Biology subjects

Bosco, D.

Publications and source records attributed to Bosco, D..

9 recordsLinked to original sources

bioRxiv2025

Reliable repurposing of antibody interactome inside the cell

In biology proximity is paramount and eighty-five percent of the human proteome has at least one documented interacting monoclonal antibody. These molecules penetrate the cytoplasm poorly and are very often non-functional within the cell. Sequence analysis of 106 antibody variable domains alongside the cytoplasmic human proteome shows charge and isoelectric point are characteristics ill adapted to intracellular monodispersity. Characterisation of forty-five single-chain variable fragment (scFv) intrabodies expressed in human cells confirmed charge to have the greatest impact on solubility. We created new interdomain linkers, optimised scFv domain orientation and found variable heavy domain framework sites to be generally positively charged, and promote insolubility, but be amenable to optimisation. This is applied in combination to reduce the search space and refine the products of AI-led inverse folding to create highly soluble, abundant and thermally stable intrabodies that maintain parent antibody epitope recognition. Over six hundred intrabody sequences are described targeting sixty cytoplasmic proteins with linear, conformational, post-translational modification or oligomeric state specificity. Interactions were validated for p53, -synuclein, SOD1, polyQ, FUS/TLS, UCHL1 and GFP. This approach removes obstacles hindering intracellular repurposing of the vast sequenced antibody interactome with applications relevant to many human disease states.

molecular biology↗

bioRxiv2025

TDP-43 dysfunction leads to impaired proteostasis and predisposes mice to worse neurological outcomes after brain injury

BackgroundPathological TAR DNA-binding protein 43 (TDP-43) dysfunction is associated with multiple neurodegenerative disorders. However, the mechanistic link between TDP-43 dysfunction and neurodegeneration is poorly understood and likely involves a combination of genetic and environmental risk factors. A major risk factor for neurodegenerative disease is exposure to traumatic brain injury (TBI). Here, we investigated the synergistic interplay between TDP-43 dysfunction and TBI in a murine model of amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). MethodsA model of TDP-43 dysfunction caused by a knock-in Q331K mutation in Tardbp was combined with a mild model of TBI. Control conditions included both WT mice and mice with sham surgery. Animals were evaluated for behavioral deficits at timepoints pre- and post-surgery. Additionally, post-mortem brain tissues were examined using RNA sequencing and mass spectrometry-based quantitative proteomics together with histological and biochemical analyses. ResultsExpression of dysfunctional TDP-43 in vivo caused deficits in multiple branches of the proteostasis network, including protein folding, protein synthesis, and protein turnover. Examples include mis-expression of chaperones and genes within the ubiquitin-proteosome pathway in mutant TDP-43 versus WT mice. Further, mutant TDP-43 expression correlated with reduced thermostability of proteins associated with the ribosome and the chaperonin containing TCP-1 complex. In response to TBI, mutant TDP-43 mice exhibited significantly worse neurological outcomes relative to WT animals. Heightened neurological deficits in mutant TDP-43 mice following TBI coincided with a robust upregulation of proteostasis- and stress-related genes at the transcript level. However, this upregulation was not detected at the protein level. ConclusionsOur data demonstrate that expression of dysfunctional TDP-43 leads to deficits within the proteostasis network in vivo at baseline. Despite an upregulation of proteostasis-related genes at the transcript level in mutant TDP-43 mice after TBI, mutant TDP-43 mice exhibit an impaired response to, and recovery from, brain trauma relative to their WT counterparts. Restoring proteostasis is expected to protect against the detrimental effects of TDP-43 dysfunction, especially under stress conditions that promote neurodegenerative disease.

neuroscience↗

bioRxiv2023

Activity of natural occurring entomopathogenic fungi on nymphal and adult stage of Philaenus spumarius

The spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the predominant vector of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) in Apulia and Europe. Current control strategies of the insect vector rely on mechanical management of nymphal stages and insecticide application against adult populations. Entomopathogenic fungi (EPF) are biological control agents naturally attacking spittlebugs and may effectively reduce population levels of host species. Different experimental trials in controlled conditions have been performed to i) identify naturally occurring EPF on P. spumarius in Northwestern Italy, and ii) evaluate the potential for biocontrol of the isolated strains on both nymphal and adult stages of the spittlebug. Four EPF species were isolated from dead P. spumarius cadavers collected in semi-field conditions: Beauveria bassiana, Conidiobolus coronatus, Fusarium equiseti and Lecanicillium aphanocladii. All the fungal isolates showed entomopathogenic potential against nymphal stages of P. spumarius ({approx} 45 % mortality), except for F. equiseti, in preliminary trials. No induced mortality was observed on the adult stage. Lecanicillium aphanocladii was the most promising fungus and its pathogenicity against spittlebug nymphs was further tested in different formulations (conidia vs blastospores) and with natural adjuvants. Blastospore formulation was the most effective in killing nymphal instars and reducing the emergence rate of P. spumarius adults, reaching mortality levels (90%) similar to those of the commercial product Naturalis(R), while no or adverse effect of natural adjuvants was recorded. The encouraging results of this study pave the way for testing EPF isolates against P. spumarius in field conditions and find new environmentally friendly control strategies against insect vectors of X. fastidiosa.

