Biology subjects

Sala, M.

Publications and source records attributed to Sala, M..

14 recordsLinked to original sources

bioRxiv2026

BATTLE-AMP: Benchmarking Antimicrobial Peptide Predictors

As antimicrobial resistance outpaces antibiotic development, antimicrobial peptides (AMPs) have emerged as a promising class of alternative antibacterials, and computational predictors are increasingly used to prioritize AMP candidates. Such predictors are typically evaluated on binary AMP/non-AMP classification, which does not test whether they can identify peptides with clinically relevant potency against specific pathogens. We present BATTLE-AMP, a benchmarking framework that evaluates AMP predictors against experimentally measured minimum inhibitory concentrations (MICs) across clinically relevant bacterial species and strains. We surveyed 48 published methods, finding fewer than 25% reproducible, and benchmarked 10 model families (21 variants) using experimental MIC data, synthetic sequence perturbations, activity cliff analyses, and all-atom molecular dynamics (MD) simulations. Four findings emerge: (i) models trained on MIC data outperform binary classifiers regardless of architecture; (ii) the best model depends on the target pathogen, so model selection must be guided by the biological question; (iii) most models cannot distinguish active peptides from inactive sequences with identical amino acid composition; and (iv) activity cliffs remain unresolved by both machine learning and MD, marking a limit of current computational methods. BATTLE-AMP is released as an open Snakemake framework at https://github.com/szczurek-lab/battleamp-snakemake for benchmarking new models and scoring novel candidate libraries.

bioinformatics↗

bioRxiv2026

Hippocampal Ring Finger Protein 10-dependent signaling supports cognitive flexibility

The ability to flexibly adapt behavior to changing environmental contingencies is a core component of brain function and relies on experience-dependent remodeling of neural circuits. While cognitive flexibility has been primarily attributed to prefrontal-striatal networks, the contribution of hippocampus and their underlying molecular substrates remains less understood. Here, we show that the dorsal hippocampus has a key role in cognitive flexibility. In particular, Ring Finger Protein 10 (RNF10)-mediated signaling, linking activation of synaptic NMDARs to specific transcriptional programs in the dorsal CA1, is necessary for cognitive flexibility. In fact, in vivo downregulation, through gene deletion and silencing of RNF10, resulting in impaired long-term synaptic plasticity, suppressed cognitive flexibility. This was reflected in the impaired ability to disengage from previously acquired contextual, visual, and spatial information and to adapt behavior to changed context. Overall, our results identified RNF10 as a key in vivo player necessary for the balance between cognitive stability and flexibility.

animal behavior and cognition↗

bioRxiv2025

Structural studies of mycobacterial HptG reveal a silent state and offer insights into TLR4 activation

HtpG of Mycobacterium tuberculosis (HtpGMtb) is an ATP-dependent chaperone that assists the correct folding of nascent and stress-accumulated misfolded proteins, in concert with other chaperones. Beside playing a role in stress response, it is able to elicit an immune response against M. tuberculosis infection by activating Dendritic Cells in a TLR4-mediated manner. However, we lack a full understanding of the molecular determinants of HtpGMtb catalytic activity and TLR4 activation, due to the lack of structural and biophysical data. Here, we report the first crystal structure of HtpGMtb, in complex with the non-hydrolysable form of ATP, AMPPNP. The crystal structure reveals that the HtpGMtb dimer adopts a conformationally silent structure, that precludes the dimerisation of the chaperone catalytic domains needed for ATP hydrolysis. Also, binding studies show that HtpGMtb directly interacts with TLR4 with a nanomolar affinity, and that this interaction allows HtpGMtb dimer to engage two TLR4 molecules. This finding suggests that activation of TLR4 by HtpGMtb is due to its ability to induce intra-cellular receptor dimerisation in an LPS-like mode.

biophysics↗

bioRxiv2025

No Risky Bets: The Brain Avoids All-In Predictions During Naturalistic Multitalker Listening

