Olive Pomace Extract Contains Low Molecular Weight Peptides and Possesses ACE Inhibitory Activity.

The aim of the present study was to determine the ACE inhibitory activity of aqueous extracts of olive pomace and to understand whether they represent a good source of bioactive LMW peptides for nutritional and pharmacological applications. We produced a water extract from olive pomace (var. Picual) and obtained its low molecular weight (LMW) fraction (<3 kDa). The calculated yield of extraction was 100.2 ± 7.9 mg of LMW peptides per 100 g of olive pomace. The olive pomace LMW fraction possessed strong ACE inhibitory activity (IC50 = 3.57 ± 0.22 µg prot/mL). The LMW fraction (<3 kDa) was analysed by nanoscale liquid chromatography-Orbitrap coupled with tandem mass spectrometry and de novo sequencing. Thirty new peptides, containing between 7-17 amino acids and molecular masses ranging 778-1354 Da, were identified by the Peaks database algorithm using the available Olea europaea (cv. Farga) genome database. Ten new peptides were also identified by Peaks de novo sequencing. The protein sources of twelve peptides detected in the database by Peaks DB were identified by BLAST search. The ACE inhibitory activity of the identified peptides was predicted by BIOPEP software. We conclude that olive pomace possesses ACE inhibitory activity and contains low molecular weight peptides with (predicted) biological activity. Olive pomace may represent a good source of peptides for nutritional and pharmaceutical applications. In our study, it has been shown that olive pomace possesses ACE inhibitory activity and contains low molecular weight peptides with (predicted) biological activity. Olive pomace may represent a good source of peptides for nutritional and pharmaceutical applications. More research is needed in order to identify the in vivo effects of olive pomace bioactive peptides.

Specific targeting of the NRF2/ß-TrCP axis promotes beneficial effects in NASH.

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease that compromises liver function, for which there is not a specifically approved medicine. Recent research has identified transcription factor NRF2 as a potential therapeutic target. However, current NRF2 activators, designed to inhibit its repressor KEAP1, exhibit unwanted side effects. Alternatively, we previously introduced PHAR, a protein-protein interaction inhibitor of NRF2/ß-TrCP, which induces a mild NRF2 activation and selectively activates NRF2 in the liver, close to normal physiological levels. Herein, we assessed the effect of PHAR in protection against NASH and its progression to fibrosis. We conducted experiments to demonstrate that PHAR effectively activated NRF2 in hepatocytes, Kupffer cells, and stellate cells. Then, we used the STAM mouse model of NASH, based on partial damage of endocrine pancreas and insulin secretion impairment, followed by a high fat diet. Non-invasive analysis using MRI revealed that PHAR protects against liver fat accumulation. Moreover, PHAR attenuated key markers of NASH progression, including liver steatosis, hepatocellular ballooning, inflammation, and fibrosis. Notably, transcriptomic data indicate that PHAR led to upregulation of 3 anti-fibrotic genes (Plg, Serpina1a, and Bmp7) and downregulation of 6 pro-fibrotic (including Acta2 and Col3a1), 11 extracellular matrix remodeling, and 8 inflammatory genes. Overall, our study suggests that the mild activation of NRF2 via the protein-protein interaction inhibitor PHAR holds promise as a strategy for addressing NASH and its progression to liver fibrosis.

Evaluation of Loop-Mediated Isothermal Amplification (LAMP) in Urine Samples for the Diagnosis of Imported Schistosomiasis.

