Biology subjects

Gill, A.

Publications and source records attributed to Gill, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A methylome-guided toolkit for genetic engineering of the fast-growing cyanobacterium UTEX 3222

Cyanobacterium aponinum UTEX 3222 is a fast-growing marine cyanobacterium that reaches high cell densities, making it an attractive chassis strain for low-cost biomanufacturing. However, past efforts to genetically engineer UTEX 3222 have been unsuccessful. We hypothesized that the complex restriction modification systems in the UTEX 3222 genome constitute a major barrier to genetic transformation. Using a combined methylation avoidance and methylation mimicking approach, we overcame this barrier and successfully transformed UTEX 3222 by natural transformation, generating a large panel of fully segregated axenic strains. Transformation method was also established for a novel shuttle vector that replicates in UTEX 3222. A large-scale promoter screen identified promoters which exhibited strong expression at high cell density. These advances establish the foundation for developing UTEX 3222 as a scalable platform for photosynthetic biomanufacturing.

synthetic biology↗

bioRxiv2026

Disease-specific differences in particulate matter handling drive pathogenic responses in human derived nasal epithelial cells

BackgroundParticulate matter (PM) exposure is associated with increased risk and exacerbation of chronic rhinosinusitis (CRS), yet underlying mechanisms remain poorly understood. ObjectiveTo define the epithelial mechanisms by which PM exposure promotes and exacerbates CRS, with a focus on epithelial remodeling, inflammation, barrier dysfunction, and cellular uptake of PM. MethodsHuman nasal epithelial cells obtained from ethmoid tissue of CRS (n = 5) and control donors (n = 4) were cultured at an air-liquid interface and exposed to PM. Single-cell RNA sequencing was performed to characterize PM-induced cellular and transcriptional changes. Protein expression, epithelial barrier integrity, cell death, and intracellular PM uptake were evaluated using biochemical, imaging, and ultrastructural approaches. ResultsUnsupervised clustering identified seven epithelial cell populations. Gene set analysis revealed baseline enrichment of inflammatory and keratinization pathways and reduced ciliogenesis in CRS compared with controls. Although PM induced inflammation and squamous differentiation in controls, the pathogenic responses were significantly amplified in CRS, including uniquely enhanced IL-1 signaling. Transcriptional changes were validated by ELISA, transepithelial electrical resistance, and immunofluorescence, demonstrating increased inflammation, epithelial barrier disruption, and cell death following PM exposure. Transmission electron microscopy revealed increased intracellular PM within membrane-bound organelles. Pre-treatment with an endocytosis inhibitor rescued PM-induced epithelial barrier dysfunction and inflammation. ConclusionCRS epithelium exhibits baseline dysfunction that may predispose it to environmental injury. PM exposure both induces CRS-like epithelial changes in controls and exacerbates disease-associated phenotypes. Key MessagesO_LICompared to controls, CRS nasal epithelium exhibits baseline inflammatory, keratinization, and ciliogenesis abnormalities. C_LIO_LIParticulate matter induces inflammation and squamous differentiation, while amplifying epithelial injury that is more robust in CRS epithelium compared to controls. C_LIO_LIInhibition of dynamin-dependent endocytosis rescues PM-induced epithelial barrier leakiness and inflammation, implicating intracellular particulate matter uptake in disease pathogenesis. C_LI Capsule SummaryParticulate matter induces CRS-like epithelial remodeling in controls and exacerbates inflammation and epithelial barrier dysfunction in CRS nasal epithelium, which can be rescued with endocytosis inhibition. This suggests a mechanistic link between baseline CRS vulnerability, intracellular uptake of particulate matter, and disease pathogenesis.

cell biology↗

bioRxiv2026

Revisiting the diversity of secondary endosymbionts in the major pest oat aphid, Rhopalosiphum padi