Listeners daily adapt to new talkers, yet how acoustic variability challenges the brains predictive mechanisms during speech processing remains unclear. Here, we used EEG and Temporal Response Function to examine neural responses to continuous speech narrated by a single talker (Single), enabling stable acoustic model formation, or multiple talkers (Multi), introducing acoustic uncertainty. We assessed whether talker variability influences phoneme recognition and predictive processing, indexed by neural responses to phonemes, phonemic and semantic surprisal. In the Multi condition, responses to phonemes increased but responses to phonemic surprisal decreased, indicating greater speech perception demands and weaker phonemic predictions. Conversely, semantic surprisal responses were stronger, suggesting increased reliance on lexical-semantic predictions. These findings reveal a trade-off in the brains predictive mechanisms, where acoustic uncertainty reduces lower-level phonemic anticipation but promotes higher-level semantic prediction. Adaptive processing underscored the brains ability to dynamically adjust predictions across linguistic levels, promoting speech comprehension in variable environments. SignificanceHumans attend to many different talkers daily, switching between them apparently without any effort. This stems from the brains ability to construct adaptive, probabilistic models of speech. In this study, we provide novel evidence that predictions in language comprehension are sensitive to acoustic uncertainty. Specifically, under conditions of increased acoustic uncertainty, listeners rely more on bottom-up information in phoneme recognition and reduce the anticipation of phonemic information. This is accompanied by enhanced semantic prediction, suggesting that listeners compensate for acoustic uncertainty by increasing reliance on higher-level contextual representations. This flexible approach allows for robust comprehension, highlighting the brains capacity to dynamically adjust its predictive processing to accommodate varying acoustic environments. TeaserTalker-driven acoustic uncertainty reduces phoneme predictions but boosts semantic inference.

neuroscience↗

bioRxiv2025

In the words of others: ERP evidence of speaker-specific phonological prediction

Prediction models usually assume that highly constraining contexts allow the pre-activation of phonological information. However, the evidence for phonological prediction is mixed and controversial. In this study, we implement a paradigm that capitalizes on the phonological errors produced by non-native speakers to investigate whether speaker-specific phonological predictions are made based on speaker identity (native-vs-foreign). EEG data was recorded from 42 healthy native Italian speakers. Participants were asked to read sentence fragments after which a final word was spoken by either a native- or a foreign-accented speaker. The spoken final word could be predictable or not, depending on the sentence context. The identity of the speaker (native-vs-foreign) may or may not be cued by an image of the face of the speaker. Our main analysis indicated that cueing the speaker identity was associated with a larger N400 predictability effect, possibly reflecting an easier processing of predictable words due to phonological preactivation. As visual inspection of the waveforms revealed a more complex pattern than initially anticipated, we used temporal EFA (Exploratory Factor Analysis) to identify and disentangle the ERP components underlying the effect observed. In the native-accent condition, predictable words elicited a posterior positivity relative to unpredictable words, possibly reflecting a P3b response, which was more pronounced when the speaker identity was cued. In the foreign-accent condition, cueing the speaker identity was associated with a smaller N1 and a larger P3a response. These results suggest that phonological prediction for native- and foreign-accented speakers likely involve different cognitive processes.

neuroscience↗

bioRxiv2024

Element partitioning, element localisation and transcriptome responses of date palms exposed to NaCl

Date palm (Phoenix dactylifera L.) is an economically important fruit crop in many (semi)arid regions. Large intra-species variation in susceptibility to NaCl has been reported. Controlled experiments were conducted to evaluate sodium (Na) and chlorine (Cl) partitioning in two-year old plants of six date palm varieties and to determine tissue location of Na and Cl and transcriptome responses to NaCl of two varieties. The largest Na concentrations were found in secondary roots, especially under NaCl treatment. By contrast, only small differences in within-plant partitioning of Cl were observed with less differences between varieties than for Na. Variety Sultana had the largest Na translocation factor. The variety Khalas exhibited better restriction of Na mobility, which may be a consequence of Na hotspots in roots, presumably immobilising Na in roots, which were absent in Sultana. In the root, shoot base and lower part of leaves, Cl accumulated in fibre bands and sclerenchyma sheaths of vascular bundles. RNAseq analysis revealed that Sultana and Khalas respond very differently to NaCl: Khalas modified expression of NaCl-specific genes in both roots and leaves, while Sultana responded manly in leaves. Gene ontology analysis showed that the expression changes for genes linked with photosynthesis and carbohydrate metabolism predominated as a response to NaCl. In Khalas, differentially expressed genes encoding for metal transporters and those reported to be involved in heat, salt and osmotic stress response were found. Large variability in Na and Cl accumulation in different date palm varieties may have practical agronomical consequences.

plant biology↗

bioRxiv2024

Using phage display for rational engineering of a higher affinity humanized 3-phosphohistidine-specific antibody

