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Interactions between amyloidogenic proteins are emerging as critical drivers of neurodegenerative diseases. Among others, in Alzheimer's disease (AD) and severe forms of chronic traumatic encephalopathy (CTE), codeposition of tau and amyloid-β (Aβ) leads to worsening of clinical outcomes and disease progression. Despite the importance of such heterotypic interactions, the underlying molecular mechanisms have proven challenging to be established. Here, we investigated the direct interaction between Aβ and tau, combining in vitro reconstruction, and in vivo models. We find that characteristic AD paired helical filament (PHF) and CTE folds catalyze the primary nucleation of Aβ42 in a fold-specific manner with enzyme-like kinetics. In particular, CTE fibrils exhibit the highest catalytic activity and constrain Aβ42 polymorphism, suggesting templating effects. Moreover, PHF and CTE tau fibrils increase Aβ42 toxicity in SH-SY5Y neuroblastoma cells and transgenic Caenorhabditis elegans, preserving fold-dependent reactivities. Our findings shed light on the molecular mechanisms of heterotypic interaction between amyloidogenic proteins in disease-relevant conditions, highlighting the role of amyloid structure and recognition mechanisms as key determinants. These results offer insights into the pathological mechanisms of multiple proteinopathies. The mechanisms described here might be used as a blueprint for structure-based design of new therapeutic agents targeting specific amyloidogenic interactions.

The conjugation of biological drugs with polyethylenglycole is widely used to reduce their immunogenicity, thereby enhancing the safety and efficacy of the drugs. For instance, some extended half-life drugs for hemophilia A patients are PEGylated FVIII products. Unfortunately, it was observed that PEG itself, whether derived from PEGylated drugs or present in everyday products (such as cosmetics or medical devices) may induce the production of anti-PEG antibodies, resulting in hypersensitivity reactions and/or loss of efficacy of PEGylated drugs. There is therefore a demand for cost-effective and rapid point-of-care (POC) measurements. To this end, a POC device based on the principle of surface plasmon resonance (SPR) is here presented. The sensor chip is functionalized directly with PEG chains via thiol chemistry and inserted into a holder manufactured in 3D printing technology. The functionalization process has been optimized to recognize anti-PEG antibodies (both IgG and IgM isotypes) and characterized using X-ray Photoelectron Spectroscopy and contact angle measurements. Dose-response curves were obtained using commercially available antibodies, both in buffer solution and in diluted human serum, achieving a limit of detection in the nanomolar range. The reusability of the optical chip was also evaluated and, as proof of concept, plasma from control subjects and hemophilia A patients treated with PEGylated FVIII were measured, comparing the results with standard techniques such as a commercial SPR instrument and an enzyme-linked immunosorbent assay. The results were obtained within 15 min from tenfold diluted human plasma, paving the way for an easy-to-use, small-sized, portable, and low-cost POC device capable of detecting anti-PEG antibodies.

Cancer cells adapt to harsh environmental conditions by inducing the Unfolded Protein Response (UPR), of which ERO1A is a mediator. ERO1A aids protein folding by acting as a protein disulfide oxidase, and under cancer-related hypoxia conditions, it favors the folding of angiogenic VEGFA, leading tumor cells to thrive and spread. The upregulation of ERO1A in cancer cells, oppositely to the dispensability of ERO1A activity in healthy cells, renders ERO1A a perfect target for cancer therapy. Here, we report the upregulation of ERO1A in a cohort of aggressive triple-negative breast cancer (TNBC) patients in which ERO1A levels correlate with a higher risk of breast tumor recurrence and metastatic spread. For ERO1A target validation and therapy in TNBC, we designed new ERO1A inhibitors in a structure-activity campaign of the prototype EN460. Cell-based screenings showed that the presence of the Micheal acceptor in the compound is necessary to engage the cysteine 397 of ERO1A but not sufficient to set out the inhibitory effect on ERO1A. Indeed, the ERO1 inhibitor must adopt a non-coplanar rearrangement within the ERO1A binding site. I2 and I3, two new EN460 analogs with different phenyl-substituted moieties, efficiently inhibited ERO1A, blunting VEGFA secretion. Accordingly, in vitro assays to measure ERO1A engagement and inhibition confirmed that I2 and I3 bind ERO1A and restrain its activity with a IC50 in a low micromolar range. EN460, I2 and I3 triggered breast cancer cytotoxicity while specifically inhibiting ERO1A in a dose-dependent manner. I2 more efficiently impaired cancer-relevant features such as VEGFA secretion and related cell migration. I2 also acted on the tumor microenvironment and viability of xenografts and syngeneic TNBC. Thus, small molecule-mediated ERO1A pharmacological inhibition is feasible and promises to lead to effective therapy for the still incurable TNBC.