Migratory flows and international travel are triggering an increase in imported cases of schistosomiasis in non-endemic countries. The present study aims to evaluate the effectiveness of the LAMP technique on patients' urine samples for the diagnosis of imported schistosomiasis in a non-endemic area in comparison to a commercial immunochromatographic test and microscopic examination of feces and urine. A prospective observational study was conducted in sub-Saharan migrants attending the Tropical Medicine Unit, Almería, Spain. For schistosomiasis diagnosis, serum samples were tested using an immunochromatographic test (Schistosoma ICT IgG-IgM). Stool and urine samples were examined by microcopy. Urine samples were evaluated by combining three LAMP assays for the specific detection of Schistosoma mansoni, S. haematobium, and for the genus Schistosoma. To evaluate the diagnostic accuracy, a latent class analysis (LCA) was performed. In total, 115 patients were included (92.2% male; median age: 28.3 years). Of these, 21 patients (18.3%) were diagnosed with schistosomiasis confirmed by microscopy, with S. haematobium being the most frequent species identified (18/115; 15.7%). The Schistosoma ICT IgG-IgM test result was 100% positive and Schistosoma-LAMP was 61.9% positive, reaching as high as 72.2% for S. haematobium. The sensitivity and specificity estimated by LCA, respectively, were: 92% and 76% for Schistosoma ICT IgG-IgM, 68% and 44% for Schistosoma-LAMP, and 46% and 97% for microscopy. In conclusion, the Schistosoma-LAMP technique presented a higher sensitivity than microscopy for the diagnosis of imported urinary schistosomiasis, which could improve the diagnosis of active infection, both in referral centers and in centers with limited experience or scarce resources and infrastructure.

Estimation of parasitaemia in imported falciparum malaria using the results of a combined rapid diagnostic test. No big help from haematological parameters.

BACKGROUND: Microscopy continues to be the mainstay for the evaluation of parasitaemia in malaria but requires laboratory support and microbiological experience. Other fast and simple methods are necessary. METHODS: A retrospective observational study of imported malaria treated from July-2007 to December-2020 was carried out to evaluate the association between the degree of parasitaemia and both rapid diagnostic tests (RDT) reactivity patterns and haematological parameters. Plasmodium falciparum monoinfections diagnosed by peripheral blood smear and/or polymerase chain reaction (PCR),which also had a positive RDT result in the same blood sample, were included in the study. RESULTS: A total of 273 patients were included. Most of them were male (n = 256; 93.8%) and visiting friends and relatives (VFR) travellers (n = 252; 92.3%). Patients with plasmodial lactate dehydrogenase (pLDH) or aldolase and histidine-rich protein 2 (HRP-2) co-reactivity (Pan/Pf pattern) had a parasitaemia range between 0 and 37% while those with just HRP-2 reactivity (P. falciparum pattern) had ranges between 0 and 1%. Not a single case of P. falciparum pattern was found for parasitaemia ranges greater than 1%, showing a negative predictive value of 100% for high parasitaemia. All the correlations between haematological parameters and parasitaemia resulted to be weak, with a maximum rho coefficient of -0.35 for lymphocytes and platelets, and of 0.40 for neutrophils-to-lymphocytes count ratio. Multivariate predictive models were constructed reflecting a poor predictive capacity. CONCLUSIONS: The reactivity pattern of RDT allows a rapid semi-quantitative assessment of P. falciparum parasitaemia in travellers with imported malaria, discriminating patients with lower parasite loads. Haematological parameters were not able to estimate parasitaemia with sufficient precision.

Come for the atmosphere, stay for the interactions: Deciphering small molecule partitioning into biomolecular condensates.

Optimizing pharmacokinetic properties remains challenging but is generally guided by a set of structural rules. However, no such rule set exists for intracellular distribution. Kilgore et al.1 have examined small molecule partitioning within biomolecular condensates, yielding findings that could open a new window in the drug design and discovery process.

Prostaglandins as Candidate Ligands for a Per-ARNT-Sim (PAS) Domain of Steroid Receptor Coactivator 1 (SRC1).

Steroid receptor coactivators (SRCs) comprise a family of three paralogous proteins commonly recruited by eukaryotic transcription factors. Each SRC harbors two tandem Per-ARNT-Sim (PAS) domains that are broadly distributed that bind small molecules and regulate interactions. Using computational docking, solution NMR, mass spectrometry, and molecular dynamics simulations, we show that the SRC1 PAS-B domain can bind to certain prostaglandins (PGs) either non-covalently to a surface that overlaps with the site used to engage transcription factors or covalently to a single, specific, conserved cysteine residue next to a solvent accessible hydrophobic pocket. This pocket is in proximity to the canonical transcription factor binding site, but on the opposite side of the domain, suggesting a potential mode of regulating transcriptional activator-coactivator interactions.