There is disagreement on whether secondary endosymbionts are found in the major cereal pest aphid, Rhopalosiphum padi. Some papers report a diversity of secondary bacterial endosymbionts while others have failed to find evidence of these bacteria in this species. Here we revisit this issue by summarizing the relevant literature and through additional sampling of the species in Australia, China and Denmark using a combination of molecular approaches. We find a general absence of secondary endosymbionts beyond the obligate endosymbiont Hamiltonella defensa in R. padi. While the inconsistency in survey results may reflect rapid changes in endosymbiont turnover in populations and/or the impact of ecological factors such as host plant type on endosymbiont diversity, we are concerned that technical issues may be at least partly responsible for inconsistencies in the literature. This leads us to emphasize the importance of multiple sources of evidence required to establish and characterize endosymbiont infections, including PCR and qPCR assays, DNA Sanger sequencing and 16SrRNA gene metabarcoding. We note that several major aphid pests show a low incidence of secondary endosymbionts which raises issues about the importance of these endosymbionts in aphids that constitute pests, even though endosymbionts can in some cases increase host fitness and therefore pest impact.

microbiology↗

bioRxiv2026

Novel but stable endosymbionts have contrasting effects on aphid dispersal and plant feeding damage in the cereal pest Diuraphis noxia

Endosymbiotic bacteria can affect many ecological attributes of their insect hosts, including (in herbivorous insects) how insects interact with plants where they feed. This raises the issue of whether deliberate endosymbiont introductions could be used to decrease crop damage caused by insect pests. Here we investigate how transinfecting Rickettsiella viridis and Regiella insecticola endosymbionts into a novel pest aphid host, the Russian wheat aphid (Diuraphis noxia), influences population growth, alate production, dispersal ability and crop damage. Both the Rickettsiella (originating from pea aphids) and Regiella (from green peach aphids) were stably maintained in their new host where they had contrasting effects. Rickettsiella increased the severity of aphid damage on wheat and barley, resulting in greater leaf loss, chlorotic streaking, and higher aphid populations, whereas Regiella reduced aphid population growth and the severity of feeding damage by aphids. Their effects on dispersal morphology also differed: Regiella had no detectable impact on alate incidence, while Rickettsiella consistently suppressed wing formation in small cages, and in larger mesocosms with multiple wheat plants this endosymbiont suppressed dispersal. Endosymbiont-mediated changes in feeding damage did not involve the main plant immune response pathways: transinfected and wild type aphids induced similar levels of jasmonic acid, jasmonic acid-isoleucine, and salicylic acid in plant tissues, even though these plant defenses were strongly activated during aphid feeding. Novel endosymbionts can therefore modulate the severity of plant feeding damage by aphids as well as influencing aphid dispersal. Potential applications in controlling pest D. noxia populations are discussed. Significance statementEndosymbiotic bacteria that live within insect cells can have wide-ranging effects on the reproduction and fitness of their insect hosts in different environments. In herbivorous insects this includes effects on host plant use. Here we test if novel endosymbionts in a pest aphid, the Russian wheat aphid, might be used to decrease crop damage and dispersal. We show that the damage caused to wheat and barley plants from aphid feeding is modulated by novel but stably transmitted introduced endosymbionts. One endosymbiont (Rickettsiella) increased the severity of damage but decreased aphid dispersal, while another (Regiella) decreased damage severity without impacting dispersal. These contrasting effects may be associated with changes in aphid population growth and wing formation but were not linked to key plant immune response pathways. We discuss implications of these findings for using endosymbionts in agricultural pest management. Classification: Applied Biological Sciences, microbiology

microbiology↗

bioRxiv2025

Hot and Bothered, Bees Gut Microbiome Shifts Under Thermal Stress and Pathogen Infection.