Deposition of abnormally phosphorylated tau aggregates is a central event leading to neuronal dysfunction and death in Alzheimer's disease (AD) and other tauopathies. Among tau aggregates, oligomers (TauOs) are considered the most toxic. AD brains show significant increase in TauOs compared to healthy controls, their concentration correlating with the severity of cognitive deficits and disease progression. In vitro and in vivo neuronal TauO exposure leads to synaptic and cognitive dysfunction, but their mechanisms of action are unclear. Evidence suggests that the cellular prion protein (PrPC) may act as a mediator of TauO neurotoxicity, as previously proposed for β-amyloid and α-synuclein oligomers. To investigate whether PrPC mediates TauO detrimental activities, we compared their effects on memory and synaptic plasticity in wild type (WT) and PrPC knockout (Prnp0/0) mice. Intracerebroventricular injection of TauOs significantly impaired recognition memory in WT but not in Prnp0/0 mice. Similarly, TauOs inhibited long-term potentiation in acute hippocampal slices from WT but not Prnp0/0 mice. Surface plasmon resonance indicated a high-affinity binding between TauOs and PrPC with a KD of 20-50 nM. Immunofluorescence analysis of naïve and PrPC-overexpressing HEK293 cells exposed to TauOs showed a PrPC dose-dependent association of TauOs with cells over time, and their co-localization with PrPC on the plasma membrane and in intracellular compartments, suggesting PrPC-may play a role in TauO internalization. These findings support the concept that PrPC mediates the detrimental activities of TauOs through a direct interaction, suggesting that targeting this interaction might be a promising therapeutic strategy for AD and other tauopathies.

Global biodiversity is under accelerating threats, and species are succumbing to extinction before being described. Madagascar's biota represents an extreme example of this scenario, with the added complication that much of its endemic biodiversity is cryptic. Here we illustrate best practices for clarifying cryptic diversification processes by presenting an integrative framework that leverages multiple lines of evidence and taxon-informed cut-offs for species delimitation, while placing special emphasis on identifying patterns of isolation by distance. We systematically apply this framework to an entire taxonomically controversial primate clade, the mouse lemurs (genus Microcebus, family Cheirogaleidae). We demonstrate that species diversity has been overestimated primarily due to the interpretation of geographic variation as speciation, potentially biasing inference of the underlying processes of evolutionary diversification. Following a revised classification, we find that crypsis within the genus is best explained by a model of morphological stasis imposed by stabilizing selection and a neutral process of niche diversification. Finally, by clarifying species limits and defining evolutionarily significant units, we provide new conservation priorities, bridging fundamental and applied objectives in a generalizable framework.

The prognosis of patients with hepatocellular cancer is substantially correlated with the abnormal expression of growing long non-coding RNA small nucleolar host gene RNA (SNHG) families in liver cancer tissues. This study aimed to examine the relationship between SNHG expression and liver cancer prognosis. After searching six internet databases, pertinent manuscripts were found based on inclusion and exclusion criteria. To determine whether SNHG expression levels affect liver cancer prognosis, raw data were collected and hazard ratios (HRs) and odds ratios (ORs) were calculated. The results were examined for potential publication bias using the sensitivity analysis and Beeg's test. Most SNHG family members were up-regulated in liver cancer tissues. High SNHG expression predicts poor liver cancer outcomes of, including overall survival (OS) (HR: 1.697, 95% confidence interval [CI]: 1.373-2.021), especially SNHG5 (the HR of OS is 4.74, 95%CI range from 1.35 to 6.64), progression-free survival (HR: 1.85, 95% CI: 1.25-2.73), tumor, node, metastasis (TNM) stage (OR: 1.696, 95% CI: 1.436-2.005), lymph node metastasis (OR: 2.383, 95% CI: 1.098-5.173), and tumor size (OR: 1363, 95% CI: 1.165-1.595). The OS results were found to be reliable and robust, as indicated by the sensitivity analysis. Additionally, Beeg's test demonstrated the absence of any potential publication bias for each result. In liver cancer tissues, most SNHGs are highly expressed, which may signal poor prognosis. SNHG has the potential to be an intriguing predictive marker and a prospective therapeutic target for liver cancer.