Synergistic Inhibition Guided Fragment-Linking Strategy and Quantitative Structure-Property Relationship Modeling To Design Inhalable Therapeutics for Asthma Targeting CSF1R.

The colony-stimulating factor-1 receptor (CSF1R) is a tyrosine-protein kinase that is a potential target for asthma therapeutics. We have applied a fragment-lead combination approach to identify small fragments that act synergistically with GW2580, a known inhibitor of CSF1R. Two fragment libraries were screened in combination with GW2580 by surface plasmon resonance (SPR). Binding affinity measurements confirmed that thirteen fragments bind specifically to the CSF1R, and a kinase activity assay further validated the inhibitory effect of these fragments. Several fragment compounds enhanced the inhibitory activity of the lead inhibitor. Computational solvent mapping, molecular docking, and modeling studies suggest that some of these fragments bind adjacent to the binding site of the lead inhibitor and further stabilize the inhibitor-bound state. Modeling results guided the computational fragment-linking approach to design potential next-generation compounds. The inhalability of these proposed compounds was predicted using quantitative structure-property relationships (QSPR) modeling based on an analysis of 71 drugs currently on the market. This work provides new insights into the development of inhalable small molecule therapeutics for asthma.

The Transcription Factor NRF2 Has Epigenetic Regulatory Functions Modulating HDACs, DNMTs, and miRNA Biogenesis.

The epigenetic regulation of gene expression is a complex and tightly regulated process that defines cellular identity and is associated with health and disease processes. Oxidative stress is capable of inducing epigenetic modifications. The transcription factor NRF2 (nuclear factor erythroid-derived 2-like 2) is a master regulator of cellular homeostasis, regulating genes bearing antioxidant response elements (AREs) in their promoters. Here, we report the identification of ARE sequences in the promoter regions of genes encoding several epigenetic regulatory factors, such as histone deacetylases (HDACs), DNA methyltransferases (DNMTs), and proteins involved in microRNA biogenesis. In this research, we study this possibility by integrating bioinformatic, genetic, pharmacological, and molecular approaches. We found ARE sequences in the promoter regions of genes encoding several HDACs, DNMTs, and proteins involved in miRNA biogenesis. We confirmed that NRF2 regulates the production of these genes by studying NRF2-deficient cells and cells treated with dimethyl fumarate (DMF), an inducer of the NRF2 signaling pathway. In addition, we found that NRF2 could be involved in the target RNA-dependent microRNA degradation (TDMD) of miR-155-5p through its interaction with Nfe2l2 mRNA. Our data indicate that NRF2 has an epigenetic regulatory function, complementing its traditional function and expanding the regulatory dimensions that should be considered when developing NRF2-centered therapeutic strategies.

Mapping the energy landscape of PROTAC-mediated protein-protein interactions.

A principal challenge in computational modeling of macromolecules is the vast conformational space that arises out of large numbers of atomic degrees of freedom. Recently, growing interest in building predictive models of complexes mediated by Proteolysis Targeting Chimeras (PROTACs) has led to the application of state-of-the-art computational techniques to tackle this problem. However, repurposing existing tools to carry out protein-protein docking and linker conformer generation independently results in extensive sampling of structures incompatible with PROTAC-mediated complex formation. Here we show that it is possible to restrict the search to the space of protein-protein conformations that can be bridged by a PROTAC molecule with a given linker composition by using a cyclic coordinate descent algorithm to position PROTACs into complex-bound configurations. We use this methodology to construct potential energy and solvation energy landscapes of PROTAC-mediated interactions. Our results suggest that desolvation of amino acids at interfaces could play a dominant role in PROTAC-mediated complex formation.

Accuracy of Three Serological Techniques for the Diagnosis of Imported Schistosomiasis in Real Clinical Practice: Not All in the Same Boat.

