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Background The main mechanism underlying cancer dissemination is the epithelial to mesenchymal transition (EMT). This process is orchestrated by cytokines like TGFβ, involving “non-canonical” AKT- or STAT3-driven pathways. Recently, the alteration of copper homeostasis seems involved in the onset and progression of cancer. Methods We expose different breast cancer cell lines, including two triple negative (TNBC) ones, an HER2 enriched and one cell line representative of the Luminal A molecular subtype, to short- or long-term copper-chelation by triethylenetetramine (TRIEN). We analyse changes in the expression of EMT markers (E-cadherin, fibronectin, vimentin and αSMA), in the levels and activity of extracellular matrix components (LOXL2, fibronectin and MMP2/9) and of copper homeostasis markers by Western blot analyses, immunofluorescence, enzyme activity assays and RT-qPCR. Boyden Chamber and wound healing assays revealed the impact of copper chelation on cell migration. Additionally, we explored whether perturbation of copper homeostasis affects EMT prompted by TGFβ. Metabolomic and lipidomic analyses were applied to search the effects of copper chelation on the metabolism of breast cancer cells. Finally, bioinformatics analysis of data on breast cancer patients obtained from different databases was employed to correlate changes in kinases and copper markers with patients’ survival. Results Remarkably, only HER2 negative breast cancer cells differently responded to short- or long-term exposure to TRIEN, initially becoming more aggressive but, upon prolonged exposure, retrieving epithelial features, reducing their invasiveness. This phenomenon may be related to the different impact of the short and prolonged activation of the AKT kinase and to the repression of STAT3 signalling. Bioinformatics analyses confirmed the positive correlation of breast cancer patients’ survival with AKT activation and up-regulation of CCS. Eventually, metabolomics studies demonstrate a prevalence of glycolysis over mitochondrial energetic metabolism and of lipidome changes in TNBC cells upon TRIEN treatment. Conclusions We provide evidence of a pivotal role of copper in AKT-driven EMT activation, acting independently of HER2 in TNBC cells and via a profound change in their metabolism. Our results support the use of copper-chelators as an adjuvant therapeutic strategy for TNBC.

The central role of eukaryotic translation initiation factor 4E (eIF4E) in controlling mRNA translation has been clearly assessed in the last decades. eIF4E function is essential for numerous physiological processes, such as protein synthesis, cellular growth and differentiation; dysregulation of its activity has been linked to ageing, cancer onset and progression and neurodevelopmental disorders, such as autism spectrum disorder (ASD) and Fragile X Syndrome (FXS). The interaction between eIF4E and the eukaryotic initiation factor 4G (eIF4G) is crucial for the assembly of the translational machinery, the initial step of mRNA translation. A well-characterized group of proteins, named 4E-binding proteins (4E-BPs), inhibits the eIF4E–eIF4G interaction by competing for the same binding site on the eIF4E surface. 4E-BPs and eIF4G share a single canonical motif for the interaction with a conserved hydrophobic patch of eIF4E. However, a second non-canonical and not conserved binding motif was recently detected for eIF4G and several 4E-BPs. Here, we review the structural features of the interaction between eIF4E and its molecular partners eIF4G and 4E-BPs, focusing on the implications of the recent structural and biochemical evidence for the development of new therapeutic strategies. The design of novel eIF4E-targeting molecules that inhibit translation might provide new avenues for the treatment of several conditions.

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

Recent data indicate that receptor for activated C kinase 1 (RACK1) is a putative prognostic marker and drug target in breast cancer (BC). High RACK1 expression is negatively associated with overall survival, as it seems to promote BC progression. In tumors, RACK1 expression is controlled by a complex balance between glucocorticoids and androgens. Given the fact that androgens and androgenic derivatives can inhibit BC cell proliferation and migration, the role of androgen signaling in regulating RACK1 transcription in mammary tumors is of pivotal interest. Here, we provide evidence that nandrolone (19-nortosterone) inhibits BC cell proliferation and migration by antagonizing the PI3K/Akt/NF-κB signaling pathway, which eventually results in RACK1 downregulation. We also show that nandrolone impairs the PI3K/Akt/NF-κB signaling pathway and decreases RACK1 expression via binding to the membrane-bound receptor, oxoeicosanoid receptor 1 (OXER1). High levels of OXER1 are observed in several BC cell lines and correlate with RACK1 expression and poor prognosis. Our data provide evidence on the role played by the OXER1-dependent intracellular pathway in BC progression and shed light on the mechanisms underlying membrane-dependent androgen effects on RACK1 regulation. Besides the mechanistic relevance, the results of the study are of interest from a translational prospective. In fact, they identify a new and actionable pathway to be used for the design of innovative and rational therapeutic strategies in the context of the personalized treatment of BC. In addition, they draw attention on nandrolone-based compounds that lack hormonal activity as potential anti-tumor agents.

