Biology subjects

Sala, C.

Publications and source records attributed to Sala, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Alterations of gut microbiota in Down syndrome and their association with Alzheimer's disease

Structured AbstractO_ST_ABSINTRODUCTIONC_ST_ABSAdults with Down syndrome (DS) have a higher risk of developing Alzheimers disease (AD). As gut microbiota (GM) alterations have been reported in AD, we investigated their association with cognitive decline and plasma AD biomarkers in DS. METHODSFecal and plasma samples were collected from 58 adults with DS (21-75 years) and 30 euploid controls (CTRL; 25-83 years). GM was profiled using 16S rRNA sequencing. Major Neurocognitive Disorder (NcD) was diagnosed according to DSM-5 criteria. Plasma levels of p-Tau181, NfL, and GFAP were measured using the Simoa platform. RESULTSCompared with CTRL, DS showed significant changes in UBA1819 and Intestinibacter genera, previously reported to be associated with mild cognitive impairment. Furthermore, DS with NcD were characterized by a reduced abundance of Roseburia genus, which was also negatively associated with plasma levels of AD biomarkers. CONCLUSIONAdults with DS display AD-associated changes in GM partially resembling those previously reported in euploid AD patients

microbiology↗

bioRxiv2024

Mechanotransduction and inflammation: an updated comprehensive representation

Mechanotransduction is the process that enables the conversion of mechanical cues into biochemical signaling. While all our cells are well known to be sensitive to such stimuli, the details of the systemic interaction between mechanical input and inflammation are not always well integrated. Often, they are considered and studied in relatively compartmentalized areas, and we therefore argue here that to understand the relationship of mechanical stimuli with inflammation - with high translational potential - it is crucial to offer and analyze a unified view of mechanotransduction. We therefore offer here a pathway representation, recollected with the standard systems biology markup language (SBML) and explored with network biology approaches. We present RAC1 as an exemplar and emerging molecule with potential for medical translation.

bioinformatics↗

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

Human monoclonal antibodies reveal subdominant gonococcal and meningococcal cross-protective antigens

Gonococcus (Gc), a bacterium resistant to most antibiotics causing more than 80 million cases of gonorrhea annually, is a WHO high priority pathogen. Recently, vaccine development prospects were boosted by reports that licensed meningococcus serogroup B (MenB) vaccines provided partial protection against Gc infection. To determine antigens responsible for cross-protection, memory B cells from 4CMenB vaccinated volunteers were single-cell sorted to identify antibodies that kill Gc in a bactericidal assay. Nine different antibodies, all deriving from the IGHV4-34 germline carrying unusually long HCDR3s, recognized the PorB protein, four recognized the lipooligosaccharide (LOS), and four unknown antigens. One of the PorB antibodies, tested in vivo, provided protection from Gc infection. The identification of PorB and LOS as key antigens of gonococcal and meningococcal immunity provides a mechanistic explanation of the cross-protection observed in the clinic and shows that isolating human monoclonal antibodies from vaccinees can be instrumental for bacterial antigen discovery.

immunology↗

bioRxiv2023

The Minimal Dataset for Cancer of the 1+Million Genomes Initiative

For a real impact on healthcare, precision cancer medicine requires accessibility and interoperability of clinical and genomic data across centres and countries. Due to the heterogeneous digitization in Europe and worldwide, the definition of models for standardised data collection and usability becomes mandatory if countries want to work together on this mission. The European Union 1+Million Genomes (1+MG) initiative, supported by the Horizon 2020 Beyond 1 Million Genomes project, aims at outlining data models, guidance, best practices, and technical infrastructures for transnational access to sequenced genomes, including cancer genomes. Within the framework of the cancer-focused Working Group 9, we developed the 1+MG-Minimal Dataset for Cancer (1+MG-MDC)-a data model encompassing 140 items and organized in eight conceptual domains for the collection of cancer-related clinical information and genomics metadata. The 1+MG-MDC, which results from a multidisciplinary effort, leverages pre-existing models and emphasizes the annotation and traceability of multiple aspects relevant to the complex longitudinal path of the cancer disease and its treatment. We strived to make the 1+MG-MDC easy to adopt, yet comprehensive, addressing the needs of both clinicians and researchers. We will periodically revise and update it to ensure it remains fit for purpose. We propose the 1+MG-MDC as a model to create homogeneous databases, which would, in turn, guide discussions on clinical and genomic features with prognostic or therapeutic value and foster real-world data research.