Histidine phosphorylation (pHis) is a non-canonical post-translational modification (PTM) that is historically understudied due to a lack of robust reagents that are required for its investigation, such as high affinity pHis-specific antibodies. Engineering pHis-specific antibodies is very challenging due to the labile nature of the phosphoramidate (P-N) bond and the stringent requirements for selective recognition of the two isoforms, 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis). Here, we present a strategy for in vitro engineering of antibodies for detection of native 3-pHis targets. Specifically, we humanized the rabbit SC44-8 anti-3-pTza (a stable 3-pHis mimetic) mAb into a scaffold (herein referred to as hSC44) that was suitable for phage display. We then constructed six unique Fab phage-displayed libraries using the hSC44 scaffold and selected high affinity 3-pHis binders. Our selection strategy was carefully designed to enrich antibodies that bound 3-pHis with high affinity and had specificity for 3-pHis versus 3-pTza. hSC44.20N32FL, the best engineered antibody, has an [~]10-fold higher affinity for 3-pHis than the parental hSC44. Eleven new Fab structures, including the first reported antibody-pHis peptide structures were solved by X-ray crystallography. Structural and quantum mechanical calculations provided molecular insights into 3-pHis and 3-pTza discrimination by different hSC44 variants and their affinity increase obtained through in vitro engineering. Furthermore, we demonstrate the utility of these newly developed high-affinity 3-pHis-specific antibodies for recognition of pHis proteins in mammalian cells by immunoblotting and immunofluorescence staining. Overall, our work describes a general method for engineering PTM-specific antibodies and provides a set of novel antibodies for further investigations of the role of 3-pHis in cell biology. Significance StatementHistidine phosphorylation is an elusive PTM whose role in mammalian cell biology is largely unknown due to the lack of robust tools and methods for its analysis. Here we report the development of antibodies with unprecedented affinity and specificity towards 3-pHis and present the first crystal structures of a pHis peptide in complex with an antibody. Finally, we show how these antibodies can be used in standard molecular biology workflows to investigate pHis-dependent biology.

synthetic biology↗

bioRxiv2024

Tks5 interactome reveals ER-associated machinery translation in invadosomes.

The ability to progress and invade through the extracellular matrix is a characteristic shared by both normal and cancer cells through the formation of structures called invadosomes gathering invadopodia and podosomes. These invadosomes are plastic and dynamic structures that can adopt different organizations depending on the cell types and the environment such as rosettes, dots or linear invadosomes. In this study, we used the specific invadosome marker Tks5 (SH3PXD2A), to identify common features in these different organizations. Tks5 immunoprecipitation coupled with mass spectrometry analysis allowed us to identify common proteins in these different models. We identified elements of the translation machinery, in particular the EIF4B protein, but also endoplasmic reticulum (ER) proteins as part of the invadosome structure. Providing new data on invadosome molecular composition through Tks5 interactome, we identified that ER-associated translation machinery is recruited to invadosome and involved in their formation, persistence and function in all types of invadosomes. SummaryInvadosomes are invasive F-actin structures exhibiting different organizations that degrade the extracellular matrix. The study uses their universal marker, Tks5, to provide new data about invadosome molecular composition and reveal the role of ER-associated translation machinery in invadosome formation and function.

cancer biology↗

bioRxiv2023

Fluorinated cGAMP analogs, which act as STING agonists and are not cleavable by poxins: structural basis of their function

The Stimulator of Interferon Genes (STING) plays a crucial role in the cGAS-STING pathway of innate immunity, detecting DNA in the cytoplasm and defending against certain cancers, viruses, and bacteria. We designed and synthesized fluorinated carbocyclic cGAMP analogs, MD1203 and MD1202D (MDs), to enhance their stability against nucleases and their affinity for STING. These compounds demonstrated exceptional activity against wild-type STING and all its allelic variations, including the hard-to-target REF isoform. Despite their distinct chemical modifications relative to the canonical CDNs, such as the substitution of guanine with hypoxanthine and the fluorination of the (pseudo)ribose ring, crystallographic analysis revealed a consistent binding mode with STING. Importantly, these compounds were resistant to cleavage by viral poxin nucleases. The crystallographic analysis of poxin/MD complexes unveiled their binding mode at the interface of poxin monomers, with dynamic adenine base orientations. Interestingly, MDs-bound poxin adopted an unliganded-like conformation, distinct from the conformation of cGAMP-bound poxin. Moreover, when MDs were in complex with poxin, they exhibited a different conformation than cGAMP when bound to poxin; in fact, it closely resembled the conformation observed when MDs were bound to STING. In conclusion, the development of MD1203 and MD1202D, showcases their potential as potent STING activators with remarkable stability against poxin-mediated degradation--a crucial characteristic for future development of antivirals.