Understanding bumble bee gut health is imperative as these vital pollinators are subjected to pathogenic infections and thermal stress from climate change. The gut microbiome serves as an indicator for health and fitness and indicates the types of stress. To investigate the combined effects of thermal stress and pathogenic infection on bee guts, we performed a two-by-two crossed design where Bombus impatiens workers were subjected to hot conditions, pathogenic infection, or both. Incubation groups were given 2-weeks of stress conditions, with infected bees initially inoculated with Crithidia bombi, a common bee gut parasite. We measured body size, quantified the infection intensity of C. bombi using qPCR, and defined the composition of the gut microbiome using full-length 16S rRNA gene amplicon sequencing on an Oxford Nanopore Technologies Mk1D. While the core gut microbiome thrived with genera such as Bombilactobacillus and Snodgrassila which were not impacted by treatment; there were notable changes in other key organisms. Asaia bogorensis spiked in control temperature infected organisms, while species of Lactobacillus were overtaken in hot temperatures by significant increases in Apilactobacillus kunkeei. Species such as Citrobacter freundii dominated in hot infected bees suggesting an increased immunocompromised state from the combined stressors impacts on bee gut health. Our novel combined effects from thermal stress and pathogenic infection strengthen existing literature and provide new directions on how to quantify the health-state of wild bees based on their gut microbiome composition. These insights enable us to better understand how bees will be further impacted in changing landscapes. ImportanceWe find significant changes in bees gut microbiome especially with an increased abundance of lactic acid bacteria. These lactic acid bacteria are often specialized: based on infection status, temperature, and the combined effects. These insights are vital to researchers, especially those studying wild bee gut health where they have an uncontrolled system and need to make assumptions about bee stress based on fitness and microbiome. Our detailed outline of relevant species will provide wild bee researchers with a baseline to determine thermal stress and recent infection status based on microbiome communities. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/687019v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1528ae6org.highwire.dtl.DTLVardef@1d100dorg.highwire.dtl.DTLVardef@8ea77org.highwire.dtl.DTLVardef@159620a_HPS_FORMAT_FIGEXP M_FIG C_FIG Overview of the experimental design where bees were either given control temperatures or thermal stress and/or an infection by Crithidia bombi. The bees microcolonies were incubated for 2-weeks before sacrificing the bees, extracting DNA, and performing downstream analysis. Full length 16S rRNA gene amplicon sequencing was performed on an Oxford Nanopore MK1D sequencer while Crithidia bombi infections were quantified using quantitative polymerase chain reaction.

ecology↗

bioRxiv2025

Structure of YbbAP-TesA: a Type VII ABC transporter lipid-hydrolase complex

Type VII ABC transporters are ATP-powered membrane protein complexes that drive key biological processes in the bacterial cell envelope. In E. coli, three of the four Type VII ABC systems have been extensively characterised including: the FtsEX-EnvC cell division complex, the LolCDE-LolA lipoprotein trafficking machinery and the MacAB-TolC efflux pump. Here we describe a fourth E. coli Type VII ABC system, YbbAP-TesA, which combines a Type VII ABC transporter with a multifunctional hydrolytic enzyme. Structures of the complete YbbAP-TesA complex, and of YbbAP with and without bound ATP analogues, capture the long-range transmembrane conformational changes that are the hallmark of this ABC superfamilys mechanotransmission mechanism. We further show that YbbAP-TesA can hydrolyse a variety of ester and thioester substrates and experimentally confirm a constellation of active site residues in TesA. Our data suggests YbbAP has a role in extracting hydrophobic molecules from the inner membrane and presenting these to TesA for hydrolysis. The work extends collective knowledge of the remarkable diversity of the ABC superfamily and establishes a new function for Type VII ABC transporters in bacterial cells. Significance StatementType VII ABC transporters are unique to bacteria and play important roles in bacterial physiology including cell division, antibiotic resistance, siderophore secretion, antibiotic sensing, toxin secretion, biofilm regulation and lipoprotein trafficking. Here we identify a complex that combines an atypical Type VII ABC transporter (YbbAP) with a periplasmic lipid hydrolase (TesA). The YbbAP-TesA complex is structurally and functionally distinct from all known Type VII ABC transporter systems including FtsEX-EnvC, LolCDE-LolA, BceAB-BceS and MacAB-TolC. Structures suggest that YbbAP-TesA uses ATP-driven mechanotransmission to extract substrates from the inner membrane and hydrolyse them in the periplasmic space. The discovery and characterisation of YbbAP-TesA highlights the structural and functional diversity of Type VII ABC transporter complexes and suggests a new function for these proteins in the bacterial cell envelope.