The eco-evolutionary history of penguins is characterised by shifting from temperate to cold environments. Breeding in Antarctica, the Emperor penguin appears as an extreme outcome of this process, with unique features related to insulation, heat production and energy management. However, whether this species actually diverged from a less cold-adapted ancestor, more ecologically similar to its sister species, the King penguin, is still an open question. As the Antarctic colonisation likely resulted in vast changes in selective pressure experienced by the Emperor penguin, the relative quantification of the genomic signatures of selection, unique to each sister species, could answer this question. Applying phylogeny-based selection tests on 7651 orthologous genes, we identified a more pervasive selection shift in the Emperor penguin than in the King penguin, supporting the hypothesis that its extreme cold adaptation is a derived state. Furthermore, among candidate genes under selection, four (TRPM8, LEPR, CRB1, and SFI1) were identified before in other cold-adapted homeotherms, like the woolly Mammoth, while other 161 genes can be assigned to biological functions relevant to cold adaptation identified in previous studies. Location and structural effects of TRPM8 substitutions in Emperor and King penguin lineages support their functional role with putative divergent effects on thermal adaptation. We conclude that extreme cold adaptation in the Emperor penguin largely involved unique genetic options which, however, affect metabolic and physiological traits common to other cold-adapted homeotherms.

Heliconiusbutterflies, a speciose genus of Müllerian mimics, represent a classic example of an adaptive radiation that includes a range of derived dietary, life history, physiological and neural traits. However, key lineages within the genus, and across the broader Heliconiini tribe, lack genomic resources, limiting our understanding of how adaptive and neutral processes shaped genome evolution during their radiation. We have generated highly contiguous genome assemblies for nine new Heliconiini, 29 additional reference-assembled genomes, and improve 10 existing assemblies. Altogether, we provide a major new dataset of annotated genomes for a total of 63 species, including 58 species within the Heliconiini tribe. We use this extensive dataset to generate a robust and dated heliconiine phylogeny, describe major patterns of introgression, explore the evolution of genome architecture, and the genomic basis of key innovations in this enigmatic group, including an assessment of the evolution of putative regulatory regions at theHeliconiusstem. Our work illustrates how the increased resolution provided by such dense genomic sampling improves our power to generate and test gene-phenotype hypotheses, and precisely characterize how genomes evolve.

Understanding how organisms adapt to extreme environments is fundamental and can provide insightful case studies for both evolutionary biology and climate-change biology. Here, we take advantage of the vast diversity of lifestyles in ants to identify genomic signatures of adaptation to extreme habitats such as high altitude. We hypothesized two parallel patterns would occur in a genome adapting to an extreme habitat: 1) strong positive selection on genes related to adaptation and 2) a relaxation of previous purifying selection. We tested this hypothesis by sequencing the high-elevation specialist Tetramorium alpestre and four other phylogenetically related species. In support of our hypothesis, we recorded a strong shift of selective forces in T. alpestre, in particular a stronger magnitude of diversifying and relaxed selection when compared with all other ants. We further disentangled candidate molecular adaptations in both gene expression and protein-coding sequence that were identified by our genome-wide analyses. In particular, we demonstrate that T. alpestre has 1) a higher level of expression for stv and other heat-shock proteins in chill-shock tests and 2) enzymatic enhancement of Hex-T1, a rate-limiting regulatory enzyme that controls the entry of glucose into the glycolytic pathway. Together, our analyses highlight the adaptive molecular changes that support colonization of high-altitude environments.

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD(+), its phosphorylated variant NAD(P)(+), and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD(+) in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD(+) pool is maintained and how it is replenished upon NAD(+) consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD(+) to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H(2)O(2) or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD(+) salvage pathway.

Fragile X Syndrome (FXS) is the most frequent cause of inherited intellectual disabilities and autism spectrum disorders, characterized by cognitive deficits and autistic behaviors. The silencing of the Fmr1 gene and consequent lack of FMRP protein, is the major contribution to FXS pathophysiology. FMRP is an RNA binding protein involved in the maturation and plasticity of synapses and its absence culminates in a range of morphological, synaptic and behavioral phenotypes. Currently, there are no approved medications for the treatment of FXS, with the approaches under study being fairly specific and unsatisfying in human trials. Here we propose peptides/peptidomimetics as candidates in the pharmacotherapy of FXS; in the last years this class of molecules has catalyzed the attention of pharmaceutical research, being highly selective and well-tolerated. Thanks to their ability to target protein-protein interactions (PPIs), they are already being tested for a wide range of diseases, including cancer, diabetes, inflammation, Alzheimer's disease, but this approach has never been applied to FXS. As FXS is at the forefront of efforts to develop new drugs and approaches, we discuss opportunities, challenges and potential issues of peptides/peptidomimetics in FXS drug design and development.