Schistosomiasis is a neglected tropical disease despite of being a major public health problem affecting nearly 240 million people in the world. Due to the migratory flow from endemic countries to Western countries, an increasing number of cases is being diagnosed in non-endemic areas, generally in migrants or people visiting these areas. Serology is the recommended method for screening and diagnosis of schistosomiasis in migrants from endemic regions. However, serological techniques have a highly variable sensitivity. The aim of this study was to evaluate retrospectively the sensitivity of three different serological tests used in real clinical practice for the screening and diagnosis of imported schistosomiasis in sub-Saharan migrant patients, using the detection of schistosome eggs in urine, faeces or tissues as the gold standard. We evaluated three different serological techniques in 405 sub-Saharan patients with confirmed schistosomiasis treated between 2004 and 2022: an enzyme-linked immunosorbent assay (ELISA), an indirect haemagglutination assay (IHA) and an immunochromatographic test (ICT). The overall sensitivity values obtained with the different techniques were: 44.4% for IHA, 71.2% for ELISA and 94.7% for ICT, respectively. According to species, ICT showed the highest sensitivity (S. haematobium: 94%, S. mansoni: 93.3%; and S. intercalatum/guineensis: 100%). In conclusion, our study shows that Schistosoma ICT has the best performance in real clinical practice, when compared to ELISA and IHA, in both S. mansoni and S. haematobium infections.

The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8+ T cells in chronic viral infection.

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

Binding sites in the epidermal growth factor receptor are responsible for bisphenol S effects on trophoblast cell invasion.

Bisphenol S (BPS) is an endocrine disrupting chemical and the second most abundant bisphenol detected in humans. We have recently demonstrated that in utero exposure to BPS reduces human placenta cell fusion by interfering with epidermal growth factor (EGF)-dependent EGF receptor (EGFR) activation. Our previous work suggests that this occurs via binding of BPS to the extracellular domain of EGFR. However, whether BPS directly binds to EGFR has not been confirmed. We evaluated the binding ability of BPA, BPF and BPS to EGFR to determine whether EGFR binding is a unique attribute of BPS. To test these hypotheses, we first exposed HTR-8/SVneo cells to BPS, BPA, or BPF, with or without EGF. When co-exposed to EGF, BPS, but not BPA nor BPF, reduced EGFR phosphorylation by ∼60%, demonstrating that only BPS can interfere with EGF-dependent EGFR activation. As this indicates that BPS binding to the extracellular domain is responsible for its effect, we performed a computational search for putative binding sites on the EGFR extracellular domain, and performed ligand docking of BPS, BPA, and BPF at these sites. We identified three sites where polar interactions between positively charged residues and the sulfonyl group of BPS could lead binding selectivity over BPA and BPF. To test whether EGFR mutations at the predicted BPS binding sites (Arg255, Lys454, and Arg297) could prevent BPS's interference on EGFR activation, mutations for each EGFR target amino acids (R255A, R297A, and K454A) were introduced. For variants with R297A or K454A mutations, BPS did not affect EGF-mediated EGFR phosphorylation or EGFR-mediated cell invasion, suggesting that these residues are needed for the BPS antagonism effect on EGFR. In conclusion, BPS, but not BPA or BPF, interferes with EGFR-mediated trophoblast cell functions through binding at Arg297 and Lys454 amino acid residues in the extracellular domain of EGFR.

Blocking interleukin-23 ameliorates neuromuscular and thymic defects in myasthenia gravis.

Acetylcholine receptor (AChR) myasthenia gravis (MG) is a chronic autoimmune disease characterized by muscle weakness. The AChR+ autoantibodies are produced by B-cells located in thymic ectopic germinal centers (eGC). No therapeutic approach is curative. The inflammatory IL-23/Th17 pathway is activated in the thymus as well as in the blood and the muscle, contributing to the MG pathogenic events. We aimed to study a potential new therapeutic approach that targets IL-23p19 (IL-23) in the two complementary preclinical MG models: the classical experimental MG mouse model (EAMG) based on active immunization and the humanized mouse model featuring human MG thymuses engrafted in NSG mice (NSG-MG). In both preclinical models, the anti-IL-23 treatment ameliorated MG clinical symptoms. In the EAMG, the treatment reduced IL-17 related inflammation, anti-AChR IgG2b antibody production, activated transduction pathway involved in muscle regeneration and ameliorated the signal transduction at the neuromuscular junction. In the NSG-MG model, the treatment reduced pathogenic Th17 cell population and expression of genes involved in eGC stabilization and B-cell development in human MG thymus biopsies. Altogether, these data suggest that a therapy targeting IL-23p19 may promote significant clinical ameliorations in AChR+ MG disease due to concomitant beneficial effects on the thymus and skeletal muscle defects.