microbiology↗

bioRxiv2025

Mechanistic computational modeling of sFLT1 secretion dynamics

Constitutively secreted by endothelial cells, soluble FLT1 (sFLT1 or sVEGFR1) binds and sequesters extracellular vascular endothelial growth factors (VEGF), thereby reducing VEGF binding to VEGF receptor tyrosine kinases and their downstream signaling. In doing so, sFLT1 plays an important role in vascular development and in the patterning of new blood vessels in angiogenesis. Here, we develop multiple mechanistic models of sFLT1 secretion and identify a minimal mechanistic model that recapitulates key qualitative and quantitative features of temporal experimental datasets of sFLT1 secretion from multiple studies. We show that the experimental data on sFLT1 secretion is best represented by a delay differential equation (DDE) system including a maturation term, reflecting the time required between synthesis and secretion. Using optimization to identify appropriate values for the key mechanistic parameters in the model, we show that two model parameters (extracellular degradation rate constant and maturation time) are very strongly constrained by the experimental data, and that the remaining parameters are related by two strongly constrained constants. Thus, only one degree of freedom remains, and measurements of the intracellular levels of sFLT1 would fix the remaining parameters. Comparison between simulation predictions and additional experimental data of the outcomes of chemical inhibitors and genetic perturbations suggest that intermediate values of the secretion rate constant best match the simulation with experiments, which would completely constrain the model. However, some of the inhibitors tested produce results that cannot be reproduced by the model simulations, suggesting that additional mechanisms not included here are required to explain those inhibitors. Overall, the model reproduces most available experimental data and suggests targets for further quantitative investigation of the sFLT1 system. Author SummaryProteins that are typically found outside cells are initially made inside cells, and later secreted into extracellular space. Many of these secreted proteins have important functions outside the cell that are well-studied; however, usually much less is known about the pre-secretion life of these molecules. Many computational models only represent the extracellular versions of secreted proteins, reducing all production and secretion steps into a single modeled process. Here, we develop a mechanistic model of the production and secretion of a specific secreted protein, sFLT1, which inhibits blood vessel growth by acting as an extracellular sponge for another set of secreted proteins, the vascular endothelial growth factors. We compare several models to existing experimentally-measured sFLT1 data, and we show that the data are most simply explained by including a delay between intracellular sFLT1 production and sFLT1 transport or degradation. This is consistent with the biology of the cells secretory pathway, where immature proteins are gradually processed into mature forms over minutes to hours. Our approach could be incorporated into improved models for any pathway involving secreted proteins, including sFLT1-regulated models of blood vessel biology.

bioinformatics↗

bioRxiv2024

A persistent bacterial Regiella transinfection in the bird cherry-oat aphid Rhopalosiphum padi increasing host fitness and decreasing plant virus transmission

The bird cherry-oat aphid, Rhopalosiphum padi, is a major pest of agriculture due to its ability to directly damage crops and transmit plant viruses. As industries move away from chemical pest control, there is interest in exploring new options to suppress the impact of this pest. Here we describe the production of a transinfected line of R. padi carrying the bacterial endosymbiont, Regiella insecticola, originating from the green peach aphid, Myzus persicae. We show that Regiella increases the fitness of its novel host despite decreasing fitness in its native host. Regiella also shows a low level of horizontal transmission. Importantly the infection suppresses the ability of R. padi to transmit the barley yellow dwarf virus which damages wheat plants. This transinfection could be released to suppress virus transmission by aphids with its ability to persist and spread, making it potentially suitable for wide area release.

microbiology↗

bioRxiv2024

Endosymbionts impact ladybird predation rates of aphids in a temperature-dependent manner

Aphids are worldwide pests causing major economic losses to growers. Current management strategies rely heavily on pesticides, but some effective pesticides are being withdrawn and the efficiency of remaining pesticides is also decreasing as aphids build up resistance. Biological control using predators can provide a sustainable alternative to pesticides under some circumstances, while the deliberate introduction of facultative bacterial endosymbionts that induce host fitness costs and reduce plant virus transmission provides another potential future strategy to combat aphid pests. However, new control options should not be antagonistic, with the concern that the effectiveness of biocontrol might be altered by endosymbiont presence in hosts. We, therefore, tested if predation by two aphidophagous ladybirds, Adalia bipunctata and Harmonia conformis, on the green peach aphid, Myzus persicae, and the oat aphid, Rhopalosiphum padi, was affected by transinfected Rickettsiella viridis and both native and transinfected Regiella insecticola endosymbionts at different temperatures. The predation rate of aphids infected by either endosymbiont was higher at 14 {degrees} C than the rate for uninfected aphids of both species, but the opposite pattern was apparent at 20 {degrees}C and for one host-endosymbiont combination at 26 {degrees}C. Overall, the results showed that higher temperatures increased predation, while differences between intermediate and low temperatures were species-dependent. No transmission of endosymbionts from aphid to ladybird through predation was detected. These findings point to a lack of consistent effects of the investigated endosymbionts on predation rates in these major aphid pests. The temperature dependence of endosymbiont-predation interactions suggests that the impact of seasonal climate should be considered when assessing the potential of endosymbionts in a biological control setting. HIGHLIGHTS- Aphid endosymbiont effects on ladybird predation rates are temperature-dependent - Ladybird predation rate of aphids increases as temperature rises - Prey (aphid) endosymbionts are not transferred to predators (ladybirds)