cancer biology↗

bioRxiv2023

Covering Hierarchical Dirichlet Mixture Models on binary data to enhance genomic stratifications in Onco-Hematology

Onco-hematological studies are increasingly adopting statistical mixture models to support the advancement of the genetically-driven classification systems for blood cancer. Targeting enhanced patients stratification based on the sole role of molecular biology attracted much interest and contributes to bring personalized medicine closer to reality. In particular, Dirichlet processes have become the preferred method to approach the fit of mixture models. Usually, the multinomial distribution is at the core of such models. However, despite their advanced statistical formalism, these processes are not to be considered black box techniques and a better understanding of their working mechanisms enables to improve their employment and explainability. Focused on genomic data in Acute Myeloid Leukemia, this work unfolds the driving factors and rationale of the Hierarchical Dirichlet Mixture Models of multinomials on binary data. In addition, we introduce a novel approach to perform accurate patients clustering via multinomials based on statistical considerations. The newly reported adoption of the Multivariate Fishers Non-Central Hypergeometric distributions reveals promising results and outperformed the multinomials in clustering both on simulated and real onco-hematological data. Author summaryExplainable models are particularly attractive nowadays since they have the advantage to convince clinicians and patients. In this work we show that a deeper understanding of the Hierarchical Dirichlet Mixture Model, a non-black box method, can lead to better data modelling. In onco-hematology Hierarchical Dirichlet Mixture Models typically help to cluster molecular alterations rather than patients. Here, an intuitive statistical approach is presented to tackle patient classification based on the Hierarchical Dirichlet Mixture Models outcome. Additionally, molecular alterations are usually modelled by Hierarchical Dirichlet Mixture Models as a mixture of multinomial distributions. This work highlights that the alternative Fishers Non-Central Hypergeometric distribution can provide even better results and can give a higher priority to rare molecular alterations for patient classification.

cancer biology↗

bioRxiv2023

High-throughput bactericidal assays for monoclonal antibody screening against antimicrobial resistant Neisseria gonorrhoeae

Neisseria gonorrhoeae (gonococcus) is an obligate human pathogen and the etiological agent of the sexually transmitted disease gonorrhea. The rapid rise in gonococcal resistance to all currently available antimicrobials has become a significant public health burden and the need to develop novel therapeutic and prophylactic tools is now a global priority. While high-throughput screening methods allowed rapid discovery of extremely potent monoclonal antibodies (mAbs) against viral pathogens, the field of bacteriology suffers from the lack of assays that allow efficient screening of large panels of samples. To address this point, we developed luminescence-based (L-ABA) and resazurin-based (R-ABA) antibody bactericidal assays that measure N. gonorrhoeae metabolic activity as a proxy of bacterial viability. Both L-ABA and R-ABA are applicable on the large scale for the rapid identification of bactericidal antibodies and were validated by conventional methods. Implementation of these approaches will be instrumental to the development of new medications and vaccines against N. gonorrhoeae and other bacterial pathogens to support the fight against antimicrobial resistance.

microbiology↗

bioRxiv2022

mRNA vaccines and hybrid immunity use different B cell germlines to neutralize Omicron BA.4 and BA.5

SARS-CoV-2 omicron BA.4 and BA.5, characterized by high transmissibility and ability to escape natural and vaccine induced immunity, are rampaging worldwide. To understand the escape mechanisms, we tested the neutralizing activity against omicron BA.4 and BA.5 of a panel of 482 human monoclonal antibodies that had been isolated from people who received two or three mRNA vaccine doses or from people that had been vaccinated after infection. None of the antibodies isolated after two vaccine doses neutralized omicron BA.4 and BA.5, while these variants were neutralized by approximately 15% of antibodies obtained from people that received three doses or had been vaccinated after infection. Remarkably, the antibodies isolated after three vaccine doses targeted mainly the receptor binding domain (RBD) Class 1/2 epitope region and were encoded by the IGHV1-69 and IGHV3-66 B cell germlines, while the antibodies isolated after infection recognized mostly the RBD Class 3 epitope region and the NTD, and were encoded by the IGHV2-5;IGHJ4-1 and IGHV1-24;IGHJ4-1 germlines. The observation that mRNA vaccination and hybrid immunity elicit a different immunity against the same antigen is intriguing and its understanding may help to design the next generation of therapeutics and vaccines against COVID-19.