How are some individuals surviving infections while others die? The answer lies in how infected individuals invest into controlling pathogen proliferation and mitigating damage, two strategies respectively called resistance and disease tolerance. Pathogen within-host dynamics (WHD), influenced by resistance, and its connection to host survival, determined by tolerance, decide the infection outcome. To grasp these intricate effects of resistance and tolerance, we used a deterministic theoretical model where pathogens interact with the immune system of a host. The model describes the positive and negative regulation of the immune response, consider the way damage accumulate during the infection and predicts WHD. When chronic, infections stabilize at a Set-Point Pathogen Load (SPPL). Our model predicts that this situation can be transient, the SPPL being then a predictor of life span which depends on initial condition (e.g. inoculum). When stable, the SPPL is rather diagnostic of non-lethal chronic infections. In lethal infections, hosts die at a Pathogen Load Upon Death (PLUD) which is almost independent from the initial conditions. As the SPPL, the PLUD is affected by both resistance and tolerance but we demonstrate that it can be used in conjunction with mortality measurement to distinguish the effect of disease tolerance from that of resistance. We validate empirically this new approach, using Drosophila melanogaster and the pathogen Providencia rettgeri. We found that, as predicted by the model, hosts that were wounded or deficient of key antimicrobial peptides had a higher PLUD, while Catalase mutant hosts, likely to have a default in disease tolerance, had a lower PLUD.

Tryptase is a tetrameric serine protease and a key component of mast cell granules. Here, we explored an integrated approach to characterize tryptase ligands, combining novel experimental binding studies using Surface Plasmon Resonance, with in silico analysis through the Exscalate platform. For this, we focused on three inhibitors previously reported in the literature, including a bivalent inhibitor and its corresponding monovalent compound. All three ligands showed concentration-dependent binding to immobilized human tryptase with the bivalent inhibitor showing the highest affinity. Furthermore, Rmax values were similar, indicating that the compounds occupy all four binding pockets of the tryptase tetramer. This hypothesis was supported by in silico computational analysis that revealed the binding mode of the monovalent ligand, one in each monomer pocket, compared with crystal structure of the bivalent one, which simultaneously occupies two binding pockets. Additionally, we solved the 2.06 Å X-ray crystal structures of human Tryptase beta-2 (hTPSB2), in both its apo form and in complex with compound #1, experimentally confirming the binding mode and the key molecular interactions predicted by docking studies for this compound. This integrated approach offers a robust framework for elucidating both the strength and mode of interaction of potential tryptase inhibitors.

Seasonal differences in insect pigmentation are attributed to the influence of ambient temperature on pigmentation development. This thermal plasticity is adaptive and heritable, and thereby capable of evolving. However, the specific genes contributing to the variation in plasticity that can drive its evolution remain largely unknown. To address this, we analysed pigmentation and pigmentation plasticity in Drosophila melanogaster. We measured two components of pigmentation in the thorax and abdomen: overall darkness and the proportion of length covered by darker pattern elements (a trident in the thorax and bands in the abdomen) in females from two developmental temperatures (17 or 28°C) and 191 genotypes. Using a GWAS approach to identify the genetic basis of variation in pigmentation and its response to temperature, we identified numerous dispersed QTLs, including some mapping to melanogenesis genes (yellow, ebony, and tan). Remarkably, we observed limited overlap between QTLs for variation within specific temperatures and those influencing thermal plasticity, as well as minimal overlap between plasticity QTLs across pigmentation components and across body parts. For most traits, consistent with selection favouring the retention of plasticity, we found that lower plasticity alleles were often at lower frequencies. The functional analysis of selected candidate QTLs and pigmentation genes largely confirmed their contributions to variation in pigmentation and/or pigmentation plasticity. Overall, our study reveals the existence and underlying basis of extensive and trait-specific genetic variation for pigmentation and pigmentation plasticity, offering a rich reservoir of raw material for natural selection to shape the evolution of these traits independently.