ecology↗

bioRxiv2024

The endosymbiont Rickettsiella viridis increases the virulence of Diuraphis noxia but reduces alate frequency

Aphids are among the worlds most economically damaging pests and carry a diverse range of bacterial endosymbionts. There is increasing interest in exploring potential applications of natural and novel strains of endosymbionts in aphid control. One endosymbiont, Rickettsiella viridis, has a large fitness cost following transfer from its natural aphid host Acyrthosiphon pisum into a novel host aphid Myzus persicae. Here, we investigated host impacts after transferring the same Rickettsiella strain to an important cereal aphid, the Russian wheat aphid Diuraphis noxia. Rickettsiella in this host resulted in modest fitness effects, with a minor increase in heat tolerance and a decrease in development time at 25{degrees} C. The infection persisted in mixed caged populations under different temperatures. Surprisingly, Rickettsiella increased aphid virulence to wheat plants and to two non-crop hosts of D. noxia, barley grass and brome grass. This was evident from sharper decreases in leaf number and leaf area, as well as an increase in chlorotic streaking when plants were exposed to Rickettsiella-infected D. noxia. Rickettsiella also reduced the proportion of alates in aphids held in small cages and in larger mesocosms containing multiple wheat plants where short-distance dispersal of aphids was impacted. These results provide compelling evidence that Rickettsiella can affect virulence - the first case of an endosymbiont transfer directly influencing aphid virulence to host plants - and highlight the species-specific impacts of endosymbiont transfers on aphids which can involve multiple traits.

microbiology↗

bioRxiv2023

PERK modulation, with GSK2606414, Sephin1 or salubrinal, failed to produce therapeutic benefits in the SOD1G93A mouse model of ALS

Amyotrophic lateral sclerosis (ALS) has been linked to overactivity of the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response (UPR) pathway, both in ALS patients and mouse models. However, attempts to pharmacologically modulate PERK for therapeutic benefit have yielded inconsistent and often conflicting results. This study sought to address these discrepancies by comprehensively evaluating three commonly used PERK modulators (GSK2606414, salubrinal, and Sephin1) in the same experimental models, with the goal of assessing the viability of targeting the PERK pathway as a therapeutic strategy for ALS. To achieve this goal, a tunicamycin-challenge assay was developed using wild-type mice to monitor changes in liver UPR gene expression in response to PERK pathway modulation. Subsequently, multiple dosing regimens of each PERK modulator were tested in standardized, well-powered, gender-matched, and litter-matched survival efficacy studies using the SOD1G93A mouse model of ALS. The alpha-2-adrenergic receptor agonist clonidine was also tested to elucidate the results obtained from the Sephin1, and of the previously reported guanabenz studies, by comparing the effects of presence or absence of -2 agonism. The results revealed that targeting PERK may not be an ideal approach for ALS treatment. Inhibiting PERK with GSK2606414 or activating it with salubrinal did not confer therapeutic benefits. While Sephin1 showed some promising therapeutic effects, it appears that these outcomes were mediated through PERK-independent mechanisms. Clonidine also produced some favorable therapeutic effects, which were unexpected and not linked to the UPR. In conclusion, this study highlights the challenges of pharmacologically targeting PERK for therapeutic purposes in the SOD1G93A mouse model and suggests that exploring other targets within, and outside, the UPR may be more promising avenues for ALS treatment.

neuroscience↗

bioRxiv2023

DNL343 is an investigational CNS penetrant eIF2B activator that prevents and reverses the effects of neurodegeneration caused by the Integrated Stress Response