immunology↗

bioRxiv2022

COVID-19 mRNA third dose induces a unique hybrid immunity-like antibody response

The continuous evolution of SARS-CoV-2 generated highly mutated variants, like omicron BA.1 and BA.2, able to escape natural and vaccine-induced primary immunity1,2. The administration of a third dose of mRNA vaccines induces a secondary response with increased protection. We investigated, at single-cell level, the longitudinal evolution of the neutralizing antibody response in four donors after three mRNA doses3. A total of 4,100 spike protein specific memory B cells were single cell sorted and 350 neutralizing antibodies were identified. The third dose increased the antibody neutralization potency and breadth against all SARS-CoV-2 variants of concern as previously observed with hybrid immunity3. However, the B cell repertoire that stands behind the response is dramatically different. The increased neutralizing response was largely due to the expansion of B cell germlines poorly represented after two doses, and the reduction of germlines predominant after primary immunization such as IGHV3-53;IGHJ6-1 and IGHV3-66;IGHJ4-1. Divergently to hybrid immunity, cross-protection after a third dose was mainly guided by Class 1/2 antibodies encoded by IGHV1-58;IGHJ3-1 and IGHV1-69;IGHJ4-1 germlines. The IGHV2-5;IGHJ3-1 germline, which induced broadly cross-reactive Class 3 antibodies after infection or viral vector vaccination, was not induced by a third mRNA dose. Our data show that while neutralizing breadth and potency can be improved by different immunization regimens, each of them has a unique molecular signature which should be considered while designing novel vaccines and immunization strategies.

immunology↗

bioRxiv2021

Structural insights of a highly potent pan-neutralizing SARS-CoV-2 human monoclonal antibody

As the coronavirus disease 2019 (COVID-19) pandemic continues, there is a strong need for highly potent monoclonal antibodies (mAbs) that are resistant against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants of concern (VoCs). Here, we evaluate the potency of a previously described mAb J08 against these variants using cell-based assays and delve into the molecular details of the binding interaction using cryo-EM. We show that mAb J08 has low nanomolar affinity against VoCs, binds high on the receptor binding domain (RBD) ridge and is therefore unaffected by most mutations, and can bind in the RBD-up and -down conformations. These findings further validate the phase II/III human clinical trial underway using mAb J08 as a monoclonal therapy. One Sentence SummaryPotent neutralizing monoclonal antibody J08 binds SARS-CoV-2 spike independent of known escape mutations.

immunology↗

bioRxiv2021

Hybrid immunity improves B cell frequency, antibody potency and breadth against SARS-CoV-2 and variants of concern

To understand the nature of the antibody response to SARS-CoV-2 vaccination, we analyzed at single cell level the B cell responses of five naive and five convalescent people immunized with the BNT162b2 mRNA vaccine. Convalescents had higher frequency of spike protein specific memory B cells and by cell sorting delivered 3,532 B cells, compared with 2,352 from naive people. Of these, 944 from naive and 2,299 from convalescents produced monoclonal antibodies against the spike protein and 411 of them neutralized the original Wuhan SARS-CoV-2 virus. More than 75% of the monoclonal antibodies from naive people lost their neutralization activity against the B.1.351 (beta) and B.1.1.248 (gamma) variants while this happened only for 61% of those from convalescents. The overall loss of neutralization was lower for the B.1.1.7 (alpha) and B.1.617.2 (delta) variants, however it was always significantly higher in those of naive people. In part this was due to the IGHV2-5;IGHJ4-1 germline, which was found only in convalescents and generated potent and broadly neutralizing antibodies. Overall, vaccination of seropositive people increases the frequency of B cells encoding antibodies with high potency and that are not susceptible to escape by any of the four variants of concern. Our data suggest that people that are seropositive following infection or primary vaccination will produce antibodies with increased potency and breadth and will be able to better control SARS-CoV-2 emerging variants.

immunology↗

bioRxiv2021

Rescuing epileptic and behavioral alterations in a Dravet syndrome mouse model by inhibiting eEF2K