Fast, reliable and point-of-care systems to detect the SARS-CoV-2 infection are crucial to contain viral spreading and to adopt timely clinical treatments. Many of the rapid detection tests currently in use are based on antibodies that bind viral proteins1. However, newly appearing virus variants accumulate mutations in their RNA sequence and produce proteins, such as Spike, that may show reduced binding affinity to these diagnostic antibodies, resulting in less reliable tests and in the need for continuous update of the sensing systems2. Here we propose a graphene field-effect transistor (gFET) biosensor which exploits the key interaction between the Spike protein and the human ACE2 receptor. This interaction is one of the determinants of host infections and indeed recently evolved Spike variants were shown to increase affinity for ACE2 receptor3. Through extensive computational analyses we show that a chimeric ACE2-Fc construct mimics the ACE2 dimer, normally present on host cells membranes, better than its soluble truncated form. We demonstrate that ACE2-Fc functionalized gFET is effective for in vitro detection of Spike and outperforms the same chip functionalized with either a diagnostic antibody or the soluble ACE2. Our sensor is implemented in a portable, wireless, point-of-care device and successfully detected both alpha and gamma virus variants in patient’s clinical samples. As incomplete immunization, due to vaccine roll-out, may offer new selective grounds for antibody-escaping virus variants4, our biosensor opens to a class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

The development of new therapeutic avenues that target the early stages of Alzheimer's disease (AD) is urgently necessary. A disintegrin and metalloproteinase domain 10 (ADAM10) is a sheddase that is involved in dendritic spine shaping and limits the generation of amyloid-β. ADAM10 endocytosis increases in the hippocampus of AD patients, resulting in the decreased postsynaptic localization of the enzyme. To restore this altered pathway, we developed a cell-permeable peptide (PEP3) with a strong safety profile that is able to interfere with ADAM10 endocytosis, upregulating the postsynaptic localization and activity of ADAM10. After extensive validation, experiments in a relevant animal model clarified the optimal timing of the treatment window. PEP3 administration was effective for the rescue of cognitive defects in APP/PS1 mice only if administered at an early disease stage. Increased ADAM10 activity promoted synaptic plasticity, as revealed by changes in the molecular compositions of synapses and the spine morphology. Even though further studies are required to evaluate efficacy and safety issues of long-term administration of PEP3, these results provide preclinical evidence to support the therapeutic potential of PEP3 in AD.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene. It is a leading monogenic cause of autism spectrum disorder and inherited intellectual disability and is often comorbid with attention deficits. Most FXS cases are due to an expansion of CGG repeats leading to suppressed expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA metabolism. We found that the previously published Fmr1 knockout rat model of FXS expresses an Fmr1 transcript with an in-frame deletion of exon 8, which encodes for the K-homology (KH) RNA-binding domain, KH1. Notably, 3 pathogenic missense mutations associated with FXS lie in the KH domains. We observed that the deletion of exon 8 in rats leads to attention deficits and to alterations in transcriptional profiles within the medial prefrontal cortex (mPFC), which map to 2 weighted gene coexpression network modules. These modules are conserved in human frontal cortex and enriched for known FMRP targets. Hub genes in these modules represent potential therapeutic targets for FXS. Taken together, these findings indicate that attentional testing might be a reliable cross-species tool for investigating FXS and identify dysregulated conserved gene networks in a relevant brain region.

MeCP2 is a fundamental protein associated with several neurological disorders, including Rett syndrome. It is considered a multifunctional factor with a prominent role in regulating chromatin structure; however, a full comprehension of the consequences of its deficiency is still lacking. Here, we characterize a novel mouse model of Mecp2 bearing the human mutation Y120D, which is localized in the methyl-binding domain. As most models of Mecp2, the Mecp2Y120D mouse develops a severe Rett-like phenotype. This mutation alters the interaction of the protein with chromatin, but surprisingly, it also impairs its association with corepressors independently on the involved interacting domains. These features, which become overt mainly in the mature brain, cause a more accessible and transcriptionally active chromatin structure; conversely, in the Mecp2-null brain, we find a less accessible and transcriptionally inactive chromatin. By demonstrating that different MECP2 mutations can produce concordant neurological phenotypes but discordant molecular features, we highlight the importance of considering personalized approaches for the treatment of Rett syndrome.