The integrated stress response (ISR) is a conserved pathway in eukaryotic cells that is activated in response to multiple sources of cellular stress. Although acute activation of this pathway restores cellular homeostasis, intense or prolonged ISR activation perturbs cell function and may contribute to neurodegeneration. DNL343 is an investigational CNS-penetrant small molecule ISR inhibitor designed to activate the eukaryotic initiation factor 2B (eIF2B) and suppress aberrant ISR activation. DNL343 reduced CNS ISR activity and neurodegeneration in a dose-dependent manner in two established in vivo models - the optic nerve crush injury and an eIF2B loss of function (LOF) mutant - demonstrating neuroprotection in both and preventing motor dysfunction in the LOF mutant mouse. Treatment with DNL343 at a late stage of disease in the LOF model reversed elevation in plasma biomarkers of neuroinflammation and neurodegeneration and prevented premature mortality. Several proteins and metabolites that are dysregulated in the LOF mouse brains were normalized by DNL343 treatment, and this response is detectable in human biofluids. Several of these biomarkers show differential levels in CSF and plasma from patients with vanishing white matter disease (VWMD), a neurodegenerative disease that is driven by eIF2B LOF and chronic ISR activation, supporting their potential translational relevance. This study demonstrates that DNL343 is a brain penetrant ISR inhibitor capable of attenuating neurodegeneration in mouse models and identifies several biomarker candidates that may be used to assess treatment responses in the clinic.

neuroscience↗

bioRxiv2023

Evaluating The Efficacy Of Endotracheal Epinephrine Administration At Standard Versus High Dose During Resuscitation Of Severely Asphyxiated Newborn Lambs: A Randomized Preclinical Study

BackgroundEpinephrine treatment is recommended during neonatal resuscitation, if ventilation and chest compressions are ineffective. Endotracheal administration is an option, if the preferred intravenous route is unavailable. We aimed to determine the efficacy of endotracheal epinephrine for achieving return of spontaneous circulation (ROSC), and maintaining physiological stability after ROSC, at standard and higher dose, in severely asphyxiated newborn lambs. MethodsNear-term fetal lambs were instrumented for physiological monitoring, and asphyxiated until asystole. Resuscitation was commenced with ventilation and chest compressions as per ILCOR recommendations. Lambs were randomly allocated to: IV Saline placebo (5 ml/kg, n=6), IV Epinephrine (20 micrograms/kg, n=9), Standard-dose ET Epinephrine (100 micrograms/kg, n=9), and High-dose ET Epinephrine (1 mg/kg, n=9). After three allocated treatment doses, rescue IV Epinephrine was administered if ROSC had not occurred. Lambs achieving ROSC were ventilated and monitored for 60 minutes before euthanasia. Brain histology was assessed for micro-hemorrhage. ResultsROSC in response to allocated treatment (without rescue IV Epinephrine) occurred in 1/6 Saline, 9/9 IV Epinephrine, 0/9 Standard-dose ET Epinephrine, and 7/9 High-dose ET Epinephrine lambs respectively. Three Saline, six Standard-dose ET Epinephrine, and one High-dose ET Epinephrine lambs achieved ROSC after rescue IV Epinephrine. Blood pressure during CPR increased after treatment with IV Epinephrine and High-dose ET Epinephrine, but not Saline or Standard-dose ET Epinephrine. After ROSC, both ET Epinephrine groups had lower pH, higher lactate, and higher blood pressure than the IV Epinephrine group. Cortex micro-hemorrhage was more frequent in the High-dose ET Epinephrine lambs (8/8 lambs examined, versus 3/8 in IV Epinephrine lambs). ConclusionsThe currently recommended dose of ET Epinephrine was ineffective in achieving ROSC. In the absence of convincing clinical or preclinical evidence of efficacy, use of ET Epinephrine at this dose may not be appropriate. High-dose ET Epinephrine requires further evaluation before clinical translation.

physiology↗

bioRxiv2023

A defined clathrin-mediated trafficking pathway regulates sFLT1/VEGFR1 secretion from endothelial cells