Chimeric antigen receptor (CAR) T cells represent a revolutionary immunotherapy that allows specific tumor recognition by a unique single-chain fragment variable (scFv) derived from monoclonal antibodies (mAbs). scFv selection is consequently a fundamental step for CAR construction, to ensure accurate and effective CAR signaling toward tumor antigen binding. However, conventional in vitro and in vivo biological approaches to compare different scFv-derived CARs are expensive and labor-intensive. With the aim to predict the finest scFv binding before CAR-T cell engineering, we performed artificial intelligence (AI)-guided molecular docking and steered molecular dynamics analysis of different anti-CD30 mAb clones. Virtual computational scFv screening showed comparable results to surface plasmon resonance (SPR) and functional CAR-T cell in vitro and in vivo assays, respectively, in terms of binding capacity and anti-tumor efficacy. The proposed fast and low-cost in silico analysis has the potential to advance the development of novel CAR constructs, with a substantial impact on reducing time, costs, and the need for laboratory animal use.

Amyloid-β1-42 (Aβ42) forms highly stable and insoluble fibrillar structures, representing the principal components of the amyloid plaques present in the brain of Alzheimer's disease (AD) patients. The involvement of Aβ42 in AD-associated neurodegeneration has also been demonstrated, in particular for smaller and soluble aggregates (oligomers). Based on these findings and on genetic evidence, Aβ42 aggregates are considered key players in the pathogenesis of AD and targets for novel therapies. Different approaches are currently used to detect the various aggregation states of Aβ peptide, including spectrophotometric methods, imaging techniques, and immunoassays, but all of these have specific limitations. To overcome them, we have recently exploited the peculiar properties of surface plasmon resonance (SPR) to develop an immunoassay capable of selectively detecting monomers and oligomers, discriminating them also from bigger fibrils in a mixture of different aggregated species, without any manipulation of the solution. In the present study, we extended these previous studies, showing that the SPR-based immunoassay makes it possible to unveil the fibril fragmentation induced mechanically, a result difficult to be conveniently and reliably assessed with other approaches. Moreover, we show that SPR-recognized fibril fragments are more toxic than the larger fibrillar structures, suggesting the relevance of the proposed SPR-based immunoassay.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

Alzheimer's disease (AD), the leading cause of dementia in older adults, is a double proteinopathy characterized by amyloid-β (Aβ) and tau pathology. Despite enormous efforts that have been spent in the last decades to find effective therapies, late pharmacological interventions along the course of the disease, inaccurate clinical methodologies in the enrollment of patients, and inadequate biomarkers for evaluating drug efficacy have not allowed the development of an effective therapeutic strategy. The approaches followed so far for developing drugs or antibodies focused solely on targeting Aβ or tau protein. This paper explores the potential therapeutic capacity of an all-D-isomer synthetic peptide limited to the first six amino acids of the N-terminal sequence of the A2V-mutated Aβ, Aβ1-6A2V(D), that was developed following the observation of a clinical case that provided the background for its development. We first performed an in-depth biochemical characterization documenting the capacity of Aβ1-6A2V(D) to interfere with the aggregation and stability of tau protein. To tackle Aβ1-6A2V(D) in vivo effects against a neurological decline in genetically predisposed or acquired high AD risk mice, we tested its effects in triple transgenic animals harboring human PS1(M146 V), APP(SW), and MAPT(P301L) transgenes and aged wild-type mice exposed to experimental traumatic brain injury (TBI), a recognized risk factor for AD. We found that Aβ1-6A2V(D) treatment in TBI mice improved neurological outcomes and reduced blood markers of axonal damage. Exploiting the C. elegans model as a biosensor of amyloidogenic proteins' toxicity, we observed a rescue of locomotor defects in nematodes exposed to the brain homogenates from TBI mice treated with Aβ1-6A2V(D) compared to TBI controls. By this integrated approach, we demonstrate that Aβ1-6A2V(D) not only impedes tau aggregation but also favors its degradation by tissue proteases, confirming that this peptide interferes with both Aβ and tau aggregation propensity and proteotoxicity.