SARS-CoV-2 epidemics quickly propagated worldwide, sorting virus genomic variants in newly established propagules of infections. Stochasticity in transmission within and between countries or an actual selective advantage could explain the global high frequency reached by some genomic variants. Using statistical analyses, demographic reconstructions, and molecular dynamics simulations, we show that the globally invasive G614 spike variant 1) underwent a significant demographic expansion in most countries explained neither by stochastic effects nor by overrepresentation in clinical samples, 2) increases the spike S1/S2 furin-like site conformational plasticity (short-range effect), and 3) modifies the internal motion of the receptor-binding domain affecting its cross-connection with other functional domains (long-range effect). Our results support the hypothesis of a selective advantage at the basis of the spread of the G614 variant, which we suggest may be due to structural modification of the spike protein at the S1/S2 proteolytic site, and provide structural information to guide the design of variant-specific drugs.

Aspergillus fumigatus is the most common opportunistic fungal pathogen and causes invasive pulmonary aspergillosis (IPA), with high mortality among immunosuppressed patients. The fungistatic activity of all-trans retinoic acid (ATRA) has been recently described in vitro., Aspergillus fumigatus is the most common opportunistic fungal pathogen and causes invasive pulmonary aspergillosis (IPA), with high mortality among immunosuppressed patients. The fungistatic activity of all-trans retinoic acid (ATRA) has been recently described in vitro. We evaluated the efficacy of ATRA in vivo and its potential synergistic interaction with other antifungal drugs. A rat model of IPA and in vitro experiments were performed to assess the efficacy of ATRA against Aspergillus in association with classical antifungal drugs and in silico studies used to clarify its mechanism of action. ATRA (0.5 and 1 mM) displayed a strong fungistatic activity in Aspergillus cultures, while at lower concentrations, synergistically potentiated fungistatic efficacy of subinhibitory concentration of amphotericin B (AmB) and posaconazole (POS). ATRA also enhanced macrophagic phagocytosis of conidia. In a rat model of IPA, ATRA reduced mortality similarly to posaconazole. Fungistatic efficacy of ATRA alone and synergistically with other antifungal drugs was documented in vitro, likely by inhibiting fungal heat shock protein 90 (Hsp90) expression and Hsp90-related genes. ATRA treatment reduced mortality in a model of IPA in vivo. Those findings suggest ATRA as a suitable fungistatic agent that can also reduce dosage and adverse reactions of classical antifungal drugs and add to the development of new therapeutic strategies against IPA and systemic fungal infections.

Ovarian carcinoma is one of the most common causes for cancer death in women; lack of early diagnosis and acquired resistance to platinum-based chemotherapy account for its poor prognosis and high mortality rate. As with other cancer types, ovarian cancer is characterized by dysregulated signaling pathways and protein synthesis, which together contribute to rapid cellular growth and invasiveness. The mechanistic/mammalian target of rapamycin (mTOR) pathway represents the core of different signaling pathways regulating a number of essential steps in the cell, among which protein synthesis and the eukaryotic initiation factor 4E (eIF4E), the mRNA cap binding protein, is one of its downstream effectors. eIF4E is a limiting factor in translation initiation and its overexpression is a hallmark in many cancers. Because its action is regulated by a number of factors that compete for the same binding site, eIF4E is an ideal target for developing novel antineoplastic drugs. Several inhibitors targeting the mTOR signaling pathway have been designed thus far, however most of these molecules show poor stability and high toxicity

Helsmoortel-Van der Aa syndrome (HVDAS) is a neurodevelopmental condition associated with intellectual disability/developmental delay, autism spectrum disorder, and multiple medical comorbidities. HVDAS is caused by mutations in activity-dependent neuroprotective protein (ADNP). A recent study identified genome-wide DNA methylation changes in 22 individuals with HVDAS, adding to the group of neurodevelopmental disorders with an epigenetic signature. This methylation signature segregated those with HVDAS into two groups based on the location of the mutations. Here, we conducted an independent study on 24 individuals with HVDAS and replicated the existence of the two mutation-dependent episignatures. To probe whether the two distinct episignatures correlate with clinical outcomes, we used deep behavioral and neurobiological data from two prospective cohorts of individuals with a genetic diagnosis of HVDAS. We found limited phenotypic differences between the two HVDAS-affected groups and no evidence that individuals with more widespread methylation changes are more severely affected. Moreover, in spite of the methylation changes, we observed no profound alterations in the blood transcriptome of individuals with HVDAS. Our data warrant caution in harnessing methylation signatures in HVDAS as a tool for clinical stratification, at least with regard to behavioral phenotypes.