Dravet Syndrome is a severe childhood pharmacoresistant epileptic disorder caused mainly by mutations in the SCN1A gene, which encodes for the 1 subunit of the type I voltage-gated sodium channel (NaV1.1), that cause imbalance between excitation and inhibition in the brain. We recently found that eEF2K knock out mice displayed enhanced GABAergic transmission and tonic inhibition and were less susceptible to epileptic seizures. In Scn1a+/- mice, a mouse model of the Dravet syndrome, we found that the activity of eEF2K/eEF2 pathway was enhanced. Then, we demonstrated that both genetic deletion and pharmacological inhibition of eEF2K were able to reduce the epileptic phenotype of Scn1a+/- mice. Interestingly we also found that motor coordination defect, memory impairments, and stereotyped behavior of the Scn1a+/- mice were reverted by eEF2K deletion. The analysis of spontaneous inhibitory postsynaptic currents (sIPSCs) suggested that the rescue of the pathological phenotype was driven by the potentiation of GABAergic synapses. Our data indicate that pharmacological inhibition of eEF2K could represent a novel therapeutic intervention for treating epilepsy and related comorbidities in the Dravet syndrome.

neuroscience↗

bioRxiv2020

Restoring Glutamate receptosome dynamics at synapses rescues Autism-like deficits in Shank3-deficient mice.

Shank3 monogenic mutations lead to Autism Spectrum Disorders (ASD). Shank3 is part of the glutamate receptosome that physically links ionotropic NMDA receptors to metabotropic mGlu5 receptors through interactions with scaffolding proteins PSD95-GKAP-Shank3-Homer. A main physiological function of the glutamate receptosome is to control NMDA synaptic function that is required for plasticity induction. Intact glutamate receptosome supports glutamate receptors activation and plasticity induction, while glutamate receptosome disruption blocks receptors activity, preventing the induction of subsequent plasticity. Despite possible impact on metaplasticity and cognitive behaviors, scaffold interaction dynamics and their consequences are poorly defined. Here we used mGlu5-Homer interaction as a biosensor of glutamate receptosome integrity to report changes of NMDA synaptic function. Combining BRET imaging and electrophysiology, we show that a transient neuronal depolarization inducing NMDA-dependent plasticity disrupts glutamate receptosome in a long-lasting manner at synapses and induces signaling required for the expression of the initiated neuronal plasticity such as ERK and mTOR pathways. Glutamate receptosome disruption also decreases NMDA/AMPA currents ratio, freezing the sensitivity of the synapse to subsequent changes of neuronal activity. These data show the importance of a fine-tuning of protein-protein interactions within glutamate receptosome, driven by changes of neuronal activity, to control plasticity. In a mouse model of ASD, a truncated mutant form of Shank3 prevents the integrity of the glutamate receptosome. These mice display altered plasticity, anxiety-like and stereotyped behaviors. Interestingly, repairing the integrity of glutamate receptosome and its sensitivity to the neuronal activity rescued synaptic transmission, plasticity and some behavioral traits of Shank3{triangleup}C mice. Altogether, our findings characterize mechanisms by which Shank3 mutations cause ASD and highlight scaffold dynamics as new therapeutic target.

neuroscience↗

bioRxiv2020

Extremely potent human monoclonal antibodies from convalescent Covid-19 patients

Human monoclonal antibodies are safe, preventive and therapeutic tools, that can be rapidly developed to help restore the massive health and economic disruption caused by the Covid-19 pandemic. By single cell sorting 4277 SARS-CoV-2 spike protein specific memory B cells from 14 Covid-19 survivors, 453 neutralizing antibodies were identified and 220 of them were expressed as IgG. Up to 65,9% of monoclonals neutralized the wild type virus at a concentration of >500 ng/mL, 23,6% neutralized the virus in the range of 100 - 500 ng/mL and 9,1% had a neutralization potency in the range of 10 - 100 ng/mL. Only 1,4% neutralized the authentic virus with a potency of 1-10 ng/mL. We found that the most potent neutralizing antibodies are extremely rare and recognize the RBD, followed in potency by antibodies that recognize the S1 domain, the S-protein trimeric structure and the S2 subunit. The three most potent monoclonal antibodies identified were able to neutralize the wild type and D614G mutant viruses with less than 10 ng/mL and are good candidates for the development of prophylactic and therapeutic tools against SARS-CoV-2. One Sentence SummaryExtremely potent neutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for prophylactic and therapeutic interventions.

immunology↗

bioRxiv2020

FasR regulates fatty acid biosynthesis and is essential for virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the worlds leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.

microbiology↗

bioRxiv2020

Identification of neutralizing human monoclonal antibodies from Italian Covid-19 convalescent patients

