Biology subjects

Roscioli, E.

Publications and source records attributed to Roscioli, E..

9 recordsLinked to original sources

bioRxiv2026

Azithromycin Derivatives to Mitigate Off-Target Inhibition of Autophagy and Retain Beneficial Host Directed Effects

Azithromycin (AZM) is central for the treatment of chronic respiratory diseases (CRD) but has divergent off-target effects. We synthesised AZM Derivatives 1 and 2 (D1/D2) that were predicted to permit autophagy and preserve AZM's anti-inflammatory effect. The 16HBE14o- airway epithelial cell model was exposed to AZM, D1 and D2 for 16 h and assessed for autophagy flux via LC3B-II:p62/SQSTM1 abundance (Western blot). Necrosis was quantified via lactate dehydrogenase release. Inflammation (IL-6 secretion) was assessed in the THP-1 macrophage model exposed to 10 ng/mL lipopolysaccharide vs co-treatment with AZM and the derivatives for 18 h. AZM-derivative antibacterial activity (vs AZM) was determined via the minimum inhibition concentration (MIC) method using methicillin sensitive Staphylococcus aureus (MSSA). Autophagy (LC3B-II and p62/SQSTM1 abundance) was not altered by the two derivatives and was indistinguishable from the control exposure (P> 0.05 for D1 and D2, each 10 and 50 ug/mL, vs control). D2 elicited a significant decrease in LPS-induced IL-6 secretion vs the LPS-only exposure (58.22 pg/ml, n=3, 95% +/- CI [6.521-109.9]). Importantly, D2 caused a similar reduction in LPS-induced IL-6 secretion, as observed for AZM (-10.30 pg/mL, n=3, 95% CI [-62.00 to 41.39]). The MIC of AZM for MSSA growth was 0.5 ug/ml, where as D1 and D2 were 1.0 and 8.0 ug/mL, respectively (P<0.05). We show for the first time that AZM can be redesigned to mitigate its potent arrest of autophagy while preserving its anti-inflammatory activity, to counter the generation of further AZM resistant strains.

cell biology↗

bioRxiv2026

A novel imaging biosensor for the detection of reversed replication forks and four-way junctions in human cells

Replication fork reversal and recombination-dependent replication protect perturbed forks by forming four-way DNA junctions. Despite their central role, detecting these intermediates in intact cells has historically required electron microscopy of bulk extracted DNA. Here, we describe a genetically encoded biosensor for four-way junctions based on nuclear-targeted bacterial RuvA tagged with GFP or Spot-tag. Combined with SIRF, RuvA selectively accumulates at nascent DNA following hydroxyurea- or camptothecin-induced replication stress. Recruitment requires fork-reversal factors and is abolished by a non-binding mutant (K84E/K119E), confirming specificity. The biosensor dynamically tracks reversed fork abundance, capturing MRE11-mediated fork degradation in BRCA2- or RAD52-deficient cells and its functional rescue. Finally, RuvA reveals RAD51-dependent four-way junctions at nucleolar rDNA arrays in unperturbed and stressed cells. This tool converts a bulk population measurement into a visualizable readout with single-cell and subnuclear resolution, enabling direct spatial analysis of DNA structures in situ.

cell biology↗

bioRxiv2024

Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

AimsPerfluorocarbons (PFCs) are inert, oxygen-rich fluids with applications in neonatal and adult liquid ventilation and potential for innovations related to hypoxic environments, such as under water and for space. Despite promising clinical applications, a laboratory model to comprehensively test PFC implications and predict human airway responses is lacking. We hypothesise that an organotypic airway epithelial cell (AEC) model is needed assess PFC impacts and provide predictive outcomes to inform the in vivo scenario that conventional submerged cultures cannot replicate. This study evaluated PFC exposure in two in vitro systems: a traditional submerged model using 16HBE14o- cells and an air-liquid interface (ALI) model using hSABCi and primary nasal AECs, cultured on transwells to mimic the human airway epithelium. MethodsPFC exposures were conducted at 2, 8, and 24 hours (submerged model) and extended to 72 hours (ALI model). We tracked gross morphological changes via microscopy, quantified apoptosis and autophagy markers through protein biochemistry, and assessed epithelial permeability using trans-epithelial electrical resistance (TEER) and tight junction protein abundance. Statistical analyses included at least three biological replicates per condition. ResultsIn the submerged 16HBE14o- model, PFC exposure led to significant apoptotic changes by 24 hours, with marked autophagic disruption. Nutrient deprivation, confirmed by starvation experiments, was a key driver of cytotoxicity due to media/PFC phase separation. Conversely, hSABCi cells in the ALI model remained viable, with no apoptosis or autophagic disruption over the exposure periods (P > 0.05 vs. control). Similarly, primary nasal AECs showed consistent viability and stability in autophagic and apoptotic markers, indicating a more accurate representation of in vivo conditions. TEER measurements and tight junction protein levels in the ALI model suggested PFC did not compromise epithelial integrity (P > 0.05 vs. control). PBS exposure, included as a liquid control, underscored baseline sensitivity in the primary nAEC model, evidenced by SQSTM1 upregulation and pronounced barrier dysfunction over 24-72 hours (P < 0.05-0.001). These findings underscore the unique biocompatibility of PFC in maintaining cellular integrity, in contrast to the disruptive effects observed with PBS. ConclusionThis is the first study to describe PFC exposure in an organotypic airway model. Results indicate that the ALI model more accurately preserves airway epithelial integrity during PFC exposure than submerged models, which are limited by nutrient depletion effects. The findings support the use of ALI cultures to replicate the human airway architecture for evaluating PFCs biological effects and offer a platform for preclinical applications in respiratory medicine. This organotypic approach may inform future therapeutic and hypoxia-related interventions and contributes significantly to the field by providing a viable model for understanding PFC interactions with airway epithelial cells. We are now trialling PFC emulsions that also contain azithromycin and steroid therapies for extended therapeutic applications. Infographic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/620983v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@a2143aorg.highwire.dtl.DTLVardef@ddf97forg.highwire.dtl.DTLVardef@127045eorg.highwire.dtl.DTLVardef@8fe405_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Autophagy is suppressed in peripheral blood mononuclear cells during chronic obstructive pulmonary disease.