The transcription factor Nrf2 coordinates a multifaceted response to various forms of stress and to inflammatory processes, maintaining a homeostatic intracellular environment. Nrf2 anti-inflammatory activity has been related to the crosstalk with the transcription factor NF-κB, a pivotal mediator of inflammatory responses and of multiple aspects of innate and adaptative immune functions. However, the underlying molecular basis has not been completely clarified. By combining into new chemical entities, the hydroxycinnamoyl motif from curcumin and the allyl mercaptan moiety of garlic organosulfur compounds, we tested a set of molecules, carrying (pro)electrophilic features responsible for the activation of the Nrf2 pathway, as valuable pharmacologic tools to dissect the mechanistic connection between Nrf2 and NF-κB. We investigated whether the activation of the Nrf2 pathway by (pro)electrophilic compounds may interfere with the secretion of pro-inflammatory cytokines, during immune stimulation, in a human immortalized monocyte-like cell line (THP-1). The capability of compounds to affect the NF-κB pathway was also evaluated. We assessed the compounds-mediated regulation of cytokine and chemokine release by using Luminex X-MAP® technology in human primary peripheral blood mononuclear cells (PBMCs) upon LPS stimulation. We found that all compounds, also in the absence of electrophilic moieties, significantly suppressed the LPS-evoked secretion of pro-inflammatory cytokines such as TNFα and IL-1β, but not of IL-8, in THP-1 cells. A reduction in the release of pro-inflammatory mediators similar to that induced by the compounds was also observed after siRNA mediated-Nrf2 knockdown, thus indicating that the attenuation of cytokine secretion cannot be directly ascribed to the activation of Nrf2 signaling pathway. Moreover, all compounds, with the exception of compound 1, attenuated the LPS-induced activation of the NF-κB pathway, by reducing the upstream phosphorylation of IκB, the NF-κB nuclear translocation, as well as the activation of NF-κB promoter. In human PBMCs, compound 4 and CURC attenuated TNFα release as observed in THP-1 cells, and all compounds acting as Nrf2 inducers significantly decreased the levels of MCP-1/CCL2, as well as the release of the pro-inflammatory cytokine IL-12. Altogether, the compounds induced a differential modulation of innate immune cytokine release, by differently regulating Nrf2 and NF-κB intracellular signaling pathways.

Gp36 is the virus envelope glycoproteins catalyzing the fusion of the feline immunodeficiency virus with the host cells. The peptide C8 is a tryptophan-rich peptide corresponding to the fragment 770W-I777 of gp36 exerting antiviral activity by binding the membrane cell and inhibiting the virus entry. Several factors, including the membrane surface charge, regulate the binding of C8 to the lipid membrane. Based on the evidence that imperceptible variation of membrane charge may induce a dramatic effect in several critical biological events, in the present work we investigate the effect induced by systematic variation of charge in phospholipid bilayers on the aptitude of C8 to interact with lipid membranes, the tendency of C8 to assume specific conformational states and the re-organization of the lipid bilayer upon the interaction with C8. Accordingly, employing a bottom-up multiscale protocol, including CD, NMR, ESR spectroscopy, atomistic molecular dynamics simulations, and confocal microscopy, we studied C8 in six membrane models composed of different ratios of zwitterionic/negatively charged phospholipids. Our data show that charge content modulates C8-membrane binding with significant effects on the peptide conformations. C8 in micelle solution or in SUV formed by DPC or DOPC zwitterionic phospholipids assumes regular β-turn structures that are progressively destabilized as the concentration of negatively charged SDS or DOPG phospholipids exceed 40%. Interaction of C8 with zwitterionic membrane surface is mediated by Trp1 and Trp4 that are deepened in the membrane, forming H-bonds and cation-π interactions with the DOPC polar heads. Additional stabilizing salt bridge interactions involve Glu2 and Asp3. MD and ESR data show that the C8-membrane affinity increases as the concentration of zwitterionic phospholipid increases. In the lipid membrane characterized by an excess of zwitterionic phospholipids, C8 is adsorbed at the membrane interface, inducing a stiffening of the outer region of the DOPC bilayer. However, the bound of C8 significantly perturbs the whole organization of lipid bilayer resulting in membrane remodeling. These events, measurable as a variation of the bilayer thickness, are the onset mechanism of the membrane fusion and vesicle tubulation observed in confocal microscopy by imaging zwitterionic MLVs in the presence of C8 peptide.

Starting in Wuhan, China, SARS-CoV-2 epidemics quickly propagated worldwide in less than three months, geographically sorting genomic variants in newly established propagules of infections. Stochasticity in transmission within and between countries and/or actual advantage in virus transmissibility could explain the high frequency reached by some genomic variants during the course of the outbreak.Using a suite of statistical, population genetics, and theoretical approaches, we show that the globally most represented spike protein variant (i.e., the G clade, A → G nucleotide change at genomic position 23,403; D → G amino acid change at spike protein position 614)i)underwent a significant demographic expansion in most countries not explained by stochastic effects or enhanced pathogenicity;ii)affects the spike S1/S2 furin-like site increasing its conformational plasticity (short range effect), andiii)modifies the internal motion of the receptor-binding domain affecting its cross-connection with other functional domains (long-range effect).Our study unambiguously links the spread of the G614 with a non-random process, and we hypothesize that this process is related to the selective advantage produced by a specific structural modification of the spike protein. We conclude that the different conformation of the S1/S2 proteolytic site is at the basis of the higher transmission rate of this invasive SARS-CoV-2 variant, and provide structural information to guide the design of selective and efficient drugs.

