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Abstract: A lipopeptide is designed that contains an epitope from simian virus T-antigen (SV40T, PKKKRKV) conjugated to an N-terminal palmitoyl (C16-) moiety, with the aim to act as an effective cell-penetrating lipopeptide, with additional aggregation propensity conferred by the lipid chain. A combination of cryo-TEM and small-angle X-ray scattering (SAXS) is used to show that the lipopeptide forms micelles, but mixtures with DNA lead to formation of fractal cluster-like co-assemblies due to intercalation of the DNA and peptide. Spectroscopic studies using fluorescence and circular dichroism (along with fiber X-ray diffraction) show that the peptide interacts with DNA and inserts into the groove. Confocal microscopy along with flow cytometry confirms delivery of DNA into both HeLa and mouse embryonic stem cells (mESCs) in pluripotent state, and the system shows excellent cytocompatibility as confirmed by MTT assays. Our data indicate that the lipopeptide may outperform the DNA transfection agent lipofectamine in DNA delivery into these stem cells and it enables DNA delivery into the cytoplasm and nucleus.

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Abstract: Iron-sulfur clusters are essential cofactors for the accurate cellular function of many proteins. In eukaryotic cells, the biogenesis of most iron-sulfur clusters occurs in the mitochondria and involves the action of the Cys desulfurase supercomplex, which is activated by the protein frataxin (FXN). The decrease of FXN expression and/or function results in Friedreich’s ataxia (FRDA). In this work, several nanobodies specific to human FXN were selected via phage display, demonstrating a wide range of effects on Cys desulfurase activity and a strong interaction with FXN. Nanobody interaction stabilized wild-type and FRDA-related FXN variants in vitro. FXN-nanobody complexes were characterized by NMR, SAXS, and X-ray crystallography. Additionally, Nanobody expression was studied in human cells. The subcellular localization, direct interaction with FXN by in situ proximity ligation assay, effect on cell viability, Fe-S-dependent enzymatic activities, and oxygen consumption rates were analyzed. Significantly, nanobody expression did not alter these key metabolic variables, suggesting that the interaction with FXN did not disrupt the pathway. As a whole, our results suggest that nanobodies can serve as binding partners for mitochondrial FXN. However, the specific effect of the nanobodies on the conformational stability of FRDA-related FXN variants in cells should be investigated.

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Abstract: Multicomponent peptide nanostructures offer a powerful platform for designing functional materials, yet controlling their co-assembly remains a key challenge. Here, we harness electrostatic molecular recognition to drive the selective co-assembly of five amphiphilic ionic peptide binary mixtures (M1–M5). Our results revealed that charge distribution governs β-sheet strand alignment (parallel vs. antiparallel), assembly kinetics, and hydrogel viscoelasticity. Mixing stoichiometry and pH significantly influences co-assembly behavior, nanofiber morphology, and network structure (self-sorted vs. hetero-aggregated). At pH 7, equimolar mixtures undergo nucleation-driven co-assembly into hetero-aggregates, immediately forming well-defined nanofibers, while non-equimolar ratios yield altered morphologies. At a slightly acidic pH of 5–7, both E and K side chains are charged, enabling complementary ionic interactions that promote co-assembly and gelation. Outside this pH range, co-assembly is impaired. Notably, M1 forms β-sheets and hydrogels at acidic pH (≤4) via independent self-assembly of its components, suggesting self-sorted fibers. Overall, we demonstrate that tuning charge complementarity, ionization state, and stoichiometry enables precise control over the molecular, nanoscale, and mechanical properties of multicomponent peptide assemblies, providing a framework for the rational design of advanced peptide-based materials.

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Abstract: Candida glabrata is the second leading cause of mortality in immunocompromised patients hospitalized for invasive candidiasis (IC). Several drugs have been available to treat this disease for decades, such as polyenes, azoles, echinocandins, flucytosine, and, in critical cases, amphotericin B. However, these antifungals’ constant and routine use have led to the development of resistance mechanisms, making the design and development of new drugs indispensable. The first step for the design and subsequent synthesis of a new chemical molecule as a potential antifungal is the identification of new therapeutic targets. In that pathway, our working group has identified moonlight-like cell wall proteins (CWPs) in different Candida species that can act as potential antifungal targets. One of these moonlight-like CWPs is phosphoglycerate kinase (Pgk) from C. glabrata. Once Pgk was identified as a potential therapeutic target in different human pathogens, the first step to perform drug design against this moonlight-like CWP was the elucidation of the three-dimensional (3D) structure since the 3D structure is key to understanding the interactions between a drug candidate and its target at the molecular level. In the present work, we aimed to elucidate the 3D structure of C. glabrata Pgk. To elucidate the 3D structure of this protein, the recombinant protein was expressed, purified, and structurally resolved by means of a structural analysis by small-angle X-ray scattering (SAXS). Additionally, in order to evaluate its potential as a therapeutic target, we have performed molecular docking studies and enzymatic activity assays with pure Pgk using known antifungals amphotericin B, nystatin, and fluconazole and with the new plausible drugs, such as nilotinib and netupitant. Our results showed some similarities and differences with orthologous Pgk proteins from other organisms, which was expected since Pgk has been observed to have evolved in the kingdoms of life. Molecular docking studies showed that Pgk interacts with all of the compounds tested. In enzyme activity assays, a change in the kinetic parameter Km on the enzyme Pgk was observed in response to its interaction with nilotinib, netupitant, and amphotericin B. Thus, our results allow us to propose Pgk from C. glabrata as a possible therapeutic target against candidiasis. We consider it essential to design and develop new molecules specifically targeting this enzyme, which will contribute to a decrease in mortality associated with IC and improve the patient’s quality of life.

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Abstract: A liquid crystal (LC) polymethylsiloxane (PMS) with rod-like aromatic side-groups attached via an alkylene spacer and bearing three n-dodecyl end-tails is found to form an unusual cubic structure. In a normal LC double gyroid (DG), the two chiral subspaces, one each side of the G-surface, are occupied by one network each. Here each such network is split into two aromatic strands that wind around the central polysiloxane bundle, forming a double helix, resulting in a four-network gyroid (4NG). While in previous normal LC DGs the network twist was assumed to follow that of the subspace, in 4NG the twist sense of the double-helix is opposite to that of the subspace., i.e., while a right-handed subspace twists by +70.5° between junctions, the double-helix “supertwists” by −109.5°, and the opposite is true for the left-handed subspace. Detailed analysis by X-ray diffraction, DSC, and depolarized fluorescence (DF) shows a gradual but significant reversible change in the degree of mixing between the aromatic side groups and the polysiloxane backbones at 120 °C–130 °C in 4NG. Also, a significant increase in the system mobility starts only at ∼40 °C above the melting point, indicating persistence of local double-helical segments even in the melt.