We developed and validated a technology platform for designing and testing peptides inhibiting the infectivity of SARS-CoV-2 spike protein-based pseudoviruses. This platform integrates target evaluation, in silico inhibitor design, peptide synthesis, and efficacy screening. We generated a cyclic peptide library derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. The cell-free validation process by ELISA competition assays and Surface Plasmon Resonance (SPR) studies revealed that the cyclic peptide c9_05, but not its linear form, binds well to ACE2. Moreover, it effectively inhibited the transduction in HEK293, stably expressing the human ACE2 receptor of pseudovirus particles displaying the SARS-CoV-2 spike in the Wuhan or UK variants. However, the inhibitory efficacy of c9_05 was negligible against the Omicron variant, and it failed to impede the entry of pseudoviruses carrying the B.1.351 (South African) spike. These variants contain three or more mutations known to increase affinity to ACE2. This suggests further refinement is needed for potential SARS-CoV-2 inhibition. Our study hints at a promising approach to develop inhibitors targeting viral infectivity receptors, including SARS-CoV-2's. This platform also promises swift identification and evaluation of inhibitors for other emergent viruses.

The effect of liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E has been evaluated on the aggregation features of different amyloidogenic proteins: human Amyloid β1-40 (Aβ1-40), transthyretin (TTR) variant S52P, human β2microglobulin (β2m) variants ΔN6 and D76N, Serum Amyloid A (SAA). The formation of fibrillar aggregates of the proteins was investigated by ThioflavinT fluorescence assay and validated by Atomic Force Microscopy. The results show that liposomes are preventing the transition of non-aggregated forms to the fibrillar state, with stronger effects on Aβ1-40, β2m ΔN6 and SAA. Liposomes also induce disaggregation of the amyloid aggregates of all the proteins investigated, with stronger effects on Aβ1-40, β2 D76N and TTR.SPR assays show that liposomes bind Aβ1-40 and SAA aggregates with high affinity (KD in the nanomolar range) whereas binding to TTR aggregates showed a lower affinity (KD in the micromolar range). Aggregates of β2m variants showed both high and low affinity binding sites. Computed Structural analysis of protein fibrillar aggregates and considerations on the multidentate features of liposomes allow to speculate a common mechanism of action, based on binding the β-stranded peptide regions responsible for the amyloid formation. Thus, multifunctional liposomes perform as pharmacological chaperones with anti-amyloidogenic activity, with a promising potential for the treatment of a number of protein-misfolding diseases.Key messageAmyloidosis is a group of diseases, each due to a specific protein misfolding.Anti-amyloidogenic nanoparticles have been gaining the utmost importance as a potential treatment for protein misfolding disorders.Liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E showed anti-amyloidogenic activity.

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The monitoring of the blood levels of therapeutic antibodies and their immune responses is proposed to guide and optimize therapy with these expensive drugs. We describe a novel Surface Plasmon Resonance (SPR)-based assay suitable for the simultaneous determination of serum concentrations of infliximab and anti-infliximab antibodies. The real-time detection by SPR avoids the incubation/washing steps of commonly used methods, thus allowing faster and more reliable measurements, in particular for low-affinity anti-drug antibodies. This method proved to be highly reproducible and may be well applied to other biotherapeutics.

Gelsolin amyloidosis (AGel) is characterized by multiple systemic and ophthalmic features resulting from pathological tissue deposition of the gelsolin (GSN) protein. To date, no cure is available for the treatment of any form of AGel. More than ten single-point substitutions in the GSN gene are responsible for the occurrence of the disease and, among them, D187N/Y is the most widespread variant. These substitutions undergo an aberrant proteolytic cascade, producing aggregation-prone peptides of 5 and 8 kDa, containing the Gelsolin Amyloidogenic Core, spanning residues 182-192 (GAC182-192). Following a structure-based approach, we designed and synthesized three novel sequence-specific peptidomimetics (LB-5, LB-6, and LB-7) built on a piperidine-pyrrolidine unnatural amino acid. LB-5 and LB-6, but not LB-7, efficiently inhibit the aggregation of the GAC182-192 amyloidogenic peptides at sub-stoichiometric concentrations. These peptidomimetics resulted also effective in vivo, in a C. elegans-based assay, in counteracting the proteotoxicity of aggregated GAC182-192. These data pave the way to a novel pharmacological strategy against AGel and also validate a toolbox exploitable in other amyloidogenic diseases.

The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug's ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.