pathology↗

bioRxiv2022

Transmission of Xylella fastidiosa subsp. pauca ST53 by the sharpshooter Cicadella viridis from different source plants and artificial diets

The sharpshooter Cicadella viridis L. (Hemiptera: Cicadellidae) is the most common sharpshooter in Europe and, given its xylem feeding behaviour, is considered a potential vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae). We tested X. fastidiosa subsp. pauca ST53 (Xfp) transmission capabilities of C. viridis adults, namely i) acquisition efficiency from four host plant species - periwinkle, milkwort, lavender, alfalfa - and from two artificial diets (PD3 and Xfm), ii) inoculation efficiency to periwinkle at different times post acquisition from different plant and artificial diet sources. The main European vector species - Philaenus spumarius - was used as a control. Cicadella viridis was able to acquire Xfp from periwinkle, milkwort, and lavender, although with low efficiency (3-16%) and from artificial diets (23-25%). Successful inoculation on periwinkle was extremely rare, being observed only three times, following feeding on milkwort plant and PD3 artificial diet sources. Our study shows that C. viridis is not a relevant vector of Xfp, given the very low transmission rate in controlled conditions and the inability to feed on olive. The low efficiency reported here sums also to ecological constraints (mainly monocots host plants, humid environments) that make difficult to forecast a relevant role in dispersing X. fastidiosa, at least within the present distribution of the exotic bacterium in Europe. However, a possible role of this species in spreading Xf in other agroecosystems, e.g. vineyard and stone fruits grown in humid areas, cannot be excluded.

ecology↗

bioRxiv2021

Scaphoideus titanus Ball feeding behaviour on three grapevine cultivars with different susceptibilities to Flavescence doree

Scaphoideus titanus (Ball) is a grapevine-feeder leafhopper, and the most important vector of Flavescence doree of grapevine (FD), a disease associated with phytoplasmas belonging to ribosomal subgroups 16Sr-V-C and -D. FD is a major constraint to viticulture in several European countries and, so far, its control has relied on roguing of infected plants and insecticide applications against the vector. Detailed knowledge on different levels of the multifaceted phytoplasma-plant-vector relationship is required to envisage and explore more sustainable ways to control the disease spread. In the present work, S. titanus feeding behaviour was described on three grapevine cultivars: Barbera (susceptible to FD), Brachetto, and Moscato (tolerant to FD) using the Electrical Penetration Graph (EPG) technique. Interestingly, no differences were highlighted in the non-phloem feeding phases, thus suggesting that the tested cultivars have no major differences in the biochemical composition or structure of the leaf cuticle, epidermis or mesophyll, that can affect the first feeding activities. On the contrary, the results showed significant differences in leafhopper feeding behaviour in terms of the duration of the phloem feeding phase, longer on Barbera and shorter on Brachetto and Moscato, and of the frequency of interruption-salivation events inside the phloem, higher on Brachetto and Moscato. These findings indicate a possible preference for the Barbera cultivar, a better host for the leafhopper. Scaphoideus titanus feeding behaviour on Barbera correlates with an enhanced FDp transmission efficiency, thus explaining, at least in part, the higher susceptibility of this cultivar to FD. The mechanisms for the possible non-preference for Brachetto and Moscato are discussed, and an antixenosis is hypothesized. We propose that breeding for resistance against FD should take into account both plant traits associated with the response to the phytoplasmas and to the vector.

animal behavior and cognition↗

bioRxiv2020

Silencing of ATP synthase β reduces phytoplasma multiplication in the leafhopper vector Euscelidius variegatus

The leafhopper Euscelidius variegatus is a natural vector of the chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of the Flavescence doree phytoplasma (FDp). Previous studies indicated a crucial role for insect ATP synthase and {beta} subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase {beta} was obtained by injection of specific dsRNAs in E. variegatus. Here we present the systemic and long-lasting nature of such silencing, its effects on the small RNA profile, the significant reduction of the corresponding protein expression, and the impact on phytoplasma acquisition capability. The specific transcript expression was silenced at least up to 37 days post injection with an average reduction of 100 times in insects injected with dsRNAs targeting ATP synthase {beta} (dsATP) compared with those injected with dsRNAs targeting green fluorescent protein (dsGFP), used as negative controls. Insects injected either with dsATP or dsGFP successfully acquired CYp and FDp during feeding on infected plants. However, the average phytoplasma amount in dsATP insects was significantly lower than that measured in dsGFP specimens, indicating a probable reduction of the pathogen multiplication rate when ATP synthase {beta} was silenced. The role of the insect ATP synthase {beta} during phytoplasma infection process is discussed.