Assessing autophagy may offer insights into the pathogenesis of chronic obstructive pulmonary disease (COPD). However, measuring the dynamic aspect of autophagy is challenging, and sample manipulation can cause signal fluctuations that deviate from physiological conditions. We applied an organotypic method to quantify autophagy in COPD, where it frequently demonstrates disease-related dysregulation. Blood from control and COPD participants were treated with or without chloroquine. LC3B-II abundance was quantified in peripheral blood mononuclear cells, and findings were validated by transmission electron microscopy. Our observations show that while basal LC3B-II abundance was similar between groups (P = 0.60), autophagic flux was significantly lower in the COPD cohort, suggesting disruption in the regulatory factors that direct autophagosome clearance (P = 0.004). This was supported by less frequent observations of autophagy-related vacuoles in the cytosol of COPD-derived PBMCs. Our findings indicate that the suppression of autophagy can be detected in the blood of individuals with COPD, which warrants further investigation into its contribution to extrapulmonary disease processes.

molecular biology↗

bioRxiv2024

Modification of Azithromycin to Mitigate its Arrest of Autophagy

Aims/hypothesisAutophagy plays a critical role in the survival and microbial clearance function of tissues that encounter the environment, such as airway epithelial cells (AECs). Contrary to the known roles of azithromycin (AZM) in promoting microbial clearance, our evidence shows that AZM is a potent inhibitor of autophagy - an effect consistent with bacterial residency. Here we investigate the structure-activity relationship of AZM vs other macrolides and AZM-3-N-oxide (AZM-[O]), to mitigate the off-target arrest of autophagy. MethodAZM-[O] was synthesised in-house via selective oxidation of the desosamine amine of AZM. Human peripheral blood mononuclear cells (PBMC) were used to assess autophagy ex vivo in an organotypic manner via transmission electron microscopy and Western blot analysis. For in vitro studies, the 16HBE14o-AEC line and Western blot was used to assess macrolide vs autophagy structure-activity relationships, and autophagic flux by quantifying the protein abundance of LC3B-II vs Sequestosome-1. Subsequent assessments of antimicrobial activity were conducted using the micro-broth dilution method. Immunomodulatory outcomes were assessed by quantifying the secretion of IL-6 in a lipopolysaccharide PMA-stimulated THP-1 macrophage model. ResultsAZM significantly inhibited autophagic flux in both the ex vivo PBMC and in vitro 16HBE14o-AEC models, evidenced by the accumulation of autophagosome-related vacuoles and LC3B-II and Sequestosome-1 protein, compared to its precursors and other macrolides including roxithromycin and clarithromycin. Notably, oxidation of AZM to produce AZM-[O] significantly alleviated this inhibitory effect on autophagy, but without completely preserving its antimicrobial and immunomodulatory functions. Conclusion/DirectionsWe show for the first time that AZM inhibits PBMC autophagy in human blood ex vivo and there is a high probability this phenomenon occurs in the clinical setting. Importantly, chemical modification of AZM to generate AZM-[O] substantially alleviated this effect, consistent with altered ionisation properties. We are now assessing clinically derived blood samples in participants treated with AZM, defining the AZM-mammalian protein interactome and developing further AZM derivatives that preserve immunomodulatory activity while minimising disruption of autophagy.

biochemistry↗

bioRxiv2024

Anti-capsule human monoclonal antibodies protect against hypervirulent and pandrug-resistant Klebsiella pneumoniae