GATA-3 is a proto-oncogene in T-cell lymphoproliferative neoplasms.

Neoplasms originating from thymic T-cell progenitors and post-thymic mature T-cell subsets account for a minority of lymphoproliferative neoplasms. These T-cell derived neoplasms, while molecularly and genetically heterogeneous, exploit transcription factors and signaling pathways that are critically important in normal T-cell biology, including those implicated in antigen-, costimulatory-, and cytokine-receptor signaling. The transcription factor GATA-3 regulates the growth and proliferation of both immature and mature T cells and has recently been implicated in T-cell neoplasms, including the most common mature T-cell lymphoma observed in much of the Western world. Here we show that GATA-3 is a proto-oncogene across the spectrum of T-cell neoplasms, including those derived from T-cell progenitors and their mature progeny, and further define the transcriptional programs that are GATA-3 dependent, which include therapeutically targetable gene products. The discovery that p300-dependent acetylation regulates GATA-3 mediated transcription by attenuating DNA binding has novel therapeutic implications. As most patients afflicted with GATA-3 driven T-cell neoplasms will succumb to their disease within a few years of diagnosis, these findings suggest opportunities to improve outcomes for these patients.

Molecular and environmental determinants of biomolecular condensate formation.

Biomolecular condensate formation has been implicated in a host of biological processes and has found relevance in biology and disease. Understanding the physical principles and underlying characteristics of how these macromolecular assemblies form and are regulated has become a central focus of the field. In this Review, we introduce features of phase-separating biomolecules from a general physical viewpoint and highlight how molecular features, including affinity, valence and a competition between inter- and intramolecular contacts, affect phase separation. We then discuss sequence properties of proteins that serve to mediate intermolecular interactions. Finally, we review how the intracellular environment can affect structural and sequence determinants of proteins and modulate physical parameters of their phase transitions. The works reviewed highlight that a complex interplay exists between structure, sequence and environmental determinants in the formation of biomolecular condensates.

Dipeptide Repeat Pathology in C9orf72-ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance.

The hexanucleotide expansion of the C9orf72 gene is found in 40% of familial amyotrophic lateral sclerosis (ALS) patients. This genetic alteration has been connected with impaired management of reactive oxygen species. In this study, we conducted targeted transcriptional profiling in leukocytes from C9orf72 patients and control subjects by examining the mRNA levels of 84 redox-related genes. The expression of ten redox genes was altered in samples from C9orf72 ALS patients compared to healthy controls. Considering that Nuclear factor erythroid 2-Related Factor 2 (NRF2) modulates the expression of a wide range of redox genes, we further investigated its status on an in vitro model of dipeptide repeat (DPR) toxicity. This model mimics the gain of function, toxic mechanisms attributed to C9orf72 pathology. We found that exposure to DPRs increased superoxide levels and reduced mitochondrial potential as well as cell survival. Importantly, cells overexpressing DPRs exhibited reduced protein levels of NRF2 and its target genes upon inhibition of the proteasome or its canonical repressor, the E3 ligase adapter KEAP1. However, NRF2 activation was sufficient to recover cell viability and redox homeostasis. This study identifies NRF2 as a putative target in precision medicine for the therapy of ALS patients harboring C9orf72 expansion repeats.

Misleading eosinophil counts in migration-associated malaria: do not miss hidden helminthic co-infections.

