control efforts. Evidence of links between the mosquito microbiota and insecticide resistance is emerging, with significant enrichment of insecticide degrading bacteria and enzymes in resistant populations. Using 16S rRNA amplicon sequencing, we characterised and compared the microbiota of Anopheles (An.) coluzzii in relation to their deltamethrin resistance and exposure profiles. ResultsComparisons between 2-3 day old deltamethrin resistant and susceptible mosquitoes, demonstrated significant differences in microbiota diversity (PERMANOVA, pseudo-F = 19.44, p=0.0015). Ochrobactrum, Lysinibacillus and Stenotrophomonas genera, each of which comprised insecticide degrading species, were significantly enriched in resistant mosquitoes. Susceptible mosquitoes had a significant reduction in alpha diversity compared to resistant individuals (Shannon index: H=13.91, q=0.0003, Faiths phylogenetic diversity: H=6.68, q=0.01), with Asaia and Serratia dominating microbial profiles. There was no significant difference in deltamethrin exposed and unexposed 5-6 day old individuals, suggesting that insecticide exposure had minimal impact on microbial composition. Serratia and Asaia were also dominant in 5-6 day old mosquitoes, regardless of exposure or phenotype, and had reduced microbial diversity compared with 2-3 day old mosquitoes. ConclusionsOur findings revealed significant alterations of An. coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for insecticide resistance surveillance. qPCR detection of Serratia and Asaia was consistent with 16S rRNA sequencing, suggesting that population level field screening of the bacterial microbiota may be feasibly integrated into wider resistance monitoring if reliable and reproducible markers associated with phenotype can be identified.

microbiology↗

Genomic and microscopic evidence of stable high density and maternally inherited Wolbachia infections in Anopheles mosquitoes

Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has been detected within populations of Anopheles (An.) malaria vectors. In the An. gambiae complex, the primary vectors in Sub-Saharan Africa, Wolbachia strains are at low density and infection frequencies in wild populations. PCR-independent evidence is required to determine whether Wolbachia strains are true endosymbionts in Anopheles given most studies to date have used nested-PCR to identify strains. Here we report high-density strains found in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti and maternal transmission was observed. Genome sequencing of both strains obtained genome depths and coverages comparable to other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present indicating these strains possess the capacity to induce the phenotype cytoplasmic incompatibility which allows Wolbachia to spread through populations. The characteristics of these two strains suggest they are ideal candidates for Wolbachia biocontrol strategies in Anopheles.

microbiology↗