Peptides and peptidomimetics are strongly re-emerging as amenable candidates in the development of therapeutic strategies against a plethora of pathologies. In particular, these molecules are extremely suitable to treat diseases in which a major role is played by protein–protein interactions (PPIs). Unlike small organic compounds, peptides display both a high degree of specificity avoiding secondary off-targets effects and a relatively low degree of toxicity. Further advantages are provided by the possibility to easily conjugate peptides to functionalized nanoparticles, so improving their delivery and cellular uptake. In many cases, such molecules need to assume a specific three-dimensional conformation that resembles the bioactive one of the endogenous ligand. To this end, chemical modifications are introduced in the polypeptide chain to constrain it in a well-defined conformation, and to improve the drug-like properties. In this context, a successful strategy for peptide/peptidomimetics design and optimization is to combine different computational approaches ranging from structural bioinformatics to atomistic simulations. Here, we review the computational tools for peptide design, highlighting their main features and differences, and discuss selected protocols, among the large number of methods available, used to assess and improve the stability of the functional folding of the peptides. Finally, we introduce the simulation techniques employed to predict the binding affinity of the designed peptides for their targets.

Background: The molecular cause of severe congenital neutropenia (SCN) is unknown in 30% to 50% of patients. SEC61A1 encodes the α-subunit of the Sec61 complex, which governs endoplasmic reticulum protein transport and passive calcium leakage. Recently, mutations in SEC61A1 were reported to be pathogenic in common variable immunodeficiency and glomerulocystic kidney disease. Objective: Our aim was to expand the spectrum of SEC61A1-mediated disease to include autosomal dominant SCN. Methods: Whole exome sequencing findings were validated, and reported mutations were compared by Western blotting, Ca2+ flux assays, differentiation of transduced HL-60 cells, in vitro differentiation of primary CD34 cells, quantitative PCR for unfolded protein response (UPR) genes, and single-cell RNA sequencing on whole bone marrow. Results: We identified a novel de novo missense mutation in SEC61A1 (c.A275G;p.Q92R) in a patient with SCN who was born to nonconsanguineous Belgian parents. The mutation results in diminished protein expression, disturbed protein translocation, and an increase in calcium leakage from the endoplasmic reticulum. In vitro differentiation of CD34+ cells recapitulated the patient's clinical arrest in granulopoiesis. The impact of Q92R-Sec61α1 on neutrophil maturation was validated by using HL-60 cells, in which transduction reduced differentiation into CD11b+CD16+ cells. A potential mechanism for this defect is the uncontrolled initiation of the unfolded protein stress response, with single-cell analysis of primary bone marrow revealing perturbed UPR in myeloid precursors and in vitro differentiation of primary CD34+ cells revealing upregulation of CCAAT/enhancer-binding protein homologous protein and immunoglobulin heavy chain binding protein UPR-response genes. Conclusion: Specific mutations in SEC61A1 cause SCN through dysregulation of the UPR., SCOPUS: ar.j, info:eu-repo/semantics/published

Introduction Dermatophytosis is a superficial fungal infection limited to the stratum corneum of the epidermis, or to the hair and nails, and constitutes an important public health problem because of its high prevalence and associated morbidity. Dermatophyte fungi, especially 2 species, Trichophyton rubrum and Trichophyton mentagrophytes, are the predominant pathogens. Topical antifungal drugs, mainly azoles or allyamines, are currently used for the treatment of dermatophytoses, although in some cases, such as in nail and hair involvement, systemic treatment is required. However, therapeutic efficacy of current antifungal agents can be limited by their side effects, costs, and the emergence of drug resistance among fungi. Plant extracts represent a potential source of active antimicrobial agents, due to the presence of a variety of chemical bioactive compounds. In the present work, we evaluated in silico and in vitro the antifungal activity of an extract of the medicinal plant Cardiospermum halicacabum against T. rubrum suggesting a potential interaction with Hsp90 as playing an important role in both pathogenicity and drug susceptibility of T. rubrum. Methods We investigated in vitro the effect of different concentrations of C. halicacabum (from 500 to 31.25 µg) against a clinical isolate of T. rubrum. Furthermore, using a computational assessment, the interaction between different C. halicacabum active compounds and the fungal Hsp90 was also investigated. Results Our results indicate a clear-cut antifungal activity of the total plant extract at the highest concentrations (500 and 250 µg). Among all tested C. halicacabum compounds, the luteolin and rutin molecules have been identified in silico as the most important potential inhibitors of Hsp90. Based on these data, luteolin and rutin were also individually assessed for their antifungal activity. Results demonstrate that both substances display an antifungal effect, even if lower than that of the total plant extract. Conclusion Our data indicate a strong fungistatic effect of C. halicacabum against T. rubrum, suggesting its potential therapeutic efficacy in the treatment of dermatophytoses. Additionally, C. halicacabum compounds, and particularly luteolin and rutin, are all possible Hsp90 interactors, explaining their fungistatic activity.