In the absence of approved drugs or vaccines, there is a pressing need to develop tools for therapy and prevention of Covid-19. Human monoclonal antibodies have very good probability of being safe and effective tools for therapy and prevention of SARS-CoV-2 infection and disease. Here we describe the screening of PBMCs from seven people who survived Covid-19 infection to isolate human monoclonal antibodies against SARS-CoV-2. Over 1,100 memory B cells were single-cell sorted using the stabilized prefusion form of the spike protein and incubated for two weeks to allow natural production of antibodies. Supernatants from each cell were tested by ELISA for spike protein binding, and positive antibodies were further tested for neutralization of spike binding to receptor(s) on Vero E6 cells and for virus neutralization in vitro. From the 1,167 memory B specific for SARS-CoV-2, we recovered 318 B lymphocytes expressing human monoclonals recognizing the spike protein and 74 of these were able to inhibit the binding of the spike protein to the receptor. Finally, 17 mAbs were able to neutralize the virus when assessed for neutralization in vitro. Lead candidates to progress into the drug development pipeline will be selected from the panel of neutralizing antibodies identified with the procedure described in this study. One Sentence SummaryNeutralizing human monoclonal antibodies isolated from Covid-19 convalescent patients for therapeutic and prophylactic interventions.

immunology↗

bioRxiv2020

Deletion of calcineurin from astrocytes reproduces proteome signature of Alzheimer's disease and epilepsy and predisposes to seizures

In astrocytes, calcineurin (CaN) is involved in neuroinflammation and gliosis, while its role in healthy CNS or in early neuro-pathogenesis is poorly understood. Here we report that in astroglial CaN KO (ACN-KO) mice, at one month of age, proteome is deranged in hippocampus and cerebellum. Bioinformatic analysis reveals association with Alzheimers disease (AD) and epilepsy. We found significant overlap with the proteome of an AD mouse model and of human subjects with drug-resistant epilepsy. In Barnes maze ACN-KO mice learned the task but adopted serial search strategy. Strikingly, from five months of age ACN-KO mice develop spontaneous seizures with an inflammatory signature of epileptic brains. These results suggest that astroglial CaN KO impairs hippocampal connectivity, produces proteome features of neurological disorders and predisposes mice to seizures. We suggest that astroglial CaN may serve as a novel Ca2+-sensitive switch which regulates protein expression and homeostasis in the CNS.

neuroscience↗

bioRxiv2019

Polarly localized EccE1is required for ESX-1 function and stabilization of ESX-1 membrane proteins in Mycobacterium tuberculosis

Mycobacterium tuberculosis is a slow-growing intracellular bacterium with the ability to induce host cell death and persist indefinitely in the human body. This pathogen uses the specialized ESX-1 secretion system to secrete virulence factors and potent immunogenic effectors required for disease progression. ESX-1 is a multi-subunit apparatus with a membrane complex that is predicted to form a pore in the cytoplasmic membrane. In M. tuberculosis this complex is composed of five membrane proteins: EccB1, EccCa1, EccCb1, EccD1, EccE1. In this study, we have characterized the membrane component EccE1 and found that deletion of eccE1 lowers the levels of EccB1, EccCa1 and EccD1 thereby abolishing ESX-1 secretion and attenuating M. tuberculosis ex vivo. Surprisingly, secretion of EspB was not affected by loss of EccE1. Furthermore, EccE1 was found to be a membrane- and cell-wall associated protein that needs the presence of other ESX-1 components to assemble into a stable complex at the poles of M. tuberculosis. Overall, this investigation provides new insights into the role of EccE1 and its localization in M. tuberculosis.\n\nIMPORTANCETuberculosis (TB), the worlds leading cause of death of humans from an infectious disease, is caused by the intracellular bacterium Mycobacterium tuberculosis. The development of successful strategies to control TB requires better understanding of the complex interactions between the pathogen and human host. We investigated the contribution of EccE1, a membrane protein, to the function of the ESX-1 secretion system, the major virulence determinant of M. tuberculosis. By combining genetic analysis of selected mutants with eukaryotic cell biology and proteomics, we demonstrate that EccE1 is critical for ESX-1 function, secretion of effector proteins and pathogenesis. Our research improves knowledge of the molecular basis of M. tuberculosis virulence and enhances our understanding of pathogenesis.

microbiology↗