The silent pandemic caused by antimicrobial resistance (AMR) requires innovative therapeutic approaches. Human monoclonal antibodies (mAbs), which are among the most transformative, safe and effective drugs in oncology and autoimmunity, are rarely used for infectious diseases and not yet used for AMR. Here we applied an antigen-agnostic strategy to isolate extremely potent human mAbs against Klebsiella pneumoniae (Kp) sequence type 147 (ST147), a hypervirulent and pandrug-resistant clonotype which is spreading globally. Isolated mAbs target the bacterial capsule and the O-antigen. Surprisingly, although both capsule- and O-antigen-specific mAbs displayed bactericidal activity in the picomolar range in vitro, only the capsule-specific mAbs were protective against fulminant ST147 bloodstream infection. Protection correlated with in vitro bacterial uptake by macrophages and enchained bacterial growth. Our study describes the only drug able to protect against pandrug-resistant Kp and provides a strategy to isolate mAbs and identify correlates of protection against AMR bacteria.

microbiology↗

bioRxiv2023

Failure to clear intra-osteocytic Staphylococcus aureus is not the result of antibiotic-mediated autophagy dysregulation

BackgroundStaphylococcus aureus is a major causative pathogen of osteomyelitis. The intracellular infection of osteocytes and related bone cells can persist despite application of gold-standard clinical interventions. The mechanisms by which intracellular S. aureus persists during antibiotic therapy are unknown. In this study, we apply S. aureus to an in vitro model of differentiated osteocytes to investigate whether antibiotic-mediated dysregulation of autophagy contributes to this phenomenon. MethodsHuman osteocyte-like cells were exposed to combinations of rifampicin, vancomycin and modulators of autophagy, in the presence or absence of S. aureus. Intracellular bacterial growth characteristics were assessed through CFU analysis, viable bacterial DNA abundance and the rate of escape into antibiotic-free medium, in parallel with measures of host cell autophagic flux. ResultsRifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of the intracellular bacterial community, concomitant with stable or increased absolute bacterial DNA levels. Both antibiotics significantly inhibited autophagic flux. However, while the modulation of autophagic flux affected bacterial culturability, this modulation did not affect viable bacterial DNA levels. ConclusionsAutophagy was shown to be a factor in the host-pathogen relationship in this model, as its modulation affected the growth state of intracellular S. aureus with respect to both their culturability and propensity to escape the intracellular niche. Whilst rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this did not explain the paradoxical response of antibiotic treatment in decreasing S. aureus culturability while failing to clear bacterial DNA and hence intracellular bacterial load. Thus, whilst rifampicin and vancomycin exhibited off-target effects that modulated autophagy in osteocyte-like cells, this could not explain the persistent infection observed for S. aureus.

microbiology↗

bioRxiv2021

Bayesian inference of multi-point macromolecular architecture mixtures at nanometre resolution

AO_SCPLOWBSTRACTC_SCPLOWGaussian spot fitting methods have significantly extended the spatial range where fluorescent microscopy can be used, with recent techniques approaching nanometre (nm) resolutions. However, small inter-fluorophore distances are systematically over-estimated for typical molecular scales ([lsim] 50nm). This bias can be corrected computationally, but current algorithms are limited to correcting distances between pairs of fluorophores. Here we present a flexible Bayesian computational approach that infers the distances and angles between multiple markers and has several advantages over these previous methods. Specifically it improves confidence intervals for small lengths, estimates measurement errors of each fluorescent marker individually and infers the correlations between polygon lengths. The latter is essential for determining the full multi-fluorophore 3D architecture. We further developed the algorithm to infer the mixture composition of a heterogeneous population of multiple polygon states. We use our algorithm to analyse the 3D architecture of the human kinetochore, a macro-molecular complex that is essential for high fidelity cell division. We examine the conformational change induced by microtubule attachment using triple fluorophore marked data and demonstrate for the first time that in metaphase kinetochore conformation is heterogeneous.

molecular biology↗

bioRxiv2019

Ensemble-level organization of human kinetochores and evidence for distinct tension and attachment sensors

Kinetochores are multi-protein machines that form dynamic attachments to microtubules and generate the forces for chromosome segregation. High-fidelity is ensured because kinetochores can monitor attachment status and tension, using this information to activate checkpoints and error correction mechanisms. To explore how kinetochores achieve this we used two and three colour subpixel fluorescence localisation to define how six protein subunits from the major kinetochore complexes CCAN, MIS12, NDC80, KNL1, RZZ and the checkpoint proteins Bub1 and Mad2 are organised in the human kinetochore. This reveals how the kinetochore outer plate is a liquid crystal-like system with high nematic order and largely invariant to loss of attachment or tension except for two mechanical sensors. Firstly, Knl1 unravelling relays tension and secondly NDC80 jack-knifes under microtubule detachment, with only the latter wired up to the checkpoint signalling system. This provides insight into how kinetochores integrate mechanical signals to promote error-free chromosome segregation.

cell biology↗