Cofilin is one of the major regulators of actin dynamics in spines where it is required for structural synaptic plasticity. However, our knowledge of the mechanisms controlling Cofilin activity in spines remains still fragmented. Here, we describe the cyclase-associated protein 2 (CAP2) as a novel master regulator of Cofilin localization in spines. The formation of CAP2 dimers through its Cys32 is important for CAP2 binding to Cofilin and for normal spine actin turnover. The Cys32-dependent CAP2 homodimerization and association to Cofilin are triggered by long-term potentiation (LTP) and are required for LTP-induced Cofilin translocation into spines, spine remodeling and the potentiation of synaptic transmission. This mechanism is specifically affected in the hippocampus, but not in the superior frontal gyrus, of both Alzheimer’s Disease (AD) patients and APP/PS1 mice, where CAP2 is down-regulated and CAP2 dimer synaptic levels are reduced. In AD hippocampi, Cofilin preferentially associates with CAP2 monomer and is aberrantly localized in spines. Taken together, these results provide novel insights into structural plasticity mechanisms that are defective in AD.

Mutations in the X-linked MECP2 gene represent the main origin of Rett syndrome, causing a profound intellectual disability in females. MeCP2 is an epigenetic transcriptional regulator containing two main functional domains: a methyl-CpG binding domain (MBD) and a transcription repression domain (TRD). Over 600 pathogenic mutations were reported to affect the whole protein; almost half of missense mutations affect the MBD. Understanding the impact of these mutations on the MBD structure and interaction with DNA will foster the comprehension of their pathogenicity and possibly genotype/phenotype correlation studies. Herein, we use molecular dynamics simulations to obtain a detailed view of the dynamics of WT and mutated MBD in the presence and absence of DNA. The pathogenic mutation Y120D is used as paradigm for our studies. Further, since the Y120 residue was previously found to be a phosphorylation site, we characterize the dynamic profile of the MBD also in the presence of Y120 phosphorylation (pY120). We found that addition of a phosphate group to Y120 or mutation in aspartic acid affect domain mobility that samples an alternative conformational space with respect to the WT, leading to impaired ability to interact with DNA. Experimental assays showing a significant reduction in the binding affinity between the mutated MBD and the DNA confirmed our predictions.

Understanding how organisms adapt to extreme environments is fundamental and can provide insightful case studies for both evolutionary biology and climate-change biology. Here, we take advantage of the vast diversity of lifestyles in ants to identify genomic signatures of adaptation to extreme habitats such as high altitude. We hypothesised two parallel patterns would occur in a genome adapting to an extreme habitat: i) strong positive selection on genes related to adaptation and, ii) a relaxation of previous purifying selection. We tested this hypothesis by sequencing the high-elevation specialist Tetramorium alpestre and four other phylogenetic related species. In support of our hypothesis, we recorded a strong shift of selective forces in T. alpestre, in particular a stronger magnitude of diversifying and relaxed selection when compared to all other ants. We further disentangled candidate molecular adaptations in both gene expression and protein-coding sequence that were identified by our genome-wide analyses. In particular, we demonstrate that T. alpestre has i) a derived level of expression for stv and other heat-shock proteins in chill shock tests, and ii) enzymatic enhancement of Hex-T1, a rate-limiting regulatory enzyme that controls the entry of glucose into the glycolytic pathway. Together, our analyses highlight the adaptive molecular changes that support colonisation of high-altitude environments.