FLT1/VEGFR1 negatively regulates VEGF-A signaling and is required for proper vessel morphogenesis during vascular development and vessel homeostasis. Although a soluble isoform, sFLT1, is often mis-regulated in disease and aging, how sFLT1 is trafficked and secreted from endothelial cells is not well understood. Here we define requirements for constitutive sFLT1 trafficking and secretion in endothelial cells from the Golgi to the plasma membrane, and we show that sFLT1 secretion requires clathrin at or near the Golgi. Perturbations that affect sFLT1 trafficking blunted endothelial cell secretion and promoted intracellular mis-localization in cells and zebrafish embryos. siRNA-mediated depletion of specific trafficking components revealed requirements for RAB27A, VAMP3, and STX3 for post-Golgi vesicle trafficking and sFLT1 secretion, while STX6, ARF1, and AP1 were required at the Golgi. Depletion of STX6 altered vessel sprouting in a 3D angiogenesis model, indicating that endothelial cell sFLT1 secretion is important for proper vessel sprouting. Thus, specific trafficking components provide a secretory path from the Golgi to the plasma membrane for sFLT1 in endothelial cells that utilizes a specialized clathrin-dependent intermediate, suggesting novel therapeutic targets.

cell biology↗

bioRxiv2022

moRphology - dEep Learning Imaging Cells (RELIC) - to Differentiate Between Normal and Pathological Kidney Exfoliated Cells

Chronic kidney disease (CKD) is characterised by progressive loss of kidney function leading to kidney failure. Significant kidney damage can occur before symptoms are detected. Currently, kidney tissue biopsy is the gold standard for evaluation of renal damage and CKD severity. This study explores how to precisely quantify morphology characteristics of kidney cells exfoliated into urine, with a view to establish a future urine-based non-invasive diagnostic for CKD. We report the development of a novel deep learning method, which was able to discover a RELIC (moRphology dEep Learning Imaging Cells) signature that can differentiate between kidney cells exfoliated in human urine of and CKD patients with varying degree of kidney damage and non-CKD controls. Exfoliated proximal tubule cells (PTCs) originating from kidneys were isolated from the urine of patients with different levels of kidney damage using previously published methods. An advanced combination of artificial intelligence techniques, deep learning, swarm intelligence, and discriminative analysis was used to discover a RELIC signature in brightfield microscopy images of exfoliated PTCs. Kidney damage in the study subjects was characterised by assessing kidney tissues obtained through a nephrectomy or kidney biopsy. A special deep learning algorithm was developed and trained to create a predictive tool. The algorithm was then used to analyse data from patients with normal and fibrotic kidneys. Data were then classified according to different groups (healthy or fibrosis) and clustering of the training and validation cells was determined for model validation. We developed a novel deep learning method, to obtain RELIC signatures and identify specific deep morphological features which can be used to differentiate urinary PTC cells shed by people with CKD (confirmed by tissue histology obtained from an invasive kidney biopsy) from those without CKD, with a discriminatory accuracy of 82%. We identified a RELIC signature which can be used on a collection of bright field images of exfoliated urinary PTCs to create a predictive tool and differentiate between normal and pathological kidney cells. This study, for the first time, provides a proof of concept that urinary exfoliated tubule cells in patients with kidney fibrosis and healthy controls differ in appearance (morphology) as observed under a basic brightfield microscope. The results suggest that morphological signatures of exfoliated PTCs have the potential to serve as a non-invasive marker of kidney fibrosis.

bioinformatics↗

bioRxiv2022

Bi-steric mTORC1-selective Inhibitors activate 4EBP1 reversing MYC-induced tumorigenesis and synergize with immunotherapy

