Biology subjects

Torelli, F.

Publications and source records attributed to Torelli, F..

8 recordsLinked to original sources

bioRxiv2026

Hypoxic Modulation of Dental Pulp Stem Cell Viability: an In Vitro Study

IntroductionTo investigate the effects of chronic hypoxic exposure simulating heavy-industrial environments on the viability, metabolic activity, stemness preservation, and osteogenic differentiation potential of human dental pulp stem cells (hDPSCs). MethodsCommercially available hDPSCs were cultured under controlled oxygen tensions representing surface atmospheric conditions (21% O2), moderate hypoxia (10% O2), deep hypoxia (5% O2), and severe hypoxia (1% O2). Cells were maintained for 1, 3, and 7 days. Cell viability was evaluated using MTT and Live/Dead assays. Reactive oxygen species (ROS) accumulation, mitochondrial membrane potential, and apoptosis were assessed using fluorescent probes and Annexin V/PI staining. Stemness marker expression (SOX2, OCT4, NANOG) and osteogenic markers (RUNX2, ALP, OCN) were analyzed via RT-qPCR. ResultsModerate hypoxia (10% O2) promoted transient increases in stemness marker expression and preserved metabolic activity. Severe hypoxia (1% O2) significantly reduced cell viability, increased ROS accumulation, disrupted mitochondrial integrity, and elevated apoptotic cell populations after prolonged exposure (p < 0.05). Osteogenic differentiation markers were significantly downregulated under severe hypoxic conditions. ConclusionsIndustrial hypoxic environments critically influence pulpal stem cell physiology and regenerative potential. While moderate oxygen reduction may transiently preserve stemness characteristics, chronic severe hypoxia impairs viability and osteogenic functionality. Chronic low-oxygen occupational environments may alter endogenous dental regenerative mechanisms and influence oral tissue healing responses.

cell biology↗

bioRxiv2026

Bioluminescent Peptide-Based Biosensors for Early Enamel Demineralization: An In Vitro Proof-of-Concept

BackgroundEarly enamel demineralization corresponding to ICDAS 0-1 is difficult to detect through routine visual-tactile examination, as initial mineral loss often precedes visible surface change. Existing optical adjuncts improve detection but frequently require specialized equipment, high costs, or ionizing radiation, limiting widespread clinical use. ObjectiveTo develop a low-cost, luciferin-inspired fluorogenic peptide biosensor capable of selectively binding early enamel porosity and producing a quantifiable green luminescent signal under standard dental blue-light activation. MethodsA calcium-affinitive peptide (P-Ca) was synthesized and functionalized with an inexpensive fluorogenic ester-quencher pair that becomes fluorescent upon conformational stabilization on partially demineralized enamel. Thirty extracted molars, collected as anonymized biowaste from orthodontic procedures, were sectioned and assigned to sound enamel, mild demineralization (pH-cycling, 48 h), or moderate demineralization (96 h). After 60 s incubation with P-Ca, specimens were illuminated using a dental curing light (450-470 nm). Emission spectra ({lambda}_max 515 {+/-} 5 nm) and fluorescence intensities were quantified and compared with quantitative light-induced fluorescence (QLF). Cytocompatibility was evaluated using an immortalized human gingival fibroblasts cell line (HGF-1). ResultsFluorescence intensity increased in accordance with demineralization severity (p < 0.001), and luminescent output strongly correlated with QLF {Delta}F values (p < 0.001). HGF-1 viability remained above 95% after 24 h exposure. ConclusionThis in vitro study supports the feasibility of a fluorogenic peptide biosensor as an inexpensive, non-radiographic adjunct for early enamel demineralization detection, with clear potential for future chairside translation.

bioengineering↗

bioRxiv2026

Gut-relevant short-chain fatty acids modulate host-pathogen dynamics of uropathogenic Escherichia coli at the colonic epithelial interface

Urinary tract infection (UTI) ranks among the most prevalent bacterial infections worldwide, affecting over 400 million people each year. Uropathogenic Escherichia coli (UPEC), the main aetiological cause of UTI, colonises the intestinal tract, which is thought to serve as a distal reservoir for gut-UTI recurrence. Despite this, the precise role of the gut in UTI recurrence is still not fully defined. Recent research investigating the gut-UTI axis has revealed that reduced abundance of gut commensals producing short-chain fatty acids (SCFAs, namely acetate, butyrate and propionate) is associated with recurrent and chronic UTI. We therefore aimed to investigate the impact of these gut commensal-derived metabolites on a diverse panel of UPEC strains, including well-studied prototypical strains (UTI89, CFT073), a non-pathogenic isolate E. coli K-12, and various clinical UTI isolates (from the urine of both symptomatic and asymptomatic individuals). We observed that SCFAs modulate bacterial growth kinetics in a concentration- and pH-dependent manner, by prolonging the lag phase without affecting final carrying capacity in vitro. These metabolites further suppressed bacterial swimming motility and biased the orientation of fimS, the phase variable switch for T1 fimbriae, under acidic conditions. In a human polarized, mucus-secreting intestinal infection model, SCFA treatment during UPEC challenge altered bacterial localization patterns, favouring planktonic over mucosal-associated populations, and preserved epithelial barrier function. Together, these in vitro findings demonstrate that SCFAs modulate key UPEC colonization-associated phenotypes and influence host-pathogen dynamics at the colonic epithelial interface. These results provide mechanistic insights into how depletion of SCFA-producing gut commensals may alter the intestinal reservoir environment in vitro and warrants further investigation into the role of gut-derived SCFAs in rUTI susceptibility.