molecular biology↗

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↗

bioRxiv2021

The quality of dissolved organic matter shapes the biogeography of the active bathypelagic microbiome

The bathypelagic ocean (1000-4000 m depth) is the largest aquatic biome on Earth but it is still largely unexplored. Due to its prevalent low dissolved organic carbon concentrations, most of the prokaryotic metabolic activity is assumed to be associated to particles. The role of free-living prokaryotes has thus been mostly ignored, except that of some chemolithoautotrophic lineages. Here we used a global bathypelagic survey of size-fractionated metagenomic and 16S (genes and transcripts) data and performed a differential abundance analysis to explore the functional traits of the different prokaryotic life-strategies, their contribution to the active microbiome, and the role that the quality of the dissolved organic matter (DOM) plays in driving this contribution. We found that free-living prokaryotes have limited capacity to uplift their metabolism in response to environmental changes and display comparatively lower growth rates than particle associated prokaryotes, but are responsible for the synthesis of vitamins in the bathypelagic. Furthermore, their contribution to the active prokaryotic microbiome increased towards waters depleted of labile DOM, which represented a large fraction of the tropical and subtropical ocean sampled stations. This points to a relevant yet overlooked role of free-living prokaryotes in DOM cycling in the vast bathypelagic desert.

ecology↗

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

Counteracting resistance to targeted therapy in melanoma by inhibiting discoidin domain receptors

Anti-BRAF plus anti-MEK are used as first-line treatment of patients with metastatic melanomas harboring BRAF V600E mutation. The main issue with targeted therapy is acquired cellular resistance. In 70% of acquired resistance, melanoma cells switch their phenotype and become more aggressive and invasive. The molecular signature of this phenotype is MITF low, AXL high associated with actin cytoskeleton reorganization. After this switch, resistant cells present an anarchic cell proliferation due to MAP kinase pathway hyper-activation. We demonstrate that resistant cell lines presenting phenotype switching overexpress DDR1 and DDR2. We show that DDR2 inhibition induces a decrease in AXL and reduces actin stress fiber formation. Once this phenotype switching is acquired, we report that both DDRs promotes tumor cell proliferation, but only DDR2 can over-activate the MAP kinase pathway in resistant invasive cells in vitro and in vivo. Therefore, DDRs inhibition could be a promising strategy for countering this resistance mechanism. SignificanceOur results show that DDR2 is a relevant target in melanoma resistance. DDR2 is required at the beginning of resistance for melanoma cell phenotype switching to occur. After phenotype switching, DDRs promote tumor cell proliferation of resistant invasive melanoma cells, but only DDR2 is able to over-activate the MAP kinase pathway. We put forward dasatinib (a DDR inhibitor) as a potential second-line treatment after targeted dual therapy for resistant patients overexpressing DDRs.

cancer biology↗

bioRxiv2019

LRRK2 G2019S kinase activity triggers neurotoxic NSF aggregation

Parkinsons disease (PD) is characterized by the progressive degeneration of dopaminergic neurons within the substantia nigra pars compacta and the presence of protein aggregates in surviving neurons. LRRK2 G2019S mutation is one the major determinant of familial PD cases and leads to late-onset PD with pleomorphic pathology, including alpha-synuclein accumulation and deposition of protein inclusions. LRRK2 binds and phosphorylates N-ethylmaleimide sensitive factor (NSF). We observed aggregates containing NSF in basal ganglia specimens from G2019S carrier PD patients and in cellular and animal models expressing the LRRK2 G2019S variant. We found that LRRK2 G2019S kinase activity induces the accumulation of NSF in toxic aggregates. Noteworthy, the induction of autophagy cleared NSF aggregation and rescued motor and cognitive impairment observed in aged hG2019S BAC mice. We suggest that LRRK2 G2019S pathological phosphorylation hampers substrate catabolism, thus causing the formation of cytotoxic protein inclusions. HighlightsO_LILRRK2 phosphorylates NSF in vivo C_LIO_LINSF aggregates in complementary LRRK2 G2019S models C_LIO_LILRRK2 G2019S kinase activity induces NSF accumulation in toxic aggregates C_LIO_LIAutophagy induction rescues hG2019S BAC mice motor and cognitive impairment C_LI

neuroscience↗