Motivation The ligand/protein binding interaction is typically investigated by docking and molecular dynamics (MD) simulations. In particular, docking-based virtual screening (VS) is used to select the best ligands from database of thousands of compounds, while MD calculations assess the energy stability of the ligand/protein binding complexes. Considering the broad use of these techniques, it is of great demand to have one single software that allows a combined and fast analysis of VS and MD results. With this in mind, we have developed the Drug Discovery Tool (DDT) that is an intuitive graphics user interface able to provide structural data and physico-chemical information on the ligand/protein interaction. Results DDT is designed as a plugin for the Visual Molecular Dynamics (VMD) software and is able to manage a large number of ligand/protein complexes obtained from AutoDock4 (AD4) docking calculations and MD simulations. DDT delivers four main outcomes: i) ligands ranking based on an energy score; ii) ligand ranking based on a ligands’ conformation cluster analysis; iii) identification of the aminoacids forming the most occurrent interactions with the ligands; iv) plot of the ligands’ center-of-mass coordinates in the Cartesian space. The flexibility of the software allows saving the best ligand/protein complexes using a number of user-defined options. Availability and implementation DDT_site_1 (alternative DDT_site_2); the DDT tutorial movie is available here. Supplementary information Supplementary data are available at Bioinformatics online.

Single molecule protein sequencing would represent a disruptive burst in proteomic research with important biomedical impacts. Due to their success in DNA sequencing, nanopore based devices have been recently proposed as possible tools for the sequencing of peptide chains. One of the open questions in nanopore protein sequencing concerns the ability of such devices to provide different signals for all the 20 standard amino acids. Here, using equilibrium all-atom molecular dynamics simulations, we estimated the pore clogging in α-Hemolysin nanopore associated to 20 different homopeptides, one for each standard amino acid. Our results show that pore clogging is affected by amino acid volume, hydrophobicity and net charge. The equilibrium estimations are also supported by non-equilibrium runs for calculating the current blockades for selected homopeptides. Finally, we discuss the possibility to modify the α-Hemolysin nanopore, cutting a portion of the barrel region close to the trans side, to reduce spurious signals and, hence, to enhance the sensitivity of the nanopore.

The G-protein bile acid receptor 1 (GPBAR1) has emerged in the last decade as prominent target for the treatment of metabolic and inflammatory diseases including type 2 diabetes, obesity, and non-alcoholic steatohepatitis. To date numerous bile acid derivatives have been identified as GPBAR1 agonists, however their clinical application is hampered by the lack of selectivity toward the other bile acid receptors. Therefore, non-steroidal GPBAR1 ligands able to selectively activate the receptor are urgently needed. With this aim, we here designed, synthesized and biologically evaluated ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl) urea derivatives as novel potent GPBAR1 agonists. Particularly, compounds 9 and 10 induce the mRNA expression of the GPBAR1 target gene pro-glucagon and show high selectivity over the other bile acid receptors FXR, LXRα, LXRβ and PXR, and the related receptors PPARα and PPARγ. Computational studies elucidated the binding mode of 10 to GPBAR1, providing important structural insights for the design of non-steroidal GPBAR1 agonists. The pharmacokinetic properties of 9 and 10 suggest that the ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureydil scaffold might be exploited to achieve effective drug candidates to treat GPBAR1 related disorders.

Purpose Fungal infections are a major complication in hematologic and neoplastic patients causing severe morbidity and mortality. Aspergillus fumigatus and Candida albicans are among the most invasive opportunistic pathogens in immunocompromised patients, and classic antifungal drugs are frequently unsuccessful in these patients. Recent reports hypothesize that the antifungal efficacy of all-trans retinoic acid (ATRA) is mainly related to its strong capacity to stimulate monocyte-mediated immunity, but no consideration was given to its potential direct fungistatic activity. Moreover, ATRA offers the opportunity for systemic therapy. Methods and results We investigated the efficacy of ATRA at different concentrations for its antifungal activity against opportunistic A. fumigatus and C. albicans obtained from clinical samples according to standard protocols. A fungistatic activity of ATRA on A. fumigatus and C. albicans at 0.5–1 mM concentration was documented up to 7 days. Conclusion This is the first evidence of a direct and strong fungistatic activity of ATRA against A. fumigatus and C. albicans. The potential adjuvant therapeutic application of ATRA might be useful in the treatment and/or prevention of systemic mycoses in immunocompromised patients. The discovery of a direct fungistatic activity, in association with its reported immunomodulatory properties, makes ATRA an excellent candidate for new combined antifungal strategies for systemic mycoses in immunocompromised and cancer patients., Video abstract

Type 1 (T1D) and type 2 (T2D) diabetes share pathophysiological characteristics, yet mechanistic links have remained elusive. T1D results from autoimmune destruction of pancreatic beta cells, whereas beta cell failure in T2D is delayed and progressive. Here we find a new genetic component of diabetes susceptibility in T1D non-obese diabetic (NOD) mice, identifying immune-independent beta cell fragility. Genetic variation in Xrcc4 and Glis3 alters the response of NOD beta cells to unfolded protein stress, enhancing the apoptotic and senescent fates. The same transcriptional relationships were observed in human islets, demonstrating the role of beta cell fragility in genetic predisposition to diabetes.