BACKGROUND: Lower eosinophil counts observed during acute malaria episodes could hide helminth-related eosinophilia. METHOD: Retrospective observational study with sub-Saharan migrants with imported malaria from May-2007 to May-2020. Absolute eosinophil count was determined upon diagnosis at hospital admission and at least once after clearance of parasitemia. Helminthic co-infections were investigated by searching for stool and urine parasites, serology for Strongyloides spp. and Schistosoma spp., and Knott and/or saponin tests for blood microfilariae. RESULTS: A total of 259 patients were included. Most of them were male (n = 237; 91.5%) and VFR travelers (n = 241; 93.1%). 131 patients (50.6%) were diagnosed with probable schistosomiasis, 15 (5.8%) with confirmed schistosomiasis, 16 (6.2%) with strongyloidiasis, 4 (1.6%) with soil-transmitted helminthiasis, and 4 (1.6%) with filariasis (Mansonella perstans). Prevalence of eosinophilia increased from 2.7% on admission to 32.5% during outpatient follow-up. Eosinophilia did not appear until several weeks after hospital discharge in up to 24% of the confirmed helminthic co-infections and in 61.1% of patients with probable schistosomiasis. Eosinophilia was associated with confirmed schistosomiasis and mansonellosis while 56.2% and 75% of cases with strongyloidiasis and soil-transmitted worms did not present eosinophilia at any time, respectively. CONCLUSIONS: Regardless of the absence of eosinophilia, patients hospitalized because of acute imported malaria might benefit from the screening of the main parasitic diseases, allowing for earlier diagnosis and treatment.

Microstrip-Fed 3D-Printed H-Sectorial Horn Phased Array.

A 3D-printed phased array consisting of four H-Sectorial horn antennas of 200 g weight with an ultra-wideband rectangular-waveguide-to-microstrip-line transition operating over the whole LMDS and K bands (24.25-29.5 GHz) is presented. The transition is based on exciting three overlapped transversal patches that radiate into the waveguide. The transition provides very low insertion losses, ranging from 0.30 dB to 0.67 dB over the whole band of operation (23.5-30.4 GHz). The measured fractional bandwidth of the phased array including the transition was 20.8% (24.75-30.3 GHz). The antenna was measured for six different scanning angles corresponding to six different progressive phases α, ranging from 0° to 140° at the central frequency band of operation of 26.5 GHz. The maximum gain was found in the broadside direction α = 0°, with 15.2 dB and efficiency η = 78.5%, while the minimum was found for α = 140°, with 13.7 dB and η = 91.2%.

A unified statistical potential reveals that amino acid stickiness governs nonspecific recruitment of client proteins into condensates.

Membraneless organelles are cellular compartments that form by liquid-liquid phase separation of one or more components. Other molecules, such as proteins and nucleic acids, will distribute between the cytoplasm and the liquid compartment in accordance with the thermodynamic drive to lower the free energy of the system. The resulting distribution colocalizes molecular species to carry out a diversity of functions. Two factors could drive this partitioning: the difference in solvation between the dilute versus dense phase and intermolecular interactions between the client and scaffold proteins. Here, we develop a set of knowledge-based potentials that allow for the direct comparison between stickiness, which is dominated by desolvation energy, and pairwise residue contact propensity terms. We use these scales to examine experimental data from two systems: protein cargo dissolving within phase-separated droplets made from FG repeat proteins of the nuclear pore complex and client proteins dissolving within phase-separated FUS droplets. These analyses reveal a close agreement between the stickiness of the client proteins and the experimentally determined values of the partition coefficients (R > 0.9), while pairwise residue contact propensities between client and scaffold show weaker correlations. Hence, the stickiness of client proteins is sufficient to explain their differential partitioning within these two phase-separated systems without taking into account the composition of the condensate. This result implies that selective trafficking of client proteins to distinct membraneless organelles requires recognition elements beyond the client sequence composition. STATEMENT: Empirical potentials for amino acid stickiness and pairwise residue contact propensities are derived. These scales are unique in that they enable direct comparison of desolvation versus contact terms. We find that partitioning of a client protein to a condensate is best explained by amino acid stickiness.