microbiology↗

bioRxiv2024

In vivo CRISPR screens identify GRA12 as a transcendent secreted virulence factor across Toxoplasma gondii strains and mouse subspecies

Toxoplasma gondii parasites exhibit extraordinary host promiscuity owing to over 250 putative secreted proteins that disrupt host cell functions, enabling parasite persistence. However, most of the known effector proteins are specific to Toxoplasma genotypes or hosts. To identify virulence factors that function across different parasite isolates and mouse strains that differ in susceptibility to infection, we performed systematic pooled in vivo CRISPR-Cas9 screens targeting the Toxoplasma secretome. We identified several proteins required for infection across parasite strains and mouse species, of which the dense granule protein 12 (GRA12) emerged as the most important effector protein during acute infection. GRA12 deletion in IFN{gamma}-activated macrophages results in collapsed parasitophorous vacuoles and increased host cell necrosis, which is partially rescued by inhibiting early parasite egress. GRA12 orthologues from related coccidian parasites, including Neospora caninum and Hammondia hammondi, complement Tg{Delta}GRA12 in vitro, suggesting a common mechanism of protection from immune clearance by their hosts.

microbiology↗

bioRxiv2024

Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens

Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-{gamma}-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While {Delta}MYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues {Delta}MYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell. SIGNIFICANCE STATEMENTPooled CRISPR screens are powerful tools to interrogate gene function in a high-throughput manner. Genes conferring fitness advantages or disadvantages upon disruption can be identified by sequencing. However, in Toxoplasma gondii pooled CRISPR screens in mice, fitness defects for some selected mutants drastically diverge from those observed in single-strain infections. Here, we show that a growth defect of a single Toxoplasma gene deletion mutant is rescued if co-infected with wildtype parasites. These results shine light on Toxoplasmas ability to subvert the host response beyond the infected cell, and highlight an important limitation of pooled CRISPR screens in mice. This limitation is probably encountered in CRISPR screens in general where paracrine effects occur.

microbiology↗

bioRxiv2023

Mapping host-microbe transcriptional interactions by dual perturb-seq

Intracellular pathogens and other endosymbionts reprogram host cell transcription to suppress immune responses and recalibrate biosynthetic pathways. This reprogramming is critical in determining the outcome of infection or colonisation. Here, we combine pooled CRISPR knockout screening with dual host-microbe single-cell RNA-sequencing to identify the molecular mediators of these transcriptional interactions, a method we term dual perturb-seq. Applying dual perturb-seq to the intracellular pathogen Toxoplasma gondii, we are able to identify previously uncharacterised effector proteins and directly infer their function from the transcriptomic data. We show that TgGRA59 contributes to the export of other effector proteins from the parasite into the host cell and identify a novel effector, TgSOS1, that is necessary for sustained host STAT6 signalling and thereby contributes to parasite immune evasion and persistence. Together, this work demonstrates a novel tool that can be broadly adapted to interrogate host-microbe transcriptional interactions and reveal mechanisms of infection and immune evasion.

molecular biology↗

bioRxiv2022

A heterotrimeric complex of Toxoplasma proteins promotes parasite survival in interferon gamma stimulated human cells

Toxoplasma gondii secretes protein effectors to subvert the human immune system sufficiently to establish a chronic infection. Relative to murine infections, little is known about which parasite effectors disarm human immune responses. Here we used targeted CRISPR screening to identify secreted protein effectors required for parasite survival in IFN{gamma}-activated human cells. Independent screens were carried out using two Toxoplasma strains which differ in virulence in mice, leading to the identification of effectors required for survival in IFN{gamma}-activated human cells. We identify the secreted protein GRA57 and two other proteins, GRA70 and GRA71, that together form a complex which enhances the ability of parasites to persist in IFN{gamma}-activated human foreskin fibroblasts (HFFs). Components of the protein machinery required for export of Toxoplasma proteins into the host cell were also found to be important for parasite resistance to IFN{gamma} in human cells, but these export components function independently of the identified protein complex. Host-mediated ubiquitination of the parasite vacuole has previously been associated with increased parasite clearance from human cells, but we find that vacuoles from GRA57, GRA70 and GRA71 knockout strains are surprisingly less ubiquitinated by the host cell. We hypothesise that deletion of this trimeric complex renders parasites hypersensitive to remaining ubiquitination, resulting in increased parasite clearance.

microbiology↗

bioRxiv2022

Toxoplasma gondii ROP1 subverts murine and human innate immune restriction

Toxoplasma gondii is an intracellular parasite that can infect many different host species and is a cause of significant human morbidity worldwide. T. gondii secretes a diverse array of effector proteins into the host cell which are critical for infection; however, the vast majority of these secreted proteins are uncharacterised. Here, we carried out a pooled CRISPR knockout screen in the T. gondii Prugniaud strain in vivo to identify secreted proteins that contribute to parasite immune evasion in the host. We identify 22 putative virulence factors and demonstrate that ROP1, the first-identified rhoptry protein of T. gondii, has a previously unrecognised role in parasite resistance to interferon gamma-mediated innate immune restriction. This function is conserved in the highly virulent RH strain of T. gondii and contributes to parasite growth in both murine and human macrophages. While ROP1 affects the morphology of rhoptries, from where the protein is secreted, it does not affect rhoptry secretion. ROP1 interacts with the host cell protein C1QBP, which appears to facilitate parasite immune evasion. In summary, we identify 22 secreted proteins which contribute to parasite growth in vivo and show that ROP1 is an important and previously overlooked effector in counteracting both murine and human innate immunity.

microbiology↗