The MYC oncogene is causally involved in the pathogenesis of most types of human cancer but it remains therapeutically untargeted. The mTORC1 protein complex regulates cap-dependent translation through 4EBP1 and S6K and thereby, downstream MYC protein expression. However, to date, agents such as rapalogs that selectively target mTORC1 (as compared to mTORC2) fail to reactivate 4EBP1 and thus, to block MYC in vivo. In contrast, agents that nonselectively inhibit both protein complexes of the mTOR pathway, mTORC1 and mTORC2, can activate 4EBP1, but often suffer from a lack of tolerability including in vivo hepatotoxicity and immunosuppression. Here, we report the anti-tumor activity of bi-steric mTORC1-selective inhibitors, including Revolution Medicines clinical candidate RMC-5552, that potently and selectively target mTORC1 over mTORC2. In an autochthonous transgenic mouse model of MYC-amplified and MYC-driven hepatocellular carcinoma (HCC), representative bi-steric mTORC1-selective inhibitors suppress translation initiation via activation of 4EBP1, thereby suppressing MYC protein expression and blocking tumor growth. Furthermore, in human HCC samples, the low levels of 4EBP1 and MYC is correlated with immune reactivation. Immunohistochemistry, CIBERSORT, and CODEX reveal that selective mTORC1 inhibition results in activation of both CD4+ T cell- and NKp46+ NK cell-mediated immune surveillance. Moreover, bi-steric mTORC1-selective inhibitors synergize with -PD-1 to induce sustained tumor regression, with immune cell degranulation and release of perforins and granzyme B. These agents also exhibit anti-tumor activity in human patient-derived xenografts of HCC, colorectal cancer, head and neck cancer, and ovarian cancer harboring genomic amplifications in MYC. We infer that selective mTORC1 inhibition is a potential therapeutic strategy to drive effective MYC inactivation in cancer, and the consequent restoration of immune surveillance against neoplasia.

cancer biology↗

bioRxiv2021

Sculpting rupture-free nuclear shapes in fibrous environments

Cytoskeleton-mediated force transmission regulates nucleus morphology. How nuclei shaping occurs in fibrous in vivo environments remains poorly understood. Here a suspended nanofiber assay of precisely-tunable (nm-m) diameters is used to quantify nucleus plasticity in fibrous environments mimicking the natural extracellular matrix. In contrast to the apical cap over the nucleus in cells on 2-dimensional surfaces, the cellular cytoskeleton of cells on fibers displays a uniform actin network caging the nucleus. The role of contractility-driven caging in sculpting nuclear shapes is investigated as cells spread on aligned single fibers, doublets, and multiple fibers of varying diameters. Cell contractility increases with fiber diameter due to increased focal adhesion clustering and density of actin stress fibers, which correlates with increased mechanosensitive transcription factor YAP translocation to the nucleus. Unexpectedly, large- and small-diameter fiber combinations lead to teardrop-shaped nuclei due to stress-fiber anisotropy across the cell. As cells spread on fibers, diameter-dependent invaginations that run the nucleuss length are formed at contact sites. The deepest and sharpest invaginations are insufficient to trigger nucleus rupture, often observed in 2D or confined systems. Overall, we describe the unknown adaptability of nuclei to fibrous environments and resultant sculpting of the nucleus shapes, with pathophysiological implications.

cell biology↗

bioRxiv2020

Persistence of the ground beetle (Coleoptera: Carabidae) microbiome to diet manipulation

Host-associated microbiomes can play important roles in the ecology and evolution of their insect hosts, but bacterial diversity in many insect groups remains poorly understood. Here we examine the relationship between host environment, host traits, and microbial diversity in three species in the ground beetle family (Coleoptera: Carabidae), a group of roughly 40,000 species that synthesize a wide diversity of defensive compounds. This study found that the ground beetle microbiome is consistent across different host food sources. We used 16S amplicon sequencing to profile three species that are phylogenetically distantly related, trophically distinct, and whose defensive chemical secretions differ: Anisodactylus similis LeConte, 1851, Pterostichus serripes (LeConte, 1875), and Brachinus elongatulus Chaudoir, 1876. Wild-caught beetles were compared to individuals maintained in the lab for two weeks on carnivorous, herbivorous, or starvation diets. Soil environment but not diet had a significant effect on bacterial diversity and composition. The three carabid species have patterns of microbial diversity similar to those previously found in other insect hosts. Metagenomic samples from two highly active tissue types -- guts, and pygidial gland secretory cells (which produce defensive compounds) -- were processed and sequenced separately from those of the remaining body. The observed similarity of the pygidial gland secretory cell microbiome across hosts suggests the possibility that it may be a conserved community, possibly due to functional interactions related to defensive chemistry. These results provide a baseline for future studies of the role of microbes in the diversification of defensive chemical biosynthesis in carabids.

ecology↗