microbiology↗

bioRxiv2020

Sorcin stimulates Activation Transcription Factor 6α (ATF6) transcriptional activity

Levels of the transcription factor ATF6, a key mediator of the unfolded protein response, that provides cellular protection during the progression endoplasmic reticulum (ER) stress, are markedly reduced in the pancreatic islet of patients with type 2 diabetes and in rodent models of the disease, including ob/ob and high fat-fed mice. Sorcin (gene name SRI) is a calcium (Ca2+) binding protein involved in maintaining ER Ca2+ homeostasis. We have previously shown that overexpressing sorcin under the rat insulin promoter in transgenic mice was protective against high fat diet-induced pancreatic beta cell dysfunction, namely preserving intracellular Ca2+ homeostasis and glucose-stimulated insulin secretion during lipotoxic stress. Additionally, sorcin overexpression was apparently activating ATF6 signalling in MIN6 cells despite lowering ER stress. Here, in order to investigate further the relationship between sorcin and ATF6, we describe changes in sorcin expression during ER and lipotoxic stress and changes in ATF6 signalling after sorcin overexpression or inactivation, both in excitable and non-excitable cells. Sorcin mRNA levels were significantly increased in response to the ER stress-inducing agents thapsigargin and tunicamycin, but not by palmitate. On the contrary, palmitate caused a significant decrease in sorcin expression as assessed by both qRT-PCR and Western blotting despite inducing ER stress. Moreover, palmitate prevented the increase in sorcin expression induced by thapsigargin. In addition, sorcin overexpression significantly increased ATF6 transcriptional activity, whereas sorcin inactivation decreased ATF6 signalling. Finally, sorcin overexpression increased levels of ATF6 immunoreactivity and FRET imaging experiments following ER stress induction by thapsigargin showed a direct sorcin-ATF6 interaction. Altogether, our data suggest that sorcin down-regulation during lipotoxicity may prevent full ATF6 activation and a normal UPR during the progression of obesity and insulin resistance, contributing to beta cell failure and type 2 diabetes.

cell biology↗

bioRxiv2020

Biological characterization of Euscelidius variegatus iflavirus 1

Virus-based biocontrol technologies represent sustainable alternatives to pesticides and insecticides. Phytoplasmas are prokaryotic plant pathogens causing severe losses to crops worldwide. Novel approaches are needed since insecticides against their insect vectors and rogueing of infected plants are the only available strategies to counteract phytoplasma diseases. A new iflavirus, named EVV-1, has been described in the leafhopper phytoplasma vector Euscelidius variegatus, raising the potential to use virus-based application strategies against phytoplasma disease. Here transmission routes of EVV-1 are characterized, and localization within the host reveals the mechanism of insect tolerance to virus infection. Both vertical and horizontal transmission of EVV-1 occur and vertical transmission was more efficient. The virus is systemic and occurs in all life-stages, with the highest loads measured in ovaries and first to third instar nymphs. The basic knowledge gained here on the biology of the virus is crucial for possible future application of iflaviruses as biocontrol agents.

microbiology↗

bioRxiv2018

FUS/TLS undergoes calcium-mediated nuclear egress during excitotoxic stress and is required for Gria2 mRNA processing

Excitotoxic levels of glutamate represent a physiological stress that is strongly linked to amyotrophic lateral sclerosis (ALS) and other neurological disorders. Emerging evidence indicates a role for neurodegenerative disease linked RNA-binding proteins (RBPs) in the cellular stress response. However, the relationships between excitotoxicity, RBP function and pathology have not been explored. Here, we found that excitotoxicity induced the translocation of select ALS-linked RBPs from the nucleus to the cytoplasm within neurons. RBPs affected by excitotoxicity include TAR DNA-binding protein 43 (TDP-43) and, most robustly, fused in sarcoma/translocated in liposarcoma (FUS/TLS). FUS translocation occurs through a calcium-dependent mechanism and coincides with striking alterations in nucleocytoplasmic transport. Further, glutamate-induced upregulation of Gria2 in neurons was dependent on FUS expression, consistent with a functional role for FUS under excitotoxic stress. These findings reveal a link between prominent factors in neurodegenerative disease, namely excitotoxicity, disease-associated RBPs and nucleocytoplasmic transport.

neuroscience↗